Young blood-mediated cerebromicrovascular rejuvenation through heterochronic parabiosis: enhancing blood-brain barrier integrity and capillarization in the aged mouse brain.

Age-related cerebromicrovascular changes, including blood-brain barrier (BBB) disruption and microvascular rarefaction, play a significant role in the development of vascular cognitive impairment (VCI) and neurodegenerative diseases. Utilizing the unique model of heterochronic parabiosis, which involves surgically joining young and old animals, we investigated the influence of systemic factors on these vascular changes. Our study employed heterochronic parabiosis to explore the effects of young and aged systemic environments on cerebromicrovascular aging in mice. We evaluated microvascular density and BBB integrity in parabiotic pairs equipped with chronic cranial windows, using intravital two-photon imaging techniques. Our results indicate that short-term exposure to young systemic factors leads to both functional and structural rejuvenation of cerebral microcirculation. Notably, we observed a marked decrease in capillary density and an increase in BBB permeability to fluorescent tracers in the cortices of aged mice undergoing isochronic parabiosis (20-month-old C57BL/6 mice [A-(A)]; 6 weeks of parabiosis), compared to young isochronic parabionts (6-month-old, [Y-(Y)]). However, aged heterochronic parabionts (A-(Y)) exposed to young blood exhibited a significant increase in cortical capillary density and restoration of BBB integrity. In contrast, young mice exposed to old blood from aged parabionts (Y-(A)) rapidly developed cerebromicrovascular aging traits, evidenced by reduced capillary density and increased BBB permeability. These findings underscore the profound impact of systemic factors in regulating cerebromicrovascular aging. The rejuvenation observed in the endothelium, following exposure to young blood, suggests the existence of anti-geronic elements that counteract microvascular aging. Conversely, pro-geronic factors in aged blood appear to accelerate cerebromicrovascular aging. Further research is needed to assess whether the rejuvenating effects of young blood factors could extend to other age-related cerebromicrovascular pathologies, such as microvascular amyloid deposition and increased microvascular fragility.

Atherosclerotic burden and cerebral small vessel disease: exploring the link through microvascular aging and cerebral microhemorrhages.

Cerebral microhemorrhages (CMHs, also known as cerebral microbleeds) are a critical but frequently underestimated aspect of cerebral small vessel disease (CSVD), bearing substantial clinical consequences. Detectable through sensitive neuroimaging techniques, CMHs reveal an extensive pathological landscape. They are prevalent in the aging population, with multiple CMHs often being observed in a given individual. CMHs are closely associated with accelerated cognitive decline and are increasingly recognized as key contributors to the pathogenesis of vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). This review paper delves into the hypothesis that atherosclerosis, a prevalent age-related large vessel disease, extends its pathological influence into the cerebral microcirculation, thereby contributing to the development and progression of CSVD, with a specific focus on CMHs. We explore the concept of vascular aging as a continuum, bridging macrovascular pathologies like atherosclerosis with microvascular abnormalities characteristic of CSVD. We posit that the same risk factors precipitating accelerated aging in large vessels (i.e., atherogenesis), primarily through oxidative stress and inflammatory pathways, similarly instigate accelerated microvascular aging. Accelerated microvascular aging leads to increased microvascular fragility, which in turn predisposes to the formation of CMHs. The presence of hypertension and amyloid pathology further intensifies this process. We comprehensively overview the current body of evidence supporting this interconnected vascular hypothesis. Our review includes an examination of epidemiological data, which provides insights into the prevalence and impact of CMHs in the context of atherosclerosis and CSVD. Furthermore, we explore the shared mechanisms between large vessel aging, atherogenesis, microvascular aging, and CSVD, particularly focusing on how these intertwined processes contribute to the genesis of CMHs. By highlighting the role of vascular aging in the pathophysiology of CMHs, this review seeks to enhance the understanding of CSVD and its links to systemic vascular disorders. Our aim is to provide insights that could inform future therapeutic approaches and research directions in the realm of neurovascular health.

Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

Riluzole treatment modulates KCC2 and EAAT-2 receptor expression and Ca2+ accumulation following ventral root avulsion injury.

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltage-activated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2.

cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Molecular Characterization of Novel Mycoviruses in Seven Umbelopsis Strains.

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or -2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization.

Biodistribution and Cellular Internalization of Inactivated SARS-CoV-2 in Wild-Type Mice.

Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enzyme 2 (ACE2) is still viewed as the main cell entry receptor mediating SARS-CoV-2 internalization. It has been reported that wild-type mice, like other rodent species of the Muridae family, cannot be infected with SARS-CoV-2 due to differences in their ACE2 receptors. On the other hand, the consensus heparin-binding motif of SARS-CoV-2's spike protein, PRRAR, enables the attachment to rodent heparan sulfate proteoglycans (HSPGs), including syndecans, a transmembrane HSPG family with a well-established role in clathrin- and caveolin-independent endocytosis. As mammalian syndecans possess a relatively conserved structure, we analyzed the cellular uptake of inactivated SARS-CoV-2 particles in in vitro and in vivo mice models. Cellular studies revealed efficient uptake into murine cell lines with established syndecan-4 expression. After intravenous administration, inactivated SARS-CoV-2 was taken up by several organs in vivo and could also be detected in the brain. Internalized by various tissues, inactivated SARS-CoV-2 raised tissue TNF-α levels, especially in the heart, reflecting the onset of inflammation. Our studies on in vitro and in vivo mice models thus shed light on unknown details of SARS-CoV-2 internalization and help broaden the understanding of the molecular interactions of SARS-CoV-2.

Survival Comes at a Cost: A Coevolution of Phage and Its Host Leads to Phage Resistance and Antibiotic Sensitivity of Pseudomonas aeruginosa Multidrug Resistant Strains.

The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (∼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection.

Characterization of Four Novel dsRNA Viruses Isolated from Mucor hiemalis Strains.

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or-2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.

Passive Transfer of Blood Sera from ALS Patients with Identified Mutations Results in Elevated Motoneuronal Calcium Level and Loss of Motor Neurons in the Spinal Cord of Mice.

Introduction: Previously, we demonstrated the degeneration of axon terminals in mice after repeated injections of blood sera from amyotrophic lateral sclerosis (ALS) patients with identified mutations. However, whether a similar treatment affects the cell body of motor neurons (MNs) remained unresolved. Methods: Sera from healthy individuals or ALS patients with a mutation in different ALS-related genes were intraperitoneally injected into ten-week-old male Balb/c mice (n = 3/serum) for two days. Afterward, the perikaryal calcium level was measured using electron microscopy. Furthermore, the optical disector method was used to evaluate the number of lumbar MNs. Results: The cytoplasmic calcium level of the lumbar MNs of the ALS-serum-treated mice, compared to untreated and healthy-serum-treated controls, was significantly elevated. While injections of the healthy serum did not reduce the number of MNs compared to the untreated control group, ALS sera induced a remarkable loss of MNs. Discussion: Similarly to the distant motor axon terminals, the injection of blood sera of ALS patients has a rapid degenerative effect on MNs. Analogously, the magnitude of the evoked changes was specific to the type of mutation; furthermore, the degeneration was most pronounced in the group treated with sera from ALS patients with a mutation in the chromosome 9 open reading frame 72 gene.

Ascorbate inactivates the oxygen-evolving complex in prolonged darkness.

Ascorbate (Asc, vitamin C) is an essential metabolite participating in multiple physiological processes of plants, including environmental stress management and development. In this study, we acquired knowledge on the role of Asc in dark-induced leaf senescence using Arabidopsis thaliana as a model organism. One of the earliest effects of prolonged darkness is the inactivation of oxygen-evolving complexes (OEC) as demonstrated here by fast chlorophyll a fluorescence and thermoluminescence measurements. We found that inactivation of OEC due to prolonged darkness was attenuated in the Asc-deficient vtc2-4 mutant. On the other hand, the severe photosynthetic phenotype of a psbo1 knockout mutant, lacking the major extrinsic OEC subunit PSBO1, was further aggravated upon a 24-h dark treatment. The psbr mutant, devoid of the PSBR subunit of OEC, performed only slightly disturbed photosynthetic activity under normal growth conditions, whereas it showed a strongly diminished B thermoluminescence band upon dark treatment. We have also generated a double psbo1 vtc2 mutant, and it showed a slightly milder photosynthetic phenotype than the single psbo1 mutant. Our results, therefore, suggest that Asc leads to the inactivation of OEC in prolonged darkness by over-reducing the Mn-complex that is probably enabled by a dark-induced dissociation of the extrinsic OEC subunits. Our study is an example that Asc may negatively affect certain cellular processes and thus its concentration and localization need to be highly controlled.

Upregulation of Nucleotide-Binding Oligomerization Domain-, LRR- and Pyrin Domain-Containing Protein 3 in Motoneurons Following Peripheral Nerve Injury in Mice.

Neuronal injuries are accompanied by release and accumulation of damage-associated molecules, which in turn may contribute to activation of the immune system. Since a wide range of danger signals (including endogenous ones) are detected by the nucleotide-binding oligomerization domain-, LRR- and pyrin domain-containing protein 3 (NLRP3) pattern recognition receptor, we hypothesized that NLRP3 may become activated in response to motor neuron injury. Here we show that peripheral injury of the oculomotor and the hypoglossal nerves results in upregulation of NLRP3 in corresponding motor nuclei in the brainstem of mice. Although basal expression of NLRP3 was observed in microglia, astroglia and neurons as well, its upregulation and co-localization with apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, suggesting inflammasome activation, was only detected in neurons. Consequently, increased production of active pro-inflammatory cytokines interleukin-1ß and interleukin-18 were detected after hypoglossal nerve axotomy. Injury-sensitive hypoglossal neurons responded with a more pronounced NLRP3 upregulation than injury-resistant motor neurons of the oculomotor nucleus. We further demonstrated that the mitochondrial protector diazoxide was able to reduce NLRP3 upregulation in a post-operative treatment paradigm. Our results indicate that NLRP3 is activated in motoneurons following acute nerve injury. Blockade of NLRP3 activation might contribute to the previously observed anti-inflammatory and neuroprotective effects of diazoxide.

Presence of Titanium and Toxic Effects Observed in Rat Lungs, Kidneys, and Central Nervous System in vivo and in Cultured Astrocytes in vitro on Exposure by Titanium Dioxide Nanorods.

BACKGROUND: Non-spherical titanium dioxide (TiO2) nanoparticles have been increasingly applied in various biomedical and technological fields. Their toxicological characterization is, however, less complete than that of roundish nanoparticles. MATERIALS AND METHODS: Anatase form TiO2 nanorods, ca. 15x65 nm in size, were applied to cultured astrocytes in vitro and to the airways of young adult Wistar rats in vivo in 5, 10, and 8 mg/kg BW dose for altogether 28 days. Presence of nanorods and cellular damage was investigated in the astrocytes and in rat lungs and kidneys. Functional damage of the nervous system was studied by electrophysiological methods. RESULTS: The treated astrocytes showed loss of viability without detectable apoptosis. In rats, TiO2 nanorods applied to the airways reached the blood and various organs including the lungs, kidneys, and the central nervous system. In lung and kidney samples, nanorods were observed within (partly damaged) phagolysosomes and attached to organelles, and apoptotic cell death was also detected. In cortical and peripheral electrophysiological activity, alterations corresponding to energy shortage (resulting possibly from mitochondrial damage) and astrocytic dysfunction were detected. Local titanium levels and relative weight of the investigated organs, apoptotic cell death in the lungs and kidneys, and changes in the central and peripheral nervous activity were mostly proportional to the applied doses, and viability loss of the cultured astrocytes was also dose-dependent, suggesting causal relationship of treatments and effects. CONCLUSION: Based on localization of the visualized nanorods, on neuro-functional changes, and on literature data, the toxic mechanism involved mitochondrial damage, oxidative stress, and apoptotic cell death. These indicate potential human toxicity and occupational risk in case of exposure to rod-shaped TiO2 nanoparticles.

Role of Protein-Water Interface in the Stacking Interactions of Granum Thylakoid Membranes-As Revealed by the Effects of Hofmeister Salts.

The thylakoid membranes of vascular plants are differentiated into stacked granum and unstacked stroma regions. The formation of grana is triggered by the macrodomain formation of photosystem II and light-harvesting complex II (PSII-LHCII) and thus their lateral segregation from the photosystem I-light-harvesting complex I (PSI-LHCI) super-complexes and the ATP-synthase; which is then stabilized by stacking interactions of the adjacent PSII-LHCII enriched regions of the thylakoid membranes. The self-assembly and dynamics of this highly organized membrane system and the nature of forces acting between the PSII-LHCII macrodomains are not well understood. By using circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and transmission electron microscopy (TEM), we investigated the effects of Hofmeister salts on the organization of pigment-protein complexes and on the ultrastructure of thylakoid membranes. We found that the kosmotropic agent (NH4)2SO4 and the Hofmeister-neutral NaCl, up to 2 M concentrations, hardly affected the macro-organization of the protein complexes and the membrane ultrastructure. In contrast, chaotropic salts, NaClO4, and NaSCN destroyed the mesoscopic structures, the multilamellar organization of the thylakoid membranes and the chiral macrodomains of the protein complexes but without noticeably affecting the short-range, pigment-pigment excitonic interactions. Comparison of the concentration- and time-dependences of SANS, TEM and CD parameters revealed the main steps of the disassembly of grana in the presence of chaotropes. It begins with a rapid diminishment of the long-range periodic order of the grana membranes, apparently due to an increased stacking disorder of the thylakoid membranes, as reflected by SANS experiments. SANS measurements also allowed discrimination between the cationic and anionic effects-in stacking and disorder, respectively. This step is followed by a somewhat slower disorganization of the TEM ultrastructure, due to the gradual loss of stacked membrane pairs. Occurring last is the stepwise decrease and disappearance of the long-range chiral order of the protein complexes, the rate of which was faster in LHCII-deficient membranes. These data are interpreted in terms of a theory, from our laboratory, according to which Hofmeister salts primarily affect the hydrophylic-hydrophobic interactions of proteins, and the stroma-exposed regions of the intrinsic membrane proteins, in particular-pointing to the role of protein-water interface in the stacking interactions of granum thylakoid membranes.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Similarities and Differences in the Effects of Toxic Concentrations of Cadmium and Chromium on the Structure and Functions of Thylakoid Membranes in Chlorella variabilis.

Trace metal contaminations in natural waters, wetlands, and wastewaters pose serious threats to aquatic ecosystems-mainly via targeting microalgae. In this work, we investigated the effects of toxic amounts of chromium and cadmium ions on the structure and function of the photosynthetic machinery of Chlorella variabilis cells. To halt the propagation of cells, we used high concentrations of Cd and Cr, 50-50 mg L-1, in the forms of CdCl2 x 2.5 H2O and K2Cr2O7, respectively. Both treatments led to similar, about 50% gradual diminishment of the chlorophyll contents of the cells in 48 h, which was, however, accompanied by a small (~10%) but statistically significant enrichment (Cd) and loss (Cr) of ß-carotene. Both Cd and Cr inhibited the activity of photosystem II (PSII)-but with more severe inhibitions with Cr. On the contrary, the PsbA (D1) protein of PSII and the PsbO protein of the oxygen-evolving complex were retained more in Cr-treated cells than in the presence of Cd. These data and the higher susceptibility of P700 redox transients in Cr-treated cells suggest that, unlike with Cd, PSII is not the main target in the photochemical apparatus. These differences at the level of photochemistry also brought about dissimilarities at higher levels of the structural complexity of the photosynthetic apparatus. Circular dichroism (CD) spectroscopy measurements revealed moderate perturbations in the macro-organization of the protein complexes-with more pronounced decline in Cd-treated cells than in the cells with Cr. Also, as reflected by transmission electron microscopy and small-angle neutron scattering, the thylakoid membranes suffered shrinking and were largely fragmented in Cd-treated cells, whereas no changes could be discerned with Cr. The preservation of integrity of membranes in Cr-treated cells was most probably aided by high proportion of the de-epoxidized xanthophylls, which were absent with Cd. It can thus be concluded that beside strong similarities of the toxic effects of Cr and Cd, the response of the photosynthetic machinery of C. variabilis to these two trace metal ions substantially differ from each other-strongly suggesting different inhibitory and protective mechanisms following the primary toxic events.

Diazoxide blocks or reduces microgliosis when applied prior or subsequent to motor neuron injury in mice.

Diazoxide (DZX), an anti-hypertonic and anti-hypoglycemic drug, was shown to have anti-inflammatory effects in several injured cell types outside the central nervous system. In the brain, the neuroprotective potential of DZX is well described, however, its anticipated anti-inflammatory effect after acute injury has not been systematically analyzed. To disclose the anti-inflammatory effect of DZX in the central nervous system, an injury was induced in the hypoglossal and facial nuclei and in the oculomotor nucleus by unilateral axonal transection and unilateral target deprivation (enucleation), respectively. On the fourth day after surgery, microglial analysis was performed on tissue in which microglia were DAB-labeled and motoneurons were labeled with immunofluorescence. DZX treatment was given either prophylactically, starting 7 days prior to the injury and continuing until the animals were sacrificed, or postoperatively only, with daily intraperitoneal injections (1.25 mg/kg; in 10 mg/ml dimethyl sulfoxide in distilled water). Prophylactically + postoperatively applied DZX completely eliminated the microglial reaction in each motor nuclei. If DZX was applied only postoperatively, some microglial activation could be detected, but its magnitude was still significantly smaller than the non-DZX-treated controls. The effect of DZX could also be demonstrated through an extended period, as tested in the hypoglossal nucleus on day 7 after the operation. Neuronal counts, determined at day 4 after the operation in the hypoglossal nucleus, demonstrated no loss of motor neurons, however, an increased Feret's diameter of mitochondria could be measured, suggesting increased oxidative stress in the injured cells. The increase of mitochondrial Feret's diameter could also be prevented with DZX treatment.

Detection and Molecular Characterization of Novel dsRNA Viruses Related to the Totiviridae Family in Umbelopsis ramanniana.

Umbelopsis ramanniana is an oleaginous fungus belonging to the Mucoromycotina subphylum. Our group had previously detected four double-stranded RNA (dsRNA) bands in the U. ramanniana NRRL 1296 strain by gel electrophoresis. Here we describe the molecular characterization of its dsRNA elements as well as the discovery of four novel dsRNA viruses: Umbelopsis ramanniana virus 1 (UrV1), Umbelopsis ramanniana virus 2 (UrV2), Umbelopsis ramanniana virus 3 (UrV3), and Umbelopsis ramanniana virus 4 (UrV4). Full genomes of UrV1, UrV3, and UrV4 were determined using the full-length amplification of cDNAs (FLAC) technique; they contain two open reading frames (ORF), which putatively encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In case of UrV2, a partial ORF encoding a partial RdRp gene could be determined. Based on the phylogeny inferred from the RdRp sequences, UrV1 and UrV4 belong to the genus Totivirus, while UrV2 may belong to the genus Victorivirus. UrV3 nested to a novel, unclassified group of Totiviridae, which is related to the genus Totivirus. Hybridization analysis revealed that the dsRNA molecules of UrV1 and UrV4 correspond to the same 5.0-kbp electrophoretic band, whilst the probe for the UrV3 hybridized to the largest, 5.3-kbp and the 3.0-kbp bands of the dsRNA pattern of U. ramanniana. Interestingly, the probe for the UrV2 sequence did not hybridized to any dsRNA bands, but it could be amplified from the isolated 3.0-kbp fragment. By transmission electron microscopy, two different isometric virus particles with about 50 and 35 nm in diameter were detected in U. ramanniana NRRL 1296 indicating that this strain harbor multiple viruses. Beside U. ramanniana, dsRNA elements were also detected in other Umbelopsis isolates with different patterns consisting of 2 to 4 discrete and different sized (0.7-5.3-kbp) dsRNA molecules. Based on a hybridization analysis with UrV1 CP and RdRp probes, the bands with the size of around 5.0-kbp, which were present in all tested Umbelopsis strains, are presumed as possible full mycovirus genomes.

Response of the neurovascular unit to brain metastatic breast cancer cells.

Therapeutic resistance of cerebral secondary tumours largely depends on unique aspects linked to the neurovascular unit, especially cerebral endothelial cells and astrocytes. By using advanced microscopy techniques, here we explored novel mechanisms related to the neurovascular unit during extravasation and proliferation of triple negative breast cancer cells in the brain. Metastatic mammary carcinoma cells arrested and elongated within one hour in cerebral microvessels, but their number decreased by almost 80% in the first two days. Interestingly, malignant cells induced vasoconstriction and development of intraluminal endothelial plugs, which isolated invading cells from the circulation. During diapedesis - which usually took place on day four and five after inoculation of the tumour cells - continuity of cerebral endothelial tight junctions remained intact, indicating migration of cancer cells through the transcellular pathway. In addition, metastatic cells induced formation of multiluminal vessels and claudin-5-positive endothelial blebs. However, even severe endothelial blebbing could be reversed and the vessel morphology was restored shortly after the tumour cells completed transendothelial migration. Similar to neuro-inflammatory leukocytes, tumour cells migrated not only through the endothelial layer, but through the glia limitans perivascularis as well. Nevertheless, along with the growth of metastatic lesions by co-option of pre-existing capillaries, astrocytes and astrocyte end-feet were gradually expelled from the vessels to the border of the tumour. Taken together, we identified previously unknown mechanisms involved in the reaction of brain resident cells to invading breast cancer cells. Our results contribute to a better understanding of the complex cross-talk between tumour cells and host cells in the brain, which is essential for the identification of new therapeutic targets in this devastating disease.

Experimental Motor Neuron Disease Induced in Mice with Long-Term Repeated Intraperitoneal Injections of Serum from ALS Patients.

In an earlier study, signs of commencing degeneration of spinal motor neurons were induced in mice with short-term intraperitoneal injections of immunoglobulin G (IgG) taken from patients with amyotrophic lateral sclerosis (ALS). Since in that study, neither weakness nor loss of motor neurons was noted, to test whether the ALS IgG in this paradigm has the potential to evoke relentless degeneration of motor neurons, treatment with repeated injections over a longer period was carried out. Mice were systematically injected intraperitoneally with serum taken from ALS patients over a 75-day period. At selected time points, the isometric force of the limbs, number of spinal motor neurons and their intracellular calcium levels were determined. Furthermore, markers of glial activation and the motoneuronal uptake of human IgG were monitored. During this period, gliosis and progressive motoneuronal degeneration developed, which led to gradual loss of spinal motor neurons, more than 40% at day 21, along with decreasing muscle strength in the limbs. The inclusion-like accumulation of IgG appeared in the perikarya with the increase of intracellular calcium in the cell bodies and motor nerve terminals. Our results demonstrate that ALS serum can transfer motor neuron disease to mice.