Muscle secreted factors enhance activation of the PI3K/Akt and ß-catenin pathways in murine osteocytes.

Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton and more recently focus has been placed on molecular/biochemical coupling of these two tissues. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5-6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 µÎµ. At 24 h post-load we observed a 2.5-fold increase in ß-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active ß-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based ß-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1-2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6-8 fold increase in pAkt compared to a 3-4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of ß-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.

Papel del 131I en el cáncer diferenciado de tiroides de bajo riesgo / Role of 131I in low-risk differentiated thyroid cancer

El cáncer diferenciado de tiroides (CDT) representa la neoplasia endocrina más frecuente, registrándose un incremento en las últimas décadas. El carcinoma papilar es el subtipo histológico más frecuente y un gran número de casos se relaciona con tumores de pequeño tamaño y con poca repercusión clínica, detectados de manera incidental o como consecuencia de la disponibilidad de las técnicas diagnósticas. El «buen pronóstico» de la mayoría de los casos han mantenido desde hace años la controversia en el abordaje de estos pacientes, especialmente en dos aspectos básicos del protocolo terapéutico: la cirugía y la administración de radioyodo. Si bien en los pacientes metastásicos y de alto riesgo, la administración de terapia con 131I está ampliamente aceptada, en los pacientes de riesgo intermedio-bajo su uso está muy cuestionado. En este trabajo realizamos una revisión de la evidencia disponible sobre la terapia con radioyodo en los pacientes de bajo riesgo (AU)

Differentiated thyroid cancer (DTC) is the most frequent endocrine neoplasm, with an incidence that has increased in recent decades. Papillary carcinoma is the most frequent histological subtype and a large number of cases are related to tumors of small size and with little clinical repercussion that are detected incidentally or as a consequence of the availability of diagnostic techniques. Due to the “good prognosis” of the majority of cases, for years the approach to these patients has remained controversial, especially in relation to two basic aspects of the therapeutic protocol: surgery and the administration of radioiodine. While in metastatic and high-risk patients, the administration of 131I therapy is widely accepted, in intermediate-low risk patients its use is highly questioned. Here we review the available evidence on radioiodine therapy in low-risk patients (AU)

[Headache associated with the use of personal protective equipment during the COVID-19 pandemic: a practical way to improve this condition]. / Cefalea asociada con el uso de equipo de protección personal durante la pandemia de la COVID-19: una forma práctica de mejorar esta condición.

TITLE: Cefalea asociada con el uso de equipo de protección personal durante la pandemia de la COVID-19: una forma práctica de mejorar esta condición.

Draft Genome Sequences of Two Epidemic OXA-48-Producing Klebsiella pneumoniae Clinical Strains Isolated during a Large Outbreak in Spain.

We report here the draft genome sequences of Klebsiella pneumoniae strains Kp1803 and Kp3380 isolated during a large outbreak at A Coruña Hospital in Spain. The final genome assemblies for Kp1803 and Kp3380 comprise approximately 6.6 and 6.1 Mb, respectively, and both strains have G+C contents of 57.2%.

Cost-effectiveness of using recombinant human thyroid-stimulating hormone before radioiodine ablation for thyroid cancer treatment in Spanish hospitals. / Coste-efectividad de la utilización de la tirotropina recombinante humana previa a la ablación con radioyodo en el tratamiento del cáncer de tiroides en hospitales españoles.

OBJECTIVES: In thyroid cancer treatment, the thyroid-stimulating hormone (TSH) must be elevated before radioiodine ablation, either by exogenous (with recombinant human thyrotropin [rhTSH]) or endogenous stimulation by thyroid hormone withdrawal (THW). The use of rhTSH avoids hypothyroidism and favours the subsequent elimination of radioiodine, but involves the cost of the product. For this reason, a cost-effectiveness analysis was performed, taking into account all costs involved and the benefits associated with the use of this therapy. MATERIAL AND METHODS: Using a Markov modelling with two analysis arms (rhTSH and THW), stratified into high (100mCi/3700 MBq) and low (30mCi/1110 MBq) radioiodine doses, and using 17 weekly cycles, the incremental cost per quality-adjusted life-year (QALY) related to the use of rhTSH was determined. The clinical inputs included in the model were based on published studies and in a treatment survey conducted in Spain. RESULTS: Radioablation preparation with rhTSH is superior to THW, showing additional benefits (0.048 AVAC), as well as cost savings (-€614.16), with an incremental cost-effectiveness rate (ICER) of -€12,795/QALY. The univariate and multivariate sensitivity analyses showed the result to be robust. CONCLUSIONS: The use of rhTSH previous to radioablation in Spain has cost savings, as well as a series of health benefits for the patient, making it highly cost-effective.

The FhaB/FhaC two-partner secretion system is involved in adhesion of Acinetobacter baumannii AbH12O-A2 strain.

Acinetobacter baumannii is a hospital-acquired pathogen that shows an extraordinary capacity to stay in the hospital environment. Adherence of the bacteria to eukaryotic cells or to abiotic surfaces is the first step for establishing an infection. The A. baumannii strain AbH12O-A2 showed an exceptional ability to adhere to A549 epithelial cells. The AbFhaB/FhaC 2-partner secretion (TPS) system involved in adhesion was discovered after the screening of the recently determined A. baumannii AbH12O-A2 strain genome (CP009534.1). The AbFhaB is a large exoprotein which transport to the bacterial surface is mediated by the AbFhaC protein. In the present study, the role of this TPS system in the AbH12O-A2 adherence phenotype was investigated. The functional inactivation of this 2-partner secretion system was addressed by analyzing the outer membrane vesicles (OMV) proteomic profile from the wild-type strain and its derivative mutant AbH12O-A2ΔfhaC demonstrating that AbFhaB is no longer detected in the absence of AbFhaC. Scanning electron microscopy (SEM) and adhesion experiments demonstrated that inactivation of the AbFhaB/FhaC system significantly decreases bacterial attachment to A549 alveolar epithelial cells. Moreover, it has been demonstrated that this 2-partner secretion system is involved in fibronectin-mediated adherence of the A. baumannii AbH12O-A2 isolate. Finally, we report that the AbFhaB/FhaC system is involved in virulence when tested using invertebrate and vertebrate hosts. These data suggest the potential role that this AbFhaB/FhaC secretion system could play in the pathobiology of A. baumannii.

Assessment of antivirulence activity of several d-amino acids against Acinetobacter baumannii and Pseudomonas aeruginosa.

OBJECTIVES: Biofilm formation and bacterial adherence are important requirements for persistence, multidrug resistance and infection. The d-amino acids play a role as modulators of bacterial growth and persistence, though their ability to inhibit biofilms is much debated. In this study, we analysed the effects of 18 different d-amino acids on the pathogens Acinetobacter baumannii and Pseudomonas aeruginosa. METHODS: In vitro assays were carried out to analyse the effect of d-amino acids on bacterial growth, biofilm formation/disassembly, capacity to attach to eukaryotic cells and cellular death. In addition, in vivo assays were performed in mice, using experimental models of sepsis and pneumonia. RESULTS: Biofilm formation was inhibited in A. baumannii by d-His, d-Cys and d-Trp (35%-86%) at 2 mM and in P. aeruginosa by d-Cys, d-Trp and d-Tyr (10%-30%) at 4 mM. Attachment to the A549 human alveolar cells was reduced in A. baumannii by d-Cys, d-His, d-Met, d-Val and d-Ser, and in P. aeruginosa by d-Arg and d-Trp. Growth was inhibited in A. baumannii by d-Cys and d-Trp, and in P. aeruginosa by d-Trp. In virulence assays, incubation of alveolar cells infected with P. aeruginosa with d-Cys, d-Trp and d-Arg reduced cell death (56%-45%). However, no significant effect of d-amino acids was observed in vivo. CONCLUSIONS: Some d-amino acids can inhibit bacterial growth, biofilm formation and adherence to eukaryotic cells in A. baumannii and P. aeruginosa, and showed a protective effect against infection of alveolar cells with P. aeruginosa. Despite the fact that some considerable protection was observed in mice, survival differences between treated and control groups were not statistically significant.

Optimisation of the Caenorhabditis elegans model for studying the pathogenesis of opportunistic Acinetobacter baumannii.

This study aimed to increase the sensitivity of Caenorhabditis elegans as an infection model for detection of minor differences in virulence or fitness between different Acinetobacter baumannii strains with known resistance and virulence mechanisms. Selected A. baumannii strains and mutants, comprising wild-type strains (ATCC 17978 and 19606), colistin-resistant strains (ATCC 19606 ΔlpxA and ATCC 19606 ΔlpxC), a clinical encapsulated isolate (AB307-0294), an imipenem-resistant strain (ATCC 17978 Δomp33-36) and an sRNA knock-out strain (ATCC 17978 Δ13573), were employed in developing killing and fertility assays in a C. elegans infection model. Because virulence levels of the strains were known, they could be used to assess assays in the nematode model for their ability to discriminate between degrees of virulence. The model was validated by microscopic analysis and in a murine sepsis infection model. The fertility assay, specifically utilising nematode growth medium, was able to detect virulence differences between the wild-type strains, ATCC 19606 ΔlpxA and isolate AB307-0294. Moreover, modification of an alternative culture medium by incremental changes in osmolarity facilitated detection of subtle virulence differences between isogenic mutants (ATCC 17978 Δomp33-36 and 17978 Δ13573). The success of the proposed fertility model depends on establishing a balance between optimal C. elegans reproduction and environmental stress leading to maximum pathogen-induced damage. This invertebrate model may reduce the need for mammalian in vivo studies of A. baumannii resistance and pathogenicity and may additionally be validated for the study of other low-virulence bacterial pathogens.

Cell aggregations in yeasts and their applications.

Yeasts can display four types of cellular aggregation: sexual, flocculation, biofilm formation, and filamentous growth. These cell aggregations arise, in some yeast strains, as a response to environmental or physiological changes. Sexual aggregation is part of the yeast mating process, representing the first step of meiotic recombination. The flocculation phenomenon is a calcium-dependent asexual reversible cellular aggregation that allows the yeast to withstand adverse conditions. Biofilm formation consists of multicellular aggregates that adhere to solid surfaces and are embedded in a protein matrix; this gives the yeast strain either the ability to colonize new environments or to survive harsh environmental conditions. Finally, the filamentous growth is the ability of some yeast strains to grow in filament forms. Filamentous growth can be attained by two different means, with the formation of either hyphae or pseudohyphae. Both hyphae and pseudohyphae arise when the yeast strain is under nutrient starvation conditions and they represent a means for the microbial strain to spread over a wide area to survey for food sources, without increasing its biomass. Additionally, this filamentous growth is also responsible for the invasive growth of some yeast.

Short communication: A comparative analysis of recombinant chymosins.

The first step in cheesemaking is the milk clotting process, in which κ-caseinolytic enzymes contribute to micelle precipitation. The best enzyme for this purpose is chymosin because of its high degree of specificity toward κ-casein. Although recombinant bovine chymosin is the most frequently used chymosin in the industry, new sources of recombinant chymosin, such as goat, camel, or buffalo, are now available. The present work represents a comparative study of 4 different recombinant chymosins (goat and buffalo chymosins expressed in Pichia pastoris, and bovine and camel chymosin expressed in Aspergillus niger). Recombinant goat chymosin exhibited the best catalytic efficiency compared with the buffalo, bovine, or camel recombinant enzymes. Moreover, recombinant goat chymosin exhibited the best specific proteolytic activity, a wider pH range of action, and a lower glycosylation degree than the other 3 enzymes. In conclusion, we propose that recombinant goat chymosin represents a serious alternative to recombinant bovine chymosin for use in the cheesemaking industry.

[Treatment protocols and follow-up of differentiated thyroid carcinoma: results of a questionnaire sent to the Spanish Metabolic Therapy Units]. / Los protocolos de tratamiento y seguimiento del carcinoma diferenciado de tiroides: resultados de un cuestionario enviado a las Unidades de Terapia Metabólica españolas.

OBJECTIVE: To know the treatment and follow-up protocols of differentiated thyroid carcinoma patients in Spanish Metabolic Therapy Units, the clinical variability between them and the adaptation to the consensus guidelines. MATERIALS AND METHODS: Analysis of the results obtained from the questionnaire submitted by E-mail to the Spanish Society of Nuclear Medicine (SEMNIM) members on the treatment and follow-up of differentiated thyroid carcinoma patients. A descriptive study was made of the qualitative variables (frequency, percentage) and quantitative variables (mean, standard deviation). RESULTS: Twenty Radiometabolic Therapy Units responded to the questionnaire. In spite of the varied origin of the patients, the Units receive sufficient clinical information and have specialized surgeons. There is variability in the surgical protocols and indication for ablation in patients with intermediate and low risk of recurrence. The Units agree on the use of (131)I doses for ablation and therapy, but show great variability regarding the preparation protocols (previous (131)I-whole body scan or other imaging techniques, (131)I-whole body scan dose, diet and radioiodine contrast prohibition, total dose per patient). Nuclear Medicine physicians perceive radioiodine adverse effects and prevention methods are used. The post-ablation follow-up protocol differs between Units. CONCLUSIONS: Treatment and follow-up protocols of differentiated thyroid carcinoma patients in the Spanish Radiometabolic Therapy Units show variability in aspects such as surgery and ablation indications, patient preparation for radioiodine therapy and follow-up. Our clinical practice differs in several aspects from the recent consensus guideline recommendations.

[The structure of long telomeres in chromosomes of the Iberian shrew].

It is shown that the size, localization, and structure of telomeres in the Iberian shrew (Sorex granarius) are not characteristic of mammals. In this species, long telomeres of an average size of 213 kb are localized on the short arms of all 32 acrocentrics; ribosomal blocks and active nucleolus-organizing regions (NORs) were also discovered there. At the remaining chromosome ends the average size of telomeres is 3.8 kb. However, in a closely related species, Sorex araneus, all telomeres have size similar to that of human telomeres, i.e., 6.8-15.2 kb. Despite the fact that some long telomeres contain ribosomal repeats in addition to telomeric ones, the long telomeres have preserved asymmetry of G- and C-rich strands as in functional telomeres. It is probable that long telomeres were formed in meiosis at the stage of chromosome bouquet as a result of global reorganization of the chromosome ends. The provoking factors for such reorganization might be the fission of several metacentrics and the necessity of telomerization of the resulting acrocentrics.

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells.

Most human tumor cells acquire immortality by activating the expression of telomerase, a ribonucleoprotein that maintains stable telomere lengths at chromosome ends throughout cell divisions. Other tumors use an alternative mechanism of telomere lengthening (ALT), characterized by high frequencies of telomeric sister chromatid exchanges (T-SCEs). Mechanisms of ALT activation are still poorly understood, but recent studies suggest that DNA hypomethylation of chromosome ends might contribute to the process by facilitating T-SCEs. Here, we show that ALT/T-SCE(high) tumor cells display low DNA-methylation levels at the D4Z4 and DNF92 subtelomeric sequences. Surprisingly, however, the same sequences retained high methylation levels in ALT/T-SCE(high) SV40-immortalized fibroblasts. Moreover, T-SCE rates were efficiently reduced by ectopic expression of active telomerase in ALT tumor cells, even though subtelomeric sequences remained hypomethylated. We also show that hypomethylation of subtelomeric sequences in ALT tumor cells is correlated with genome-wide hypomethylation of Alu repeats and pericentromeric Sat2 DNA sequences. Overall, this study suggests that, although subtelomeric DNA hypomethylation is often coincident with the ALT process in human tumor cells, it is not required for T-SCE.

Studying telomere replication by Q-CO-FISH: the effect of telomestatin, a potent G-quadruplex ligand.

Telomere replication is a critical process for preserving genome integrity. The telomere replication fork proceeds unidirectionally from the last subtelomeric origin towards the end of the chromosome, replicating the 5'-3' G-rich strand by lagging mechanisms and the complementary C-rich strand by leading mechanisms. It has been proposed that the G-rich nature of telomeres may favor the formation of secondary structures such as G-quadruplexes during replication and that specific mechanisms must prevent this to allow the fork to progress unimpeded. The potential of G-quadruplex formation by telomeric sequences has been clearly demonstrated in vitro but it is not known whether these structures form in vivo. We tested the effect of a potent and specific G-quadruplex ligand, telomestatin (TMS), on telomere replication using a novel quantitative approach applied to CO-FISH. We show that TMS, although it penetrates and persists within cells, does not affect telomere replication after short or long-term treatments of mouse embryonic fibroblasts. It does however affect the hybridization efficiency of FISH telomeric probes that recognize the G-rich strand. Our work illustrates the use of a novel technique to measure telomere replication efficiency and suggests that G-quadruplex ligands do not affect telomere replication in a non tumoral context.

Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13.

AIM: The study of a milk-clotting protease secreted by Bacillus licheniformis strain USC13. METHODS AND RESULTS: Growth of B. licheniformis USC13 in LB medium resulted in the production of a serine protease with a molecular weight of 62 kDa processed to its mature form of 34 kDa, both forms were found in the extracellular medium. The enzyme exhibited typical milk-clotting kinetics. CONCLUSIONS: The capacity of this protease to produce milk curds could make it useful as a new source of milk coagulants. SIGNIFICANCE AND IMPACT OF THE STUDY: Cheese-making industry seeks for novel enzyme sources, and microbial coagulants have several advantages over animal and plant counterparts. The protease from B. licheniformis has the ability to produce milk curds although more studies about quality of both the enzyme and the milk curds formed should be carried out in the future to confirm its usefulness in the dairy industry.

Cloning and expression of the XPR2 gene from Yarrowia lipolytica in Pichia pastoris.

Yarrowia lipolytica is a dimorphic yeast able to secrete different types of proteases depending on the pH of the environment. At neutral pH, the production of an extracellular alkaline protease (AEP) is induced. This protease could be useful in the leather, detergent, or food industries. The XPR2 gene, coding for AEP, was extracted from the pINA154 vector and cloned into the pHIL-D2 vector to obtain a new protease-producing recombinant Pichia pastoris strain. The gene was efficiently integrated in the P. pastoris genome and expressed from the AOX1 promoter actively induced by methanol. Finally, the protease was successfully secreted by P. pastoris GS115.

Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases.

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using kappa-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.