Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae)

The family Nematotaeniidae, tapeworms commonly found in the small intestines of amphibians and reptiles, includes 27 recognised species distributed among four genera: Bitegmen Jones, Cylindrotaenia Jewell, Distoichometra Dickey and Nematotaenia Lühe. The taxonomy of these cestodes is poorly defined, due in part to the difficulties of observing many anatomical traits. This study presents and describes a new genus and species of nematotaeniid parasite found in cane toads (Rhinella marina) from eastern Brazilian Amazonia. The cestodes were collected during the necropsy of 20 hosts captured in the urban area of Belém, Pará. The specimens were fixed and processed for light microscopy, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. Samples were also collected for molecular analyses. The specimens presented a cylindrical body, two testes and paruterine organs. However, they could not be allocated to any of the four existing nematotaeniid genera due to the presence of two each of dorsal compact medullary testes, cirri, cirrus pouches, genital pores, ovaries and vitelline glands per mature segment. Lanfrediella amphicirrus gen. nov. sp. nov. is the first nematotaeniid studied using Historesin analysis, SEM and 3D reconstruction, and it is the second taxon for which molecular data have been deposited in GenBank.     

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.     

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.     

PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.     

Enhanced programmed death 1 (PD-1) and PD-1 ligand (PD-L1) expression in patients with actinic cheilitis and oral squamous cell carcinoma

PD-1 and PD-L1 can be involved in tumor escape, and little is known about the role of these molecules in oral tumors or pre-malignant lesions. In the present study, we investigated the expression of PD-1 and PD-L1 in the blood and lesion samples of patients with actinic cheilitis (AC) and oral squamous cell carcinoma (OSCC). Our results showed that lymphocytes from peripheral blood and tissue samples exhibited high expression of PD-1 in both groups analyzed. Patients with AC presented higher percentage as well as the absolute numbers of CD4⁺PD-1⁺ and CD8⁺PD-1⁺ lymphocytes in peripheral blood mononuclear cells (PBMC) than healthy individuals, while patients with OSCC presented an increased frequency of CD8⁺PD1⁺ in PBMC when compared with controls. On the other hand, increased frequency of CD4⁺ and CD8⁺ T cells expressing PD-1⁺ accumulate in samples from OSCC, and the expression of PD-L1 was intense in OSCC and moderate in AC lesion sites. Lower levels of IFN-γ and higher levels of TGF-β were detected in OSCC samples. Our data demonstrate that PD-1 and PD-L1 molecules are present in blood and samples of AC and OSCC patients. Further studies are required to understand the significance of PD-1 and PD-L1 in oral tumors microenvironment.     

Pregnancy rate of Nelore females inseminated with male-sexed semen

Pregnancy rates of Nelore females inseminated with male-sexed semen and conventional semen from the same bulls were evaluated. The females included 433 heifers (2 years old) and 230 non-suckling cows, totaling 663 animals. Average body condition score was 3.5 (1-5 scale). Estrus was induced with prostaglandin F2α. The total pregnancy rate of females inseminated with male-sexed semen of bulls A, B and C was 38.8% (131/338) less (P<0.0001) than the total pregnancy rate observed for females inseminated with conventional semen from the same bulls (57.9% [188/325]). Pregnancy rates of non-suckling cows inseminated with male-sexed semen was 43.3% (49/113), which was similar (P≥0.05) to the values found for heifers inseminated with male-sexed semen from the same bulls (36.4% [82/225]). The pregnancy rate of females inseminated with male-sexed semen was less compared with females inseminated with conventional semen. In addition, there was no significant difference in the pregnancy rate of heifers versus non-suckling cows.     

Physical performance in kidney transplanted patients: a study on desert trekking

Physical performance of kidney transplanted patients in challenging environments, such as deserts, has been poorly studied. Six kidney transplanted (T: 5 males, 1 female; 45±6 yrs) and 8 control (C: 5 males, 3 females; 49±13 yrs) subjects participated in a 5-day desert trek. Blood pressure, hydration status (Height2/Rz by bioimpedance), heart rate, energy expenditure (by SenseWear Pro Armband) and walking velocities were recorded during each daily trekking stage (GPS-assisted wearable devices). Systo-diastolic blood pressure did not differ between C (119/77±12/8 mmHg) and T (121/77±10/6 mmHg) groups throughout the study. The hydration status was stable from day 1 (Ht2/Rz: 64±13 cm2/Ohm in T and 59±12 cm2/Ohm in C subjects) to day 5 (66±11 cm2/Ohm in T and 61±13 cm2/Ohm in C subjects) in both groups. Two patients on steroid treatment showed a relative hyperhydration. Mean heart rate did not differ between T (135±10 bpm) and C (136±5 bpm) subjects throughout the study, although a reduction from day 1 to day 5 was observed in T subjects only (p<0.05 vs C group). No differences were found between T and C group in walking velocity (1.7±0.6 km/h in T and 1.7±0.5 km/h in C group); mean intensity of physical activity was 3.4±0.5 METs in T and 3.3±0.6 METs in C group during each trekking stage. Negligible differences were observed in cardiovascular, metabolic and hydration status adaptations to desert trekking between selected T and C individuals. T subjects with creatinine clearance > 55 ml/min showed acceptable physical performance and acclimatization to desert environment, suggesting a good long-term outcome of transplantation.     

Chilling-induced changes of vacuolar proton pumps in hypocotyls of <Emphasis Type="Italic">Vigna unguiculata</Emphasis>

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H⁺ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H⁺ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.     

Functional characterization of a nonmammalian IL-21: rainbow trout Oncorhynchus mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFN-gamma, IL-10, and IL-22

In mammals, IL-21 is a common γ chain cytokine produced by activated CD4(+) T cells and NKT cells that acts on multiple lineages of cells. Although IL-21 has also been discovered in birds, amphibians, and fish, to date, no functional studies have been reported for any nonmammalian IL-21 molecule. We have sequenced an IL-21 gene (tIL-21) in rainbow trout, which has a six-exon/five-intron structure, is expressed in immune tissues, and is induced by bacterial and viral infection and the T cell stimulant PHA. In contrast to mammals, calcium ionophore and PMA act synergistically to induce tIL-21. Recombinant tIL-21 (rtIL-21) induced a rapid and long-lasting (4-72 h) induction of expression of IFN-γ, IL-10, and IL-22, signature cytokines for Th1-, Th2-, and Th17-type responses, respectively, in head kidney leukocytes. However, rtIL-21 had little effects on the expression of other cytokines studied. rtIL-21 maintained the expression of CD8α, CD8β, and IgM at a late stage of stimulation when their expression was significantly decreased in controls and increased the expression of the Th cell markers CD4, T-bet, and GATA3. Intraperitoneal injection of rtIL-21 confirmed the in vitro bioactivity and increased the expression of IFN-γ, IL-10, IL-21, IL-22, CD8, and IgM. Inhibition experiments revealed that the activation of JAK/STAT3, Akt1/2, and PI3K pathways were responsible for rtIL-21 action. This study helps to clarify the role of IL-21 in lower vertebrates for the first time, to our knowledge, and suggests IL-21 is a likely key regulator of T and B cell function in fish.