A putative Type IIS restriction endonuclease GeoICI from <Emphasis Type="BoldItalic">Geobacillus</Emphasis> sp. – A robust,thermostable alternative to mezophilic prototype BbvI

Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers’ biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5′-GCAGC(N_8/12)-3′ DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-methyltransferases (MTases) and as a result, another putative REase-MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-MTases was detected.     

The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation

Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host’s health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.     

Homeostasis of chosen bioelements in organs of rats receiving lithium and/or selenium

Lithium is an essential trace element, widely used in medicine and its application is often long-term. Despite beneficial effects, its administration can lead to severe side effects including hyperparathyroidism, renal and thyroid disorders. The aim of the current study was to evaluate the influence of lithium and/or selenium treatment on magnesium, calcium and silicon levels in rats’ organs as well as the possibility of using selenium as an adjuvant in lithium therapy. The study was performed on rats divided into four groups (six animals each): control-treated with saline; Li-treated with Li₂CO₃ (2.7 mg Li/kg b.w.); Se-treated with Na₂SeO₃·H₂O (0.5 mg Se/kg b.w.); Se + Li-treated simultaneously with Li₂CO₃ and Na₂SeO₃·H₂O (2.7 mg Li/kg b.w. and of 0.5 mg Se/kg b.w., respectively). The administration was performed in form of water solutions by stomach tube once a day for 3 weeks. In the organs (liver, kidney, brain, spleen, heart, lung and femoral muscle) the concentrations of magnesium, calcium and silicon were determined. Magnesium was increased in liver of Se and Se + Li given rats. Lithium decreased tissue Ca and co-administration of selenium reversed this effect. Silicon was not affected by any treatment. The beneficial effect of selenium on disturbances of calcium homeostasis let suggest that further research on selenium application as an adjuvant in lithium therapy is worth being performed.     

New Rapid Method of DNA Isolation from Milk Somatic Cells

Isolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses.     

Dehalogenation Activity of Selected Fungi Toward δ‐Iodo‐γ‐Lactone Derived from trans,trans‐Farnesol

Time‐course of biotransformation of racemic trans‐4‐((E)‐4′,8′‐dimethylnona‐3′,7′‐dien‐1‐yl)‐5‐iodomethyl‐4‐methyldihydrofuran‐2‐one ( 1 ) in fungal and yeast cultures was investigated. In these conditions, the substrate 1 was enantioselectively dehalogenated yielding 4‐((E)‐4′,8′‐dimethylnona‐3′,7′‐dien‐1‐yl)‐4‐methyl‐5‐methylenedihydrofuran‐2‐one ( 2 ) and its structure was established based on the spectroscopic data. The most effective biocatalyst used was Didymosphaeria igniaria, which catalyzed the process with highest rate and enantioselectivity (ee of product = 76%). The antiproliferative activity of δ‐iodo‐γ‐lactone 1 , product of its biotransformation 2 , and starting substrate (farnesol) were evaluated toward two cancer cell lines: A549 (human lung adenocarcinoma) and HL‐60 (human promyelocytic leukemia).     

Perfusion-CT - Can We Predict Acute Pancreatitis Outcome within the First 24 Hours from the Onset of Symptoms?

Purpose

Severe acute pancreatitis (AP) is still a significant clinical problem which is associated with a highly mortality. The aim of this study was the evaluation of prognostic value of CT regional perfusion measurement performed on the first day of onset of symptoms of AP, in assessing the risk of developing severe form of acute pancreatitis.

Material and Methods

79 patients with clinical symptoms and biochemical criteria indicative of acute pancreatitis (acute upper abdominal pain, elevated levels of serum amylase and lipase) underwent perfusion CT within 24 hours after onset of symptoms. The follow-up examinations were performed after 4–6 days to detect progression of the disease. Perfusion parameters were compared in 41 people who developed severe form of AP (pancreatic and/or peripancreatic tissue necrosis) with parameters in 38 consecutive patients in whom course of AP was mild. Blood flow, blood volume, mean transit time and permeability surface area product were calculated in the three anatomic pancreatic subdivisions (head, body and tail). At the same time the patient''s clinical status was assessed by APACHE II score and laboratory parameters such as CRP, serum lipase and amylase, AST, ALT, GGT, ALP and bilirubin were compared.

Results

Statistical differences in the perfusion parameters between the group of patients with mild and severe AP were shown. Blood flow, blood volume and mean transit time were significantly lower and permeability surface area product was significantly higher in patients who develop severe acute pancreatitis and presence of pancreatic and/or peripancreatic necrosis due to pancreatic ischemia. There were no statistically significant differences between the two groups in terms of evaluated on admission severity of pancreatitis assessed using APACHE II score and laboratory tests.

Conclusions

CT perfusion is a very useful indicator for prediction and selection patients in early stages of acute pancreatitis who are at risk of developing pancreatic and/or peripancreatic necrosis already on the first day of the onset of symptoms and can be used for treatment planning and monitoring of therapy of acute pancreatitis. Early suspicion of possible pancreatic necrosis both on the basis of scores based on clinical status and laboratory tests have low predictive value.     

Mycorrhizas influence functional traits of two tallgrass prairie species

Over the past decade, functional traits that influence plant performance and thus, population, community, and ecosystem biology have garnered increasing attention. Generally lacking, however, has been consideration of how ubiquitous arbuscular mycorrhizas influence plant allometric and stoichiometric functional traits. We assessed how plant dependence on and responsiveness to mycorrhizas influence plant functional traits of a warm‐season, C4 grass, Andropogon gerardii Vitman, and the contrasting, cool‐season, C3 grass, Elymus canadensis L. We grew both host species with and without inoculation with mycorrhizal fungi, across a broad gradient of soil phosphorus availabilities. Both host species were facultatively mycotrophic, able to grow without mycorrhizas at high soil phosphorus availability. A. gerardii was most dependent upon mycorrhizas and E. canadensis was weakly dependent, but highly responsive to mycorrhizas. The high dependence of A. gerardii on mycorrhizas resulted in higher tissue P and N concentrations of inoculated than noninoculated plants. When not inoculated, E. canadensis was able to take up both P and N in similar amounts to inoculated plants because of its weak dependence on mycorrhizas for nutrient uptake and its pronounced ability to change root‐to‐shoot ratios. Unlike other highly dependent species, A. gerardii had a high root‐to‐shoot ratio and was able to suppress colonization by mycorrhizal fungi at high soil fertilities. E. canadensis, however, was unable to suppress colonization and had a lower root‐to shoot ratio than A. gerardii. The mycorrhiza‐related functional traits of both host species likely influence their performance in nature: both species attained the maximum responsiveness from mycorrhizas at soil phosphorus availabilities similar to those of tallgrass prairies. Dependence upon mycorrhizas affects performance in the absence of mycorrhizas. Responsiveness to mycorrhizal fungi is also a function of the environment and can be influenced by both mycorrhizal fungus species and soil fertility.     

Comprehensive Expression of Wnt Signaling Pathway Genes during Development and Maturation of the Mouse Cochlea

Background

In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points.

Results

We identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity.

Conclusion

A large number of Wnt signaling molecules were dynamically expressed during cochlear development and in the early postnatal period, suggesting complex regulation of Wnt transduction. The data revealed several potential key regulators for further study.