Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

Treating COVID-19: Review of Drug Hypersensitivity Reactions.

The disease caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ie, coronavirus disease 2019 (COVID-19), has become a global pandemic since it was first reported in Wuhan, China in December 2019. Its severe clinical manifestations, which often necessitate admission to intensive care units, and high mortality rate represent a therapeutic challenge for the medical community. To date, no drugs have been approved for its treatment, and various therapeutic options are being assayed to address the pathophysiological processes underlying the clinical manifestations experienced by patients. New and old drugs administered as monotherapy or in combination to immunologically compromised patients may favor the development of adverse drug reactions, including drug hypersensitivity reactions, which must be identified and managed accordingly. Given the lack of herd immunity and the high rate of viral contagion, new cases are expected to emerge in the coming months. Thus, the probability of more adverse reactions or even new clinical manifestations may increase in parallel. Allergists must receive updated information on these treatments, as well as on the management of possible drug hypersensitivity reactions.

Simultaneous nitrogen and dissolved methane removal from an upflow anaerobic sludge blanket reactor effluent using an integrated fixed-film activated sludge system.

One of the main drawbacks of upflow anaerobic sludge blanket (UASB) reactors that treat low-strength sewage at room temperature is related to the low quality of their effluents in terms of dissolved methane, organic matter, and nitrogen content. The present study aims to evaluate the feasibility of using an integrated fixed-film activated sludge (IFAS) system as an alternative post-treatment technology to mitigate the environmental impact of such effluents. For this purpose, a pilot plant composed of a UASB (120 L) followed by an IFAS (66 L) system was operated for 407 days. Special attention was paid to the suspended biomass retention capacity and the dissolved methane and nitrogen removal potential of the IFAS post-treatment system. Furthermore, the role of carriers on denitrification and nitrification processes and the microbial communities present in the biofilm were also analyzed. Average total chemical oxygen demand (CODT) and ammonium removal efficiencies of 92 ± 3% and around 57 ± 16% were attained throughout the entire operation, respectively. During a first period in which biomass was maintained in both biofilms and suspension, and nitrite was the main electron acceptor, maximum nitrogen removal and methane removal efficiencies of 32.5 mg TN L-1 and 93% were observed in the IFAS system, respectively. However, throughout the second period, in which suspended biomass was completely washed out from the IFAS system, and nitrate became the main electron acceptor, these values decreased to 18 ± 4 mg TN Lfeed-1 and 77 ± 12%, respectively. Surprisingly, throughout the entire operation, it was observed that around 50 and 41% of the total nitrogen and methane removals observed in the IFAS system, respectively, were carried out in the aerobic compartment. Aerobic methane oxidizers and anammox were detected with significant relative abundances in the biofilm carriers used in the anoxic and aerobic compartments using 16S rRNA gene amplicon sequencing analysis. Therefore, the use of an IFAS system could be suited to diminish greenhouse gas emissions and nutrients concentration for those sewage treatment plants that used UASB systems, especially in countries with temperate and warm climates.

Comprehensive Kinetics of Hydrolysis of Organotriethoxysilanes by 29Si NMR.

The kinetics of several representative hybrid precursors were studied via 29Si NMR: three alkyl precursors, methyltriethoxysilane, ethyltriethoxysilane, and propyltriethoxysilane; as well as two unsaturated radicals, vinyltriethoxysilane and phenyltriethoxysilane. The reaction rate is related to the chemical shift of 29Si in the NMR spectra, which gives information about the electronic density of the Si atoms and the inductive effects of substituents. The concentration of the precursors decreased exponentially with time, and the intermediate products of hydrolysis and the beginning of the condensation reactions showed curves characteristic of sequential reactions, with a similar distribution of the species as a function of the fractional conversion. For all of the precursors, condensation started when the most hydrolyzed species reached a maximum concentration of 0.30 M, when the precursor had run out. A prediction following the developed mathematical model fits the experimental results in line with a common pathway described by eight parameters.

Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

Esophageal dysfunction and immunological changes induced by grass sublingual immunotherapy.

Sublingual immunotherapy frequently causes local oropharyngeal adverse events which are usually of mild severity, and tend to be self-limited and disappear within the first weeks of therapy. The mechanism of action involves changes in the specific humoral response to allergens, with increases in allergen-specific immunoglobulin G4 (IgG4) and blunting of the seasonal increase in allergen-specific IgE. We describe the case of a 25-year-old man diagnosed with grass pollen induced allergic rhinoconjunctivitis, who was treated with a lyophilisate of Phleum pratense by sublingual route. After 5 weeks of therapy he developed repeatedly intense symptoms of esophageal dysfunction immediately after the administration. Symptoms recurred every day, subsided in some hours without treatment and disappeared with the termination of therapy. The episode coincided with a marked elevation of total and specific IgE. The immunological changes gradually declined during the three years of follow up. The reported case suggests the need to evaluate the role of the immunological changes detected after the first weeks of sublingual therapy with Phleum pratense, in the induction of esophageal disorders.

Enhancement of struvite pellets crystallization in a full-scale plant using an industrial grade magnesium product.

A full-scale struvite crystallization system was operated for the treatment of the centrate obtained from the sludge anaerobic digester in a municipal wastewater treatment plant. Additionally, the feasibility of an industrial grade Mg(OH)2 as a cheap magnesium and alkali source was also investigated. The struvite crystallization plant was operated for two different periods: period I, in which an influent with low phosphate concentration (34.0 mg P·L-1) was fed to the crystallization plant; and period II, in which an influent with higher phosphate concentration (68.0 mg P·L-1) was used. A high efficiency of phosphorus recovery by struvite crystallization was obtained, even when the effluent treated had a high level of alkalinity. Phosphorus recovery percentage was around 77%, with a phosphate concentration in the effluent between 10.0 and 30.0 mg P·L-1. The experiments gained struvite pellets of 0.5-5.0 mm size. Moreover, the consumption of Mg(OH)2 was estimated at 1.5 mol Mg added·mol P recovered-1. Thus, industrial grade Mg(OH)2 can be an economical alternative as magnesium and alkali sources for struvite crystallization at industrial scale.

On interfacial properties of tetrahydrofuran: Atomistic and coarse-grained models from molecular dynamics simulation.

We have determined the interfacial properties of tetrahydrofuran (THF) from direct simulation of the vapor-liquid interface. The molecules are modeled using six different molecular models, three of them based on the united-atom approach and the other three based on a coarse-grained (CG) approach. In the first case, THF is modeled using the transferable parameters potential functions approach proposed by Chandrasekhar and Jorgensen [J. Chem. Phys. 77, 5073 (1982)] and a new parametrization of the TraPPE force fields for cyclic alkanes and ethers [S. J. Keasler et al., J. Phys. Chem. B 115, 11234 (2012)]. In both cases, dispersive and coulombic intermolecular interactions are explicitly taken into account. In the second case, THF is modeled as a single sphere, a diatomic molecule, and a ring formed from three Mie monomers according to the SAFT-γ Mie top-down approach [V. Papaioannou et al., J. Chem. Phys. 140, 054107 (2014)]. Simulations were performed in the molecular dynamics canonical ensemble and the vapor-liquid surface tension is evaluated from the normal and tangential components of the pressure tensor along the simulation box. In addition to the surface tension, we have also obtained density profiles, coexistence densities, critical temperature, density, and pressure, and interfacial thickness as functions of temperature, paying special attention to the comparison between the estimations obtained from different models and literature experimental data. The simulation results obtained from the three CG models as described by the SAFT-γ Mie approach are able to predict accurately the vapor-liquid phase envelope of THF, in excellent agreement with estimations obtained from TraPPE model and experimental data in the whole range of coexistence. However, Chandrasekhar and Jorgensen model presents significant deviations from experimental results. We also compare the predictions for surface tension as obtained from simulation results for all the models with experimental data. The three CG models predict reasonably well (but only qualitatively) the surface tension of THF, as a function of temperature, from the triple point to the critical temperature. On the other hand, only the TraPPE united-atoms models are able to predict accurately the experimental surface tension of the system in the whole temperature range.

Hsp90 activity is necessary to acquire a proper neuronal polarization.

Chaperones are critical for the folding and regulation of a wide array of cellular proteins. Heat Shock Proteins (Hsps) are the most representative group of chaperones. Hsp90 represents up to 1-2% of soluble protein. Although the Hsp90 role is being studied in neurodegenerative diseases, its role in neuronal differentiation remains mostly unknown. Since neuronal polarity mechanisms depend on local stability and degradation, we asked whether Hsp90 could be a regulator of axonal polarity and growth. Thus, we studied the role of Hsp90 activity in a well established model of cultured hippocampal neurons using an Hsp90 specific inhibitor, 17-AAG. Our present data shows that Hsp90 inhibition at different developmental stages disturbs neuronal polarity formation or axonal elongation. Hsp90 inhibition during the first 3h in culture promotes multiple axon morphology, while this inhibition after 3h slows down axonal elongation. Hsp90 inhibition was accompanied by decreased Akt and GSK3 expression, as well as, a reduced Akt activity. In parallel, we detected an alteration of kinesin-1 subcellular distribution. Moreover, these effects were seconded by changes in Hsp70/Hsc70 subcellular localization that seem to compensate the lack of Hsp90 activity. In conclusion, our data strongly suggests that Hsp90 activity is necessary to control the expression, activity or location of specific kinases and motor proteins during the axon specification and axon elongation processes. Even more, our data demonstrate the existence of a "time-window" for axon specification in this model of cultured neurons after which the inhibition of Hsp90 only affects axonal elongation mechanisms.

Collagen type IV-related nephropathies in Portugal: pathogenic COL4A5 mutations and clinical characterization of 22 families.

Alport syndrome (AS) is caused by pathogenic mutations in the genes encoding α3, α4 or α5 chains of collagen IV (COL4A3/COL4A4/COL4A5), resulting in hematuria, chronic renal failure (CRF), sensorineural hearing loss (SNHL) and ocular abnormalities. Mutations in the X-linked COL4A5 gene have been identified in 85% of the families (XLAS). In this study, 22 of 60 probands (37%) of unrelated Portuguese families, with clinical diagnosis of AS and no evidence of autosomal inheritance, had pathogenic COL4A5 mutations detected by Sanger sequencing and/or multiplex-ligation probe amplification, of which 12 (57%) are novel. Males had more severe and earlier renal and extrarenal complications, but microscopic hematuria was a constant finding irrespective of gender. Nonsense and splice site mutations, as well as small and large deletions, were associated with younger age of onset of SNHL in males, and with higher risk of CRF and SNHL in females. Pathogenic COL4A3 or COL4A4 mutations were subsequently identified in more than half of the families without a pathogenic mutation in COL4A5. The lower than expected prevalence of XLAS in Portuguese families warrants the use of next-generation sequencing for simultaneous COL4A3/COL4A4/COL4A5 analysis, as first-tier approach to the genetic diagnosis of collagen type IV-related nephropathies.

30 years of preventive studies of uterine cervical cancer 1982-2012.

The studies for the prevention of uterine cervical cancer in Panama City began in a private institute, impelled by the high incidence of cancer. The preventive programs were initiated with the support of the Obstetrics and Gynecology Institute of the University of Padua in Italy. In these studies, we applied the methodological diagnostics of this Institute with certain modifications adapted to our needs. The diagnosis, treatment, and follow-up were carried out under the protocol of the University of Padua. We achieved a diagnosis of oncogenic risk (OR) in 6,411 patients which corresponded to 5,498 cases of human papillomavirus (HPV), 1,150 cases of dysplasia, 210 cases of cancer, and 794 cases of OR. From 2011, polymerase chain reaction (PCR) was also applied with the aim to improve the accuracy of the diagnosis. With this method the prevalence of pathologies were HPV infections both in healthy patients or in patients cured from HPV. Patients were treated by means of local destructive treatments (LDT), basically with cryotherapy and cauterization. We can consider these results as successful prevention and suggest to extend a preventive program to all the population.

30 years of colposcopic studies: validity of local destructives treatments.

The effectiveness of local destructive treatments (LDT) applied in patients due to cervical pathology oncogenic risk (OR), were followed and verified in 396 patients who came to our attention, focusing on the type of pathology, type of treatment received, diagnosis clinical evolutionary, and results of the new study applying polymerase chain reaction (PCR), and above all, the time between the diagnosis, treatment, and PCR findings. The clinical evolution of the same reports achieved a healing rate of 82% followed by persistence 8.3%; improvement 4.8%, recurrence 2.8%, and only one case of progression 0.2%. The elapsed time in initial care and treatment was almost immediate, as the pathology diagnosis was considered on an emergency basis. Successive controls of these indicated that 119 studies of routine colposcopy were carried out, on an average of the first three years and with a maximum follow up of 30 years, with over 30 routine colposcopies that achieved healing in most of these. In 2011 and 2012, we added to the usual diagnostic methodology, molecular biology, and 119 studies were performed in those patients, resulting in only five negative cases. Most studies were classified as high risk papillomavirus (HR-HPV), corresponding to subtypes 31, 35, 18, and 16.

Oestradiol signalling through the Akt-mTORC1-S6K1.

The oestradiol plays an important role in normal brain development and exerts neuroprotective actions. Oestradiol is mainly produced in the ovary and in addition is locally synthesised in the brain. Most of the oestradiol functions have been associated with its capacity to directly bind and dimerize "classical oestrogen receptors" (ERs), alpha and beta. The ERs' actions have been classified as "genomic" and "non-genomic" depending on whether protein synthesis occurs through ER driven transcription or not. Indeed, recent evidence suggests that oestrogen may also act as a more general "trophic factor". Hence, we have studied the capacity of oestradiol to activate the PI3K/Akt pathway and its implication in axonal growth and neuronal morphogenesis. Our data show that when oestrogen receptors are blocked the axonal and dendritic lengths are reduced in mouse primary neurons. We found that Akt/Rheb/mTORC1 responds to ER activation in neurons and that some elements of this pathway are able to restore a normal neuronal morphology even in the presence of oestrogen receptor antagonist. All these data demonstrate a new mechanism regulated by oestradiol, at least in neuronal morphogenesis.

Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease.

AIMS: Transmissible spongiform encephalopathies, also called prion diseases, are characterized by the cerebral accumulation of misfolded prion protein (PrP(SC) ) and subsequent neurodegeneration. However, despite considerable research effort, the molecular mechanisms underlying prion-induced neurodegeneration are poorly understood. Here, we explore the hypothesis that prions induce dysfunction of the PI3K/Akt/GSK-3 signalling pathway. METHODS: We employed two parallel approaches. Using cell cultures derived from mouse primary neurones and from a human neuronal cell line, we identified common elements that were modified by the neurotoxic fragment of PrP(106-126) . These studies were then complemented by comparative analyses in a mouse model of prion infection. RESULTS: The presence of a polymerized fragment of the prion protein (PrP(106-126) ) or of a prion strain altered PI3K-mediated signalling, as evidenced by Akt inhibition and GSK-3 activation. PI3K activation by the addition of insulin or the expression of a constitutively active Akt mutant restored normal levels of Akt and GSK-3 activity. These changes were correlated with a reduction in caspase activity and an increase in neuronal survival. Moreover, we found that activation of caspase 3, Erk and GSK-3 are common features of PrP(106-126) -mediated neurotoxicity in cellular systems and prion infection in the mouse cerebellum, while activation of caspase 12 and JNK was observed in cellular models. CONCLUSIONS: Our findings in cell culture and in vivo models of prion disease demonstrate marked alterations to the PI3K/Akt/GSK-3 pathway and suggest that two additional pathways contribute to PrP-induced neurotoxicity as responsible of JNK and caspase 12 activation.

New highly polymorphic microsatellite markers for the aquatic angiosperm Ruppia cirrhosa reveal population diversity and differentiation.

Ruppia cirrhosa is a clonal monoecious plant phylogenetically associated to seagrass families such as Posidoniaceae and Cymodoceaceae. It inhabits shallow waters that are important for productivity and as a biodiversity reservoir. In this study, we developed 10 polymorphic microsatellite loci for R. cirrhosa. Additionally, we obtained cross-amplification for two microsatellites previously described for Ruppia maritima. These 12 markers were tested in four R. cirrhosa populations from the southwest of Europe. The number of alleles per locus was high for most of the markers, ranging from 4 to 13. Two populations (Sicily and Cádiz) showed heterozygote deficit (p < 0.001). The four populations (Sicily, Murcia, Cádiz, and Tavira) were significantly differentiated (F(ST) ≠ 0; p < 0.001), corroborating the usefulness of these microsatellites on R. cirrhosa population genetics.

A quasi-classical trajectory study of the OH + SO reaction: the role of ro-vibrational energy.

A study of the OH + SO → H + SO2 reaction using a quasi-classical trajectory method is presented with the aim of investigating the role of the ro-vibrational energy of the reactants in the reactivity. The calculations were carried out using a previously reported global potential energy surface for HSO2((2)A). Different initial conditions with one and both reactants ro-vibrationally excited were studied. The reactive cross sections, for each studied combination, are calculated and then fitted to a capture-like model combined with a factor accounting for the recrossing effects. The Vibrational Energy Quantum Mechanical Threshold of the Complex method was used to correct for the zero-point vibrational energy leakage of the classical calculations. State specific and averaged rate constants are reported. The reactivity is affected when ro-vibrational energy of either of the reactants is changed. The present calculations provide a theoretical support for the experimental rate constant for temperatures below 550 K, but fail to account for the significant fall in the observed rate constant upon increasing the temperature above this value.