Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

Parasin I (PI) is a 19 amino acid peptide with potent antimicrobial activities against a broad spectrum of microorganisms and is a good candidate for development as a novel antimicrobial agent. The objective of this study was to express and characterize a codon optimized parasin I peptide fused with human lysozyme (hLY). A 513 bp cDNA fragment encoding the mature hLY protein and parasin I peptide was designed and synthesized according to the codon bias of Pichia pastoris. A 4 × Gly flexible amino acid linker with an enterokinase cleavage (DDDDK) was designed to link the PI to the C-terminal of hLY. The codon optimized recombinant hLY-PI was cloned into the pPICZαA vector and expressed in P. pastoris. The over-expressed extracellular rehLY-PI was purified using Ni sepharose affinity column and exhibited a molecular mass of approximately 18 kDa. After digested with enterokinase the rehLY-PI protein release its corresponding rehLY and rePI, with molecular mass of 16 kDa and 2 kDa, respectively, on Tricine-SDS-PAGE. The released rehLY exhibited similar lytical activity against Micrococcus lysodeikticus to its commercial hLY. The digested rehLY-PI product exhibited antimicrobial activities against Bacillus subtilis, Staphylococcus aureus and Escherichia coli, and synergism has been found between the released rePI and rehLY. In conclusion, we successfully optimized a rehLY-PI fusion protein encoding gene and over-expressed the rehLY-PI in P. pastoris. The recombination protein digested with enterokinase released functional hLY and antimicrobial parasin I, which demonstrates a potential for future use as an animal feed additive to partly replace antibiotic.     

Activity of recombinant GST in Escherichia coli grown on glucose and glycerol

We have investigated production, solubility and activity of recombinant glutathione-S-transferase (GST) expressed in Escherichia coli BL21 grown in defined media with glucose or glycerol as carbon source. GST was predominantly expressed as a soluble protein on both carbon sources, and 83–84% was found in the supernatant after cell lysis. In cultures grown on glucose, only 32 ± 9% of the GST was active, while 76 ± 13% of the GST was active in cultures grown on glycerol. This shows that glycerol has the potential to increase the activity of soluble GST in E. coli cultures in vivo.     

Biological treatment of n-hexane and methanol in trickle bed air biofilters under acidic conditions

This study focuses on evaluating the degradation of n-hexane/methanol mixture in trickle-bed-air-biofilters (TBABs). Two different concentration ratios of methanol:n-hexane were evaluated (3:1) for TBAB “A” and (5:1) for TBAB “B”. Both TBABs were run and fed with nutrients buffered at pH 4 for encouraging the growth of fungi. The TBABs were loaded with pelletized diatomaceous earth support media and were run at an empty bed residence time of 120 s. n-Hexane loading rates (LRs) ranged from 0.9 to 13.2 g/m³ h for both TBABs. The corresponding methanol LRs varied from 2.3 to 37.7 g/m³ h and from 4.6 to 64.5 g/m³ h for TBABs “A” and “B”, respectively. Experimental results have shown that the degradation of n-hexane in presence of methanol is enhanced for n-hexane LRs less than 10.6 g/m³ h as compared to previous study for sole-fed n-hexane, but for n-hexane LRs of 13.2 g/m³ h, the performance of TBABs in eliminating n-hexane depended on the methanol to n-hexane ratios. The impact was less severe for TBAB “A” (RE 85%) as compared to TBAB “B” (RE 72%). This is attributed to the high LRs of methanol in TBAB “B”. n-Hexane performance stability was another advantage attained.     

Rapid torque development in older female fallers and nonfallers: A comparison across lower-extremity muscles

The objective of this study was to compare reaction time, joint torque, rate of torque development, and magnitude of neuromuscular activation of lower-extremity muscles in elderly female fallers and nonfallers. Participants included 11, elderly, female fallers (71.3 ± 5.4 years) and twelve nonfallers (71.3 ± 6.2 years) who completed a fall risk questionnaire. Then, maximal, voluntary, isometric contractions of the knee and ankle muscles were performed in reaction to a visual cue to determine joint torque, rate of torque development, reaction time, and nervous activation of agonists and antagonists. Results indicated that significantly more fallers reported “dizziness upon rising”, “use of balance altering medications”, “stress or depression”, “not enough sleep”, “arthritis in lower body”, “chronic pain in lower body”, and “tiring easily while walking” (all P < 0.05). Normalized dorsiflexion and plantarflexion strength scores (summation of peak torque, rate of torque development and impulse) were lower in fallers than in nonfallers (P < 0.05). When summed across lower-extremity muscle groups, fallers demonstrated 19% lower peak torque and 29% longer motor time (P < 0.05). In conclusion, comprehensive fall risk screening and prevention programs should address both neuromuscular and non-muscular factors, and, weakness of the ankle dorsiflexors and plantarflexors should be further studied as potential contributors to falls in older adults.     

Molecular cloning and characterization of two peptide toxins from the spider Araneus ventricosus

Spider toxins have great potential in the development of biopesticides. Here, we report the molecular cloning and characterization of two peptide toxins from the spider Araneus ventricosus. Two cDNAs encoding peptide toxins were cloned from A. ventricosus. Analysis of the cDNA sequence shows that the mature peptides of AvT-39 and AvT-48 consist of 39-amino acid residues and 48-amino acid residues, respectively. Both of the mature peptides include six conserved cysteine residues and a principal structural motif typical of spider toxins. The AvT-39 and AvT-48 cDNAs, which encode the mature peptide, were expressed in baculovirus-infected insect cells. AvT-39 and AvT-48 expression in insect cells significantly decreased cell viability. Additionally, the median lethal time (LT₅₀) of Spodoptera exigua larvae inoculated with recombinant AcNPV expressing AvT-48 was approximately 1 day shorter than that of larvae expressing wild-type AcNPV, demonstrating that the recombinant virus reduced LT₅₀ by approximately 25%. Taken together, our findings describe the molecular characterization of two peptide toxins from A. ventricosus and demonstrate the potential for these toxins to be used as biopesticides.     

Delinking indicators on regional industry development and carbon emissions: Beijing–Tianjin–Hebei economic band case

Based on the two-level Logarithmic Mean Divisia Index (LMDI) method and Tapio index, this paper constructed an expanded decomposition model for decoupling elasticity and effort index of industrial carbon emissions, and further quantitatively analyzed the delinking indicators on industry growth and environmental pressures in Beijing–Tianjin–Hebei (BTH) economic band from 1996 to 2010. The results indicate that: (1) together with the rapid economic growth, industrial energy-related carbon emissions in BTH region rose from 44477.43 × 10⁴ t in 1996 to 105602.33 × 10⁴ t in 2006 and then declined to 64426.71 × 10⁴ t in 2010, with an average annual growth rate of 2.94%. Our results also show that carbon emissions in BTH region were dominated by the secondary industry, which accounted for about 80% of total carbon emissions. (2) “Weak decoupling” was the main characteristic during the 9th Five-Year Plan period (1996–2000) and 11th Five-Year Plan period (2006–2010), particularly “strong decoupling” in 2007; while “weak coupling” was the main characteristic during the 10th Five-Year Plan period (2001–2005). (3) The rapid economic growth was the main factor responsible for industrial decoupling blocking. The energy structure and energy intensity made significant contributions to the industrial decoupling progress. The industrial structure effect had varied considerably over the years without showing a clear trend.     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.     

Estimating the contribution of N-sulfocarbamoyl paralytic shellfish toxin analogs GTX6 and C3 + 4 to the toxicity of mussels (Mytilus galloprovincialis) over a bloom of Gymnodinium catenatum

Gymnodinium catenatum, a dinoflagellate species with a global distribution, is known to produce paralytic shellfish poisoning (PSP) toxins. The profile of toxins of G. catenatum is commonly dominated by sulfocarbamoyl analogs including the C3 + 4 and GTX6, which to date has no commercial certified reference materials necessary for their quantification via chemical methods, such as liquid chromatography. The aim of this study was to assess the presence of C3 + 4 and GTX6 and their contribution to shellfish toxicity. C3 + 4 and GTX6 were indirectly quantified via pre-column oxidation liquid chromatography with fluorescence detection after hydrolysis conversion into their carbamate analogs. Analyses were carried out in mussel samples collected over a bloom of G. catenatum (>63 × 10³ cells l⁻¹) in Aveiro lagoon, NW Portuguese coast. Concentration levels of sulfocarbamoyl toxin analogs were two orders of magnitude higher than decarbamoyl toxins, which were in turn one order of magnitude higher than carbamoyl toxins. Among the sulfocarbamoyl toxins, C1 + 2 were clearly the dominant compounds, followed by C3 + 4 and GTX6. The least abundant sulfocarbamoyl toxin was GTX5. The most important compounds in terms of contribution for sample toxicity were C1 + 2, which justified 26% of the PSP toxicity. The lesser abundant dcSTX constitutes the second most important compound with similar % of toxicity to C1 + 2, C3 + 4 and GTX6 were responsible for approximately 11% and 13%, respectively. The median of the sum of C3 + 4 and GTX6 was 27%. These levels reached a maximum of 60% as was determined for the sample collected closest to the G. catenatum bloom. This study highlights the importance of these low potency PSP toxin analogs to shellfish toxicity. Hydrolysis conversion of C3 + 4 and GTX6 is recommended for determination of PSP toxicity when LC detection methods are used for PSP testing in samples exposed to G. catenatum.     

In vivo skin dose measurement using MOSkin detectors in tangential breast radiotherapy

The purpose of this study is to measure patient skin dose in tangential breast radiotherapy. Treatment planning dose calculation algorithm such as Pencil Beam Convolution (PBC) and in vivo dosimetry techniques such as radiochromic film can be used to accurately monitor radiation doses at tissue depths, but they are inaccurate for skin dose measurement. A MOSFET-based (MOSkin) detector was used to measure skin dose in this study. Tangential breast radiotherapies (“bolus” and “no bolus”) were simulated on an anthropomorphic phantom and the skin doses were measured. Skin doses were also measured in 13 patients undergoing each of the techniques. In the patient study, the EBT2 measurements and PBC calculation tended to over-estimate the skin dose compared with the MOSkin detector (p < 0.05) in the “no bolus radiotherapy”. No significant differences were observed in the “bolus radiotherapy” (p > 0.05). The results from patients were similar to that of the phantom study. This shows that the EBT2 measurement and PBC calculation, while able to predict accurate doses at tissue depths, are inaccurate in predicting doses at build-up regions. The clinical application of the MOSkin detectors showed that the average total skin doses received by patients were 1662 ± 129 cGy (medial) and 1893 ± 199 cGy (lateral) during “no bolus radiotherapy”. The average total skin doses were 4030 ± 72 cGy (medial) and 4004 ± 91 cGy (lateral) for “bolus radiotherapy”. In some cases, patient skin doses were shown to exceed the dose toxicity level for skin erythema. Hence, a suitable device for in vivo dosimetry is necessary to accurately determine skin dose.     

The effect of inspiratory and expiratory loads on abdominal muscle activity during breathing in subjects “at risk” for the development of chronic obstructive pulmonary disease and healthy

The abdominal muscle activity has been shown to be variable in subjects with chronic obstructive pulmonary disease (COPD) when respiratory demand increases and their recruitment pattern may change the mechanics, as well as the work and cost of breathing. The scientific evidence in subjects “at risk” for the development of COPD may be important to understand the natural history of this disease. This study aims to evaluate the effect of inspiratory and expiratory loads on the abdominal muscle activity during breathing in subjects “at risk” for the development of COPD and healthy. Thirty-one volunteers, divided in “At Risk” for COPD (n = 17; 47.71 ± 5.11 years) and Healthy (n = 14; 48.21 ± 6.87 years) groups, breathed at the same rhythm without load and with 10% of the maximal inspiratory or expiratory pressures, in standing. Surface electromyography was performed to assess the activation intensity of rectus abdominis (RA), external oblique and transversus abdominis/internal oblique (TrA/IO) muscles, during inspiration and expiration. During inspiration, in “At Risk” for COPD group, RA muscle activation was higher with loaded expiration (p = 0.016); however, in Healthy group it was observed a higher activation of external oblique and TrA/IO muscles (p < 0.050). During expiration, while in “At Risk” for COPD group, RA muscle activation was higher with loaded inspiration (p = 0.009), in Healthy group TrA/IO muscle showed a higher activation (p = 0.025). Subjects “at risk” for the development of COPD seemed to have a specific recruitment of the superficial layer of ventrolateral abdominal wall for the mechanics of breathing.     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

The “deduction” approach: A non-invasive method for estimating secondary production of earthworm communities

Secondary production is an important parameter for the study of population dynamics and energy flow through animal communities. Secondary production of earthworm communities has been determined with the size-frequency and instantaneous growth rate methods, whereby earthworm populations are repeatedly sampled at regular intervals and the change in biomass of cohorts or individuals between sampling dates is determined. The major disadvantage of repeated sampling is that it disturbs the soil and permanently removes earthworms from the study area. The “deduction” approach is a theoretical model that partitions individuals into defined pools and makes assumptions about the growth, recruitment and mortality of each pool. In 2004 and 2005, earthworms were added to undisturbed field enclosures and the “deduction” approach was used to estimate secondary production of the indigenous and added earthworm populations during the crop growing period (17–18 weeks) in each year. Secondary production estimates made by the “deduction” approach were similar to estimates from direct earthworm sampling in temperate agroecosystems. The “deduction” approach is an indirect method that estimates population dynamics and secondary production, and is appropriate for manipulation experiments where removal of organisms and physical disturbance of the habitat by repeated sampling could bias results.     

Reproductive toxicity in boron exposed workers in Bandirma, Turkey

Boric acid and sodium borates have been considered as being “toxic to reproduction and development”, following results of animal studies with high doses. However unfavorable effects of boron exposure on reproduction and development have not been proved in epidemiological studies so far. The aim of the present study was to investigate the reproductive toxicity indicators in highly exposed workers employed in a boric acid production plant in Band?rma, Turkey. Two hundred and four workers participated in this study. The mean blood boron concentration of the high exposure group of workers was 223.89 ± 69.49 (152.82–454.02) ng/g. Unfavorable effects of boron exposure on the reproductive toxicity indicators were not observed.     

Biodegradability and mechanical properties of polycaprolactone composites encapsulating phosphate-solubilizing bacterium Bacillus sp. PG01

This study examined the feasibility of using polycaprolactone (PCL) and its composites (with starch and/or clay) in encapsulating cells of phosphate-solubilizing bacteria (PSB) for the development of biodegradable and “controlled-release” bacterial fertilizer. The PSB used in this work was an indigenous Bacillus sp. PG01 isolate. The results show that the PG01 strain was able to degrade all the cell-loaded capsules made of PCL and PCL composites, resulting in a continual cell release. Morphology observation indicates that severe disruption of the capsule structure occurred after incubation for 15–20 days. The biodegradability of the capsules decreased in the order of PCL/starch (20 wt%) > PCL/starch (20 wt%)/cay (7 wt%) > PCL alone > PCL/clay (7 wt%). Similar trends were also observed for the decrease in tensile strength and elongation at break, suggesting strong connections between biodegradability and the mechanical properties. Addition of starch appeared to enhance the biodegradability of the capsules, whereas the clay-blended composites were less biodegradable. The amount and rate of cell release from cell-encapsulated PCL-based capsules were positively dependent on the biodegradability and on the decrease in the mechanical strength. Nevertheless, the pattern of cell release was quite similar for all types of capsules. The outcome of this work seems to suggest that by proper manipulation of composite compositions, the controlled release of the bacterial fertilizer (i.e., Bacillus sp. PG01 cells) might be achievable.     

Heterotrimeric G protein α-subunit is localized in the plasma membrane of Pinus bungeana pollen tubes

Heterotrimeric G proteins are involved in a variety of cellular responses, but relatively little is known about their function and biochemistry in plant pollen. In this paper, we establish the presence of a G protein associated with the plasma membranes of Pinus bungeana pollen tube. A 40 kDa polypeptide is detected and immunolocalized predominantly in pollen tube plasma membranes by polyclonal antisera directed against conserved peptides of mammalian Gα-subunit during pollen tube development. Cholera and pertussis toxins exhibited biphasic actions on tube growth, that is to say, inhibited pollen tube growth and result in rupture of tubes at concentrations less than 400 ng mL⁻¹, whereas stimulated pollen tube growth at concentration over 500 ng mL⁻¹. Fourier transform-infrared (FT-IR) spectra showed that the two toxins at concentrations of 400 ng mL⁻¹ resulted in enhanced synthesis of phenolics and reduced synthesis of cellulose, hemicellulose, and xylan of pollen tube wall, which may account for incidental rupture of pollen tubes at the concentration. These results suggest that the two toxins possibly affect pollen tube growth via downstream pertussis or cholera toxin-sensitive functional proteins, which regulate tube wall biosynthesis than at the Gα-subunit in P. bungeana tube growth.     

Stress as adaptation? A test of the adaptive boost hypothesis among Batswana men

Many studies have found elevated cortisol linked to negative events (“stress”) and subsequent negative outcomes, such as reduced immunity and stunted growth, leading to the conclusion that high cortisol is “bad.” However, a growing number of studies have found more advantaged groups showing relatively elevated cortisol. For example, higher morning cortisol followed by a steeper diurnal decline among Caucasians compared to ethnic minorities has been interpreted as a context-specific “adaptive boost” to meet daily demands. We tested the adaptive boost hypothesis using data on socioeconomic status, depressive affect and salivary cortisol among adult men (n = 32) in Botswana. Three findings emerged: (i) depressive affect was associated with lower morning cortisol (r = ? 0.43, p = 0.014); (ii) depressive affect was associated with a diurnal increase in cortisol when comparing morning and evening samples (r = 0.49, p = 0.004); and (iii) depressive affect was associated with lower income (r = ? 0.55, p = 0.001). Findings are consistent with the adaptive boost hypothesis and add to a growing body of evidence that elevated cortisol is not universally bad. Hypothalamic–pituitary adrenal axis (HPAA) activity, such as cortisol, may be adaptive depending on a person's contextual circumstances. Based on our findings and those of previous studies, we develop a “person-in-context” model of the threat appraisal process. Integrated with life history theory, our model facilitates testable hypotheses about intra- and inter-individual variability in HPAA and adaptive consequences.