Engineering multiple properties of a protein by combinatorial mutagenesis

A method for simultaneously engineering multiple properties of a protein, based on the observed additivity of effects of individual mutations, is presented. We show that, for the gene V protein of bacteriophage f1, effects of double mutations on both protein stability and DNA binding affinity are approximately equal to the sums of the effects of the constituent single mutations. This additivity of effects implies that it is possible to deliberately construct mutant proteins optimized for multiple properties by combination of appropriate single mutations chosen from a characterized library.     

Physiological deterioration of cassava roots

Rapid post-harvest physiological deterioration of cassava roots (Manihot esculenta Crantz) appears to be due essentially to wound responses, comparable to those observed in other plant storage organs. The wound responses observed include: increased activity of phenylalanine ammonia lyase, an enzyme associated with phenol biosynthesis; increased activity of peroxidase and polyphenol oxidase; formation of phenols/polyphenols including leucoanthocyanidins, catechins, sco-poletin and condensed tannins, and often the formation of a wound periderm. In cassava, the responses did not remain localised at wound surfaces in roots when held at low storage humidity but spread through the roots causing a discolouration of the vascular tissue and storage parenchyma. Roots stored at high humidity showed a more typical wound response with localised production of phenols and periderm formation.     

High dietary calcium level decreases colonic phytate degradation in pigs fed a rapeseed diet

The degradation of phytate (inositol hexaphosphate) in rapeseed meal diet not containing phytase activity was studied in 15 growing ileum-fistulated pigs. Stomach and small intestinal degradation and total gastrointestinal degradation were compared. The effect of addition of calcium carbonate to the rapeseed meal diet at two levels (9.2 and 18.5 g/kg diet) was investigated. A commercial barley-wheat-soybean diet with intrinsic phytase activity was used as reference. Phytate and its hydrolysis products in diets, ileal digesta and feces were determined by HPLC ion-pair chromatography. Hydrolysis of phytate in the stomach and small intestine was 35-45% in pigs fed the rapeseed meal diet independent of calcium addition, and 65% in pigs fed the reference diet. Total gastrointestinal degradation of phytate in pigs fed the rapeseed diet was 97, 77 and 42% (P < 0.001) when calcium intakes were 4.5, 9.9 and 15 g/d, respectively; total gastrointestinal degradation was 72% in pigs fed the reference diet. The intestinal phytate degradation pattern, when rapeseed diet was fed, indicated the activity of an unspecific phosphatase, whereas that of the reference diet indicated intrinsic dietary phytase activity. We conclude that dietary supplementation of calcium carbonate decreases the phytate degradation in the colon of pigs, but not in the stomach and small intestine.     

Improved methods for evaluating the molar mass distributions of cellulose in kraft pulp

Multi‐angle laser light scattering (MALLS) was used to characterize birch kraft pulps with respect to their absolute molecular mass distributions (MMDs). The pulps were dissolved in lithium chloride/N,N‐dimethylacetamide and separated by size exclusion chromatography (SEC). The weight‐average and number‐average molecular masses of the cellulose fractions of the pulps obtained from the absolute MALLS measurements were compared with the molar masses obtained by direct‐standard‐calibration relative pullulan standards. Discrepancies between the two detection methods were found, and two ways of correlating the relative pullulan molar masses to the absolute molar masses were examined. In the first method, the correlation was made over a large range of molecular masses. The second method correlated the molecular masses of the standards to the molecular masses of samples by the calculation of fictitious, cellulose‐equivalent molar masses of the standards. With the preferred second method, a more correct MMD of kraft pulp samples could, therefore, be obtained from an SEC system calibrated with narrow standards. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 1170–1179, 2003     

Mitigating Detrimental Effect of Self-Doping Near the Anode in Highly Efficient Organic Solar Cells

Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) has been one of the most established hole transport layers (HTL) in organic solar cells (OSCs) for several decades. However, the presence of PSS⁻ ions is known to deteriorate device performance via a number of mechanisms including diffusion to the HTL-active layer interface and unwanted local chemical reactions. In this study, it is shown that PSS⁻ ions can also result in local p-doping in the high efficiency donor:non-fullerene acceptor blends – resulting in photocurrent loss. To address these issues, a facile and effective approach is reported to improve the OSC performance through a two-component hole transport layer (HTL) consisting of a self-assembled monolayer of 2PACz ([2-(9H-Carbazol-9-yl)ethyl]phosphonic acid) and PEDOT:PSS. The power conversion efficiency (PCE) of 17.1% using devices with PEDOT:PSS HTL improved to 17.7% when the PEDOT:PSS/2PACz two-component HTL is used. The improved performance is attributed to the overlaid 2PACz layer preventing the formation of an intermixed p-doped PSS⁻ ion rich region (≈5–10 nm) at the bulk heterojunction-HTL contact interface, resulting in decreased recombination losses and improved stability. Moreover, the 2PACz monolayer is also found to reduce electrical shunts that ultimately yield improved performance in large area devices with PCE enhanced from 12.3% to 13.3% in 1 cm² cells.     

Correction of congenital diaphragmatic hernia in utero IX: fetuses with poor prognosis (liver herniation and low lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion

BACKGROUND/PURPOSE: Fetuses with congenital diaphragmatic hernia (CDH) who have a "poor prognosis" with postnatal treatment now can be identified on the basis of liver herniation, early diagnosis (before 25 weeks' gestation) and a low lung-to-head ratio (LHR). Because complete in utero repair proved unsuccessful for this group, the strategy of temporary tracheal occlusion was developed to gradually enlarge the hypoplastic fetal lung. The purpose of this study is to compare the outcome of patients in the poor-prognosis group treated by one of three methods: (1) standard postnatal care, (2) fetal tracheal occlusion via open hysterotomy, and (3) the recently developed video-fetoscopic (Fetendo) technique of tracheal occlusion without hysterotomy. METHODS: In the past 3 years, 34 of 86 fetuses with an isolated left CDH met criteria for the poor-prognosis group. Thirteen families chose postnatal treatment at an extracorporeal membrane oxygenation (ECMO) center, 13 underwent open fetal tracheal occlusion, and eight underwent fetoscopic tracheal occlusion. RESULTS: The survival rate was 38% in the group treated by standard postnatal therapy, 15% in the open tracheal occlusion group, and 75% in the Fetendo group. There were less postoperative pulmonary complications noted in mothers who underwent the Fetendo procedure versus the open tracheal occlusion. All but one Fetendo clip patient had a striking physiological response demonstrated by sonographic enlargement of the small left lung that was documented postnatally by plain radiographs and its subjective appearance during repair of the CDH. In contrast, only 5 of the 13 open tracheal occlusion patients demonstrated lung growth. CONCLUSION: Fetuses with a left CDH who have liver herniation and a low LHR are at high risk of neonatal demise and appear to benefit from temporary tracheal occlusion when performed fetoscopically, but not when performed by open fetal surgery.     

Deadlock free routing algorithms for irregular mesh topology NoC systems with rectangular regions

The simplicity of regular mesh topology Network on Chip (NoC) architecture leads to reductions in design time and manufacturing cost. A weakness of the regular shaped architecture is its inability to efficiently support cores of different sizes. A proposed way in literature to deal with this is to utilize the region concept, which helps to accommodate cores larger than the tile size in mesh topology NoC architectures. Region concept offers many new opportunities for NoC design, as well as provides new design issues and challenges. One of the most important among these is the design of an efficient deadlock free routing algorithm. Available adaptive routing algorithms developed for regular mesh topology cannot ensure freedom from deadlocks. In this paper, we list and discuss many new design issues which need to be handled for designing NoC systems incorporating cores larger than the tile size. We also present and compare two deadlock free routing algorithms for mesh topology NoC with regions. The idea of the first algorithm is borrowed from the area of fault tolerant networks, where a network topology is rendered irregular due to faults in routers or links, and is adapted for the new context. We compare this with an algorithm designed using a methodology for design of application specific routing algorithms for communication networks. The application specific routing algorithm tries to maximize adaptivity by using static and dynamic communication requirements of the application. Our study shows that the application specific routing algorithm not only provides much higher adaptivity, but also superior performance as compared to the other algorithm in all traffic cases. But this higher performance for the second algorithm comes at a higher area cost for implementing network routers.     

Steady-state bioavailability and day-to-day variability of a multiple-unit (CR/ZOK) and a single-unit (OROS) delivery system of metoprolol after once-daily dosing

Home blood pressure (BP) monitoring is useful in the clinical management of patients with hypertension and the identification of those with "white-coat" hypertension; i.e. high readings in the clinic but normal BP at home. In the process of evaluating this technique, we compared self-measured home BP with intra-arterial BP. Healthy white men (n = 40) of 20-40 years of age and body weight below 95 kg were recruited by advertising in the local newspaper. Following a standardized procedure, performed within 2-4 weeks of a response to the advertisement, BP was measured by a physician at a clinic screening, by the subject at home (14 readings in 7 days) and finally in the clinic concomitantly intra-arterially and oscillometrically. The correlation coefficient for mean (M) home BP (r = 0.73) and oscillometric BP (r = 0.74) against intra-arterial BP were slightly higher than for screening BP (r = 0.65). However, in plots of the differences for individual MBP between the methods against the average of the methods, it appears that at levels of average MBP above 100 mmHg, screening BP overestimates the BP level, while this was not the case for home BP or oscillometric BP. Thus, by using intra-arterial measurement as standard of comparison, subject self-measured home BP is a reliable method of estimating blood pressure level in young men. Home BP measured shortly after screening and recruitment provides useful information of resting BP in subjects who potentially may have initial anxiety about BP measurement.     

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.