Czech Academy of Sciences, Institute of Experimental Medicine

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is approximately 20% and the tortuosity is approximately 1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain.

The aim of the study was to correlate the sonographic [ultrasound (US)] and color-Doppler (CFD) findings with the results of US-guided fine needle aspiration biopsy (FNA) and of pathologic staging of resected carcinomas to establish: 1) the relative importance of US features as risk factors of malignancy; and 2) a cost-effective management of nonpalpable thyroid nodules. Four hundred ninety-four consecutive patients with nonpalpable thyroid nodules (8-15 mm) were evaluated by US, CFD, and US-FNA. Ninety-two patients with inadequate cytology were excluded from the study. All patients with suspicious or malignant cytology underwent surgery, whereas subjects with benign cytology had clinical and US control 6 months later. Thyroid malignancies were observed in 18 of 195 (9.2%) solitary thyroid nodules and in 13 of 207 (6.3%) multinodular goiters. Cancer prevalence was similar in nodules greater or smaller than 10 mm (9.1 vs. 7.0%). Extracapsular growth (pT(4)) was present in 35.5%, and nodal involvement in 19.4% of neoplastic lesions, with no significant differences between tumors greater or smaller than 10 mm. At US cancers presented a solid hypoechoic appearance in 87% of cases, irregular or blurred margins in 77.4%, an intranodular vascular pattern in 74.2%, and microcalcifications in 29.0%. Irregular margins (RR 16.83), intranodular vascular spots (RR 14.29), and microcalcifications (RR 4.97) were independent risk factors of malignancy. FNA performed on hypoechoic nodules with at least one risk factor was able to identify 87% of the cancers at the expence of cytological evaluation of 38.4% of nonpalpable lesions. The majority of nonpalpable thyroid tumors can be identified by cytological evaluation of lesions presenting hypoechoic appearance in conjunction with one independent risk factor. Due to the nonnegligible prevalence of extracapsular growth and nodal metastasis, US-FNA should be performed on all 8-15 mm hypoechoic nodules with irregular margins, intranodular vascular spots or microcalcifications. Nonpalpable lesions of the thyroid without risk factors should be followed by means of clinical and US evaluation.

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial "molecular mimicry" of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.

Abstract Physical activity has been associated with lower risks of breast and colorectal cancer in epidemiological studies; however, it is unknown if these associations are causal or confounded. In two-sample Mendelian randomisation analyses, using summary genetic data from the UK Biobank and GWA consortia, we found that a one standard deviation increment in average acceleration was associated with lower risks of breast cancer (odds ratio [OR]: 0.51, 95% confidence interval [CI]: 0.27 to 0.98, P-value = 0.04) and colorectal cancer (OR: 0.66, 95% CI: 0.48 to 0.90, P-value = 0.01). We found similar magnitude inverse associations for estrogen positive (ER +ve ) breast cancer and for colon cancer. Our results support a potentially causal relationship between higher physical activity levels and lower risks of breast cancer and colorectal cancer. Based on these data, the promotion of physical activity is probably an effective strategy in the primary prevention of these commonly diagnosed cancers.

Over the last decade or so, a large number of studies have investigated the possible adverse effects of ambient air pollution on birth outcomes. We reviewed these studies, which were identified by a systematic search of the main scientific databases. Virtually all reviewed studies were population based, with information on exposure to air pollution derived from routine monitoring sources. Overall, there is evidence implicating air pollution in adverse effects on different birth outcomes, but the strength of the evidence differs between outcomes. The evidence is sufficient to infer a causal relationship between particulate air pollution and respiratory deaths in the postneonatal period. For air pollution and birth weight the evidence suggests causality, but further studies are needed to confirm an effect and its size and to clarify the most vulnerable period of pregnancy and the role of different pollutants. For preterm births and intrauterine growth retardation (IUGR) the evidence as yet is insufficient to infer causality, but the available evidence justifies further studies. Molecular epidemiologic studies suggest possible biologic mechanisms for the effect on birth weight, premature birth, and IUGR and support the view that the relation between pollution and these birth outcomes is genuine. For birth defects, the evidence base so far is insufficient to draw conclusions. In terms of exposure to specific pollutants, particulates seem the most important for infant deaths, and the effect on IUGR seems linked to polycyclic aromatic hydrocarbons, but the existing evidence does not allow precise identification of the different pollutants or the timing of exposure that can result in adverse pregnancy outcomes.

Abstract Background Morbidity and mortality from COVID‐19 caused by novel coronavirus SARS‐CoV‐2 is accelerating worldwide, and novel clinical presentations of COVID‐19 are often reported. The range of human cells and tissues targeted by SARS‐CoV‐2, its potential receptors and associated regulating factors are still largely unknown. The aim of our study was to analyze the expression of known and potential SARS‐CoV‐2 receptors and related molecules in the extensive collection of primary human cells and tissues from healthy subjects of different age and from patients with risk factors and known comorbidities of COVID‐19. Methods We performed RNA sequencing and explored available RNA‐Seq databases to study gene expression and co‐expression of ACE2, CD147 ( BSG ), and CD26 ( DPP4 ) and their direct and indirect molecular partners in primary human bronchial epithelial cells, bronchial and skin biopsies, bronchoalveolar lavage fluid, whole blood, peripheral blood mononuclear cells (PBMCs), monocytes, neutrophils, DCs, NK cells, ILC1, ILC2, ILC3, CD4 + and CD8 + T cells, B cells, and plasmablasts. We analyzed the material from healthy children and adults, and from adults in relation to their disease or COVID‐19 risk factor status. Results ACE2 and TMPRSS2 were coexpressed at the epithelial sites of the lung and skin, whereas CD147 ( BSG ), cyclophilins ( PPIA and PPIB ), CD26 ( DPP4 ), and related molecules were expressed in both epithelium and in immune cells. We also observed a distinct age‐related expression profile of these genes in the PBMCs and T cells from healthy children and adults. Asthma, COPD, hypertension, smoking, obesity, and male gender status generally led to the higher expression of ACE2‐ and CD147‐related genes in the bronchial biopsy, BAL, or blood. Additionally, CD147‐related genes correlated positively with age and BMI. Interestingly, we also observed higher expression of CD147‐related genes in the lesional skin of patients with atopic dermatitis. Conclusions Our data suggest different receptor repertoire potentially involved in the SARS‐CoV‐2 infection at the epithelial barriers and in the immune cells. Altered expression of these receptors related to age, gender, obesity and smoking, as well as with the disease status, might contribute to COVID‐19 morbidity and severity patterns.

Synopsis Studies of 3 populations of F 2 families in the F 3 and F 4 generations grown at 2 locations revealed the following factors: genotype year interactions relatively large; seed yield generally more heritable than total yield; heritability of vigor lower for scores on July 1 than those on Aug. 15; and exceptionally low genetic coefficient of variation and heritability of vigor scores for the population segregating for procumbency.

Introduction: Intravenous(IV) immunoglobulin(Ig) treatment is known to alleviate behavioral deficits in the experimentally induced model of sepsis. To delineate the mechanisms by which IVIg treatment prevents neuronal dysfunction, an array of immunological and apoptosis markers was investigated. Methods: Sepsis was induced by cecal ligation perforation(CLP) in rats. The animals were divided into five groups; sham, control, CLP + saline, CLP + immunoglobulin G IgG(250 mg/kg,iv), and CLP + immunoglobulins enriched with immunoglobulin M-IgGAM(250 mg/kg,iv). Blood and brain samples were taken in two sets of experiments after CLP to see the early(24 hrs) and late(10 days) effects of treatment. Total complement activity, complement 3(C3) and soluble complement C5b-9 levels were measured in sera of rats using ELISA-based methods. Cerebral complement content was analyzed by Western Blot. Immune cell infiltration and gliosis were examined by immunohistochemistry using cluster of differentiation 3, CD4, CD8, CD11b, CD19 and glial fibrillary acidic protein antibodies. Apoptotic neuronal death was investigated by TUNEL staining and Western Blot-based semi-quantitative evaluation of brain homogenates by bax and bcl-2 antibodies. Results: IV IgG and IgGAM administration significantly reduced systemic complement activity but increased serum C3 and soluble C5b-9 levels. Likewise, Western Blot data showed slightly increased C5b-9 expression and significantly reduced C1q expression in brain samples of IgGAM-treated but not IgG-treated septic rats especially in the first day of administration. No cerebral cellular infiltrates were observed in treated and non-treated septic rats. By contrast, IV IgG and IgGAM treatment induced considerable amelioration in glial cell proliferation which was increased in non-treated rats. IgG and IgGAM treated rats exhibited significantly reduced numbers of apoptotic neurons and cerebral expression levels of bax and bcl-2 as compared to nontreated rats. Conclusions: We suggest that IV IgG and IgGAM administration ameliorates neuronal dysfunction and behavioral deficits by reducing apoptotic cell death and glial cell proliferation. IgGAM treatment might be suppressing classical complement pathway by reducing C1q expression.

Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.

The relationship between intrauterine growth retardation (IUGR) and exposure to particulate matter [less than/equal to] 10 microm (PM(10)) and particulate matter [less than and equal to] 2.5 microm (PM(2.5))( )in early pregnancy was recently studied in the highly polluted district of Teplice (Northern Bohemia). From this observation rose the question about the possible role of the carcinogenic fraction of polycyclic aromatic hydrocarbons (c-PAHs), which are usually bound to fine particles. The impact of c-PAHs and fine particles on IUGR was analyzed in Teplice and in Prachatice, a region with similarly high c-PAH but low particle levels. All European, single live births occurring in a 4-year period in Teplice (n = 3,378) and Prachatice (n = 1,505) were included. Detailed personal data were obtained via questionnaires and medical records. Mean PM(10), PM(2.5,) and c-PAHs levels during the 9 gestational months (GM) were estimated for each mother. Adjusted odds ratios (AORs) of IUGR for three levels of c-PAHs (low, medium, and high) and for continuous data were estimated after adjustment for a range of covariates using logistic regression models. In the present 4-year sample from Teplice, previously published results about increasing IUGR risk after exposure to particles in the first GM were fully confirmed, but no such effects were found in Prachatice. The AOR of IUGR for fetuses from Teplice exposed to medium levels of c-PAHs in the first GM was 1.60 [confidence interval (CI), 1.06-2. 15], and to high levels 2.15 (CI, 27-3.63). An exposure-response relationship was established by analyzing the continuous data. For each 10 ng increase of c-PAHs in the first GM, the AOR was 1.22 (CI, 1.07-1.39). About the same relationship was observed in Prachatice in spite of the low particle levels. The results prove that exposure to c-PAHs in early gestation may influence fetal growth. The particulate matter-IUGR association observed earlier may be at least partly explained by the presence of c-PAHs on particle surfaces.

A plethora of neurological disorders shares a final common deadly pathway known as excitotoxicity. Among these disorders, ischemic injury is a prominent cause of death and disability worldwide. Brain ischemia stems from cardiac arrest or stroke, both responsible for insufficient blood supply to the brain parenchyma. Glucose and oxygen deficiency disrupts oxidative phosphorylation, which results in energy depletion and ionic imbalance, followed by cell membrane depolarization, calcium (Ca 2+ ) overload, and extracellular accumulation of excitatory amino acid glutamate. If tight physiological regulation fails to clear the surplus of this neurotransmitter, subsequent prolonged activation of glutamate receptors forms a vicious circle between elevated concentrations of intracellular Ca 2+ ions and aberrant glutamate release, aggravating the effect of this ischemic pathway. The activation of downstream Ca 2+ -dependent enzymes has a catastrophic impact on nervous tissue leading to cell death, accompanied by the formation of free radicals, edema, and inflammation. After decades of "neuron-centric" approaches, recent research has also finally shed some light on the role of glial cells in neurological diseases. It is becoming more and more evident that neurons and glia depend on each other. Neuronal cells, astrocytes, microglia, NG2 glia, and oligodendrocytes all have their roles in what is known as glutamate excitotoxicity. However, who is the main contributor to the ischemic pathway, and who is the unsuspecting victim? In this review article, we summarize the so-far-revealed roles of cells in the central nervous system, with particular attention to glial cells in ischemiainduced glutamate excitotoxicity, its origins, and consequences.

BACKGROUND: The primary objective of Tissue engineering is a regeneration or replacement of tissues or organs damaged by disease, injury, or congenital anomalies. At present, Tissue engineering repairs damaged tissues and organs with artificial supporting structures called scaffolds. These are used for attachment and subsequent growth of appropriate cells. During the cell growth gradual biodegradation of the scaffold occurs and the final product is a new tissue with the desired shape and properties. In recent years, research workplaces are focused on developing scaffold by bio-fabrication techniques to achieve fast, precise and cheap automatic manufacturing of these structures. Most promising techniques seem to be Rapid prototyping due to its high level of precision and controlling. However, this technique is still to solve various issues before it is easily used for scaffold fabrication. In this article we tested printing of clinically applicable scaffolds with use of commercially available devices and materials. Research presented in this article is in general focused on "scaffolding" on a field of bone tissue replacement. RESULTS: Commercially available 3D printer and Polylactic acid were used to create originally designed and possibly suitable scaffold structures for bone tissue engineering. We tested printing of scaffolds with different geometrical structures. Based on the osteosarcoma cells proliferation experiment and mechanical testing of designed scaffold samples, it will be stated that it is likely not necessary to keep the recommended porosity of the scaffold for bone tissue replacement at about 90%, and it will also be clarified why this fact eliminates mechanical properties issue. Moreover, it is demonstrated that the size of an individual pore could be double the size of the recommended range between 0.2-0.35 mm without affecting the cell proliferation. CONCLUSION: Rapid prototyping technique based on Fused deposition modelling was used for the fabrication of designed scaffold structures. All the experiments were performed in order to show how to possibly solve certain limitations and issues that are currently reported by research workplaces on the field of scaffold bio-fabrication. These results should provide new valuable knowledge for further research.

Synopsis Plot error variances were large for lint yield, bolls per plant, seed per boll, and boll weight; small for lint percentage, seed and tint index, and fiber length, strength and fineness. Genotype x environment interaction variances were generally small for all traits except yield and bolls per plant. Lint yield was highly positively correlated with lint percentage and bolls per plant and negatively correlated with seed index and weight per boll.

AIM: To characterize the gut bacterial microbiota of patients with primary sclerosing cholangitis (PSC) and ulcerative colitis (UC). METHODS: = 11) concomitant inflammatory bowel disease, 32 UC patients, and 31 healthy controls. The V3 and V4 regions of the 16S ribosomal RNA gene were sequenced on Illumina MiSeq platform to cover low taxonomic levels. Data were further processed in QIIME employing MaAsLin and LEfSe tools for analysis of the output data. RESULTS: . Low levels of serum albumin were significantly correlated with enrichment of order Actinomycetales. CONCLUSION: PSC is associated with specific gut microbes independently of concomitant IBD and several bacterial taxa clearly distinguish IBD phenotypes (PSC-IBD and UC).

It has become generally accepted that new neurones are added and integrated mainly in two areas of the mammalian CNS, the subventricular zone and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus, which is of central importance in learning and memory. The newly generated cells display neuronal morphology, are able to generate action potentials and receive functional synaptic inputs, i.e. their properties are similar to those found in mature neurones. Alzheimer's disease (AD) is the primary and widespread cause of dementia and is an age-related, progressive and irreversible neurodegenerative disease that deteriorates cognitive functions. Here, we have used male and female triple transgenic mice (3xTg-AD) harbouring three mutant genes (beta-amyloid precursor protein, presenilin-1 and tau) and their respective non-transgenic (non-Tg) controls at 2, 3, 4, 6, 9 and 12 months of age to establish the link between AD and neurogenesis. Using immunohistochemistry we determined the area density of proliferating cells within the SGZ of the DG, measured by the presence of phosphorylated Histone H3 (HH3), and their possible co-localisation with GFAP to exclude a glial phenotype. Less than 1% of the HH3 labeled cells co-localised with GFAP. Both non-Tg and 3xTg-AD showed an age-dependent decrease in neurogenesis. However, male 3xTg-AD mice demonstrated a further reduction in the production of new neurones from 9 months of age (73% decrease) and a complete depletion at 12 months, when compared to controls. In addition, female 3xTg-AD mice showed an earlier but equivalent decrease in neurogenesis at 4 months (reduction of 63%) with an almost inexistent rate at 12 months (88% decrease) compared to controls. This reduction in neurogenesis was directly associated with the presence of beta-amyloid plaques and an increase in the number of beta-amyloid containing neurones in the hippocampus; which in the case of 3xgTg females was directly correlated. These results suggest that 3xTg-AD mice have an impaired ability to generate new neurones in the DG of the hippocampus, the severity of which increases with age and might be directly associated with the known cognitive impairment observed from 6 months of age onwards . The earlier reduction of neurogenesis in females, from 4 months, is in agreement with the higher prevalence of AD in women than in men. Thus it is conceivable to speculate that a recovery in neurogenesis rates in AD could help to rescue cognitive impairment.

Astrocytes dynamically interact with neurons to regulate synaptic transmission. Although the gap junction proteins connexin 30 (Cx30) and connexin 43 (Cx43) mediate the extensive network organization of astrocytes, their role in synaptic physiology is unknown. Here we show, by inactivating Cx30 and Cx43 genes, that astroglial networks tone down hippocampal synaptic transmission in CA1 pyramidal neurons. Gap junctional networking facilitates extracellular glutamate and potassium removal during synaptic activity through modulation of astroglial clearance rate and extracellular space volume. This regulation limits neuronal excitability, release probability, and insertion of postsynaptic AMPA receptors, silencing synapses. By controlling synaptic strength, connexins play an important role in synaptic plasticity. Altogether, these results establish connexins as critical proteins for extracellular homeostasis, important for the formation of functional synapses.

Traditionally the auditory system was considered a hard-wired sensory system; this view has been challenged in recent years in light of the plasticity of other sensory systems, particularly the visual and somatosensory systems. Practical experience in clinical audiology together with the use of prosthetic devices, such as cochlear implants, contributed significantly to the present view on the plasticity of the central auditory system, which was originally based on data obtained in animal experiments. The loss of auditory receptors, the hair cells, results in profound changes in the structure and function of the central auditory system, typically demonstrated by a reorganization of the projection maps in the auditory cortex. These plastic changes occur not only as a consequence of mechanical lesions of the cochlea or biochemical lesions of the hair cells by ototoxic drugs, but also as a consequence of the loss of hair cells in connection with aging or noise exposure. In light of the aging world population and the increasing amount of noise in the modern world, understanding the plasticity of the central auditory system has its practical consequences and urgency. In most of these situations, a common denominator of central plastic changes is a deterioration of inhibition in the subcortical auditory nuclei and the auditory cortex. In addition to the processes that are elicited by decreased or lost receptor function, the function of nerve cells in the adult central auditory system may dynamically change in the process of learning. A better understanding of the plastic changes in the central auditory system after sensory deafferentation, sensory stimulation, and learning may contribute significantly to improvement in the rehabilitation of damaged or lost auditory function and consequently to improved speech processing and production.