Rejection Limit Research Articles

Molecular-docking study of malaria drug target enzyme transketolase in Plasmodium falciparum 3D7 portends the novel approach to its treatment.

BackgroundMalaria has been a major life threatening mosquito borne disease from long since. Unavailability of any effective vaccine and recent emergence of multi drug resistant strains of malaria pathogen Plasmodium falciparum continues to cause persistent deaths in the tropical and sub-tropical region. As a result, demands for new targets for more effective anti-malarial drugs are escalating. Transketolase is an enzyme of the pentose phosphate pathway; a novel pathway which is involved in energy generation and nucleic acid synthesis. Moreover, significant difference in homology between Plasmodium falciparum transketolase (Pftk) and human (Homo sapiens) transketolase makes it a suitable candidate for drug therapy. Our present study is aimed to predict the 3D structure of Plasmodium falciparum transketolase and design an inhibitor against it.ResultsThe primary and secondary structural features of the protein is calculated by ProtParam and SOPMA respectively which revealed the protein is composed of 43.3 % alpha helix and 33.04 % random coils along with 15.62 % extended strands, 8.04 % beta turns. The three dimensional structure of the transketolase is constructed using homology modeling tool MODELLAR utilizing several available transketolase structures as templates. The structure is then subjected to deep optimization and validated by structure validation tools PROCHECK, VERIFY 3D, ERRAT, QMEAN. The predicted model scored 0.74 for global model reliability in PROCHECK analysis, which ensures the quality of the model. According to VERIFY 3D the predicted model scored 0.77 which determines good environmental profile along with ERRAT score of 78.313 which is below 95 % rejection limit. Protein-protein and residue–residue interaction networks are generated by STRING and RING server respectively. CASTp server was used to analyze active sites and His 109, Asn 108 and His 515 are found to be more positive site to dock the substrate, in addition molecular docking simulation with Autodock vina determined the estimated free energy of molecular binding was of −6.6 kcal/mol for most favorable binding of 6′-Methyl-Thiamin Diphosphate.ConclusionThis predicted structure of Pftk will serve first hand in the future development of effective Pftk inhibitors with potential anti-malarial activity. However, this is a preliminary study of designing an inhibitor against Plasmodium falciparum 3D7; the results await justification by in vitro and in vivo experimentations.

Low-Temperature Solid-State Bonding Using Hydrogen Radical Treated Solder for Optoelectronic and MEMS Packaging

Many optoelectronic and MEMS devices require hermetic sealing for protection of the optoelectronic elements and movable fragile parts to ensure stable and reliable performance characteristics. Most commonly used bonding techniques for hermetic sealing are glass-frit bonding, anodic bonding, solder bonding, and eutectic bonding. For many applications, low temperature bonding is desirable because the temperature sensitive materials or components degrade and thermally induced stresses can be created during bonding. Furthermore, since the hermetic sealing is typically the final processing step in device fabrication, bonding method that is tolerant to surface roughness is desirable.In this study, a low-temperature solid-state bonding process at bonding temperature lower than 200 °C using hydrogen radical treated Sn-3.0Ag-0.5Cu (wt%) solder without flux is investigated for potential application as hermetic packaging. The interfacial reaction between solder and Au is studied by means of scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX). The bonding quality is evaluated by means of die-shear test and leak rate test.The re-oxidation of hydrogen radical treated Sn was studied by X-ray photoelectron spectroscopy (XPS). Fig. 1 shows the typical XPS spectra of Sn plate (purity: 5N) treated with hydrogen radicals (a) and argon fast atom beam (FAB) (b). The XPS Sn (3d2/5) spectra have been used to follow the surface oxide growth on Sn surfaces as a function of exposure time to air after removal of surface oxide. Two peaks are due to the presence of Sn metal and SnOx. The surface of Sn plate treated with Ar FAB contained a large amount of SnOxthan that with hydrogen radicals after exposure time to air of over 20 h. These results indicate differences between the removal of surface oxide by chemical reaction of hydrogen radicals and that by physical bombardment using fast atom beams. The inhibitory effect of re-oxidation of hydrogen radical treated solder [1] was confirmed by XPS measurement.Fig. 2 shows the process flow of the low-temperature solid-state bonding. Sn-Ag-Cu solder paste (melting point: 217 °C) is deposited by screen printing technique. Then, the surface oxides of Sn-Ag-Cu particles in the paste are reduced by hydrogen radical treatment at 180 ºC. After that, the solder is reflowed at 230 °C for 90 s with additional hydrogen radical treatment for 3 sec. During the reflow process, the Sn-Ag-Cu particles completely coalesce and solder pattern is formed on the wettable metal parts of the substrate [2]. Then, the Sn-Ag-Cu patterns are exposed to air and bonded with Au films without flux at temperatures below the melting point.Successful bonding was obtained at a bonding temperature above 150 °C for 10 min. Fig. 3 shows the SEM image and EDX map of the cross section of the bonded interface under the application of a bonding pressure of 150 MPa and held at 170 °C for 30 min. The Sn-Ag-Cu solder pattern was deformed to achieve enough contact with Au thin film (thickness: 500 nm) and Au-Sn intermetallic compounds were formed during bonding process as shown in Fig. 3(b). No clear interface corresponding to the original Au surfaces is visible. To evaluate the bonding strength, die-shear test was performed and the die shear strength was over 30 MPa. Fracture mainly occurred in the inside of solder layer, which indicates that solder layer was well bonded with Au.The Sn-Ag-Cu sealing ring pattern (width: 100 μm, size: 2.4 mm × 2. 4 mm, thickness: 5-10 μm) was formed on a glass substrate (2.8 × 2.8 mm2) by hydrogen radical reflow process for hermetic leak rate testing. The cavity structure (cavity volume: 1.9 × 10-3 cm3) with Ti (thickness: 50 nm)/Au (thickness: 500 nm) sealing ring was also fabricated on a Si substrate. Hermeticity was evaluated by measuring the resonance characteristics of photothermally excited microcantilevers inside the cavity [3]. The samples bonded at the low temperature of 170 °C under the application of a bonding pressure of 150 MPa showed leakage rates of less than 1.0 × 10−9 Pa·m3 s−1, which is the rejection limit defined by the MIL-STD-883G specification.In conclusion, hydrogen radical treated Sn-Ag-Cu solder patterns were bonded with Au thin films at a lower temperature than the melting point of Sn-Ag-Cu without flux. This process is expected to be applicable for chip-size packaging of optoelectronic and MEMS devices.

ABR in newborns: Effects of electrode configuration, stimulus rate, and EEG rejection levels on test efficiency

Objective: This study investigated the effect of electrode configuration, stimulus rate, and EEG rejection level on the efficiency of ABR testing in babies. Design: ABR to click stimuli at 40 dB nHL were simultaneously recorded from two electrode configurations, ipsilateral mastoid to high forehead (Mi-Fh) and nape to high forehead (N-Fh), with two EEG rejection levels (± 5 μV and ± 10 μV). Stimulus rates were between 39.1 and 69.1 per second. Efficiency was measured by confidence in the ABR for a given test time. Study sample: Thirty babies who had passed a targeted newborn hearing screen with ABR thresholds ≤ 40 dB nHL. Results: The N-Fh configuration, as expected, gave on average a larger response amplitude compared to the Mi-Fh configuration but was only marginally significantly better in terms of test efficiency. There was no significant effect of stimulus rate on test efficiency between 39.1/s and 59.1/s. The lower ± 5 μV EEG rejection level was more test efficient. Conclusions: This study provides some evidence that, for ABR threshold testing in babies, alternatives of ipsilateral mastoid or nape electrode and a range of stimulus rates have little or no effect on test efficiency. The results support the use of low EEG rejection limits.

Cu–Cu Bond Quality Enhancement Through the Inclusion of a Hermetic Seal for 3-D IC

In this paper, we investigate the formation of hermetic seal, electrical contact, and mechanical support simultaneously through Cu-Cu bonding. Two types of test vehicles are designed and fabricated using wafer-to-wafer Cu thermocompression bonding to characterize the hermeticity and electrical contact of the Cu-Cu bonds. Excellent helium-leak rate is found and it is 30× lower than the reject limit (5×10-8atm.cm3/sec) with the application of seal ring with a width of ≥20 μm. This result shows an outstanding bonding quality against harsh environment for hermetic encapsulation in a 3-D integration application. In addition, the electrical stability of Kelvin structure is greatly improved with the inclusion of the Cu-Cu seal. FIB analysis shows that the Cu-Cu bond surrounded by the Cu-Cu seal results in remarkable reduction in imperfections at the bonding interface. Finally, high-density Cu-Cu bonding structures exhibit advantageous stability with the protection of the hermetic seal. This promotes Cu-Cu bonding as a reliable option for wafer level 3-D integration.

Lung xenogenic injury: does anticoagulation help?

The shortage of human organ donors remains a major limitation to the field of transplantation [1], and can potentially be solved using xenografts. During the past decade, many advances have been made to address the major initial immunological obstacle to “discordant” pig‐to‐human solid organ transplantation: hyperacute rejection (HAR). As organs from unmodified pigs were evaluated in animal models, HAR was found to be primarily a consequence of the recipient's preformed anti‐pig antibodies binding on porcine vascular endothelial cells leading to subsequent complement activation, thrombosis and graft failure [2]. This process occurs within minutes to hours of human blood perfusion in porcine organs. Since the carbohydrate structure Galactose‐α(1,3‐Galactose (Gal) is recognized by over 80% of anti‐pig antibodies found in man, genetically modified galactosyl transferase knock‐out (GalTKO) pig organs have been developed [3,4]. Endothelium and parenchymal cells from GalTKO animals lack the Galα1,3Gal epitope. As predicted, heart and kidney transplant studies in baboons showed that the GalTKO phenotype is associated with decreased antibody binding, reduced activation of the complement cascade and prolonged graft survival [5–8]. However, delayed xenograft rejection, consumptive coagulopathy and microangiopathy limit long‐term outcomes. Xenogenic lungs are even more sensitive to xenogenic injury [9, 10], even with additional expression of complement regulatory protein human CD46 [Burdorf et al]. Immunohistology and biochemical evidence and work by others implicated inflammation and coagulation cascade activation as residual xenogenic injury pathways. To determine the role of these pathways in lung xenogenic injury, our group is evaluating new transgenic pigs [11] and various pharmacologic approaches. New transgenes include human thrombomodulin (hTM), endothelial protein C receptor (EPCR) and ectonucleoside triphosphate diphosphohydrolase‐1 (CD39). Pharmacologic interventions consist in targeting platelet receptors (GPIb, GPIIbIIIa), thrombin (hirulog) and adding exogenous activated protein C to perfused human blood and administering desmopressin to lung donor pigs. The differential effects of these interventions on lung physiological parameters, platelet and coagulation activation will be summarized. Future work will place emphasis on combined targeting of these pathways.Acknowledgments: Funded in part by NIH (IU19A1090959, 1U01AI066335) and by gifts from Revivicor and United Therapeutics.

Effect of bonding temperature on hermetic seal and mechanical support of wafer-level Cu-to-Cu thermo-compression bonding for 3D integration

Hermetic seal and mechanical support of wafer-level Cu-to-Cu thermo-compression bonding with different bonding temperature are analyzed in this work. The investigation consists of two parts: hermetic seal study using helium bomb test and mechanical support study using four-point bending method. The wafer pairs are bonded at 250, 300 and 350 °C, respectively, under a bonding force of 5,500 N for a duration of 1 h in vacuum (~2.5 × 10−4 mbar). The bonding medium consists of Cu (300 nm) bonding layer and Ti (50 nm) barrier layer. Excellent helium leak rate, which is smaller than the reject limit defined by MIL-STD-883E standard (method 1014.10), and outstanding interfacial adhesion energy are detected for all samples. The cavities sealed at 300 °C present an excellent reliability of temperature cycling test up to 500 cycles. Cu-to-Cu thermo-compression bonding at low temperature (≤300 °C) presents an attractive hermetic seal and a robust mechanical support for 3D integration application.

Wafer-level hermetic packaging of 3D microsystems with low-temperature Cu-to-Cu thermo-compression bonding and its reliability

Low-temperature wafer-level Cu-to-Cu thermo-compression bonding and its reliability for hermetic sealing application have been investigated in this work. The volume of the encapsulated cavities is about 1.4×10−3 cm3 in accordance with the MIL-STD-883E standard prescribed for microelectronics packaging hermeticity measurement. The samples under test are bonded at 300 °C under a bonding force of 5500 N for 1 h in vacuum (∼2.5 × 10−4 mbar) with a 300 nm thick Cu diffusion layer and 50 nm thick Ti barrier layer which are deposited in an e-beam evaporator. The reliability test is accomplished through a temperature cycling test (TCT) from −40 to 125 °C up to 1000 cycles and a humidity test based on IPC/JEDEC J-STD-020 standard. In addition, an immersion in acid/base solution is applied to verify the corrosion resistance of the Cu frame for hermetic application. Excellent helium leak rate which is smaller than the reject limit defined by the MIL-STD-883E standard (method 1014.10) is detected for all the samples. These excellent helium leak rates show an outstanding bonding quality against harsh environment for hermetic encapsulation in 3D integration applications.

Three-Dimensional Wafer Stacking Using Cu–Cu Bonding for Simultaneous Formation of Electrical, Mechanical, and Hermetic Bonds

Wafer-on-wafer stacking is demonstrated successfully using bumpless Cu-Cu bonding for the simultaneous formation of electrical connection, mechanical support, and hermetic frame for 3-D IC application. The ohmic behavior of the Cu-Cu bond is verified. A daisy chain of at least 44 000 contacts at a 15-μm pitch is connected successfully and sustains thermal cycling. Postbonding delamination is found to be strongly affected by the wafer curvature and bond strength. The Cu-Cu hermetic seal ring shows a helium leak rate more than ten times lower than the reject limit (5 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-8</sup> atm · cm/s based on MIL-STD-883E standard) without underfill. This provides a robust IC-to-IC connection density of 4.4 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">5</sup> cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-2</sup> , suitable for future wafer-level 3-D integration.

Low-temperature hermetic packaging for microsystems using Au–Au surface-activated bonding at atmospheric pressure

Low-temperature hermetic bonding based on surface activation is useful for optical microsystem packaging because high bonding temperatures may degrade microsystem performance and sensitivity. However, surface-activated bonding (SAB) is usually performed under ultra-high-vacuum conditions, and the bonding environment cannot be chosen freely. In this study, thin Au sealing rings (300–500 nm thick, and 100 μm wide) were used as bonding layers for SAB at atmospheric pressure. A sufficiently high die-shear strength was achieved via surface activation using an argon radio-frequency plasma treatment. On examination of the fracture surfaces of the broken seal after the die-shear test, we observed that the fractures typically occurred at the deposited interface or partially inside the bulk substrates. Hermeticity was evaluated by measuring the resonance characteristics of photothermally excited microcantilevers inside the cavities. The samples bonded at the low temperature of 150 °C under the application of a bonding pressure of 313 MPa for 30 s showed leakage rates of less than 5.0 × 10−9 Pa·m3 s−1, which is the rejection limit defined by the MIL-STD-883G specification.

Low temperature wafer-level bonding for hermetic packaging of 3D microsystems

Metallic copper–copper (Cu–Cu) thermo-compression bonding, oxide–oxide (SiO2–SiO2) fusion bonding and silicon–silicon (Si–Si) direct bonding are investigated for potential application as hermetic seal in 3D microsystem packaging. Cavities are etched to a volume of 1.4 × 10−3 cm3 in accordance with the MIL-STD-883E standard prescribed for microelectronics packaging. In the case of metal bonding, a clean Cu layer with a thickness of 300 nm and a Ti barrier layer with an underlying thickness of 50 nm are used. The wafer pair is bonded at 300 °C under the application of a bonding force of 5500 N for 1 h. On the other hand, Si–Si bonding and SiO2–SiO2 bonding are initiated at room ambient after surface activation, followed by annealing in inert ambient at 300 °C for 1 h. The bonded cavities are stored in a helium bomb chamber and the leak rate is measured with a mass spectrometer. An excellent helium leak rate below 5 × 10−9 atm cm3 s−1 is detected for all cases and this is at least ten times better than the reject limit.

Quality changes of yellow grouper(&lt;I&gt;Epinephelus awoara&lt;/I&gt;) fillets stored under vacuum packaging at 0 ℃

Sensory attributes,total number of bacteria,physical and chemical parameters ( proximate composition,texture analysis,color measurements,pH,TBA,TMA-N,TVB-N,K-value,Ca2 +-ATPase activity, SDS-PAGE) changes of yellow grouper ( Epinephelus awoara) stored under vacuum packaging at 0 ℃ were investigated.The results showed that yellow grouper was a fish with low level of fat but high level of protein.During the storage,sensory attributes( odor and taste) exhibited significant variations and correlations in storage time( r=0.94 and 0.98,respectively) .Taste score increased faster than the odor score,however,neither of them reached the reject limit of 3 scores.There were significant linear increases in total viable counts( TVC) in the storage( r=0.97) .TVC on the 12th day was lower than 4 Log CFU/g,indicating the fillets kept at first grade of freshness,and was 4.34 Log CFU/g at the end of 15 days storage,kept at second grade of freshness.With regards to the texture profile,hardness( r =-0.89) ,gumminess( r =-0.73) ,chewiness( r =-0.87) showed a significant and negative correlation with storage time.L and the chroma values decreased,and b values increased,reflecting the colour changes experienced by the fillets over time.No significant differences were found in pH and TBA concentrations during the storage and they displayed a weak correlation with time, so they may not be the suitable indicators for reflecting the quality changes of yellow grouper fillets during the storage.The amount of TVB-N and TMA-N significantly increased with time,at the final of storage,TVB-N and TMA-N reached 12.94 mg N/100 g and 0.75 mg N/100 g respectively,and both of them were lower than limit.The proteolytic degradation of muscle protein was observed by SDS-PAGE,the results showed that MHC and Actin were hydrolyzed continuously throughout storage.Ca2 +-ATPase activity declined fast from the initial value of 0.25 μg Pi/( min·mg) to the final value of 0.07 μg Pi/( min·mg) .K value increased fast with time ( r =0.98) and reached 54.91% at the end of storage,keeping the middle level of freshness.The results suggested that total viable counts( TVC) increased slowly under vacuum packaging at 0 ℃,and it kept good sensory characters,inhibited the changes of the paraments( TVB-N value,etc.) and hydrolysis of protein.The sensory attributes,microbiological counts,texture( hardness,cohesiveness and chewiness) ,colour( L ,b ,and Cab) ,TVB-N,TMA-N,Ca2 +-APTase activity and K value exhibited strong correlations with storage time and were sensitive.Therefore,they may be considered as good indicators for evaluating yellow grouper spoilage during the storage under vacuum packaging at 0 ℃ for 15 days,but the consideration of shelf-life should be combined with all those indicators.

Valuation of quality parameters for rearing Aceria malherbae Nuzzaci (Acari: Eriophyidae), a biological control agent of field bindweed, Convolvulus arvensis L.

ABSTRACT Field bindweed (Convolvulus arvensis) is one of the 10 most problematic weeds in the world. Its control using chemical herbicides is not completely effective. Aceria malherbae is a mite used successfully as a biological agent to control field bindweed. Effective implementation of this kind of biocontrol requires high-quality production of these mites. The purpose of this study was to investigate different quality indicators for effectively rearing A. malherbae under greenhouse conditions. The indicators considered were number of gallmites (adults, immature stages, and eggs), number of developed stems per plant, and number of galls per stem. We used 25 plots with 10 pots per plot, testing a sub-sample from one-fourth of these pots. The Shewhart chart or process–behavior chart was applied to determine basic statistics (means and range) and acceptance and rejection limits. Rejection limits for inferior plots were set at 3.9 developed stems per plant, 5.2 galls per stem, 7.4 adults, 32 nymphs, and 11 eggs, per plot. We also propose for quality control that the number of developed stems per plant and the number of galls per stem be routinely counted to detect any reduced quality of mite production. The results suggest that these indicators could be used for assessing quality control in large-scale reproduction of A. malherbae.

Low Temperature Cu-to-Cu Bonding for Wafer-Level Hermetic Encapsulation of 3D Microsystems

Summary and Conclusion Cu thermocompression wafer level bonding at low temperature(250 and 300 ◦ C) is investigated for potential application as hermeticsealinmicrosystemspackaging.Heliumleaktestdemonstratesthatallsamples(bondingbetweennon-functionalcavitywaferandcapwafer)present superior leak rate below 5.0×10 −9 atm·cm 3 /sec, which is atleast one order of magnitude smaller than the reject limit of 5×10 −8 atm·cm 3 /sec defined by the MILSTD-883E standard. Leak rate inthe order of 10 −10 atm·cm 3 /sec is detected for some samples. Thecavities sealed by double ring exhibit better hermeticity than thosesealed by single ring as expected. The experimental results show thepossibility for simultaneous formation of hermetic cavities duringCu-Cu bonding in 3D microsystems. Acknowledgment This work has been carried out as part of the Nanyang Assis-tant Professorship supported by Nanyang Technological University(NTU). Partial funding is also provided by Defence Science and Tech-nology Agency (DSTA), Singapore. Authors are grateful for supportprovidedbythemanagementandtechnicalstaffintheNanyangNano-Fabrication Center at NTU.

Protocolo para captação dos potenciais evocados auditivos de longa latência

Os potenciais evocados auditivos de longa latência (PEALLs) se referem a uma série de mudanças elétricas, ocorridas no sistema nervoso central, resultante da estimulação da via sensorial auditiva. Muitos estudos abordam o uso destes potenciais, controlando o artefato gerado pelo movimento ocular com a utilização de equipamentos com grande número de canais. Porém, na prática clínica nacional, a realidade é diferente, havendo disponibilidade de equipamentos com número reduzido de canais. OBJETIVO: Comparar dois métodos de controle do artefato gerado pelo movimento ocular durante a captação dos PEALLs usando dois canais de registro. MATERIAL E MÉTODO: Estudo prospectivo pela aplicação de dois métodos de captação dos PEALLs (subtração do artefato ocular e controle do limite de rejeição) em 10 adultos ouvintes normais. RESULTADOS: Não foi observada diferença estatisticamente significante entre os valores de latência obtidos com o uso dos dois métodos, apenas entre os valores de amplitude. CONCLUSÃO: Os dois métodos foram eficientes para a captação dos PEALLs e para o controle do artefato do movimento ocular. O método do controle do limite de rejeição promoveu maiores valores de amplitude.

Protocol to collect late latency auditory evoked potentials

SummaryLong Latency Auditory Evoked Potentials (LLAEP) represents a number of electrical changes occurring in the central nervous system, resulting from stimulation of the auditory sensorial pathways. Many studies approach the use of these potentials controlling the artifact created by eye movement with the use of equipment with a large number of channels. However, what happens is very different in Brazilian clinical practice, where the equipment used has a very limited number of channels.Aimto compare the two methods used to control the artifacts created by eye movements during LLAEP capture using two recording channels.Materials and Methodsthis is a prospective study with the application of two LLAEP capturing methods (eye artifact subtraction and rejection limit control) in 10 normal hearing individuals.Resultswe did not observe statistically significant differences concerning the latency values obtained with the use of both methods, only concerning amplitude values.Conclusionboth methods were efficient to capture the LLAEP and to control the eye movement artifact. The rejection limit control method produced greater amplitude values.

Microbiological, chemical and sensory assessment of (I) whole ungutted, (II) whole gutted and (III) filleted tilapia (Oreochromis niloticus) during refrigerated storage

SummaryThe effects of gutting and filleting on microbiological, chemical, and sensory properties of tilapia (Oreochromis niloticus) stored under refrigeration were studied. All the tilapia samples were analysed periodically for microbiological (APC), chemical (pH, total volatile basic nitrogen, 2‐thiobarbituricacid, K‐value), and sensory characteristics. Results indicated that sensory data were in good agreement with microbiological data (APC rejection limit of 107 CFU g−1) and K‐value (rejection limit of 60%). Storage life of (I) whole ungutted, (II) whole gutted and (III) filleted tilapia stored under refrigeration (5 ± 1 °C) can be expected to be 12, 10, and 6 days, respectively.

Cold Adaptation of Zinc Metalloproteases in the Thermolysin Family from Deep Sea and Arctic Sea Ice Bacteria Revealed by Catalytic and Structural Properties and Molecular Dynamics

Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.

Analytical Methods for Quantification of Modified Fatty Acids and Sterols Formed as a Result of Processing

Fats and oils are often submitted to technological treatments before being consumed. Some treatments like refining, hydrogenation, and frying often lead to the formation of modified fatty acids such as cyclic fatty acid monomers (CFAM), geometrical fatty acid isomers, and/or oxidized fatty acids and sterols (cholesterol and phytosterols). Both cholesterol oxidation products (COP) and phytosterol oxidation products (POP), may be present in foods. As some of the newly formed components may present some adverse effects upon consumption, methods have been developed to analyze these compounds in food products and biological samples. Gas liquid chromatography (GC) on long polar columns (100m) is a good choice to quantify trans mono- and poly-unsaturated fatty acids. In some cases a pre-fractionation step using silver nitrate thin layer chromatography (AgNO3-TLC) may be necessary to avoid GC overlapping of cis and trans isomers. Analysis of CFAM usually involves transformation of the sample in fatty acid methyl esters (FAME) which after addition of an internal standard (IS) are further hydrogenated. An enrichment step using reverse phase high performance liquid chromatography (RP-HPLC) permits to obtain a fraction which consists of a mixture of CFAM and the IS. This fraction is further analyzed by GC on a polar column. The analysis of oxidized triacylglycerol monomers (oxTG) as a group was feasible by a combination of adsorption and size-exclusion chromatography. Quantification in used frying fats and oils around the limit of rejection for human consumption (25% polar compounds) has shown that the amount of oxTG range 5.9–9.4% expressed on fat or oil weight. In foods and biological tissues, the level of oxidized sterols (SOP) is often a very small fraction of their unoxidized forms. Analysis of SOP involved extraction of lipids, saponification or transesterification, enrichment, and subsequent qualitative and quantitative determination by GC and GC-MS, or HPLC and HPLC-MS. In addition, enrichment of SOP requires complete separation from the unoxidized sterols in order to separate these compounds even by high resolution GC capillary columns.

Gluing ability of plywood produced with DMDHEU-modified veneers of Fagus sp., Betula sp., and Picea sp.

The shear strength, the cohesive wood failure, and the delamination were determined in plywood constructed with veneers of Fagus sp., Betula sp., and Picea sp. impregnated with 0.8, 1.3, and 2.3M 1,3-dimethylol-4,5-dihydroxyethyleneurea (DMDHEU). The plywood production process consisted of two steps: firstly impregnation and curing of the veneers, and secondly assembly of the panel. The adhesive used was a phenolic resin. The values obtained for shear strength were above the standard limit of rejection (1Nmm−2), and therefore the samples met the European requirements of plywood designed for exterior conditions. The phenolic resin improved its adhesive ability when the samples were heated for 72h at 95°C. The highest shear strength for modified wood was 3.97±0.45Nmm−2 at 0.8M, 3.39±0.26Nmm−2 at 1.3M, and 2.35±0.23Nmm−2 at 2.3M, for Betula sp., Fagus sp., and Picea sp., respectively. The samples constructed with unmodified veneers presented higher shear strengths and higher cohesive wood failures than the samples constructed with modified veneers. The possible effect of the two-steps production of the plywood is discussed. The delamination test of DMDHEU-modified Betula sp. and Fagus sp. plywood showed no open glue lines, while the samples of Picea sp. treated with the higher concentration of DMDHEU suffered delamination.

Immobilized yeasts in κ-carragenate to prevent browning in white wines

Yeasts immobilized in κ-carragenate gel beads of variable size were used to delay accelerated browning of sherry pale white wines at 35°C. Based on the results, the wines maintained into contact with no gel took 27 days to reach the browning limit level for commercial wines of this type (A420 value of 0.180 au). However, the wines containing gel beads 1, 2 or 3 mm thick reached such a level 24, 30 and 33 days later, respectively, than the previous ones. After 27 days of contact with gel beads, the wines exhibited decreased contents in phenolic acids, hydroxycinnamic esters and flavan-3-ol monomers and dimers. On the other hand, the yeast gels substantially delayed browning of wines stored at 19°C in bottles for 1 year. Thus, while the wine stored without gels reached the commercial rejection limit for A420, those stored in contact with gel beads exhibited virtually no browning and only slight changes in their sensory properties, although they were still acceptable for consumption.