Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV

The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)₆], or a combination of GST and (His)₆ [GST-(His)₆] were compared to native EcoRV with respect to expression level, susceptability to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)₆-EcoRV was not observed and 2-D immunoblots did not show heterogenous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)₆-EcoRV fusion protein was intensive when cells were grown at 37°C but not at 30°C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)₆-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.     

Simple and inexpensive devices to measure heart rates of incubating birds

ABSTRACT Heart rate is a useful physiological index for studies of stress, locomotion, and activity patterns. Measuring heart rates of birds without the need to handle individuals is desirable when trapping is problematic or may cause unwanted disturbance. Heart‐rate recorders housed in dummy eggs offer an effective solution, but the usefulness of previously described devices is limited by their size, complex construction, and reliance on analog media. We constructed egg‐based, heart‐rate monitors through simple modifications of inexpensive, commercially available MP3 players and Bluetooth headsets. We compared the merits of each device during tests in the laboratory, an aviary, and the field in 2008. Field testing was undertaken at Presqu’ile Provincial Park, Ontario, Canada, where we recorded heart rates of Common Terns (Sterna hirundo), Caspian Terns (Hydroprogne caspia), and Ring‐billed Gulls (Larus delawarensis). Birds incubated dummy eggs normally, with no differences in behavior (all P > 0.05) when incubating either wired (MP3 players) or wireless (Bluetooth) devices. MP3 devices were more reliable in the field. Bluetooth devices often lost pairing with laptop computers (33% of files analyzed contained no signal), produced files with more obscuring noise, and only two could be deployed simultaneously with a single computer; there was no limit on how many MP3 devices could be deployed simultaneously. Common Terns, the smallest of our three focal species, had significantly higher (P < 0.001) mean heart rates (268.6 ± 9.3 beats per min [bpm]) than either Ring‐billed Gulls (198.0 ± 7.1 bpm) or Caspian Terns (204.2 ± 8.0 bpm). Heart rates of all three species were consistent with those reported or predicted from previous studies. The MP3 devices we describe provide investigators with a simple, inexpensive, and minimally invasive way to digitally record heart rates of birds of almost any size.     

Increasing temperatures increase the risk of reproductive failure in a near threatened alpine ground-nesting bird,the Cape Rockjumper Chaetops frenatus

A major cause of reproductive failure in birds is nest predation. Predation risk depends on predator type, as predators vary in their ecology and sensory modalities (e.g. visual vs. olfactory). Snakes (generally olfactory predators) are a major nest predator for small birds, with predation strongly associated with higher temperatures. We investigated nest survival in a ground-nesting alpine species, the Cape Rockjumper Chaetops frenatus, endemic to alpine fynbos in southwestern South Africa. We collected 3 years of nest data, testing whether nest survival was related to (1) habitat stage (early post-fire vs. late post-fire habitat, ≤ 3 and > 3 years since fire respectively), (2) nest concealment and (3) temperature. We found that nests had better survival at lower temperatures, with snake predation (our main source of predation) increasing in higher temperatures.     

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia

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Heat production during sleep

Heat production during sleep was studied by continuous indirect calorimetry with simultaneous electroencephalographic monitoring. Controlling for gross influences on heat production, comparisons of heat production during different sleep stages showed heat production in stage 4 sleep to be significantly lower than in other sleep stages. There appeared to be a gradation in heat production in non-rapid-eye-movement stages of sleep with stage 2 higher and stage 4 lower than stage 3. Heat production in stage 4 was less variable than in any other sleep stage. Both the level and variability of heat production was similar in stage 2 and rapid-eye-movement sleep. Heat production during the night was 9% lower than during resting wakefulness. The average heat production in stage 4 sleep was 14.4% lower than resting wakeful values.     

Relationship between Breathlessness and Anxiety in Asthma and Bronchitis: A Comparative Study

Two personality testing forms, the Eysenck Personality Inventory Form A and the Cattell Self Analysis Form, were completed on 471 hospital patients who fell within the general diagnostic range of asthma, bronchitis, or both. Respiratory diagnoses were based on the standard M.R.C. questionary. All categories of patients showed a tendency towards neuroticism, anxiety, and introversion, and the scores were slightly higher for bronchitics than for asthmatics. Neuroticism and anxiety increased with increasing respiratory disability. Variations in these scores with age of onset of symptoms and length of history were small.