Physical activity and long COVID: findings from the Prospective Study About Mental and Physical Health in Adults cohort.

OBJECTIVES: The study investigated the longitudinal association between physical activity and the risk of long COVID in patients who recovered from COVID-19 infection. STUDY DESIGN: We analyzed longitudinal data of the Prospective Study About Mental and Physical Health cohort, a prospective cohort study with adults living in Southern Brazil. METHODS: Participants responded to an online, self-administered questionnaire in June 2020 (wave 1) and June 2022 (wave 4). Only participants who self-reported a positive test for COVID-19 were included. Physical activity was assessed before (wave 1, retrospectively) and during the pandemic (wave 1). Long COVID was assessed in wave 4 and defined as any post-COVID-19 symptoms that persisted for at least 3 months after infection. RESULTS: A total of 237 participants (75.1% women; mean age [standard deviation]: 37.1 [12.3]) were included in this study. The prevalence of physical inactivity in baseline was 71.7%, whereas 76.4% were classified with long COVID in wave 4. In the multivariate analysis, physical activity during the pandemic was associated with a reduced likelihood of long COVID (prevalence ratio [PR]: 0.83; 95% confidence interval [CI]: 0.69-0.99) and a reduced duration of long COVID symptoms (odds ratio: 0.44; 95% CI: 0.26-0.75). Participants who remained physically active from before to during the pandemic were less likely to report long COVID (PR: 0.74; 95% CI: 0.58-0.95), fatigue (PR: 0.49; 95% CI: 0.32-0.76), neurological complications (PR: 0.47; 95% CI: 0.27-0.80), cough (PR: 0.40; 95% CI: 0.22-0.71), and loss of sense of smell or taste (PR: 0.43; 95% CI: 0.21-0.87) as symptom-specific long COVID. CONCLUSION: Physical activity practice was associated with reduced risk of long COVID in adults.

Characterization of a DRC1 null variant associated with primary ciliary dyskinesia and female infertility.

PROPOSE: We here present a female case with primary ciliary dyskinesia (PCD) and infertility. In this report, we also present the evaluation of the patient family, including her twin sister, also with PCD and infertility. METHODS: Confirmation of the PCD clinical diagnosis was performed through assessment of cilia motility, by high-speed video microscopy (HSVM), axoneme ultrastructure, by transmission electron microscopy (TEM), and genetic characterization, by whole-exome sequence (WES). Gene expression studies used qPCR for mRNA expression and immunofluorescence to determine cell protein localization. RESULTS: We identified a homozygous nonsense variant in the DRC1 gene (NM 145038.5:c.352C>T (p.Gln118Ter)) in the female patient with PCD and infertility that fit the model of autosomal recessive genetic transmission. This variant eventually results in a dyskinetic ciliary beat with a lower frequency and a partial lack of both dynein arms as revealed by TEM analysis. Moreover, this variant implies a decrease in the expression of DRC1 mRNA and protein. Additionally, expression analysis suggested that DRC1 may interact with other DRC elements. CONCLUSIONS: Our findings suggest that the DRC1 null variant leads to PCD associated with infertility, likely caused by defects in axoneme from Fallopian tube cilia. Overall, our outcomes contribute to a better understanding of the genetic factors involved in the pathophysiology of PCD and infertility, and they highlight the interaction of different genes in the patient phenotype, which should be investigated further because it may explain the high heterogeneity observed in PCD patients.

Point prevalence audit surveys of respiratory tract infection consultations and antibiotic prescribing in primary care before and during the COVID-19 pandemic in Ireland.

BACKGROUND: Respiratory tract infections (RTIs) are the most common reason for prescribing antibiotics in general practice. The COVID-19 pandemic has impacted on antibiotic prescribing and delivery of primary care in Ireland. OBJECTIVES: To assess the quality of antibiotic prescribing, the impact of the COVID-19 pandemic and identify opportunities for antimicrobial stewardship (AMS) in Ireland. METHODS: Point prevalence audit surveys for RTI consultations were conducted as part of a European study at three time periods: January-February 2020, March-May 2020 and March-May 2021. Antibiotic prescribing was assessed and comparisons made between the three time periods. RESULTS: In total, 765 consultations were recorded, which were mainly face to face before the pandemic, but changed to predominantly remote consultations during the pandemic surveys in 2020 and 2021 (82% and 75%). Antibiotics were prescribed in 54% of RTI consultations before the pandemic. During pandemic surveys, this dropped to 23% in 2020 and 21% in 2021. There was a decrease in prescribing of Red (reserve) agents in 2021. Assessment against indication-specific quality indicators showed a high proportion of consultations for bronchitis and tonsillitis resulting in an antibiotic prescription (67% and 85%). Point-of-care testing (POCT) to aid diagnosis of RTIs were utilized in less than 1% of consultations. CONCLUSIONS: During the COVID-19 pandemic, there was a reduction in antibiotic prescribing. Opportunities identified to support AMS in primary care in Ireland are targeted initiatives to reduce antibiotic prescribing for bronchitis and tonsillitis and introducing POCT to support appropriate antibiotic prescribing.

The Silent Threat-Alkali Foot Burn: A Case Report.

Despite current workplace protection measures, chemical burn accidents are not uncommon. Among these, alkali burn is the most challenging due to its silent behaviour and aggressive mechanism. Characterized by its initial painless onset, allowing a prolonged exposure, it tends to result in deeper and more destructive burns, creating major reconstructive and therapeutic challenges. This case concerns a 53-year-old male who sustained a full thickness burn of his left instep foot after prolonged contact with an alkali substance. The wound was submitted to several surgical debridement procedures, with preservation of the major tendinous and vascular-nervous structures. The skin defect was then repaired with skin graft. Early recognition and prompt management with copious and prolonged wound irrigation is paramount. As in this type of burn it is difficult to initially assess its true depth, even after initial surgical debridement, a more cautious approach is recommended. Chronic pain is associated with chemical burns and it should be treated early in the process with the use of multimodal analgesia in order to prevent future complications. No matter the absence of major complaints in the 4 week-postoperative evaluation, the possible long-term consequences are still unknown. Despite the prolonged exposure time and the initial presentation with a deep burn, after several surgical debridement procedures, preservation of major tendon and neurovascular structures was assured, which allowed a plain approach for reconstruction of the wound with a skin graft. The case illustrates different challenges associated with evaluation and treatment of patients with deep alkali burns. Also, usage auditing and awareness of regular users appear to be essential.

Les brûlures chimiques au travail restent relativement fréquentes malgré les mesures de protection. Les atteintes par agent alcalin sont particulièrement problématiques car elles sont insidieuses bien que très destructrices. Le temps de contact est prolongé et les destructions sont majeures, posant de difficiles problèmes de reconstruction, en raison de leur caractère initialement indolore. Nous présentons le cas d'un homme de 53 ans victime d'une brûlure du cou de pied gauche après contact prolongé avec un agent alcalin. Les excisions successives ont permis de préserver les éléments nobles et de greffer secondairement la zone touchée. Il est indispensable de diagnostiquer précisément l'atteinte et de laver la région atteinte à grande eau. La profondeur réelle est difficile à apprécier initialement si bien que les excisions doivent être prudentes. Les douleurs chroniques ne sont pas rares dans ces cas et peuvent être prévenues par la qualité de l'analgésie initiale, multimodale. A un mois, le patient ne se plaint de rien, ce qui ne préjuge pas de complications plus tardives. Malgré un temps de contact prolongé et un aspect initial profond, les éléments nobles on pu être préservés, permettant une couverture par simple greffe. Ce cas illustre les difficultés dans le diagnostic et le traitement des brûlures profondes par base. Il est essentiel d'éduquer et surveiller les usagers réguliers de ces produits.

N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes.

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 µg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

Fusion of Tumor-Derived Exosomes with Long-Circulating and pH-Sensitive Liposomes Loaded with Doxorubicin for the Treatment of Breast Cancer.

Doxorubicin (DOX) is a chemotherapeutic agent that has been used in the treatment of breast cancer. However, serious toxic effects have limited its use, mainly cardiotoxicity. To minimize the adverse effects, liposomal preparations containing DOX have been developed. These preparations can reach the target in the tumor region as well as bypass the resistance-related problems. An alternative to increased therapeutic efficacy may be the fusion of liposomes with exosomes released from tumor cells to facilitate membrane and fusion interactions, achieving greater cell uptake. Thus, the purpose of this study was the fusion of exosomes derived from breast tumor cells with long-circulating and pH-sensitive liposomes loading DOX (ExoSpHL-DOX) for the treatment of breast cancer. The mean diameter of ExoSpHL-DOX was 100.8 ± 7.8 nm, the polydispersity index was 0.122 ± 0.004, and the encapsulated DOX content was equal to 83.5 ± 2.5%. The fusion of exosomes with long-circulating and pH-sensitive liposomes was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, and nano-flow cytometry. The physicochemical characteristics of ExoSpHL-DOX were maintained for 60 days, at 4 °C. The study of the release of DOX from ExoSpHL-DOX in dilution media with different pH values showed the pH sensitivity characteristic of the nanosystem, since 96.6 ± 0.2% of DOX was released from ExoSpHL-DOX at pH 5.0, while at pH 7.4, the release was 70.1 ± 1.7% in the medium. The cytotoxic study against the breast cancer cell line demonstrated that ExoSpHL-DOX treatment significantly reduced the cancer cell viability.

N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

Functional analysis of ldlr genetic variants identified in hypercholesterolemic patients in brazil, USING CRISPR/CAS9

BACKGROUND AND AIMS: Introduction: The familial hypercholesterolemia (FH) is one of the main causes of cardiovascular diseases, and it is mainly caused by genetic variants at the low-density lipoprotein receptor (LDLR). Although ultrasequencing technology has allowed the identification of several genetic variants, few of them was functional analyzed. The CRISPR/ Cas9 tool promotes precise genetic editing and allows the creation of experimental models, therefore contributing to the functional validation process. Aim: To use the CRISPR/Cas9 tool to perform in vitro functional analysis of LDLR variants identified in FH patients. METHODS: Two missense LDLR variants were selected within a group of variants identified in FH patients, based on in silico data, the affected protein domain and MAF. Three sgRNAs were designed for each of the variants c.551G>A and c.1118G>A, to analyze the accuracy of the sgRNAs. The sgRNAs were inserted on PX458 plasmid, cloned, purified in E. coli DH5a, and then co-transfected with the DNA template at HepG2 cells. The DNAs templates were designed to contain the selected variants. RESULTS: HepG2 cells co-transfected with PX458 constructs and DNA templates showed considerably transfection rate, being possible to visualize it at fluorescence microscopy. However, it was noted that single transfection of sgRNAs showed a higher transfection efficiency than cotransfection. CONCLUSIONS: We designed sgRNA for c.551G>A and c.1118G>A variants, being able to analyze the transfection efficiency. In further steps, we will select new sgRNAs for LDLR variants that have not been described yet, and functional analysis will be performed to determine the clinical relevance of these variants.

Effect of Terbufos (Organophosphate) on the Cadaveric Colonization Process: Implications for Postmortem Interval Calculation.

The determination of necrophagous fly specie's development time is considered an accurate method for estimating postmortem interval (PMI). However, pesticides and other chemicals can alter the flies' life cycle, inducing errors in PMI estimation. Thus, this work aimed to evaluate the effect of different doses of Terbufos (Organophosphates) on the temporal dispersion pattern and development of immature dipterans associated with decaying rat carcasses. For this, 150 g female Wistar rats received, via gavage, 200 µl of Terbufos (5 or 10 mg/kg) or distilled water (control) and, after 30 min of administration, the animals were euthanized and distributed in suspended traps to decompose under environmental conditions. The dispersing immatures were collected daily, and their development time was monitored until the emergence of adult flies. After data analysis, it was observed that Terbufos altered 1) the temporal pattern of larval dispersion; 2) the composition and structure of the colonizing assemblage (emerged adults); 3) species' development time, accelerating or delaying their cycle, depending on the dose used; and 4) the califorids and sarcophagids emergence rate, increasing the mortality of pupae from intoxicated carcasses. Thus, this work demonstrates experimentally that Terbufos directly influences the development of flies with forensic potential and discusses the implications for PMI estimation, which can assist in future investigative processes with suspected poisoning by this organophosphate.

Aesthetic perception in children with molar incisor hypomineralization.

PURPOSE: To investigate the relationship between aesthetic perception and molar incisor hypomineralization (MIH) in schoolchildren aged 8-12 years. METHODS: The study included 467 pupils enrolled in elementary schools. Once authorization was received, the students were examined for DMFT and dmft indexes (WHO criteria), and for MIH (EAPD criteria). The aesthetic perceptions were verified by the Child Perceptions Questionnaire About Tooth Appearance (CQATA), a questionnaire divided into several items, including physical, psychological and social domains; perception about tooth pleasantness, tooth alignment, tooth discoloration and tooth health; and reported pleasant colour. Data analysis was carried out using descriptive statistics, the Chi-square, the Mann-Whitney and the Kruskal-Wallis tests, and linear regression at a significance level of 0.05. RESULTS: The tooth health perception was worse when the child had MIH, and the tooth alignment and tooth discoloration perceptions, when MIH affected the incisors. The number of teeth affected by MIH was associated with higher averages in the tooth discoloration perception. CONCLUSIONS: Children with MIH showed more negative perceptions toward tooth health, tooth alignment and tooth discoloration. However, none of the clinically investigated variables negatively impacted the reported pleasant colour question.

A systematic review of instruments measuring grief after perinatal loss and factors associated with grief reactions.

OBJECTIVE: Expectant parents who live through perinatal loss experience intense grief, which is not always acknowledged or accepted. A screening tool to detect bereaved parents' grief reactions can guide professionals, including perinatal palliative care teams, to provide follow-up for those in need. This review's goal is to identify and synthesize the international published literature on existent instruments specifically measuring the grieving process after any perinatal loss and to identify factors that could moderate grief reactions. METHOD: Systematic review (PROSPERO # CRD42018092555) with critical synthesis. PUBMED, Cochrane, and PsycINFO databases were searched in English language articles using the keywords "perinatal" AND ("grief" OR "bereavement" OR mourning) AND ("scale" OR "questionnaire" OR "measure" OR "assessment") up to May 2018. Eligibility criteria included every study using a measure to assess perinatal grief after all kinds of perinatal losses, including validations and translations to other languages and interventions designed to alleviate grief symptoms. RESULTS: A total of 67 papers met inclusion criteria. Seven instruments measuring perinatal grief published between 1984 and 2002 are described. The Perinatal Grief Scale (PGS) was used in 53 of the selected studies. Of those, 39 analyzed factors associated with grief reactions. Six articles used PGS scores to evaluate pre- and post-bereavement interventions. Studies in English language only might have limited the number of articles. SIGNIFICANCE OF RESULTS: The PGS is the most used standardized measures to assess grief after perinatal loss. All parents living through any kind of perinatal loss should be screened.

Double membrane based on lidocaine-coated polymyxin-alginate nanoparticles for wound healing: In vitro characterization and in vivo tissue repair.

The aim of this study was to develop and characterize a double layer biomembrane for dual drug delivery to be used for the treatment of wounds. The membrane was composed of chitosan, hydroxypropyl methylcellulose and lidocaine chloride (anesthetic drug) in the first layer, and of sodium alginate-polymyxin B sulphate (antibiotic) nanoparticles as the second layer. A product with excellent thickness (0.01-0.02 mm), adequate mechanical properties with respect to elasticity, stiffness, tension, and compatible pH for lesion application has been successfully obtained. The incorporation of the drugs was confirmed analysing the membrane cross-sections by scanning electron microscopy. A strong interaction between the drugs and the functional groups of respective polymers was confirmed by Fourier-Transform Infrared Spectroscopy, thermal analysis and X-ray diffraction. Microbiological assays showed a high antimicrobial activity when polymyxin B was present to act against the Staphylococcus aureus and Pseudomonas aeruginosa strains. Low cytotoxicity observed in a cell viability colorimetric assay and SEM analysis suggest biocompatibility between the developed biomembrane and the cell culture. The in vivo assay allowed visualizing the healing potential by calculating the wound retraction index and by histological analysis. Our results confirm the effectiveness of the developed innovative biomaterial for tissue repair and regeneration in an animal model.

Insects Associated to Crime Scenes in the Northeast of Brazil: Consolidation of Collaboration Between Entomologists and Criminal Investigation Institutes.

In Brazil, many studies on Forensic Entomology analyze the activity and succession of flies in animal models. Data on human corpses are always collected and evaluated in isolated cases. This study aimed to list the insect species associated with crime scenes investigated by the Technical-Scientific Institute of criminal expertise of the State of Rio Grande do Norte (ITEP-RN), in the Northeast of Brazil, a region exposed to high homicide rates. In total, 10 cases were investigated, of which 50% were in the initial stage of decomposition. The examined bodies were colonized by species of three orders of insects, Diptera, Coleoptera and Hymenoptera. The order Diptera represented 96% of the total insects, being represented by the following species: Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae), Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae), Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae), and Cochliomyia macellaria, (Fabricius) (Diptera: Calliphoridae); Musca domestica (Linnaeus) (Diptera: Muscidae); and unidentified females of the Sarcophagidae family. Among beetles, the occurrence of Dermestes maculatus (DeGeer) (Coleoptera: Dermestidae), Necrobia rufipes (De Geer) (Coleoptera: Cleridae), and Onthophagus sp. (Scarabaeidae), as well as unidentified specimens of the families Tenebrionidae and Histeridae were recorded. In addition, specimens of Ectatomma sp. (Formicidae) were also recorded. Considering that the Rio Grande do Norte state presents a high homicide rate and the last cadaver study was conducted over a decade ago, these records update the list of species associated with cadaveric decomposition and contribute to consolidate forensic entomology in the Northeast region.

Antibacterial activity of chitosan/collagen membranes containing red propolis extract.

In this study we developed a mucoadhesive polymeric membrane wound dressing incorporating red propolis extract (HERP). Membranes were made using a casting method employing collagen, chitosan, polyethylene glycol (15, 20, and 30v%), and hydroethanolic extract of EtOH-H2O 70v% - 30v% (v/v) of HERP (0.5, 1.0, and 1.5%). Membranes were extensively characterized to assess the thickness, pH, morphology using Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), mechanical properties, swelling, in vitro mucoadhesion, cytotoxicity, and minimum inhibitory concentration (MIC). Assessment of the thickness and mechanical properties of the membranes containing HERP revealed that the most significant thickness obtained was 40.7 µm; thermal analysis suggests suggesting the hydrogen bonds between hydroxyl groups of isoflavones and the free amine present in the region of chitosan. Cell viability decreased as the amount of HERP increased. Finally, the MICs were 7.8 and 1.9 µg.mL-1 for Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853, respectively. These results were suggesting that the 0.5 % HERP membrane has the potential for future studies for wound application.

Characterization of CCDC103 expression profiles: further insights in primary ciliary dyskinesia and in human reproduction.

PROPOSE: To study CCDC103 expression profiles and understand how pathogenic variants in CCDC103 affect its expression profile at mRNA and protein level. METHODS: To increase the knowledge about the CCDC103, we attempted genotype-phenotype correlations in two patients carrying novel homozygous (missense and frameshift) CCDC103 variants. Whole-exome sequencing, quantitative PCR, Western blot, electron microscopy, immunohistochemistry, immunocytochemistry, and immunogold labelling were performed to characterize CCDC103 expression profiles in reproductive and somatic cells. RESULTS: Our data demonstrate that pathogenic variants in CCDC103 gene negatively affect gene and protein expression in both patients who presented absence of DA on their axonemes. Further, we firstly report that CCDC103 is expressed at different levels in reproductive tissues and somatic cells and described that CCDC103 protein forms oligomers with tissue-specific sizes, which suggests that CCDC103 possibly undergoes post-translational modifications. Moreover, we reported that CCDC103 was restricted to the midpiece of sperm and is present at the cytoplasm of the other cells. CONCLUSIONS: Overall, our data support the CCDC103 involvement in PCD and suggest that CCDC103 may have different assemblies and roles in cilia and sperm flagella biology that are still unexplored.

Study of Efficacy and Replication of Recombinant Vector Vaccines by Using Turkey Herpesvirus Combined with Other Marek's Disease Vaccines.

Several recombinant turkey herpesviruses (rHVTs) have been developed within the past decades, and they are now used commercially worldwide. In broiler chickens, rHVTs are usually administered alone, but in long-living birds they are used in combination with Marek's disease (MD) vaccines of other serotypes (i.e., CVI988). The objectives of this work were to 1) evaluate protection against MD conferred by HVT and two rHVTs when combined with CVI988 and 2) optimize the use of rHVT in combination with CVI988 to maximize replication of rHVT without compromising MD protection. Various vaccine protocols, all using rHVT or HVT at the recommended dose (RD), were evaluated. Protocols evaluated included in ovo vaccination with HVT+CVI988 or rHVT+CVI988 (using either the double dose [DD] or the RD of CVI988), day of age vaccination of rHVT+CVI988 at DD, and revaccination protocols using rHVT in ovo followed by CVI988 at DD at day of age. Our results show that, when combined with CVI988, HVT and rHVTs confer a similar level of protection against MD (>90%) regardless of whether CVI988 was used at RD or at DD. However, the combination of rHVT with CVI988 at DD resulted in reduced replication rates of rHVT (60%-76% vs. 95%-100%). Our results show that such a negative effect could be avoided without jeopardizing MD protection by administering CVI988 at RD (if combined in ovo with rHVT) or administered rHVT first in ovo followed by CVI988 at DD at day of age.

Estudio de la eficacia y replicación de vacunas con vectores recombinantes mediante el uso del virus del herpes del pavo combinado con otras vacunas contra la enfermedad de Marek. Varios herpesvirus de pavo recombinantes (rHVT) se han desarrollado en las últimas décadas y ahora se utilizan comercialmente en todo el mundo. En pollos de engorde, los rHVT generalmente se administran solos, pero en aves de vida larga se usan en combinación con vacunas contra la enfermedad de Marek (MD) de otros serotipos (especialmente, CVI988). Los objetivos de este trabajo fueron 1) evaluar la protección contra la enfermedad de Marek conferida por herpesvirus de pavo (HVT9 y por dos rHVT cuando se combinan con la cepa CVI988 y 2) optimizar el uso de rHVT en combinación con la cepa CVI988 para maximizar la replicación de rHVT sin comprometer la protección contra la enfermedad de Marek. Se evaluaron varios protocolos de vacunas, todos con rHVT o con HVT a la dosis recomendada (RD). Los protocolos evaluados incluyeron la vacunación in ovo con HVT + CVI988 o rHVT + CVI988 (usando la dosis doble o la dosis recomendada de la cepa CVI988), la vacunación al día de la edad con rHVT + CVI988 con dosis doble, y los protocolos de revacunación usando rHVT seguido por la cepa CVI988 con dosis doble al día de edad. Los resultados muestran que cuando se combinan con CVI988, HVT y rHVT confieren un nivel de protección similar contra la enfermedad de Marek (> 90%) independientemente de que la cepa CVI988 se haya usado a la dosis recomendada o con dosis doble. Sin embargo, la combinación de rHVT con la cepa CVI988 con doble dosis produjo una reducción en las tasas de replicación de rHVT (60% ­76% vs. 95% ­100%). Estos resultados muestran que dicho efecto negativo podría evitarse sin poner en peligro la protección contra la enfermedad de Marek administrando la cepa CVI988 a la dosis recomendada (si se combina in ovo con rHVT) o administrando rHVT primero in ovo, seguido de CVI988 con dosis doble al día de la edad.

Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the intestinal villus to crypt ratios of Ross 708 broilers during early post-hatch development1,2,3.

Villus to crypt ratio (VCR) is used to quantify the microanatomical response of the intestine to various treatments. In early age chickens, comparative effects of the in ovo (i.o.) and s.c. methods of administration (moa) of the Marek's disease (MD) vaccine on 2 types of measurement of small intestinal VCR at 0 and 4 h post-hatch (poh) were investigated. The effects of moa and 4 and 18 h pre-placement holding times (pht) on the VCR measurements at 168 h (7 d) poh were also investigated. In the jejunum of the small intestine, a standard method for VCR determination, based on 10 villus and crypt length measurements, was utilized for the calculation of villus to crypt length ratio (VCLR). In that same region, a single histomorphometric determination of the crypt and total mucosa areas using image analysis software was also used. Subtraction of the crypt area from the total mucosa area provided the villus area, allowing for calculation of the villus to crypt area ratio (VCAR). Across 0, 4, and 18 h of poh bird age, the VCLR of birds that received an s.c. vaccination was higher in comparison to that of those that received an i.o. vaccination. The highest and lowest VCAR values were observed in the s.c. treatment at 0 h poh and in the i.o. treatment at 4 h poh, respectively. Furthermore, at 168 h poh, VCLR values in the 18 h pht and s.c. vaccination group were higher than those in the 4 h pht and s.c. vaccination or 18 h and i.o. vaccination groups. In conclusion, the effects of pht and MD vaccine moa on VCR were dependent on the use of either the VCLR or VCAR method of measurement. However, regardless of method, s.c. injection overall led to a higher VCR through 4 h poh in Ross 708 broilers, and the effects of moa on VCLR at 168 h were influenced by pht.

Molecular basis of growth hormone daily mRNA and protein synthesis in rats.

AIMS: Daily and seasonal rhythms coordinate the endocrine and metabolic functions. The pituitary gland is the master regulator of several endocrine activities, and its function is classically regulated by endocrine signals from its target glands as well as from the hypothalamus. The growth hormone (GH) produced and secreted by the anterior pituitary presents a pulsatile secretion throughout the 24-hour cycle. However, the molecular mechanisms regulating the daily pattern of GH secretion are still unclear. Herein we investigated whether circadian GH mRNA and protein synthesis is modulated by acute adjustments in the stability and expression of GH mRNA. MAIN METHODS: GH mRNA and protein content were evaluated by real-time PCR and Western blotting, respectively, in pituitary gland of rats euthanized every 3 h during a 24-h period at the Zeitgeber times (ZT3 to ZT24). The GH mRNA poly(A) tail length was determined by RACE-PAT assay. KEY FINDINGS: We identified two main peaks of GH mRNA level in the pituitary gland of rats; one in the middle of the light-cycle and another in the middle of the dark-cycle. The latter was associated with an increase in pituitary GH protein content. Interestingly, an increment in the poly(A) tail length of the GH transcript was observed in association to reduced migration rate of the GH transcript and increased mRNA content in the dark-cycle period. SIGNIFICANCE: Our findings provide evidence that changes in the GH mRNA poly(A) length may underlie the circadian pattern of GH mRNA and protein levels in the pituitary gland of rats.