Temporal synchronization of CA1 pyramidal cells by high-frequency, depressing inhibition, in the presence of intracellular noise

The Sharp Wave-associated Ripple is a high-frequency, extracellular recording observed in the rat hippocampus during periods of immobility. During the ripple, pyramidal cells synchronize over a short period of time despite the fact that these cells have sparse recurrent connections. Additionally, the timing of synchronized pyramidal cell spiking may be critical for encoding information that is passed on to post-hippocampal targets. Both the synchronization and precision of pyramidal cells is believed to be coordinated by inhibition provided by a vast array of interneurons. This dissertation proposes a minimal model consisting of a single interneuron which synapses onto a network of uncoupled pyramidal cells. It is shown that fast decaying, high-frequency, depressing inhibition is capable of rapidly synchronizing the pyramidal cells and modulating spike timing. In addition, these mechanisms are robust in the presence of intracellular noise. The existence and stability of synchronous, periodic solutions using geometric singular perturbation techniques are proven. The effects of synaptic strength, synaptic recovery, and inhibition frequency are discussed. In contrast to prior work, which suggests that the ripple is produced by homogeneous populations of either pyramidal cells or interneurons, the results presented here suggest that cooperation between interneurons and pyramidal cells is necessary for ripple genesis

Synthetically recoded virus sCPD9 – A tool to accelerate SARS-CoV-2 research under biosafety level 2 conditions

Research with infectious SARS-CoV-2 is complicated because it must be conducted under biosafety level 3 (BSL-3) conditions. Recently, we constructed a live attenuated SARS-CoV-2 virus by rational design through partial recoding of the SARS-CoV-2 genome and showed that the attenuated virus, designated sCPD9, was highly attenuated in preclinical animal models. The recoded sequence was designed by codon pair deoptimization and is located at the distal end of gene ORF1ab. Codon pair deoptimization involves recoding of the viral sequence with underrepresented codon pairs but without altering the amino acid sequence of the encoded proteins. Thus, parental and attenuated viruses produce exactly the same proteins. In Germany, the live attenuated SARS-CoV-2 mutant sCPD9 was recently classified as a BSL-2 pathogen based on its genetic stability and strong attenuation in preclinical animal models. Despite its high attenuation in vivo, sCPD9 grows to high titers in common cell lines, making it suitable as substitute for virulent SARS-CoV-2 in many experimental setups. Consequently, sCPD9 can ease and accelerate SARS-CoV-2 research under BSL-2 conditions, particularly in experiments requiring replicating virus, such as diagnostics and development of antiviral drugs

The Effect of Alkaloids on Fluorescence in Solutions

The quenching of fluorescence of different substances by a series of alkaloids has \u27 been studied. The alkaloids generally exert a quenching action on the fluorescence. In evaluating these effects, in certain cases it is necessary to take into consideration the absorptii. on of exciting light by the molecules of the quencher. The quenching effect of the anions of ailka loid salts should also be taken into account. By .mathematical corrections of experimental data for the absorption of the exciting light and the quenching by anions, the constants and semi-concentrations of quenching are obtained for the cation:ic part of the alkaloid. It may be assumed that the quenching effect of alkaloids involves an external static mechanism, i. e. the formation of molecular compounds between the quencher and the fluorescent substance

The effect of alkaloids on the chemiluminescence of luminol

Istraživana je djelovanje niza alkaloida na kemiluminescenciju luminola (3-aminoftalhidrazida) za vrijeme oksidacione reakcije u prisutnosti klorhemina, odnosno methemoglobina ili kalijevog heksacijanoferata(III) kao aktivatora luminescencije. Fotoelektričnim mjerenjima ustanovljene su vremenske krivulje intenziteta kemiluminescencije za različite koncentracije alkaloida. Pretežno su nađeni efekti gašenja kemiluminescencije utjecajem alkaloida. Očito se radi o stvaranju neaktivnih molekularnih spojeva između aktivatora (hematina) i alkaloida. Jednadžba za kompetitivnu inhibiciju enzimatsko kataliziranih biokemijskih reakcija može se dobro primijeniti na ustanovljene efekte gašenja luminescencije. U prisutnosti heksacijanoferata(III) kao aktivatora luminescencije odnosi su znatno zamršeniji. Neki alkaloidi ne gase tako aktiviranu kemiluminescenciju, drugi je gase tek u znatno većoj koncentraciji, odnosno povisuju jakost luminescencije.The effect of certain alkaloids on the chemiluminescent oxidation of luminol in the presence of chlorochemin, methemoglobin and potassium ferricyanide as activators has been studied. The curves of chemiluminescence (intensity of luminescence vs. reaction time) for various concentrations of alkaloids were obtained by photoelectric measurements. In most cases a quenching effect of alkaloids has been observed. The numerical values of the half concentrations of the quenching effect were determined. It is apparent that inactive molecular compounds are formed by the activator (hematin) and alkaloids. The equation of competitive inhibition of biochemical reactions catalyzed by enzymes - which is in fact identical with Stern Volmer\u27s equation for the quenching of fluorescence by addition of foreign substances - can be applied to the observed effects of quenching. In the presence of potassium ferricyanide as activator of luminescence, the reactions involved are much more complicated. Some of the alkaloids studied do not quench the luminescence activated by potassium ferricyanide. Others produce an activating effect in small concentrations and a quenching effect in higher concentrations only

The Roborovski Dwarf Hamster Is A Highly Susceptible Model for a Rapid and Fatal Course of SARS-CoV-2 Infection

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated an unprecedented and yet-unresolved health crisis worldwide. Different mammals are susceptible to SARS-CoV-2; however, few species examined so far develop robust clinical disease that mirrors severe human cases or allows testing of vaccines and drugs under conditions of severe disease. Here, we compare the susceptibilities of three dwarf hamster species (Phodopus spp.) to SARS-CoV-2 and introduce the Roborovski dwarf hamster (P. roborovskii) as a highly susceptible COVID-19 model with consistent and fulminant clinical signs. Particularly, only this species shows SARS-CoV-2-induced severe acute diffuse alveolar damage and hyaline microthrombi in the lungs, changes described in patients who succumbed to the infection but not reproduced in any experimentally infected animal. Based on our findings, we propose the Roborovski dwarf hamster as a valuable model to examine the efficacy and safety of vaccine candidates and therapeutics, particularly for use in highly susceptible individuals

Isolation and characterization of new Puumala orthohantavirus strains from Germany

Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies

A phylogenomic analysis of Marek's disease virus reveals independent paths to virulence in Eurasia and North America

Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10−5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV

Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection

Fast-forwarding evolution—Accelerated adaptation in a proofreading-deficient hypermutator herpesvirus

Evolution relies on the availability of genetic diversity for fitness-based selection. However, most deoxyribonucleic acid (DNA) viruses employ DNA polymerases (Pol) capable of exonucleolytic proofreading to limit mutation rates during DNA replication. The relative genetic stability produced by high-fidelity genome replication can make studying DNA virus adaptation and evolution an intensive endeavor, especially in slowly replicating viruses. Here, we present a proofreading-impaired Pol mutant (Y547S) of Marek’s disease virus that exhibits a hypermutator phenotype while maintaining unimpaired growth in vitro and wild-type (WT)-like pathogenicity in vivo. At the same time, mutation frequencies observed in Y547S virus populations are 2–5-fold higher compared to the parental WT virus. We find that Y547S adapts faster to growth in originally non-permissive cells, evades pressure conferred by antiviral inhibitors more efficiently, and is more easily attenuated by serial passage in cultured cells compared to WT. Our results suggest that hypermutator viruses can serve as a tool to accelerate evolutionary processes and help identify key genetic changes required for adaptation to novel host cells and resistance to antiviral therapy. Similarly, the rapid attenuation achieved through adaptation of hypermutators to growth in cell culture enables identification of genetic changes underlying attenuation and virulence, knowledge that could practically exploited, e.g. in the rational design of vaccines