Quantitative analysis of short-chain acyl-coenzymeAs in plant tissues by LC–MS–MS electrospray ionization method

Because acyl-CoAs play major roles in numerous anabolic and catabolic pathways, the quantitative determination of these metabolites in biological tissues is paramount to understanding the regulation of these metabolic processes. Here, we report a method for the analysis of a collection of short-chain acyl-CoAs (<6 carbon chain length) from plant extracts. Identification of each individual acyl-CoA was conducted by monitoring specific mass-fragmentation ions that are derived from common chemical moieties of all Coenzyme A (CoA) derivatives, namely the adenosine triphosphate nucleotide, pantothenate and acylated cysteamine. This method is robust and quick, enabling the quantitative analysis of up to 12 different acyl-CoAs in plant metabolite extracts with minimal post-extraction processing, using a 30 min chromatographic run-time.     

Exposure assessment of boron in Bandırma boric acid production plant

Boric acid and sodium borates have been considered as being “toxic to reproduction and development”, following results of animal studies with high doses. Experimentally, a NOAEL of 17.5 mg B/kg-bw/day (corresponds to ～2020 ng boron/g blood) has been identified for the (male) reproductive effects of boron in a multigenerational study of rats, and a NOAEL for the developmental effects in rats was identified at 9.6 mg B/kg-bw/day (corresponds to 1270 ng boron/g blood). These values are being taken as the basis of current EU safety assessments. The present study was conducted to assess the boron exposure under extreme exposure conditions in a boric acid production plant located in Band?rma, Turkey. The mean blood boron concentrations of low and high exposure groups were 72.94 ± 15.43 (48.46–99.91) and 223.89 ± 60.49 (152.82–454.02) ng/g respectively. The mean blood boron concentration of the high exposure group is still ～6 times lower than the highest no effect level of boron in blood with regard to the developmental effects in rats and ～9 times lower than the highest no effect level of boron in blood with regard to the reprotoxic effects in male rats. In this context, boric acid and sodium borates should not be considered as toxic to reproduction for humans in daily life.     

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol^− 1 and 8.93 kcal mol^− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol^− 1 and 9.09 kcal mol^− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.     

Rapid determination of 25-hydroxy vitamin D3 in swine tissue using an isotope dilution HPLC-MS assay

A rapid method for quantification of 25-hydroxy vitamin D3 in different swine tissues based on isotope dilution HPLC-MS has been developed and validated. Six times deuterated analyte is used as internal standard. The method is fast and can be performed with only 1 g sample. Sample preparation for kidney, liver, muscle and spleen requires only homogenisation and extraction with methanol. An additional enzymatic digest is required for skin, and clean-up of the extract by solid-phase extraction (SPE) is used for adipose tissue and skin. The lower limit of detection varies from 1 ng/g (muscle) to 5 ng/g (adipose and skin). The method has been successfully applied to various tissue samples of pigs fed for 119 days either 2000 IU of vitamin D3 or 50 μg of 25-hydroxy vitamin D3 per kg feed. For animals ingesting 25-OH-D3 supplements the highest tissue contents were observed in the skin (24.8 ± 3.5 ng/g), followed by kidney (14.2 ± 1.5 ng/g), liver and muscle (5.7 ± 0.6 ng/g). The 25-OH-D3 content in the skin was significantly higher in animals ingesting 2000 IU/kg of vitamin D3 (39.5 ± 13.4 ng/g). Levels in selected tissues of some animals were below the lower limit of quantification. No measurable amounts of 25-OH-D3 were found in spleen, abdominal fat and subcutaneous fat of the animals of both groups as well as in the liver, kidney and muscle of the animals ingesting 2000 IU/kg of vitamin D3.     

Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system

Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.     

Inductive reasoning and forecasting of population dynamics of Cylindrospermopsis raciborskii in three sub-tropical reservoirs by evolutionary computation

Seven-day-ahead forecasting models of Cylindrospermopsis raciborskii in three warm-monomictic and mesotrophic reservoirs in south-east Queensland have been developed by means of water quality data from 1999 to 2010 and the hybrid evolutionary algorithm HEA. Resulting models using all measured variables as inputs as well as models using electronically measurable variables only as inputs forecasted accurately timing of overgrowth of C. raciborskii and matched well high and low magnitudes of observed bloom events with 0.45 ≤ r² > 0.61 and 0.4 ≤ r² > 0.57, respectively. The models also revealed relationships and thresholds triggering bloom events that provide valuable information on synergism between water quality conditions and population dynamics of C. raciborskii. Best performing models based on using all measured variables as inputs indicated electrical conductivity (EC) within the range of 206–280 mS m⁻¹ as threshold above which fast growth and high abundances of C. raciborskii have been observed for the three lakes. Best models based on electronically measurable variables for the Lakes Wivenhoe and Somerset indicated a water temperature (WT) range of 25.5–32.7 °C within which fast growth and high abundances of C. raciborskii can be expected. By contrast the model for Lake Samsonvale highlighted a turbidity (TURB) level of 4.8 NTU as indicator for mass developments of C. raciborskii.Experiments with online measured water quality data of the Lake Wivenhoe from 2007 to 2010 resulted in predictive models with 0.61 ≤ r² > 0.65 whereby again similar levels of EC and WT have been discovered as thresholds for outgrowth of C. raciborskii. The highest validity of r² = 0.75 for an in situ data-based model has been achieved after considering time lags for EC by 7 days and dissolved oxygen by 1 day. These time lags have been discovered by a systematic screening of all possible combinations of time lags between 0 and 10 days for all electronically measurable variables. The so-developed model performs seven-day-ahead forecasts and is currently implemented and tested for early warning of C. raciborskii blooms in the Wivenhoe reservoir.     

Biomechanical characterization of cervical spinal manipulation in living subjects and cadavers

BackgroundCervical spinal manipulative therapy (cSMT) is a common therapeutic modality used in the treatment of neck pain and headaches. Cadaveric necks have been used as a model for assessing the effects of cSMT on vertebral artery mechanics. However, there have been no previous studies comparing the biomechanical indices of cSMT performed in living subjects versus cadavers.MethodsThe preload force, peak force and duration of cSMT performed by two chiropractors were recorded in 28 subjects with and without neck pain, and in five cadavers.ResultsThere were no statistical differences in terms of the preload, peak force and duration of cSMT in living subjects with versus without neck pain. However, all three parameters differed statistically in living subjects versus cadavers; and both preload and peak forces were significantly higher for cadaveric cSMT; the average peak force was 190.3 ± 85.5 N (mean ± SD) in living subjects, versus 283.9 ± 53.6 N in cadavers. Furthermore, the duration was significantly faster for cadaveric cSMT (175 ± 100 ms in living subjects versus 120 ± 30 ms in cadavers. These observations were consistent for both chiropractors.ConclusionsWhen performed in cadavers, cSMT tends to be more “aggressive” in terms of all biomechanical indices used to describe cSMT.     

Skeletal muscle pericyte subtypes differ in their differentiation potential

Neural progenitor cells have been proposed as a therapy for central nervous system disorders, including neurodegenerative diseases and trauma injuries, however their accessibility is a major limitation. We recently isolated Tuj1 + cells from skeletal muscle culture of Nestin–GFP transgenic mice however whether they form functional neurons in the brain is not yet known. Additionally, their isolation from nontransgenic species and identification of their ancestors is unknown. This gap of knowledge precludes us from studying their role as a valuable alternative to neural progenitors. Here, we identified two pericyte subtypes, type-1 and type-2, using a double transgenic Nestin–GFP/NG2–DsRed mouse and demonstrated that Nestin–GFP +/Tuj1 + cells derive from type-2 Nestin–GFP +/NG2–DsRed +/CD146 + pericytes located in the skeletal muscle interstitium. These cells are bipotential as they generate either Tuj1 + cells when cultured with muscle cells or become “classical” α-SMA + pericytes when cultured alone. In contrast, type-1 Nestin–GFP ?/NG2–DsRed +/CD146 + pericytes generate α-SMA + pericytes but not Tuj1 + cells. Interestingly, type-2 pericyte derived Tuj1 + cells retain some pericytic markers (CD146 +/PDGFRβ +/NG2 +). Given the potential application of Nestin–GFP +/NG2–DsRed +/Tuj1 + cells for cell therapy, we found a surface marker, the nerve growth factor receptor, which is expressed exclusively in these cells and can be used to identify and isolate them from mixed cell populations in nontransgenic species for clinical purposes.     

2-Heteroarylidene-1-indanone derivatives as inhibitors of monoamine oxidase

In the present study a series of fifteen 2-heteroarylidene-1-indanone derivatives were synthesised and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. These compounds are structurally related to series of heterocyclic chalcone derivatives which have previously been shown to act as MAO-B specific inhibitors. The results document that the 2-heteroarylidene-1-indanones are in vitro inhibitors of MAO-B, displaying IC₅₀ values of 0.0044–1.53 μM. Although with lower potencies, the derivatives also inhibit the MAO-A isoform with IC₅₀ values as low as 0.061 μM. An analysis of the structure-activity relationships for MAO-B inhibition indicates that substitution with the methoxy group on the A-ring leads to a significant enhancement in MAO-B inhibition compared to the unsubstituted homologues while the effect of the heteroaromatic substituent on activity, in decreasing order is: 5-bromo-2-furan > 5-methyl-2-furan > 2-pyridine ≈ 2-thiophene > cyclohexyl > 3-pyridine ≈ 2-furan. It may therefore be concluded that 2-heteroarylidene-1-indanone derivatives are promising leads for the design of MAO inhibitors for the treatment of Parkinson’s disease and possibly other neurodegenerative disorders.     

Alkaline phosphatase conductometric biosensor for heavy-metal ions determination

Alkaline phosphatase conductometric biosensors consisting of interdigitated gold electrodes and enzyme membranes have been used for assessment of heavy-metal ions in water. These analytes act as enzyme inhibitors. Enzyme residual activity has been measured in Tris-nitrate buffer without metal preincubation in the presence of Mg²⁺ ions as activator. The results indicate that the toxicity of the various metals tested toward immobilized phosphatase is ranged as follows: Cd²⁺ > Co²⁺ > Zn²⁺ > Ni²⁺ > Pb²⁺. Detection limits were about 0.5 ppm for Cd²⁺, 2 ppm for both Zn²⁺ and Co²⁺, 5 ppm for Ni²⁺ and 40 ppm for lead ions. In addition, the responses during 10 h were stable (RSD 4%) and a drift of about 7% per day was observed. The storage stability in buffer solution at 4 °C remained stable for more than one month.     

Questions and answers to edibility problem of the Canavalia ensiformis seeds – A review

The seed of Canavalia ensiformis (jackbean), a highly productive large-seeded tropical legume, contains about 300 g crude protein and 600 g carbohydrates kg⁻¹ dry matter. It, however, contains toxic and antinutritional factors which limit its use as human food or animal feed. The trypsin inhibitors in the seed have, however, been reported to be easily inactivated by moist heat. The toxic alkaline non-protein amino acid, canavanine (present at about 50 g kg⁻¹ seed dry matter), a naturally occuring analog of l-arginine, has also been reported to induce reduced feed intake in non-ruminants but at the equivalent of about 300 g kg⁻¹ dietary level of the raw seed, or in the condition of dietary arginine deficiency. The concentrations of the specialized secondary plant biomolecules – cyanogenic glycosides, saponins, alkaloids and terpenoids – have also been shown to drop below detectable levels following 1 h of cooking. Urease and canatoxin which were isolated from the seed are highly toxic if injected into experimental animals but exert no toxic effects if orally administered, and thus cannot be classified as antinutritional factors. Concanavalin A (Con A), the most studied of plant lectins, appears to be the most important toxic and antinutritional factor in the seed, being highly resistant to heat treatments and to proteolytic digestion in the gut. It requires 3 h of cooking at 96°C or 45 min of pressure cooking; 48 h of soaking the seed in water prior to cooking for 2 h; 72 h of soaking in water prior to ordinary cooking for 1 h; pressure-cooking for 15 min, to completely eliminate it from the seed. Complete inactivation of Con A in the seed can, however, be easily achieved if the seed is broken into pieces and cooked for 1 h or pressure-cooked for 15 min.     

Developmental variation of the blow fly Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) by different substrate tissue types

The green bottle blow fly Lucilia sericata is recognized as being among the first wave of the faunal succession on human cadavers. Thus, it is used to estimate the postmortem interval (PMI). The nutritional intake of larvae is likely to vary subject to the part of a corpse on which they are feeding. A study was therefore conducted to investigate the effect for the type of food substrate on larval growth in this species. Larvae were reared on cow liver, brain, heart, lung, kidney, intestine or minced meat. Results showed significant differences in the duration of the feeding, postfeeding larval, and pupal stages from different substrata (F = 42.43; 74.71 and 655.71; P < 0.0001, respectively). Larval growth measured as length varied significantly between different tissues (F = 3.56; P < 0.05). Larvae that were reared on heart were smaller than those reared on other tissues. Also, there was a significant effect for the type of tissue on the adult size of males (F = 2.41, P < 0.05) and females (F = 2.85, P < 0.05). These results may have important implications for forensic entomologists, since initial infestations commonly occur in wounds or in the cranial area. Therefore, the position at which larvae have been feeding on a body will be a crucial observation at a crime scene and is very important in PMI estimation.     

Descriptive analysis of kinematics and kinetics of catchers throwing to second base from their knees

In order to decrease the amount of time that it takes the catcher to throw the ball, a catcher may chose to throw from the knees. Upper extremity kinematics may play a significant role in the kinetics about the elbow observed in catchers throwing from the knees. If relationships between kinematics and kinetics exist then the development of training and coaching instruction may help in reduced upper extremity injury risk. Twenty-two baseball and softball catchers (14.36 ± 3.86 years; 165.11 ± 17.54 cm; 65.67 ± 20.60 kg) volunteered. The catchers exhibited a less trunk rotation (5.6 ± 16.2°), greater elbow flexion (87.9 ± 21.4°) and decreased humeral elevation (71.1 ± 12.3°) at the event of maximum shoulder external rotation as compared to what has previously reported in catchers. These variables are important, as they have previously been established as potential injury risk factors in pitchers, however it is not yet clear the role these variables play in catchers’ risk of injury. A positive relationship between elbow varus torque during the deceleration phase and elbow flexion at MIR was observed (r = 0.609; p = 0.003). Throwing from the knees reduces a catcher’s ability to utilize the proximal kinetic chain and this may help to explain why their kinematics and kinetics differ from what has previously been presented in the literature.     

Cenozoic long-term terrestrial climatic evolution in Germany tracked by δ18O of rodent tooth phosphate

Even though the Cenozoic has been recognized as a period of important climate change, long-term climatic changes that took place in the continental domain are still questioned. For an area, southern Germany, for which other long-term palaeoclimatic records exist, analysis of oxygen isotope composition of small mammal teeth has been carried out for localities ranging in age from the late Eocene (c.36 Ma) to the middle Miocene (c.10 Ma). Comparison of this long-term continental δ¹⁸O record with the marine record reveals comparable trends. The major Cenozoic climatic events are thus recognized in the continental oxygen isotope record.Through comparison with other quantitative palaeoclimatic reconstructions available for the studied area, temperatures in southern Germany are broadly estimated to have fluctuated between 12 °C and 25 °C from late Eocene to early late Miocene. According to the different available chronological frameworks, either a ～ 2 °C (considering the classical biostratigraphy) or a ～ 6 °C (considering a revised biostratigraphy) decrease in mean air temperature is estimated for the Eocene/Oligocene boundary.     

Simultaneous determination of quinolones for veterinary use by high-performance liquid chromatography with electrochemical detection

A selective method based on high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed to enable simultaneous determination of three fluoroquinolones (FQs), namely danofloxacin (DANO), difloxacin (DIFLO) and sarafloxacin (SARA). The fluoroquinolones are separated on a Novapack C-18 column and detected in a high sensitivity amperometric cell at a potential of +0.8 V. Solid-phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 10 to 150 ng g^?1 for danofloxacin, from 25 to 100 ng g^?1 for sarafloxacin and from 50 to 315 ng g^?1 for difloxacin, respectively. The method presents detection limits under 10 ng g^?1 and recoveries around 90% for the three analytes have been obtained in the experiments with fortified samples. This HPLC-ECD approach can be useful in the routine analysis of antibacterial residues being less expensive and less complicated than other more powerful tools as hyphenated techniques.     

Voluntary activation of the trapezius muscle in cases with neck/shoulder pain compared to healthy controls

Subjects reporting neck/shoulder pain have been shown to generate less force during maximal voluntary isometric contractions (MVC) of the shoulder muscles compared to healthy controls. This has been suggested to be caused by a pain-related decrease in voluntary activation (VA) rather than lack of muscle mass. The aim of the present study was to investigate VA of the trapezius muscle during MVCs in subjects with and without neck/shoulder pain by use of the twitch interpolation technique.Ten cases suffering from pain and ten age and gender matched, healthy controls were included in the study. Upper trapezius muscle thickness was measured using ultrasonography and pain intensity was measured on a 100 mm visual analog scale (VAS). VA was calculated from five maximal muscle activation attempts. Superimposed stimuli were delivered to the accessory nerve at peak force and during a 2% MVC following the maximal contraction.Presented as mean ± SD for cases and controls, respectively: VAS; 16.0 ± 14.4 mm and 2.1 ± 4.1 mm (P = 0.004), MVC; 545 ± 161 N and 664 ± 195 N (P = 0.016), upper trapezius muscle thickness; 10.9 ± 1.9 mm and 10.4 ± 1.5 mm (P = 0.20), VA; 93.6 ± 14.2% and 96.3 ± 6.0% (P = 0.29).In spite of significantly eight-fold higher pain intensity and ∼20% lower MVC for cases compared to controls, no difference was found in VA. Possible explanations for the reduction in MVC could be differences in co-activation of antagonists and synergists as well as muscle quality.     

Rapid phosphorylation of MAP kinase-like proteins in two species of Arctic kelps in response to temperature and UV radiation stress

Mitogen-activated protein kinases (MAPKs) are a group of cytoplasmic phosphoproteins that constitute the central core of the signalling network to respond to stress in most organisms. Their role in stress responses has been extensively studied in organisms from yeast to humans, and recently, their presence has also been described in higher plants as well as in micro- and macroalgae. In this study, we demonstrate via short experiments (1 h in duration), the rapid activation of two MAPKs similar to p38 and JNK of mammalian cells, in the Arctic kelps Laminaria solidungula and Saccharina latissima exposed to temperature and UV stress. The molecular mass of p38 is 40 kDa in L. solidungula and 42 kDa in S. latissima, while two JNKs were detected in both species, of 36 and 42 kDa in L. solidungula, and 36 and 40 kDa in S. latissima. These MAPKs are highly phosphorylated in response to temperature and UV light. In S. latissima, both p38 and the JNK showed higher phosphorylation at 2 °C than at 7 °C, while the reverse response was shown for L. solidungula. In addition, a significant increase in phosphorylation of both kinases was found following exposure to UV radiation (UVR). Exposure to PAR + UVA + UVB induced higher phosphorylation than PAR + UVA in L. solidungula, especially at 7 °C. In S. latissima, this response occurred only with JNK, and no differences in p38 phosphorylation between PAR + UVA and PAR + UVA + UVB at any temperature were observed. These results indicate the possible participation of MAPK-like proteins in response to stress in Arctic kelps, and that their activation is species-specific.     

Synthesis and evaluation of benzo[b]thiophene derivatives as inhibitors of alkaline phosphatases

Presence of basic calcium phosphate in knee joints of osteoarthritis patients could be prevented by inhibiting tissue non-specific alkaline phosphatase (TNAP) activity. Levamisole or the L stereoisomer of tetramisole (a known TNAP inhibitor) has been used as a treatment for curing rheumatoid arthritis but its therapeutical use is limited due to side effects. We report the synthesis and the TNAP inhibition property of benzo[b]thiophene derivatives, among which benzothiopheno-tetramisole and benzothiopheno-2,3-dehydrotetramisole, which could be involved in a drug therapy for osteoarthritis. Two water soluble racemic benzothiopheno-tetramisole and -2,3-dehydrotetramisole with apparent inhibition constants K_i = 85 ± 6 μM and 135 ± 3 μM (n = 3) comparable to that of enantiomeric levamisole 93 ± 4 μM were found. Several novel derivatives showed more pronounced inhibition properties towards intestinal alkaline phosphatase than TNAP.     

Operation of a bioelectrochemical system as a polishing stage for the effluent from a two-stage biohydrogen and biomethane production process

Anaerobic bioenergy production processes including fermentative biohydrogen (BioH₂), anaerobic digestion (AD) and bioelectrochemical system have been investigated for converting municipal waste or various biomass feedstock to useful energy carriers. However, the performance of a microbial fuel cell (MFC) fed on the effluent from a two-stage biogas production process has not yet been investigated extensively in continuous reactor operation on complex substrates. In this study we have investigated the extent to which a microbial fuel cell (MFC) can reduce COD and recover further energy from the effluent of a two-stage biohydrogen and biomethane system. The performance of a four-module tubular MFC was determined at six different organic loadings (0.036–6.149 g sCOD L⁻¹ d⁻¹) in terms of power generation, COD removal efficiency, coulombic efficiency (CE) and energy conversion efficiency (ECE). A power density of 3.1 W m⁻³ was observed at the OLR = 0.572 g sCOD L⁻¹ d⁻¹, which resulted in the highest CE (60%) and ECE (0.8%), but the COD removal efficiency decreased at higher organic loading rates (35.1–4.4%). The energy recovery was 92.95 J L⁻¹ and the energy conversion efficiency, based on total influent COD was found to be 0.48–0.81% at 0.572 g sCOD L⁻¹ d⁻¹. However, the energy recovery by the MFC is only reported for a four-module reactor and improved performance can be expected with an extended module count, as chemical energy remained available for further electrogenesis.