In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network.

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.

Using demand mapping to assess the benefits of urban green and blue space in cities from four continents.

The benefits of urban green and blue infrastructure (UGI) are widely discussed, but rarely take into account local conditions or contexts. Although assessments increasingly consider the demand for the ecosystem services that UGI provides, they tend to only map the spatial pattern of pressures such as heat, or air pollution, and lack a wider understanding of where the beneficiaries are located and who will benefit most. We assess UGI in five cities from four continents with contrasting climate, socio-political context, and size. For three example services (air pollution removal, heat mitigation, accessible greenspace), we run an assessment that takes into account spatial patterns in the socio-economic demand for ecosystem services and develops metrics that reflect local context, drawing on the principles of vulnerability assessment. Despite similar overall levels of UGI (from 35 to 50% of urban footprint), the amount of service provided differs substantially between cities. Aggregate cooling ranged from 0.44 °C (Leicester) to 0.98 °C (Medellin), while pollution removal ranged from 488 kg PM2.5/yr (Zomba) to 48,400 kg PM2.5/yr (Dhaka). Percentage population with access to nearby greenspace ranged from 82% (Dhaka) to 100% (Zomba). The spatial patterns of pressure, of ecosystem service, and of maximum benefit within a city do not necessarily match, and this has implications for planning optimum locations for UGI in cities.

Low erythropoietin plasma levels during exacerbations of COPD.

BACKGROUND: It is known that pro-inflammatory cytokines suppress in vitro the gene expression and protein production of erythropoietin (Epo). We hypothesized that systemic inflammation in patients with chronic obstructive pulmonary disease (COPD) may influence Epo production, particularly during episodes of exacerbation of the disease (ECOPD) where an inflammatory burst is known to occur. OBJECTIVES: We compared the plasma levels of Epo and high-sensitivity (hs) C-reactive protein (hsC-RP) in patients hospitalized because of ECOPD (n = 26; FEV(1): 48 +/- 15% predicted), patients with clinically stable COPD (n = 31; FEV(1): 49 +/- 17% predicted), smokers with normal lung function (n = 9), and healthy never smokers (n = 9). METHODS: Venous blood samples were taken between 9 and 10 a.m. after an overnight fast into tubes with EDTA (10 ml) or without EDTA (10 ml). Plasma levels of Epo (R&D Systems Inc., Minneapolis, Minn., USA) and hsC-RP (BioSource, Belgium) were determined by ELISA. RESULTS: Log-Epo plasma levels were significantly lower (0.46 +/- 0.32 mU/ml) in ECOPD than in stable COPD (1.05 +/- 0.23 mU/ml), smokers (0.95 +/- 0.11 mU/ml) and never smokers with normal lung function (0.92 +/- 0.19 mU/ml) (p < 0.01, each). In a subset of 8 COPD patients who could be studied both during ECOPD and clinical stability, log-Epo increased from 0.49 +/- 0.42 mU/ml during ECOPD to 0.97 +/- 0.19 mU/ml during stability (p < 0.01). In patients with COPD log-Epo was significantly related to hsC-RP (r = -0.55, p < 0.0001) and circulating neutrophils (r = -0.48, p < 0.0001). CONCLUSIONS: These results show that the plasma levels of Epo are reduced during ECOPD likely in relation to a burst of systemic inflammation.

Abnormal levels of circulating endothelial progenitor cells during exacerbations of COPD.

Cardiovascular morbidity and mortality is increased in patients with chronic obstructive pulmonary disease (COPD). Reduced levels of circulating endothelial progenitor cells (EPCs) are associated with increased risk of death in patients with stable coronary artery disease (CAD). Likewise, during acute events of CAD, the number of circulating EPCs increases under the influence of vascular endothelial growth factor (VEGF) and systemic inflammation. Abnormal levels of circulating EPCs have been reported in patients with COPD. However, the response of EPCs to episodes of exacerbation of the disease (ECOPD) has not been investigated yet. We hypothesized that similar to what occurs during acute events of CAD, levels of circulating EPCs would increase during ECOPD. We compared levels of circulating EPCs (assessed by the % of CD34(+)KDR(+) cells determined by flow cytometry) in patients hospitalized because of ECOPD (n = 35; 65 +/- 9 years [mean +/- SD]; FEV(1) = 46 +/- 15% predicted), patients with stable COPD (n = 44; 68 +/- 8 years; FEV(1) = 49 +/- 17% predicted), smokers with normal lung function (n = 10; 60 +/- 9 years), and healthy never smokers (n = 10; 62 +/- 4 years). To investigate potential mechanisms of EPC regulation, we assessed both VEGF and high-sensitivity C-reactive protein (hsC-RP) in plasma. Our results show that EPC levels were higher (p < 0.05) in patients with ECOPD (1.46 +/- 1.63%) than in those with stable disease (0.68 +/- 0.83%), healthy smokers (0.65 +/- 1.11%), and healthy never smokers (1.05 +/- 1.36%). The percentage of circulating EPCs was positively related to VEGF plasma levels during ECOPD (r = 0.51, p = 0.003). In a subset of 12 patients who could be studied during both ECOPD and clinical stability, the EPCs levels increased during ECOPD. We conclude that EPC levels are increased during ECOPD, likely in relation to VEGF upregulation.

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples.

Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RT-qPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples.

Agrobacterium tumefaciens-Mediated Transformation of Fonsecaea monophora and Fonsecaea erecta for Host-Environment Interaction Studies.

The fungal genus Fonsecaea contains etiological agents of human chromoblastomycosis, a (sub)tropical, (sub)cutaneous implantation disease caused by plant contact. The invasive potential differs significantly between species. Infections by Fonsecaea monophora are believed to originate from the environment and the species has been reported as one of the main causative agents of the disease, but also of cases of primary brain infection. The epidemiology of the disease has not been fully elucidated and questions related to its infection route and virulence are still to be clarified. The environmental species Fonsecaea erecta was isolated from organic material and living plants in endemic areas for chromoblastomycosis in Brazil. The present paper describes Agrobacteriumtumefaciens-mediated transformation (AMT) of the environmental species F. erecta and the pathogenic species F. monophora. We propose the use of Agrobacterium transformation for future gene function studies related to Fonsecaea virulence and pathogenicity. We evaluated the co-cultivation ratios 1:1, 10:1 and 100:1 (Agrobacterium:conidia) at 28 °C during 72 h. pAD1625 and pCAMDsRed plasmids were inserted into both species. Confirmation of transformation was realized by hph gene amplification and Southern blot determined the amount of foreign DNA integrated into the genome. In order to evaluate a potential link between environmental and clinical strains, we obtained red fluorescent transformants after pCAMDsRed insertion. We observed by confocal fluorescence microscopy that both F. monophora and F. erecta were able to colonize the palm Bactris gasipaes, penetrating the epidermis. These results contribute to understanding the ability of Fonsecaea species to adapt to different environmental and host conditions.

Biological Marker Analysis as Part of the CIBERES-RTIC Cancer-SEPAR Strategic Project on Lung Cancer.

The aim of the Clinical and Molecular Staging of Stage I-IIp Lung Cancer Project is to identify molecular variables that improve the prognostic and predictive accuracy of TMN classification in stage I/IIp non-small cell lung cancer (NSCLC). Clinical data and lung tissue, tumor and blood samples will be collected from 3 patient cohorts created for this purpose. The prognostic protein signature will be validated from these samples, and micro-RNA, ALK, Ros1, Pdl-1, and TKT, TKTL1 y G6PD expression will be analyzed. Tissue inflammatory markers and stromal cell markers will also be analyzed. Methylation of p16, DAPK, RASSF1a, APC and CDH13 genes in the tissue samples will be determined, and inflammatory markers in peripheral blood will also be analyzed. Variables that improve the prognostic and predictive accuracy of TNM in NSCLC by molecular staging may be identified from this extensive analytical panel.

Hepatitis a virus: molecular detection and typing.

Hepatitis A virus (HAV) infection is the leading cause of viral hepatitis throughout the world. HAV infection is mainly propagated via the fecal-oral route, and waterborne and foodborne outbreaks of the disease have been reported.HAV, the prototype of the genus Hepatovirus, belongs to the family Picornaviridae. Its 7.5-kb single-stranded RNA genome bears different distinct regions: the 5' and 3' noncoding regions (NCR), the P1 region, which encodes the structural proteins VP1, VP2, VP3, and a putative VP4, and the P2 and P3 regions encoding nonstructural proteins associated with replication. A single HAV serotype has been described, although seven genotypes have been defined. Since environmental samples usually contain low numbers of viral particles, sensitive methods such as molecular techniques based on nucleic acid amplification are required for their detection. However, even with the adoption of these techniques, the choice of the most adequate target is of relevant importance. The target region should be highly conserved, to increase the chance of detection, and should have an appropriate structure and length to allow sensitivity high enough for these kind of samples. As a target region, we have chosen a fragment of the 5'NCR flanked by highly conserved sequences that have been used for the primer design (forward primer from position 68 to position 85; reverse primer from position 222 to position 240 in the HM175 strain of HAV; GenBank accession number M14707). The internal part of this region, however, may present a certain degree of variation mainly owing to insertions and/or deletions, causing a variable size of the amplimer obtained, i.e., the wild-type HM175 strain gives a size of 174 bp whereas the cell-adapted pHM175 strain gives a size of 186 bp. For this reason it is extremely important to include a confirmative method such as Southern blot hybridization with an internal probe from a region not affected by the insertions/deletions.

Análisis de marcadores biológicos en el Proyecto Estratégico de Cáncer de Pulmón CIBERES-RTIC Cáncer-SEPAR / Biological Marker Analysis as Part of the CIBERES-RTIC Cancer-SEPAR Strategic Project on Lung Cancer

El proyecto «Estadificación clínica y molecular del cáncer de pulmón estadios I-IIp», tiene por objetivo la identificación de variables moleculares que mejoren la capacidad pronóstica y predictiva de la clasificación TNM en el cáncer de pulmón no célula pequeña (NSCLC) estadio I/IIp. Para ello se han creado tres cohortes de pacientes de los que se recoge información clínica y muestras biológicas pulmonares y de sangre. En las muestras se validará una firma pronóstica proteica, y se analizarán micro-RNA, ALK, Ros1, Pdl-1, y niveles de expresión de TKT, TKTL1 y G6PD. Asimismo se analizarán marcadores de inflamación en tejido y marcadores de estroma. Se determinará la metilación de los genes p16, DAPK, RASSF1a, APC y CDH13 en la muestra tisular y se analizarán marcadores inflamatorios en sangre periférica. Este amplio panel analítico puede permitir la identificación de variables que mejoren la capacidad pronóstica y predictiva del TNM en el NSCLC mediante la estadificación molecular

The aim of the Clinical and Molecular Staging of Stage I-IIp Lung Cancer Project is to identify molecular variables that improve the prognostic and predictive accuracy of TMN classification in stage I/IIp nonsmall cell lung cancer (NSCLC). Clinical data and lung tissue, tumor and blood samples will be collected from 3 patient cohorts created for this purpose. The prognostic protein signature will be validated from these samples, and micro-RNA, ALK, Ros1, Pdl-1, and TKT, TKTL1 y G6PD expression will be analyzed. Tissue inflammatory markers and stromal cell markers will also be analyzed. Methylation of p16, DAPK, RASSF1a, APC and CDH13 genes in the tissue samples will be determined, and inflammatory markers in peripheral blood will also be analyzed. Variables that improve the prognostic and predictive accuracy of TNM in NSCLC by molecular staging may be identified from this extensive analytical panel

Pulmonary and systemic hepatocyte and keratinocyte growth factors in patients with chronic obstructive pulmonary disease.

BACKGROUND: The potential role of growth factors in chronic obstructive pulmonary disease (COPD) has begun to be addressed only recently and is still poorly understood. For this study, we investigated potential abnormalities of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in patients with COPD. METHODS: To this end, we compared the levels of HGF and KGF, measured by enzyme-linked immunosorbent assay (ELISA), in bronchoalveolar lavage (BAL) fluid and in serum in 18 patients with COPD (62 +/- 9 yrs, forced expiratory volume in one second [FEV1] 57 +/- 12% ref, X +/- standard deviation of mean), 18 smokers with normal lung function (58 +/- 8 yrs, FEV1 90 +/- 6% ref) and 8 never smokers (67 +/- 9 yrs, 94 +/- 14% ref). RESULTS: We found that in BAL, HGF levels were higher in patients with COPD than in the other two groups whereas, in serum, HGF concentration was highest in smokers with normal lung function (p < 0.01). KGF levels were not significantly different between groups, neither in blood nor in BAL (most values were below the detection limit). CONCLUSIONS: These results highlight a different response of HGF in BAL and serum in smokers with and without COPD that may be relevant for tissue repair in COPD.

Relative fitness and replication capacity of a multinucleoside analogue-resistant clinical human immunodeficiency virus type 1 isolate with a deletion of codon 69 in the reverse transcriptase coding region.

Deletions, insertions, and amino acid substitutions in the beta3-beta4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Delta69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Delta69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Delta69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Delta69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.

Evidence for preferential genotyping of a minority human immunodeficiency virus population due to primer-template mismatching during PCR-based amplification.

Human immunodeficiency virus type 1 (HIV-1) genotyping assays have come to be widely used for monitoring antiretroviral drug resistance. We report a case in which primer-template mismatches during nested PCR-based amplification biased the composition of the original viral population in the sample, magnifying a distinct minority HIV-1 population. This observation might help to explain some unexpected HIV-1 genotypes.

Group A rotavirus in sewage samples from Barcelona and Cairo: emergence of unusual genotypes.

The presence of rotavirus strains in sewage samples from Cairo, Egypt (November 1998 to October 1999), and Barcelona, Spain (November 1998 to December 2002), was investigated by using a generic molecular detection method based on amplification of a VP6 gene fragment. Overall, 85.7 and 66.9% of the sewage samples from Cairo and Barcelona, respectively, were positive. Positive samples were characterized further, and VP7 and VP4 genotypes were determined. Although 30% of the positive samples from Cairo were G untypeable, the distribution of G types in the positive samples was 69.6% G1, 13% G3, 8.7% G4, and 8.7% G9. The percentage of untypeable samples was much higher for the Barcelona samples (56.5%), and the distribution in the positive samples was 56.4% G1, 31.5% G3, 6% G9, 4% G2, and 2% G5. When the P types were examined, 26.7% of the positive samples from Cairo were untypeable, and the distribution of types in the positive samples was 53.3% P[8], 30% P[6], and 16.6% P[4]. In Barcelona, 27.2% of the samples were P untypeable, and the frequencies of the types detected were 49.7% P[8], 37.2% P[4], 8.8% P[6], and 4.2% P[9]. The distribution for strains from Cairo was 38.5% P[8]G1, 27% P[6]G1, 11.5% P[4]G1, 11.5% P[8]G3, 7.7% P[6]G4, and 3.8% P[8]G9. Strikingly, equivalent frequencies of common and uncommon strains were observed for Barcelona samples, and the distribution was 38.8% P[8]G1, 30.6% P[4]G1, 11.6% P[8]G3, 6.6% P[4]G3, 5.8% P[6]G1, 1.6% P[6]G3, 1.6% P[9]G1, 0.8% P[4]G2, 0.8% P[6]G9, 0.8% P[8]G9, and 0.8% P[8]G5. Additionally, two P[-]G5 strains were isolated in Barcelona, and the porcine or human origin of these strains was unclear. Rotavirus variability exhibited not only a geographic pattern but also a temporal pattern.

Molecular epidemiology of astrovirus infection in Barcelona, Spain.

A 3-year study involving 2,347 gastroenteritis samples was conducted to determine the prevalence, time distribution, and medical significance of human astrovirus infection in Barcelona, Spain. The overall incidence of astrovirus was found to be 4.9%. Mixed infections with other enteric agents were detected in 17.2% of all astrovirus-positive samples. During the 3-year period, the highest astrovirus incidence was reported in the winter months, although infections also occurred in summer. The peak detection rate was observed in children between 2 and 4 years of age. Overall, HAstV-1 was the most prevalent type, followed by HAstV-4, HAstV-3, HAstV-8, and HAstV-2. HAstV-5, HAstV-6, and HAstV-7 were not detected during these 3 years. From our serotype data for each age group, we observed that HAstV-1, HAstV-2, and HAstV-3 affected mostly children younger than 3 years of age, while HAstV-4 and HAstV-8 had a greater impact in older children. Genetic variability was analyzed between astroviruses isolated in Barcelona and strains isolated in other parts of the world. A fourth lineage was described for HAstV-1, most likely due to the large number of assayed samples, which may also explain the high level of genetic variability observed in the astrovirus isolates.