Ivana Gadjanski

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification.

Objective: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degen-eration during autoimmune optic neuritis. Methods: Calcium ion (Ca 2) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using-conotoxin GVIA, an N-type specific blocker. Results: We observed that pathological Ca 2 influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of 1B , the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. Interpretation: We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca 2 influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage.

Neuritis of the optic nerve is one of the most frequent early symptoms of multiple sclerosis. There are only scarce data correlating magnetic resonance imaging (MRI) contrast alterations with the underlying pathology, that is inflammation, demyelination, and axonal damage. Here we studied optic neuritis in a rat model of experimental autoimmune encephalomyelitis by comparing in vivo MRI findings from multiple techniques (T1, T2, proton density, magnetization transfer) to histo-pathology. We further assessed a breakdown of the blood–brain barrier by using Gd-DTPA and indirectly estimated the intracellular accumulation of calcium as a consequence of axonal damage by using manganese-enhanced MRI. Hyperintensity on T2-weighted images and signal enhancement after Gd-DTPA were highly sensitive to lesions of the optic nerve but did not differentiate between mild, moderate, and severe damage. Signal reduction on T1-weighted images was less sensitive but correlated well with the severity of tissue damage. No significant changes in magnetization transfer ratio were observed. Manganese ions tended to accumulate in the central parts of the inflamed optic nerve. The resulting signal enhancement at 24 h after administration positively correlated with the severity of axonal loss. Thus, manganese might be an indicator of intracellular calcium accumulation that is known to be associated with axon damage. Although none of the methods alone distinguished between inflammation, demyelination, and reduced axon density, their specific capabilities should prove useful for future in vivo MRI studies of optic neuritis in both animal models and humans.

A major hurdle in treating osteochondral (OC) defects is the different healing abilities of two types of tissues involved - articular cartilage and subchondral bone. Biomimetic approaches to OC-construct engineering, based on recapitulation of biological principles of tissue development and regeneration, have potential for providing new treatments and advancing fundamental studies of OC tissue repair. Areas covered: This review on state of the art in hierarchical OC tissue graft engineering is focused on tissue engineering approaches designed to recapitulate the native milieu of cartilage and bone development. These biomimetic systems are discussed with relevance to bioreactor cultivation of clinically sized, anatomically shaped human cartilage/bone constructs with physiologic stratification and mechanical properties. The utility of engineered OC tissue constructs is evaluated for their use as grafts in regenerative medicine, and as high-fidelity models in biological research. Expert opinion: A major challenge in engineering OC tissues is to generate a functionally integrated stratified cartilage-bone structure starting from one single population of mesenchymal cells, while incorporating perfusable vasculature into the bone, and in bone-cartilage interface. To this end, new generations of advanced scaffolds and bioreactors, implementation of mechanical loading regimens and harnessing of inflammatory responses of the host will likely drive the further progress.

Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

ABSTRACT In habitually loaded tissues, dynamic loading can trigger ATP (adenosine-5'-triphosphate) release to the extracellular environment, and result in calcium signaling via ATP binding to purine P2 receptors.(1) In the current study, the purinergic responses (ATP release) of two types of cells: bovine chondrocytes (bCHs) and human mesenchymal stem cells (hMSCs) that were encapsulated in agarose and subjected to dynamic loading were compared. Both cell types were cultured under chondrogenic conditions, and their responses to loading were evaluated by an ATP release assay in combination with a connexin (Cx)-sensitive fluorescent dye (lucifer yellow - LY) and a Cx-hemichannel blocker (flufenamic acid - FFA). In response to dynamic loading, the chondrogenic hMSCs released significantly higher amounts of ATP (5-fold) in comparison to the bCHs early in culture (day 2). The triggering of LY uptake in the bCHs and hMSCs by dynamic loading implies opening of the Cx-hemichannels. However, the number of LY-positive cells in the hMSC-constructs was 2.5-fold lower compared to the loaded bCH-constructs, suggesting utilization of additional mechanisms of ATP release. Cx-reactive sites were detected in both the bCHs and hMSCs-constructs. FFA application led to reduced ATP release in both the bCHs and hMSCs, which confirmed the involvement of connexin hemichannels, with more prominent effects in the bCHs than in the hMSCs, further implying the existence of additional mechanisms of ATP release in chondrogenic hMSCs. Taken together, these results indicate a stronger purinergic response to dynamic loading of the chondrogenic hMSCs than that of primary chondrocytes, by activation of connexin hemichannels and additional mechanisms of ATP release.

Optic neuritis is one of the most common clinical manifestations of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). After an episode of optic neuritis, 30–50% of patients develop persistent impairment of vision caused by ...

The aim of the present study was to evaluate the ability and accuracy of spectral domain optical coherence tomography (OCT) for in vivo monitoring of retinal ganglion cell degeneration in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. First, OCT imaging was established for imaging of all retinal layers in Brown Norway rats. Second, thickness measurements of retinal nerve fiber layer (RNFL) were performed by periodically imaging during the development and progression of autoimmune optic neuritis. Third, the reproducibility of OCT measurements was determined by comparing RNFL measurements of two independent investigators. Fourth, OCT data were correlated with histopathology obtained ex vivo after the final imaging session. Results showed that RNFL thickness declined significantly before clinical manifestation of the disease and decline progresses continuously during the disease course. RNFL thickness measured by OCT had good repeatability and also corresponded with histomorphometric measurements. The reproducibility was limited because of the post-processing analyses performed by manual measurements. In summary, it is shown here for the first time that OCT can reliably monitor neurodegeneration in an experimental model of autoimmune optic neuritis in rodents. Moreover, in comparing RNFL thickness decline with histopathological analyses of the optic nerve, these results suggest an early, and in part, inflammation-independent process of RNFL degeneration in autoimmune optic neuritis.

Heterogeneity in clinical disease course and histopathology complicates the treatment of multiple sclerosis. We detected important differences in neurodegeneration in various subtypes of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis. Dark Agouti (DA) rats showed a significantly higher survival of retinal ganglion cells in comparison to Brown Norway rats. After surgical transection of the optic nerve neuronal loss was similar in both rat strains. We identified an increased expression of interleukin 1beta and glial cell line-derived neurotrophic factor in DA rats as the possible mechanism of the observed endogenous neuroprotection in MOG-induced optic neuritis.

In multiple sclerosis (MS), post-mortem studies of human brain tissue as well as data from animal models have shown that apoptosis of neurons occurs to a significant extent during this disease. As neurodegeneration in MS correlates with permanent neurological deficits in patients, understanding the mechanisms would be an important pre-condition for designing appropriate neuroprotective therapies. Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis often affects the optic nerve and leads to consecutive apoptosis of retinal ganglion cells (RGCs), the neurons that form its axons. In this study, we fused Bcl-XL to the protein transduction domain of the HIV-transactivator of transcription. Thereby, this anti-apoptotic member of the Bcl-2 family was delivered into RGCs of rats with electrophysiologically diagnosed optic neuritis. Transduction of Bcl-XL in our study led to significant rescue of RGCs indicating the relevance of this pathway for neuronal survival under autoimmune inflammatory conditions.

In order to engineer biomimetic osteochondral (OC) construct, it is necessary to address both the cartilage and bone phase of the construct, as well as the interface between them, in effect mimicking the developmental processes when generating hierarchical scaffolds that show gradual changes of physical and mechanical properties, ideally complemented with the biochemical gradients. There are several components whose characteristics need to be taken into account in such biomimetic approach, including cells, scaffolds, bioreactors as well as various developmental processes such as mesenchymal condensation and vascularization, that need to be stimulated through the use of growth factors, mechanical stimulation, purinergic signaling, low oxygen conditioning, and immunomodulation. This chapter gives overview of these biomimetic OC system components, including the OC interface, as well as various methods of fabrication utilized in OC biomimetic tissue engineering (TE) of gradient scaffolds. Special attention is given to addressing the issue of achieving clinical size, anatomically shaped constructs. Besides such neotissue engineering for potential clinical use, other applications of biomimetic OC TE including formation of the OC tissues to be used as high-fidelity disease/ healing models and as in vitro models for drug toxicity/efficacy evaluation are covered. Highlights Biomimetic OC TE uses " smart " scaffolds able to locally regulate cell phenotypes and dual-flow bioreactors for two sets of conditions for cartilage/ bone Protocols for hierarchical OC grafts engineering should entail mesenchymal condensation for cartilage and vascular component for bone Immunomodulation, low oxygen tension, purinergic signaling, time dependence of stimuli application are important aspects to consider in biomimetic OC TE