Structural and mechanistic insights into Streptococcus pneumoniae NADPH oxidase.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including Streptococcus pneumoniae NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function.

Mortalidad intrahospitalaria de pacientes con COVID-19 complicados con neumonías bacterianas en asistencia respiratoria mecánica en Cuidados Intensivos de Adultos en un Hospital del Paraguay / In-hospital mortality of patients with COVID-19 complicated by bacterial pneumonia on mechanical ventilation in Adult Intensive Care in a Hospital in Paraguay

Introducción: los pacientes con COVID-19 ingresan en mayor proporción a asistencia respiratoria mecánica, aumentando: el riesgo de neumonía asociada a ventilador (NAV) las tasas de mortalidad, los días de permanencia en las unidades de terapia intensiva (UCI) y los costos sanitarios. Objetivo: determinar la Mortalidad intrahospitalaria de pacientes con COVID-19 complicados con neumonías bacterianas en asistencia respiratoria mecánica en Cuidados Intensivos de Adultos en un Hospital del Paraguay durante los años 2020 a 2021. Metodología: estudio analítico de tipo cohorte retrospectiva. Se registraron variables demográficas, comorbilidades, puntajes en scores de gravedad como el APACHE II al ingreso, la cifra más baja de oxigenación durante la internación expresado por la PaO2 / FIO2, días de ventilación, colocación en decúbito prono, traqueotomía, medidas terapéuticas farmacológicas y no farmacológicas, días de internación, así como las complicaciones y la mortalidad. Resultados: fueron incluidos 214 pacientes, 135 ingresaron a asistencia respiratoria mecánica (ARM) de los cuales 58 (42,9 %) desarrollaron NAV, con edad mediana de 52 años (40-60). Los microorganismos de NAV fueron cocos Gram negativos en 98,3 %, incluyendo Acinetobacter baumanii en 46,5 %, Klebsiella pneumoniae en 22,8 %, Pseudomona aeruginosa en 15,5 % y 5,2 % Stenotrophomona maltofilia. La mortalidad intrahospitalaria fue del 44,8 %. Los menores de 50 años tienen una sobrevida mayor que los mayores (34 días vs 22 días, con p de 0,026). Conclusión: la mortalidad intrahospitalaria fue del 44,8 %. La edad fue un factor de riesgo independiente para la mortalidad en pacientes con NAV, por lo que los profesionales de la salud deben estar atentos a la posibilidad de NAV en pacientes que requieren asistencia respiratoria mecánica, especialmente en pacientes mayores de 50 años.

Introduction: patients with COVID-19 are more likely to require mechanical ventilation, which increases the risk of ventilator-associated pneumonia (VAP), mortality rates, length of stay in intensive care units (ICUs), and healthcare costs. Objective: to determine the in-hospital mortality of patients with COVID-19 complicated by bacterial pneumonia on mechanical ventilation in Adult Intensive Care in a Hospital in Paraguay during the years 2020 to 2021. Methodology: this is a retrospective cohort analytical study. Demographic variables, comorbidities, severity scores such as APACHE II on admission, the worst oxygenation during hospitalization expressed by PaO2/FiO2, days of ventilation, prone position, tracheostomy, pharmacological and non-pharmacological therapeutic measures, days of hospitalization, as well as complications and mortality were recorded. Results: a total of 214 patients were included, 135 were admitted to mechanical ventilation (MRA), of which 58 (42.9%) developed VAP, with a median age of 52 years (40-60). VAP microorganisms were Gram-negative cocci in 98.3%, including Acinetobacter baumanii in 46.5%, Klebsiella pneumoniae in 22.8%, Pseudomona aeruginosa in 15.5%, and Stenotrophomona maltophilia in 5.2%. In-hospital mortality was 44.8%. Those under 50 years of age have a longer survival than those older (34 days vs. 22 days, with p of 0.026). Conclusion: the overall mortality rate was 44.8%. Age was an independent risk factor for mortality in patients with VAP, so healthcare professionals should be aware of the possibility of VAP in patients who require mechanical ventilation, especially in patients over 50 years of age.

Effectiveness of COVID-19 vaccines in Ecuador: A test-negative design.

Background: The COVID-19 pandemic poses a significant global health threat, characterized by high morbidity, severity, and the emergence of concerning variants. Latin America has been greatly affected, with high infection and mortality rates. Vaccination plays a crucial role in mitigating severe disease and controlling the pandemic. This study aims to assess the effectiveness of COVID-19 vaccines in preventing SARS-CoV-2 severe acute respiratory infections (SARI) in hospitalized vaccination target groups in Ecuador. Methods: This is a test-negative design study. We used data reported through sentinel surveillance of SARI between May 2021 and March 2022 in Ecuador. Patients with case criteria of SARI and hospitalized for a minimum of 24 hours were included in the study. Cases were defined as patients with SARI with a positive RT-qPCR test for SARS-CoV-2 and controls were those with a negative result. Information on vaccination status was obtained from the national vaccination registry, a valid dose of vaccination was considered when it was administered at least 14 days prior to symptom onset. Vaccine effectiveness (VE) (1-OR/OR) was calculated using a logistic regression. Results: A total of 1,277 patients were included in the analysis of VE. The adjusted vaccine effectiveness (aVE) in preventing hospitalization, adjusted for sex, age group, presence of one or more comorbidities, and period of the predominance of the omicron variant, was 44.5% for the partial primary schedule, 74.7% for the complete primary schedule, and 79.9% for the complete primary schedule plus booster doses. The aVE in avoiding ICU admissions was close to 80% with both the complete primary schedule and the booster doses, and in avoiding deaths, the aVE was 89% and 98%, respectively. Conclusions: In Ecuador, COVID-19 vaccination prevents hospitalizations, ICU admissions, and deaths. The effectiveness of the vaccines improves with more doses, offering increased protection across all age groups.

Challenges in radiobiology - technology duality as a key for a risk-free α/ß ratio.

Since radiotherapy discovery, prediction of biological response to ionizing radiation remains a major challenge. Indeed, several radiobiological models appeared through radiotherapy history. Nominal single dose so popular in the 1970s, was tragically linked to the dark years in radiobiology by underestimating the late toxicity of the high-dose fractions. The actual prominent linear-quadratic model continues to prove to be an effective tool in radiobiology. Mainly with its pivotal α/ß ratio, which gives a reliable estimate of tissues sensitivity to fractions. Despite these arguments, this model experiences limitations with substantial doubts of α/ß ratio values. Interestingly, the story of radiobiology since X-ray discovery is truly instructive and teaches modern clinicians to refine fractionation schemes. Many fractionation schemes have been tested with successes or dramas. This review retraces radiobiological models' history, and confronts these models to new fractionation schemes, drawing a preventive message.

hTERT and IGF-1R Proteins Expression in Response to Treatment in Patients with HPV Alpha 9-Positive Cervical Cancer.

Human papillomavirus (HPV) infection is strongly associated with cervical cancer (CC). Genomic alterations caused by viral infection and subsequent dysregulation of cellular metabolism under hypoxic conditions could influence the response to treatment. We studied a possible influence of IGF-1Rb, hTERT, HIF1a, GLUT1 protein expression, HPV species presence and relevant clinical parameters on the response to treatment. In 21 patients, HPV infection and protein expression were detected using GP5+/GP6+PCR-RLB and immunohistochemistry, respectively. The worse response was associated with radiotherapy alone compared with chemoradiotherapy (CTX-RT), anemia and HIF1a expression. HPV16 type was the most frequent (57.1%) followed by HPV-58 (14.2%) and HPV-56 (9.5%). The HPV alpha 9 species was the most frequent (76.1%) followed by alpha 6 and alpha 7. IGF-1Rb (85.7%), HIF1a (61.9%), GLUT1 (52.3%), and hTERT expression [cytoplasm and nucleus (90.4%)] were detected. The MCA factorial map showed different relationships, standing out, expression of hTERT and alpha 9 species HPV, expression of hTERT and IGF-1Rb expression [Fisher's exact test (P = 0.04)]. A slight trend of association was observed between, GLUT1 and HIF1 a expression, hTERT and GLUT1 expression. A noteworthy finding was the subcellular localization of hTERT in the nucleus and cytoplasm of CC cells and its possible interaction with IGF-1R in presence of HPV alpha 9 species. Our findings suggest that the expression of HIF1a, hTERT, IGF-1Rb and GLUT1 proteins that interact with some HPV species may contribute to cervical cancer development, and the modu lation of treatment response.

Impact of Radiation Therapy on Biological Parameters in Cancer Patients: Sub-analysis from the RIT Prospective Epidemiological Study.

Scarce data investigate the impact of radiotherapy (RT) on biology markers. An analysis of ancillary study of RIT (Radiation Impact on Thromboembolic events) prospective trial was carried out. All patients with non-metastatic solid tumors and treated with radiotherapy and/or brachytherapy in curative and consenting to have blood samples were included. A significant decrease in white blood count, (i.e. lymphocytes, monocytes, neutrophils and basophils) and platelet counts was observed after RT and maintained at 6 months. Whereas, eosinophils, D-dimers and hemoglobin levels were affected respectively 3 months and 6 months after RT initiation. Conversely, red cells count and CRP level were not affected by RT. This study is an advocacy to develop an understanding of basic immune system in relation with RT.

Therapeutic hypothermia for the management of hypoxic-ischemic encephalopathy in asphyxiated newborns: Clinical, electrographic and radiologic features of newborns treated at Instituto Materno Perinatal / Therapeutic hypothermia for the management of hypoxic-ischemic encephalopathy in asphyxiated newborns: Clinical, electrographic and radiological features of newborns treated at Instituto Materno Perinatal

La encefalopatía hipóxico-isquémica (EHI) es el síndrome neurológico causado por la asfixia perinatal. La hipotermia terapéutica (HT) ha demostrado reducir la mortalidad y morbilidad asociadas a EHI. Se realizó un estudio descriptivo retrospectivo con 30 recién nacidos con EHI moderada y severa que recibieron HT en la Unidad de Cuidados Intensivos del Instituto Materno Perinatal desde setiembre de 2017 a noviembre de 2020. Nueve de los casos fueron severos (30 %). El tiempo promedio de ingreso a HT fue 3.4 horas de vida. No se registraron efectos adversos importantes atribuibles a HT. Todos los pacientes severos tuvieron crisis epilépticas, ecografías cerebrales de ingreso y resonancias con anormalidades. La mortalidad fue de 20.0 %, aunque fue significativamente menor en el grupo con EHI moderada. Se identificaron las características de presentación clínica, electrográfica y radiológica de los neonatos con EHI que recibieron hipotermia terapéutica, la cual se muestra como un procedimiento seguro y efectivo.

Hipotermia terapéutica para tratamiento de encefalopatía hipóxico-isquémica del recién nacido asfixiado: Características clínicas, radiológicas y electrográficas de los neonatos atendidos en el Instituto Nacional Materno Perinatal

La encefalopatía hipóxico-isquémica (EHI) es el síndrome neurológico causado por la asfixia perinatal. La hipotermia terapéutica (HT) ha demostrado reducir la mortalidad y morbilidad asociadas a EHI. Se realizó un estudio descriptivo retrospectivo con 30 recién nacidos con EHI moderada y severa que recibieron HT en la Unidad de Cuidados Intensivos del Instituto Materno Perinatal desde setiembre de 2017 a noviembre de 2020. Nueve de los casos fueron severos (30 %). El tiempo promedio de ingreso a HT fue 3.4 horas de vida. No se registraron efectos adversos importantes atribuibles a HT. Todos los pacientes severos tuvieron crisis epilépticas, ecografías cerebrales de ingreso y resonancias con anormalidades. La mortalidad fue de 20.0 %, aunque fue significativamente menor en el grupo con EHI moderada. Se identificaron las características de presentación clínica, electrográfica y radiológica de los neonatos con EHI que recibieron hipotermia terapéutica, la cual se muestra como un procedimiento seguro y efectivo.

Hypoxic-ischemic encephalopathy (HIE) is the neurological syndrome caused by perinatal asphyxia. Therapeutic hypothermia (TH) has been shown to reduce HIE-associated morbidity and mortality. A descriptive and retrospective study with 30 newborns with moderate and severe HIE who underwent TH in the Intensive Care Unit (ICU) of Instituto Materno Perinatal, from September 2017 until November 2020. Nine patients were severely affected (30%). The average tome for being admitted in the ICU was at 3.4 hours of life. No important adverse effects attributable to TH were observed. All severely affected patients experienced epileptic crises, and abnormal cerebral ultrasonography and magnetic resonance imaging studies on admission. Mortality was 20.0%, but it was significantly lower in the group with moderate HIE. Clinical, electrographic, and radiological characteristics of neonates with HIE who underwent therapeutic hypothermia were identified. This procedure has been shown to be safe and effective.

KRAS Promoter Methylation Status and miR-18a-3p and miR-143 Expression in Patients With Wild-type KRAS Gene in Colorectal Cancer.

BACKGROUND/AIM: Although some mutations of KRAS proto-oncogene, GTPase (KRAS) have been associated with the prognosis and therapeutic management of colorectal cancer (CRC), the epigenetic mechanisms (DNA methylation and microRNA expression) that regulate wild-type KRAS expression in patients with CRC are poorly known. The aim of this study was to establish whether there is a relationship between the expression of the wild-type KRAS gene, the methylation status of its distal promoter, and miR-143 and miR-18a-3p levels in samples of sporadic CRC. PATIENTS AND METHODS: A total of 51 cases of sporadic CRC with wild-type KRAS were analyzed. The expression levels of KRAS mRNA, miR-18a-3p, miR-143, and KRAS protein, as well as methylation in the distal promoter of the KRAS gene were evaluated. RESULTS: In the analyzed cases, KRAS mRNA expression was detected in 51.1%; wild-type KRAS protein was found in the membrane in 31.4% and in the cytoplasm in 98% of cases. An inverse relationship of marginal significance was observed between miR-18a-3p and KRAS protein expression in the cytoplasm (odds ratio=0.14, 95% confidence interval=0.012-1.092; p=0.08). The methylation status of the distal promoter of KRAS at four CpG islands was analyzed in 30 cases (58.8%): partial methylation of the four CpG islands evaluated was observed in two cases (6.7%). In these cases, KRAS protein expression was not evidenced at the membrane level; miR-18a-3p expression was not detected either but high expression of miR-143 was observed. CONCLUSION: No association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.

Erector Spinae Plane Block in pediatric cancer pain: Case report / Bloqueo del plano erector espinal en dolor oncológico pediátrico: reporte de caso

Abstract The advent of the erector spinae plane block brought a new therapeutic option in a multimodal analgesia strategy, as evidenced in this case, which describes a five-year old pre-school patient who presented with severe abdominal cancer pain, secondary to an abdominal neuroblastoma, with partial high-dose opioid response, undergoing bilateral erector spinal plane block. The technique used did not give rise to complications and proved to be effective in blocking pain and reducing the opioid dosage 36 hours after the procedure. The paper discusses the variables involved in the administration mode (continuous infusion vs. bolus) and the benefit for optimal analgesia in the pediatric oncology setting.

Resumen Con la aparición del bloqueo del plano erector espinal surgen nuevas alternativas terapéuticas dentro de una estrategia de analgesia multimodal, tal como se puede apreciar en este caso, en el cual se describe un paciente preescolar de cinco años, quien cursó con dolor abdominal oncológico intenso secundario a neuroblastoma abdominal con respuesta parcial a opioides en dosis altas y en el que se empleó el bloqueo mencionado aplicado bilateralmente. La técnica empleada no generó complicaciones y demostró ser efectiva al permitir el control del dolor y la disminución de las dosis de opioides en las 36 horas posteriores a su colocación. Se plantea la discusión de variables con relación a la forma de administración (infusión continua vs. bolo) y la utilidad en la optimización analgésica en el contexto oncológico pediátrico.

Dose to pelvic lymph nodes in locally advanced cervical cancer during high-dose-rate brachytherapy with tandem-ring applicators.

Purpose: The present study aimed to assess the correlation between dose to pelvic lymph nodes and to point B with tandem-ring (TR) applicators for intra-cavitary brachytherapy treatment of locally advanced cervical cancer. Material and methods: Cervical cancer patients treated at brachytherapy department of Lucien Neuwirth Cancer Center, from 2015 to 2018, were included. Target delineation was performed in compliance with GEC-ESTRO guidelines. Revised American Brachytherapy Society (ABS) point A was determined (ARN (right) and ALN (left)) as well as Manchester point B. Prescription dose was 25-35 Gy in 5 fractions. Pelvic lymph nodes were delineated, then dose to points A and B, and dose-volume histogram (DVH) parameters of delineated lymph nodes were extracted. Significant relationships or correlations between lymph nodes reference points, dosage to points B, and their DVH parameters were investigated. Results: The mean dose and mean percentage of the prescription dose to the left and right points B were 4.6 ±0.18 Gy and 82.08 ±0.72%, respectively. Pearson correlation coefficient R = 0.81 (p-value = 0.00) between dose to ARN and ALN points and prescription dose was obtained. Negative correlation between CTVHR volume and difference between French and ABS prescription points was found. Conclusions: Dose to point B can be a moderate surrogate for maximum, minimum, and median dose to the internal iliac and presacral lymph node, but cannot be for maximum dose to the obturator lymph node. Points B cannot be a reliable substitute for common and external iliac chains.

A Review of the Role of Hypoxia in Radioresistance in Cancer Therapy.

Hypoxia involves neoplastic cells. Unlike normal tissue, solid tumors are composed of aberrant vasculature, leading to a hypoxic microenvironment. Hypoxia is also known to be involved in both metastasis initiation and therapy resistance. Radiotherapy is the appropriate treatment in about half of all cancers, but loco-regional control failure and a disease recurrence often occur due to clinical radioresistance. Hypoxia induces radioresistance through a number of molecular pathways, and numerous strategies have been developed to overcome this. Nevertheless, these strategies have resulted in disappointing results, including adverse effects and limited efficacy. Additional clinical studies are needed to achieve a better understanding of the complex hypoxia pathways. This review presents an update on the mechanisms of hypoxia in radioresistance in solid tumors and the potential therapeutic solutions.

Multiomics Profiling of Alzheimer's Disease Serum for the Identification of Autoantibody Biomarkers.

New biomarkers of Alzheimer's disease (AD) with a diagnostic value in preclinical and prodromal stages are urgently needed. AD-related serum autoantibodies are potential candidate biomarkers. Here, we aimed at identifying AD-related serum autoantibodies using protein microarrays and mass spectrometry-based methods. To this end, an untargeted complementary screening using high-density (42,100 antigens) and low-density (384 antigens) planar protein-epitope signature tag (PrEST) arrays and an immunoprecipitation protocol coupled to mass spectrometry analysis were used for serum autoantibody profiling. From the untargeted screening phase, 377 antigens corresponding to 338 proteins were selected for validation. Out of them, IVD, CYFIP1, and ADD2 seroreactivity was validated using 128 sera from AD patients and controls by PrEST-suspension bead arrays, and ELISA or luminescence Halotag-based bead immunoassay using full-length recombinant proteins. Importantly, IVD, CYFIP1, and ADD2 showed in combination a noticeable AD diagnostic ability. Moreover, IVD protein abundance in the prefrontal cortex was significantly two-fold higher in AD patients than in controls by western blot and immunohistochemistry, whereas CYFIP1 and ADD2 were significantly down-regulated in AD patients. The panel of AD-related autoantigens identified by a comprehensive multiomics approach may provide new insights of the disease and should help in the blood-based diagnosis of Alzheimer's disease. Mass spectrometry raw data are available in the ProteomeXchange database with the access number PXD028392.

Genetic Analysis in Anal and Cervical Cancer: Exploratory Findings About Radioresistance in the ProfiLER Database.

BACKGROUND/AIM: This study aimed to describe genomic alterations on squamous cell cervical and anal carcinomas. MATERIALS AND METHODS: From 2013 to 2019, 3,269 patients were included in the molecular screening ProfiLER trial. Only patients with non-metastatic cervical or anal cancer, and those initially treated with radiotherapy in a curative intent were selected. Genetic analyses were performed by next generation sequencing (NGS). RESULTS: Genomic alterations were observed in most patients: 5 patients out of 15 (33.3%) had at least one mutation on NGS and 4 out of 15 (26.7%) had at least one aberration of the number of copies of genes in the comparative genomic hybridation (CGH) analysis. The most common mutated gene was PIK3CA. CONCLUSION: All omic approaches must be integrated in the locally advanced cancer setting by new clinical trial design to develop two routes in the treatment strategy: intensification or de-escalation treatment strategy according to omic markers.

Phage Microarrays for Screening of Humoral Immune Responses.

Chronic diseases are the leading cause of disability and responsible for about 63% of deaths worldwide. Among the noninfectious chronic diseases with the highest incidence are cancer and neurodegenerative diseases. Although they have been extensively studied in the last years, there is still an urgent need to find and elucidate the molecular mechanisms underlying their formation and progression to get an early diagnosis and find new therapeutic targets of intervention. Beyond other microarray-based proteomic techniques more extensively used because of their commercial availability, such as protein and antibody microarrays, phage microarrays are another kind of protein microarrays useful for the identification and characterization of disease-specific humoral immune responses and to get further insights into these devastating diseases. Here, we describe the integration and utilization of phage microarrays, which offer such a combination of sensitivity and cost-effective multiplexing capabilities that makes them an affordable strategy for the characterization of humoral immune responses in multiple diseases.

Epitope Mapping of Allergenic Lipid Transfer Proteins.

Food allergy is becoming a great problem in industrialized countries. Thus, there is the need for a robust understanding of all aspects characterizing IgE response to allergens. The epitope mapping of B-cell epitopes has the potential to become a fundamental tool for food allergy diagnosis and prognosis and to lead to a better understanding of the pathogenesis. Using this approach, we have worked on epitope mapping of the most important plant food allergens identified in the Mediterranean area. The final aim of this study is to define the immune response regarding B epitopes and its clinical relevance in LTP allergy. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of allergenic lipid transfer proteins.

Evaluación de la metilación del DNA en tejidos de cáncer colorrectal fijados en formalina y embebidos en parafina (FFPE) / Evaluation of DNA methylation in formalin-fixed, paraffin embedded colorectal cancer tissues (FFPE)

Resumen Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación, a partir de tejidos fijados en formalina y embebidos en parafina (FFPE), representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica, describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS, a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.

Abstract Alterations in the methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer that has potential use as a biomarker. Its evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues represents a great challenge given the partial degradation, crosslinking, and low amounts of the obtained DNA. In this technical note we describe a protocol for the study of the methylation status of the distal promoter of the K-RAS proto-oncogene from several samples obtained from two 11-years old FFPE tissues of colorectal cancer. An alternative bisulfite conversion protocol to the usual one was used; a modified DNA polymerase and a nested PCR were used and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation states in other genes and types of cancer.

An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation.

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.

hTERT Protein Expression in Cytoplasm and Nucleus and its Association With HPV Infection in Patients With Cervical Cancer.

BACKGROUND: Few studies have analyzed the association between human telomerase reverse transcriptase (hTERT) protein expression (nuclear and cytoplasmic localization), hTERT methylation status, and human papillomavirus (HPV) genotype infection in cervical cancer. PATIENTS AND METHODS: One hundred seventy-three patients with cervical cancer were analyzed. hTERT protein expression was detected by immunohistochemistry. hTERT DNA methylation analysis was performed using a PCR-RLB-hTERT assay, targeting two regions of the hTERT promoter. Type specific HPV infection was detected by using GP5+/GP6+PCR-RLB. RESULTS: hTERT protein expression was found in both cytoplasm and nucleus (78.0% of the samples showed a cytoplasmic localization and 79.8% had a nuclear localization). A statistically significant association was found between alpha 9 and 7 HPV species with a non-methylation pattern of the hTERT promoter and between these species and high expression of hTERT protein with nuclear localization. CONCLUSION: hTERT protein is found in both the nucleus and cytoplasm of patients with cervical cancer and confirm the relationship between the non-methylated status of hTERT promoter and some HPV species as well as the relationship between these species and hTERT protein expression.

Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen Ole e 7 with lipids.

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways.