Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin

We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.     

The effects of different roster schedules on sleep in miners

Shiftwork involving early morning starts and night work can affect both sleep and fatigue. This study aimed to assess the impact of different rostering schedules at an Australian mine site on sleep and subjective sleep quality. Participants worked one of four rosters;

4 × 4 (n = 14) 4D4O4N4O

7 × 4 (n = 10) 7D4O7N40

10 × 5 (n = 17) 5D5N50

14 × 7 (n = 12) 7D7N70

Sleep (wrist actigraphy and sleep diaries) was monitored for a full roster cycle including days off. Total sleep time (TST) was longer on days off (7.0 ± 1.9) compared to sleep when on day (6.0 ± 1.0) and nightshifts (6.2 ± 1.6). Despite an increase in TST on days off, this may be insufficient to recover from the severe sleep restriction occurring during work times. Restricted sleep and quick shift-change periods may lead to long-term sleep loss and associated fatigue.     

DynaMICs: Comprehensive Support for Run-Time Monitoring

Software engineering strives to enable the economic construction of software systems that behave reliably, predictably, and safely. In other engineering disciplines, safety is assured in part by detailed monitoring of processes. In software, we may achieve some level of confidence in the operation of programs by monitoring their execution. DynaMICs is a software tool that facilitates the collection and use of constraints for software systems. In addition, it supports traceability by mapping constraints to system artifacts. Constraint specifications are stored separately from code; constraint-monitoring code is automatically generated from the specifications and inserted into the program at appropriate places; and constraints are verified at execution time. These constraint checks are triggered by changes made to variable values. We describe the architecture of DynaMICs, discuss alternative verification techniques, and outline research directions for the DynaMICs project.     

Simulated train driving: fatigue, self-awareness and cognitive disengagement

Fatigue is a serious issue for the rail industry, increasing inefficiency and accident risk. The performance of 20 train drivers in a rail simulator was investigated at low, moderate and high fatigue levels. Psychomotor vigilance (PVT), self-rated performance and subjective alertness were also assessed. Alertness, PVT reaction times, extreme speed violations (>25% above the limit) and penalty brake applications increased with increasing fatigue level. In contrast, fuel use, draft (stretch) forces and braking errors were highest at moderate fatigue levels. Thus, at high fatigue levels, errors involving a failure to act (errors of omission) increased, whereas incorrect responses (errors of commission) decreased. The differential effect of fatigue on error types can be explained through a cognitive disengagement with the virtual train at high fatigue levels. Interaction with the train reduced dramatically, and accident risk increased. Awareness of fatigue-related performance changes was moderate at best. These findings are of operational concern.     

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.     

Computer tracking of objects moving in space

A method is developed to represent movement of convex blocks in three-dimensional space from a sequence of two-dimensional camera images. The goals are to determine the objects' movement toward or away from the camera as well as left/right and up/down movement in the image plane and to build models of the blocks. The movement information is used as part of a hierarchical matching process that determines the correspondence of blocks between scenes.     

Synthesis and isolation of <Emphasis Type="Italic">trans</Emphasis>-7, <Emphasis Type="Italic">cis</Emphasis>-9 octadecadienoic acid and other CLA isomers by base conjugation of partially hydrogenated γ-linolenic acid

CLA is of considerable interest because of reported potentially beneficial effects in animal studies. CLA, while not yet unambiguously defined, is a mixture of octadecadienoic acids with conjugated double bonds. The major isomer in natural products is generally considered to be cis-9,trans-11-octadecadienoic acid (c9, t11), which represents >75% of the total CLA in most cases. Other isomers are drawing increased attention. The t7,c9 isomer, which is often the second-most prevalent CLA in natural products, has been reported to represent as much as 40% of total CLA in milk from cows fed a high-fat diet. The need for a reference material became apparent in a recent study directed specifically at measuring t7,c9-CLA in milk, plasma, and rumen. A suitable standard mixture was produced by stirring 0.5 g of γ-linolenic acid (all cis-6,9, 12-C_18∶3) with 100 mL of 10% hydrazine hydrate in methanol for 2.5 h at 45°C. The solution was diluted with H₂O and acidified with HCl. The resulting partially hydrogenated FA were extracted with ether/petroleum ether, dried with Na₂SO₄, and conjugated by adding of 6.6% KOH in ethlylene glycol and heating for 1.5 h at 150–160°C. Approximately 20 mg each of cis-6, trans-8; trans-7, cis-9; cis-9, trans-11; and rans-10, cis-12 were obtained along with other FA. Methyl esters (FAME) of these four cis/trans isomers were resolved by Ag⁺HPLC (UV 233) and partially resolved by GC/(MS or FID) (CP-Sil 88). Treatment of these FAME with I₂ yielded all possible cis/trans (geometric) isomers for the four positions 6,8; 7,9; 9,11; and 10,12.