CsgA, a putative signal molecule of the myxobacterium Stigmatella aurantiaca involved in fruiting : characterization of the csgA gene and influence of csgA inactivation on development

Cell-cell interaction is a prerequisite for multi-cellular development and cellular differentiation of the Gram-negative bacterium Stigmatella aurantiaca. For the elucidation of the temporal and spatial coordination of the physiology and motility of the cells during development, isolation and characterization of molecules involved in cell-cell signalling is needed. The best studied intercellular signal of Myxococcus xanthus is the C signal, which is the gene product of the csgA gene. Using the M. xanthus csgA gene as probe a homologous gene was previously isolated from S. aurantiaca (Butterfass, 1992). Inactivation of the gene by insertional mutagenesis caused alterations in S. aurantiaca fruiting. A XhoI fragment harbouring the csgA gene and flanking regions was isolated. Sequence analysis revealed additional putative start codons located upstream of the proposed csgA GTG translational start. Based on the homology data of the M. xanthus csgA gene the best reading frame indicates that the csgA translational start ATG codon is located 189 bp upstream of GTG. It specifies a protein of 236 amino acids with an estimated molecular mass of 26 kDa. The CsgA protein appears to be a member of the SRD family. The putative catalytic site (Ser139, Tyr158 and Lys162) is highly conserved in CsgA, as well as the putative coenzyme binding domain of the protein. Further, a new ORF, the protoporphyrinogene oxidase gene, was found upstream of csgA in the opposite orientation, 242 bp apart from csgA. The deduced amino acid sequence of this ORF has significant similarity with protoporphyrinogene oxidase from M. xanthus. An unknown ORF, orf2, was found upstream of csgA in same orientation and 251 bp apart from csgA, which encodes a polypeptide of 247 amino acids. No similarity was found between the deduced amino acid sequence of orf2 product and known proteins. An ORF, fprA, was found downstream of csgA in the opposite orientation. It overlaps with csgA (55 bp). The fprA gene specifies a protein of 223 amino acids with significant similarity to the flavin associated protein from M. xanthus. Due to the strong evidences supporting the role of CsgA in M. xanthus intercellular signalling and the close phylogenetic relationship between Stigmatella and Myxococcus it was speculated that the CsgA protein plays a role in communication between S. aurantiaca cells during development. A S. aurantiaca csgA insertion-mutant was constructed. CsgA mutant cells show an altered developmental behaviour as compared with wild type cells. The motility behaviour of the cells during development was changed and their ability to stay more closely together in the early stages of development. Inactivation of the csgA gene completely abolished rippling of the cells. This indicates the crucial role of the CsgA protein in regulating this rhythmic behaviour. S. aurantiaca csgA mutant cells do not produce CsgA but they are able to respond to it when mixed with wild type cells. Mixing the cells of the S.aurantiaca csgA mutant with those of a mutant that expresses the green fluorescence protein, resulted in wild-type fruiting body with an intermediate colour (orange / green). The csgA promoter seems to be very weak. Promoter activity of csgA was studied using a promoterless DtrpA-lacZ gene as reporter gene. b_galactosidase activity was very low and increased weakly at the beginning of the starvation induced development. A 0,6 kbp putative promoter region is sufficient for csgA expression. The concentration of the CsgA protein is low. This is a consequence of weak expression of the csgA gene. CsgA probably acts in the pM range as a signal per se or has an enzymatic function to convert a substrate into the signal molecule

A rapid screening method to evaluate the impact of nanoparticles on macrophages

Nanotechnology is an emerging and highly promising field to develop new approaches for biomedical applications. There is however at present an unmet need for a rapid and universal method to screen nanoparticles (NP) for immunocompatibility at early stages of their development. Indeed, although many types of highly diverse NP are currently under investigation, their interaction with immune cells remains fairly unpredictable. Macrophages which are professional phagocytic cells are believed to be among the first cell types that take up NP, mediating inflammation and thus immunological responses. The present work describes a highly reproducible screening method to study the NP interaction with macrophages. Three essential questions are answered in parallel, in a single multiwell plate: Are the NP taken up by macrophages? Do the NP cause macrophage cell death? Do the NP induce inflammatory reactions? This assay is proposed as a standardized screening protocol to obtain a rapid overview of the impact of different types of NP on macrophages. Due to high reproducibility, this method also allows quality control assessment for such aspects as immune-activating contaminants and batch-to-batch variability

Taylor dispersion of nanoparticles

The ability to detect and accurately characterize particles is required by many fields of nanotechnology, including materials science, nanotoxicology, and nanomedicine. Among the most relevant physicochemical properties of nanoparticles, size and the related surface-to-volume ratio are fundamental ones. Taylor dispersion combines three independent phenomena to determine particle size: optical extinction, translational diffusion, and sheer-enhanced dispersion of nanoparticles subjected to a steady laminar flow. The interplay of these defines the apparent size. Considering that particles in fact are never truly uniform nor monodisperse, we rigorously address particle polydispersity and calculate the apparent particle size measured by Taylor dispersion analysis. We conducted case studies addressing aqueous suspensions of model particles and large-scale-produced “industrial” particles of both academic and commercial interest of various core materials and sizes, ranging from 15 to 100 nm. A comparison with particle sizes determined by transmission electron microscopy confirms that our approach is model-independent, non-parametric, and of general validity that provides an accurate account of size polydispersity—independently on the shape of the size distribution and without any assumption required a priori

Istorijat gajenja krompira u Crnoj Gori

The beginning of potato growing is associated to Incas. This ancient civilization cultivated potatoes of all shapes, colours and flavours 10,000 years ago in the Andes. For communities that lived in the Andes at that time, these plants were the most important source of food. For the rest of the world, until the XVI century, potato was unknown culture. Spanish conqueror Pizarro brought potatoes from South America to Europe in 1562. As a botanical curiosity, first potatoes arrived to Spain, and were later on spread to other countries of Europe. For many years after introduction, potato was grown as an ornamental plant, until the Europeans accepted it as an edible plant. Growing of potatoes in Montenegro begins in late eighteenth century, similar as in other Balkan countries. Bishop Petar I Petrović Njegoš brought potatoes in Montenegro probably in early 1786. Due to favourable climatic and soil conditions, the areas planted with potatoes were quickly spread in Montenegro. Expansion of the areas planted with potatoes significantly improved the life of local population, and this culture, due to its enormous importance as a food, quickly got the epithet of “life-saving food”. Therefore, potatoes became, for a very short time, the leading agricultural crop and this status is maintained up to the present day.Početak gajenja krompira vezuje se za Inke. Ova drevna civilizacija je prije 10.000 godina, na Andima, gajila krompir svih oblika, boja i ukusa. Za zajednice koje su u to vrijeme živjele na Andima ova biljna vrsta je bila najznačajniji izvor hrane. Za ostatak svijeta, sve do XVI vijeka, krompir je bio nepoznata kultura. Iz Južne Amerike u Evropu krompir je donio španski osvajač Pizarro 1562. godine. Kao botanički kuriozitet krompir je prvo stigao u Španiju, a odatle se kasnije proširio i u ostale zemlje Evrope. Godinama nakon unošenja krompir je gajen kao ukrasna biljka, sve dok ga Evropljani nijesu prihvatili kao jestivu namirnicu. Gajenje krompira u Crnoj Gori počinje krajem XVIII vijeka, manje-više kao i u ostalim balkanskim državama. Krompir je u Crnu Goru donio vladika Petar I Petrović Njegoš, najvjerovatnije početkom 1786. godine. Zahvaljujući veoma povoljnim klimatskim i zemljišnim uslovima površine pod krompirom u Crnoj Gori su se brzo širile. Širenjem površina pod krompirom značajno je poboljšan život lokalnog stanovništva, pa je ova kultura, zbog svog ogromnog prehrambenog značaja, vrlo brzo dobila epitet spasonosne hrane. Zbog toga je krompir, za veoma kratko vrijeme, postao vodeća poljoprivredna kultura, a taj status je zadržao i do današnjih dana

Involvement of two uptake mechanisms of gold and iron oxide nanoparticles in a co-exposure scenario using mouse macrophages

Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold (AuNPs) and iron oxide nanoparticles (FeOxNPs) either alone or combined. Environmental scanning electron microscopy revealed that single NPs of both types bound within minutes on the cell surface but with a distinctive difference between FeOxNPs and AuNPs. Uptake analysis studies based on laser scanning microscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry revealed intracellular appearance of both NP types in all exposure scenarios and a time-dependent increase. This increase was higher for both AuNPs and FeOxNPs during co-exposure. Cells treated with endocytotic inhibitors recovered after co-exposure, which additionally hinted that two uptake mechanisms are involved. Cross-talk between uptake pathways is relevant for toxicological studies: Co-exposure acts as an uptake accelerant. If the goal is to maximize the cellular uptake, e.g., for the delivery of pharmaceutical agents, this can be beneficial. However, co-exposure should also be taken into account in the case of risk assessment of occupational settings. The demonstration of co-exposure-invoked pathway interactions reveals that synergetic nanoparticle effects, either positive or negative, must be considered for nanotechnology and nanomedicine in particular to develop to its full potential

Induction of NTPDase1/CD39 by Reactive Microglia and Macrophages Is Associated With the Functional State During EAE

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE

Beyond global charge: role of amine bulkiness and protein fingerprint on nanoparticle–cell interaction

Amino groups presented on the surface of nanoparticles are well‐known to be a predominant factor in the formation of the protein corona and subsequent cellular uptake. However, the molecular mechanism underpinning this relationship is poorly defined. This study investigates how amine type and density affect the protein corona and cellular association of gold nanoparticles with cells in vitro. Four specific poly(vinyl alcohol‐co‐N‐vinylamine) copolymers are synthesized containing primary, secondary, or tertiary amines. Particle cellular association (i.e., cellular uptake and surface adsorption), as well as protein corona composition, are then investigated. It is found that the protein corona (as a consequence of “amine bulkiness”) and amine density are both important in dictating cellular association. By evaluating the nanoparticle surface chemistry and the protein fingerprint, proteins that are significant in mediating particle–cell association are identified. In particular, primary amines, when exposed on the polymer side chain, are strongly correlated with the presence of alpha‐2‐HS‐ glycoprotein, and promote nanoparticle cellular association

Artificial lysosomal platform to study nanoparticle long-term stability

Nanoparticles (NPs) possess unique properties useful for designing specific functionalities for biomedi- cal applications. A prerequisite of a safe-by-design and effective use in any biomedical application is to study NP–cell interactions to gain a better understanding of cellular consequences upon exposure. Cellular uptake of NPs results mainly in the localization of NPs in the complex environment of lysosomes, a compartment which can be mimicked by artificial lysosomal fluid. In this work we showed the applicability of lysosomal fluid as a platform for a fast assessment of gold, iron oxide and silica NP stability over 24 h in a relevant biological fluid, by using multiple analytical methods

A rational and iterative process for targeted nanoparticle design and validation

The lack of understanding of fundamental nano-bio interactions, and difficulties in designing particles stable in complex biological environments are major limitations to their translation into biomedical clinical applications. Here we present a multi- parametric approach to fully characterize targeted nanoparticles, and emphasizes the significant effect that each detail in the synthetic process can have on downstream in vitro results. Through an iterative process, particles were designed, synthesized and tested for physico-chemical and bio-interactive properties which allowed the optimization of nanoparticle functionality. Taken together all interative steps demonstrate that we have synthesized a multifunctional gold nanoparticles that can detect ERBB2-positive breast cancer cells while showing stealth-like behavior toward ERBB2-negative cells and excellent physicochemical stability