Molecularly generated rat hepatitis E virus strains from human and rat show efficient replication in a human hepatoma cell line.

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1-4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro-transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV.

Strain-Specific Interactions between the Viral Capsid Proteins VP4, VP7 and VP6 Influence Rescue of Rotavirus Reassortants by Reverse Genetics.

Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. To gain insight into the factors that restrict reassortment, we utilized reverse genetics and tested the generation of simian RVA strain SA11 reassortants carrying the human RVA strain Wa capsid proteins VP4, VP7, and VP6 in all possible combinations. VP7-Wa, VP6-Wa, and VP7/VP6-Wa reassortants were effectively rescued, but the VP4-Wa, VP4/VP7-Wa, and VP4/VP6-Wa reassortants were not viable, suggesting a limiting effect of VP4-Wa. However, a VP4/VP7/VP6-Wa triple-reassortant was successfully generated, indicating that the presence of homologous VP7 and VP6 enabled the incorporation of VP4-Wa into the SA11 backbone. The replication kinetics of the triple-reassortant and its parent strain Wa were comparable, while the replication of all other rescued reassortants was similar to SA11. Analysis of the predicted structural protein interfaces identified amino acid residues, which might influence protein interactions. Restoring the natural VP4/VP7/VP6 interactions may therefore improve the rescue of RVA reassortants by reverse genetics, which could be useful for the development of next generation RVA vaccines.

Coronavirus-mimicking nanoparticles (CorNPs) in artificial saliva droplets and nanoaerosols: Influence of shape and environmental factors on particokinetics/particle aerodynamics.

Severe acute respiratory syndrome coronavirus 2, abbreviated as SARS-CoV-2, has been associated with the transmission of infectious COVID-19 disease through breathing and speech droplets emitted by infected carriers including asymptomatic cases. As part of SARS-CoV-2 global pandemic preparedness, we studied the transmission of aerosolized air mimicking the infected person releasing speech aerosol with droplets containing CorNPs using a vibrating mesh nebulizer as human patient simulator. Generally speech produces nanoaerosols with droplets of <5 µm in diameter that can travel distances longer than 1 m after release. It is assumed that speech aerosol droplets are a main element of the current Corona virus pandemic, unlike droplets larger than 5 m, which settle down within a 1 m radius. There are no systemic studies, which take into account speech-generated aerosol/droplet experimental validation and their aerodynamics/particle kinetics analysis. In this study, we cover these topics and explore role of residual water in aerosol droplet stability by exploring drying dynamics. Furthermore, a candle experiment was designed to determine whether air pollution might influence respiratory virus like nanoparticle transmission and air stability.

Host Range, Morphology and Sequence Analysis of Ten Temperate Phages Isolated from Pathogenic Yersinia enterocolitica Strains.

Yersinia enterocolitica is a heterogeneous species comprising highly pathogenic, weakly pathogenic and non-pathogenic strains. Previous data suggest that gene exchange may occur in Yersinia. Only scarce information exists about temperate phages of Y. enterocolitica, even though many prophage sequences are present in this species. We have examined 102 pathogenic Y. enterocolitica strains for the presence of inducible prophages by mitomycin C treatment. Ten phages were isolated from nine strains belonging to the bio (B)/serotypes (O) B2/O:5,27, B2/O:9 and 1B/O:8. All phages are myoviruses showing lytic activity only at room temperature. Whole-genome sequencing of the phage genomes revealed that they belong to three groups, which, however, are not closely related to known phages. Group 1 is composed of five phages (type phage: vB_YenM_06.16.1) with genome sizes of 43.8 to 44.9 kb, whereas the four group 2 phages (type phage: vB_YenM_06.16.2) possess smaller genomes of 29.5 to 33.2 kb. Group 3 contains only one phage (vB_YenM_42.18) whose genome has a size of 36.5 kb, which is moderately similar to group 2. The host range of the phages differed significantly. While group 1 phages almost exclusively lysed strains of B2/O:5,27, phages of group 2 and 3 were additionally able to lyse B4/O:3, and some of them even B2/O:9 and 1B/O:8 strains.

Monitoring and imaging pH in biofilms utilizing a fluorescent polymeric nanosensor.

Biofilms are ubiquitous in nature and in the man-made environment. Given their harmful effects on human health, an in-depth understanding of biofilms and the monitoring of their formation and growth are important. Particularly relevant for many metabolic processes and survival strategies of biofilms is their extracellular pH. However, most conventional techniques are not suited for minimally invasive pH measurements of living biofilms. Here, a fluorescent nanosensor is presented for ratiometric measurements of pH in biofilms in the range of pH 4.5-9.5 using confocal laser scanning microscopy. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with pH-inert dye Nile Red and is surface functionalized with a pH-responsive fluorescein dye. Its performance was validated by fluorometrically monitoring the time-dependent changes in pH in E. coli biofilms after glucose inoculation at 37 °C and 4 °C. This revealed a temperature-dependent decrease in pH over a 4-h period caused by the acidifying glucose metabolism of E. coli. These studies demonstrate the applicability of this nanosensor to characterize the chemical microenvironment in biofilms with fluorescence methods.

Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany.

Infection with hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. The HEV genotype 3 can be zoonotically transmitted from animals to humans, with wild boars representing an important reservoir species. Cell culture isolation of HEV is generally difficult and mainly described for human isolates so far. Here, five sera and five liver samples from HEV-RNA-positive wild boar samples were inoculated onto PLC/PRF/5 cells, incubated for 3 months and thereafter passaged for additional 6 weeks. As demonstrated by RT-qPCR, immunofluorescence and immune electron microscopy, virus was successfully isolated from two liver samples, which originally contained high HEV genome copy numbers. Both isolates showed slower growth than the culture-adapted HEV strain 47832c. In contrast to this strain, the isolated strains had no insertions in their hypervariable genome region. Next generation sequencing using an HEV sequence-enriched library enabled full genome sequencing. Strain Wb108/17 belongs to subtype 3f and strain Wb257/17 to a tentative novel subtype recently described in Italian wild boars. The results indicate that HEV can be successfully isolated in cell culture from wild boar samples containing high HEV genome copy numbers. The isolates may be used further to study the zoonotic potential of wild boar-derived HEV subtypes.

Hepatitis E virus persists in the ejaculate of chronically infected men.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate. METHODS: The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing. RESULTS: In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact. CONCLUSIONS: The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles. LAY SUMMARY: Enveloped hepatitis E virus particles could be identified by PCR and electron microscopy in the ejaculate of immunosuppressed chronically infected patients, but not in immunocompetent experimentally infected pigs or in patients with acute self-limiting hepatitis E.

3D Printing - Evaluating Particle Emissions of a 3D Printing Pen.

Three-dimensional (3D) printing as a type of additive manufacturing shows continuing increase in application and consumer popularity. The fused filament fabrication (FFF) is an inexpensive method used most frequently by consumers. Studies with 3D printers have shown that during the printing process particulate and volatile substances are released. Handheld 3D printing pens also use the FFF method but the consumer's proximity to the 3D pens gives reason to higher exposure compared to a 3D printer. At the same time, 3D printing pens are often marketed for children who could be more sensitive to the printing emission. The aim of this study was to implement a low cost method to analyze the emissions of 3D printing pens. Polylactide (PLA) and acrylonitrile butadiene styrene (ABS) filaments of different colors were tested. In addition, filaments containing metal and carbon nanotubes (CNTs) were analyzed. An 18.5 L chamber and sampling close to the emission source was used to characterize emissions and concentrations near the breathing zone of the user. Particle emissions and particle size distributions were measured and the potential release of metal particles and CNTs investigated. Particle number concentrations were found in a range of 105 - 106 particles/cm3, which is comparable to previous reports from 3D printers. Transmission electron microscopy (TEM) analysis showed nanoparticles of the different thermoplastic materials as well as of metal particles and CNTs. High contents of metal were observed by inductively coupled plasma mass spectrometry (ICP-MS). These results call for a cautious use of 3D pens due to potential risk to the consumers.

TatS: a novel in vitro tattooed human skin model for improved pigment toxicology research.

Reports of tattoo-associated risks boosted the interest in tattoo pigment toxicity over the last decades. Nonetheless, the influence of tattoo pigments on skin homeostasis remains largely unknown. In vitro systems are not available to investigate the interactions between pigments and skin. Here, we established TatS, a reconstructed human full-thickness skin model with tattoo pigments incorporated into the dermis. We mixed the most frequently used tattoo pigments carbon black (0.02 mg/ml) and titanium dioxide (TiO2, 0.4 mg/ml) as well as the organic diazo compound Pigment Orange 13 (0.2 mg/ml) into the dermis. Tissue viability, morphology as well as cytokine release were used to characterize TatS. Effects of tattoo pigments were compared to monolayer cultures of human fibroblasts. The tissue architecture of TatS was comparable to native human skin. The epidermal layer was fully differentiated and the keratinocytes expressed occludin, filaggrin and e-cadherin. Staining of collagen IV confirmed the formation of the basement membrane. Tenascin C was expressed in the dermal layer of fibroblasts. Although transmission electron microscopy revealed the uptake of the tattoo pigments into fibroblasts, neither viability nor cytokine secretion was altered in TatS. In contrast, TiO2 significantly decreased cell viability and increased interleukin-8 release in fibroblast monolayers. In conclusion, TatS emulates healed tattooed human skin and underlines the advantages of 3D systems over traditional 2D cell culture in tattoo pigment research. TatS is the first skin model that enables to test the effects of pigments in the dermis upon tattooing.

Potential of avian and mammalian species A rotaviruses to reassort as explored by plasmid only-based reverse genetics.

Species A rotavirus (RVA) is an important gastrointestinal pathogen that is widely distributed in humans, mammalian animals and birds. The RVA genome consists of eleven double-stranded RNA segments, enabling the generation of novel strains with new pathogenic or antigenic features by genetic reassortment. While reassortants between human and mammalian animal RVAs have been repeatedly described, data on the reassortment potential of avian RVA strains are rare. To investigate genome segment exchanges between avian and mammalian RVA strains, a plasmid-based reverse genetics strategy originally developed for the simian RVA strain SA11 was used here. All eleven genome segments of the chicken RVA strain 02V0002G3 were cloned into similar plasmids as in the SA11 system. However, in contrast to SA11, no infectious virus could be generated by transfection of the eleven 02V0002G3 plasmids into cell culture under the same conditions. In another series of experiments, each of the genome segments of 02V0002G3 was transfected together with the remaining ten genome segments of SA11. Viable mono-reassortants were only retrieved for the avian VP3 and VP4 genes. The reassortant viruses were structurally indistinguishable from their parental viruses, but grew to slightly lower titers in cell culture. The results indicate that the VP3 and VP4 genes, but not the other genes of avian RVA, can functionally substitute their mammalian homologs and create viable reassortants. Further research should focus on the reasons behind the reassortment incompatibility and on the optimization of the system for the generation of viable avian RVA rescued entirely from cloned avian RVA genome segments.

Whole genome sequence analysis of cell culture-adapted rotavirus A strains from chicken.

Rotavirus A (RVA) is a major cause of gastroenteritis in humans and mammalian animals, and has also been abundantly detected in avian species. Avian RVA infection is associated with diarrhea, reduced growth and increased mortality, leading to economic losses in the poultry industry. Avian RVA forms a unique genetic clade within the whole RVA species. However, up to now, only a few full-length avian RVA genomes have been published and only a small number of avian RVA strains have been adapted to grow in cell culture for subsequent studies. Here, the four cell culture-adapted chicken RVA strains 02V0002G3, 04V0027G6, 05V0500F6 and 06V0661G1 were characterized in more detail. Transmission electron microscopy of the viruses derived from culture supernatant showed a typical triple-layered morphology of rotavirus particles; in addition, strain 06V0661G1 showed a high proportion of double-layered particles. The (nearly) complete genome sequences of the viruses were determined using next-generation sequencing (NGS). The resulting sequences were compared to full-length or partial sequences of the strains previously determined using Sanger sequencing; and a few nucleotide mismatches, some of them resulting in amino acid substitutions, were identified. The genomes of strains 02V0002G3, 04V0027G6 and 05V0500F6 were closely related to each other showing a G19-P[30]-I11-R6-C6-M7-A16-N6-T8-E10-H8 genotype constellation. Strain 06V0661G1 carries the VP4 genotype P[31] in the same genetic backbone like the other strains. However, further sequence analysis showed that the genes of this strain, especially that encoding NSP3, clustered more separately from the other strains in phylogenetic trees. The characterized cell culture-adapted chicken RVA strains may be useful for future studies investigating genetic diversity and replication of avian rotaviruses, as well as for the development of vaccines and diagnostic tools.

Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics.

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.

Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c.

The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.

Slow Ca2+ Efflux by Ca2+/H+ Exchange in Cardiac Mitochondria Is Modulated by Ca2+ Re-uptake via MCU, Extra-Mitochondrial pH, and H+ Pumping by FOF1-ATPase.

Mitochondrial (m) Ca2+ influx is largely dependent on membrane potential (ΔΨm), whereas mCa2+ efflux occurs primarily via Ca2+ ion exchangers. We probed the kinetics of Ca2+/H+ exchange (CHEm) in guinea pig cardiac muscle mitochondria. We tested if net mCa2+ flux is altered during a matrix inward H+ leak that is dependent on matrix H+ pumping by ATPm hydrolysis at complex V (FOF1-ATPase). We measured [Ca2+]m, extra-mitochondrial (e) [Ca2+]e, ΔΨm, pHm, pHe, NADH, respiration, ADP/ATP ratios, and total [ATP]m in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca2+ after adding DNP and CaCl2 is dependent on whether the ΔpHm gradient is/is not maintained by reciprocal outward H+ pumping by complex V. We found that adding CaCl2 enhanced DNP-induced increases in respiration and decreases in ΔΨm while [ATP]m decreased, ΔpHm gradient was maintained, and [Ca2+]m continued to increase slowly, indicating net mCa2+ influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl2 caused larger declines in ΔΨm as well as a slow fall in pHm to near pHe while [Ca2+]m continued to decrease slowly, indicating net mCa2+ efflux in exchange for H+ influx (CHEm) until the ΔpHm gradient was abolished. The kinetics of slow mCa2+ efflux with slow H+ influx via CHEm was also observed at pHe 6.9 vs. 7.6 by the slow fall in pHm until ΔpHm was abolished; if Ca2+ reuptake via the MCU was also blocked, mCa2+ efflux via CHEm became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHEm activity is driven largely by the ΔpHm chemical gradient with H+ leak, while mCa2+ entry via MCU depends largely on the charge gradient ΔΨm. A fall in ΔΨm with excess mCa2+ loading can occur during cardiac cell stress. Cardiac cell injury due to mCa2+ overload may be reduced by temporarily inhibiting FOF1-ATPase from pumping H+ due to ΔΨm depolarization. This action would prevent additional slow mCa2+ loading via MCU and permit activation of CHEm to mediate efflux of mCa2+. HIGHLIGHTS -We examined how slow mitochondrial (m) Ca2+ efflux via Ca2+/H+ exchange (CHEm) is triggered by matrix acidity after a rapid increase in [Ca2+]m by adding CaCl2 in the presence of dinitrophenol (DNP) to permit H+ influx, and oligomycin (OMN) to block H+ pumping via FOF1-ATP synthase/ase (complex V).-Declines in ΔΨm and pHm after DNP and added CaCl2 were larger when complex V was blocked.-[Ca2+]m slowly increased despite a fall in ΔΨm but maintained pHm when H+ pumping by complex V was permitted.-[Ca2+]m slowly decreased and external [Ca2+]e increased with declines in both ΔΨm and pHm when complex V was blocked.-ATPm hydrolysis supports a falling pHm and redox state and promotes a slow increase in [Ca2+]m.-After rapid Ca2+ influx due to a bolus of CaCl2, slow mCa2+ efflux by CHEm occurs directly if pHe is low.

Electroporation loading of membrane-impermeable molecules to investigate intra- and intercellular Ca2+ signaling.

Electroporation is a technique that temporarily induces pores in the plasma membranes of cells, thereby allowing plasma membrane-impermeable substances to enter the cells. This loading method requires an electrical drive circuit providing an electroporation signal, an electrode to apply the signal to a localized zone in a cell monolayer, and a special solution that has a low electrical conductivity. To avoid impairment of cell function and cell death from the electroporation procedure itself, the applied electrical signal should ideally be a high-frequency oscillating signal (50 kHz) without any direct current (DC) component. Here, we describe the detailed procedure of electroporation loading.

Flash photolysis of caged IP3 to trigger intercellular Ca2+ waves.

Caged IP3 is an inactive form of the second messenger IP3, consisting of the biologically active molecule linked to a cage group through a photolabile bond. This bond is cleaved by exposure to brief "flashes" of ultraviolet (UV) light, thereby releasing the active IP3 molecule. The protection of caged IP3 against metabolic transformation in combination with a defined time point of fast photoliberation of IP3 provides an efficient way to temporally and spatially control the cytosolic release of IP3 and subsequent increase of cytoplasmic Ca(2+). These properties make it an ideal method for kinetic studies and also a well-suited procedure to initiate intercellular Ca(2+) waves from a point source of IP3. This protocol describes cell loading with membrane impermeable caged IP3 and the UV flash illumination procedure.

Electroporation loading and flash photolysis to investigate intra- and intercellular Ca2+ signaling.

Many cellular functions are driven by variations in the intracellular Ca(2+) concentration ([Ca(2+)]i), which may appear as a single-event transient [Ca(2+)]i elevation, repetitive [Ca(2+)]i increases known as Ca(2+) oscillations, or [Ca(2+)]i increases propagating in the cytoplasm as Ca(2+) waves. Additionally, [Ca(2+)]i changes can be communicated between cells as intercellular Ca(2+) waves (ICWs). ICWs are mediated by two possible mechanisms acting in parallel: one involving gap junctions that form channels directly linking the cytoplasm of adjacent cells and one involving a paracrine messenger, in most cases ATP, that is released into the extracellular space, leading to [Ca(2+)]i changes in neighboring cells. The intracellular messenger inositol 1,4,5-trisphosphate (IP3) that triggers Ca(2+) release from Ca(2+) stores is crucial in these two ICW propagation scenarios, and is also a potent trigger to initiate ICWs. Loading inactive, "caged" IP3 into cells followed by photolytic "uncaging" with UV light, thereby liberating IP3, is a well-established method to trigger [Ca(2+)]i changes in single cells that is also effective in initiating ICWs. We here describe a method to load cells with caged IP3 by local electroporation of monolayer cell cultures and to apply flash photolysis to increase intracellular IP3 and induce [Ca(2+)]i changes, or initiate ICWs. Moreover, the electroporation method allows loading of membrane-impermeable agents that interfere with IP3 and Ca(2+) signaling.

Genetically determined mitochondrial preservation and cardioprotection against myocardial ischemia-reperfusion injury in a consomic rat model.

Cardioprotection may be genome dependent. One example is the increased tolerance to cardiac ischemia-reperfusion (IR) in Brown Norway (BN) compared with Dahl salt-sensitive (SS) rats. By narrowing the genetic difference to chromosome 6 only, we found the consomic SS(6BN) to be similarly IR tolerant as BN. We hypothesized that better preserved mitochondrial structure and function are genetically determined and therefore critically linked to myocardial IR tolerance associated with BN chromosome 6. Langendorff-prepared BN, SS, and SS(6BN) rat hearts were subjected to IR, while corresponding controls were continuously perfused. Though largely equal in nonischemic controls, assessment of functional data and ventricular infarct size in IR experiments confirmed that BN and SS(6BN) have an equally higher tolerance to IR than SS hearts. This was complemented by equally better preserved mitochondrial structure, oxidative phosphorylation, and calcium retention capacity in BN and SS(6BN) vs. SS hearts. For the first time, our data indicate that SS(6BN) are as resistant to IR injury as BN hearts in mitochondrial and myocardial function and viability compared with SS hearts. These findings not only link myocardial and mitochondrial protection in a genetic model but also suggest that genetic information on rat chromosome 6 is critical for mitochondrial preservation and IR tolerance.

Endothelial calcium dynamics, connexin channels and blood-brain barrier function.

Situated between the circulation and the brain, the blood-brain barrier (BBB) protects the brain from circulating toxins while securing a specialized environment for neuro-glial signaling. BBB capillary endothelial cells exhibit low transcytotic activity and a tight, junctional network that, aided by the cytoskeleton, restricts paracellular permeability. The latter is subject of extensive research as it relates to neuropathology, edema and inflammation. A key determinant in regulating paracellular permeability is the endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)]i) that affects junctional and cytoskeletal proteins. Ca(2+) signals are not one-time events restricted to a single cell but often appear as oscillatory [Ca(2+)]i changes that may propagate between cells as intercellular Ca(2+) waves. The effect of Ca(2+) oscillations/waves on BBB function is largely unknown and we here review current evidence on how [Ca(2+)]i dynamics influence BBB permeability.