Identification and characterization of the 285L and K145R proteins of African swine fever virus.

African swine fever (ASF) is a lethal disease of domestic pigs and wild boar, against which no vaccines are available to date. The large dsDNA genome of African swine fever virus (ASFV) contains up to 167 ORFs predicted to encode proteins. The functions and antigenic properties of many of these proteins are still unknown, which impedes vaccine development. Based on the results of mass spectrometry-based proteome analyses of ASFV-infected cells, two highly abundant but previously uncharacterized viral proteins, p285L and pK145R, were investigated in detail. To this end, monospecific rabbit antisera and corresponding gene deletion mutants of ASFV were prepared. RNA and immunoblot analyses revealed that p285L is an early gene product expressed prior to viral DNA replication, whereas pK145R is a true late protein. The predicted membrane protein p285L could be localized in purified ASFV particles. In contrast, pK145R was not detectable in virions, but accumulated diffusely in the cytoplasm of infected cells. Deletion of 285L or K145R from the genome of a virulent ASFV strain from Armenia did not significantly affect spread and productive growth in a permissive wild boar lung cell line, nor in primary macrophage cultures. Future studies must elucidate, whether p285L and pK145R, although non-essential for in vitro propagation of ASFV, are relevant for replication or virulence in swine. Furthermore, it remains to be investigated whether deletion of the abundant ASFV proteins p285L or pK145R might support serological differentiation from wild-type-infected animals.

Acute glycemic and pressure responses of continuous and interval aerobic exercise in patients with type 2 diabetes.

BACKGROUND: Aerobic training has been widely indicated to patients with type 2 diabetes. However, there are still few studies comparing acute glycemic and blood pressure effects of different methods of aerobic training. The aim is to compare glycemic and pressure acute responses of continuous aerobic exercise to interval aerobic exercise in patients with type 2 diabetes. MATERIALS AND METHODS: This study is a randomized, crossover clinical trial. Fourteen patients with type 2 diabetes performed two sessions of aerobic training with different methods (continuous and interval). Continuous session had duration of 35 minutes with intensity of 85-90% of heart rate corresponding to anaerobic threshold (HRAT), while interval session had 45 minutes, with stimulus in intensity of 85-90% of HRAT with recovery in intensity under 85% of HRAT. Capillary glycemia, systolic and diastolic blood pressure were analyzed before and after the sessions. RESULTS: Patients were 63.5 ± 9.8 years old. Glycemia was reduced in both sessions (p < 0.001). Only glycemia measured at 25 minutes after continuous session was not lower than pre-session values. Systolic blood pressure was also reduced in both sessions (p = 0.010) with similar behavior between them. In the diastolic blood pressure, there were differences only between the values measured immediately after exercise and the values measured 20 minutes (p = 0.002) and 30 minutes after exercise (p = 0.008). CONCLUSION: Both continuous and interval aerobic exercise, in a same intensity, are effective for glycemic and pressure acute reductions in individuals with type 2 diabetes. For patients with greater risk of hypertension, we believe that the interval method is safer.

Potential use of bixin beyond function as a natural colourant in food and cosmetics.

Bixin is a carotenoid found in the covering of Bixa orellana seeds and widely used as a dye, mainly in food products and cosmetics. This compound is insoluble in water, but technologies have been developed for its use in aqueous preparations, as well as a component for new formulations to disperse other lipophilic ingredients. It has recently aroused the interest of researchers in areas such as Pharmacology, Endocrinology and Oncology, but also new applications in Food Technology. This work aimed to review the main studies in the period from 1911 to 2023, but we emphasised aspects in technologies related to the field of health such as Pharmacology and Pharmacy. We used 'bixin' as a keyword to search for articles on the Web of Science and obtained 488 results, most of which were original articles and 20 were reviews. The analysis demonstrated a continuing large number of studies in Food Technology, but a rapid growth in areas related to health aspects.

Safety and Photoprotective Efficacy of a Sunscreen System Based on Grape Pomace (Vitis vinifera L.) Phenolics from Winemaking.

In winemaking, a large amount of grape pomace is produced that is rich in polyphenolics and highly beneficial for human health, as phenols are useful for skin ultraviolet (UV) protection. In this investigation, we evaluated the safety and clinical efficacy of a sunscreen system containing a grape pomace extract from Vitis vinifera L. as a bioactive ingredient. The recovery of phenolics in the waste was performed by percolation. Nine emulsions were developed using a factorial design and two were evaluated clinically: Formulation E, containing only UV filters (butylmethoxydibenzoyl methane, ethylhexyl methoxycinnamate and ethylhexyl dimethyl PABA), and F, with the extract at 10.0% w/w + UV filters. The antioxidant activity was determined by the DPPH assay and the in vitro efficacy was established by sun protection factor (SPF) measurements (Labsphere UV-2000S). Clinical tests were performed to determine safety (human repeated insult patch test) and to confirm efficacy (photoprotective effectiveness in participants). The results showed a synergistic effect between the sunscreen system and the extract on UVB protection and antioxidant activity. Both samples were considered safe. Formulation F was 20.59% more efficient in protecting skin against UVB radiation, taking approximately 21% more time to induce erythema compared to the extract-free sample.

Detection of Two Highly Diverse Peribunyaviruses in Mosquitoes from Palenque, Mexico.

The Peribunyaviridae family contains the genera Orthobunyavirus, Herbevirus, Pacuvirus, and Shangavirus. Orthobunyaviruses and pacuviruses are mainly transmitted by blood-feeding insects and infect a variety of vertebrates whereas herbeviruses and shangaviruses have a host range restricted to insects. Here, we tested mosquitoes from a tropical rainforest in Mexico for infections with peribunyaviruses. We identified and characterized two previously unknown viruses, designated Baakal virus (BKAV) and Lakamha virus (LAKV). Sequencing and de novo assembly of the entire BKAV and LAKV genomes revealed that BKAV is an orthobunyavirus and LAKV is likely to belong to a new genus. LAKV was almost equidistant to the established peribunyavirus genera and branched as a deep rooting solitary lineage basal to herbeviruses. Virus isolation attempts of LAKV failed. BKAV is most closely related to the bird-associated orthobunyaviruses Koongol virus and Gamboa virus. BKAV was successfully isolated in mosquito cells but did not replicate in common mammalian cells from various species and organs. Also cells derived from chicken were not susceptible. Interestingly, BKAV can infect cells derived from a duck species that is endemic in the region where the BKAV-positive mosquito was collected. These results suggest a narrow host specificity and maintenance in a mosquito-bird transmission cycle.

The Synergistic Behavior of Antioxidant Phenolic Compounds Obtained from Winemaking Waste's Valorization, Increased the Efficacy of a Sunscreen System.

Grape pomace retains polyphenols in the peels and in the seeds after winemaking, which is indicative of the high valorization potential of this industrial waste. There is strong evidence that phenolics are robust antioxidants and confer photoprotection; thus, it is rational to apply these active compounds from winemaking waste to sunscreens, in order to increase UV protection. Despite the importance of this class of cosmetics to public health, more efficacious strategies are still needed to overcome the problems caused by the photoinstability of some UV filters. The hydroethanolic extract of Vitis vinifera L. grapes was obtained by percolation and then lyophilized. Six formulations were developed: Type I-cosmetic base and UV filters; Type II-cosmetic base and extract; and Type III-cosmetic base, extract and UV filters. Each formulation was prepared in the pHs 5 and 7. The antioxidant activities of the samples were measured by DPPH• and expressed in Trolox® equivalents (TE), and their photostability and in vitro sun protection factor (SPF) were analyzed by diffuse reflectance spectrophotometry. The anti-radical efficiencies observed in the formulations with grape extract were: (II) 590.12 ± 0.01 µmol TE g-1 at pH 5 and 424.51 ± 0.32 µmol TE g-1 at pH 7; (III) 550.88 ± 0.00 µmol TE g-1 at pH 5 and 429.66 ± 0.10 µmol TE g-1, at pH 7, demonstrating that the UV filters, butylmethoxydibenzoyl methane, ethylhexyl methoxycinnamate and ethylhexyl dimethyl 4-aminobenzoic acid had no influence on this effect. The photoprotective efficacy and the photostability of formulation III containing the extract and UV filters at pH 5 suggested that a synergism between the active molecules provided an 81% increase in SPF. Additionally, this was the only sample that maintained a broad spectrum of protection after irradiation. These results confirmed that the grape pomace extract has multifunctional potential for cosmetic use, mainly in sunscreens, granting them superior performance.

A Deep-Sequencing Workflow for the Fast and Efficient Generation of High-Quality African Swine Fever Virus Whole-Genome Sequences.

African swine fever (ASF) is a severe disease of suids caused by African swine fever virus (ASFV). Its dsDNA genome (170-194 kbp) is scattered with homopolymers and repeats as well as inverted-terminal-repeats (ITR), which hamper whole-genome sequencing. To date, only a few genome sequences have been published and only for some are data on sequence quality available enabling in-depth investigations. Especially in Europe and Asia, where ASFV has continuously spread since its introduction into Georgia in 2007, a very low genetic variability of the circulating ASFV-strains was reported. Therefore, only whole-genome sequences can serve as a basis for detailed virus comparisons. Here, we report an effective workflow, combining target enrichment, Illumina and Nanopore sequencing for ASFV whole-genome sequencing. Following this approach, we generated an improved high-quality ASFV Georgia 2007/1 whole-genome sequence leading to the correction of 71 sequencing errors and the addition of 956 and 231 bp at the respective ITRs. This genome, derived from the primary outbreak in 2007, can now serve as a reference for future whole-genome analyses of related ASFV strains and molecular approaches. Using both workflow and the reference genome, we generated the first ASFV-whole-genome sequence from Moldova, expanding the sequence knowledge from Eastern Europe.

Efficient transgene insertion in a pseudorabies virus vector by CRISPR/Cas9 and marker rescue-enforced recombination.

For development of vectored vaccines against porcine pathogens the genome of the pseudorabies virus vaccine strain Bartha (PrV-Ba) was previously cloned as an infectious bacterial artificial chromosome (BAC), containing the bacterial replicon and a reporter gene cassette encoding enhanced green fluorescent protein (EGFP) at the nonessential glycoprotein G locus. To facilitate substitution of this insertion, this BAC was now modified by deletion of the adjacent promoter and initiation codon of the essential glycoprotein D (gD) gene of PrV-Ba. Furthermore, rabbit kidney (RK13) cells stably expressing Cas9 nuclease and an EGFP gene-specific guide RNA were prepared to induce site specific cleavage of the BAC DNA. After co-transfection of these cells with the modified BAC and recombination plasmids containing expression cassettes for new transgenes flanked by PrV DNA sequences including the intact 5'-end of the gD gene, >95% of the recombinants exhibited the desired gene substitutions, while no EGFP-expressing progeny virus was detectable. This approach was used for insertion and expression of the open reading frames E199L, CP204L (p30) and KP177R (p22) of African swine fever virus. The studies revealed that codon adaptation significantly enhanced expression of E199L, and that the chimeric CAG promoter increased transgene expression compared to cytomegalovirus immediate-early promoters.

Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L).

African swine fever is a devastating viral disease of domestic and wild pigs against which no vaccine or therapy is available. Therefore, we applied the CRISPR (clustered regularly interspaced short palindromic repeats) - Cas9 nuclease system to target the double-stranded DNA genome of African swine fever virus (ASFV). To this end, a permissive wild boar lung (WSL) cell line was modified by stable transfection with a plasmid encoding Cas9 and a guide RNA targeting codons 71 to 78 of the phosphoprotein p30 gene (CP204L) of ASFV. Due to targeted Cas9 cleavage of the virus genome, plaque formation of ASFV was completely abrogated and virus yields were reduced by four orders of magnitude. The specificity of these effects could be demonstrated by using a natural ASFV isolate and escape mutants possessing nucleotide exchanges within the target sequence, which were not inhibited in the Cas9-expressing cell line. Growth of the cell line was not affected by transgene expression which, as well as virus inhibition, proved to be stable over at least 50 passages. Thus, CRISPR-Cas9 mediated targeting of the ASFV p30 gene is a valid strategy to convey resistance against ASF infection, which may also be applied in its natural animal host.

Acute and chronic glycemic effects of aerobic training in patients with type 2 diabetes / Efeitos glicêmicos agudos e crônicos do treinamento aeróbio em pacientes com diabetes tipo 2

The aim of the present study was to analyze acute glycemic effects in different moments of an aerobic training, as well as to analyze the chronic effect of training, in patients with type 2 diabetes mellitus (T2D). The participants performed 16 weeks of interval aerobic training with three weekly sessions. The main part of each session consisted of nine blocks of five minutes, in which four minutes consist-ed of stimulus between 85% and 95% of the anaerobic threshold heart rate (ATHR) and one minute consisted of recovery below 85% of the ATHR, totalizing 45 minutes. Capillary glucose was assessed before, immediately after and 30 minutes after the first and the last training sessions. Glycated hemoglobin (HbA1c) was assessed before and after the intervention. Paired t-test and Generalized Estimating Equations were performed for the analyses; α = 5%. The participants were seven individ-uals (four women) aged 59.60 ± 6.69 years. In the first session, glucose values immediately after and 30 minutes after exercise were lower than pre-exercise values. On the other hand, in the last training session, only the glucose values immediately after exercise were lower than pre-exercise values. Ana-lyzing the glycemic reductions, the first session presented a greater reduction immediately after (p = 0.042) and 30 minutes after exercise (p = 0.010). Regarding chronic glycemic effects, an increase (p = 0.010) in HbA1c levels was observed after training. It is concluded that, after 16 weeks of training without progression of duration and intensity, the exercise loses its acute glycemic effect, and may be even insufficient to reduce HbA1c levels

O objetivo do estudo foi analisar os efeitos glicêmicos agudos em diferentes momentos de um treinamento aeróbio, bem como o efeito glicêmico crônico deste treinamento, em pacientes com diabetes tipo 2 (DM2). Os participantes realizaram16 semanas de treinamento aeróbio intervalado, com três sessões semanais, sendo a parte principal de cada sessão composta de nove blocos de cinco minutos, tendo cada bloco quatro minutos de estímulo a 85% a 90% da frequência cardíaca referente ao limiar anaeróbio (FCLAN) e um minuto de recu-peração abaixo de 85% da FCLAN, totalizando 45 minutos. A glicemia capilar foi avaliada antes, imediata-mente e 30 minutos após a primeira e a última sessão de treinamento. A hemoglobina glicada (HbA1c) foi avaliada antes e após a intervenção. Teste t pareado e Equações de Estimativas Generalizadas foram usados para as análises; α = 5%. Participaram sete indivíduos (59,60 ± 6,69 anos; quatro mulheres). Na primeira sessão, os valores glicêmicos imediatamente e 30 minutos após o exercício foram menores que os valores pré--exercício. Já na última sessão de treinamento, somente os valores glicêmicos imediatamente após o exercício foram menores que os valores pré-exercício. Analisando as reduções glicêmicas, a primeira sessão apresentou maior redução tanto imediatamente após (p = 0,042) como 30 minutos após o exercício (p = 0,027). Em relação ao efeito glicêmico crônico, observou-se aumento (p = 0,010) dos níveis de HbA1c após o treinamento. Conclui-se que após 16 semanas de treinamento sem progressão de duração e intensidade, o exercício perde efeito glicêmico agudo, podendo inclusive ser ineficiente na redução dos níveis de HbA1c

Hydrolyzed collagen interferes with in vitro photoprotective effectiveness of sunscreens

ABSTRACT The chronological skin aging is a progressive and natural process with genetic and physiological changes. However, ultraviolet (UV) radiation may accelerate the oxidative stress, generating carcinogenesis and photoaging. Natural compounds and their applications are considered a trend in the cosmetic market. The protein-based film-forming compounds play an important role, once it collaborates for the better distribution of sunscreens on the skin. Here we investigated the in vitro photoprotective effectiveness of sunscreens containing the hydrolyzed collagen associated with UVA, UVB and/or inorganic filters. Sunscreens were developed with octocrylene (7.5%), butyl methoxydibenzoylmethane (avobenzone) (3.0%) and/or titanium dioxide (5.0%), associated or not with the hydrolyzed collagen (3.0%). In vitro photoprotective effectiveness was determined in a Labsphere(r) UV2000S by the establishment of the sun protection factor (SPF) and critical wavelength (nm) values. Physicochemical and organoleptic characteristics were also assayed. The hydrolyzed collagen subjectively improved the formulation sensory characteristics. However, this bioactive compound led to a decrease of the SPF values of the photoprotective formulations containing octocrylene alone and octocrylene + butyl methoxydibenzoylmethane + TiO2. This inadequate interaction may be considered during the development of new sunscreens intended to contain protein-based components.