Strategies for high titre plasmid DNA production in Escherichia coli DH5α

The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD₆₀₀ 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD₆₀₀ 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD₆₀₀ 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 °C), low dO₂) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °C, glucose-/dO₂-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD₆₀₀ of 80.     

The influence of cryopreservation and quick-freezing on the mechanical properties of tendons

In order to maintain their native properties, cryopreserved tendons are usually used in biomechanical research and in transplantation of allogenic tendon grafts. The use of different study protocols leads to controversy in literature and thus complicates the evaluation of the current literature. The aim of this study consisted in examining the influence of different freezing and thawing temperatures on the mechanical properties of tendons. 60 porcine tendons were frozen at either −80 °C or −20 °C for 7 days and thawed at room or body temperature for 240 or 30 min, respectively. A subgroup of ten tendons was quick-frozen with liquid nitrogen (−196 °C) for 2 s before cryopreservation. Biomechanical testing was performed with a material testing machine and included creep, cyclic and load-to-failure tests. The results showed that freezing leads to a reduced creep strain after constant loading and to an increased secant modulus. Freezing temperature of −80 °C increased the secant modulus and decreased the strain at maximum stress, whereas thawing at room temperature reduced the maximum stress, the strain at initial tendon failure and the Young’s Modulus. Quick-freezing led to increased creep strain after constant loading, increased strain at initial failure in the load-to-failure test, and decreased strain at maximum stress. When cryopreserving, tendons for scientific or medical reasons, freezing temperature of −20 °C and thawing temperature of 37.5 °C are recommended to maintain the native properties of tendons. A treatment with liquid nitrogen in the sterilization process of tendon allografts is inadvisable because it alters the tendon properties negatively.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Stability of the intra- and extracellular toxins of Prymnesium parvum using a microalgal bioassay

Prymnesium parvum produces a variety of toxic compounds, which affect other algae, grazers and organisms at higher trophic levels. Here we provide the method for development of a sensitive algal bioassay using a microalgal target, Teleaulax acuta, to measure strain variability in P. parvum toxicity, as well as the temporal stability of both the intracellular and the extracellular lytic compounds of P. parvum. We show high strain variation in toxicities after 3 h incubation with LC₅₀s ranging from 24 to 223 × 10³ cells ml⁻¹. Most importantly we prove the necessity of testing physico-chemical properties of P. parvum toxins before attempting to isolate and characterize them. The extracellular toxin in the supernatant is highly unstable, and it loses significant lytic effects after 3 days despite storage at −20 °C and after only 24 h stored at 4 °C. However, when stored at −80 °C, lytic activity is more easily maintained. Reducing oxidation by storing the supernatant with no headspace in the vials significantly slowed loss of activity when stored at 4 °C. We show that the lytic activity of the intracellular toxins, when released by sonication, is not as high as the extracellular toxins, however the stability of the intracellular toxins when kept as a cell pellet at −20 °C is excellent, which proves this is a sufficient storage method for less than 3 months. Our results provide an ecologically appropriate algal bioassay to quantify lytic activity of P. parvum toxins and we have advanced our knowledge of how to handle and store the toxins from P. parvum so as to maintain biologically relevant toxicity.     

Bacteriophage phi11 lysin: Physicochemical characterization and comparison with phage phi80α lysin

Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11 (LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The objectives of the study were to investigate the stability of lysins of phages phi11 and phi80α in storage and functioning conditions, to identify optimum storage conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and electrophoresis were used to identify causes of inactivation. At 37 °C, pH 7.5 and concentration of NaCl not higher than 150 mM, LysPhi11 molecules contain a high percentage of random coils (43%). However, in spite of this the enzyme has high activity (0.4–0.8 OD_600 nm s⁻¹ mg⁻¹). In storage conditions (4 °C and 22 °C, pH 6.0–9.0, 10–500 mM NaCl) LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for LysPhi11 (4 °C, pH 6.0–7.5, 10 mM NaCl) were selected under which the time of the enzyme half-inactivation is 120–160 days. LysPhi80α stability is insufficient: at 37 °C the enzyme loses half of its activity almost immediately; at 4 °C and 22 °C the time of half-inactivation of LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent.     

Solution for promoting egl3 gene of Trichoderma reesei high-efficiency secretory expression in Escherichia coli and Lactococcus lactis

The objective of this study was to develop a solution for promoting egl3 gene of Trichoderma reesei (coding β-1,4-endoglucanase, EGIII) high-efficiency secretory expression in Escherichia coli and Lactococcus lactis and to investigate the effect of the best recombinant on degrading paper and wheat straw. The coding sequence of the egl3 gene fused with a gene fragment of Usp45 (usp45) of L. lactis was cloned to pMG36e and was expressed in E. coli DH 5α (DH 5α) and L. lactis subsp. lactis MG1363 (MG1363). The maximal productivity in recombinant DH 5α was 226 mU mL⁻¹ for extracellular EGIII and 535 mU mL⁻¹ for intracellular EGIII. The maximal productivity in recombinant MG1363 was 1118 mU mL⁻¹ for extracellular EGIII and 761 mU mL⁻¹ for intracellular EGIII. The plasmid stability in recombinant MG1363 was higher than 85% at 60 generations. Recombinant MG1363 vigorously degraded paper and wheat straw and produced sufficient acids. This study provided EGIII transgenic lactic acid bacteria for processing agricultural byproducts.     

Effect of initial freezing temperature on the semen characteristics of frozen-thawed ram spermatozoa in a semi-arid tropical environment

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including initial freezing temperature. The present study was conducted to observe the effect of initial freezing temperature on post-thawing motility of ram spermatozoa of native and crossbred rams maintained in a semi-arid tropical environment. Good quality semen obtained from native Malpura and crossbred Bharat Merino rams were pooled within breed and diluted at a rate of 1000 million spermatozoa per milliliter in TEST—yolk–glycerol extender. Diluted semen samples were loaded in 0.25 ml straws and cooled to −25, −75 or −125 °C freezing temperature at the rate of −25 °C/min under controlled conditions before plunging into liquid nitrogen for storage. The thawing of straws was performed at 50 °C in a water bath for 10 s and motility characteristics of the frozen-thawed spermatozoa were assessed by a computer-assisted spermatozoa analysis technique. Initial freezing temperature significantly affected the post-thawing motility of sperm in both the breeds. The post-thawing % motility and rapid motile spermatozoa were significantly higher at initial freezing temperature of −125 °C and lower at −25 or −75 °C. The percentage medium motile sperm were similar at all three initial freezing temperatures. The percentage of slow motile and linearity of sperm varied (P<0.01) between the different freezing temperatures. The curvilinear velocity, average path velocity and straight line velocity of spermatozoa were higher (P<0.01) at −125 °C than −25 or −75 °C. Although the lateral head displacement of spermatozoa did not vary significantly between the different initial freezing temperatures, the stroke frequency was significantly lower at −25 °C than −75 or −125 °C. Except for % linearity, the average path velocity and straight line velocity, other spermatozoa characteristics were not significantly different between breeds. The interaction between freezing temperature and breed was significant only for the % motility and linearity of the spermatozoa. The study indicates that initial freezing temperature has a significant effect on spermatozoa motility and velocity following post-thawing. The best motile spermatozoa following thawing were achieved at −125 °C freezing temperature.     

Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the “in cooling” period (5 °C)

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100 × 10⁶ sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (−20 °C/min to −100 °C and stored at −196 °C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters – by CASA –, and viability (VIAB), viable and non-apoptotic status (YOPRO−), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) – by flow cytometry –. In Experiment 1, we assessed different storage times (0, 0.5, 1 – control –, 4–5, 7–8 and 11–12 h) at 5 °C from final dilution to freezing. After thawing, non-equilibrated samples (0 h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72 h) at 5 °C before freezing using storage for 1 h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48–72 h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24 h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.     

Validation of a new whole-body cryotherapy chamber based on forced convection

Whole-body cryotherapy (WBC) and partial-body cryotherapy (PBC) are two methods of cold exposure (from −110 to −195 °C according to the manufacturers). However, temperature measurement in the cold chamber during a PBC exposure revealed temperatures ranging from −25 to −50 °C next to the skin of the subjects (using isolating layer placed between the sensor and the skin). This discrepancy is due to the human body heat transfer. Moreover, on the surface of the body, an air layer called the boundary layer is created during the exposure and limits heat transfer from the body to the cabin air. Incorporating forced convection in a chamber with a participant inside could reduce this boundary layer. The aim of this study was to explore the use of a new WBC technology based on forced convection (frontal unilateral wind) through the measurement of skin temperature. Fifteen individuals performed a 3-min WBC exposure at −40 °C with an average wind speed of 2.3 m s⁻¹. The subjects wore a headband, a surgical mask, underwear, gloves and slippers. The skin temperature of the participants was measured with a thermal camera just before exposure, just after exposure and at 1, 3, 5, 10, 15 and 20 min after exposure. Mean skin temperature significantly dropped by 11 °C just after exposure (p<0.001) and then significantly increased during the 20-min post exposure period (p<0.001). No critically low skin temperature was observed at the end of the cold exposure. This decrease was greater than the mean decreases in all the cryosauna devices with reported exposures between −140 °C and −160 °C and those in two other WBC devices with reported exposures between −60 °C and −110 °C. The use of this new technology provides the ability to reach decreases in skin temperature similar to other technologies. The new chamber is suitable and relevant for use as a WBC device.     

Adult plasticity of cold tolerance in a continental-temperate population of Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21 °C; control) and induced plasticity by rapid cold-hardening (RCH, 1 h at 0 °C followed by 1 h recovery), cold acclimation (CA, 5 days at 6 °C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between −16 and −23 °C, and were chill-susceptible. 80% of control flies were killed after 1 h at −7.2 °C (males) or −7.5 °C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0 °C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CT_min) at approximately −1.7 °C, which was ca. 0.5 °C colder than control flies; RCH and CA increased the CT_min compared to controls. Control and RCH flies exposed to 0 °C for 8 h took 30–40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity.     

Effects of sample handling,processing, storage,and hemolysis on measurements of key energy metabolites in ovine blood

Four experiments were conducted using mature Suffolk ewes to evaluate the effects of blood sample handling, processing and storage on measurements of the energy metabolites, β-hydroxybutyrate, total cholesterol, glucose, l-lactate, nonesterified fatty acid (NEFA), and triacylglycerol. In experiment 1 the effects of anticoagulants on metabolite measurements and packed-cell volume (PCV) were evaluated. Blood samples (n=12) were collected into one of four evacuated blood-collecting tubes: no anticoagulant (SER; yields serum), and plasma-yielding sodium heparin (HEP), sodium fluoride and potassium oxalate (NAF), and tripotassium ethylenediaminetetraacetic acid (K₃EDTA). Anticoagulant affected (P<0.05) metabolite values, with total cholesterol, triacylglycerol, and l-lactate highest in SER, and glucose highest in HEP; NEFA and β-hydroxybutyrate measurements were not affected (P>0.10) by anticoagulant. In addition, among the plasma-yielding tubes, PCV was highest in HEP and lowest in NAF (P<0.01). Experiment 2 investigated the effects of repetitive freezing-thawing cycles of plasma on metabolite levels. Blood samples (n=20) were collected using NAF tubes, and plasma was stored at −20 °C and thawed in a water bath (21 °C) 0, 1, 2, and 3 times within 18 h after collection. Compared with fresh samples (Thaw 0), by thaw 2, l-lactate increased (P<0.01) 5%, and glucose and total cholesterol decreased (P<0.001) 13 and 4%, respectively. Plasma NEFA increased 10% (P<0.01) between thaws 0 and 1, but returned to fresh levels (Thaw 0) with subsequent freeze-thaw cycles (P<0.05). Plasma β-hydroxybutyrate decreased (P<0.01) between thaws 0 and 1, but there was no further decline with subsequent freeze-thaw cycles (P<0.05). Experiment 3 evaluated the effects of plasma storage temperature (−20 °C versus −80 °C) and length (0–180 days) on metabolite levels in blood samples (n=12) collected in NAF tubes. All plasma metabolites were affected by storage length (day effect, P<0.01) but only total cholesterol values were affected by storage temperature, with values being higher in plasma stored at −20 than −80 °C (P<0.01). Glucose measurements were higher in samples stored at −20 °C for up to 30 days, but were higher thereafter in samples stored at −80 °C (storage length × temperature effect, P<0.01). Experiment 4 evaluated the effects of anticoagulant (SER versus NAF) and whole blood storage (4 °C) for 0, 1, 3, and 7 days on metabolite concentrations. Glucose was higher (P<0.0001) in NAF, possibly the result of the presence of the glycolytic inhibitor, sodium fluoride, whereas l-lactate, NEFA, total cholesterol and β-hydroxybutyrate were higher in SER (P<0.01). Total cholesterol, l-lactate, and NEFA increased, whereas β-hydroxybutyrate decreased with days in storage (P<0.01). Development of hemolysis in the samples artifactually elevated glucose and NEFA values by as much as 41 and 230%, respectively. Attention to proper blood handling, processing, and storage procedures, and avoidance of hemolysis are important in blood clinical analyses and in the proper interpretation of experimental results.     

Individualising the exposure of −110 °C whole body cryotherapy: The effects of sex and body composition

The purpose of this study was to investigate the effects of whole body cryotherapy (WBC) on a range of thermoregulatory measures. We also sought to examine the influence of sex and body composition. A convenience sample of 18 healthy participants (10 males and 8 females) (27±6 yr) volunteered for this study. Temperature (core, tympanic, skin and mean body), heart rate, blood pressure, and thermal comfort and sensation were recorded pre- and post- (immediately and every 5 min until 35 min post) exposure to a single bout of WBC (30 s at −60 °C, 150 s at 110 °C). Anthropometric data (height, weight, body surface area, body mass index, fat mass and fat free mass) were also recorded. No significant differences in temperature (core, tympanic, skin and mean body), heart rate, blood pressure, or thermal comfort / sensation were observed between male and females at baseline. Immediately post WBC mean body (male:31.9±0.8 °C; female:31.0±0.9 °C; ∆ mean body temperature:0.9±0.1 °C; P≤0.05, d=0.64) and mean skin (male:22.1±2.2 °C; female:19.6±2.8 °C; ∆ mean skin temperature:−2.5±0.6 °C; d=0.99, P≤0.05) temperature was significantly different between sexes. Sex differences were also observed in regional skin temperature (male thigh, 20.8±1.1 °C; female thigh, 16.7±1.1 °C, ∆ mean thigh skin temperature:−4.1 °C; d=3.72; male calf, 20.5±1.1 °C; female calf, 18.2±1 °C, ∆ mean calf skin temperature:−2.3±0.1 °C; d=3.61; male arm, 21.7±1 °C; female arm, 19±0.4 °C, ∆ mean arm skin temperature: −2.7±0.3 °C; d=3.54; P≤0.05). Mean arterial pressure was significantly different over time (P≤0.001) and between sexes (male 0 mins:94±10 mmHg; female 0 mins:85±7 mmHg; male 35 mins:88±7 mmHg; female 35 mins:80±6 mmHg; P≤0.05). Combined data set indicated a strong negative relationship between skin temperature and body fat percentage 35 min’ post WBC (r=−0.749, P≤0.001) and for core temperature and body mass index in males only (r=0.726, P≤0.05) immediately after WBC. There were no significant differences between sexes in any other variables (heart rate, tympanic and perceptual variables). We observed sex differences in mean skin and mean body temperature following exposure to whole body cryotherapy. In an attempt to optimise treatment, these differences should be taken into account if whole body cryotherapy is prescribed.     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

Sharper angle,higher risk? The effect of cutting angle on knee mechanics in invasion sport athletes

IntroductionCutting is an important skill in team-sports, but unfortunately is also related to non-contact ACL injuries. The purpose was to examine knee kinetics and kinematics at different cutting angles.Material and methods13 males and 16 females performed cuts at different angles (45°, 90°, 135° and 180°) at maximum speed. 3D kinematics and kinetics were collected. To determine differences across cutting angles (45°, 90°, 135° and 180°) and sex (female, male), a 4 × 2 repeated measures ANOVA was conducted followed by post hoc comparisons (Bonferroni) with alpha level set at α ≤ 0.05 a priori.ResultsAt all cutting angles, males showed greater knee flexion angles than females (p < 0.01). Also, where males performed all cutting angles with no differences in the amount of knee flexion −42.53° ± 8.95°, females decreased their knee flexion angle from −40.6° ± 7.2° when cutting at 45° to −36.81° ± 9.10° when cutting at 90°, 135° and 180° (p < 0.01). Knee flexion moment decreased for both sexes when cutting towards sharper angles (p < 0.05). At 90°, 135° and 180°, males showed greater knee valgus moments than females. For both sexes, knee valgus moment increased towards the sharper cutting angles and then stabilized compared to the 45° cutting angle (p < 0.01). Both females and males showed smaller vGRF when cutting to sharper angles (p < 0.01).ConclusionIt can be concluded that different cutting angles demand different knee kinematics and kinetics. Sharper cutting angles place the knee more at risk. However, females and males handle this differently, which has implications for injury prevention.     

Evaluation of hexavalent chromium reduction by chromate-resistant moderately halophile,Nesterenkonia sp. strain MF2

A Gram-positive moderately halophilic chromate reducing bacterial strain was isolated from effluents of tanneries, and identified as Nesterenkonia sp. strain MF2 by phenotypic characterization and 16S rRNA analysis. The strain could tolerate up to 600 mM of chromate and completely reduced 0.2 mM highly toxic and soluble Cr(VI) (as CrO₄²⁻) into almost non-toxic and insoluble Cr(III) in 24 h under aerobic condition.The maximum chromate removal was exhibited in 1.5 M NaCl at 35 °C and pH 8.0. Initial Cr(VI) concentration until 0.4 mM did not have a significant effect on Cr(VI) reduction. The isolate was capable of chromate reduction in the presence of various concentrations of salts. The chromate reduction corresponded with growth of bacteria and reached a maximum level at the end of exponential phase.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

Production of recombinant ghost bacterial vaccine against streptococcal disease of olive flounder

The simple fed-batch fermentation was carried out to produce E. coli XL1-Blue/pHCE-InaN-GAPDH Ghost 37 SDM as a ghost bacterial vaccine (GBV). The fermentation was carried out in four phases of batch fermentation (phase 1), fed-batch fermentation with intermittent feeding strategy (phase 2), thermal induction by temperature increase to 42 °C for the expression of lysis E gene (GBV formation, phase 3) and high temperature holding phase to increase the efficiency of GBV formation (phase 4). After the high temperature holding phase at 47 °C, efficiency of the GBV formation reached 99.7% with the culture OD₆₀₀ of 57.9. The maximum GBV of 22 g dcw/l was obtained. The protective efficacy of GBV was determined by a challenge test to immunized olive flounder using live Streptococcus iniae. In 14 days of challenge test, the positive and E. coli strain control groups showed 100% cumulative mortalities. Test groups immunized by formalin killed cell (FKC) vaccine, GBV with 42 °C and 47 °C heat shock showed 66%, 54% and 54% of cumulative mortalities, respectively. These results suggest that GBV showed the effectiveness for the protection from the streptococcal infection and had higher potential to induce protective antibodies than FKC vaccine.     

Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats

AimsCardiovascular disease (CVD) is common in chronic kidney disease (CKD) patients. Indoxyl sulfate (IS) is a nephrovascular uremic toxin that induces oxidative stress in kidney and vascular system. The present study aimed to examine the effect of IS on fibrosis and oxidative stress in rat heart.Main methodsThe effects of IS on heart were examined by Masson's trichrome (MT) staining and immunohistochemistry using: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive IS-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive IS-administered rats (DH + IS).Key findingsDH + IS rats showed significantly increased heart weight and left ventricle weight compared with DN. DH and DH + IS rats showed significantly increased diameter of cardiomyocytes, increased MT-positive fibrotic area, increased staining for transforming growth factor (TGF)-β1, α-smooth muscle actin (SMA), type 1 collagen, NADPH oxidase Nox 4, malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and decreased staining for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the heart compared with DN. More notably, DH + IS rats showed significantly increased diameter of cardiomyocytes, increased fibrotic area, increased staining for TGF-β1, SMA, type 1 collagen, Nox4, 8-OHdG and MDA, and decreased staining for Nrf2 and HO-1 in the heart compared with DH.SignificanceIS aggravates cardiac fibrosis and cardiomyocyte hypertrophy with enhanced oxidative stress and reduced anti-oxidative defense in hypertensive rats.     

Effect of heating rate on pDNA production by E. coli

The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (Q_P) and average pDNA yield (Y_p/x) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Y_p/x remained relatively constant, whereas Q_P tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.