Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His216-Gln219-Tyr221 motif in cellulose- and chitin-oxidizing MaAA10B toward Arg-Glu-Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H2O2, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H2O2. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

Current Evidence: Seizures in Extremely Low Gestational Age Newborns (ELGANs).

Extremely low gestational age newborns (ELGANs) are born at or below 28 weeks of gestational age. Despite improved obstetric care, the incidence of preterm birth continues to rise in advanced countries. Preterm birth remains a major cause of infant mortality, and for infants who survive, neonatal seizures are a significant predictor of later neurologic morbidity. However, little is known about risk factors for neonatal seizures in ELGANs. Understanding the association between neonatal seizures and the development of other neurologic disorders is important given the increasing prevalence of ELGANs. Identifying risk factors that contribute to the development of neonatal seizures in ELGANs may offer insights into novel mechanisms of epileptogenesis in the developing brain and improvements in the prevention or treatment of seizures in preterm infants, including ELGANs. In this literature review, we outline the limitations of epidemiologic studies of neonatal seizures in ELGANs and discuss risk factors for neonatal seizures.

Enhanced Bone Healing in Critical-Sized Rabbit Femoral Defects: Impact of Helical and Alternate Scaffold Architectures.

This study investigates the effect of scaffold architecture on bone regeneration, focusing on 3D-printed polylactic acid-bioceramic calcium phosphate (PLA-bioCaP) composite scaffolds in rabbit femoral condyle critical defects. We explored two distinct scaffold designs to assess their influence on bone healing and scaffold performance. Structures with alternate (0°/90°) and helical (0°/45°/90°/135°/180°) laydown patterns were manufactured with a 3D printer using a fused deposition modeling technique. The scaffolds were meticulously characterized for pore size, strut thickness, porosity, pore accessibility, and mechanical properties. The in vivo efficacy of these scaffolds was evaluated using a femoral condyle critical defect model in eight skeletally mature New Zealand White rabbits. Then, the results were analyzed micro-tomographically, histologically, and histomorphometrically. Our findings indicate that both scaffold architectures are biocompatible and support bone formation. The helical scaffolds, characterized by larger pore sizes and higher porosity, demonstrated significantly greater bone regeneration than the alternate structures. However, their lower mechanical strength presented limitations for use in load-bearing sites.

Population dynamics of potentially harmful haplotypes: a pedigree analysis.

BACKGROUND: The identification of low-frequency haplotypes, never observed in homozygous state in a population, is considered informative on the presence of potentially harmful alleles (candidate alleles), putatively involved in inbreeding depression. Although identification of candidate alleles is challenging, studies analyzing the dynamics of potentially harmful alleles are lacking. A pedigree of the highly endangered Gochu Asturcelta pig breed, including 471 individuals belonging to 51 different families with at least 5 offspring each, was genotyped using the Axiom PigHDv1 Array (658,692 SNPs). Analyses were carried out on four different cohorts defined according to pedigree depth and at the whole population (WP) level. RESULTS: The 4,470 Linkage Blocks (LB) identified in the Base Population (10 individuals), gathered a total of 16,981 alleles in the WP. Up to 5,466 (32%) haplotypes were statistically considered candidate alleles, 3,995 of them (73%) having one copy only. The number of alleles and candidate alleles varied across cohorts according to sample size. Up to 4,610 of the alleles identified in the WP (27% of the total) were present in one cohort only. Parentage analysis identified a total of 67,742 parent-offspring incompatibilities. The number of mismatches varied according to family size. Parent-offspring inconsistencies were identified in 98.2% of the candidate alleles and 100% of the LB in which they were located. Segregation analyses informed that most potential candidate alleles appeared de novo in the pedigree. Only 17 candidate alleles were identified in the boar, sow, and paternal and maternal grandparents and were considered segregants. CONCLUSIONS: Our results suggest that neither mutation nor recombination are the major forces causing the apparition of candidate alleles. Their occurrence is more likely caused by Allele-Drop-In events due to SNP calling errors. New alleles appear when wrongly called SNPs are used to construct haplotypes. The presence of candidate alleles in either parents or grandparents of the carrier individuals does not ensure that they are true alleles. Minimum Allele Frequency thresholds may remove informative alleles. Only fully segregant candidate alleles should be considered potentially harmful alleles. A set of 16 candidate genes, potentially involved in inbreeding depression, is described.

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO).

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Long-Term Effects of a 6-Week Power-Based Resistance Training and Fast Walking Interval Training Program on Physical Function, Muscle Power, Disability, and Frailty in Pre-Frail and Frail Older Adults.

INTRODUCTION: Concurrent training has been shown to be a beneficial approach to improve overall health status in older adults. However, little is known about the adaptations of this type of training in the long term (i.e., after cessation of exercise), even less in older people affected by frailty syndrome. Therefore, this study aimed (i) to assess the effects of a 6-week concurrent training program composed of power-oriented resistance training and fast walking interval training on physical function, muscle power, disability in activities of daily living and frailty in pre-frail and frail older people, and (ii) to assess the effects of a 6-month detraining period on these outcomes. METHODS: A total of 59 pre-frail and frail older adults (>75 years old; Frailty Phenotype >1) were allocated into intervention (INT; n = 32; 81.8 years; 21 women) or control (CON; n = 27; 82.5 years; 19 women) groups. Primary outcomes of this study were Short Physical Performance Battery (SPPB), relative sit-to-stand (STS) power, Barthel index, Lawton scale and Frailty Phenotype. Assessments were performed at baseline (PRE), after the concurrent training programme (POST) and after 6 months of follow-up (DET) in both groups. Mixed model repeated measures ANOVA with Bonferroni's post hoc tests was used. RESULTS: Immediately after the intervention (∆ = POST-PRE), INT improved SPPB (∆ = 3.0 points; p < 0.001), relative STS power (∆ = 0.87 W·kg-1; p < 0.001) and reduced their frailty levels (∆ = -1.42 criteria; p < 0.001), while no changes were observed in CON. After 6 months of detraining (∆ = DET-PRE), INT showed higher SPPB (∆ = 2.2 points; p < 0.001), higher relative STS power (∆ = 0.73 W·kg-1; p < 0.001) and lower frailty (∆ = -1.24 criteria; p < 0.001) values than those reported at baseline, which were significantly different than those reported by CON. Both, Barthel index and Lawton scale values were not modified during the study in either group. CONCLUSIONS: The 6-week concurrent training program improved physical function, muscle power and reduced frailty in pre-frail and frail older people and these improvements were maintained above baseline levels after 6 months of detraining. However, due to the individual variability found, future studies of long-term responders versus non-responders in frail populations are required.

Power-oriented resistance training combined with high-intensity interval training in pre-frail and frail older people: comparison between traditional and cluster training set configurations on the force-velocity relationship, physical function and frailty.

OBJECTIVES: To analyse the force-velocity relationship changes in response to two different training programmes differing in the set configuration (cluster vs. traditional), and their impact on physical function and frailty in pre-frail and frail older adults. METHODS: 43 pre-frail and frail (Frailty Phenotype ≥ 1 criteria) older adults (81.4 ± 5.1 years) participated in this study. Participants were assigned to cluster (CT; n = 10; 10-s intra-set rest), traditional (TT; n = 13; no intra-set rest) or control (CON; n = 20) groups. Force-velocity relationship (F0, V0 and Pmax), physical function (Short Physical Performance Battery, SPPB) and frailty (Frailty Phenotype, FP) were assessed at baseline and after the training programme. RESULTS: Both CT and TT groups showed similar improvements in Pmax after training (CT = + 36.7 ± 34.2 W; TT = + 33.8 ± 44.6 W; both p < 0.01). V0 was improved by both CT (+ 0.08 ± 0.06 m s-1; p < 0.01), and TT (+ 0.07 ± 0.15 m s-1, p > 0.05). F0 remained unchanged in CT (+ 68.6 ± 224.2 N, p > 0.05) but increased in TT (+ 125.4 ± 226.8 N, p < 0.05). Finally, SPPB improved in both training conditions (CT = + 2.3 ± 1.3 points; TT = + 3.0 ± 1.2 points; both p < 0.05) and in the CON group (+ 0.9 ± 1.4 points, p < 0.05). CT and TT reduced their FP (CT = - 1.1 criteria; TT = - 1.6 criteria; both p < 0.01), while no changes were observed in the CON group (- 0.2 criteria, p = 0.38). CONCLUSIONS: Both training methods were equally effective for improving Pmax, physical function and reducing frailty in pre-frail and frail older people. TT may be effective for improving both force and velocity parameters, while CT may be effective for improving velocity parameters alone, although further research is required to confirm these findings.

If the Patient Does Not Take the Medication, It Will Not Work.

COGNITIVE AND BEHAVIORAL COMORBIDITIES: AN UNWANTED EFFECT OF ANTIEPILEPTIC DRUGS IN CHILDREN: Adriana Ulate-Campos, Iván Sánchez Fernández Seminars in Pediatric Neurology Volume 24, Issue 4, November 2017, Pages 320-330 Epilepsy is one of the most common neurological disorders and, despite optimally chosen and dosed antiepileptic drugs (AEDs), approximately 20%-30% of patients will continue to have seizures. Behavior and cognition are negatively impacted by seizures, but AEDs are also a major contributor to behavioral and cognitive deficits. However, the cognitive and behavioral effect of AEDs in children is insufficiently emphasized in the literature. This review summarizes the cognitive and behavioral effects of AEDs in the pediatric population with the objective of helping pediatricians and pediatric neurologists to select the AEDs with the best profile for their individual patient's needs.

Understanding cost effectiveness in neurology.

The USA spends more in healthcare per capita than any other country in the world, but ranks last among high-income industrialized nations in major markers of healthcare effectiveness such as life expectancy, maternal mortality, neonatal mortality, and infant mortality. Unlike other high-income industrialized nations, the USA does not have a national agency that systematically evaluates the cost-effectiveness of health care interventions and negotiates their price accordingly. This manuscript aims to introduce the rationale, terminology, advantages, and limitations of cost-effectiveness analysis. Cost-effectiveness analysis compares health interventions and evaluates their incremental value and their incremental cost compared with already existing healthcare interventions. Cost-effectiveness analysis integrates the best available evidence with patients' preferences to inform clinical decision making. Patients with neurological conditions are facing increasing challenges to access healthcare and prescription drugs. Cost-effectiveness analysis may help improve access to the most effective healthcare interventions and prescription drugs while containing healthcare costs.

Approaching autozygosity in a small pedigree of Gochu Asturcelta pigs.

BACKGROUND: In spite of the availability of single nucleotide polymorphism (SNP) array data, differentiation between observed homozygosity and that caused by mating between relatives (autozygosity) introduces major difficulties. Homozygosity estimators show large variation due to different causes, namely, Mendelian sampling, population structure, and differences among chromosomes. Therefore, the ascertainment of how inbreeding is reflected in the genome is still an issue. The aim of this research was to study the usefulness of genomic information for the assessment of genetic diversity in the highly endangered Gochu Asturcelta pig breed. Pedigree depth varied from 0 (founders) to 4 equivalent discrete generations (t). Four homozygosity parameters (runs of homozygosity, FROH; heterozygosity-rich regions, FHRR; Li and Horvitz's, FLH; and Yang and colleague's FYAN) were computed for each individual, adjusted for the variability in the base population (BP; six individuals) and further jackknifed over autosomes. Individual increases in homozygosity (depending on t) and increases in pairwise homozygosity (i.e., increase in the parents' mean) were computed for each individual in the pedigree, and effective population size (Ne) was computed for five subpopulations (cohorts). Genealogical parameters (individual inbreeding, individual increase in inbreeding, and Ne) were used for comparisons. RESULTS: The mean F was 0.120 ± 0.074 and the mean BP-adjusted homozygosity ranged from 0.099 ± 0.081 (FLH) to 0.152 ± 0.075 (FYAN). After jackknifing, the mean values were slightly lower. The increase in pairwise homozygosity tended to be twofold higher than the corresponding individual increase in homozygosity values. When compared with genealogical estimates, estimates of Ne obtained using FYAN tended to have low root-mean-squared errors. However, Ne estimates based on increases in pairwise homozygosity using both FROH and FHRR estimates of genomic inbreeding had lower root-mean-squared errors. CONCLUSIONS: Parameters characterizing homozygosity may not accurately depict losses of variability in small populations in which breeding policy prohibits matings between close relatives. After BP adjustment, the performance of FROH and FHRR was highly consistent. Assuming that an increase in homozygosity depends only on pedigree depth can lead to underestimating it in populations with shallow pedigrees. An increase in pairwise homozygosity computed from either FROH or FHRR is a promising approach for characterizing autozygosity.

Evolution of antiseizure medication use and cost in the United States of America 2006-2021.

OBJECTIVE: To describe the evolution in use and cost of antiseizure medications (ASM) in the United States of America (USA). METHODS: Retrospective descriptive study using the IBM MarketScan Commercial Database (data of privately-insured patients) for the years 2006 to 2021. We identified patients with epilepsy who were on ASM. We adjusted cost for inflation with the Gross Domestic Product Implicit Price Deflator. RESULTS: We evaluated 347,158 patients (46.9 % males; median (p25-p75) age: 33 (17-49) years; 28 % with pediatric-onset epilepsy and 72 % with adult-onset epilepsy) with a total of 1,385,382 person-years and 588,285,065 ASM prescription days. The most commonly prescribed (as percentage of prescription days) ASMs in 2006 were valproate (18 %) and lamotrigine (17 %) in pediatric-onset epilepsy and phenytoin (21 %) and carbamazepine (17 %) in adult-onset epilepsy, but starting in the 2010s, levetiracetam and lamotrigine became the most commonly prescribed ASMs in both pediatric-onset (in 2021, levetiracetam 25 %, lamotrigine 16 %) and adult-onset (in 2021, levetiracetam 27 %, lamotrigine 20 %) epilepsy. The proportion of generic ASM use increased 3.6-fold: from 23 % of prescription days in 2006 to 83 % of prescription days in 2021. The median (p25-p75) average wholesale price (AWP) per person-year increased by 102 % from $2,684 ($990-$5,509) in 2006 to $5,417 ($2,858-$12,310) in 2021. The increases were greater in absolute terms for brand-name ASMs by 419 %: $3,109 ($1,564-$5,068 in 2006 and $16,149 ($12,950-$23,377) in 2021 than for generic ASMs by 462 %: $699 ($457-$1,678) in 2006 and $3,931 ($2,618-$6,081) in 2021. The costs directly borne by the patient (copay, coinsurance, deductibles, and pharmacy processing fees) increased by 69 % for brand-name ASMs from $393 ($246-$570) in 2006 to $665 ($335-$1,308) in 2021, but decreased by 37 % for generic ASMs from $147 ($98-$213) in 2006 to $92 ($51-$141) in 2021. CONCLUSIONS: The median cost of ASMs per person-year approximately doubled from 2006 to 2021. The increase in use of generic ASMs probably helped buffer the growing costs of ASMs. However, generic ASMs already represent 83 % of prescription days in 2021, with limited room to further contain costs by just increasing the proportion of generics.

Ovine placental explants: A new ex vivo model to study host‒pathogen interactions in reproductive pathogens.

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

Comparing assembly strategies for third-generation sequencing technologies across different genomes.

The recent advent of long-read sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore technology (ONT), has led to substantial accuracy and computational cost improvements. However, de novo whole-genome assembly still presents significant challenges related to the computational cost and the quality of the results. Accordingly, sequencing accuracy and throughput continue to improve, and many tools are constantly emerging. Therefore, selecting the correct sequencing platform, the proper sequencing depth and the assembly tools are necessary to perform high-quality assembly. This paper evaluates the primary assembly reconstruction from recent hybrid and non-hybrid pipelines on different genomes. We find that using PacBio high-fidelity long-read (HiFi) plays an essential role in haplotype construction with respect to ONT reads. However, we observe a substantial improvement in the correctness of the assembly from high-fidelity ONT datasets and combining it with HiFi or short-reads.

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

Copy Number Variation Regions Differing in Segregation Patterns Span Different Sets of Genes.

Copy number variations regions (CNVRs) can be classified either as segregating, when found in both parents, and offspring, or non-segregating. A total of 65 segregating and 31 non-segregating CNVRs identified in at least 10 individuals within a dense pedigree of the Gochu Asturcelta pig breed was subjected to enrichment and functional annotation analyses to ascertain their functional independence and importance. Enrichment analyses allowed us to annotate 1018 and 351 candidate genes within the bounds of the segregating and non-segregating CNVRs, respectively. The information retrieved suggested that the candidate genes spanned by segregating and non-segregating CNVRs were functionally independent. Functional annotation analyses allowed us to identify nine different significantly enriched functional annotation clusters (ACs) in segregating CNVR candidate genes mainly involved in immunity and regulation of the cell cycle. Up to five significantly enriched ACs, mainly involved in reproduction and meat quality, were identified in non-segregating CNVRs. The current analysis fits with previous reports suggesting that segregating CNVRs would explain performance at the population level, whereas non-segregating CNVRs could explain between-individuals differences in performance.

Use of 3D-printed polylactic acid/bioceramic composite scaffolds for bone tissue engineering in preclinical in vivo studies: A systematic review.

3D-printed composite scaffolds have emerged as an alternative to deal with existing limitations when facing bone reconstruction. The aim of the study was to systematically review the feasibility of using PLA/bioceramic composite scaffolds manufactured by 3D-printing technologies as bone grafting materials in preclinical in vivo studies. Electronic databases were searched using specific search terms, and thirteen manuscripts were selected after screening. The synthesis of the scaffolds was carried out using mainly extrusion-based techniques. Likewise, hydroxyapatite was the most used bioceramic for synthesizing composites with a PLA matrix. Among the selected studies, seven were conducted in rats and six in rabbits, but the high variability that exists regarding the experimental process made it difficult to compare them. Regarding the results, PLA/Bioceramic composite scaffolds have shown to be biocompatible and mechanically resistant. Preclinical studies elucidated the ability of the scaffolds to be used as bone grafts, allowing bone growing without adverse reactions. In conclusion, PLA/Bioceramics scaffolds have been demonstrated to be a promising alternative for treating bone defects. Nevertheless, more care should be taken when designing and performing in vivo trials, since the lack of standardization of the processes, which prevents the comparison of the results and reduces the quality of the information. STATEMENT OF SIGNIFICANCE: 3D-printed polylactic acid/bioceramic composite scaffolds have emerged as an alternative to deal with existing limitations when facing bone reconstruction. Since preclinical in vivo studies with animal models represent a mandatory step for clinical translation, the present manuscript analyzed and discussed not only those aspects related to the selection of the bioceramic material, the synthesis of the implants and their characterization. But provides a new approach to understand how the design and perform of clinical trials, as well as the selection of the analysis methods, may affect the obtained results, by covering authors' knowledgebase from veterinary medicine to biomaterial science. Thus, this study aims to systematically review the feasibility of using polylactic acid/bioceramic scaffolds as grafting materials in preclinical trials.

Study of the cross-talk between Fasciola hepatica juveniles and the intestinal epithelial cells of the host by transcriptomics in an in vitro model.

Fasciolosis is a globally widespread trematodiasis with a major economic and veterinary impact. Therefore, this disease is responsible for millions of dollars in losses to the livestock industry, and also constitutes an emerging human health problem in endemic areas. The ubiquitous nature of Fasciola hepatica, the main causative agent, is one of the key factors for the success of fasciolosis. Accordingly, this parasite is able to subsist in a wide variety of ecosystems and hosts, thanks to the development of a plethora of strategies for adaption and immune evasion. Fasciolosis comprises a growing concern due to its high prevalence rates, together with the emergence of strains of the parasite resistant to the treatment of choice (triclabendazole). These facts highlight the importance of developing novel control measures which allow for an effective protection against the disease before F. hepatica settles in a niche inaccessible to the immune system. However, knowledge about the initial phases of the infection, including the migration mechanisms of the parasite and the early innate host response, is still scarce. Recently, our group developed an in vitro host-parasite interaction model that allowed the early events to be unveiled after the first contact between the both actors. This occurs shortly upon ingestion of F. hepatica metacercariae and the emergence of the newly excysted juveniles (FhNEJ) in the host duodenum. Here, we present a transcriptomic analysis of such model using an approach based on RNA sequencing (RNA-Seq), which reveals changes in gene expression related to proteolysis and uptake of metabolites in FhNEJ. Additionally, contact with the parasite triggered changes in host intestinal cells related to pseudogenes expression and host defence mechanisms, including immune response, among others. In sum, these results provide a better understanding of the early stages of fasciolosis at molecular level, and a pool of targets that could be used in future therapeutic strategies against the disease.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

Pediatric status epilepticus management by Emergency Medical Services (the pSERG cohort).

PURPOSE: Delayed treatment in status epilepticus (SE) is independently associated with increased treatment resistance, morbidity, and mortality. We describe the prehospital management pathway and Emergency Medical Services (EMS) timeliness in children who developed refractory convulsive status epilepticus (RCSE). METHODS: Retrospective multicenter study in the United States using prospectively collected observational data from June 2011 to March 2020. We selected pediatric patients (one month-21 years) with RCSE initiated outside the hospital and transported to the hospital by EMS. RESULTS: We included 91 patients with a median (percentile25-percentile75) age of 3.0 (1.5-7.3) years. The median time from seizure onset to hospital arrival was 45 (30-67) minutes, with a median time cared for by EMS of 24 (15-36) minutes. Considering treatment by caregivers and EMS before hospital arrival, 20 (22%) patients did not receive any anti-seizure medications (ASM) and 71 (78%) received one to five doses of benzodiazepines (BZD), without non-BZD ASM. We provided the prehospital treatment flow path of these patients through caregivers and EMS including relevant time points. Patients with a history of SE were more likely to receive the first BZD in the prehospital setting compared to patients without a history of SE (adjusted HR 3.25, 95% CI 1.72-6.12, p<0.001). CONCLUSION: In this multicenter study of pediatric RCSE, prehospital treatment may be streamlined further. Patients with a history of SE were more likely to receive prehospital rescue medication.

Residual effects of 12 weeks of power-oriented resistance training plus high-intensity interval training on muscle dysfunction, systemic oxidative damage, and antioxidant capacity after 10 months of training cessation in older people with COPD.

OBJECTIVE: This study aimed to assess the residual effects of a 12-week concurrent training program (power training + high-intensity interval training) in older adults with chronic obstructive pulmonary disease (COPD). METHODS: A total of 21 older adults with COPD [intervention (INT), n = 8; control (CON), n = 13; 76.9 ± 6.8 years] were assessed at baseline and 10 months after the completion of the intervention by the short physical performance battery (SPPB), health-related quality of life (EQ-5D-5L), vastus lateralis muscle thickness (MT), peak pulmonary oxygen uptake (peak VO2 ) and peak work rate (Wpeak ), early and late isometric rate of force development (RFD), leg and chest press maximum muscle power (LPmax and CPmax ), and systemic oxidative damage and antioxidant capacity. RESULTS: Compared to baseline, after 10 months of detraining, the INT group presented increased SPPB (∆ = 1.0 point), health-related quality of life (∆ = 0.07 points), early RFD (∆ = 834 N∙s-1 ), LPmax (∆ = 62.2 W), and CPmax (∆ = 16.0 W) (all p < 0.05). In addition, a positive effect was noted in INT compared to CON regarding MT and Wpeak (both p < 0.05). No between-group differences were reported in peak VO2 , late RFD, systemic oxidative damage, and antioxidant capacity from baseline to 10 months after the completion of the intervention (all p > 0.05). CONCLUSIONS: Twelve weeks of concurrent training were enough to ensure improved physical function, health-related quality of life, early RFD and maximum muscle power and to preserve MT and Wpeak but not peak VO2 , late RFD, systemic oxidative damage and antioxidant capacity in the subsequent 10 months of detraining in older adults with COPD.