Functional metagenomic screening approach for discovery of new glycoside phosphorylases

Glycoside phosphorylases (GPs) have recently been recognized as potentially useful biocatalysts for the synthesis and biotransformation of glycans. These enzymes ordinarily carry out phosphorolysis of the glycosidic linkage by transferring a glycosyl moiety from the non-reducing end of a di- or polysaccharide substrate onto inorganic phosphate, thereby cleaving the glycosidic bond and generating a sugar-1-phosphate. GPs distinguish themselves from most carbohydrate-active enzymes in that the hydrolytic free energy associated with the ester-linkage of the sugar-1-phosphate product is roughly equivalent to that of the glycosidic linkage in the glycan substrate. Therefore, the equilibrium position can be tipped in favour of glycoside synthesis by manipulation of reaction conditions. GPs thus have considerable potential for the assembly of glycans, especially since their reversibility would allow the use of one GP to degrade an inexpensive glycan to produce a pool of sugar-1-phosphates, while a second GP could be deployed to use those sugar-1-phosphates as donors to synthesize a different, more valuable target glycan. The bottleneck in this approach, however, is the limited range of GPs available, which restricts the classes of glycan that can be assembled. To help increase the spectrum of known GPs available, we have turned to metagenomics as a means to discover new enzymes belonging to this class. We have adapted the molybdenum blue reaction to a high-throughput plate-based metagenomic screen for the discovery of GPs. Our method utilizes the reverse phosphorolysis ability of GPs by coupling inorganic phosphate released during glycan synthesis to the molybdenum blue reaction. Therefore, GP activity can be identified by incubating metagenomic clones with appropriate donor sugar-1-phosphates and acceptor glycans, then monitoring inorganic phosphate accumulation by measuring formation of molybdenum blue. Our pilot screen was optimized to identify cellulose degrading GPs and yielded 7 novel GP ORFs, all from CAZy family GH94. To our knowledge this is the first high-throughput functional metagenomic screen for GP activity. Looking ahead, we have planned to further adapt the screening method so it may identify GP activity from other CAZy families, beyond GH94. The activities that can be identified are dependent on the combination of donor and acceptor substrates used. By mixing and matching different substrates we will be able to narrow or broaden the scope of activities that can be detected within a single screen

Generation of internal stress and its effects

Internal stresses may be generated continually in many polycrystalline materials. Their existence is manifested by changes in crystal defect concentration and arrangement, by surface observations, by macroscopic shape changes and particularly by alteration of mechanical properties when external stresses are simultaneously imposed

Why Is It So Challenging to Measure Residual Stresses ?

BACKGROUND: Residual stresses have a “hidden” character because they exist in a material without the presence of any external loads. They cannot easily be added or subtracted in a quantified manner, as is done when measuring applied stresses, and so are much more challenging to measure. OBJECTIVE: The objective here is to identify and describe the various features that make residual stress measurement methods challenging and to consider the ways that these challenges can be addressed in practice. METHODS: Various of the most common residual stress measurements methods are considered and the challenges associated with them are identified and classified. RESULTS: Five major challenges for residual stress measurements, and the approaches used for their resolution, are identified. CONCLUSIONS: Despite the various challenges that need to be overcome, residual stress measurements can be successfully undertaken in practice. The most significant feature for success is a highly skilled and knowledge practitioner

Obesity-related perivascular adipose tissue damage is reversed by sustained weight loss in the rat

Objective – Perivascular adipose tissue (PVAT) exerts an anticontractile effect in response to various vasoconstrictor agonists and this is lost in obesity. A recent study reported that bariatric surgery reverses the damaging effects of obesity on PVAT function. However, PVAT function has not been characterised following weight loss induced by caloric restriction, which is often the first line treatment for obesity. Approach and Results – Contractility studies were performed using wire myography on small mesenteric arteries with and without PVAT from control, diet-induced obese, calorie restricted and sustained weight loss rats. Changes in the PVAT environment were assessed using immunohistochemistry. PVAT from healthy animals elicited an anticontractile effect in response to norepinephrine. This was abolished in diet-induced obesity through a mechanism involving increased local TNFα and reduced nitric oxide bioavailability within PVAT. Sustained weight loss led to improvement in PVAT function associated with restoration of adipocyte size, reduced TNFα and increased nitric oxide synthase function. This was associated with reversal of obesity-induced hypertension and normalisation of plasma adipokine levels, including leptin and insulin. Conclusions – We have shown that diet-induced weight loss reverses obesity-induced PVAT damage through a mechanism involving reduced inflammation and increased nitric oxide synthase activity within PVAT. These data reveal inflammation and nitric oxide synthase, particularly eNOS, as potential targets for the treatment of PVAT dysfunction associated with obesity and the metabolic syndrome

Modularised process-based modelling of phosphorus loss at farm and catchment scale

In recent years, a co-ordinated programme of data collection has resulted in the collation of sub-hourly time-series of hydrological, sediment and phosphorus loss data, together with soil analysis, cropping and management information for two small (< 200 ha) headwater agricultural catchments in the UK Midlands (Rosemaund, Herefordshire and Cliftonthorpe, Leicestershire). These data sets have allowed the dynamics of phosphorus loss to be characterised and the importance of both storm runoff and drainflow to be identified, together with incidental losses following manure and fertiliser additions in contributing to total annual loss. A modularised process-based model has been developed to represent current understanding of the dynamics of phosphorus loss. Modules describing runoff and sediment generation and associated phosphorus adsorption/desorption dynamics are described and tested. In the model, the effect of a growing crop on sediment detachment processes is represented and the stability of topsoil is considered so that, overall, the model is responsive to farm management factors. Importantly, using data sets available from national-scale survey programmes to estimate model parameters, a transferable approach is presented, requiring only sub-hourly rainfall data and field-specific landcover information for application of the model to new sites. Results from application of the model to the hydrological year 1998–99 are presented. Assessment of performance, which suggests that the timing of simulated responses is acceptable, has focused attention on quantifying landscape and in-stream retention and remobilisation processes.</b></p> <p style='line-height: 20px;'><b>Keywords: </b>phosphorus, erosion, process-based modelling, agricultur

Inhibition of Cellular Respiration by Doxorubicin

Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O(2)]. In solutions containing glucose and cells, [O(2)] declined linearly with time, showing that the kinetics of oxygen consumption was zero order. Complete inhibition of oxygen consumption by cyanide indicated that oxidations occurred in the respiratory chain. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2-4 h of incubation with 20 microM doxorubicin, paralleling the decline in respiration rate. Thus, cells incubated with doxorubicin exhibit caspase-mediated inhibition of oxidative phosphorylation

ExoMars entry, descent and landing science

The entry, descent and landing of ExoMars offer a rare (once-per-mission) opportunity to perform in situ investigation of the martian environment over a wide altitude range. Entry, Descent and Landing System (EDLS) measurements can provide essential data for atmospheric scientific investigations. We intend to perform atmospheric science measurements by exploiting data from EDLS engineering sensors and exploiting their readings beyond the expected engineering information

Fermi surface nesting in several transition metal dichalcogenides

By means of high-resolution angle resolved photoelectron spectroscopy (ARPES) we have studied the fermiology of 2H transition metal dichalcogenide polytypes TaSe2, NbSe2, and Cu0.2NbS2. The tight-binding model of the electronic structure, extracted from ARPES spectra for all three compounds, was used to calculate the Lindhard function (bare spin susceptibility), which reflects the propensity to charge density wave (CDW) instabilities observed in TaSe2 and NbSe2. We show that though the Fermi surfaces of all three compounds possess an incommensurate nesting vector in the close vicinity of the CDW wave vector, the nesting and ordering wave vectors do not exactly coincide, and there is no direct relationship between the magnitude of the susceptibility at the nesting vector and the CDW transition temperature. The nesting vector persists across the incommensurate CDW transition in TaSe2 as a function of temperature despite the observable variations of the Fermi surface geometry in this temperature range. In Cu0.2NbS2 the nesting vector is present despite different doping level, which lets us expect a possible enhancement of the CDW instability with Cu-intercalation in the CuxNbS2 family of materials.Comment: Accepted to New J. Phy