Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis

Polyethylene terephthalate (PET) hydrolase from Ideonella sakaiensis (IsPETase) can be used to degrade PET. In order to use IsPETase in industry, we studied the enzymatic activity of IsPETase in different conditions containing environmental and physicochemical factors commonly found in nature. We observed that salts and glycerol enhanced the enzymatic activity, while detergents and organic solvents reduced the enzymatic activity. IsPETase hydrolyzed p-nitrophenyl (p-NP) esters instead of naphthyl esters. To make IsPETase an enzyme capable of hydrolyzing naphthyl esters, site-directed mutagenesis was carried out based on the structural information provided by the crystal structure. We found that the IsPETase^S93M, IsPETase^W159F, and IsPETase^N241F mutants can hydrolyze naphthyl esters. IsPETase engineering can direct researchers to use this α/β-hydrolase protein scaffold to design enzymes that can hydrolyze a variety of polyesters.     

Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular‐shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut‐shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.      

Optimization of peptidic HIV‐1 fusion inhibitor T20 by phage display

The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage‐displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five‐helix bundle (5HB) mimetic of HIV‐1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at noncontact points to fine‐tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV‐1 variants, including a common T20‐escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV‐1 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering.     

Integrating the intrinsic conformational preferences of noncoded α-amino acids modified at the peptide bond into the noncoded amino acids database

Recently, we reported a database (Noncoded Amino acids Database; http://recerca.upc.edu/imem/index.htm) that was built to compile information about the intrinsic conformational preferences of nonproteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, the experimentally established conformational propensities, and applications (Revilla-López et al., J Phys Chem B 2010;114:7413-7422). The database initially contained the information available for α-tetrasubstituted α-amino acids. In this work, we extend NCAD to three families of compounds, which can be used to engineer peptides and proteins incorporating modifications at the--NHCO--peptide bond. Such families are: N-substituted α-amino acids, thio-α-amino acids, and diamines and diacids used to build retropeptides. The conformational preferences of these compounds have been analyzed and described based on the information captured in the database. In addition, we provide an example of the utility of the database and of the compounds it compiles in protein and peptide engineering. Specifically, the symmetry of a sequence engineered to stabilize the 3(10)-helix with respect to the α-helix has been broken without perturbing significantly the secondary structure through targeted replacements using the information contained in the database.     

Hexose monophosphate shunt,the role of its metabolites and associated disorders: A review

The hexose monophosphate (HMP) shunt acts as an essential component of cellular metabolism in maintaining carbon homeostasis. The HMP shunt comprises two phases viz. oxidative and nonoxidative, which provide different intermediates for the synthesis of biomolecules like nucleotides, DNA, RNA, amino acids, and so forth; reducing molecules for anabolism and detoxifying the reactive oxygen species during oxidative stress. The HMP shunt is significantly important in the liver, adipose tissue, erythrocytes, adrenal glands, lactating mammary glands and testes. We have researched the articles related to the HMP pathway, its metabolites and disorders related to its metabolic abnormalities. The literature for this paper was taken typically from a personal database, the Cochrane database of systemic reviews, PubMed publications, biochemistry textbooks, and electronic journals uptil date on the hexose monophosphate shunt. The HMP shunt is a tightly controlled metabolic pathway, which is also interconnected with other metabolic pathways in the body like glycolysis, gluconeogenesis, and glucuronic acid depending upon the metabolic needs of the body and depending upon the biochemical demand. The HMP shunt plays a significant role in NADPH₂ formation and in pentose sugars that are biosynthetic precursors of nucleic acids and amino acids. Cells can be protected from highly reactive oxygen species by NADPH ₂. Deficiency in the hexose monophosphate pathway is linked to numerous disorders. Furthermore, it was also reported that this metabolic pathway could act as a therapeutic target to treat different types of cancers, so treatments at the molecular level could be planned by limiting the synthesis of biomolecules required for proliferating cells provided by the HMP shunt, hence, more experiments still could be carried out to find additional discoveries.     

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.     

Soil nutrient budgets following projected corn stover harvest for biofuel production in the conterminous United States

Increasing demand for food and biofuel feedstocks may substantially affect soil nutrient budgets, especially in the United States where there is great potential for corn (Zea mays L) stover as a biofuel feedstock. This study was designed to evaluate impacts of projected stover harvest scenarios on budgets of soil nitrogen (N), phosphorus (P), and potassium (K) currently and in the future across the conterminous United States. The required and removed N, P, and K amounts under each scenario were estimated on the basis of both their average contents in grain and stover and from an empirical model. Our analyses indicate a small depletion of soil N (?4 ± 35 kg ha^?1) and K (?6 ± 36 kg ha^?1) and a moderate surplus of P (37 ± 21 kg ha^?1) currently on the national average, but with a noticeable variation from state to state. After harvesting both grain and projected stover, the deficits of soil N, P, and K were estimated at 114–127, 26–27, and 36–53 kg ha^?1 yr^?1, respectively, in 2006–2010; 131–173, 29–32, and 41–96 kg ha^?1 yr^?1, respectively, in 2020; and 161–207, 35–39, and 51–111 kg ha^?1 yr^?1, respectively, in 2050. This study indicates that the harvestable stover amount derived from the minimum stover requirement for maintaining soil organic carbon level scenarios under current fertilization rates can be sustainable for soil nutrient supply and corn production at present, but the deficit of P and K at the national scale would become larger in the future.     

Sustainable limits to crop residue harvest for bioenergy: maintaining soil carbon in Australia's agricultural lands

The use of crop residues for bioenergy production needs to be carefully assessed because of the potential negative impact on the level of soil organic carbon (SOC) stocks. The impact varies with environmental conditions and crop management practices and needs to be considered when harvesting the residue for bioenergy productions. Here, we defined the sustainable harvest limits as the maximum rates that do not diminish SOC and quantified sustainable harvest limits for wheat residue across Australia's agricultural lands. We divided the study area into 9432 climate‐soil (CS) units and simulated the dynamics of SOC in a continuous wheat cropping system over 122 years (1889 – 2010) using the Agricultural Production Systems sIMulator (APSIM). We simulated management practices including six fertilization rates (0, 25, 50, 75, 100, and 200 kg N ha^?1) and five residue harvest rates (0, 25, 50, 75, and 100%). We mapped the sustainable limits for each fertilization rate and assessed the effects of fertilization and three key environmental variables – initial SOC, temperature, and precipitation – on sustainable residue harvest rates. We found that, with up to 75 kg N ha^?1 fertilization, up to 75% and 50% of crop residue could be sustainably harvested in south‐western and south‐eastern Australia, respectively. Higher fertilization rates achieved little further increase in sustainable residue harvest rates. Sustainable residue harvest rates were principally determined by climate and soil conditions, especially the initial SOC content and temperature. We conclude that environmental conditions and management practices should be considered to guide the harvest of crop residue for bioenergy production and thereby reduce greenhouse gas emissions during the life cycle of bioenergy production.     

Offsetting high water demands with high productivity: Sorghum as a biofuel crop in a high irradiance arid ecosystem

High irradiance arid environments are promising, yet understudied, areas for biofuel production. We investigated the productivity and environmental trade‐offs of growing sorghum (Sorghum bicolor) as a biofuel feedstock in the low deserts of California (CA). Using a 5.3 ha experimental field in the Imperial Valley, CA, we measured aboveground biomass production and net ecosystem exchange of CO₂ and H₂O via eddy covariance over three growing periods between February and November 2012. Environmental conditions were extreme, with high irradiance, vapor pressure deficit (VPD), and air temperature throughout the growing season. Air temperature peaked in August with a maximum of 45.7 °C. Sorghum produced an annual aboveground biomass yield of 43.7 Mg per hectare. Net ecosystem exchange (NEE) was highest during the summer growth period and reached a maximum of ?68 μmol CO₂ m^?2 s^?1. Water use efficiency, or biomass water ratio (BWR), was high (4.0 g dry biomass kg^?1 H₂O) despite high seasonal evapotranspiration (1094 kg H₂O m^?2). The BWR of sorghum surpassed that of many C4 biofuel candidate crops in the United States, as well as that of alfalfa which is currently widely grown in the Imperial Valley. Sorghum also outperformed many US biofuel crops in terms of radiation use efficiency (RUE), achieving 1.5 g dry biomass MJ^?1. We found no evidence of saturation of NEE at high levels of photosynthetically active radiation (PAR) (up to 2250 μmol m^?2 s^?1). In addition, we found no evidence that NEE was inhibited by either high VPD or air temperature during peak photosynthetic phases. The combination of high productivity, high BWR, and high RUE suggests that sorghum is well adapted to this extreme environment. The biomass production rates and efficiency metrics spanning three growing periods provide fundamental data for future Life Cycle Assessments (LCA), which are needed to assess the sustainability of this sorghum biofuel feedstock system.     

大肠杆菌全局调控转录因子环腺苷酸受体蛋白质在代谢工程中的应用

环腺苷酸受体蛋白质(Cyclic amp receptor protein,CRP)是原核生物共有的一类全局转录因子,调控原核生物大肠杆菌Escherichia coli中近一半基因的转录。通过易错PCR或DNA shuffling的方法可以获得CRP突变体文库,然后经过筛选可以得到目的细胞表型——"抗逆性"增强。本研究综述了突变CRP在细胞表型:耐氧化压力、耐渗透胁迫、耐有机溶剂(甲苯)、发酵中耐醋酸盐类和生物醇生产中耐生物醇中的应用。推论出CRP同样可以应用于相似微生物的表型培育,并展望了CRP的巨大应用潜力。