Automated ‘X‐Y’ robot for peptide synthesis with microwave heating: application to difficult peptide sequences and protein domains

Precise microwave heating has emerged as a valuable method to aid solid‐phase peptide synthesis (SPPS). New methods and reliable protocols, as well as their embodiment in automated instruments, are required to fully use this potential. Here we describe a new automated robotic instrument for SPPS with microwave heating, report protocols for its reliable use and report the application to the synthesis of long sequences, including the β‐amyloid 1‐42 peptide. The instrument is built around a valve‐free robot originally developed for parallel peptide synthesis, where the robotic arm transports reagents instead of pumping reagents via valves. This is the first example of an ‘X‐Y’ robotic microwave‐assisted synthesizer developed for the assembly of long peptides. Although the instrument maintains its capability for parallel synthesis at room temperature, in this paper, we focus on sequential peptide synthesis with microwave heating. With this valve‐free instrument and the protocols developed for its use, fast and efficient syntheses of long and difficult peptide sequences were achieved. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Response to commentary: does before bedtime body warming by bathing or other means attenuate sleep-time arterial blood pressure?

ABSTRACT

The commentary by Dr. Smolensky provides an insightful assessment of our study from the circadian point of view. In the present letter, we have responded to the commentary by Dr. Smolensky et al., suggesting interpretation of our analytic results and providing additional statistical data regarding the timing of antihypertensive drug use. In addition, we have discussed the terms “nighttime” and “sleep-time” period used in previous researches in relation to ambulatory blood pressure.     

A review of antenna designs for percutaneous microwave ablation

Microwave (MW) antenna is a key element in microwave ablation (MWA) treatments as the means that energy is delivered in a focused manner to the tumor and its surrounding area. The energy delivered results in a rise in temperature to a lethal level, resulting in cell death in the ablation zone. The delivery of energy and hence the success of MWA is closely dependent on the structure of the antennas. Therefore, three design criteria, such as expected ablation zone pattern, efficiency of energy delivery, and minimization of the diameter of the antennas have been the focus along the evolution of the MW antenna. To further improve the performance of MWA in the treatment of various tumors through inventing novel antennas, this article reviews the state-of-the-art and summarizes the development of MW antenna designs regarding the three design criteria.     

Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.     

Amino acid sequences and solution structures of manganese stabilizing protein that affect reconstitution of Photosystem II activity

This minireview presents a summary of information available on the secondary and tertiary structure of manganese stabilizing protein (MSP) in solution, and on the identity of amino acid residues that affect binding and functional assembly of this protein into Photosystem II. New data on the secondary structure of C-terminal mutants and 90 °C-heated manganese stabilizing protein, along with earlier data on the secondary structure of N-terminal mutants and the tertiary structure of all modified MSP species, allow for an evaluation of models for spinach MSP secondary and tertiary structure. This summary of previous and new information better documents the natively unfolded behavior of the protein in solution. A two-step mechanism for binding of manganese stabilizing protein to Photosystem II is discussed and possible solution three-dimensional conformations of the wild-type protein and some of its unfolded mutants, are proposed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of cooling, cryopreservation and heating on sperm proteins, nuclear DNA, and fertilization capability in mouse

In the present study, we used confocal microscopy and electrophoresis to study the effects of heating to 5 or 100 degrees C or cooling to 4 degrees C or -- 196 degrees C on the stability of sperm proteins and DNA. We used intracytoplasmic sperm injection (ICSI) to determine the fertilizing capability of treated spermatoza. It was shown that sperm cryopreservation at - 196 degrees C or cooling at 4 degrees C altered neither protein and DNA profiles nor the sperm fertilization capability, while the protein and DNA profiles of sperm heated at 100 degrees C were irreversibly degraded and inactivated. The proteins of sperm were severely damaged while the nuclear DNA still maintained its integrity when heated to 58 degrees C. Observation by laser confocal microscopy showed that after being heated to 58 degrees C and 100 degrees C, the nuclear of mouse sperm lost their ability to activate oocytes and they could not transform to male pronuclei though the membrane of some sperm could degrade and induce the formation of sperm asters in ICSI oocytes. The results indicate that the use of 58 degrees C heating only causes the degradation of sperm proteins, while the 100 degrees C heating elicits the irreversible degradation of both sperm proteins and nuclear DNA, and the damage of sperm proteins is primarily responsible for the observed decrease in sperm fertilizing capability.     

Convective water exchanges during differential cooling and heating: implications for dissolved constituent transport

Convective water exchange patterns, determined from water temperature variations, were examined in the Minky Creek embayment of Guntersville Reservoir, Alabama (USA), during the month of September, 1990. During periods of differential cooling, cooler water originating from shallow stations moved as an underflow current toward the center of the embayment, while warmer water moved as an overflow current toward the shore. During periods of differential heating, convective exchange was much more shallow, occurring in the upper 3 m of the water column. As warmer water from shallow regions moved out as an overrflow current, this water was replaced by a return flow of cooler water originating from the pelagic epilimnion. Wind speed appeared to influence convective exchange patterns during differential heating phases by affecting the depth of the surface mixed layer. The potential for convective exchanges during periods of differential cooling and heating occurred on 54% and 74% of the days, respectively, in September. This high potential for horizontal water exchanges in Guntersville Reservoir has strong implications for the lateral transport of dissolved constituents.     

Influence of microwave irradiation on enzymatic properties: applications in enzyme chemistry

Although microwave-assisted reactions are widely applied in various domains of organic chemistry, their use in the area of enzyme chemistry has been rather limited, due to the high temperatures associated with the microwave heating: Because current technology, allows a good control of reaction parameters, several examples of microwave-assisted enzyme chemistry have been reported, using stable and effective biocatalysts (modified enzymes). The purpose of this review is to highlight the applications and studies on the influence of microwave irradiation on enzymatic properties and their application in enzyme chemistry.     

Climate dependent heating efficiency in the common lizard

Regulation of body temperature is crucial for optimizing physiological performance in ectotherms but imposes constraints in time and energy. Time and energy spent thermoregulating can be reduced through behavioral (e.g., basking adjustments) or biophysical (e.g., heating rate physiology) means. In a heterogeneous environment, we expect thermoregulation costs to vary according to local, climatic conditions and therefore to drive the evolution of both behavioral and biophysical thermoregulation. To date, there are limited data showing that thermal physiological adjustments have a direct relationship to climatic conditions. In this study, we explored the effect of environmental conditions on heating rates in the common lizard (Zootoca vivipara). We sampled lizards from 10 populations in the Massif Central Mountain range of France and measured whether differences in heating rates of individuals correlated with phenotypic traits (i.e., body condition and dorsal darkness) or abiotic factors (temperature and rainfall). Our results show that heat gain is faster for lizards with a higher body condition, but also for individuals from habitats with higher amount of precipitation. Altogether, they demonstrate that environmentally induced constraints can shape biophysical aspects of thermoregulation.     

Integrated generation of solid fuel and biogas from green cut material from landscape conservation and private households

Green cut material is a potential source of renewable energy which is not fully exploited through conventional energy recovery systems. A new energy conversion process, the integrated generation of solid fuel and biogas from biomass (IFBB), which includes mechanical separation after hydro-thermal conditioning, was investigated. Ash softening temperature and lower heating value of the solid fuel were increased through the IFFB process in comparison to the untreated raw material. The net energy yield of IFBB at 40 °C conditioning temperature ranged between 1.96 and 2.85 kWh kg(-1) dry matter (DM) and for the direct combustion between 1.75 and 2.65 kWh kg(-1) DM. Conversion efficiencies for the IFBB system were 0.42-0.68 and for direct combustion 0.42-0.63. The IFBB system produces storable energy from material which is nowadays not used for energy conversion.