Cu-BTC Functional Microdevices as Smart Tools for Capture and Preconcentration of Nerve Agents.

Cu-based metal-organic framework (MOF) microdevices are applied in sampling and preconcentration of nerve agents (NAs) diluted in gaseous streams. An in situ electrochemical-assisted synthesis of a Cu-benzene-1,3,5-tricarboxylate (BTC) thick film is carried out to functionalize a Cu-modified glass substrate. This simple, rapid, reproducible, and easy-to-integrate MOF synthesis approach enables the microfabrication of functional micro-preconcentrators with a large Brunauer-Emmett-Teller (BET) surface area (above 2000 cm2) and an active pore volume (above 90 nL) for the efficient adsorption of nerve agent molecules along the microfluidic channel 2.5 cm in length. The equilibrium adsorption capacity of the bulk material has been characterized through thermogravimetric analysis after exposure to controlled atmospheres of a sarin gas surrogate, dimethyl methylphosphonate (DMMP), in both dry and humid conditions (30% RH at 293 K). Breakthrough tests at the ppm level (162 mg/m3) reveal equilibrium adsorption capacities up to 691 mg/g. The preconcentration performance of such µ-devices when dealing with highly diluted surrogate atmosphere, i.e., 520 ppbV (2.6 mg/m3) at 298 K, leads to preconcentration coefficients up to 171 for sample volume up to 600 STP cm3. We demonstrate the potentialities of Cu-BTC micro-preconcentrators as smart first responder tools for "on-field" detection of nerve agents in the gas phase at relevant conditions.

[Estimated Effect of COVID-19 Lockdown on Skin Tumor Size and Survival: An Exponential Growth Model]. / Estimación del efecto en el tamaño y la supervivencia de los tumores cutáneos debido al confinamiento por COVID-19: modelo basado en un crecimiento exponencial.

BACKGROUND AND OBJECTIVES: Spain is in a situation of indefinite lockdown due to the ongoing coronavirus disease 2019 (COVID-19) pandemic. One of the consequences of this lockdown is delays in medical and surgical procedures for common diseases. The aim of this study was to model the impact on survival of tumor growth caused by such delays in patients with squamous cell carcinoma (SCC) and melanoma. MATERIAL AND METHODS: Multicenter, retrospective, observational cohort study. We constructed an exponential growth model for both SCC and melanoma to estimate tumor growth between patient-reported onset and surgical excision at different time points. RESULTS: Data from 200 patients with SCC of the head and neck and 1000 patients with cutaneous melanoma were included. An exponential growth curve was calculated for each tumor type and we estimated tumor size after 1, 2, and 3 months of potential surgical delay. The proportion of patients with T3 SCC (diameter >4cm or thickness >6 mm) increased from 41.5% (83 patients) in the initial study group to an estimated 58.5%, 70.5%, and 72% after 1, 2, and 3 months of delay. Disease-specific survival at 2, 5, and 10 years in patients whose surgery was delayed by 3 months decreased by 6.2%, 8.2%, and 5.2%, respectively. The proportion of patients with ultrathick melanoma (>6 mm) increased from 6.9% in the initial study group to 21.9%, 30.2%, and 30.2% at 1, 2, and 3 months. Five- and 10-year disease-specific survival both decreased by 14.4% in patients treated after a potential delay of 3 months. CONCLUSIONS: In the absence of adequate diagnosis and treatment of SCC and melanoma in the current lockdown situation in Spain, we can expect to see to a considerable increase in large and thick SCCs and melanomas. Efforts must be taken to encourage self-examination and facilitate access to dermatologists in order to prevent further delays.

Cloning of the gp63 surface protease of Leishmania infantum. Differential post-translational modifications correlated with different infective forms.

The Leishmania cell surface virulence factor gp63 is a protease family that plays an important role in the survival of the parasite protozoon into the host macrophages. We have cloned and characterised the gp63 gene from L. infantum. The sequence analysis of the gene indicates the existence of a high degree of conservation with the other old world species L. major and L. donovani. The similarity is lower with new world species with the exception of L. chagasi which shows a strikingly high percentage of identity (99-100%). In L. infantum the gp63 gene expresses two polypeptides of 58 and 60 kDa, respectively, which show a similar proteolytic activity. The 60 kDa polypeptide is expressed during the whole life cycle of the promastigote form of the parasite with a moderate increase at the stationary phase of growth while the 58 kDa product, although slightly present in the logarithmic phase, notable increases its expression during the highly infectious stationary phase. RNA analysis showed that the presence in L. chagasi of these two polypeptides correlates with two RNA molecules and with the degree of parasite infectivity, whereas in the case of L. infantum a single 3 kb messenger RNA is detected through the whole promastigote life cycle. Our data indicate that in L. infantum, the differences in gene expression of the gp63 protease family according to parasite phase of growth seem to be due to a differential pattern of glycosilation of the polypeptides which correlates with the different infective forms of the promastigote form of the parasite.

Estimación del efecto en el tamaño y la supervivencia de los tumores cutáneos debido al confinamiento por COVID-19: Modelo basado en un crecimiento exponencial / Estimated Effect of COVID-19 Lockdown on Skin Tumor Size and Survival: AnExponential Growth Model

BACKGROUND AND OBJECTIVES: Spain is in a situation of indefinite lockdown due to the ongoing coronavirus disease 2019 (COVID-19) pandemic. One of the consequences of this lockdown is delays in medical and surgical procedures for common diseases. The aim of this study was to model the impact on survival of tumor growth caused by such delays in patients with squamous cell carcinoma (SCC) and melanoma. MATERIAL AND METHODS: Multicenter, retrospective, observational cohort study. We constructed an exponential growth model for both SCC and melanoma to estimate tumor growth between patient-reported onset and surgical excision at different time points. RESULTS: Data from 200 patients with SCC of the head and neck and 1000 patients with cutaneous melanoma were included. An exponential growth curve was calculated for each tumor type and we estimated tumor size after 1, 2, and 3 months of potential surgical delay. The proportion of patients with T3 SCC (diameter > 4 cm or thickness > 6 mm) increased from 41.5% (83 patients) in the initial study group to an estimated 58.5%, 70.5%, and 72% after 1, 2, and 3 months of delay. Disease-specific survival at 2, 5, and 10 years in patients whose surgery was delayed by 3 months decreased by 6.2%, 8.2%, and 5.2%, respectively. The proportion of patients with ultrathick melanoma (> 6 mm) increased from 6.9% in the initial study group to 21.9%, 30.2%, and 30.2% at 1, 2, and 3 months. Five-and 10-year disease-specific survival both decreased by 14.4% in patients treated after a potential delay of 3 months. CONCLUSIONS: In the absence of adequate diagnosis and treatment of SCC and melanoma in the current lockdown situation in Spain, we can expect tosee to a considerable increase in large and thick SCCs and melanomas. Efforts must be taken to encourage self-examination and facilitate access to dermatologists in order to prevent further delays

ANTECEDENTES Y OBJETIVOS: La pandemia del coronavirus COVID-19 ha provocado un confinamiento indefinido. Una posible consecuencia de esta situación es un retraso en los procedimientos asistenciales de las patologías comunes. El objetivo de este estudio es estimar el hipotético impacto en la supervivencia que tendría el aumento del tamaño tanto para los carcinomas de células escamosas (CCE) como de los melanomas. MATERIAL Y MÉTODO: Estudio observacional retrospectivo de cohortes multicéntrico. Se desarrolló un modelo de crecimiento exponencial para cada tumor basado en el tiempo de evolución que refiere el paciente. RESULTADOS: Se incluyeron un total de 200 pacientes con CCEs localizados en la cabeza y el cuello y 1000 pacientes con melanoma cutáneo. Se calculó una curva de crecimiento exponencial para cada tumor y se estimó el tamaño del tumor tras 1, 2 y 3 mes tras el diagnóstico. En la muestra, los CCE mayores de 4 cm o > 6 mm de grosor (definidos como T3) pasaron de 83 (41.5%) en el grupo de estudio real a una estimación de 58,5%, 70,5% y 72% tras 1, 2 y 3 meses de retraso quirúrgico estimado. Se estimó una disminución de la supervivencia específica de enfermedad (SEE) de un 6,2%, 8,2% y 5,2% a los 2, 5 y 10 años, respectivamente, tras tres meses de retraso. Para los melanomas, los melanomas ultragruesos (> 6 mm) pasaron del 6,9% en el grupo de estudio al 21,9%, 30,2% y 30,2% tras 1,2 y 3 meses de demora. La SEE a los 5 y 10 años del grupo de estudio descendió un 14,4% en ambos tiempos. CONCLUSIONES: En ausencia de un adecuado diagnóstico y tratamiento de los pacientes con CCE y melanoma en la actual situación de confinamiento en España, podemos llegar a asistir a un considerable aumento de los casos de CCE y melanomas gruesos y de gran tamaño. Se deben fomentar los esfuerzos para promocionar la autoexploración y facilitar el acceso a los dermatólogos para no aumentar la demora de estos pacientes. Palabras clave: melanoma, pronóstico, diagnóstico precoz, carcinoma de células escamosas cutáneo, COVID-19, confinamiento

Feasibility of using an oleate-rich diet to reduce the susceptibility of low-density lipoprotein to oxidative modification in humans.

Oxidized low-density lipoprotein (LDL) is more atherogenic than native LDL. The initial step in the oxidation is the peroxidation of polyunsaturated fatty acids. Thus, decreasing the concentration of polyunsaturated fatty acids should reduce the susceptibility of LDL to oxidation. Therefore, we tested the possibility that diets enriched in oleate might result in LDL that is less susceptible to oxidative modification. LDL isolated from subjects consuming an oleate-enriched diet, compared with LDL from subjects on a linoleate-enriched diet, contained significantly more oleate (28.7% vs 11.5%) and less linoleate (31.9% vs 50.9%). Generation of conjugated dienes was significantly lower in the LDL from the oleate group. Most important, after incubation with endothelial cells, LDL from the oleate group underwent less degradation by macrophages. These studies demonstrate the feasibility of altering the diet in a way that will not raise LDL cholesterol concentrations and yet will decrease the susceptibility of LDL to oxidative modification.

Vectors for the genetic manipulation of African swine fever virus.

Plasmid vectors designed to facilitate the genetic manipulation of African swine fever virus (ASFV) are described. Our results demonstrate that the beta-glucuronidase enzyme (GUS) can be used to follow gene expression in ASFV-infected cells. Infectious plaques formed by ASFV expressing GUS are visually detectable, thus providing a simple and highly sensitive method for the selection of ASFV recombinants. These and previous results have allowed us to construct two chimeric gene cassettes that constitute the basic tools for the generation of vectors to carry out the deletion of multiple target sequences from the ASFV genome. These cassettes, formed by: (a) a virus promoter; (b) the coding sequence of a reporter gene, either Lac Z or gusA; and (c) a strong signal for the 3' end formation of ASFV mRNAs, can be easily isolated by endonuclease restriction from their corresponding plasmid vectors. A general insertion/coexpression plasmid vector, pEPV2, has also been constructed. pEPV2 facilitates the insertion of foreign genes, together with the Lac Z reporter, into the thymidine kinase locus of ASFV. The functionality of pEPV2 has been tested by generating a recombinant ASFV expressing the luciferase gene. The vectors presented in this report constitute the first reported set of tools for the genetic manipulation of ASFV.

Coronavirus derived expression systems.

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (>20 microg ml(-1)) and stable (>20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.

Induction of aggregation in porcine lymphoid cells by antibodies to CD46.

CD46 is a major transmembrane glycoprotein that belongs to the regulator of complement activation (RCA) family. Recently, mAbs to human CD46 were shown to suppress IL-12 production. Here, we describe that mAbs against different porcine CD46 epitopes induced a marked adhesion of normal lymphocytes. Addition of low amounts of antibody to freshly isolated lymphocytes or thymocytes resulted in the clustering of the cells. Cross-linking of CD46 molecules seems essential since Fab fragments failed to induce aggregation. This aggregation was dependent on active cell metabolism and on the presence of divalent cations and required a functional cytoskeleton. It was not inhibited by antibodies to CD18, CD29, CD2, CD11a and CD11b. Staurosporine, an inhibitor of protein kinases, partially blocked the aggregation. This finding is indicative of a role of protein kinases in the transduction of the signal generated by CD46 engagement.

[The current situation of adolescents with cancer in pediatric hematology-oncology units in Spain. Results of a national survey]. / Situación actual de los adolescentes con cáncer en las unidades de hemato-oncología pediátrica españolas.

Little attention was paid to adolescents with Cancer in Spain up to 2010. In 2011 an "Adolescents with Cancer Committee" was established by the Spanish Society of Pediatric Hemato-Oncology (SEHOP) to care for the needs of these patients. The aim of this national survey was to outline the present situation of adolescents with cancer in Spanish Pediatric Hemato-Oncology units. A web based survey assessed institutional management of adolescents with cancer. The survey was personally sent to one member of the staff of each Pediatric Hemato-Oncology unit in Spain. It included questions about epidemiology, management, psycho-social coverage, specific facilities, and follow up of these patients. A total of 40 institutions out of 41 responded to the survey (overall response rate 98%). Fifty-six percent of the institutions had patients over 14, but only 36% of the institutions treated patients up to 18 years old. Only 25.6% of the units have more than 40 new pediatric cases every year. The percentage of patients between 14 and 18 years of age is below 10% in most of the units (77%). In 30.8% and 48.7% of the institutions, pediatric hemato-oncologists treat adolescents with hematological and solid tumors, respectively. The rest of the patients are seen by adult oncologists. There is only one institution that has a physician specifically dedicated to adolescent patients, and only two units have a "teenager's room". Only 2 units have a psychologist specifically trained to treat adolescents with cancer. The survey shows that most adolescents with cancer in Spain between 14 and 18 years of age are treated by adult oncologists. Most pediatric institutions still do not have specific facilities and psychosocial support for adolescents. The SEHOP is working hard in order to improve the quality of cancer care, and the quality of survival of this population.

Proteolytic processing in African swine fever virus: evidence for a new structural polyprotein, pp62.

We have identified an open reading frame (ORF), CP530R, within the EcoRI C' fragment of the African swine fever virus (ASFV) genome that encodes a polyprotein of 62 kDa (pp62). Antisera raised against different regions of ORF CP530R recognized a polypeptide of 62 kDa in ASFV-infected cells during the late phase of virus replication, after the onset of viral DNA synthesis. Pulse-chase experiments showed that polyprotein pp62 is posttranslationally processed to give rise to two proteins of 35 kDa (p35) and 15 kDa (p15). This proteolytic processing was found to take place at the consensus sequence Gly-Gly-X through an ordered cascade of proteolytic cleavages like that which also occurs with ASFV polyprotein pp220 (C. Simón-Mateo, G. Andrés, and E. Viñuela, EMBO J. 12:2977-2987, 1993). Immunofluorescence studies showed that polyprotein pp62 is localized in the viral factories. In addition, immunoprecipitation analysis of purified virus particles showed that mature products p35 and p15 are major structural proteins. According to these results, polyprotein processing represents an essential strategy for the maturation of ASFV structural proteins.

Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72.

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

Multigene families in African swine fever virus: family 110.

The genome of African swine fever virus was screened for the existence of repetitive sequences by hybridization between different cloned restriction fragments covering the viral DNA. Several sets of repeated sequences were detected in fragments located close to the DNA ends. One of these groups of repetitions involved fragments located at both ends of the genome. The remaining groups involved fragments that were located exclusively at the left end. The sequence of a 3.2-kilobase segment spanning from 7.5 to 11 kilobases from the left DNA end, which showed a complex pattern of cross-hybridizations, was determined. Two short and three long blocks of direct repeated sequences were found in this DNA region, which accounted for the hybridization results. The repeated sequences formed a family of five homologous genes with an average length of 116 codons (multigene family 110), one of which had a dimeric structure. Transcripts of the five members of the family were detected both in RNA synthesized in vitro by purified African swine fever virions and in RNA isolated at early times after infection. Comparison of the predicted protein sequences revealed a striking conservation of a cysteine-rich domain in the central part of the proteins. In addition, a highly hydrophobic NH2-terminal sequence present in all the proteins suggests that these proteins are processed through the endoplasmic reticulum.

The vaccinia virus superoxide dismutase-like protein (A45R) is a virion component that is nonessential for virus replication.

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.

The African swine fever virus IAP homolog is a late structural polypeptide.

The analysis of the complete nucleotide sequence of the African swine fever virus genome has revealed the existence of a number of genes potentially capable of modifying the host's response to the virus infection. In this report, we describe the results of the characterization of the A224L gene that encodes a novel member of the family of apoptosis inhibitors known as IAP proteins. A224L is expressed during the late phase of the infectious cycle, and its polypeptide product is assembled into virus particles.

African swine fever virus encodes a CD2 homolog responsible for the adhesion of erythrocytes to infected cells.

We have identified an open reading frame, EP402R, within the EcoRI E' fragment of the African swine fever virus genome that encodes a polypeptide of 402 amino acid residues homologous to the adhesion receptor of T cells, CD2. Transcription of EP402R takes place during the late phase of virus replication. The disruption of EP402R, achieved through the replacement of a 354-bp-long fragment from within EP402R by the marker gene lacZ, does not affect the virus growth rate in vitro but abrogates the ability of the virus to induce the adsorption of pig erythrocytes to the surface of infected cells. This result demonstrates that the protein encoded by EP402R is directly involved in the hemadsorption phenomenon induced by the infection of susceptible cells with African swine fever virus.

Transcriptional mapping of a late gene coding for the p12 attachment protein of African swine fever virus.

The transcriptional characterization of the gene coding for the p12 attachment protein of the African swine fever virus is presented. The results obtained have been used to generate the first detailed transcriptional map of an African swine fever virus late gene. Novel experimental evidence indicating the existence of major differences between the mechanisms controlling the transcription of late genes in African swine fever virus and poxviruses is provided.