[Bacterial coinfection in the critically-ill COVID-19 patient: incidence, impact and need for antimicrobial therapy]. / Coinfección bacteriana en el paciente COVID-19 crítico: incidencia, impacto y necesidad de tratamiento antibiótico.

OBJECTIVE: To assess the frequency of bacterial coinfection upon ICU admission in SARS-CoV-2 pneumonia patients, its microbiology, and impact on prognosis.The secondary objective was to identify risk factors for coinfection on admission. METHODS: Retrospective study, including patients with SARS-CoV-2 pneumonia admitted to the ICU.We defined bacterial coinfection by respiratory symptoms, radiological data, positive and clinically significant microbiological results in samples obtained in the first 48 h of admission and/or a determination of procalcitonin ≥ 0.5 ng/mL in the first 48 h.We evaluated demographic variables, comorbidities, SARS-CoV-2 infection data, severity scores, treatments received, need for respiratory support and outcomes (ICU and hospital mortality). RESULTS: A total of 182 patients were analyzed, 62 (34.1%) with bacterial coinfection.The most frequent microbiology was S. pneumoniae and M. pneumoniae. 96.1% of the patients received antibiotic therapy on admission, 98.9% corticosteroids, 27.5% tocilizumab, and 7.7% remdesivir.85.7% required invasive mechanical ventilation.The SOFA score (OR: 1.315, 95% CI1.116-1.548) and the delay in ICU admission (OR: 0.899, 95% CI 0.831-0.972) were related to the risk of coinfection. Bacterial coinfection increases the risk of death in hospital (OR 2.283; 95% CI 1.011.5.151; p=0.047). CONCLUSIONS: Bacterial coinfection is common in COVID patients admitted to the ICU and increases the risk of death. It is not possible to identify with certainty, at the time of admission, which patients do not benefit from antibiotic treatment.

Analysis of Genetic Variation in CD40 and CD40L: Relationship with mRNA Relative Expression and Soluble Proteins in Acute Coronary Syndrome.

Acute coronary syndrome (ACS) can be triggered by the presence of inflammatory factors which promote the activation of immune cells by costimulatory molecules such as CD40 and its ligand CD40L. Environmental and genetic factors are involved in the etiology of the ACS. The aim of this study was to explore the gene and protein expression associated with CD40 and CD40L genetic variants in ACS patients from the western Mexican population. A total of 620 individuals from western Mexico were recruited: 320 ACS patients and 300 individuals without a history of ischemic cardiopathy were evaluated. The genotype was determined using TaqMan SNP genotyping assays. CD40 and CD40L expressions at the mRNA level were quantified using TaqMan Gene Expression Assays. Soluble protein isoforms were measured by enzyme-linked immunosorbent assay. We did not find evidence of association between CD40 (rs1883832, rs4810485, and rs11086998) and CD40L (rs3092952 and rs3092920) genetic variants and susceptibility to ACS, although rs1883832 and rs4810485 were significantly associated with high sCD40 plasma levels. Plasma levels of sCD40L can be affected by gender and the clinical spectrum of acute coronary syndrome. Our results do not suggest a functional role of CD40 and CD40L genetic variants in ACS. However, they could reflect the inflammatory process and platelet activation in ACS patients, even when they are under pharmacological therapy. Due to the important roles of the CD40-CD40L system in the pathogenesis of ACS, longitudinal studies are required to determine if soluble levels of CD40 and CD40L could be clinically useful markers of a recurrent cardiovascular event after an ACS.

Influence of airflow intensity on phytase production by solid-state fermentation.

Phytase production by Aspergillus niger F3 by solid state fermentation (SSF) on citrus peel was evaluated at pilot scale under different aeration conditions. The best airflow intensity was 1 VkgM (Lair kg medium(-1) min(-1)), which allowed to produce 65 units of phytases per gram in dry basis (65 Ug(-1) d.b.) as it removed the metabolic heat generated by the microorganism, Agitation did not improve heat removal. Airflow intensity was considered as scale-up criterion. When the airflow intensity was maintained at 1 VkgM for SSF with 2 and 20 kg of medium, the kinetics parameters for biomass and enzyme concentration at the end of fermentation differed by less than 2. The air flow intensity was required to maintain the temperature and cool the SSF and to provide oxygen for microbial growth. Air flow intensity is a key a factor that must be considered when scale-up of SSF is attempted.

The behavior of kinetic parameters in production of pectinase and xylanase by solid-state fermentation.

Solid-state fermentation (SSF) is defined as the growth of microbes without a free-flowing aqueous phase. The feasibility of using a citrus peel for producing pectinase and xylanase via the SSF process by Aspergillus niger F3 was evaluated in a 2 kg bioreactor. Different aeration conditions were tested to optimize the pectinase and xylanase production. The best air flow intensity was 1 V kg M (volumetric air flow per kilogram of medium), which allowed a sufficient amount of O2 for the microorganism growth producing 265 U/g and 65 U/g pectinases and xylanases, respectively. A mathematical model was applied to determine the different kinetic parameters related to SSF. The specific growth rate and biomass oxygen yield decreased during fermentation, whereas an increase in the maintenance coefficient for the different employed carbon sources was concurrently observed.

Comparison of lures loaded with codlemone and pear ester for capturing codling moths, Cydia pomonella, in apple and pear orchards using mating disruption.

Abstract Studies were conducted in apple, Malus domestica Borkhausen and pear, Pyrus communis L. (Rosales: Rosaceae), orchards to evaluate the attractiveness of grey halobutyl septa loaded with 1 (L2) and 10 (Mega) mg of codlemone, 8E, 10E-dodecadien-1-ol, 3 mg of pear ester, ethyl (E,Z)-2,4-decadienoate (DA2313), and 3 mg of pear ester plus 3 mg of codlemone (Combo) to adult codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). All studies were conducted in orchards treated with pheromone mating disruption. All four lures were tested on diamond-shaped sticky traps placed in 60 plots of apple and 40 plots of pears in 2003/04, and in 62 plots of apples and 30 of pears in 2004–05. Combo lures attracted significantly more moths (males + females) than all the others in both years. Comparisons among flights showed significant differences mainly for flight 1 and 2, but not always for flight 3. Mega lures provided no significant improvement compared with L2 lures during both seasons regarding the total number of moths. Combo and DA2313 lures attracted fewer females than males during the whole season. For most sample dates, more virgin than mated females were attracted to Combo lures, except during the third flight, and the overall ratio was 60:40, although the difference was not statistically significant. We conclude that the Combo lures are better indicators of codling moth activity in pheromone treated orchards, regardless of pest population level, when compared with similar lures containing codlemone or pear ester alone.

The embryo MADS domain factor AGL15 acts postembryonically. Inhibition of perianth senescence and abscission via constitutive expression.

AGL15 (AGAMOUS-like 15), a member of the MADS domain family of regulatory factors, accumulates preferentially throughout the early stages of the plant life cycle. In this study, we investigated the expression pattern and possible roles of postembryonic accumulation of AGL15. Using a combination of reporter genes, RNA gel blot analysis, and immunochemistry, we found that the AGL15 protein accumulates transiently in the shoot apex in young Arabidopsis and Brassica seedlings and that promoter activity is associated with the shoot apex and the base of leaf petioles throughout the vegetative phase. During the reproductive phase, AGL15 accumulates transiently in floral buds. When AGL15 was expressed in Arabidopsis under the control of a strong constitutive promoter, we noted a striking increase in the longevity of the sepals and petals as well as delays in a selected set of age-dependent developmental processes, including the transition to flowering and fruit maturation. Although ethylene has been implicated in many of these same processes, the effects of AGL15 could be clearly distinguished from the effects of the ethylene resistant1-1 mutation, which confers dominant insensitivity to ethylene. By comparing the petal breakstrength (the force needed to remove petals) for flowers of different ages, we determined that ectopic AGL15 had a novel effect: the breakstrength of petals initially declined, as occurs in the wild type, but was then maintained at an intermediate value over a prolonged period. Abscission-associated gene expression and structural changes were also altered in the presence of ectopic AGL15.

The MADS-domain protein AGAMOUS-like 15 accumulates in embryonic tissues with diverse origins.

AGL15 (AGAMOUS-like 15), a member of the MADS-domain family of regulatory factors, accumulates preferentially in the organs and tissues derived from double fertilization in flowering plants (i.e. the embryo, suspensor, and endosperm). The developmental role of AGL15 is still undefined. If it is involved in embryogenesis rather than some other aspect of seed biology, then AGL15 protein should accumulate whenever development proceeds in the embryonic mode, regardless of the origin of those embryos or their developmental context. To test this, we used AGL15-specific antibodies to analyze apomictic embryogenesis in dandelion (Taraxacum officinale), microspore embryogenesis in oilseed rape (Brassica napus), and somatic embryogenesis in alfalfa (Medicago sativa). In every case, AGL15 accumulated to relatively high levels in the nuclei of the embryos. AGL15 also accumulated in cotyledon-like organs produced by the xtc2 (extra cotyledon2) mutant of Arabidopsis and during precocious germination in oilseed rape. Furthermore, the subcellular localization of AGL15 appeared to be developmentally regulated in all embryogenic situations. AGL15 was initially present in the cytoplasm of cells and became nuclear localized before or soon after embryogenic cell divisions began. These results support the hypothesis that AGL15 participates in the regulation of programs active during the early stages of embryo development.

Developmental basis of homeosis in precociously germinating Brassica napus embryos: phase change at the shoot apex.

Precociously germinating Brassica napus (oilseed rape) embryos produce extra cotyledons or chimeric organs with sectors of cotyledon and leaf tissue, rather than leaves, at the shoot apex. To investigate this phenomenon in more detail, scanning electron microscopy was used to examine the development of organ primordia at the shoot apex. In situ hybridizations with molecular markers of the embryonic phase were used to assess the status of individual cells in the shoot apex with regard to the transition between embryonic and vegetative phases. The results indicate that, under conditions that support precocious germination, primordia develop at the shoot apex in the mode characteristic of postgerminative growth, i.e. they arise sequentially in a spiral phyllotaxy. Cells in the rest of the embryo, however, can continue to express molecular markers of the embryonic phase for several weeks after the start of culture. When patterns of gene expression and the fate of individual primordia were compared, a strong correlation was found between organ identity and the status of cells in the vicinity of the meristem with regard to phase. Primordia that develop in situations where neighboring cells are in the embryonic phase always produce organs with cotyledon morphology. Primordia that develop in situations where neighboring cells have exited the embryonic phase produce leaves. Based on an examination of situations where chimeric organs are produced, I propose that short range interactions or signalling are likely to be involved in communicating information about phase to developing primordia.

The MADS domain protein AGL15 localizes to the nucleus during early stages of seed development.

Little is known about regulatory factors that act during the earliest stages of plant embryogenesis. The MADS domain protein AGL15 (for AGAMOUS-like) is expressed preferentially during embryogenesis and accumulates during early seed development in monocotyledonous and dicotyledonous flowering plants. AGL15-specific antibodies and immunohistochemistry were used to demonstrate that AGL15 accumulates before fertilization in the cytoplasm in the cells of the egg apparatus and moves into the nucleus during early stages of development in the suspensor, embryo, and endosperms. Relatively high levels of AGL15 are present in the nuclei during embryo morphogenesis and until the seeds start to dry in Brassica, maize, and Arabidopsis. AGL15 is associated with the chromosomes during mitosis, and gel mobility shift assays were used to demonstrate that AGL15 binds DNA in a sequence-specific manner. To assess whether AGL15 is likely to play a role in specifying the seed or embryonic phase of development, AGL15 accumulation was examined in Arabidopsis mutants that prematurely exit embryogenesis. lec1-2 mutants show an embryo-specific loss of AGL15 at the transition stage, suggesting that AGL15 interacts with regulators in the leafy cotyledons pathway.

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.

Effects of amphetamine on influenza virus infection in mice.

Several experiments were conducted to evaluate the effects of chronic amphetamine on the influenza A (PR-8/34) virus specific immune injury in CD-1 mice. Treatment with amphetamine resulted in a significant increase of lung virus titers and pulmonary vascular permeability. Amphetamine also increased the lethality of infected mice.

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos.

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.

A single-blind, placebo-controlled study of glycosaminoglycan-peptide complex ('Rumalon') in patients with osteoarthritis of the hip or knee.

A randomized, single-blind, placebo-controlled trial was carried out in 62 patients (30 with osteoarthritis of the hip, 32 with osteoarthritis of the knee) to examine the efficacy of glycosaminoglycan-peptide complex in the treatment of osteoarthritis. Patients received 8-week courses of trial medication, each consisting of intramuscular injections of 3 x 2 ml ampoules per week, alternating with 8-week periods free of trial medication, in addition to conventional drug therapy and physiotherapy, as required. After 2-years' treatment, glycosaminoglycan-peptide-treated patients showed significant improvements, as compared with placebo, in relation to night pain, pain during the day, joint mobility and walking ability. Similar results were seen with both osteoarthritis of the hip and knee. In osteoarthritis of the knee it was also possible to assess joint swelling and this also showed a significant improvement. There were no significant changes in range of joint movement except for a significant decrease in active flexion in the patients with osteoarthritis of the knee treated with placebo. In contrast with many anti-osteoarthritic drugs, glycosaminoglycan-peptide complex was very well tolerated. These results suggest that glycosaminoglycan-peptide complex may be a valuable alternative form of long-term therapy for patients with osteoarthritis.

Immunogold Localization of the L3 Protein of Maize Lipid Bodies during Germination and Seedling Growth.

We have used antibodies directed against the 16.5 kilodalton protein L3, the most abundant integral protein of maize (Zea mays L. cv Mo 17) lipid bodies, to follow the fate of this protein in scutellar parenchyma cells of maize during germination and subsequent seedling growth. Using gel electrophoresis and immunoblotting as well as immunocytochemical electron microscopy, we found that the amount of L3 decreases gradually during the first 3 to 4 days of seedling growth and more rapidly over the course of the next several days. Immunogold localization of the protein on thin sections indicated that L3 is found exclusively in the surface phospholipid monolayer of lipid bodies. The density of L3 in the surface layer of individual lipid bodies does not change during seedling growth; therefore, the decrease in the amount of L3 can be attributed to a decrease in the number of lipid bodies rather than to selective removal of protein components from the surface of all lipid bodies. Thus, L3 is apparently degraded at the same time as the matrix lipid of each lipid body. Unlike lipase, L3 does not appear to be transferred to other cellular compartments such as vacuoles during late stages of seedling growth.

Does gibberellic Acid induce the transfer of lipase from protein bodies to lipid bodies in barley aleurone cells?

We have examined the effect of gibberellic acid (GA(3)) on the distribution of the enzyme responsible for mobilizing storage triacylglycerol in aleurone cells of Hordeum vulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA(3) for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism.

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion.

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s(-1)) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of α-amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active α-amylase secretion in gibberellic acid (GA3)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA3 also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 µm(-2) to 3800 µm(-2) because of an increase of particles in the 3-8.5-nm size range. A slight decrease in 9-11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.