Abstract
Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous ...subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.
Subchondroplasty is a new minimally invasive surgical technique developed to treat bone marrow lesions (BML) and early osteoarthritis (OA). During the procedure, engineered calcium phosphate compound ...(CPC) is injected. It is claimed by the manufacturer that during the healing process, the CPC is replaced with new bone. The purpose of this study was to verify the replacement of CPC with new bone after subchondroplasty for the first time in humans. A 76-year old woman was referred for resistant medial knee pain. Standing radiographs showed varus knee OA and magnetic resonance imaging (MRI) revealed BML. She was treated with subchondroplasty of medial femoral condyle. Excellent relief of pain was achieved after procedure. Afterwards, the pain worsened, the radiographs confirmed the OA progression and the patient was treated with a total knee arthroplasty (TKA) 4 years after primary procedure. The resected bone was examined histologically and with micro-computed tomography (CT). Histologically, bone trabeculae of subcortical bone were embedded in the amorphous mass. However, no signs of CPC resorption and/or bone replacement have been found with micro-CT. In short term, excellent pain relief could be expected after the subchondroplasty procedure. However, there was no replacement of CPC with bone and the technique probably did not influence the natural process of knee OA.
Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (
). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it ...forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.
Nanocomposites with a high, uniform loading of magnetic nanoparticles are very desirable for applications such as electromagnetic shielding and cancer treatment based on magnetically induced ...hyperthermia. In this study, a simple and scalable route for preparing nanocomposites with a high, uniform loading of magnetic nanoparticles is presented. The magnetic iron-oxide nanoparticles were functionalized with a methacrylate-based monomer that copolymerized in a toluene solution with the methyl methacrylate (MMA) monomer. The resulting suspension of magnetic nanoparticles decorated with poly(methyl methacrylate) (PMMA) chains in toluene were colloidal, even in the presence of a magnetic field gradient. Nanocomposites were precipitated from these suspensions. The transmission electron microscopy investigation of the prepared nanocomposites revealed that the magnetic nanoparticles were homogeneously dispersed in the PMMA matrix, even in amounts up to 53 wt %. The uniform dispersion of the nanoparticles in the PMMA matrix was attributed to the good solvation of the grafted PMMA chains from the magnetic nanoparticles by the PMMA chains of the matrix. The nanocomposites were superparamagnetic and exhibited large values for the saturation magnetization of up to 36 emu/g. Moreover, the nanocomposite with the largest amount of incorporated nanoparticles exhibited relatively large values for the specific power loss when subjected to alternating magnetic fields, giving this material great potential for the magnetically induced hyperthermia-based treatment of cancer.
Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized
model would ...reinforce studies of the MG microbiota development. We aimed to establish and characterize an
cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for
studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for
studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC
tool also offers new opportunities for cancer research.
With only 43 described stygobionts and only two isopod species the obligate groundwater fauna of Iran, a vast country with over 10% of limestone surface, is inadequately known. Here, we report the ...discovery of
Asellus ismailsezarii
sp. nov. from Zagros mountains, the first eyeless and depigmented asellid isopod from Iran. The new species is morphologically similar to
Asellus monticola
Birstein, 1932, but it is eyeless and fully depigmented, has a slightly curved pereopod IV and does not bear any setae on proximal margins of exopodite of pleopods IV and V. Species phylogenetic relationships using original and datamined mitochondrial DNA and nuclear rDNA, and estimation of molecular divergences with other
Asellus
species, suggest that
A. ismailsezarii
sp. nov. is sister to a larger clade that also contains the European
A. aquaticus
species complex. Surface populations of
Asellus
have colonized groundwater at multiple occasions and localities, both in Europe and Asia, giving rise to species and subspecies that have evolved troglomorphisms, such as depigmentation and loss of eyes. Of the 37 formally described species and subspecies of
Asellus
, 15 are from groundwater, including
A. ismailsezarii
sp. nov. We predict that many more obligate groundwater
Asellus
taxa are yet to be discovered in Asia.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The neurotropic and extremophilic black yeast
(
) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is ...polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of
on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of
biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of
with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by
hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's.
(1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been ...conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in
, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.
The surface structures and mucus layers that form an interface between the epithelial layer of organisms and their external environment were studied in the bloom-forming moon jellyfish (Aurelia ...solida, Scyphozoa) from the northern Adriatic. The surface of the polyps revealed epithelial ciliated cells and numerous nematocysts, both non-discharged and discharged. Cilia were also the most prominent features on the surface of adult medusa, protruding from the epidermal cells and with microvilli surrounding the base. Histochemical methods and various microscopy techniques (light/epifluorescence and electron microscopy) confirmed the presence of abundant mucus around polyps and on the surfaces of adult medusa, and that the mucus contained acidic and neutral mucins. The observed mucus secretions on the exumbrella surface of the medusae were in the form of granules, flocs, and sheets. Scanning electron microscopy and transmission electron microscopy analyses confirmed the presence of various microbes in the mucus samples, but not on the epithelial surfaces of the polyps or the exumbrella of the medusae.
The olm (Proteus anguinus), an endemic amphibian of the Dinarides' underground waters (Europe), is one of the world's most widely known subterranean species. Although various aspects of olm biology ...have been extensively studied, the data on their behavior in the wild remain scarce mostly due to inaccessibility of their natural habitat. Yet, olms also occur in several karstic springs during nighttime. These are easier to access and present an exciting opportunity to study olm behavior in nature. Here, we report on systematic observations of olms in one such spring in Slovenia, where we observed them for nine consecutive summer nights, coupling direct on-site observations with IR camera trap recordings. We used IR camera trap recordings to construct simple ethograms, as well as to quantify olm movement activity by video-tracking. Olms regularly occurred on the surface during the night, and dawn appeared to be a key stimulus for their retreat underground. They were constantly active, but rarely swam far from the spring. Despite the short-term nature of the study, we collected new occurrence and movement data, and at the same time tested the usability of IR cameras for surveying olm presence and behavior in nature. Experience gained through the study may prompt long-term and more complex behavioral studies using similar approaches.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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