New Synergistic Combination Therapy of Mupirocin and α-Pinene Against Multidrug-Resistant Clinical Strains of Methicillin-Resistant Staphylococcus aureus.

OBJECTIVE: Increased mupirocin use leads to mupirocin resistance and is associated with persistence of methicillin-resistant Staphylococcus aureus (MRSA) carriers, prolonged hospitalization, and significant economic burdens for health systems. The study aimed to investigate the antimicrobial activity of compounds of Salvia rosmarinus L. ("rosemary", formerly Rosmarinus officinalis), alone or in combination with mupirocin, against multidrug resistant MRSA using isolates obtained from pediatric patients. METHODS: The in vitro antibacterial activity of the monoterpene α-pinene (α-Pi), a rosemary essential oil constituent, alone and in combination with mupirocin, was evaluated by determining the minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs) and the fractional inhibitory concentration indices (FICIs) and fractional bactericidal concentration indices against multidrug-resistant clinical MRSA strains. The in vivo efficacy of α-Pi, alone and in combination with mupirocin, to eradicate MRSA infection was determined using an optimized mouse model of MRSA-infected wounds. Mouse skin samples (obtained via biopsy) were assessed for toxicity, and rabbit skin samples for irritation. RESULTS: Both in vitro and in vivo, α-Pi was active against MRSA strains and acted synergistically with mupirocin against MRSA strains. Mupirocin-monoterpene combinations exhibited FICI values of 0.2 to 0.4, reducing the MBC of topical mupirocin 33-fold. A topical formulation containing α-Pi and mupirocin enhanced the efficacy of mupirocin in an in vivo MRSA-infected mouse skin model without significantly harming the skin of mice and rabbits. CONCLUSIONS: A topical formulation combining mupirocin and α-Pi may aid in the development of innovative agents for treating MRSA infections.

Development and Validation of UPLC Tandem Mass Spectrometry Assay for Ceftibuten and Sulbactam in Human Plasma.

A sensitive and rapid ultra-performance liquid chromatography coupled with -tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine ceftibuten (CTB) and sulbactam (SUL) in human plasma. An ACQUITY UPLC HSS T3 C18 (2.1 × 100 mm), 1.8 µm column with gradient elution of water (0.1% formic acid) and acetonitrile was used for separation at a flow rate of 0.2 mL/min. This method involves a simple sample preparation with acetonitrile. The calibration curves of CTB and SUL in plasma showed good linearity over the concentration range of 0.50-25 µg/mL and with a correlation coefficient (r2) >0.99. This method was validated in terms of selectivity, linearity, precision, accuracy and stability. High precision was obtained with coefficients of variation <15%. Excellent recovery in the range of 90-104% was achieved for CTB and SUL was 86-110%. The method has the potential utility to support pharmacometric modeling in clinical practice and biopharmaceutic studies.

Efectos de la pandemia por COVID-19 en determinantes sociales de familias del área metropolitana de Bucaramanga

Introducción: La pandemia por COVID-19 trajo consigo grandes cambios a nivel socioeconómico, producto de las medidas tomadas para mitigar su expansión, que implicó cierre de colegios y dificultad para el acceso a servicios de salud. Sin embargo, no se cuenta con mucha información respecto al impacto que estas medidas han tenido en la salud y el bienestar de niños y adolescentes, por lo cual se desarrolló una encuesta virtual para conocer la dimensión de los efectos de la pandemia en el bienestar integral de los menores y sus familias. Metodología: Estudio observacional de corte transversal que se realizó mediante la aplicación de una encuesta en formato electrónico a padres de familia de niños y adolescentes del Área Metropolitana de Bucaramanga. Resultados: Se obtuvieron 960 respuestas. El 25,63 % de los encuestados refieren cancelación de citas médicas. El 98 % de los estudiantes pudo continuar las actividades académicas durante el aislamiento. El factor económico fue la principal causa de preocupación en el periodo de la encuesta. Discusión: Durante el periodo de aislamiento, los problemas de salud mental, las dificultades para el acceso a herramientas para la educación virtual y las barreras para la atención, propias de la emergencia sanitaria, causaron efectos significativos en la calidad de vida de los menores. Conclusiones: Ante emergencias sanitarias, se deben mantener los servicios de atención en salud de la misma forma que se hacía previo a la ocurrencia del evento, como los programas de vacunación, crecimiento y desarrollo, promoción y prevención, además de la continuidad de la escolaridad.

Introduction: The COVID-19 pandemic has brought great changes along with, some of those were at the socioeconomic level, as a result of the actions taken to mitigate the virus expansion, which involved the closure of schools and restriction in accessing to some health services. However, there is not much information regarding the impact that these measures have had on the health and well-being of children and adolescents, for this reason, a virtual survey was developed to find out the dimension of the pandemic's effect on the comprehensive welfare of minors and families. Methodology: Cross-sectional observational study, which was carried out by applying a survey in electronic format to parents of children and adolescents in the Metropolitan Area of Bucaramanga. Results: A total of 960 responses were obtained. Of those surveyed, 25,63% refer cancellation of medical appointments. The 98% of students were able to continue academic activities during isolation. The economic factor was the main cause of concern in the survey period. Discussion: During the isolation period, mental health problems, difficulties in accessing tools for virtual education and barriers to care, typical of the health emergency, caused a significant effects on the quality of life of youngsters. Conclusions: In the event of health emergencies, health care services should be maintained in the same way as before the occurrence of the event, such as vaccination, growth and development, promotion and prevention programs, in addition to the continuity of schooling.

Biomimetic Protocells Featuring Macrophage-Like Capture and Digestion of Protein Pathogens.

Modern medical research develops interest in sophisticated artificial nano- and microdevices for future treatment of human diseases related to biological dysfunctions. This covers the design of protocells capable of mimicking the structure and functionality of eukaryotic cells. The authors use artificial organelles based on trypsin-loaded pH-sensitive polymeric vesicles to provide macrophage-like digestive functions under physiological conditions. Herein, an artificial cell is established where digestive artificial organelles (nanosize) are integrated into a protocell (microsize). With this method, mimicking crossing of different biological barriers, capture of model protein pathogens, and compartmentalized digestive function are possible. This allows the integration of different components (e.g., dextran as stabilizing block) and the diffusion of pathogens in simulated cytosolic environment under physiological conditions. An integrated characterization approach is carried out, with identifying electrospray ionization mass spectrometry as an excellent detection method for the degradation of a small peptide such as ß-amyloid. The degradation of model enzymes is measured by enzyme activity assays. This work is an important contribution to effective biomimicry with the design of cell-like functions having potential for therapeutic action.

Model-informed precision dosing of antimicrobial drugs in pediatrics: experiences from a pilot scale program.

Antibiotics are among the most utilized drugs in pediatrics. Nonetheless, there is a lack in pharmacokinetics information for this population, and dosing criteria may vary between healthcare centers. Physiological variability associated with maturation in pediatrics makes it challenging to reach a consensus on adequate dosing, which is further accentuated in more vulnerable groups, such as critically ill or oncology patients. Model-informed precision dosing is a useful practice that allows dose optimization and attainment of antibiotic-specific pharmacokinetic/pharmacodynamic targets. The aim of this study was to evaluate the needs of model-informed precision dosing of antibiotics in a pediatrics unit, at a pilot scale. Pediatric patients under antibiotic treatment were monitored with either a pharmacokinetic/pharmacodynamic optimized sampling scheme or through opportunistic sampling. Clindamycin, fluconazole, linezolid, meropenem, metronidazole, piperacillin, and vancomycin plasma concentrations were quantified through a liquid chromatography coupled to mass spectrometry method. Pharmacokinetic parameters were estimated using a Bayesian approach to verify pharmacokinetic/pharmacodynamic target attainment. A total of 23 pediatric patients aged 2 to 16 years were included, and 43 dosing regimens were evaluated; 27 (63%) of them required adjustments as follows: 14 patients were underdosed, 4 were overdosed, and 9 patients needed infusion rate adjustments. Infusion rate adjustments were mostly recommended for piperacillin and meropenem; daily doses were augmented for vancomycin and metronidazole, meanwhile linezolid was adjusted for under- and overdosing. Clindamycin and fluconazole regimens were not adjusted at all.  Conclusion: Results showcase a lack of antibiotic pharmacokinetic/pharmacodynamic target attainment (particularly for linezolid, vancomycin, meropenem, and piperacillin), and the need for model-informed precision dosing in pediatrics. This study provides pharmacokinetic evidence which can further improve antibiotic dosing practices. What is Known: • Model-informed precision dosing is performed in pediatrics to optimize the treatment of antimicrobial drugs such as vancomycin and aminoglycosides, while its usefulness is debated for other groups (beta-lactams, macrolides, etc.). What is New: • Vulnerable pediatric subpopulations, such as critically ill or oncology patients, can benefit the most from model-informed precision dosing of antibiotics. • Model-informed precision dosing of linezolid, meropenem, piperacillin, and vancomycin is particularly useful in pediatrics, and further research may improve dosing practices altogether.

A new methodology based on TRU resin to measure U-, Th-isotopes and 210Po by alpha-particle spectrometry.

This report presents a new methodology to isolate and measure 210Po, as well as uranium and thorium isotopes. This new methodology reduces the standard time of operation, the minimum amount of chemical reagents and the quantity of resin used in comparison with other standard and well-established procedures for alpha spectrometry. Thus, the amount of chemicals reagent was lower than the amount used in other standard radiochemical processes: only 6 mL of 1 M HCl was used for the thorium elution, and 2 mL of H2O and 1 mL of Ammonium Oxalate (0.05 M) (3 mL in total) for the uranium elution. Likewise, many samples of various activities and materials (liquids and solids) were used to validate the method.

The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection.

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis. IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress.

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e., tachyzoites) and establishing chronic infection (i.e., bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here, we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with downregulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores, leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

Multi-organ pathological findings associated with COVID-19 in postmortem needle core biopsies in four patients and a review of the current literature.

Coronavirus disease 2019 (COVID-19) has spread worldwide, resulting in significant morbidity and mortality. Histopathological findings are essential in understanding its pathogenesis and we present our findings from postmortem core needle biopsies in an attempt to share information that may shed some light on this severe pandemic. Different organ samples from four patients with PCR-confirmed COVID-19 at the Infanta Sofía Hospital (Madrid) were studied during the months of April and May, 2020 by six pathologists using routine stains, histochemistry and immunohistochemistry. Results were compared with other reported cases. All patients had a clinical diagnosis of pneumonia and biopsies revealed lung damage in the majority. Heart, liver, spleen and kidney were also studied and abnormalities were found in all cases and are extensively described. The histopathology of organs affected by COVID-19 is vital to the understanding of this disease and its sequelae.

A Radiogenomics Ensemble to Predict EGFR and KRAS Mutations in NSCLC.

Lung cancer causes more deaths globally than any other type of cancer. To determine the best treatment, detecting EGFR and KRAS mutations is of interest. However, non-invasive ways to obtain this information are not available. Furthermore, many times there is a lack of big enough relevant public datasets, so the performance of single classifiers is not outstanding. In this paper, an ensemble approach is applied to increase the performance of EGFR and KRAS mutation prediction using a small dataset. A new voting scheme, Selective Class Average Voting (SCAV), is proposed and its performance is assessed both for machine learning models and CNNs. For the EGFR mutation, in the machine learning approach, there was an increase in the sensitivity from 0.66 to 0.75, and an increase in AUC from 0.68 to 0.70. With the deep learning approach, an AUC of 0.846 was obtained, and with SCAV, the accuracy of the model was increased from 0.80 to 0.857. For the KRAS mutation, both in the machine learning models (0.65 to 0.71 AUC) and the deep learning models (0.739 to 0.778 AUC), a significant increase in performance was found. The results obtained in this work show how to effectively learn from small image datasets to predict EGFR and KRAS mutations, and that using ensembles with SCAV increases the performance of machine learning classifiers and CNNs. The results provide confidence that as large datasets become available, tools to augment clinical capabilities can be fielded.

Ca2+ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V-H+ -ATPase in Toxoplasma gondii.

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V-H+ -ATPase, a pump previously shown to transport protons to the extracellular medium, and to control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca2+ at physiological levels and for Ca2+ influx. Proton transport was also important for Ca2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca2+ and concentrates in acidocalcisomes. This was supported by the co-localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V-ATPase in acidocalcisomes. Our work shows that proton transport regulates plasma membrane Ca2+ transport and control acidocalcisome polyP and Ca2+ content, impacting Ca2+ signaling and downstream stimulation of motility and egress in T. gondii.

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.

Clinical utility of ABCB1 and ABCG2 genotyping for assessing the clinical and pathological response to FAC therapy in Mexican breast cancer patients.

PURPOSE: Resistance to neoadjuvant chemotherapy with 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC) in some patients with locally advanced breast cancer remains one of the main obstacles to first-line treatment. We investigated clinical and pathological responses to FAC neoadjuvant chemotherapy in Mexican women with breast cancer and their possible association with SNPs present in ABC transporters as predictors of chemoresistance. MATERIALS: A total of 102 patients undergoing FAC neoadjuvant chemotherapy were included in the study. SNP analysis was performed by RT-PCR from genomic DNA. Two SNPs were analyzed: ABCB1 rs1045642 (3435 C > T) and ABCG2 rs2231142 (421 G > T). RESULTS: In clinical response evaluation, significant associations were found between the ABCB1 C3435T genotype and breast cancer chemoresistant and chemosensitive patients (p < 0.05). In the early clinical response, patients with genotype C/C or C/T were more likely to be chemosensitive to neoadjuvant therapy than patients with genotype T/T (OR = 4.055; p = 0.0064). Association analysis between the ABCB1 gene polymorphism and the pathologic response to FAC chemotherapy showed that the C/C + C/T genotype was a protective factor against chemoresistance (OR = 3.714; p = 0.0104). Polymorphisms in ABCG2 indicated a lack of association with resistance to chemotherapy (p = 0.2586) evaluating the clinical or pathological response rate to FAC neoadjuvant chemotherapy. CONCLUSION: The early clinical response and its association with SNPs in the ABCB1 transporter are preserved until the pathological response to neoadjuvant chemotherapy; therefore, it could be used as a predictor of chemoresistance in locally advanced breast cancer patients of the Mexican population.

DOTA Glycodendrimers as Cu(II) Complexing Agents and Their Dynamic Interaction Characteristics toward Liposomes.

Copper (Cu)(II) ions, mainly an excess amount, play a negative role in the course of several diseases, like cancers, neurodegenerative diseases, and the so-called Wilson disease. On the contrary, Cu(II) ions are also capable of improving anticancer drug efficiency. For this reason, it is of great interest to study the interacting ability of Cu(II)-nanodrug and Cu(II)-nanocarrier complexes with cell membranes for their potential use as nanotherapeutics. In this study, the complex interaction between 1,4,7,10-tetraazacyclododecan-N,N',N'',N'''-tetraacetic acid (DOTA)-functionalized poly(propyleneimine) (PPI) glycodendrimers and Cu(II) ions and/or neutral and anionic lipid membrane models using different liposomes is described. These interactions were investigated via dynamic light scattering (DLS), ζ-potential (ZP), electron paramagnetic resonance (EPR), fluorescence anisotropy, and cryogenic transmission electron microscopy (cryo-TEM). Structural and dynamic information about the PPI glycodendrimer and its Cu(II) complexes toward liposomes was obtained via EPR. At the binding site Cu-N2O2 coordination prevails, while at the external interface, this coordination partially weakens due to competitive dendrimer-liposome interactions, with only small liposome structural perturbation. Fluorescence anisotropy was used to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer, while DLS and ZP allowed us to determine the distribution profile of the nanoparticle (PPI glycodendrimer and liposomes) size and surface charge, respectively. From this multitechnique approach, it is deduced that DOTA-PPI glycodendrimers selectively extract Cu(II) ions from the bioenvironment, while these complexes interact with the liposome surface, preferentially with even more negatively charged liposomes. However, these complexes are not able to cross the cell membrane model and poorly perturb the membrane structure, showing their potential for biomedical use.

A population approach of rifampicin pharmacogenetics and pharmacokinetics in Mexican patients with tuberculosis.

The aim of this study was to develop a population pharmacokinetic model of rifampicin (RMP) in Mexican patients with tuberculosis (TB) to evaluate the influence of anthropometric and clinical covariates, as well as genotypic variants associated with MDR1 and OATP1B1 transporters. A prospective study approved by Research Ethics Committee was performed at Hospital Central in San Luis Potosí, Mexico. TB patients under DOTS scheme and who signed informed consent were consecutively included. Anthropometric and clinical information was retrieved from medical records. Single nucleotide polymorphisms in MDR1 (C3435T) and SLCO1B1 (A388G and T521C) genes were evaluated. RMP plasma concentrations and time data were assessed with NONMEM software. A total of 71 Mexican TB patients from 18 to 72 years old were included for RMP quantification from 0.3 to 12 h after dose; 329 and 97 plasma concentrations were available for model development and validation, respectively. Sequential process includes a typical lag time of 0.25 h prior to absorption start with a Ka of 1.24 h-1 and a zero-order absorption of 0.62 h to characterize the gradual increase in RMP plasma concentrations. Final model includes total body weight in volume of distribution (0.7 L/kg, CV = 26.8%) and a total clearance of 5.96 L/h (CV = 38.5%). Bioavailability was modified according to time under treatment and generic formulation administration. In conclusion, a population pharmacokinetic model was developed to describe the variability in RMP plasma concentrations in Mexican TB patients. Genetic variants evaluated did not showed significant influence on pharmacokinetic parameters. Final model will allow therapeutic drug monitoring at early stages.

Chromatin remodeling and transcription of the TPK1 subunit of PKA during stress in Saccharomyces cerevisiae.

In response to environmental changes cells rapidly rearrange their gene expression pattern in order to adapt to the new conditions. Chromatin remodeling is critical for this process playing a major role in the induction of genes involved in stress responses. We demonstrated previously that TPK1, encoding one of the catalytic subunits of PKA from Saccharomyces cerevisiae, is upregulated under heat shock. Herein, we investigate the chromatin remodeling of the TPK1, TPK2 and TPK3 promoters under heat stress. The TPK1 promoter is the only one that presents three positioned nucleosomes. Upon heat stress or osmostress these nucleosomes are evicted in clear correlation with promoter activation and upregulation of TPK1 mRNA levels. We find that remodelers SWI/SNF, RSC, INO80 and ISW1 participate in chromatin remodeling of the TPK1 promoter under thermal stress conditions. RSC and INO80 are necessary for nucleosomes positioning and contribute to repression of the TPK1 promoter under normal conditions while SWI/SNF participates in the eviction of nucleosomes after heat stress. SWI/SNF complex is recruited to the TPK1 promoter upon heat shock in a Msn2/4-dependent manner. Finally, both Tpk1 and Tpk2 catalytic subunits are recruited to the TPK1 promoter with opposite association patterns. Tpk1 catalytic activity is necessary for nucleosome rearrangement on the TPK1 promoter while Tpk2 and Tpk3 inhibit the promoter activity and maintain a repressive chromatin conformation. This work enlightens the mechanism of regulation of TPK1 expression during heat-stress, contributing to the knowledge of specificity in fine-tuning the cAMP-PKA signaling circuit.

Generation Dependent Effects and Entrance to Mitochondria of Hybrid Dendrimers on Normal and Cancer Neuronal Cells In Vitro.

Dendrimers as drug carriers can be utilized for drugs and siRNA delivery in central nervous system (CNS) disorders, including various types of cancers, such as neuroblastomas and gliomas. They have also been considered as drugs per se, for example as anti-Alzheimer's disease (AD), anti-cancer, anti-prion or anti-inflammatory agents. Since the influence of carbosilane-viologen-phosphorus dendrimers (SMT1 and SMT2) on the basic cellular processes of nerve cells had not been investigated, we examined the impact of two generations of these hybrid macromolecules on two murine cell lines-cancer cell line N2a (mouse neuroblastoma) and normal immortalized cell line mHippoE-18 (embryonic mouse hippocampal cell line). We examined alterations in cellular responses including the activity of mitochondrial dehydrogenases, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, and morphological modifications and fractions of apoptotic and dead cells. Our results show that both dendrimers at low concentrations affected the cancer cell line more than the normal one. Also, generation-dependent effects were found: the highest generation induced greater cytotoxic effects and morphological modifications. The most promising is that the changes in mitochondrial membrane potential and transmission electron microscopy (TEM) images indicate that dendrimer SMT1 can reach mitochondria. Thus, SMT1 and SMT2 seem to have potential as nanocarriers to mitochondria or anti-cancer drugs per se in CNS disorders.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids.

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Postsurgical intestinal occlusion ileum-ileum double intussusception in eleven-month girl.

BACKGROUND: Postoperative intussusception is an unusual complication in children; the incidence is 0.01-0.25%. CLINICAL CASE: Female of 11 months who underwent colostomy, 48 h later presents abdominal pain, vomiting gastrobiliar and abdominal distension. A laparotomy was performed, finding a double ileo-ileal invagination 60 cm from the ileocecal valve. DISCUSSION: The postoperative intestinal invaginations are extremely rare and most of them occur within the first 7 days. The etiology of postoperative intussusception is unknown. CONCLUSIONS: Postoperative children with intestinal occlusion, intussusception should be suspected and an immediate laparotomy should be performed.

ANTECEDENTES: La invaginación posoperatoria es una complicación inusual en niños. Su incidencia es del 0.01-0.25%. CASO CLÍNICO: Niña de 11 meses a quien se realizó colostomía y 48 horas después presentó dolor abdominal, vómito gastrobiliar y distensión abdominal. Se realizó laparotomía y se encontró una doble invaginación íleo-ileal a 60 cm de la válvula ileocecal. DISCUSIÓN: Las invaginaciones intestinales posoperatorias son extremadamente raras y la mayoría se presentan dentro de los primeros 7 días. La etiología de la invaginación posoperatoria es desconocida. CONCLUSIONES: En niños operados con oclusión intestinal debe sospecharse invaginación intestinal y realizar una laparotomía inmediata.

[Anemia increases mortality risk associated with frailty or disability in older adults. The FRADEA Study]. / La anemia aumenta el riesgo de mortalidad debido a fragilidad y discapacidad en mayores: Estudio FRADEA.

OBJECTIVE: To analyze if anemia increases 10-year mortality risk associated to frailty and disability in older adults. DESIGN: Substudy of the FRADEA population-based concurrent cohort study (Frailty and dependence in Albacete), with a 10-year follow-up (2007-2017) in people older than 69years. SETTING: Albacete city, Spain. PARTICIPANTS: Of the 993 participants included in the first wave, 790 were selected with valid data on function (frailty and disability), anemia and vital status at 10years. MAIN MEASUREMENTS: Anemia was defined according to the criteria of the World Health Organization (hemoglobin <13g/dL in men and <12g/dL in women). A functional classification variable was created, including frailty and disability, identifying four progressive functional levels: robust, prefrail, frail and disabled in basic activities of daily life, using frailty phenotype and Barthel index respectively. A new eight categories variable was constructed combining the four functional groups with the presence or absence of anemia. The association with mortality was determined by Kaplan-Meier and Cox proportional hazards analysis adjusted for age, sex, comorbidity, polypharmacy, institutionalization and creatinine. RESULTS: Mean age was 79years and 59.6% were women. 393 participants (49.7%) died during the follow-up period. The median survival was 98.4months (interquartile range 61). The risk of mortality increased from the levels with better functionality to those with worse functionality, and for each subgroup it was higher in the participants with anemia. Prefrail without anemia HR [hazard ratio] 1.59 (95%CI 1.07-2.36) and with anemia HR 2.37 (95%CI 1.38-4.05). Frail without anemia HR 3.18 (95%CI 1.68-6.02) and with anemia HR 4.42 (95%CI 1.99-9.84). Disabled without anemia HR 3.81 (95%CI 2.45-5.84) and with anemia HR 5.48 (95%CI 3.43-8.76). CONCLUSION: Anemia increases the risk of mortality associated with frailty and disability in older adults.