Production and purification of self-assembling peptides in Ralstonia eutropha

Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.     

Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound

The blood-brain barrier (BBB) is a persistent obstacle for the local delivery of macromolecular therapeutic agents to the central nervous system (CNS). Many drugs that show potential for treating CNS diseases cannot cross the BBB and there is a need for a non-invasive targeted drug delivery method that allows local therapy of the CNS using larger molecules. We developed a non-invasive technique that allows the image-guided delivery of antibody across the BBB into the murine CNS. Here, we demonstrate that subsequent to MRI-targeted focused ultrasound induced disruption of BBB, intravenously administered dopamine D(4) receptor-targeting antibody crossed the BBB and recognized its antigens. Using MRI, we were able to monitor the extent of BBB disruption. This novel technology should be useful in delivering macromolecular therapeutic or diagnostic agents to the CNS for the treatment of various CNS disorders.     

Chronotype and postmenopausal breast cancer risk among women in the California Teachers Study

ABSTRACT

Chronotype is the behavioral manifestation of an individual’s underlying circadian rhythm, generally characterized by one’s propensity to sleep at a particular time during the 24 hour cycle. Evening chronotypes (“night owls”) generally suffer from worse physical and mental health compared to morning chronotypes (“morning larks”) – for reasons that have yet to be explained. One hypothesis is that evening chronotypes may be more susceptible to circadian disruption, a condition where the coordinated timing of biologic processes breaks down. The role of chronotype as an independent or modifying risk factor for cancer has not been widely explored. The objective of the current study was to evaluate the risk of breast cancer associated with chronotype in a case-control study nested within the California Teachers Study (CTS) cohort. The study population consisted of 39686 post-menopausal CTS participants who provided information on chronotype by completing a questionnaire in 2012–2013. 2719 cases of primary invasive breast cancer diagnosed from 1995/1996 through completion of the chronotype questionnaire were identified by linkage of the CTS to the California Cancer Registry. 36967 CTS participants who had remained cancer-free during this same time period served as controls. Chronotype was ascertained by responses to an abbreviated version of the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ) and was characterized into five categories: definite morning, more morning than evening, neither morning or evening, more evening than morning, definite evening. Multivariable unconditional logistic regression analyses were performed to estimate the odds ratios (ORs) and 95% confidence intervals (95% CIs) for each of the chronotypes, adjusted for established breast cancer risk factors. Compared to definite morning types, definite evening types had an increased risk of breast cancer with elevated ORs that were statistically significant in both the crude (OR = 1.24, 95% CI: 1.10–1.40) and fully-adjusted models (OR = 1.20, 95% CI: 1.06–1.35). The risk estimates in the fully-adjusted model for all other chronotypes did not significantly differ from one. These results suggest that evening chronotype may be an independent risk factor for breast cancer among a population of women who are not known to have engaged in any substantial night shift work. Further research in other populations of non-shift workers is warranted.     

Cysteine proteinases substitute for serine proteinases in the midgut glands of Crangon crangon and Crangon allmani (Decapoda: Caridea)

The utilization of dietary proteins in crustaceans is facilitated by a set of peptide hydrolases which are often dominated by “trypsin-like” serine proteinases. As expected, the North Sea shrimps Crangon crangon and Crangon allmani showed in their midgut glands high proteolytic activities. However, the majority of animals lacked trypsin and chymotrypsin. Conversely, a minority of about 10% of the animals had elevated trypsin activities. The appearance of trypsin was neither related to the mode of feeding nor to the nutritive state of the animals. When present, trypsin was expressed in both species as a single isoform of apparently 20 kDa. The lack of serine proteinases was also confirmed by inhibitor assays. AEBSF, a serine proteinase inhibitor, slightly reduced total proteinase activity by less than 10%. In contrast E 64, a cysteine proteinase inhibitor, caused a reduction of more than 70% of total proteinase activity, indicating that a substantial share of proteolytic activity is caused by cysteine proteinases. Cathepsin L-like proteinases were identified as major cysteine proteinases.A comparison with the eucarid crustaceans Pandalus montagui, Pagurus bernhardus, Cancer pagurus and Euphausia superba showed a similar high level of total proteinase activity in all species. Trypsin, however, varied significantly between species showing lowest activities in Caridea and the highest activity in E. superba. E 64 suppressed total proteinase activity by more than 70% in Crangon species but not in C. pagurus and E. superba. In contrast, the serine proteinase inhibitor AEBSF had only little effect in Caridea but was most effective in P. bernhardus, C. pagurus and E. superba. The results may indicate different traits of food utilization strategies in some eucarid crustaceans. Caridea may express predominantly cysteine proteinase, while in Anomura, Brachyura and Euphausiacea, serine proteinases may prevail.     

Evidence for a blockwise distribution of acetyl groups onto homogalacturonans from a commercial sugar beet (Beta vulgaris) pectin

Commercial acid-extracted sugar beet pectin was extensively hydrolysed using an endo-polygalacturonase (AnPGI from Aspergillus niger or AnPGII from A. niger or FmPG from Fusarium moniliforme) in combination with Aspergillus aculeatus pectin methyl-esterase (AaPME). The homogalacturonan-derived oligogalacturonates released were quantified by high-performance anion-exchange chromatography and their structure determined by mass spectrometry. The different endo-polygalacturonases exhibited variable tolerance towards acetyl groups. AnPGI was the most active and FmPG the less. A hypothetical homogalacturonan was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account FmPG observed requirements and hydrolysis products. A blockwise repartition of the acetyl groups onto sugar beet pectin homogalacturonan is proposed.     

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.     

Toxoplasma CRISPR/Cas9 constructs are functional for gene disruption in Neospora caninum

Herein we describe, to our knowledge for the first time the use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system for genome editing of Neospora caninum, an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide. By using plasmids containing the CRISPR/Cas9 components adapted to the closely related parasite Toxoplasma gondii, we successfully knocked out a green fluorescent protein (GFP) in an Nc-1 GFP-expressing strain, and efficiently disrupted the NcGRA7 gene in the Nc-Spain7 isolate by insertion of a pyrimethamine resistance cassette. The successful use of this technology in N. caninum lays the foundation for an efficient, targeted gene modification tool in this parasite.     

Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis,an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China,was completed in 2002.However,in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system.In order to establish such a system,the plasmid pBOLOl containing the replication origin of the T.tengcongensis chromosome and a kanamycin resistance cassette,in which kanamycin resistance gene expression was controlled by the ttel482 promoter from T.tengcongensis,was constructed and introduced into T.tengcongensis via electroporation.Subsequently,the high transformation efficiency occurred when using freshly cultured T.tengcongensis cells without electroporation treatment,suggesting that T.tengcongensis is naturally competent under appropriate growth stage.A genetic transformation system for this strain was then established based on these important components,and this system was proved to be available for studying physiological characters of T.tengcongensis in vivo by means of hisG gene disruption and complementation.     

Toxoplasma gondii: Over-expression of lactate dehydrogenase enhances differentiation under alkaline conditions

Toxoplasma gondii, an intracellular parasite, has two distinctive growth stages, namely rapidly growing tachyzoites and slowly growing bradyzoites. Here we report a unique physiological function of the last committed glycolytic enzyme of T. gondii, lactate dehydrogenase (TgLDH), which is present in two isoforms and expressed in a stage-specific manner. TgLDH1 is present in tachyzoites while TgLDH2 is found in bradyzoites. Using clonal transgenic parasites over-expressing either TgLDH1 or TgLDH2, we showed that the enzymatic activity, growth, and virulence of tachyzoites were unaffected by the presence of the recombinant protein. Interestingly, under alkaline conditions the presence of the recombinant TgLDH proteins increased the differentiation, as detected by the formation of cyst structures in vitro, while green fluorescent protein did not. The differentiation enhancement of the recombinant TgLDH1 and TgLDH2 strongly suggests that TgLDH1 and TgLDH2 have an important physiological function, in addition to being glycolytic enzymes and differentiation markers.     

Monitoring codling moth (Lepidoptera: Tortricidae) in orchards treated with pear ester and sex pheromone combo dispensers

Monitoring codling moth, Cydia pomonella (L.), with its sex pheromone (E,E)‐8,10‐dodecadien‐1‐ol (codlemone) alone or in combination with plant volatiles (PH‐plant volatile lures) with and without the addition of an acetic acid (AA) lure was compared in apple and walnut orchards treated with PVC Cidetrak^® dispensers (CM‐DA Combo) loaded with 60 mg pear ester, ethyl (E, Z)‐2,4‐decadienoate (PE) and 185 mg codlemone. Lures were also evaluated in untreated apple and walnut orchards and apple orchards treated with Cidetrak^® dispensers (CM) loaded with 120 mg codlemone. Traps with codlemone lures caught more moths than PE plus AA but similar numbers as traps with the PH‐PE lure in untreated apple and walnut orchards. The PH‐PE outperformed codlemone lures in apple orchards treated with CM dispensers. Codlemone outperformed PH‐PE lures in apple but not in walnut orchards treated mid‐season with CM‐DA Combo dispensers. Combining AA with PE significantly increased moth catch and together they performed similarly to the PH‐PE lure in all dispenser treatments in apple orchards. However, adding AA with the PH‐PE lure did not increase moth catches. The addition of AA to any of the lures tested except codlemone alone significantly increased the catch of females. Adding AA to either PE or the PH‐PE lure created a similarly attractive lure in orchards treated with CM‐DA Combo dispensers. Combining (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT) with codlemone in a combinational lure plus AA performed as well as any lure tested in apple orchards treated with CM‐DA Combo dispensers. Combinational lures loaded with either (E,E)‐farnesol, (E)‐β‐farnesene or DMNT performed significantly better than the PH‐PE lure in apple orchards treated with CM‐DA Combo dispensers only in the first generation. Both PH‐DMNT and PH‐farnesol lures were comparable to PH‐PE, except that the PH‐farnesol lure was less attractive to female moths.