A method for the temperature-controlled extraction of DNA from ancient bones v1

We here provide a protocol for the decontamination of ancient bones and teeth that is based on a temperature-controlled, sequential release of DNA. DNA can be extracted from all fractions generated with this method and the fraction with the highest proportion of endogenous DNA identified for further analysis. The protocol proceeds through repeated incubation of the sample powder in phosphate buffer at 37, 60 and 90 °C, followed by the complete lysis of the residual sample powder. As DNA is denatured at high temperature, subsequent DNA extraction and library preparation has to be performed using methods optimized for single-stranded DNA.

A temperate phage 5W infecting multidrug-resistant Acinetobacter baumannii

We isolated 5W, a temperate phage that infects multidrug-resistant Acinetobacter baumannii from pond water, using an enrichment method that involves the addition of host bacteria. 5W is a long-tailed phage with a narrow host range lysed four of 19 A. baumannii clinical isolates tested, and complete lysis was observed for A. baumannii clinical isolate Ab1. 5W adsorbed rapidly to its Ab1 host and > 80% adsorption was observed after 2 min of mixing. The one-step growth curve showed that 5W has a 20 min latent period and a ~ 100 min rise period, with a burse size of ~ 180 PFU/cell. 5W contains a dsDNA genome 43,032 bp in length, with 61 open reading frames and a GC content of 39.85%. The genome lacks any known virulence and drug resistance genes, but encodes an N-acetyl-β-D-muramidase with numerous positively charged amino acids at its C-terminus that belongs to the GH_108 family. The M/S subunits of the restriction endonuclease are inserted in the lysogenic gene cluster. The first and second halves of the 5W genome are highly homologous with prophages phiABCR01-03 and phiABCR01-02 in the A. baumannii ABCR01 genome, respectively, which suggests that 5W may be a product of recombination between the two prophages.

Use of a Specific Phage Cocktail for Soft Rot Control on Ware Potatoes: A Case Study

Using bacteriophages (bacterial viruses) to control pathogenic bacteria is a promising approach in horticulture. However, the application of this strategy in real conditions requires compliance with particular technological and environmental restraints. The presented paper concerns the process of phage selection to create a cocktail that is efficient against the circulating causal agents of potato soft rot. The resulting phage cocktail causes a complete lysis of a mixture of circulating pectobacterial strains in vitro. In the context of being used to treat ware potatoes during off-season storage, the protocol of phage application via the humidity maintenance system was designed. The phage cocktail was shown to reduce the population of Pectobacterium spp. 10–12-fold, achieving a population that was below a symptomatic threshold.

Green Biosynthesized Selenium Nanoparticles by Cinnamon Extract and Their Antimicrobial Activity and Application as Edible Coatings with Nano-Chitosan

Bioactive nanocomposites were constructed, containing chitosan (Cht), extracted from shrimps’ wastes, and transformed into nanoparticles (NPs) using ionic-gelation. Selenium NPs (Se-NPs) were phytosynthesized using cinnamon (Cinnamomum zeylanicum) bark extract (CIE), characterized and evaluated with Cht-NPs as antimicrobial composites against bacterial food-borne pathogens “Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes” and as potential edible coating (EC) basements. The CIE-phytosynthesized Se-NPs had well-distributed and spherical shapes with 23.2 nm mean diameter. The CIE, CIE/Se-NPs, and innovative CIE/Se/Cht-NP composites exhibited distinguished antibacterial actions toward the entire screened pathogens; CIE/Se/Cht-NP composite was significantly the most potent. The formulated ECs from CIE/Se/Cht-NP nanocomposites had matching antibacterial manner, which was strengthened with CIE/Se-NP percentage increments. Scanning micrographs indicated the attachment of CIE/Se-NPs to bacterial cells to cause their complete lysis and death after 10 h of exposure. CIE/Se/Cht-NP composites are proposed as effectual control agents toward food-borne pathogens using efficient biological carriers and eco-friendly phytosynthesis protocol.

Features of obtaining the protein complex of vegetative cultures Bacillus anthracis for proteomic mapping of strains

Introduction. The study of the protein composition of the causative agent of anthrax — Bacillus anthracis, allows you to identify both general species and individual characteristics of strains that differ in phenotypic properties, manifested mainly in the vegetative form and which are important for virulence, immunogenicity and the ability to adapt to different vegetation conditions.Purpose of the work. In the group of anthrax microbe strains having different plasmid composition and virulence, different methods of extraction of the total proteome from vegetative bacillus cells have been tested.Results. In the course of the work, it was shown that the rate of spore formation varies significantly between individual strains of the anthrax microbe and can have a significant impact on the efficiency of extraction and the composition of the protein complex. Preliminary treatment with lysozyme, which affects the cell membrane, promotes a more complete lysis of cells, and ultramicrocentrifuge filtration provides complete specific sterility of the obtained samples.Conclusion. A culture preparation scheme was developed for B. anthracis, which allows one to obtain a culture in the vegetative phase of the life cycle and to efficiently extract proteins in combination with reliable disinfection of samples.

Phytosynthesis of selenium nanoparticles using the costus extract for bactericidal application against foodborne pathogens

Selenium (Se) as a bioactive micronutrient could be augmented via transforming into nanoparticles (NPs), especially using biogenic protocols, for usage as an antimicrobial element. The reducing power of costus (Saussurea costus) root extract (SCE) was employed for phytosynthesis of Se-NPs through a simple and rapid protocol that included stirred mixing of 10 mM Na2SeO3 with 1.0% SCE solution for 4 h. The phytosynthesized SCE/Se-NP composite was obtained with a mean diameter of 6.13 nm and a zeta potential of −42.8 mV. Infrared analyses revealed the involvement of many SCE phytogroups in Se-NP synthesis, whereas transmission microscopy displayed well distribution and spherical shapes of the phytosynthesized NPs. The antibacterial assessments against foodborne pathogens (Escherichia coli, Salmonella typhimurium and Staphylococcus aureus) revealed the superior powers of SCE/Se-NPs and the elevated potentialities of SCE and Se-NPs for inhibition of bacterial pathogens. The scanning micrographs indicated that SCE/Se-NPs were attached to bacterial cells and led to their complete lysis/explosion with exposure prolongation. The SCE/Se-NP composites are recommended for the effective control of foodborne bacterial pathogens, applying a simple and eco-friendly phytosynthesis protocol.

Biosynthesis and Antimicrobial Activity of Pseudodesmin and Viscosinamide Cyclic Lipopeptides Produced by Pseudomonads Associated with the Cocoyam Rhizosphere

Pseudomonas cyclic lipopeptides (CLPs) are encoded non-ribosomally by biosynthetic gene clusters (BGCs) and possess diverse biological activities. In this study, we conducted chemical structure and BGC analyses with antimicrobial activity assays for two CLPs produced by Pseudomonas strains isolated from the cocoyam rhizosphere in Cameroon and Nigeria. LC-MS and NMR analyses showed that the Pseudomonas sp. COR52 and A2W4.9 produce pseudodesmin and viscosinamide, respectively. These CLPs belong to the Viscosin group characterized by a nonapeptidic moiety with a 7-membered macrocycle. Similar to other Viscosin-group CLPs, the initiatory non-ribosomal peptide synthetase (NRPS) gene of the viscosinamide BGC is situated remotely from the other two NRPS genes. In contrast, the pseudodesmin genes are all clustered in a single genomic locus. Nano- to micromolar levels of pseudodesmin and viscosinamide led to the hyphal distortion and/or disintegration of Rhizoctonia solani AG2-2 and Pythium myriotylum CMR1, whereas similar levels of White Line-Inducing Principle (WLIP), another member of the Viscosin group, resulted in complete lysis of both soil-borne phytopathogens. In addition to the identification of the biosynthetic genes of these two CLPs and the demonstration of their interaction with soil-borne pathogens, this study provides further insights regarding evolutionary divergence within the Viscosin group.

Novel Protocol for Estimating Viruses Specifically Infecting the Marine Planktonic Diatoms

Since their discovery, at least 15 diatom viruses have been isolated and characterised using a culture method with two cycles of extinction dilution. However, the method is time consuming and laborious, and it isolates only the most dominant virus in a water sample. Recent studies have suggested inter-species host specificity of diatom viruses. Here, we describe a new protocol to estimate previously unrecognised host-virus relationships. Host cell cultures after inoculation of natural sediment pore water samples were obtained before complete lysis. The proliferated viral genomes in the host cells were amplified using degenerate primer pairs targeting protein replication regions of single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) viruses, and then sequenced. Diverse ssRNA virus types within known diatom virus group were detected from inoculated Chaetoceros tenuissimus and C. setoensis cells. A previously unknown ssDNA virus type was detected in inoculated C. tenuissimus cells, but not in C. setoensis cells. Despite the possible protocol biases, for example non-specific adsorptions of virions onto the host cells, the present method helps to estimate the viruses infectious to a single host species. Further improvements to this protocol targeting the proliferated viral genomes might reveal unexpected diatom–virus ecological relationships.

Spontaneous Necrosis of a Large Choroidal Melanoma: Unusual Presentation in a 49-Year-Old Male

Purpose: To demonstrate a case of massive vitreous haemorrhage obscuring the underlying diagnosis of a large mixed-cell choroidal melanoma which had undergone spontaneous necrosis. Case Report: A 49-year-old man in good general health suddenly lost vision in his right eye due to an extensive vitreous haemorrhage 1 day after a workout at the gym. He reported good vision prior to that without any symptoms of flashes, floaters, or shadows. He was referred to the vitreoretinal department of a tertiary eye hospital, where he presented with a drop in vision to light perception only in the right phakic eye. Pars plana vitrectomy was performed in the right eye, which revealed intraoperatively massive retinal ischemia and choroidal haemorrhage, but no obvious tumour mass that could have been biopsied. The vitrectomy cassette specimen was sent for histopathology, where “ghost-like” melanoma cells were identified. The eye was subsequently enucleated, revealing an extensively necrotic and haemorrhagic choroidal melanoma of mixed cell type with only small viable tumour foci at the base and almost complete lysis of the detached retina. Conclusion: Some uveal melanomas (UMs) undergo spontaneous necrosis due to rapid growth, with the centre of the tumour outstripping its established blood supply in the “watershed area” of the eye, and becoming hypoxic with associated necrosis of intraocular structures. Such UMs are often associated with haemorrhage and/or inflammation and usually cause significant destruction of ocular tissues, resulting in enucleation as the only treatment option.

The acceleration of collagen biodegradation after adsorption of mesenchymal multipotent stromal cells in experiment

Background. The scientific literature contents clearly not enough data at interaction of collagen materials with a living organism and about influence of multipotent stromal cells (MSC) on this process. There are controversies about the collagen degradation and the development of foreign body reactions. However, without account these results, it is impossible to estimate the timeframes for complete lysis of such materials, to progress effective methods for the prevention and treatment of developing complications. Objective. To study the features of the collagen-based material degradation after implantation with adsorbed autologous mesenchymal MSC of bone marrow origin (AMMSCBMO) in the experiment. Design and methods. In different times the condition of tissues around the implanted collagen membrane with adsorbed AMMSCBMO was studied by method of light microscopy. Results. The number of vessels and cellular elements in the collagen material implanted without AMMSCBMO increases to 3 weeks and remains at this level until the end of the observation. A distinctive features of the use of collagen membrane with adsorbed AMMSCBMO are increased vascularization and cellular infiltration of the material in the first 2 weeks after surgery. This effect further leads to a more rapid degradation of all implanted collagen, including its more dense parts, but does not prevent the formation of multinuclear macrophages with fused cytoplasm. Conclusion. As a result of more significant vascularization and cellular infiltration caused by AMMSCBMO adsorbed on the surface of the collagen material, to the 4th week the entire implant is full destroyed and absorbed, and dense fibrous connective tissue is formed in its place. For the implantation into the body, one should choose the most homogeneous collagen materials, without areas of different density, as slowly degrading fragments can cause the development of granulomatous inflammation and the failure of the implantation procedure.