Development of an improved selective agar medium for isolation of Yersinia pestis

Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. We report the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions. The final BIN formulation is based on brain heart infusion agar, to which the selective agents irgasan, cholate salts, crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar). The advantage of BIN over other media has been also demonstrated in recovering virulent Y. pestis from the mixed bacterial populations found in decaying carcasses of infected mice. The BIN medium is suggested as a selective medium for isolation and recovery of Y. pestis from various backgrounds.     

Lipid peroxidation and scavenging enzyme levels in the liver of streptozotocin-induced diabetic rats

In this study, alterations in the liver antioxidant enzymes status and lipid peroxidation in short-term (8-weeks) and long-term (24-weeks) diabetic rats were examined. Glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) levels were significantly increased, but superoxide dismutase (SOD) activity was significantly reduced in 8-weeks diabetic rats, compared to control. Catalase (CAT) activity, however, was found unchanged. In 24-weeks diabetic rats, while GSH-Px activity was unchanged, but SOD and CAT activities and MDA levels were significantly increased, compared to control. These results suggest that diabetes-induced alterations in tissue antioxidant system may reflect a generalized increase in tissue oxidative stress. It can be concluded that lipid peroxidation and antioxidant enzyme levels are elevated in diabetic condition. Hence, diabetes mellitus, if left untreated, may increase degenerative processes due to accumulation of oxidative free radicals.     

Changes along the pituitary-gonadal axis during maturation of the black carp, Mylopharyngodon piceus

The black carp, Mylopharyngodon piceus, is a late-maturing cyprinid reaching sexual maturity at the age of 6-7 years. The present work attempted to define nonfunctional sites along the pituitary-gonadal axis in immature fish utilizing in vivo and in vitro challenge experiments. Two- and 3-year old fish injected with salmon gonadotropin-releasing hormone analog (sGnRHa; 10 microg/kg) and metoclopramide (20 mg/kg) did not reveal any increase in circulating gonadotropin (cGtH) or estradiol (E(2)) level. Furthermore, cGtH release from cultured pituitary cells of fish at these ages did not increase in response to sGnRH (0.1 nM - 1 microM) but was augmented when exposed to TPA (12.5 nM). However, 4-year old female fish did respond to the above treatments both in vivo and in vitro. These results suggest the existence of nonfunctional site(s) proximal to the activation of PKC in the immature black carp gonadotrophs, probably at the level of GnRH receptors. These site(s) start to become functional in 4-year old fish. Two- and 3-year old fish injected with common carp pituitary extract (CPE) containing 350 microg cGtH/kg did not show any increase in circulating E(2). In addition, the estrogen secretion from fragments of the rudimentary gonads did not increase after exposure to CPE containing cGtH (0.5-4 microg/ml) but was elevated dose-dependently by exposure to dbcAMP (0.3-3 mM). However, the ovaries of 4-year old fish did respond to the gonadotropic stimulation, both in vivo and in vitro. These results suggest the existence of other non-functional site(s) in the immature black carp, proximal to the formation of cAMP in the gonads, probably at the level of GtH receptors. These site(s) start to become functional in 4-year old females. Another source of E(2) was discovered in the immature black carp: namely, the fat pad adjacent to the gonads. In contrast to the visceral adipose tissue, the fat pad secretes estrogen in response to cAMP elevation in 2- and 3-year old fish while in 4-year old fish it also responds to gonadotropic stimulation. Due to its large mass and high steroidogenic potency, it is assumed that the gonadal fat pad is involved in the process of puberty in the black carp. J. Exp. Zool. 286:405-413, 2000.     

Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys₁₀), which is frequently affected by oxidative post‐translational modifications. As Cys₁₀ is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys₁₀ may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys₁₀ modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys₁₀ seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.     

Gastroprotective and Antioxidant Effects of Lobaria pulmonaria and Its Metabolite Rhizonyl Alcohol on Indomethacin‐Induced Gastric Ulcer

Two lichen metabolites, rhizonaldehyde ( 1 ) and rhizonyl alcohol ( 2 ), were isolated from the acetone extract of Lobaria pulmonaria by chromatographic methods, and their chemical structures were determined by UV/VIS, IR, and 1D‐ and 2D‐NMR spectroscopic methods. The gastroprotective and in vivo antioxidant activities of extracts of L. pulmonaria and its metabolites, 1 and 2 , were investigated in indomethacin‐induced ulcer models in rats. The gastric lesions were significantly reduced by acetone, hexane, and CHCl₃ extracts, with 75.3–41.5% inhibition. Rhizonyl alcohol ( 2 ) significantly reduced the gastric lesions with an inhibition rate of 84.6–42.8%, whereas rhizonaldehyde ( 1 ) significantly increased the gastric lesions. Antioxidant parameters and myeloperoxidase activities were also evaluated in the gastric tissues of the rats. Indomethacin caused oxidative stress, which resulted in lipid peroxidation in gastric tissues by decreasing the levels of the antioxidants as compared to healthy rat tissues. In contrast to indomethacin, all extracts and rhizonyl alcohol ( 2 ) caused a significant decrease in lipid peroxidation levels and an increase in antioxidant parameters, superoxide dismutase, glutathione peroxidase, and glutathione‐S‐transferase, and reduced glutathione in gastric tissues. The administration of rhizonyl alcohol ( 2 ) also resulted in a decrease in gastric myeloperoxidase activity increased by indomethacin. The gastroprotective effect of rhizonyl alcohol ( 2 ) can be attributed to its antioxidant properties and its suppressing effect on neutrophil infiltration into gastric tissues.     

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01196871","term_id":"NCT01196871"}}NCT01196871     

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice

Background

Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates.

Aim

To examine the role of CD24 in the wound healing process.

Methods

An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA ^-/-) compared to wild-type (WT) mice.

Results

Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA ^+/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA ^-/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice.

Conclusions

Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.     

Fatal Systemic Cladosporiosis in a Merino Sheep Flock

Systemic cladosporiosis is described in 25 merino sheep from a flock consisting 250 animals. The fungal pneumonia appeared after an intensive antibiotic treatment, because of a respiratory system disorder. The pen of the flock was humid and crowded, and animals had signs of respiratory distress, coughing, fever and anorexia. All of the ill animals died, and necropsy was performed on 10 sheep. The lesions were characterized by a multifocal pyogranulomatous pneumonia and an abomasitis. Severe hemorrhages were observed in the lungs. At the histopathological examination, severe vasculitis with thrombosis was observed in various organs, especially in the lungs and abomasums, suggestive for a hematogenous dissemination of the infection in these organs. Numerous PAS-positive fungal elements were seen in the pyogranulomatous foci. Dark green fungal colonies were seen in the blood agar and Sabouraud dextrose agar that were identified as Cladosporium cladosporioides. This report highlights that phaeohyphomycosis can cause a severe systemic and fatal disease in merino sheep under insufficient management conditions.