Patterned PVA Hydrogels with 3D Petri Dish® Micro-Molds of Varying Topography for Spheroid Formation of HeLa Cancer Cells: In Vitro Assessment.

Cell spheroids are an important three-dimensional (3D) model for in vitro testing and are gaining interest for their use in clinical applications. More natural 3D cell culture environments that support cell-cell interactions have been created for cancer drug discovery and therapy applications, such as the scaffold-free 3D Petri Dish® technology. This technology uses reusable and autoclavable silicone micro-molds with different topographies, and it conventionally uses gelled agarose for hydrogel formation to preserve the topography of the selected micro-mold. The present study investigated the feasibility of using a patterned Poly(vinyl alcohol) hydrogel using the circular topography 12-81 (9 × 9 wells) micro-mold to form HeLa cancer cell spheroids and compare them with the formed spheroids using agarose hydrogels. PVA hydrogels showed a slightly softer, springier, and stickier texture than agarose hydrogels. After preparation, Fourier transform infrared (FTIR) spectra showed chemical interactions through hydrogen bonding in the PVA and agarose hydrogels. Both types of hydrogels favor the formation of large HeLa spheroids with an average diameter of around 700-800 µm after 72 h. However, the PVA spheroids are more compact than those from agarose, suggesting a potential influence of micro-mold surface chemistry on cell behavior and spheroid formation. This was additionally confirmed by evaluating the spheroid size, morphology, integrity, as well as E-cadherin and Ki67 expression. The results suggest that PVA promotes stronger cell-to-cell interactions in the spheroids. Even the integrity of PVA spheroids was maintained after exposure to the drug cisplatin. In conclusion, the patterned PVA hydrogels were successfully prepared using the 3D Petri Dish® micro-molds, and they could be used as suitable platforms for studying cell-cell interactions in cancer drug therapy.

The Fluorescent Cell Line SW620-GFP Is a Valuable Model to Monitor Magnetic Hyperthermia.

In this work, the cell line SW620-GFP has been used in a complete magnetic hyperthermia assay, from the preparation of the ferrofluid with folate-coated iron oxide nanoparticles to in vivo experiments. The physical and chemical characterization of the nanoparticles evidenced their superparamagnetic behaviour, an average diameter of 12 ± 4 nm, a 2 nm coat thickness, and a high-power loss density. The main innovation of the work is the exclusive capability of viable SW620-GFP cells to emit fluorescence, enabling fast analysis of both, cell viability in vitro with an epifluorescence microscope and tumour size and shape in vivo in a non-invasive manner using the iBox technology. Moreover, with this imaging technique, it was possible to demonstrate the successful tumour size reduction in mice applying magnetic hyperthermia three times a week over 3 weeks.

Cobimetinib Plus Vemurafenib in Patients With Solid Tumors With BRAF Mutations: Results From the Targeted Agent and Profiling Utilization Registry Study.

PURPOSE: The Targeted Agent and Profiling Utilization Registry Study is a phase II basket study evaluating antitumor activity of commercially available targeted agents in patients with advanced cancers with genomic alterations known to be drug targets. The results in a cohort of patients with solid tumors with BRAF mutations treated with cobimetinib plus vemurafenib are reported. METHODS: Eligible patients had measurable disease (RECIST v.1.1), Eastern Cooperative Oncology Group performance status 0-2, adequate organ function, and no standard treatment options. The primary end point was disease control (DC), defined as complete response (CR) or partial response (PR) or stable disease of at least 16-weeks duration (SD16+). Low-accruing histology-specific cohorts with BRAF mutations treated with cobimetinib plus vemurafenib were collapsed into a single histology-pooled cohort for this analysis. The results were evaluated on the basis of a one-sided exact binomial test with a null DC rate of 15% versus 35% (power, .82; α, .10). The secondary end points were objective response (OR), progression-free survival, overall survival, duration of response, duration of stable disease, and safety. RESULTS: Thirty-one patients with solid tumors with BRAF mutations were enrolled. Twenty-eight patients were evaluable for efficacy. Patients had tumors with BRAF V600E (n = 26), K601E (n = 2), or other (n = 3) mutations. Two patients with CR (breast and ovarian cancers; V600E), 14 with PR (13 V600E, one N581I), and three with SD16+ (two V600E, one T599_V600insT) were observed with a DC rate of 68% (P < .0001; one-sided 90% CI, 54 to 100) and an OR rate of 57% (95% CI, 37 to 76). Nineteen patients experienced ≥one drug-related grade 3-5 adverse event or serious adverse event including one death attributed to treatment-related kidney injury. CONCLUSION: Cobimetinib plus vemurafenib showed antitumor activity in patients with advanced solid tumors with BRAF V600E mutations; additional study is warranted to confirm the antitumor activity in tumors with non-V600E BRAF mutations.

The Value of CXCL1, CXCL2, CXCL3, and CXCL8 as Potential Prognosis Markers in Cervical Cancer: Evidence of E6/E7 from HPV16 and 18 in Chemokines Regulation.

Cervical cancer (CC) is a serious global health issue, and it is well-known that HPV infection is the main etiological factor that triggers carcinogenesis. In cancer, chemokine ligands and receptors are involved in tumor cell growth, metastasis, leukocyte infiltration, and angiogenesis; however, information on the role played by E6/E7 of HPV16/18 in the modulation of chemokines is very limited. Therefore, this study aimed to determine whether chemokines are differentially expressed in CC-derived cell lines; if E6/E7 oncoproteins from HPV16 and 18 are capable of mediating chemokine expression, what is the expression profile of chemokines in tissues derived from CC and what is their impact on the overall survival of patients with this pathology? For this purpose, RNA sequencing and real-time PCR were performed on SiHa, HeLa, and C33A tumorigenic cell lines, on the non-tumorigenic HaCaT cells, and the E6/E7 HPV-transduced HaCaT cell models. Furthermore, chemokine expression and survival analysis were executed on 304 CC and 22 normal tissue samples from The Cancer Genome Atlas (TCGA) repository. The results demonstrate that CXCL1, CXCL2, CXCL3, and CXCL8 are regulated by E6/E7 of HPV16 and 18, are overexpressed in CC biopsies, and that their higher expression is related to a worse prognostic survival.

Embedded Living HER2+ Cells in a 3D Gelatin-Alginate Hydrogel as an In Vitro Model for Immunotherapy Delivery for Breast Cancer.

Epidermal growth factor receptor 2 (HER2) is the second target molecule most commonly used in breast cancer treatment. Both recurrence and metastasis are still deadly for HER2+ breast cancer patients. Hydrogels can be an option for developing three-dimensional (3D) cell culture systems that resemble tumor features better than monolayer cultures and could be used for preclinical screening for new biotherapeutics. Biopolymers (gelatin and alginate) were used to develop a hydrogel capable of encapsulating living HER2+ breast cancer cells BT-474/GFP. The hydrogel was physicochemically characterized, and the viability of embedded cells was evaluated. The hydrogel developed had suitable physical properties, with swelling of 38% of its original mass at 20 h capacity and pore sizes between 20 and 125 µm that allowed cells to maintain their morphology in a 3D environment, in addition to being biocompatible and preserving 90% of cell viability at 10 days. Furthermore, encapsulated BT-474/GFP cells maintained HER2 expression that could be detected by the Trastuzumab-fluorescent antibody, so this hydrogel could be used to evaluate new HER2-targeted therapies.

Tratamiento de sonrisa gingival con planificación digital. Reporte de caso / Treatment of gummy smile with digital planning. Case report

La sonrisa gingival (SG) es una condición clínica de etiología multifactorial, caracterizada por la sobreexposición de la encía maxilar, mayor de 3 mm, mientras el paciente sonríe. Es considerada una de las alteraciones más comunes en la población con una prevalencia de 10,5-29%, 7% en hombres y 14% en mujeres, en el grupo etário de 20-30 años. La literatura científica reporta diversos tratamientos quirúrgicos invasivos y mínimamente invasivos que pueden ser acompañados con planificación digital. El objetivo de este reporte de caso fue describir el tratamiento de SG por erupción pasiva alterada tipo I, subcategoría A e hipermovilidad de músculo elevador del labio superior, en paciente femenino corregido con procedimientos quirúrgicos y métodos conservadores como la toxina botulínica planificado digitalmente. El uso de planificación digital en la reducción de SG acompañada de técnicas conservadoras como toxina botulínica permiten realizar procedimientos con postoperatorios favorables, disminución en errores de la técnica con resultados predecibles, seguros, rápidos y consistentes de acuerdo a las expectativas del paciente

Gummy smile (GS) is a clinical condition of multifactorial etiology, characterized by overexposure of the maxillary gingiva greater than 3mm while the patient smiles. It is considered one of the most common alterations among the population with a prevalence between 10,5-29%, 7% in men and 14% in women, in the age group of 20-30 years-old. The scientific literature reports several invasive and minimally invasive surgical treatments that can be accompanied with digital planning. The aim of this case report was to describe the treatment of GS due to altered passive eruption type I subcategory A and levator labii superioris muscle hypermobility in a female patient corrected with surgical procedures and conservative methods such as digital planned botulinum toxin. The use of digital planning in GS reduction accompanied by conservative techniques such as botulinum toxin allows performing procedures with favorable postoperative results, less technical errors with predictable, safe, fast and consistent results according to patient's expectations.

A Ferrofluid with Surface Modified Nanoparticles for Magnetic Hyperthermia and High ROS Production.

A ferrofluid with 1,2-Benzenediol-coated iron oxide nanoparticles was synthesized and physicochemically analyzed. This colloidal system was prepared following the typical co-precipitation method, and superparamagnetic nanoparticles of 13.5 nm average diameter, 34 emu/g of magnetic saturation, and 285 K of blocking temperature were obtained. Additionally, the zeta potential showed a suitable colloidal stability for cancer therapy assays and the magneto-calorimetric trails determined a high power absorption density. In addition, the oxidative capability of the ferrofluid was corroborated by performing the Fenton reaction with methylene blue (MB) dissolved in water, where the ferrofluid was suitable for producing reactive oxygen species (ROS), and surprisingly a strong degradation of MB was also observed when it was combined with H2O2. The intracellular ROS production was qualitatively corroborated using the HT-29 human cell line, by detecting the fluorescent rise induced in 2,7-dichlorofluorescein diacetate. In other experiments, cell metabolic activity was measured, and no toxicity was observed, even with concentrations of up to 4 mg/mL of magnetic nanoparticles (MNPs). When the cells were treated with magnetic hyperthermia, 80% of cells were dead at 43 °C using 3 mg/mL of MNPs and applying a magnetic field of 530 kHz with 20 kA/m amplitude.

Green Metallic Nanoparticles for Cancer Therapy: Evaluation Models and Cancer Applications.

Metal-based nanoparticles are widely used to deliver bioactive molecules and drugs to improve cancer therapy. Several research works have highlighted the synthesis of gold and silver nanoparticles by green chemistry, using biological entities to minimize the use of solvents and control their physicochemical and biological properties. Recent advances in evaluating the anticancer effect of green biogenic Au and Ag nanoparticles are mainly focused on the use of conventional 2D cell culture and in vivo murine models that allow determination of the half-maximal inhibitory concentration, a critical parameter to move forward clinical trials. However, the interaction between nanoparticles and the tumor microenvironment is not yet fully understood. Therefore, it is necessary to develop more human-like evaluation models or to improve the existing ones for a better understanding of the molecular bases of cancer. This review provides recent advances in biosynthesized Au and Ag nanoparticles for seven of the most common and relevant cancers and their biological assessment. In addition, it provides a general idea of the in silico, in vitro, ex vivo, and in vivo models used for the anticancer evaluation of green biogenic metal-based nanoparticles.

Immunoprotection evaluation of the recombinant N-terminal domain of Cys-loop receptors against Rhipicephalus (Boophilus) microplus tick infestation.

Rhipicephalus (Boophilus) microplus ticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the application of chemical acaricides; however, their uncontrolled use has increased resistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective. Therefore, there is a need to characterize novel antigens in order to improve immunological protection. The aim of this work was to evaluate Cys-loop receptors as vaccine candidates. N-terminal domains of a glutamate receptor and of a glycine-like receptor were recombinantly produced in Escherichia coli. Groups of BALB/c mice were independently immunized with four doses of each recombinant protein emulsified with Freund's adjuvant. Both vaccine candidates were immunogenic in mice as demonstrated by western blot analysis. Next, recombinant proteins were independently formulated with the adjuvant Montanide ISA 50 V2 and evaluated in cattle infested with Rhipicephalus microplus tick larvae. Groups of three European crossbred calves were immunized with three doses of each adjuvanted protein. ELISA test was used to evaluate the IgG immune response elicited against the recombinant proteins. Results showed that vaccine candidates generated a moderate humoral response on vaccinated cattle. Vaccination significantly affected the number of engorged adult female ticks, having no significant effects on tick weight, egg weight and egg fertility values. Vaccine efficacies of 33% and 25% were calculated for the glutamate receptor and the glycine-like receptor, respectively.

TITLE: Évaluation de l'immunoprotection du domaine N-terminal recombinant des récepteurs Cys-loop contre l'infestation par les tiques Rhipicephalus (Boophilus) microplus. ABSTRACT: Les tiques Rhipicephalus (Boophilus) microplus sont des ectoparasites hématophages obligatoires des bovins et agissent comme vecteurs de micro-organismes pathogènes. Le contrôle conventionnel des tiques est basé sur l'application d'acaricides chimiques, mais leur utilisation incontrôlée a augmenté les populations de tiques résistantes ainsi que la contamination des aliments et de l'environnement. Le contrôle immunologique alternatif des tiques s'est avéré partiellement efficace. Par conséquent, il est nécessaire de caractériser de nouveaux antigènes afin d'améliorer la protection immunologique. Le but de ce travail était d'évaluer les récepteurs Cys-loop comme candidats vaccins. Les domaines N-terminaux d'un récepteur du glutamate et d'un récepteur de type glycine ont été produits par recombinaison chez Escherichia coli. Des groupes de souris BALB/c ont été immunisés indépendamment avec quatre doses de chaque protéine recombinante émulsionnée avec l'adjuvant de Freund. Les deux vaccins candidats étaient immunogènes chez la souris, comme l'a démontré l'analyse par transfert Western. Ensuite, des protéines recombinantes ont été formulées indépendamment avec l'adjuvant Montanide ISA 50 V2 et évaluées chez des bovins infestés de larves de tiques Rhipicephalus microplus. Des groupes de trois veaux croisés européens ont été immunisés avec trois doses de chaque protéine avec adjuvant. Le test ELISA a été utilisé pour évaluer la réponse immunitaire IgG induite contre les protéines recombinantes. Les résultats ont montré que les candidats vaccins généraient une réponse humorale modérée sur les bovins vaccinés. La vaccination a affecté de manière significative le nombre de tiques femelles adultes engorgées mais n'a eu aucun effet significatif sur le poids des tiques, le poids des œufs et les valeurs de fertilité des œufs. Des efficacités vaccinales de 33 % et 25 % ont été calculées pour le récepteur du glutamate et le récepteur de type glycine, respectivement.

Serum Analysis of Women with Early-Stage Breast Cancer Using a Mini-Array of Tumor-Associated Antigens.

Background: Several studies have shown that patients with cancer have antibodies in serum that react with cellular autoantigens, known as Tumor-Associated Antigens (TAA). The present work aimed to determine whether a mini-array comprising four recombinant TAA increases the detection of specific serum antibodies for the diagnosis of early-stage breast cancer. Methods: The mini-array included Alpha 1-AntiTrypsin (A1AT), TriosePhosphate Isomerase 1 (TPI1), Peptidyl-Prolyl cis-trans Isomerase A (PPIA), and PeroxiReDoXin 2 (PRDX2) full-length recombinant proteins. The proteins were produced after gene cloning, expression, and purification, and were verified by Western blot assays. Then, Dot-Blot was performed to find antibodies against the four TAA in 12 sera from women with early-stage breast cancer (stage II) and 12 sera from healthy women. Results: Antibody detection against individual TAA in early-stage breast cancer sera ranged from 58.3% to 83.3%. However, evaluation of the four TAA showed that there was a positive antibody reaction reaching a sensitivity of 100% and a specificity of 85% in early-stage breast cancer, suggesting that this mini-array must be evaluated as a clinical diagnostic tool for early-stage breast cancer in a larger sample size. Conclusion: Our results suggest that TAA mini-arrays may provide a promising and powerful method for improving the detection of breast cancer in Mexican women.

Molecular subtypes and clinicopathological features of breast cancer in Mexican women. / Subtipos moleculares y características clinicopatológicas de cáncer de mama en mujeres mexicanas.

BACKGROUND: Breast cancer is a multifactorial and heterogeneous disease with distinct molecular features and histopathologic subtypes involving different therapeutic responses and clinical outcomes. Classification of breast cancer in molecular subtypes has made possible an approach to develop therapeutic strategies in order to have a better understanding of the breast cancer development. Due to the heterogeneity of the disease, there are still features to be elucidated in the behavior, etiology and clinical outcomes of each molecular subtype in breast cancer. METHODS: Variables measured in 1,695 cases of invasive breast carcinoma were age, histopathological diagnosis, histopathological grade, expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), cell proliferation marker (Ki67) and basal cytokeratins (CK 5/6). P values were obtained using Chi square test and hazard ratios were calculated with 95% confidence intervals. RESULTS: An increase of aggressive molecular subtypes of breast cancer was observed. The mean age of incidence of breast cancer patients is decreasing, and breast cancer Patients younger than 40-years-old showed higher risk to exhibit Triple negative and Basal-like tumors. CONCLUSIONS: The mean age for this pathology is decreasing in our population and there is predominance in the differential occurrence of etiologically distinct entities of breast cancer affecting to the young women.

INTRODUCCIÓN: El cáncer de mama es una enfermedad heterogénea y multifactorial. Presenta distintas características clínicas, moleculares e histopatológicas, las cuales se asocian con la respuesta a los esquemas terapéuticos, así como al resultado clínico. La clasificación en subtipos moleculares (luminales, HER2, triple negativo y basales) ha permitido el desarrollo y aplicación de estrategias terapéuticas particulares. Sin embargo, dada la gran heterogeneidad de la enfermedad, existen aún características por elucidar en el comportamiento, etiología y resultados clínicos de cada subtipo molecular de cáncer de mama. MÉTODOS: Se obtuvieron datos de 1695 casos de cáncer de mama invasor. Se realizaron correlaciones entre las siguientes variables: edad, diagnóstico histopatológico, grado histológico, expresión del receptor de estrógenos (ER), receptor de progesterona (PR), receptor 2 del factor de crecimiento epidérmico humano (HER2), marcador de proliferación celular (Ki67) y citoqueratinas basales (CK 5/6). Los valores de p fueron calculados utilizando Chi cuadrada y el cociente de riesgo fue calculado con un intervalo de confianza de 95%. RESULTADOS: Se observó un incremento en la frecuencia de los subtipos moleculares más agresivos, así como una disminución en el valor de la media de la edad en las pacientes diagnosticadas con cáncer de mama. El análisis de la información indica que en pacientes menores de 40 años existe mayor riesgo a la presencia de tumores triple negativo o basales. CONCLUSIONES: En población mexicana, la media de edad para el diagnóstico primario de cáncer de está disminuyendo y hay mayor frecuencia de subtipos moleculares más agresivos en pacientes jóvenes.

Fenómeno de no reflujo en la angioplastia coronaria: incidencia, características clínico-angiográficas y eficacia del tratamiento / No-reflow phenomenon in coronary angioplasty: incidence, clinical-angiographic characteristics and efficacy of treatment

Resumen: Introducción: el fenómeno de no reflujo (NR) coronario se define como la persistencia de un flujo inadecuado menor a TIMI 3 durante la angioplastia coronaria (ATC), en ausencia de obstáculo macroscópico en las arterias coronarias epicárdicas. Objetivo principal: determinar la incidencia de NR en pacientes tratados con ATC por cualquier indicación. Objetivos secundarios: describir las características clínicas y angiográficas de la población con NR, características del procedimiento de angioplastia, así como el tratamiento y el valor pronóstico del NR. Métodos: estudio observacional, prospectivo, unicéntrico, sobre el total de ATC realizadas en un centro de cardiología intervencionista entre octubre de 2016 y enero de 2017, excluyendo la reestenosis intrastent y la enfermedad de injertos venosos. Se definieron dos grupos: control (flujo normal) y con fenómeno de NR. Se analizaron variables clínicas, angiográficas, terapéuticas y pronósticas. Resultados: se incluyó un total de 322 pacientes, 291 control y 31 NR. La incidencia de NR fue de 9,6%, presentándose en 20,7% de los síndromes coronarios agudos con elevación de ST (SCAcST), en 6,3% de los síndromes coronarios agudos sin elevación de ST (SCAsST) y en 3% de las anginas estables. La adenosina a dosis altas fue el tratamiento más utilizado. Se comprobó una mejoría significativa del flujo coronario evaluado por TIMI Frame Count asociado al tratamiento instituido (39,4±1,6 pretratamiento vs 21,9±1,2 postratamiento, p<0,001). A tres meses, en los pacientes con NR vesus control se observó una mayor incidencia de ángor (6,6% vs 1,1% respectivamente, p=0,001), nueva coronariografía (10% vs 1,1%, p=0,017) y nueva ATC (19% vs 1,1%, p=0,009). Conclusiones: la incidencia de NR en pacientes sometidos a ATC fue de 9,6%, presentándose más frecuentemente en el SCAcST. La adenosina intracoronaria a dosis altas fue el tratamiento más frecuentemente instituido y fue eficaz para mejorar significativamente el flujo coronario.

Summary: Introduction: no-reflow phenomenon is defined as persistence of inadequate flow less than TIMI 3 during coronary angioplasty in the absence of an obstacle in epicardial coronary arteries. Primary endpoint: to determine the incidence of no-reflow in patients treated with coronary angioplasty performed for any indication. Secondary endpoints: to describe the clinical and angiographic characteristics, procedural characteristics, treatment and prognostic value of no-reflow phenomenon. Methods: this is an observational, prospective and unicentric trial including all coronary angioplasties performed in an interventional Cardiology center between October 2016 and January 2017, excluding instent reestenosis and coronary vein grafts disease. Two groups were defined: control (normal coronary flow) and no-reflow phenomenon. Clinical, angiographic and prognostic variables were analyzed. Results: 322 patients were included, 291 control and 31 no-reflow. No-reflow incidence was 9.6%, occurring in 20.7% of acute coronary syndromes with ST segment elevation, 6.3% in non-ST segment elevation syndromes and 3% in stable coronary artery disease. High dose adenosine was the most frequently used agent for no-reflow treatment. No-reflow treatment was associated with a significant improvement in coronary flow measured by TIMI Frame Count (39.4±1.6 pretreatment vs 21.9±1.2 postreatment, p<0.001). At 3 month follow up, no-reflow patients vs control had a higher incidence of angina pectoris (6.6% vs 1.1% respectively, p=0.001), coronary angiography (10% vs 1.1%, p=0.017) and coronary angioplasty (19% vs 1.1%, p=0.009). Conclusions: no-reflow incidence in patients treated with coronary angioplasty was 9.6%, occurring more frequently in ST segment elevation syndromes. High dose adenosine was the most frequently used agent for no-reflow treatment and significantly improved coronary flow.

An immunoconjugated up-conversion nanocomplex for selective imaging and photodynamic therapy against HER2-positive breast cancer.

Photodynamic therapy represents a very attractive therapeutic tool considered to be effective, minimally invasive and minimally toxic. However, conventional photodynamic therapy actually has two main constraints: the limited penetration depth of visible light needed for its activation, and the lack of selectivity. Considering this, this work reports the synthesis and evaluation of a novel nanoconjugate for imaging and selective photodynamic therapy against HER2-positive breast cancer, a particularly aggressive form of the disease. It was demonstrated that upon 975 nm near infrared light exposure, the red emission of the NaYF4:Yb,Er up-conversion nanoparticles (UCNPs) can be used for optical imaging and simultaneously represent the source for the excitation of a covalently bound zinc tetracarboxyphenoxy phthalocyanine (ZnPc), a photosensitizer that in turn transfers energy to ground state molecular oxygen to produce cytotoxic singlet oxygen. The specificity of our nanoconjugates was achieved by immunoconjugation with Trastuzumab (Tras), a specific monoclonal antibody for selective detection and treatment of HER2-overexpressing malignant breast cancer cells. Selective tracking of SKBR-3 HER2-positive cells was verified by confocal microscopy analysis, and the photodynamic therapy effect was considerably improved when Trastuzumab was incorporated into the nanoconjugate, the UCNPs-ZnPc-Tras being practically inert in the absence of infrared light exposure but reducing the HER2-positive cell viability up to 21% upon 5 min of the irradiation. This theranostic nanoconjugate represents a valuable alternative for HER2-positive breast cancer imaging and selective photodynamic therapy.

Characterization of B7H6, an endogenous ligand for the NK cell activating receptor NKp30, reveals the identity of two different soluble isoforms during normal human pregnancy.

B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of human NK cells. In contrast, the release of soluble B7H6 has been proposed as a novel mechanism by which tumors might evade NK cell-mediated recognition. Since NK cells are critical for the maintenance of early pregnancy, it is not illogical that soluble B7H6 might also be an important factor in directing NK cell activity during normal pregnancy. Thus, this study was focused on the characterization of soluble B7H6 during the development of normal pregnancy. Serum samples were obtained from healthy pregnant women who were experiencing their second pregnancies (n=36). Additionally, 17 of these pregnant participants were longitudinally studied for the presence of B7H6 during their second and third trimesters. Age-matched healthy non-pregnant women served as controls (n=30). The presence of soluble B7H6 was revealed by Western blotting. A further characterization was performed using an immunoproteomic approach based on 2DE-Western blotting combined with MALDI-MS. The results show that sera from all pregnant women were characterized by the presence of two novel isoforms of B7H6, both with lower MW than the reported of 51kDa. These isoforms were either a heavy (∼37kDa) or a light isoform (∼30kDa) and were mutually exclusive. N-glycosylation did not completely explain the different molecular weights exhibited by the two isoforms, as was demonstrated by enzymatic deglycosylation with PNGase F. The confirmation of the identity and molecular mass of each isoform indicates that B7H6, while maintaining the C- and N-termini, is most likely released during pregnancy by a mechanism distinct from proteolytic cleavage. We found that both isoforms, but mainly the heavier B7H6, were released via exosomes; and that the lighter isoform was also released in an exosome-free manner that was not observed in the heavy isoform samples. In conclusion, we find that soluble B7H6 is constitutively expressed during pregnancy and that, moreover, the soluble B7H6 is present in two new isoforms, which are released by exosomal and exosome-free mechanisms.

30. Fenómeno de no reflujo en la angioplastia coronaria. Incidencia, tratamiento y eficacia con vasodilatadores coronarios

Antecedentes: el fenómeno de no reflujo (NR) se define como la reducción del flujo coronario

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Identification of immunogenic proteins from ovarian tissue and recognized in larval extracts of Rhipicephalus (Boophilus) microplus, through an immunoproteomic approach.

Rhipicephalus (Boophilus) microplus ticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the use of chemical acaricides; however, their uncontrolled use has increased tSresistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective. The only anti-tick vaccine commercially available at present in the world is based on intestinal Bm86 protein, and shows a variable effectiveness depending on tick strains or geographic isolates. Therefore, there is a need to characterize new antigens in order to improve immunological protection. The aim of this work was to identify immunogenic proteins from ovarian tissue extracts of R. microplus, after cattle immunization. Results showed that ovarian proteins complexed with the adjuvant Montanide ISA 50 V generated a strong humoral response on vaccinated cattle. IgG levels peaked at fourth post-immunization week and remained high until the end of the experiment. 1D and 2D SDS-PAGE-Western blot assays with sera from immunized cattle recognized several ovarian proteins. Reactive bands were cut and analyzed by LC-MS/MS. They were identified as Vitellogenin, Vitellogenin-2 precursor and Yolk Cathepsin. Our findings along with bioinformatic analysis indicate that R. microplus has several Vitellogenin members, which are proteolytically processed to generate multiple polypeptide fragments. This apparent complexity of vitellogenic tick molecular targets gives the opportunity to explore their potential usefulness as vaccine candidates but, at the same time, imposes a challenge on the selection of the appropriate set of antigens.

A case of successful restoration of a tropical wetland evaluated through its Odonata (Insecta) larval assemblage

etlands are important wildlife habitats that also provide vital services for human societies. Unfortunately, they have been disappearing due to human activities such as conversion to farmland, pollution, habitat fragmentation, invasion of alien species, and inappropriate management, resulting in declines in species diversity, wildlife habitat quality, and ecosystem functions and services. In some countries, many programs and actions have been undertaken to reverse the rate of wetland loss by restoring, creating and constructing new wetlands. We report on the assessment of Odonata larvae from a tropical and putatively restored wetland located in the La Mancha Biological Station, CICOLMA (LM, Ramsar site #1336), Veracruz, Mexico. Larval surveys were performed during the 2010 and 2011 dry and rainy seasons in both LM and a reference site, Cansaburro (CB), located approximately 2 km South of LM. Twelve samples were collected during each survey using a D-frame aquatic net (0.2 mm mesh size), sweeping 1 m2 areas along shorelines using a random design. The effect of site, season and year on Odonata larval abundance was explored and diversity and abundance patterns of the assemblages were compared. A total of 3 718 larvae from 25 species (five Zygoptera and 20 Anisoptera) in 14 genera and three families were collected from both wetlands. Species number was equal in both wetlands although abundance was significantly higher in LM. Renyi´s diversity profiles and species abundance patterns (rank abundance curves) in both sites were similar, suggesting an apparent recovery at LM. Differences in species composition (sites shared 13 species), and species dominance between both assemblages were observed and were related to differences in the aquatic plant structure between both wetlands as a result of extensive plant management in LM and cattle grazing in CB. Most evidence derived from this work shows that the LM wetland may be recovered. Rev. Biol. Trop. 63 (4): 1043-1058. Epub 2015 December 01.

os humedales son hábitats importantes para la vida silvestre y también proporcionan servicios vitales para la sociedad que, por desgracia, han ido desapareciendo debido al uso del suelo que produce una disminución en la diversidad de especies, la calidad del hábitat de vida silvestre, y de la función de los ecosistemas. En algunos países se han llevado a cabo programas y acciones para revertir la tasa de pérdida de los humedales mediante la restauración, la creación y la construcción de nuevos humedales. En este trabajo se reporta un estudio numérico de las larvas de odonatos de un humedal tropical asumido como restaurado, localizado en la Estación de Biología La Mancha, CICOLMA (LM, sitio Ramsar # 1336), Veracruz, México. Los muestreos de las larvas se realizaron durante las estaciones de secas y lluvias de 2010 y 2011 tanto en LM como en el sitio de referencia Cansaburro (CB) situado a unos 2 km al sur de LM. Se tomaron doce muestras al azar en las orillas durante cada recolecta en cada sitio utilizando una red acuática D-net (abertura de malla de 0.2 mm) barriendo una área aproximada de 1 m2. El efecto del sitio, la temporada y el año sobre la abundancia de larvas fue explorado y los patrones de diversidad y abundancia de ambas comunidades fueron comparadas. Un total de 3 718 larvas de 25 especies (cinco Zygoptera y 20 Anisoptera) de 14 géneros y tres familias se obtuvieron de ambos humedales. El número de especies fue igual en ambos humedales aunque la abundancia fue significativamente mayor en LM. Los perfiles de diversidad de Renyi y los patrones de abundancia de especies (curvas de rango abundancia) en ambos sitios fueron similares sugiriendo una evidente recuperación de LM. Las diferencias observadas en la composición de especies (los sitios compartieron 13 especies) y la dominancia de las especies entre ambas comunidades se explica con base en las diferencias en la estructura vegetal acuática entre los dos humedales que es principalmente el resultado de una amplio manejo en LM y del pastoreo de ganado en CB. La mayoría de las pruebas derivadas de este trabajo muestran que el humedal de LM puede estar recuperado.

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach.

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

Mannheimia haemolytica et Histophilus somni sont fréquemment isolées de bovins atteints de maladies respiratoires bovines (MRB). Ces agents compromettent la fonction pulmonaire et les réponses immunitaires générées ne permettent pas de limiter l'infection. L'identification d'antigènes spécifiques et immunogènes qui permettraient le développement de vaccins, représente un grand défi actuellement. Les protéines immunogènes ont été identifiées par une approche immunoproteomique en utilisant des sérums provenant de bovins immunisés par des vaccins commerciaux de M. haemolytica et H. somni. Les protéines de M. haemolytica ont été identifiées comme étant un transporteur ABC, une protéine de liaison du fer et une hypothétique protéine impliquée dans la biosynthèse de la capsule. Celles de H. somni correspondent à une porine, à un transporteur ABC d'acides aminés, à une hypothétique protéine de membrane externe, à la cystéine synthase et à la protéine membranaire P6. Bien que ces antigènes présentent une forte homologie avec des protéines provenant d'autres pathogènes d'animaux, les protéines du système ABC sont associées à la virulence et pourraient être considérées comme des candidats potentiels pour l'élaboration de vaccins contre les MBR.(Traduit par Docteur Patricia Dupre).

The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG.

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.