Novel Herbal Formulation Jing Si Exhibits Multiple Functions to Inhibit Replication Activity and Subsides Viral Load of COVID-19 Variants

Background: SARS-CoV-2 is susceptible to frequent mutations and gets transformed into variants therefore identifying novel multi targeting remedies is necessary in formulating strategies to overcome the pandemic. Methods: Traditional Chinese medicine based formula Jing Si herbal (JSH) was screened and analyzed by HPLC to evaluate its ability to act against infection by SARS-CoV-2 variants. The 3CL protease and RdRp assay kit were utilized to detect the enzyme activity. In order to determine the effect of JSH on the binding efficiency and viral penetration of SARS-CoV-2 variants, Calu-3 lung cells and Caco-2 colon cells were infected with fluorescent SARS-CoV-2 pseudo type lentiviruses. In addition, the effect of JSH (16.22 mg /mice/day and 48.66 mg/mice/day) on the viral load in SKH1J mice exposed to inhalation of luminescent SARS-CoV-2 variants for three days was determined. Results: The JSH was found to be effective in inhibiting the viral entry into Calu-3 and Caco-2 cells and in mice pre-treated with JSH for 3 days also inhibited the viral load exposed to different SARS-CoV-2 variants. Interestingly, JSH also decreased 3cL and RdRp activity thereby revealing the multi targeting nature of JSH and therefore will be a potential preventive SARS-CoV-2 infection.Conclusion: Taken together, our present results revealed that JSH could be a potential candidate for COVID-19 treatment.

Direct detection of SARS-CoV-2 antisense and sense genomic RNA in human saliva by semi-autonomous fluorescence in situ hybridization: A proxy for contagiousness?

Saliva is a matrix which may act as a vector for pathogen transmission and may serve as a possible proxy for SARS-CoV-2 contagiousness. Therefore, the possibility of detection of intracellular SARS-CoV-2 in saliva by means of fluorescence in situ hybridization is tested, utilizing probes targeting the antisense or sense genomic RNA of SARS-CoV-2. This method was applied in a pilot study with saliva samples collected from healthy persons and those presenting with mild or moderate COVID-19 symptoms. In all participants, saliva appeared a suitable matrix for the detection of SARS-CoV-2. Among the healthy, mild COVID-19-symptomatic and moderate COVID-19-symptomatic persons, 0%, 90% and 100% tested positive for SARS-CoV-2, respectively. Moreover, the procedure allows for simultaneous measurement of viral load (‘presence’, sense genomic SARS-CoV-2 RNA) and viral replication (‘activity’, antisense genomic SARS-CoV-2 RNA) and may yield qualitative results. In addition, the visualization of DNA in the cells in saliva provides an additional cytological context to the validity and interpretability of the test results. The method described in this pilot study may be a valuable diagnostic tool for detection of SARS-CoV-2, distinguishing between ‘presence’ (viral load) and ‘activity’ (viral replication) of the virus. Moreover, the method potentially gives more information about possible contagiousness.

Archaeal Orc1 protein interacts with T-rich single-stranded DNA

Objective The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study. Results Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction.

Population Bottlenecks and Intra-host Evolution During Human-to-Human Transmission of SARS-CoV-2

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.

Tuning cell behavior with nanoparticle shape

We investigated how the shape of polymeric vesicles, made by the exact same material, impacts the replication activity and metabolic state of both cancer and non-cancer cell types. First, we isolated discrete geometrical structures (spheres and tubes) from a heterogeneous sample using density-gradient centrifugation. Then, we characterized the cellular internalization and the kinetics of uptake of both types of polymersomes in different cell types (either cancer or non-cancer cells). We also investigated the cellular metabolic response as a function of the shape of the structures internalized and discovered that tubular vesicles induce a significant decrease in the replication activity of cancer cells compared to spherical vesicles. We related this effect to the significant up-regulation of the tumor suppressor genes p21 and p53 with a concomitant activation of caspase 3/7. Finally, we demonstrated that combining the intrinsic shape-dependent effects of tubes with the delivery of doxorubicin significantly increases the cytotoxicity of the system. Our results illustrate how the geometrical conformation of nanoparticles could impact cell behavior and how this could be tuned to create novel drug delivery systems tailored to specific biomedical application.

Kinetics and Fidelity of Psychrophilic DNA Polymerases

The discovery of extremophiles has enabled development of groundbreaking biotechnology. While most of the extremophile research is focused on thermophiles, organisms that adapt to living in cold temperature, known as psychrophiles, remain under-studied. We expressed and purified DNA polymerases PIPI and PIPB from Psychromonas ingrahamii, a psychrophile that can grow below water’s freezing temperature. We demonstrate that they have in vitro DNA replication activity at temperatures as low as −19°C. To our knowledge, this is the coolest DNA polymerization reaction ever carried out in a laboratory. In exploring the behavior of a variety of polymerases as a function of temperature, we found that reaction temperature substantially increases substitution and deletion error rates of both psychrophilic and mesophilic DNA polymerases. Motif analysis further reveals that the substitution error profiles cluster according to phylogenetic similarity of polymerases. Our results provide a useful reference for how reaction temperature, a crucial parameter of biochemistry, can affect the fidelity of DNA polymerases adapted to a wide range of environment.

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.Author summaryIn this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early- and late-replicating euchromatin

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes acrossSaccharomyces cerevisiaechromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.

P1815The impact of PVB19 presence in myocardium in recent onset dilated cardiomyopathy

Introduction The most common virus in myocardium of patients with recent onset dilated cardiomyopathy (RODCM) is PVB19. Opinions on its pathogenicity are different. According to some, viral load has to be evaluated and if it is low, the PVB19 has no potential to induce inflammation. Others recommend assessment of viral replication, while “innocent” are those cases where replication activity is low. Purpose To compare the development of echocardiographic parameters in patients with RODCM with isolated presence of PVB19 in low viral load (below 500 copies per μg nucleic acid) against a group in which no virus was detected in the myocardium. Patient and methods 243 patients with RODCM, in 151 cases (62%), the virus was detected in the myocardium (PCR+ group), in 92 cases the finding was negative (PCR−; ie 38%). In the PCR+ group, PVB19 was captured in 135 cases (89% of all PCR+), in 121 cases of them was PVB19 the only one isolated virus (PCRPVB19+; 80% of all PCR+). The viral presence was assessed by real time PCR. Results In the PCR− group, the left ventricular ejection fraction (LVEF) was 23.5±7.2%, in the PCRPVB19+ group 24.6±7.1% (p=0.26). Left ventricle end-diastolic diameter (LVEDD) in PCR− was 66.4±8.9mm vs 65.5±7.9mm in PCRPVB19+ (p=0.42), right ventricle diameter (RV) 33.1±5.7mm vs. 33.4±5.2mm (p=0.69), TAPSE 19.5±4.1mm vs 18.9±4.0mm (p=0.28) and E/é 14.0±5.7 vs. 14.9±7.0 (p=0.32). In the 12-month control, LVEF in the PCR− was 35.5±12.3% (p<0.001 for comparison with baseline), in PCRPVB19+ 35.3±12.3% (p<0.001 for comparison with baseline; p=0.92 for difference between groups); LVEDD in PCR− was 62.0±9.3mm vs 62.1±9.3mm in PCRPVB19+ (both p<0.001 for comparison with baseline, p=0.91 for difference between groups), RV 31.6±5.5mm vs. 31.6±5.4mm (p=0.033, resp. p=0.003 for comparison with baseline; p=0.98 for difference between groups), TAPSE 20.7±4.3mm vs. 20.7±4.4mm (p=0.026, resp. p<0.001 for comparison with baseline; p=0.98 for difference between groups), and E/e' 10.3±4.1 vs. 10.4±4.1 (both p<0.001 for comparison with baseline; p=0.93 for difference between groups). Conclusion PVB19 presence in myocardium had no effect on the development of echocardiographic parameters in RODCM patients in one-year follow-up. Acknowledgement/Funding Supported by Ministry of Health of Czech Republic AZV Grant 16-30537A

CrispR method for efficient tagging in S2 cells

Here we present a modified technique for improved efficient tagging in Drosophila S2 cells. it is a combination of a technique developed by Forstemann lab (Mainly homologous recombination design using pMH3, pMH4 and modified pMH to include UAS sequence and Myc tag) and Bassett lab for the sgRNA maufacture using pAc-sgRNA-Cas9 vector and following their protocol. Design of the homologous recombination templatenew vectors for Homologous recombination have also been engineered in order to get new tags: Replacement of GFP by excision of GFP from pMH3 using and replacement by UAS sequence or myc tag. UAS tags have been eficiently inserted at a specific location in the genome in order to analyse the binding of replication proteins and their effect on initiation of replication activity.