Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH₄Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.     

Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.     

Pilot-scale production of mosquitocidal toxins by Bacillus thuringiensis and Lysinibacillus sphaericus under solid-state fermentation

Mosquitocidal toxins of Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaericus 14N1 (Ls14N1) were produced under solid-state fermentation using agro-industrial wastes. Sugar beet pulp–sesame meal (1:1) and wheat germ meal–linen meal (1:1) at 9% were the efficient substrate mixtures for the growth and toxin production of Bti and Ls14N1, respectively. Bti was more active after the addition of beef extract (0.2%) or yeast extract (0.5%) to the medium. On the other hand, the addition of yeast extract (0.2%) or NYSM salts (2%) significantly enhanced the toxicity produced by Ls14N1. The optimum conditions for the maximum toxicity of Bti were at pH 7–8, 20–30% moisture, 4–10% inoculum and 7 days incubation. For Ls14N1, the best conditions were pH 6.5–7.5, 20–30% moisture, 4–10% inoculum and 5 days incubation. It was found that the best thickness of carrier-substrates in the plate (15?cm in diameter) for the maximum mosquitocidal activity was about 0.5?cm for Bti and 0.5–1?cm for Ls14N1. Pilot-scale production in aluminium trays applying the above conditions showed a decrement of toxicity of fermented cultures and some plates were contaminated. These problems were dissolved by reducing the moisture content to 15%, increasing inoculum to 10% and manual agitation of trays every-day.     

Identifying delayed left ventricular lateral wall activation in patients with non-specific intraventricular conduction delay using coronary venous electroanatomical mapping

Background

Delayed left ventricular (LV) lateral wall activation is considered the electrical substrate that characterises patients suitable for cardiac resynchronisation therapy (CRT). Although typically associated with left bundle branch block, delayed LV lateral wall activation may also be present in patients with non-specific intraventricular conduction delay (IVCD). We assessed LV lateral wall activation in a cohort of CRT candidates with IVCD using coronary venous electroanatomical mapping, and investigated whether baseline QRS characteristics on the ECG can identify delayed LV lateral wall activation in this group of patients.

Methods

Twenty-three consecutive CRT candidates with IVCD underwent intra-procedural coronary venous electroanatomical mapping using EnSite NavX. Electrical activation time was measured in milliseconds from QRS onset and expressed as percentage of QRS duration. LV lateral wall activation was considered delayed if maximal activation time measured at the LV lateral wall (LVLW-AT) exceeded 75 % of the QRS duration. QRS morphology, duration, fragmentation, axis deviation, and left anterior/posterior fascicular block were assessed on baseline ECGs.

Results

Delayed LV lateral wall activation occurred in 12/23 patients (maximal LVLW-AT = 133 ± 20 ms [83 ± 5 % of QRS duration]). In these patients, the latest activated region was consistently located on the basal lateral wall. QRS duration, and prevalence of QRS fragmentation and left/right axis deviation, and left anterior/posterior fascicular block did not differ between patients with and without delayed LV lateral wall activation.

Conclusion

Coronary venous electroanatomical mapping can be used at the time of CRT implantation to determine the presence of delayed LV lateral wall activation in patients with IVCD. QRS characteristics on the ECG seem unable to identify delayed LV lateral wall activation in this subgroup of patients.     

Overproduction of a mosquitocidal chloramphenicol-resistant Lysinibacillus sphaericus mutant obtained through UV irradiation

A highly active mosquitocidal mutant UV-chloramphenicol-resistant mutant (UCR-146) of Lysinibacillus sphaericus 2362 was isolated by UV irradiation and selected through resistance to chloramphenicol which was inhibitory for growth of the parent strain. The growth of UCR-146 in NYSMCL at different concentrations of chloramphenicol (20–100 µg/ml) showed high mosquitocidal activity against Culex pipiens larvae with the optimum concentration of 35µg/ml. At this level, LC₅₀ of UCR-146 was decreased about five times from that of L. sphaericus 2362. Comparative efficiency of mosquitocidal activity of UCR-146 and L. sphaericus 2362 within 28 days of consecutive growth exhibited notable stability of both cultures through seven cycles of growth in their optimal media. UCR-146 was grown in industrial by-products media for production of the binary toxin under economic conditions. Offal medium at 2% showed the highest mosquitocidal activity of UCR-146 with LC₅₀ of 0.53 PPM which was 16.6, 13, 12.3 and 3.4 times less than that produced with L. sphaericus 2362 grown in NYSM, L. sphaericus 2362 grown in offal, UCR-146 grown in NYSM and UCR-146 grown in NYSMCL, respectively. Hence, the mosquitocidal activity of L. sphaericus 2362 increased several times through ultraviolet (UV) irradiation followed by chloramphenicol resistance selection and then growth in 2% offal medium. Finally, this procedure for selecting UV-chloramphenicol-resistant mutant and the medium used could potentially be a simple and cost effective approach for obtaining and producing a highly active mosquitocidal mutant.     

Production of a chloramphenicol-resistant mutant of Lysinibacillus sphaericus by solid state fermentation

A highly active mosquitocidal mutant of Lysinibacillus sphaericus Ahmed 2362, namely, UCR-146, was efficiently produced on cottonseed meal (CSM) medium, using sand as a carrier under solid state fermentation (SSF). The optimum CSM concentration for the highest sporulation and toxin formation was 12%. The maximum toxicity of the tested organism against second instar larvae of Culex pipiens was obtained at 25% moisture content, initial pH 6–7, 1% sodium acetate, 18.9×10⁶ CFU/g inoculum and 6 days incubation period at 30°C. Pilot scale production of UCR-146 under the optimum SSF conditions was assessed in aluminium trays. Spore count, mortality of larvae and LC₅₀ of the final product were 5.5×10¹⁰ CFU/g, 72% at 1 part per million (PPM) and 0.54 PPM, respectively. These results were comparable with those obtained from bench-scale production (in flasks). The cost of 1 kg of this bio-larvicide was estimated at US $0.34.     

Evaluation of biological protein-based collagen scaffolds in cartilage and musculoskeletal tissue engineering--a systematic review of the literature

The term tissue engineering is the technology that combines cells, engineering and biological/synthetic material in order to repair, replace or regenerate biological tissues such as bone, muscle, tendons and cartilage. The major human applications of tissue engineering are: skin, bone, cartilage, corneas, blood vessels, left mainstem bronchus and urinary structures. In this systematic review several criteria were identified as the most desirable characteristics of an ideal scaffold. These state that an ideal scaffolds needs to be biodegradable, possess mechanical strength, be highly porous, biocompatible, non-cytotoxic, non antigentic, stuitable for cell attachment, proliferation and differentiation, flexible and elastic, three dimensional, osteoconductive and support the transport of nutrients and metabolic waste. Subsequently, studies reporting on the various advantages and disadvantages of using collagen based scaffolds in musculoskeletal and cartilage tissue engineering were identified. The purpose of this review is to 1) provide a list of ideal characteristics of a scaffold as identified in the literature 2) identify different types of biological protein-based collagen scaffolds used in musculoskeletal and cartilage tissue engineering 3) assess how many of the criteria each scaffold type meets 4) weigh different scaffolds against each other according to their relative properties and shortcomings. The rationale behind this approach is that the ideal scaffold material has not yet been identified. Hence, this review will define how many of the identified ideal characteristics are fulfilled by natural collagen-based scaffolds and address the shortcomings of its use as found in the literature.     

Efficient mosquitocidal toxin production by Bacillus sphaericus using cheese whey permeate under both submerged and solid state fermentations

Whey permeate (WP) was used efficiently for production of mosquitocidal toxin by Bacillus sphaericus 2362 (B. sphaericus 2362) and the Egyptian isolate, B. sphaericus 14N1 (B. sphaericus 14N1) under both submerged and solid state fermentation conditions. Under submerged fermentation, high mosquitocidal activity was produced by B. sphaericus 2362 and B. sphaericus 14N1 at 50-100% and 25-70% WP, respectively. Initial pH of WP was a critical factor for toxin production by both tested organisms. The highest toxicity was obtained at initial pH 7. Egyptian isolate, B. sphaericus 14N1 was tested for growth and toxin production under solid state fermentation conditions (SSF) by using WP as moistening agent instead of distilled water. The optimum conditions for production of B. sphaericus 14N1 on wheat bran-WP medium were 10 g wheat bran/250 ml flask moistened with 10-70% WP at 50% moisture content, inoculum size ranged between 17.2 × 10⁷ and 34.4 × 10⁷ and 6 days incubation under static conditions at 30 °C. Preliminary pilot-scale production of B. sphaericus 14N1 under SSF conditions in trays proved that wheat bran-WP medium was efficient and economic for industrial production of mosquitocidal toxin by B. sphaericus.