Live imaging of transcription sites using an elongating RNA polymerase II–specific probe

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody’s specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.

Doxycycline Inhibition of a Pseudotyped Virus Transduction Does Not Translate to Inhibition of SARS-CoV-2 Infectivity

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity

The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials.

Pre-Clinical Evaluation of the Nanoliposomal antiPCSK9 Vaccine in Healthy Non-Human Primates

Background: Our previous studies showed the safe preventive and therapeutic effects of immunization using the nanoliposomal antiPCSK9 vaccine called “Liposomal Immunogenic Fused PCSK9-Tetanus plus Alum adjuvant” (L-IFPTA), in mouse models of atherosclerosis. Here we aimed to ascertain the immunogenicity and safety of the L-IFPTA vaccine in a pre-clinical study in healthy non-human primates. Methods: Five male rhesus macaque monkeys were subcutaneously immunized with the L-IFPTA vaccine, four times with bi-weekly intervals. To evaluate immunogenicity, the plasma antiPCSK9 antibody in immunized monkeys was detected and quantified using the ELISA method. The functionality of the induced antiPCSK9 antibodies was determined by the PCSK9/LDLR in vitro binding assay kit. The safety of the vaccine was tested using the evaluation of several major circulating indicators including plasma lipid alterations, inflammatory biomarkers and organ injury biomarkers. Results: The resultant data indicated that the L-IFPTA vaccine significantly and highly induced the generation of functional and safe antiPCSK9 antibodies in immunized monkeys. Plasma levels of specific biomarkers indicating organ performance including creatinine, urea, uric acid, bilirubin, ALP, AS, ALT and TSH were not significantly altered. After immunization in healthy monkeys, non-prespecified endpoints (plasma levels of TC, LDL-C, VLDL-C and TG) were non-significantly reduced by 11.6 ± 36%; 16 ± 28%; 22 ± 53% and 24 ± 51%, respectively, while HDL-C was slightly increased by 2 ± 64%. There were also no significant changes in plasma levels of pro- and anti-inflammatory biomarkers. Conclusion: The L-IFPTA vaccine could efficiently stimulate the host humoral immune response to produce active antibodies that inhibit plasma PCSK9 while not provoking systemic inflammation and not adversely affecting organ performance.

Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing

Antisense oligonucleotide (ASO)-based therapy is one of the next-generation therapy, especially targeting neurological disorders. Many cases of ASO-dependent gene expression suppression have been reported. Recently, we developed a tocopherol conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO) as a new type of drug. Toc-HDO is more potent, stable, and efficiently taken up by the target tissues compared to the parental ASO. However, the detailed mechanisms of Toc-HDO, including its binding proteins, are unknown. Here, we developed native gel shift assays with fluorescence-labeled nucleic acids samples extracted from mice livers. These assays revealed two Toc-HDO binding proteins, annexin A5 (ANXA5) and carbonic anhydrase 8 (CA8). Later, we identified two more proteins, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and flap structure-specific endonuclease 1 (FEN1) by data mining. shRNA knockdown studies demonstrated that all four proteins regulated Toc-HDO activity in Hepa1–6, mouse hepatocellular cells. In vitro binding assays and fluorescence polarization assays with purified recombinant proteins characterized the identified proteins and pull-down assays with cell lysates demonstrated the protein binding to the Toc-HDO and ASO in a biological environment. Taken together, our findings provide a brand new molecular biological insight as well as future directions for HDO-based disease therapy.

Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription “factories” in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters.SummaryThe authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

Microtubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.