An unexpected cell‐penetrating peptide from Bothrops jararaca venom identified through a novel size exclusion chromatography screening

Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell‐penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography‐MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP – interestingly, a classical bradykinin‐potentiating peptide – in the peptide‐rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence‐labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell‐cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label‐free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Understanding the function of nocturnal song in ovenbirds: males do not countersing at night

Many diurnal bird species vocalize at night, however the function of nocturnal song is, generally, still poorly understood. Previous research has suggested that nocturnal song may serve a social function and is influenced by environmental factors. To test whether males attend to the nocturnal song of conspecifics, we experimentally exposed ovenbirds Seiurus aurocapilla to nocturnal flight songs, and recorded their response both during the night and during the following dawn chorus. We compared latency to song and vocal output before and after playback exposure to determine if males altered their vocalizations in response to exposure to flight songs from an unknown male. We found no evidence of counter singing or change in nocturnal song output, nor a change in vocal output during the dawn chorus following playback exposure. Our results suggest that, in ovenbirds, nocturnal song does not serve as an intraspecific social function. Nocturnal song, through rare, may be significant in the mating systems of some diurnal bird species, and requires additional study.     

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group (1/2). These results suggest different origins for these strains.     

Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.     

Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria

The proteomes of the venoms of the Brazilian wandering "armed" spiders Phoneutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi, were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21(A) automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2-27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca(2+), Na(+) and K(+)) channels and NMDA-type glutamate receptors. In addition two new families of small (3-4 kDa) toxins, some larger protein (>10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.     

Aspects of the bionomics of Phlebotomus sergenti sandflies from an endemic area of anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria

Aspects of the bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied from June to November 2005 in three foci of anthroponotic cutaneous leishmaniasis (ACL) in Aleppo Governorate, Syria, where the agent Leishmania tropica (Wright) (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus sergenti Parrot. Syria has been designated by the World Health Organization as one of four countries in the Old World where cutaneous leishmaniasis is hyperendemic, but little is known about the biology of local vector populations. Standard collections by sticky traps showed two peaks in density, in June and late August. In total, 1840 sandflies were caught, comprising five species: Phlebotomus papatasi (Scopoli) (68.0%); P. sergenti (25.4%); Sergentomyia minuta (Rondani) (6.4%); Phlebotomus tobbi Adler & Theodor (0.1%), and Phlebotomus mascittii canaaniticus Adler & Theodor (0.1%). Similar numbers of P. sergenti were caught indoors (246 specimens) and outdoors (222), whereas P. papatasi was significantly more abundant indoors (1096 specimens) than outdoors (156) (χ² = 241, P < 0.01). In total, 212 blood-fed females were tested for host blood determination, of which 176 (83.0%) reacted with anti-species reagent. Results from 20 P. sergenti suggest that this species is an opportunistic feeder, imbibing human, ovine, avian, bovine and feline blood, although more bloodmeals were taken from humans and cattle than expected in relation to the relative proportions of potential hosts present (the forage ratio, FR). The bionomics of P. sergenti are discussed in relation to the inefficacy of control campaigns based on indoor spraying with residual insecticides that have been implemented by the Syrian Ministry of Health to control the epidemics of ACL in the Aleppo Governorate.     

Performance of polyvoltine silkworm Bombyx mori L. on different mulberry plant varieties

Five cultivated mulberry plant varieties, BSRM‐34, BSRM‐56, BSRM‐57, BSRM‐58 and BSRM‐59, were used to evaluate the effects of plant variety on larval and cocoon characteristics of mulberry silkworm Bombyx mori L. Silkworm larvae undergo their entire larval periods on leaves of mulberry varieties. Different larval characteristics of larval weight, larval length and larval breadth were measured in different growth stages. After rearing, the fifth instar larvae were transferred into a mountage (Chandraki) structure for cocoon rearing for spinning and cocoon production. The cocoons were then harvested and different cocoon characteristics like cocoon weight, shell weight and shell percentage; and other characters like pupal weight and effective rate of rearing were measured. The results showed statistically significant differences among different varieties on larval and cocoon characteristics. The highest performance was observed by feeding the variety BSRM‐34 in respect of the average weight of 10 mature larvae (40.5 g), single mature larval length (7.6 cm) and breadth (1.0 cm), single cocoon weight (1.4 g), shell weight (0.2 g) and pupal weight (1.2 g) while the average performance was recorded by feeding varieties BSRM‐57 and BSRM‐59. The poorest performance was showed by feeding the variety BSRM‐56 (in case of larval characters) and BSRM‐58 (in case of cocoon characteristics). These two varieties also showed the poorest performance for silk yield. Through comparing all the above parameters it was concluded that BSRM‐34 is the best mulberry variety and it is recommended that Bangladeshi farmers adopt that variety for better yield of silk.     

Biofloc Technology: Emerging Microbial Biotechnology for the Improvement of Aquaculture Productivity

With the significant increases in the human population, global aquaculture has undergone a great increase during the last decade. The management of optimum conditions for fish production, which are entirely based on the physicochemical and biological qualities of water, plays a vital role in the prompt aquaculture growth. Therefore, focusing on research that highlights the understanding of water quality and breeding systems’ stability is very important. The biofloc technology (BFT) is a system that maximizes aquaculture productivity by using microbial biotechnology to increase the efficacy and utilization of fish feeds, where toxic materials such as nitrogen components are treated and converted to a useful product, like a protein for using as supplementary feeds to the fish and crustaceans. Thus, biofloc is an excellent technology used to develop the aquaculture system under limited or zero water exchange with high fish stocking density, strong aeration, and biota. This review is highlighted on biofloc composition and mechanism of system work, especially the optimization of water quality and treatment of ammonium wastes. In addition, the advantages and disadvantages of the BFT system have been explained. Finally, the importance of contemporary research on biofloc systems as a figure of microbial biotechnology has been emphasized with arguments for developing this system for better production of aquaculture with limited natural resources of water.Key words: biofloc, BFT, aquaculture, microbes, water quality, wastes     

Localization of filipin-sterol complexes in cell membranes of eosinophils

The polyene antibiotic filipin was used as a probe for the detection of cholesterol in the cell membranes of eosinophils isolated from the peritoneal exudate of rats. A homogenous distribution of filipin-sterol complexes was observed, both in thin sections and freeze-fracture replicas throughout the whole plasma membrane but not in the membrane of pynocytic vesicles, Golgi complex, endoplasmic reticulum, mitochondria and the nucleus. Few complexes were seen in freeze-fracture replicas showing the membrane of the specific granules. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.