Adsorption of temperate phages of Lactobacillus delbrueckii strains and phage resistance linked to their cell diversity

Aims: The aim of this work was to study the adsorption step of two new temperate bacteriophages (Cb1/204 and Cb1/342) of Lactobacillus delbrueckii and to isolate phage‐resistant derivatives with interesting technological properties. Methods and Results: The effect of divalent cations, pH, temperature and cell viability on adsorption step was analysed. The Ca²⁺ presence was necessary for the phage Cb1/342 but not for the phage Cb1/204. Both phages showed to be stable at pH values between 3 and 8. Their adsorption rates decreased considerably at pH 8 but remained high at acid pH values. The optimum temperatures for the adsorption step were between 30 and 40°C. For the phage Cb1/342, nonviable cells adsorbed a lower quantity of phage particles in comparison with the viable ones, a fact that could be linked to disorganization of phage receptor sites and/or to the physiological cellular state. The isolation of phage‐resistant derivatives with good technological properties from the sensitive strains and their relationship with the cell heterogeneity of the strains were also made. Conclusions: Characterization of the adsorption step for the first temperate Lact. delbrueckii phages isolated in Argentina was made, and phage‐resistant derivatives of their host strains were obtained. Significance and Impact of the Study: Some phage‐resistant derivatives isolated exhibited good technological properties with the prospective to be used at industrial level.     

Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti

D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.     

Permian macroburrows as microhabitats for meiofauna organisms: an ancient behaviour common in extant organisms

Meiofaunal organisms are indirectly influenced by the activity of benthic macroinvertebrates within the sediment, which plays a role in modifying physical and chemical characteristics of the habitat. The association of meiofaunal organisms and macroburrows is well known in modern environments, but the record of this relationship in the geological record is still incipient. This study documents diminutive burrows (Helminthoidichnites tenuis) associated with the surface of macroburrows (Palaeophycus tubularis) in Early Permian deposits. The cylindrical shape and meandering to loop trajectory of the diminutive burrows indicate that they were produced by small free‐living meiofaunal nematodes. Apparently, P. tubularis (open burrow) constituted a favourable microhabitat for nematodes, providing the following: (1) protection against erosive processes and meiofauna predators; (2) oxygen access to more in‐depth layers within the sediment; (3) temperature stabilization; and (4) food supply due to mucus impregnation in the macroburrow walls by the Palaeophycus tracemaker. The association between H. tenuis and P. tubularis constitutes the first fossil record of a symbiotic relationship between meiofaunal nematodes and macrobenthic organisms (polychaetes). It also suggests that ecological strategies such as mutualism or commensalism, which are common between extant nematodes and macrobenthic invertebrates, were available in the behavioural programme of these organisms since the Early Permian.     

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.     

Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.     

Abrogation of hepatocyte apoptosis and early appearance of liver dysplasia in ethanol-fed p53-deficient mice

Ethanol consumption represents a major risk factor for cancer development, and a significant fraction of hepatocarcinomas arises in alcoholic liver cirrhosis. Increasing evidence indicates that ethanol acts as a tumor promoter on genetically initiated cells, by increasing the intracellular concentration of reactive oxygen species and promoting tissue necrosis/regeneration and cell proliferation. The tumor suppressor p53 restrains the expansion of carcinogen-initiated cells by inducing cell cycle arrest and apoptosis; accordingly, p53-deficient mice develop spontaneous and chemically induced neoplasms at a much higher frequency than normal mice. In normal mice exposed to a subacute (3 weeks) ethanol intoxication, a significant increase in the number of apoptotic hepatocytes was observed in concomitance with the up-regulation of the mitochondrial superoxide scavenger MnSOD, a reliable indicator of oxidative stress. Cell death occurred in the absence of liver inflammation and necrosis. Ethanol-induced hepatocyte apoptosis was completely abrogated in the p53 null background, suggesting that the tumor suppressor is necessary for hepatocyte death by ethanol. Accordingly, p53 -/- MEF were, unlike wild type cells, completely insensitive up to 0.5M ethanol in the culture medium. Strikingly, marked and widespread signs of dysplasia, with nuclear pleomorphisms and initial loss of normal architecture, heralding malignant transformation, were scored in all the mutant mice exposed to ethanol, but not in the control-fed littermates nor in ethanol-fed normal mice. These observations suggest that p53-dependent apoptosis restrains the tumorigenic effect of ethanol on liver cells, in agreement with the frequent loss of p53 function in HCC, and reveal an unexpected carcinogenic potential of alcohol which appears to be independent from the induction of cirrhosis and hepatocyte regeneration.     

Recovery of the pubic symphysis on primiparous young and multiparous senescent mice at postpartum

It has been observed that parturition has a significant effect on female skeletal architecture and that age alters musculoskeletal tissues and their functions. We therefore hypothesized that multiparity affects the recovery of the pubic symphysis in senescent mice at postpartum and the morphology of the interpubic tissues. The pubic symphysis of primiparous young, virgin senescent (VS) and multiparous senescent (MS) Swiss mice was examined by light microscopy, transmission electron microscopy, morphometric analysis and immunohistochemistry. The mouse pubic symphysis was remodeled during the first pregnancy: the cellular phenotype and morphology changed to ensure a structurally safe birth canal, followed by recovery of the interpubic articulation after birth. The morphology of the pubic symphysis in the VS group was maintained in a state similar to that observed in virgin young mice. In contrast, MS mice exhibited an interpubic ligament characterized by extended fibrocyte-like cells, an opened interpubic articulation gap, compacted and thin collagen fibrils and scarce galectin-3-positive cells. Thus, we found that the cellular and extracellular characteristics of the pubic symphysis were altered by multiparity in senescent mice. These particular tissue characteristics of the MS group might be associated with an impaired recovery process at postpartum. Thus, a better understanding of the alterations that occur in the birth canal, including the pubic symphysis, due to multiparity in reproductively aged mice may contribute to our comprehension of the biological mechanisms that modify the skeleton and pelvic ligaments and even play a role in the murine model of pelvic organ prolapse.     

Ulva lactuca and U. flexuosa (Chlorophyta,Ulvophyceae) cultivation in Brazilian tropical waters: recruitment,growth, and ulvan yield

Ulva spp. are used in a wide range of commercial applications, including bioremediation, food, bioenergy, pharmaceuticals, and agriculture. The sulfated polysaccharide ulvan obtained from Ulva spp. is of interest for triggering plant defenses against disease. However, the cultivation of Ulva spp. is still in its infancy. This study verified the feasibility of cultivating Ulva lactuca and Ulva flexuosa at two sites on the tropical Brazilian coast. We investigated the following: (a) methods to induce sporulation, (b) comparison of seeding ropes inoculated in vitro versus seeding at sea over 40 days, (c) production and harvest cycles at 15 and 30 days, (d) growth productivity of U. flexuosa at sea and in outdoor tanks, and (e) comparison of ulvan yields from biomass cultivated in tanks and the sea. High nutrient treatment was the most efficient method to induce sporulation (7,540?±?3,133 spores m^?1). Sea-based cultivation of U. flexuosa was only successful at one site. Seeding of ropes in vitro was more efficient than seeding at sea (0.31?±?0.20 g m^?2 day^?1), and 15-day harvest cycles were most efficient (20.1?±?1.8 % day^?1; 0.46?±?0.11 g m^?2 day^?1). Despite differences in plant growth in tanks (27.9?±?4.4 % day^?1) and at sea (20.1?±?1.8 % day^?1), the dry biomass and ulvan yields (17.7?±?5.0 %) did not differ between these systems. Cultivation of U. flexuosa was feasible at sea using in vitro seeding with a production cycle of 15 days in Brazilian tropical waters and tanks with high irradiance and enriched seawater.