Genetic and morphological identification of filarial worm from Iberian hare in Portugal.

The Iberian hare (Lepus granatensis) is an endemic species of the Iberian Peninsula and the only hare species found in Portugal, although also being present in some areas of Spain. The reduction of wild hare populations due to several ecological and sanitary factors, has been raising growing concerns in the recent years. Despite different helminth species were already described in Iberian hares in Portugal, to this date, no filarial worms have been identified in this species. Furthermore, only a few studies on lagomorphs' onchocercid worms are available, referring to other hosts species of hares and/or rabbits. In this study, we describe the presence of filarial worms in the blood vessels of two adult Iberian hares collected in 2019 in continental Portugal. Morphology and sequencing data from the 12S rRNA, coxI, 18S rRNA, myoHC, hsp70 and rbp1 genes, showed that the filaroid species were genetically related with Micipsella numidica. However, the extension of the genetic differences found with M. numidica suggests that the filaroids specimens under study belong to a new species, that we provisionally named Micipsella iberica n. sp.. The body location of this putative new parasite species and its physiological implications indicate that it may constitute a potential menace to the already fragile Iberian hare justifying, therefore, further investigation regarding the morphological characterization, prevalence and real clinical impact of this new parasite in hares.

Acquired bedaquiline resistance in Karakalpakstan, Uzbekistan.

BACKGROUND: The WHO recommends the use of bedaquiline (BDQ) in longer, as well as shorter, multidrug-resistant TB (MDR-TB) treatment regimens. However, resistance to this new drug is now emerging. We aimed to describe the characteristics of patients in Karakalpakstan, Uzbekistan, who were treated for MDR-TB and acquired BDQ resistance during treatment.METHODS: We performed a retrospective study of routinely collected data for patients treated for MDR-TB in Karakalpakstan between January 2015 and December 2020. We included patients on BDQ-containing regimens with baseline susceptibility to BDQ who developed BDQ resistance at any point after treatment initiation. Patients resistant to BDQ at baseline or with no confirmed susceptibility to BDQ at baseline were excluded.RESULTS: Of the 523 patients who received BDQ-containing regimens during the study period, BDQ resistance was detected in 31 patients (5.9%); 20 patients were excluded-16 with no prior confirmation of BDQ susceptibility and 4 who were resistant at baseline. Eleven patients with acquired BDQ resistance were identified. We discuss demographic variables, resistance profiles, treatment-related variables and risk factors for unfavourable outcomes for these patients.CONCLUSION: Our programmatic data demonstrated the acquisition of BDQ resistance during or subsequent to receiving a BDQ-containing regimen in a patient cohort from Uzbekistan. We highlight the need for individualised treatment regimens with optimised clinical and laboratory follow up to prevent resistance acquisition.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize / Índices zootécnicos e digestibilidade em juvenis de tambaqui Colossoma macropomum alimentados com dieta contendo milho particulado

Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 μm) had better results digestibility. Smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 μm of corn be used for tambaqui.

Três experimentos foram realizados com diferentes tamanhos de partícula de ração para o desempenho zootécnico, taxa de aprovação e digestibilidade aparente de tambaqui juvenil (Colossoma macropomum). No primeiro, foram utilizados 200 juvenis e distribuídos em 20 tanques (220 L), 10 peixes por unidade (quatro repetições). O sistema experimental utilizado para registrar o tempo de passagem foi composto por cinco incubadoras com 200 L. No segundo, 75 juvenis foram utilizados por 55 dias (três repetições). No terceiro, 75 juvenis foram utilizados e distribuídos em cinco incubadoras de 200 L. Todos os experimentos foram realizados aleatoriamente. Diferentes tratamentos de comportamento linear foram observados para digestibilidade aparente da proteína bruta; e o menor tamanho de partícula (150 μm) apresentou melhor digestibilidade dos resultados. menor tamanho de partícula do milho apresentou melhores resultados, afetou o desempenho de crescimento de tambaquis e a digestibilidade aparente da proteína bruta e extrato etéreo. Portanto, recomenda-se o uso de um tamanho de partícula de 150 μm de milho para o tambaqui.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize

Abstract Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 m) had better results digestibility. smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 m of corn be used for tambaqui.

Resumo Três experimentos foram realizados com diferentes tamanhos de partícula de ração para o desempenho zootécnico, taxa de aprovação e digestibilidade aparente de tambaqui juvenil (Colossoma macropomum). No primeiro, foram utilizados 200 juvenis e distribuídos em 20 tanques (220 L), 10 peixes por unidade (quatro repetições). O sistema experimental utilizado para registrar o tempo de passagem foi composto por cinco incubadoras com 200 L. No segundo, 75 juvenis foram utilizados por 55 dias (três repetições). No terceiro, 75 juvenis foram utilizados e distribuídos em cinco incubadoras de 200 L. Todos os experimentos foram realizados aleatoriamente. Diferentes tratamentos de comportamento linear foram observados para digestibilidade aparente da proteína bruta; e o menor tamanho de partícula (150 m) apresentou melhor digestibilidade dos resultados. menor tamanho de partícula do milho apresentou melhores resultados, afetou o desempenho de crescimento de tambaquis e a digestibilidade aparente da proteína bruta e extrato etéreo. Portanto, recomenda-se o uso de um tamanho de partícula de 150 m de milho para o tambaqui.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize / Índices zootécnicos e digestibilidade em juvenis de tambaqui Colossoma macropomum alimentados com dieta contendo milho particulado

Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 µm) had better results digestibility. smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 µm of corn be used for tambaqui.

Três experimentos foram realizados com diferentes tamanhos de partícula de ração para o desempenho zootécnico, taxa de aprovação e digestibilidade aparente de tambaqui juvenil (Colossoma macropomum). No primeiro, foram utilizados 200 juvenis e distribuídos em 20 tanques (220 L), 10 peixes por unidade (quatro repetições). O sistema experimental utilizado para registrar o tempo de passagem foi composto por cinco incubadoras com 200 L. No segundo, 75 juvenis foram utilizados por 55 dias (três repetições). No terceiro, 75 juvenis foram utilizados e distribuídos em cinco incubadoras de 200 L. Todos os experimentos foram realizados aleatoriamente. Diferentes tratamentos de comportamento linear foram observados para digestibilidade aparente da proteína bruta; e o menor tamanho de partícula (150 µm) apresentou melhor digestibilidade dos resultados. menor tamanho de partícula do milho apresentou melhores resultados, afetou o desempenho de crescimento de tambaquis e a digestibilidade aparente da proteína bruta e extrato etéreo. Portanto, recomenda-se o uso de um tamanho de partícula de 150 µm de milho para o tambaqui.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize.

Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 µm) had better results digestibility. smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 µm of corn be used for tambaqui.

Antibacterial activity of Barbatimão (Stryphnodendron adstringens) against Staphylococcus aureus: in vitro and in silico studies.

We evaluated the activity of the aqueous fraction and the ethyl acetate fraction of Stryphnodendron adstringens against Staphylococcus aureus and proposed their mechanism of action. The antibacterial activity of S. adstringens fractions was evaluated against S. aureus and the cell targets were rated by docking. The fractions showed moderate antibacterial activity against S. aureus without toxicity on two mammalian cell lines. They also showed synergistic antibacterial activity with tannic acid (TA). In silico assays indicated FabG, FabZ and FabI as probable targets. The metabolic pathway for fatty acid biosynthesis in S. aureus was affected by components of S. adstringens. The synergistic effect when combining TA with S. adstringens fractions suggests a natural alternative to S. aureus control. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing the possible targets of action of Stryphnodendron adstringens on Staphylococcus aureus. Molecular dynamics simulations showed that the components of S. adstringens affected the metabolic pathway for fatty acid biosynthesis (FAS II) in S. aureus, inhibiting the FabI, FabG and FabZ enzymes. As tannic acid (TA) is a known inhibitor of some targets identified, we showed synergistic antibacterial activity of S. adstringens in combination with TA. This combination did not show toxicity against HaCaT and Vero cells and based on all these results we suggest that S. adstringens can be a natural and sustainable alternative to S. aureus control.

First description of a herpesvirus infection in genus Lepus.

During the necropsies of Iberian hares obtained in 2018/2019, along with signs of the nodular form of myxomatosis, other unexpected external lesions were also observed. Histopathology revealed nuclear inclusion bodies in stromal cells suggesting the additional presence of a nuclear replicating virus. Transmission electron microscopy further demonstrated the presence of herpesvirus particles in the tissues of affected hares. We confirmed the presence of herpesvirus in 13 MYXV-positive hares by PCR and sequencing analysis. Herpesvirus-DNA was also detected in seven healthy hares, suggesting its asymptomatic circulation. Phylogenetic analysis based on concatenated partial sequences of DNA polymerase gene and glycoprotein B gene enabled greater resolution than analysing the sequences individually. The hare' virus was classified close to herpesviruses from rodents within the Rhadinovirus genus of the gammaherpesvirus subfamily. We propose to name this new virus Leporid gammaherpesvirus 5 (LeHV-5), according to the International Committee on Taxonomy of Viruses standards. The impact of herpesvirus infection on the reproduction and mortality of the Iberian hare is yet unknown but may aggravate the decline of wild populations caused by the recently emerged natural recombinant myxoma virus.

In vitro and in vivo activity of a possible novel antifungal small molecule against Candida albicans.

Nosocomial infections by fungi are important causes of morbidity and mortality, and the adhesion capacity of yeast on abiotic and biotic surfaces has been considered an important step in this process. Als3 proteins are widely studied for their ability to allow Candida albicans to bind to various surfaces. The objective of the present study was to verify, with more details, the action of F2768-0318 in relation to its antifungal activity as well as its ability to act on C. albicans virulence factors related to adhesion and biofilm formation in vitro and in vivo by inhibiting the Als3 protein. F2768-0318 was assessed in tests of biofilm formation and adhesion on abiotic surfaces (polystyrene plates) and adherence on biotic surfaces, including human endocervical (HeLa) cells, human umbilical vein endothelial cells (HUVECs), and fresh buccal epithelial cells (BEC). Our results showed F2768-0318 was useful in reducing the adhesion and biofilm formation of C. albicans on abiotic surfaces, indicating the possibility of treating hospital materials and preventing biofilm formation on these types of equipment. Further studies are still needed, including optimization of the molecule to allow this molecule to be effective on other types of surfaces, such as human cells.

GASMoC method: a phenol-free technique to detect acid-fast bacilli.

The genre Mycobacterium includes a series of pathological species, such as M. tuberculosis, which is important for pathology laboratories to detect for a correct diagnosis. The Ziehl-Neelsen technique (ZNT) is the most commonly histochemical method used to detect these bacilli and uses a heated mixture of carbol-fuchsine, which contains basic fuchsine and phenol. Whereas the former component is responsible for the pinkish staining of acid-fast mycobacteria, the role of phenol is not completely understood and it has been suggested that its exclusion does not impact the detection ability of the ZNT. Since phenol is highly toxic and induces several injuries, the goal of this study is to determine the detection capacity of mycobacteria through a method that excludes the use of phenol. Accordingly, the GASMoC method, a modified ZNT that employs a solution of aqueous basic fuchsine heated at 37 °C, was tested on histological samples positive for mycobacteria and the results were compared to that of the ZNT. Data demonstrated that the GASMoC method was able to detect acid-fast bacilli (AFB) in all analyzed cases. Remarkably, microscopic inspection of mycobacteria was easier when the GASMoC method was applied. In conclusion, our study demonstrates that AFB detection does not require phenol and that the GASMoC method, a phenol-free technique, may substitute the ZNT in pathology laboratories.

Preparation of dissociated mouse primary neuronal cultures from long-term cryopreserved brain tissue.

BACKGROUND: Dissociated primary neuronal cultures are widely used as a model system to investigate the cellular and molecular properties of diverse neuronal populations and mechanisms of action potential generation and synaptic transmission. Typically, rodent primary neuronal cultures are obtained from freshly-dissociated embryonic or postnatal brain tissue, which often requires intense animal husbandry. This can strain resources when working with genetically modified mice. NEW METHOD: Here we describe an experimental protocol for frozen storage of mouse hippocampi, which allows fully functional dissociated primary neuronal cultures to be prepared from cryopreserved tissue. RESULTS: We show that thawed hippocampal neurons have functional properties similar to those of freshly dissociated neurons, including neuronal morphology, excitability, action potential waveform and synaptic neurotransmitter release, even after cryopreservation for several years. COMPARISON TO THE EXISTING METHODS: In contrast to the existing methods, the protocol described here allows for efficient long-term storage of samples, allowing researchers to perform functional experiments on neuronal cultures from brain tissue collected in other laboratories. CONCLUSIONS: We anticipate that this method will facilitate collaborations among laboratories based at distant locations and will thus optimise the use of genetically modified mouse models, in line with the 3Rs (Replacement, Reduction and Refinement) recommended for scientific use of animals in research.

FATAL cryptococcal meningitis in a child with hyper-immunoglobulin M syndrome, with an emphasis on the agent.

Following a fatal case of Cryptococcus neoformans meningitis in a child with X-linked hyper-immunoglobulin M syndrome (XHIGM), we evaluated the fungal isolate in an experimental infection in a mouse model with respect to microbiology, epidemiology, virulence and response to therapy. The minimum inhibitory concentrations for antifungals in the susceptibility test were 0.5mg/L for amphotericin B, 4.0mg/L for fluconazole and 0.12mg/L for voriconazole. Evaluation of pathogenicity by means of an experimental infection in BALB/c mice showed that fungus isolated from the blood and cerebrospinal fluid of the child was able to disseminate, reaching the spleen, lungs and brain, where it caused significant macroscopic alterations in the size and texture of each organ. Treatment of infected mice with amphotericin B reduced the fungal load in the spleen and lungs, but not in the brain.

A new small-molecule KRE2 inhibitor against invasive Candida parapsilosis infection.

AIM: To investigate the antifungal activity of MOL3, a small molecule that was selected by virtual screening, against Candida spp. MATERIALS & METHODS: The antifungal activity of MOL3 was evaluated using standard strains and clinical isolates. Activity was evaluated in both in vitro tests and animal models. RESULTS: The minimum fungicidal concentration of MOL3 against Candida spp. ranged from 16 to 128 mg/l. MOL3 at the sub-minimum fungicidal concentration inhibited hyphal elongation. The remaining yeast cells presented morphological changes and were metabolically inactive. MOL3 was toxicologically inert both in vitro and in the animal model. MOL3 also reduced experimental systemic infection by C. parapsilosis in mice. CONCLUSION: The selection of MOL3 by virtual screening was successful, revealing a promising antifungal candidate.

Metionina+cistina digestível para juvenis de tambaqui / Digestible methionine+cystine for tambaqui juveniles

Avaliou-se o efeito dos níveis de metionina+cistina digestível para juvenis de tambaqui na fase inicial de desenvolvimento (3,03±0,06g) durante 45 dias. Foram utilizados 160 juvenis de tambaqui distribuídos em DIC, com cinco tratamentos (níveis de metionina+cistina digestível = 0,66; 0,73; 0,80; 0,87; 0,94%), quatro repetições e oito animais por unidade experimental. As dietas utilizadas foram isoproteicas (25%) e isoenergéticas (3200kcal.kg-1). Os resultados obtidos foram submetidos aos testes de homogeneidade, normalidade, à análise de variância e à análise de regressão. Os parâmetros físicos e químicos da água do sistema de recirculação ficaram dentro da faixa de tolerância para a espécie. Os níveis de metionina+cistina digestíveis avaliados não influenciaram (P>0,05) a sobrevivência, o índice hepatossomático, o índice de gordura visceral e o consumo de ração. Entretanto, ganho de peso e taxa de eficiência proteica apresentaram aumento linear (P<0,05) com aumento da metionina + cistina na ração. Verificou-se que os níveis de metionina + cistina para o tambaqui não foram completamente atendidos, sendo recomendada a realização de novos estudos.(AU)

This study aimed to determine digestible methionine+cystine requirement of tambaqui juveniles in the early stage of development (3.03 ± 0.06 g) lasting 45 days. 160 tambaqui juveniles were distributed into DIC, with five treatments (methionine + cystine = 0.66; 0.73; 0.80; 0.87; 0.94%), four replications and eight animals per experimental unit. The basal diets were isonitrogenous (25% crude protein) and isocaloric (3200kcal.kg-1). The results were submitted to the homogeneity test, normality test, analysis of variance, and regression analysis. The physical and chemical parameters of water from the water recirculation system were within those recommended for the species. Digestible methionine+cystine levels did not influence (P>0.05) the survival, hepatosomatic index, visceral fat index and feed intake. However, weight gain and protein efficiency ratio exhibit a linear increase (P<0.05) with increase in methionine and cystine in the feed. Our results showed that the methionine + cystine levels for tambaqui were not fully met, and new studies are recommended.(AU)

Tongue coating frequency and its colonization by yeasts in chronic kidney disease patients.

The objective of this study was to investigate tongue coating (TC) frequency and its colonization by yeasts in a group of chronic kidney disease (CKD) patients. Clinical examination of the oral mucosa of 33 CKD patients was performed to investigate oral and tongue lesions. TC was diagnosed according to its clinical characteristics. Stimulated saliva and TC samples were collected to verify the salivary flow, and determine yeast frequency, species and counts. TC was found in 18/33 of the patients (54.55 %) and was the most frequent oral lesion found. Of 18 patients with TC, 13 (72.22 %) presented positive cultures for yeasts on the tongue dorsum, and one (5.55 %) in the saliva only. Yeasts were significantly more frequent in the tongue dorsum when compared to the saliva (p = 0.0106). The most frequent yeast species found was C. albicans (55.55 %), while C. parapsilosis comprised 50 % of non-albicans Candida species. This study demonstrated high amounts of yeasts on the cultures from TC samples of CKD patients, strongly suggesting that TC is a clinical representation of a polymicrobial biofilm, which could serve as a gateway for disseminated infection in immunosuppressed patients undergoing frequent hospitalization.

Spermatogenesis and nucleolar behavior in Triatoma vandae and Triatoma williami (Hemiptera, Triatominae).

This study describes spermatogenesis in Triatoma vandae and the nucleolar behavior of T. vandae and Triatoma williami, with a cytotaxonomic focus. Analysis of mitotic and meiotic metaphases of T.vandae confirms the species karyotype. T. vandae presents some characteristics during meiosis that differentiate it from T. williami, including the presence of a chromocenter with two sex chromosomes individualized during early prophase, and the presence of a bi- or tripartite corpuscle inpolyploid nuclei. It was possible to observe the compaction of chromatin during prophase resulting in holocentric chromosomes. During metaphase,the autosomes presented a ring shape and the sex chromosomes were in the center of the ring. These chromosomes were separated in anaphase. Although it is common, we did not observe the phenomenon of late migration of the sex chromosomes. By means of silver ion impregnation it was possible to describe nucleologenesis in T. vandae and T. williami. In both species we observed persistence of the nucleolar material duringmeiosis. In addition to the cells in meiotic division, we also observed the presence of polyploid nuclei in the seminiferous tubule walls that nourish the cells during cell division. The nucleolar markings reflect their capacity for synthetic activity. T. vandae and T. williami presented only one nucleolar corpuscle, which reflects low synthetic activity. This study confirms the karyotype of T. vandae, describes characteristics that differentiate T. vandae and T. williami during meiosis, and describes the phenomenon of nucleolar persistence in both species.

Flow cytometry reliability analysis and variations in sugarcane DNA content.

The aim of this study was to evaluate the reliability of flow cytometry analysis and the use of this technique to differentiate species and varieties of sugarcane (Saccharum spp) according to their relative DNA content. We analyzed 16 varieties and three species belonging to this genus. To determine a reliable protocol, we evaluated three extraction buffers (LB01, Marie, and Tris·MgCl2), the presence and absence of RNase, six doses of propidium iodide (10, 15, 20, 25, and 30 µg), four periods of exposure to propidium iodide (0, 5, 10, and 20 min), and seven external reference standards (peas, beans, corn, radish, rye, soybean, and tomato) with reference to the coefficient of variation and the DNA content. For statistical analyses, we used the programs Sisvar(®) and Xlstat(®). We recommend using the Marie extraction buffer and at least 15 µg propidium iodide. The samples should not be analyzed immediately after the addition of propidium iodide. The use of RNase is optional, and tomato should be used as an external reference standard. The results show that sugarcane has a variable genome size (8.42 to 12.12 pg/2C) and the individuals analyzed could be separated into four groups according to their DNA content with relative equality in the genome sizes of the commercial varieties.

Optical spectrum of bottom-up graphene nanoribbons: towards efficient atom-thick excitonic solar cells.

Recently, atomically well-defined cove-shaped graphene nanoribbons have been obtained using bottom-up synthesis. These nanoribbons have an optical gap in the visible range of the spectrum which make them candidates for donor materials in photovoltaic devices. From the atomistic point of view, their electronic and optical properties are not clearly understood. Therefore, in this work we carry out ab-initio density functional theory calculations combine with many-body perturbation formalism to study their electronic and optical properties. Through the comparison with experimental measurements, we show that an accurate description of the nanoribbon's optical properties requires the inclusion of electron-hole correlation effects. The energy, binding energy and the corresponding excitonic transitions involved are analyzed. We found that in contrast to zigzag graphene nanoribbons, the excitonic peaks in the absorption spectrum are a consequence of a group of transitions involving the first and second conduction and valence bands. Finally, we estimate some relevant optical properties that strengthen the potential of these nanoribbons for acting as a donor materials in photovoltaic.