Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14.

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.

Nuclear Rac1 controls nuclear architecture and cell migration of glioma cells.

Rac1 (Ras-related C3 botulinum toxin substrate 1) protein has been found in the cell nucleus many years ago, however, its nuclear functions are still poorly characterized but some data suggest its nuclear accumulation in cancers. We investigated nuclear Rac1 in glioma cancer cells nuclei and compared its levels and activity to normal astrocytes, and also characterized the studied cells on various nuclear properties and cell migration patterns. Nuclear Rac1 indeed was found accumulated in glioma cells, but only a small percentage of the protein was in active, GTP-bound state in comparison to healthy control. Altering the nuclear activity of Rac1 influenced chromatin architecture and cell motility in GTP-dependent and independent manner. This suggests that the landscape of Rac1 nuclear interactions might be as complicated and wide as its well-known, non-nuclear signaling.

Study of silicone hydrogel contact lenses' surface reflection characteristics using confocal microscopy.

The physical and chemical properties of contact lenses (CLs) differ significantly from one another. This is already covered by the FDA classification, which divides soft lenses into groups and subgroups for additional characteristics. The differences relate to both the interior and surface of the lens. Several differences in the surface characteristics of individual contact lenses have been studied and demonstrated to date. However, one of their fundamental physical properties, that is light reflection or, quantitatively, reflectance has not been compared. This paper describes the surface differences of a range of silicone-hydrogel (SiHy) lenses using reflectance confocal microscopy. It shows the relationship between the amount of light reflected from the lens surface and the material parameters. Common SiHy lens materials were used in the study, including two lenses with surface modifications. Light incident at the interface between two media (phosphate-buffered saline and lens) with different refractive indices is partially reflected. The normalized results show significant differences between the reflection signals (1 vs 0.07), and that they are not correlated with the refractive index (R2 = 0.5536). For the water content (%H2O), a general trend was observed that the higher the %H2O, the lower the reflection signal is (R2 = 0.8105). The reflection signal and surface modulus show the best correlation. (R2 = 0.9883). The proposed CLs analysis method, using reflectance confocal microscopy, provides data to differentiate between lenses with and without surface modifications.

Influence of Yokukansan on the refractive index of neuroblastoma cells.

Yokukansan (YKS) is a traditional Japanese herbal medicine that is increasingly being studied for its effects on neurodegenerative diseases. In our study, we presented a novel methodology for a multimodal analysis of the effects of YKS on nerve cells. The measurements of 3D refractive index distribution and its changes performed by holographic tomography were supported with an investigation by Raman micro-spectroscopy and fluorescence microscopy to gather complementary morphological and chemical information about cells and YKS influence. It was shown that at the concentrations tested, YKS inhibits proliferation, possibly involving reactive oxygen species. Also substantial changes in the cell RI after few hours of YKS exposure were detected, followed by longer-term changes in cell lipid composition and chromatin state.

The Analysis of Mechanical Properties and Geometric Accuracy in Specimens Printed in Material Jetting Technology.

The purpose of this research was to analyze polymer materials based on mechanical properties and geometrical parameters, such as the smallest material deviations and the best printing texture after three-dimensional (3D) printing in two methods of Material Jetting technology: PolyJet and MultiJet. This study covers checks for Vero Plus, Rigur, Durus, ABS, and VisiJet M2R-WT materials. Thirty flat specimens were printed both for 0 and 90 raster orientations. Specimen scans were superimposed on the 3D model from CAD software. Each of them was tested, paying attention to the accuracy and the layer thickness effect of printed components. Then, all specimens were subjected to tensile tests. The obtained data-Young's modulus and Poisson's ratio-were compared using statistical methods, focusing on the two most important parameters: the isotropy of the printed material in two directions and the characteristics close to linear. It was found that unitary surface deviation with general dimensional accuracy equal to ±0.1 mm was the common feature of printed models. Some small areas had lower accuracy depending on the material and printer device. Rigur material obtained the highest mechanical properties. Dimensional accuracy in Material Jetting technology as a function of layer parameters such as layer thickness and raster orientation was checked. The materials were checked in terms of relative isotropy and linearity. Additionally, similarities and differences between PolyJet and MultiJet methods were covered.

The association between kidney function, cognitive function, and structural brain abnormalities in community-dwelling individuals aged 50+ is mediated by age and biomarkers of cardiovascular disease.

AIMS: Cognitive impairment has been associated with kidney function and chronic kidney disease. Whether this association is due to accelerated cardiovascular disease (CVD) or an independent specific kidney function effect related to toxins is unclear. We investigated the impact of an array of clinical factors, inflammatory biomarkers, and cardiovascular biomarkers on the association between kidney function, cognitive function, and structural brain abnormalities. METHODS AND RESULTS: We used data from the first and third waves of the TILDA Study, a population-representative prospective cohort of Irish adults aged 50 years and over, based on stratified random sampling (n = 3774). The MRI sub-study included participants who consented to MRI brain imaging in addition to the health assessment. Multivariable linear and mixed-effect longitudinal regression models were fitted separately for each kidney marker/estimated glomerular filtration rate (eGFR) equation after adjusting for baseline age and demographics, clinical vascular risk factors, and biomarkers. Unadjusted analyses showed an association between low eGFR, cognitive dysfunction, and cognitive decline (P < 0.001 for all kidney markers). Kidney function markers were also associated with white matter disease [OR = 3.32 (95% CI: 1.11, 9.98)], total grey matter volume (ß = -0.17, 95% CI -0.27 to -0.07), and regional grey matter volumes within areas particularly susceptible to hypoxia (P < 0.001 for all). All the associations decreased after adjusting for age and were also diminished after adjusting for CVD biomarkers. Age and CVD-biomarker score were significant mediators of the adjusted associations between eGFR and cognitive status. These results remained consistent for cross-sectional and longitudinal outcomes and specific cognitive domains. CONCLUSION: Decreased kidney function was associated with cerebrovascular disease. The association appeared to be mediated predominantly by age and the combination of CVD markers [namely N-terminal pro-B-type natriuretic peptide (NT-proBNP) and Growth Differentiation Factor 15 (GDF15)], supporting the idea that shared biological pathways underline both diseases. Further mechanistic studies of the specific molecular mechanisms that lead to both kidney and cognitive decline are warranted.

Method to analyze effects of low-level laser therapy on biological cells with a digital holographic microscope.

Low-level laser therapy (LLLT) is a therapeutic tool that uses the photobiochemical interaction between light and tissue. Its effectiveness is controversial due to a strong dependence on dosimetric parameters. In this work, we demonstrate that digital holographic microscopy is an effective label-free imaging technique to analyze the effects of LLLT on biological cells, and we propose the full methodology to create correct synthetic aperture phase maps for further extensive, highly accurate statistical analysis. The proposed methodology has been designed to provide a basis for many other biological experiments using quantitative phase imaging. We use SHSY-5Y and HaCaT cells irradiated with different doses of red light for the experiment. The analysis shows quantitative changes in cell dry mass density and the projected cell surface in response to different radiation doses.

P2X7 receptor: the regulator of glioma tumor development and survival.

P2X7 is an ionotropic nucleotide receptor, forming the cation channel upon ATP stimulation. It can also function as a large membrane pore as well as transmit ATP-dependent signal without forming a channel at all. P2X7 activity in somatic cells is well-known, but remains poorly studied in glioma tumors. The current paper presents the comprehensive study of P2X7 activity in C6 and glioma cell line showing the wide range of effects the receptor has on glioma biology. We observed that P2X7 stimulation boosts glioma cell proliferation and increases cell viability. P2X7 activation promoted cell adhesion, mitochondria depolarization, and reactive oxygen species overproduction in C6 cells. P2X7 receptor also influenced glioma tumor growth in vivo via activation of pro-survival signaling pathways and ATP release. Treatment with Brilliant Blue G, a selective P2X7 antagonist, effectively inhibited glioma tumor development; decreased the expression of negative prognostic cancer markers pro-survival and epithelial-mesenchymal transition (EMT)-related proteins; and modulated the immune response toward glioma tumor in vivo. Finally, pathway-specific enrichment analysis of the microarray data from human patients also showed an upregulation of P2X7 receptor in gliomas from grades I to III. The presented results shed more light on the role of P2X7 receptor in the biology of this disease.

The Nephroprotective Properties of Extracellular Vesicles in Experimental Models of Chronic Kidney Disease: a Systematic Review.

Extracellular vesicle (EV)-based therapy was hypothesized as a promising regenerative approach which has led to intensive research of EVs in various pathologies. In this study, we performed a comprehensive systematic review of the current experimental evidence regarding the protective properties of EVs in chronic kidney disease (CKD). We evaluated the EV-based experiments, EV characteristics, and effector molecules with their involvement in CKD pathways. Including all animal records with available creatinine or urea data, we performed a stratified univariable meta-analysis to assess the determinants of EV-based therapy effectiveness. We identified 35 interventional studies that assessed nephroprotective role of EVs and catalogued them according to their involvement in CKD mechanism. Systematic assessment of these studies suggested that EVs had consistently improved glomerulosclerosis, interstitial fibrosis, and cell damage, among different CKD models. Moreover, EV-based therapy reduced the progression of renal decline in CKD. The stratified analyses showed that the disease model, administered dose, and time of therapeutic intervention were potential predictors of therapeutic efficacy. Together, EV therapy is a promising approach for CKD progression in experimental studies. Further standardisation of EV-methods, continuous improvement of the study quality, and better understanding of the determinants of EV effectiveness will facilitate preclinical research, and may help development of clinical trials in people with CKD.

Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery.

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex "pretzel-like" structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.

Muscle-derived exophers promote reproductive fitness.

Organismal functionality and reproduction depend on metabolic rewiring and balanced energy resources. However, the crosstalk between organismal homeostasis and fecundity and the associated paracrine signaling mechanisms are still poorly understood. Using Caenorhabditis elegans, we discovered that large extracellular vesicles (known as exophers) previously found to remove damaged subcellular elements in neurons and cardiomyocytes are released by body wall muscles (BWM) to support embryonic growth. Exopher formation (exopheresis) by BWM is sex-specific and a non-cell autonomous process regulated by developing embryos in the uterus. Embryo-derived factors induce the production of exophers that transport yolk proteins produced in the BWM and ultimately deliver them to newly formed oocytes. Consequently, offspring of mothers with a high number of muscle-derived exophers grew faster. We propose that the primary role of muscular exopheresis is to stimulate reproductive capacity, thereby influencing the adaptation of worm populations to the current environmental conditions.

Protective factors as biomarkers and targets for prevention and treatment of diabetic nephropathy: From current human evidence to future possibilities.

Although hyperglycemia, high blood pressure and aging increase the risk of developing kidney complications, some diabetes patients exposed to these risk factors do not develop kidney disease, suggesting the presence of endogenous protective factors. There is a growing need to understand these factors determining protection of the kidney in order to improve the design of preventive strategies and to enhance the processes responsible for renoprotection. The aim of this review was to present the existing molecular and epidemiological data on factors showing protective effects in diabetic kidney disease, and to summarize the evidence regarding their potential in the area of future clinical diagnostics, therapeutics and early preventive strategies. These include transcriptomic and proteomic studies regarding the anti-inflammatory, anti-fibrotic and regenerative factors that were associated with slower progression of renal function loss. Another focus is the new evidence regarding the evaluation of alterations in the regulatory epigenome and its involvement in the risk of diabetic kidney disease. Further effort is required to validate and extend these findings, and to define their potential for clinical implementation in the future.

P2X7 receptor activity landscape in rat and human glioma cell lines.

P2X7 is a commonly expressed purinergic receptor, which functions as a cation-permeable channel in the plasma membrane. In certain circumstances, the receptor may also form a large transmembrane pore what results in cell death. P2X7 receptors control numerous physiological and pathological cellular processes and their overexpression is often associated with cancer progression. As nucleotides are important signaling molecules in the central nervous system, P2X7 plays also an important but ambiguous role in glioma biology with contrary observations originating from different glioma models. Therefore, the aim of our research was to investigate P2X7 receptor expression and functions in three human (U-87 MG, U-138 MG, U-251 MG) and one rat (C6) glioma cell lines. Although the receptor mRNA and protein were present in all the studied cells, we found profound differences in their level. We also encountered a problem with one human cell lines authenticity (U-87 MG) and excluded it from most of the experiments. Interestingly, there was no clear dependency between P2X7 receptor level, calcium signal and pore formation ability in the studied glioma lines. In U-138 human cell line, the receptor seemed to be inactive, while in U-251 human and C6 rat cell line its activation resulted in calcium influx and large pore formation. However, the viability of studied cells upon the administration of specific P2X7 agonist - BzATP - was not affected for U-138 and U-251, whereas for C6 cells a stimulatory effect was observed. Our results stress the variability of P2X7 signaling in glioma models and the need for future research which would take into account the complicated landscape of the receptor signaling in the brain.

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling.

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cß, sarco-endoplamatic reticulum calcium ATP-ase and sodium­calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.

TNFα stimulates NO release in EA.hy926 cells by activating the CaMKKß-AMPK-eNOS pathway.

Previously we showed that a mild stimulation of EA.hy926 cells with tumour necrosis factor alpha (TNFα) activated mitochondrial biogenesis, probably as a mechanism preventing cell death. This was accompanied by an increased phosphorylation of eNOS and elevation of NO release. The aim of the present study was to explain the biochemical basis of this effect. Our results indicate that eNOS is the only enzyme catalysing NO generation in EA.hy926 cells, and TNFα stimulates its activity by activating AMP-activated protein kinase (AMPK). Inhibition of AMPK with Compound C prevents the TNFα-induced activatory phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and reduces the NO release. AMPK is activated by phosphorylation catalysed by liver kinase B1 (LKB1) and calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß), which are phosphorylated and thereby activated in the presence of TNFα. Moreover, CaMKKß catalyses an activatory phosphorylation of sirtuin 1, which could deacetylate and activate eNOS both directly and indirectly by an elevating the LKB1 activity. TNFα hardly increases the nuclear fraction of sirtuin 1, thus its major activity is probably attributed to the cytosolic pool. This is in line with the elevated activity of eNOS. We conclude that the increased AMPK-dependent phosphorylation of eNOS at least partially explains the stimulation of NO generation by TNFα in EA.hy926 cells.

Organic matter decomposition before arsenic speciation analysis of water sample - "Soft decomposition" using nano-photocatalysts.

The applicability of photolysis in the speciation analysis of arsenic is investigated. The use of nano scale semiconductors (Fe2O3/WO3/Fe2O3 at pH 6) as an active film during solar light irradiation of a water sample, containing some surfactants (SDS), results in the simplification of the organic matter and gives no speciation change in the arsenic. The reproducibility of active layer is shown to be high and the surface roughness of each photoactive sample and photocurrent do not differ by more than 6 and less than 8%, respectively. The procedure of sample pretreatment caused a minimum (8-10%) amount of speciation change, whilst the irradiation is no longer that 2 h. The study indicates that "soft decomposition" can be performed for as long as 4 h, and still give photostable arsenates (III) and methylarsenate species. However, the saturation of the water sample with Ar is required (to reduce the oxygen content) for the longer the decomposition time being applied.

Markers of early progressive renal decline in type 2 diabetes suggest different implications for etiological studies and prognostic tests development.

To identify determinants of early progressive renal decline in type 2 diabetes a range of markers was studied in 1032 patients enrolled into the 2nd Joslin Kidney Study. eGFR slopes estimated from serial measurements of serum creatinine during 5-12 years of follow-up were used to define early renal decline. At enrollment, all patients had normal eGFR, 58% had normoalbuminuria and 42% had albuminuria. Early renal decline developed in 6% and in 18% patients, respectively. As determinants, we examined baseline values of clinical characteristics, circulating markers: TNFR1, KIM-1, and FGF23, and urinary markers: albumin, KIM-1, NGAL, MCP-1, EGF (all normalized to urinary creatinine) and the ratio of EGF to MCP-1. In univariate analysis, all plasma and urinary markers were significantly associated with risk of early renal decline. When analyzed together, systolic blood pressure, TNFR1, KIM-1, the albumin to creatinine ratio, and the EGF/MCP-1 ratio remained significant with the latter having the strongest effect. Integration of these markers into a multi-marker prognostic test resulted in a significant improvement of discriminatory performance of risk prediction of early renal decline, compared with the albumin to creatinine ratio and systolic blood pressure alone. However, the positive predictive value was only 50% in albuminuric patients. Thus, markers in plasma and urine indicate that the early progressive renal decline in Type 2 diabetes has multiple determinants with strong evidence for involvement of tubular damage. However, new, more informative markers are needed to develop a better prognostic test for such decline that can be used in a clinical setting.

Increased plasma kidney injury molecule-1 suggests early progressive renal decline in non-proteinuric patients with type 1 diabetes.

Progressively decreasing glomerular filtration rate (GFR), or renal decline, is seen in patients with type 1 diabetes (T1D) and normoalbuminuria or microalbuminuria. Here we examined the associations of kidney injury molecule-1 (KIM-1) in plasma and urine with the risk of renal decline and determine whether those associations are independent of markers of glomerular damage. The study group comprised patients with T1D from the 2nd Joslin Kidney Study of which 259 had normoalbuminuria and 203 had microalbuminuria. Serial measurements over 4 to 10 years of follow-up (median 8 years) of serum creatinine and cystatin C were used jointly to estimate eGFRcr-cys slopes and time of onset of CKD stage 3 or higher. Baseline urinary excretion of IgG2 and albumin were used as markers of glomerular damage, and urinary excretion of KIM-1 and its plasma concentration were used as markers of proximal tubular damage. All patients had normal renal function at baseline. During follow-up, renal decline (eGFRcr-cys loss 3.3% or more per year) developed in 96 patients and 62 progressed to CKD stage 3. For both outcomes, the risk rose with increasing baseline levels of plasma KIM-1. In multivariable models, elevated baseline plasma KIM-1 was strongly associated with risk of early progressive renal decline, regardless of baseline clinical characteristics, serum TNFR1 or markers of glomerular damage. Thus, damage to proximal tubules may play an independent role in the development of early progressive renal decline in non-proteinuric patients with T1D.

Evaluation of the quality of life in subjects with a history of severe anaphylactic reaction to the Hymenoptera venom.

INTRODUCTION: Sensitization to the Hymenoptera venom is one of the main causes of anaphylaxis in Poland. Venom immunotherapy is the only effective treatment in such cases. Comprehensive patient care includes also education. The aim of our study was to assess the state of knowledge and to evaluate the quality of life and the anxiety level in patients allergic to the Hymenoptera venom after anaphylactic reaction. MATERIAL AND METHODS: The survey was carried out in the period of the insects flight in 61 adult subjects (35 wasp and 26 bee allergic), using a validated Vespid Allergy Quality of Life Questionnaire (VQLQ), Hospital Anxiety and Depression Scale, and subjective assessment of anxiety level. The majority of respondents received venom immunotherapy. RESULTS: Sensitized to the wasp venom had significantly impaired quality of life (VQLQ score) as compared to the bee venom allergic (p = 0.014). The intensity of anxiety decreased with the duration of immunotherapy (p = 0.01). The majority of subjects knew how to recognize and treat anaphylaxis, but only 8% employed an identification card and about 50% implemented rules of the pre-exposition prophylaxis. CONCLUSIONS: History of a severe anaphylaxis to the Hymenoptera venom affected the quality of life. Venom immunotherapy reduced anxiety. We hope that presented surveys and their results might be useful in qualifying for immunotherapy in clinically uncertain cases.