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.

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.

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.

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.

[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.

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.

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%.

Mutated telomeres sensitize tumor cells to anticancer drugs independently of telomere shortening and mechanisms of telomere maintenance.

Telomerase is a ribonucleoprotein complex that maintains the stability of chromosome ends and regulates replicative potential. Telomerase is upregulated in over 85% of human tumors, but not in adjacent normal tissues and represents a promising target for anticancer therapy. Most telomerase-based therapies rely on the inhibition of telomerase activity and require extensive telomere shortening before inducing any antiproliferative effect. Disturbances of telomere structure rather than length may be more effective in inducing cell death. Telomerase RNA subunits (hTRs) with mutations in the template region reconstitute active holoenzymes that incorporate mutated telomeric sequences. Here, we analysed the feasibility of an anticancer approach based on the combination of telomere destabilization and conventional chemotherapeutic drugs. We show that a mutant template hTR dictates the synthesis of mutated telomeric repeats in telomerase-positive cancer cells, without significantly affecting their viability and proliferative ability. Nevertheless, the mutant hTR increased sensitivity to anticancer drugs in cells with different initial telomere lengths and mechanisms of telomere maintenance and without requiring overall telomere shortening. This report is the first to show that interfering with telomere structure maintenance in a telomerase-dependent manner may be used to increase the susceptibility of tumor cells to anticancer drugs and may lead to the development of a general therapy for the treatment of human cancers.

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.

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.

[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.

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.

The pattern of chromosome-specific variations in telomere length in humans shows signs of heritability and is maintained through life.

This paper characterizes the distribution of telomere length on individual chromosome arms in humans. By fluorescent in situ hybridization (FISH), followed by computer-assisted analysis of digital images, it is shown that the distribution of telomere length on individual chromosome arms is not random, but that humans have a common telomere profile. This profile exists in lymphocytes, amniocytes and fibroblasts, and seems to be conserved during life. A closer look at the overall pattern of the profile shows that the length of the telomeres in general follows the total chromosome length. In addition to the common profile, it is found that each person has specific characteristics, which are also conserved throughout life. Studying both twins and families we have obtained indications that these individual characteristics are at least partly inherited. Altogether, our results suggest that the length of individual telomeres might occasionally play a role in the heritability of life span.

Differences in telomere length between homologous chromosomes in humans.

Telomeres are important structures for DNA replication and chromosome stability during cell growth. Telomere length has been correlated with the division potential of human cells and has been found to decrease with age in healthy individuals. Nevertheless, telomere lengths within the same cell are heterogeneous and certain chromosome arms typically have either short or long telomeres. Both the origin and the physiological consequences of this heterogeneity in telomere length remain unknown. In this study we used quantitative telomeric FISH combined with a method to identify the parental origin of chromosomes to show that significant differences in relative telomere intensities are frequently observed between chromosomal homologs in short-term stimulated cultures of peripheral blood lymphocytes. These differences appear to be stable for at least 4 months in vivo, but disappear after prolonged proliferation in vitro. The telomere length differences are also stable during in vitro growth of telomerase-negative fibroblast cells but can be abolished by exogenous telomerase expression in these cells. These findings suggest the existence of a mechanism maintaining differences in telomere length between chromosome homologs that is independent of telomere length itself.

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.

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.

Catecholamine levels in practitioners of the transcendental meditation technique.

With the aim of evaluating the sympathetic-adrenal medulla system in subjects practicing transcendental meditation (TM), their plasma catecholamine levels were determined at two different times of day. The study group consisted of 19 subjects who regularly practice either TM or Sidhi-TM technique, with a control group made up of 16 healthy subjects who had not previously used any relaxation technique. Catecholamine plasma levels were determined by high performance liquid chromatography, at 0900 and 2000 h. Morning and evening norepinephrine (NE) levels and morning epinephrine (E) levels were significantly lower in the TM group than in the control subjects (morning NE levels, pg/ml, mean+/-S.E.: TM group 136.6+/-13.0, control 236.8+/-21.0, P=.0001; evening NE levels: TM group 119.7+/-10.8, control 175.6+/-17.4, P=.009; morning E levels, pg/ml: TM group 140.2+/-10.6, control 196.7+/-23.8, P=.019). No differences were recorded for evening E levels and dopamine (DA) levels. No significant differences were found for catecholamine levels measured at different times of day in the TM group, demonstrating a lack of daily hormonal rhythm. Anxiety levels were similar in both groups. Based on the results obtained, it can be considered that the regular practice of TM has a significant effect on the sympathetic-adrenal medulla system. A low hormonal response to daily stress caused by sympathetic tone regulation through regular TM could explain our results, as well as the physiological and other effects related to the field of health described in those who practice meditation.

CA 549 and SP2 in postoperative breast cancer patients. Comparison with CA 15.3, CEA and TPA.

The levels of CA 549 and SP2 were measured in 430 subjects: 100 healthy blood donors, 130 patients with benign diseases and 200 postoperative breast cancer patients. In the latter group, the serum levels of CA 15.3, CEA and TPA were also measured. The Kolmogorov-Smirnov, Mann Whitney and McNemar tests were used for statistical analysis. The upper normal limits were established on the basis of the values obtained in the healthy blood donors group, the benign diseases group and R.O.C. analysis of the breast cancer group. They were: CA 549 = 13 U/ml, SP2 = 14 U/ml, CA 15.3 = 35 U/ml, CEA = 5 ng/ml and TPA = 110 U/ml. The sensitivity, specificity and accuracy in the breast cancer group were, respectively: CA 549 = 78.1%, 97.1% and 88%; SP2 = 21.9%, 90.4% and 57.5%; CEA = 66.7%, 95.2% and 81.5%; CA 15.3% = 80.2%, 98.1% and 89.5%, and TPA = 73.9%, 78.8% and 76.5%. Statistical analysis showed significant differences only between CA 15.3, the marker which gave the best results, and SP2 (p < 0.001). There was no significant differences with the association of two or three tumor markers.

Evaluation of palpable breast masses with 201Tl scintigraphy.

39 female patients (age range: 31-84 years) with palpable breast masses detected by physical examination, underwent 201Tl scintigraphy in order to assess its value in the detection of breast carcinomas and to differentiate them from benign breast masses. Planar images were carried out at 20-30 min and 2-3 h after intravenous administration of 111-185 MBq (3-5 mCi) of 201Tl chloride. In 12 patients single photon emission tomography (SPET) studies were also performed. In 18 patients the scintigraphic studies were positive and in 17 of these cases, breast carcinomas were confirmed. Tumour sizes ranged from 1.3 to 6 cm in diameter. In the remaining patient a false positive result was obtained where there was benign breast change. In three of seven cases, malignant axillary nodes were also detected. All 21 patients with negative scintigraphy had benign breast lesions. There were no differences between images obtained at 20-30 min and 2-3 h or between planar images and SPET studies. In 10 patients there was disagreement between mammography and 201Tl scans. 201Tl scans confirmed the presence of carcinoma in three cases and discarded malignancy in the other six cases. In the remaining case, 201Tl scan was false positive. 201Tl scintigraphy is useful in distinguishing malignant from benign breast masses, even when compared with mammography.