Bioreactor Study Employing Bacteria with Enhanced Activity toward Cyanobacterial Toxins Microcystins

An important aim of white (grey) biotechnology is bioremediation, where microbes are employed to remove unwanted chemicals. Microcystins (MCs) and other cyanobacterial toxins are not industrial or agricultural pollutants; however, their occurrence as a consequence of human activity and water reservoir eutrophication is regarded as anthropogenic. Microbial degradation of microcystins is suggested as an alternative to chemical and physical methods of their elimination. This paper describes a possible technique of the practical application of the biodegradation process. The idea relies on the utilization of bacteria with a significantly enhanced MC-degradation ability (in comparison with wild strains). The cells of an Escherichia coli laboratory strain expressing microcystinase (MlrA) responsible for the detoxification of MCs were immobilized in alginate beads. The degradation potency of the tested bioreactors was monitored by HPLC detection of linear microcystin LR (MC-LR) as the MlrA degradation product. An open system based on a column filled with alginate-entrapped cells was shown to operate more efficiently than a closed system (alginate beads shaken in a glass container). The maximal degradation rate calculated per one liter of carrier was 219.9 µg h⁻¹ of degraded MC-LR. A comparison of the efficiency of the described system with other biological and chemo-physical proposals suggests that this new idea presents several advantages and is worth investigating in future studies.     

Assessment of the haemostatic system in patients surgically treated for ruptured cerebral aneurysm

BACKGROUND AND PURPOSE: The aim of the study was to evaluate selected markers of thrombin generation and subsequent fibrinolysis in patients with aneurysmal subarachnoid haemorrhage (SAH) and to assess the relationship between thrombin generation/fibrinolysis and clinical course and outcome. MATERIAL AND METHODS: This prospective study included 72 patients after aneurysmal SAH who underwent surgery within 72 hours after onset of symptoms. The results were compared with 84 control patients without SAH. Selected markers of thrombin generation (thrombin-antithrombin complexes, TAT), fibrinolysis (D-dimer) and fibrinogen level were examined in blood just after admission and on day 7 after surgery. The relationship between levels of those markers and selected clinical and radiological data, and outcome at 3-6 months after surgery, were assessed. RESULTS: On admission, patients with SAH had higher levels of TAT (p<0.001), D-dimer (p=0.048), and fibrinogen than the control group (p<0.001). Also, patients with severe bleeding demonstrated higher TAT (p<0.001) and D-dimer (p=0.04) levels. The admission level of TAT (higher than 24 g/l; odds ratio = 10.8) and the elevated blood fibrinogen level (odds ratio = 1.2) showed a strong correlation with mortality. Furthermore, a level of TAT higher than 24 g/l (odds ratio = 9.98) and the level of fibrinogen (odds ratio = 1.3) strongly correlated with poor outcome. There was no significant correlation between markers of coagulation on the 7th day after surgery for SAH and the outcome. CONCLUSIONS: Activation of blood coagulation as well as the fibrinolytic system occurred early in the course of SAH. Such activation was associated with poor clinical status of patients on admission, greater amount of subarachnoid blood, and poor clinical outcome. Thus, blood levels of TAT and fibrinogen are independent factors associated with mortality and morbidity after aneurysmal SAH.     

KCNQ/M-currents contribute to the resting membrane potential in rat visceral sensory neurons

The M-current is a slowly activating, non-inactivating potassium current that has been shown to be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of M-current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from rats through immunocytochemical studies. All neurons showed expression of each of the three proteins. In voltage clamp studies, the cognition-enhancing drug linopirdine (1–50 μ m ) and its analogue, XE991 (10 μ m ), quickly and irreversibly blocked a small, slowly activating current that had kinetic properties similar to KCNQ/M-currents. This current activated between −60 and −55 mV, had a voltage-dependent activation time constant of 208 ± 12 ms at −20 mV, a deactivation time constant of 165 ± 24 ms at −50 mV and V _1/2 of −24 ± 2 mV, values which are consistent with previous reports for endogenous M-currents. In current clamp studies, these drugs also led to a depolarization of the resting membrane potential at values as negative as −60 mV. Flupirtine (10–20 μ m ), an M-current activator, caused a 3–14 mV leftward shift in the current–voltage relationship and also led to a hyperpolarization of resting membrane potential. These data indicate that the M-current is present in nodose neurons, is activated at resting membrane potential and that it is physiologically important in regulating excitability by maintaining cells at negative voltages.     

Duplex Steels Used in Building Structures and Their Resistance to Chloride Corrosion

Welded structures made of duplex steels are used in building applications due to their resistance to local corrosion attack initiated by chlorides. In this paper, the material and technological factors determining the corrosion resistance are discussed in detail. Furthermore, recommendations are formulated that allow, in the opinion of the authors, to obtain a maximum corrosion resistance for welded joints. The practical aspects of corrosion resistance testing are also discussed, based on the results of qualification tests. This work is of a review character. The conclusions and practical recommendations are intended for contractors and investors of various types of structures made of the duplex steel. The recommendations concern the selection and use of duplex steels, including the issues of metallurgy, welding techniques, and corrosion protection.     

Heterologous expression and characterisation of microcystinase

The first enzyme in the microcystin (MC) degradation pathway identified in bacterial strains is coded by mlrA gene and is referred to as microcystinase. To date, there has been no biochemical characterisation of this enzyme. The results presented herein show a successful heterologous expression of MlrA as well as mutational studies, partial purification and biochemical characterisation of the enzyme. The mutation and inhibition study confirmed previous ideas that MlrA is a metalloprotease and allowed to calculate the inhibition parameters. Moreover, the kinetic parameters of MC-LR linearization were measured showing that MlrA exhibits a positive cooperativity towards MC-LR. Furthermore, in vitro experiments with Escherichia coli cells expressing MlrA indicated the potency of the heterologous host to eliminate MCs with very high efficiency. This study reports a new approach to the analysis of a microcystin degrading enzyme, extends the knowledge about MC biodegradation and opens broad scope for future study.     

CA 125 regression after two cycles of neoadjuvant chemotherapy as prognostic factor in patients with advanced ovarian cancer and primary peritoneal serous cancer who underwent interval surgical cytoreduction

OBJECTIVES: The aim of our study was to assess the prognostic role of CA 125 regression during neoadjuvant chemotherapy (NAC) in patients with ovarian cancer (OC) or primary peritoneal serous carcinoma (PPSC) that underwent interval debulking surgery (IOC). MATERIAL AND METHODS: Thirty one patients with advanced OC or PPSC (FIGO stage IIIC and IV) who underwent initial exploratory surgery, followed by NAC containing platinum analogs, have been analyzed, retrospectively. We have used a regression coefficient (RCA 125), which was calculated as following: log10 (CA 125 level measured after two cycles of NAC/baseline CA 125) for statistical analysis. The median value of RCA 125 reached -0.788 and has been used to dichotomize. Optimal IOC has been performed in 67.74% (21/31) patients, suboptimal in 25.81% (8/31) patients and 6.45% (2/31) of patients did not undergo IOC due to the progression of the disease. RESULTS: We have noted significant correspondence between time to progression and RCA 125 in univariate analysis, which we have also confirmed in multivariate analysis (HR 0.27; 95% CI, 0.15-0.96; p = 0.0178). Similarly, we have observed significant relationship between overall survival, RCA 125 and extension IOC in univariate analysis. Multivariate analysis confirmed that RCA 125 was independent prognostic factor, HR-0.18 (95% CI, 0.07-0.56; p = 0.004). In case of patients with high RCA 125, a greater rate of optimal debulking cytoreduction (p = 0.0278, U = 50.0) has been observed. CONCLUSIONS: RCA 125 after two courses of NAC appears to be an important prognostic factor in patients with OC or PPSC, who underwent IOC High RCA 125 during NAC seems to be a good predictive factor in order to achieve optimal IOC.     

Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins

Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.     

Intraoperative contrast-enhanced ultrasound in traumatic brain surgery

ObjectiveThe objective was to assess intraoperative contrast-enhanced ultrasound (CEUS) in traumatic brain surgery.MethodsWe prospectively performed intraoperative conventional ultrasound (IOUS) and CEUS in 32 patients who underwent emergency surgery for the treatment of traumatic brain injury (TBI). Sonographic appearance including echogenicity, border, and size of the traumatic lesion and adjacent brain tissue on CEUS were compared with those on IOUS using surgical results as the gold standard. The differences in the size and contrast enhancement parameters of the lesions between IOUS and CEUS were analyzed with a paired t test.ResultsThe accuracy of CEUS in assessing TBI was 100%, whereas IOUS was 51%. The absolute peak intensity (API) varied depending on the severity of brain injury. Lower API was observed in severely damaged brain tissue, whereas high API was seen in normal brain tissue or the brain tissue with mild injury. The border of the trauma lesion was more clearly defined on CEUS when compared to IOUS. The size of the lesions measured on CEUS was significantly larger than that on IOUS (P< .01). Importantly, small vessels supplying blood to the tissue in traumatic lesions, as an indication of possible brain vitality, were optimized on CEUS during the surgery. Based on the parameter of time intensity curve and appearances of the lesions on CEUS, the severity of lesions was reclassified and surgical intervention was redesigned in 21 (21/32, 66%) cases.ConclusionIntraoperative CEUS improves accuracy in classification of traumatic brain injury, which helps neurosurgeons to effectively remove hematoma, preserve normal brain tissue, and prevent damaging the vessels during surgical intervention.     

Exfoliative Toxins of Staphylococcus aureus

Staphylococcus aureus is an important pathogen of humans and livestock. It causes a diverse array of diseases, ranging from relatively harmless localized skin infections to life-threatening systemic conditions. Among multiple virulence factors, staphylococci secrete several exotoxins directly associated with particular disease symptoms. These include toxic shock syndrome toxin 1 (TSST-1), enterotoxins, and exfoliative toxins (ETs). The latter are particularly interesting as the sole agents responsible for staphylococcal scalded skin syndrome (SSSS), a disease predominantly affecting infants and characterized by the loss of superficial skin layers, dehydration, and secondary infections. The molecular basis of the clinical symptoms of SSSS is well understood. ETs are serine proteases with high substrate specificity, which selectively recognize and hydrolyze desmosomal proteins in the skin. The fascinating road leading to the discovery of ETs as the agents responsible for SSSS and the characterization of the molecular mechanism of their action, including recent advances in the field, are reviewed in this article.