Bacterial expression of larval peritrophins of Chryosomya bezziana

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and –42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies.   Key words: Chrysomya, peritrophic membrane, peritrophin, recombinant protein, hexaHis tag, GS

Purification of recombinant peritrophic membrane proteins of the Old World Screwworm fly, Chrysomya bezziana

To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike.   Key words: Screwworm fly, Chrysomya bezziana, recombinant proteins, immobilized metal affinity chromatograph

Unpacking the proteome and metaproteome of the black soldier fly larvae: Efficacy and complementarity of multiple protein extraction protocols

The larvae of the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), have demonstrated the ability to efficiently bioconvert organic waste into a sustainable source of food and feed, but fundamental biology remains to be discovered to exploit their full biodegradative potential. Herein, LC-MS/MS was used to assess the efficiency of eight differing extraction protocols to build foundational knowledge regarding the proteome landscape of both the BSF larvae body and gut. Each protocol yielded complementary information to improve BSF proteome coverage. Protocol 8 (liquid nitrogen, defatting, and urea/thiourea/chaps) was better than all other protocols for the protein extraction from larvae gut samples, and the exclusion of defatting steps yielded the highest number of proteins for the larval body samples. Protocol-specific functional annotation using protein level information has shown that the selection of extraction buffer can affect protein detection and their associated functional classes within the measured BSF larval gut proteome. A targeted LC-MRM-MS experiment was performed on the selected enzyme subclasses to assess the influence of protocol composition using peptide abundance measurements. Metaproteome analysis of the BSF larvae gut has uncovered the prevalence of two bacterial phyla: actinobacteria and proteobacteria. We envisage that using complementary extraction protocols and investigating the proteome from the BSF body and gut separately will expand the fundamental knowledge of the BSF proteome and thereby provide translational opportunities for future research to enhance their efficiency for waste degradation and contribution to the circular economy

The Effect of Supplementing Mannan Oligosaccharide or Finely Ground Fiber, during the Summer on Body Temperature, Performance, and Blood Metabolites of Finishing Steers

Crossbred beef steers (12 pens, n=96) were used to determine the effect of adding Agrimos or 5% ground (1 in.) wheat straw compared to a control on body temperature, panting score and performance. Th ere were no differences in final BW, ADG, and DMI among treatments. Feed conversion was increased for cattle fed 5% additional ground straw when compared to control and Agrimos. Hot carcass weight, dressing %, LM area, and marbling score were not different among treatments. Cattle fed the control had greater 12th rib fat depth and USDA yield grade than cattle fed straw or Agrimos. Both average and maximum body temperatures were slightly greater for cattle fed Agrimos than for cattle fed control or added straw. Panting scores were decreased slightly for cattle fed the extra straw when compared to control and Agrimos. The addition of Agrimos or wheat straw to the diet had minimal effects on heat stress measures

Calculation of metal buildings with structural elements of corrugated trunks

100 σ.Στα πλαίσια της παρούσας διπλωματικής εργασίας μελετάται η κατασκευή ενός απλού μονώροφου κτιρίου από χάλυβα S235, με χρήση δοκών με αυλακωτούς κορμούς. Συνήθως για πλαίσια με μεγάλα ανοίγματα, γίνεται χρήση δικτυωτής δοκού, όπου μέσω μεταβολής του εμβαδού των ράβδων και του στατικού ύψους, επιτυγχάνεται η βελτιστοποίηση του βάρους. Όμως, η μείωση του κόστους συνδέεται με πρόσθετα μέτρα όσον αφορά την οργάνωση, την παραγωγή και την ανέγερση του έργου. Η χρήση δικτυωμάτων για τα συνήθη ανοίγματα και στατικά ύψη συνίσταται αντιοικονομική λόγω κόστους των συνδέσεων των ράβδων. Έτσι, αποφασίστηκε χρήση δοκών με αυλακωτούς κορμούς για τις δοκούς και τα υποστυλώματα του υπό μελέτη κτιρίου. Οι δοκοί με αυλακωτούς κορμούς είναι συγκολλητές διατομές, η παραγωγή των οποίων γίνεται τελείως αυτόματα. Η κάμψη των ελασμάτων των κορμών, η σύνδεσή τους και η συγκόλληση τους με τα πέλματα γίνεται σε μια φάση εργασίας. Αρχικά, γίνεται υπολογισμός των δράσεων του φέροντος οργανισμού του κτιρίου, που προέρχονται από μόνιμα (ίδιον βάρος κατασκευής, φορτία αναρτήσεων, φορτία επικαλύψεων), κινητά (χιόνι, άνεμος) και σεισμικά φορτία. Στην συνέχεια γίνεται διαστασιολόγηση της δοκού και του υποστυλώματος για τους επιμέρους ελέγχους που απαιτούνται, όσον αφορά τη χρήση δοκών με αυλακωτούς κορμούς, όπως έλεγχος έναντι διατμητικού λυγισμού (κύρτωση) της ορθότροπης πλάκας, αντοχή έναντι τέμνουσας, έναντι συγκεντρωμένου φορτίου και συνδυαστική αντοχή έναντι αξονικών δυνάμεων και ροπών. Αναλυτικά, ξετάστηκαν δύο περιπτώσεις πλαισίων, ένα με δοκούς με κυματοειδής κορμούς και ένα με δοκούς με τραπεζοειδείς κορμούς. Στη συνέχεια, προχωρήσαμε στη σύνδεση δοκού και υποστυλώματος μέσω μια κοχλίωσης. Αρχικά τοποθετήθηκε μια μετωπικά πλάκα, πάνω στην οποία συγκολλήθηκε η δοκός και στη συνέχεια με χρήση κοχλιώσεων έγινε η σύνδεση δοκού και υποστυλώματος. Ο χάλυβας που χρησιμοποιήθηκε είναι S235, ενώ οι κοχλίες είναι Μ24 και ποιότητας 10.9. Η πραγμάτωση των παραπάνω ελέγχων για τον φέροντα οργανισμό και την κοχλίωση έγινε με χρήση του προγράμματος Microsoft Excel, ενώ η σχεδίαση της λεπτομέρειας της κοχλίωσης έγινε με Autocad.Ιn the present of this diploma thesis , we deal with the construction of a simple storey metal building, using beams with corrugated trunks. For frames with large openings, use is made of lattice ginder, wherein by varying the area of the bars and the static height, we can achieve weight optimization. However, the cost savings are associated with additional measures regarding the organization, production and construction of the project. Τhe use of a netting for standard openings and static heights is uneconomical due to cost of connections of rods. Thus, it was decided using beams with corrugated trunks for beams and columns of the studied building. The beams with corrugated trunks are welded sections, the production of which is fully automatic. The bending of plates of logs, connecting a bonding with soles are made in a working phase. First, we start with a calculation of the actions of the metal structure of the building from permanent (self-weight construction, suspension loads, loads of coatings), mobile (snow, wind) and seismic loads. Then becomes dimensioning of the beam and the columns for the individual controls required in the use of beams with corrugated trunks, as control versus shear buckling of orthotropic plate, shear resistance and concentrated load versus combined resistance to axial forces and torques. Analytically, we examined two cases of frames, one with undulatory beams and a corrugated web beams with trapezoidal trunks. Then we proceeded to connect beams and columns via a screw. Initially, we placed frontally a plate, on which the beam was annealed, and then by using bolting we ade the connection of the beam and the column. The steel used is S235 , while the screws are M24 and 10.9 quality. The making of these checks for load bearing and bolting was performed by using the program Microsoft Excel, while the design detail of the screw was performed by using Autocad.Δημήτριος Σ. Χονδρόπουλο

Secretion of the type 2 peritrophic matrix protein, peritrophin-15, from the cardia

The midgut of most insects is lined with a peritrophic matrix, which is thought to facilitate digestion and protect the midgut digestive epithelial cells from abrasive damage and invasion by ingested micro-organisms. The type 2 peritrophic matrix is synthesised by a complex and highly specialised organ called the cardia typically located at the junction of the cuticle-lined foregut and midgut. Although the complex anatomy of this small organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2 peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and secretion of the intrinsic peritrophic matrix protein, peritrophin-15 has been followed in the cardia of Lucilia cuprina larvae using immuno-gold localisations. The protein is synthesised by cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with the plasma membrane at the base of the microvilli and release peritrophin-15 into the inter-microvilli spaces. The protein then becomes associated with the nascent peritrophic matrix, which lies along the tips of the epithelial cell microvilli. It is proposed that peritrophin-15 binds to the ends of chitin fibrils present in the nascent peritrophic matrix, thereby protecting the fibril from the action of exochitinases

Identification of putative DnaN-binding motifs in plasmid replication initiation proteins

Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the β subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation. Crow

Bacterial expression of larval peritrophins of Chryosomya bezziana

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and –42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies

Purification of recombinant peritrophic membrane proteins of the Old World Screwworm fly, Chrysomya bezziana

To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike