Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein

Aims: To study effects on cellular innate immune responses to novel genes ORF8 and ORF10, and the more conserved Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, either alone, or in combination with cannabidiol (CBD). Main Methods: HEK293 cells were transfected with a control plasmid, or plasmids expressing ORF8, ORF10, or M protein, and assayed for cell number and markers of apoptosis at 24 h, and expression of interferon and interferon-stimulated genes at 14 h. Key findings: A significant reduction in cell number, and increase in early and late apoptosis, was found after 24 h in cells where expression of viral genes was combined with 1-2 μM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. CBD (2 μM) augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2'-5'-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL, in cells expressing ORF8, ORF10, and M protein. CBD also augmented expression of these genes in control cells not expressing viral genes, without enhancing apoptosis. Significance: Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but suggest an augmented innate anti-viral response to these genes in the presence of CBD. Furthermore, our results indicate that CBD may prime components of the innate immune system, increasing readiness to respond to viral infection without activating apoptosis, and therefore could be studied for potential in prophylaxis.

New Histoplasma Diagnostic Assays Designed via Whole Genome Comparisons

Histoplasmosis is a systemic fungal disease caused by the pathogen Histoplasma spp. that results in significant morbidity and mortality in persons with HIV/AIDS and can also affect immunocompetent individuals. Although some PCR and antigen-detection assays have been developed, conventional diagnosis has largely relied on culture, which can take weeks. Our aim was to provide a proof of principle for rationally designing and standardizing PCR assays based on Histoplasma-specific genomic sequences. Via automated comparisons of aligned genome contigs/scaffolds and gene (sub)sequences, we identified protein-coding genes that are present in existing sequences of Histoplasma strains but not in other genera. Two of the genes, PPK and CFP4, were used for designing primer sets for conventional and real-time PCR assays. Both resulted in a 100% analytical specificity in vitro and detected 62/62 H. capsulatum isolates using purified DNA. We also obtained positive detections of 2/2 confirmed H. capsulatum clinical FFPE (formalin-fixed paraffin-embedded) samples using both primer sets. Positive control plasmid 10-fold serial dilutions confirmed the analytical sensitivity of the assays. The findings suggest that these novel primer sets should allow for detection sensitivity and reduce false positive results/cross-reactions. New assays for detecting pathogenic fungi, constructed along these lines, could be simple and affordable to implement.

Lead Drives Complex Dynamics of a Conjugative Plasmid in a Bacterial Community

Plasmids carrying metal resistance genes (MRGs) have been suggested to be key ecological players in the adaptation of metal-impacted microbial communities, making them promising drivers of bio-remediation processes. However, the impact of metals on plasmid-mediated spread of MRGs through selection, plasmid loss, and transfer is far from being fully understood. In the present study, we used two-member bacterial communities to test the impact of lead on the dispersal of the IncP plasmid pKJK5 from a Pseudomonas putida KT2440 plasmid donor and two distinct recipients, Variovorax paradoxus B4 or Delftia acidovorans SPH-1 after 4 and 10 days of mating. Two versions of the plasmid were used, carrying or not carrying the lead resistance pbrTRABCD operon, to assess the importance of fitness benefit and conjugative potential for the dispersal of the plasmid. The spread dynamics of metal resistance conveyed by the conjugative plasmid were dependent on the recipient and the lead concentration: For V. paradoxus, the pbr operon did not facilitate neither lead resistance nor variation in plasmid spread. The growth gain brought by the pbr operon to D. acidovorans SPH-1 and P. putida KT2440 at 1 mM Pb enhanced the spread of the plasmid. At 1.5 mM Pb after 4 days, the proteomics results revealed an oxidative stress response and an increased abundance of pKJK5-encoded conjugation and partitioning proteins, which most likely increased the transfer of the control plasmid to D. acidovorans SPH-1 and ensured plasmid maintenance. As a consequence, we observed an increased spread of pKJK5-gfp. Conversely, the pbr operon reduced the oxidative stress response and impeded the rise of conjugation- and partitioning-associated proteins, which slowed down the spread of the pbr carrying plasmid. Ultimately, when a fitness gain was recorded in the recipient strain, the spread of MRG-carrying plasmids was facilitated through positive selection at an intermediate metal concentration, while a high lead concentration induced oxidative stress with positive impacts on proteins encoding plasmid conjugation and partitioning.

The detection efficiency of digital PCR for the virulence genes of waterborne pathogenic bacteria

Waterborne pathogens are the primary concern for the safe reuse of wastewater. Although digital PCR (dPCR) is considered promising for absolutely quantitating genes, the detection efficiency of dPCR is affected by many factors. This study tested 8 virulence genes of pathogenic bacteria on a control plasmid and reclaimed water samples with reported primer-probe sets and designed ones on quantitative PCR (qPCR) and dPCR. Probe efficiency, data analysis, and PCR inhibition, were found to affect the detection efficiency of dPCR. Firstly, poor probe quality, which is determined by probe quenching and activation efficiencies, was the main cause of PCR failure. Secondly, even if the PCR was successful, the probe quality and signal intensity could still affect the quantitative process. Manual analysis of dPCR data on the weak signal intensity would significantly reduce errors. And lastly, the sensitivity of PCR inhibition was lower in dPCR than qPCR, but inhibition still existed. The dPCR produced various detection efficiencies for different targets in one sample indicating inconstant inhibitory effects. Dilution was still the proper approach to overcome inhibition, but decreased detection limit. More studies are required to ensure accurate waterborne pathogen quantitation by dPCR.

Endogenous 2μ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-copy system by insertion of the target DNA element and elimination of the original pE2μ plasmid. The resulting plasmid pE2μRAF1 and pE2μREP2 demonstrated higher copy number and slower loss rate than a pC2μ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2μRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2μ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2μ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2μRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2μ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2μ due to the more stable pE2μ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

Selenoprotein V Protects Against Oxidative Stress, Endoplasmic Reticulum Stress, and Apoptosis

Objectives Selenoprotein V (SELENOV) contains a thioredoxin-like fold and a conserved CxxU motif with a potential redox function. Three experiments were performed to assess its in vivo and in vitro roles and mechanisms in coping with different oxidant insults. Methods In Expt.1, SELENOV knockout (KO) and wildtype (WT) mice (male, 8-wk old) were given an IP injection of saline, diquat (DQ, 12.5 mg/kg), or acetaminophen (APAP, 300 mg/kg) (n = 10), and killed 5 h after the injection to collect liver and blood. In Expt. 2, primary hepatocytes were isolated from the 2 genotypes, cultured in complete Williams's medium E, and treated with DQ (0, 0.25 and 0.75 mM) and APAP (0, 1, 3, and 6 mM) for 12 h. In Expt. 3, 293 T cells were transfected with a control plasmid (GFP) or the plasmid containing Selenov gene (full length, OE) and treated with APAP (0, 1, 2, and 4 mM) for 24 h or H2O2 (0.1, 0.2, and 0.4 mM) for 12 h. Results In Expt. 1, the DQ and APAP injections caused greater (P < 0.05) rises in serum alanine aminotransferase activities, hepatic malondialdehyde (MDA) and carbonyl contents, endoplasmic reticulum (ER) stress-related proteins (BIP and CHOP), apoptosis-related proteins (FAK and caspase 9), and 3-nitrotyrosine, along with lower total anti-oxidizing-capability (T-AOC) and severer hepatocyte necrosis in the central lobular areas, in the KO than in the WT. In Expt. 2, the DQ and APAP treatments induced elevated (P < 0.05) cell death (20–40%), MDA contents (25–35%), and decreased (P < 0.05) T-AOC (50–65%) in the KO hepatocytes than in the WT cells. The KO hepatocytes treated with APAP displayed a sharp decline (P < 0.05) in cellular total respiration ability than the WT cells. In Expt. 3, the OE cells had greater viability and T-AOC and lower reactive oxygen species, MDA, and carbonyl contents after the APAP and H2O2 exposures (all at P < 0.05) than the controls. Moreover, the OE cells had greater (P < 0.05) redox enzyme activities (GPX, TrxR, and SOD), and lower (P < 0.05) expressions of ER stress-related genes (Atf4, Atf6, Bip, Xpp1t, Xbp1s, and Chop) and proteins (BIP, CHOP, FAK, caspase 9) than the controls after the treatment of H2O2 (0.4 mM). Conclusions Our data revealed the in vivo and in vitro roles and mechanisms of SELENOV in protecting against oxidative stress, ER stress, and apoptosis induced by pro-oxidants. Funding Sources This research is supported in part by an NSFC grant #31,320,103,920.

SAT-632 Involvement of NF-κB-p65 in BAG3 Regulation After Stress Stimuli

We previously identified the limb salvage QTL1 (LSQ-1) on mouse chromosome 7 as a locus that offered protection against ischemic injury following induction of hind limb ischemia (HLI) a model of experimental peripheral arterial disease (PAD) in mice. To better understand the role of the LSQ-1 locus in post ischemic adaptation we characterized several genes within this locus and identified a number of genes that were important in tissue adaptation to ischemia, including BCL2-associated athanogene 3 (BAG3). BAG3 is an anti-apoptotic protein that plays an important role in cell survival through the regulation of autophagy. BAG3 expression is induced in the gastrocnemius muscles of mice after hind limb ischemia but how ischemia regulates BAG3 expression is poorly understood. Additionally, the activation of NF-κB transcription factor is essential for cell survival after stress stimuli. We hypothesized that BAG3 upregulation following stress stimuli is regulated by NF-κB. We determined whether NF-κB is involved in BAG3-mediated survival of primary human skeletal muscle cells (HSMC) during ischemia and diabetic conditions. Within 6 hours of treatment, ischemia induced BAG3 mRNA (no ischemia vs ischemia: 1.0 ±0.09 vs 1.41±0.02; p<0.01) and protein expression (BAG3/total protein, no ischemia vs ischemia 1.0±0.01 vs 1.38±0.06; p<0.01). Knockdown of BAG3 expression by shRNA induced early cell damage in HMSC under ischemic conditions as measured by HMGB1 expression (HMGB1/total protein; control plasmid vs shRNA, 1.0±0.09 vs 1.71±0.04; p<0.01). Knockdown of p65 subunit of the NF-κB by shRNA significantly diminished BAG3 mRNA expression after ischemia (control plasmid vs shRNA: 2.11±0.18 vs 1.48±0.05; p<0.05). Moreover, treatment of HSMC with 750 uM palmitic acid (PA) and 100nM insulin for 3 days to mimic diabetic conditions significantly increased the expression of BAG3 mRNA (control vs PAL+Ins, 1.0±0.14 vs 2.27±0.08; p<0.01) and protein (BAG3/total protein, control vs PAL+Ins, 1.0±0.03 vs 1.39±0.11, p<0.01). Knocking down p65 attenuated these increase in BAG3 mRNA (PAL+Ins vs shRNA+PAL+Ins, 2.27±0.08 vs 1.56±0.02 p<0.01) and protein (BAG3/total protein; PAL+Ins vs shRNA+PAL+Ins, 1.39±0.11 vs 0.99±0.1; p<0.05) Thus, 1) BAG3 expression is important in cell survival under ischemic conditions, and 2) NF-kB plays a key role in upregulating the expression of BAG3 under diabetic and ischemic conditions.

The mechanism of hsa-miR-424-5 combining PD-1 through mTORC signaling pathway to stimulate immune effect and participate in Type 1 diabetes

In the present study, hsa-miR-424-5p mimic plasmid and hsa-mir-424-5p inhibitor plasmid were designed and injected into rats respectively, and miRNA control plasmid was also constructed. Models of Type 1 diabetes (T1D) were built. After successful modeling, the expression of hsa-miR-424-5p in lymphocytes was analyzed by RT-PCR. The expression of protein PD-1, T-bet, CXCR3, STING in Th1 lymphocytes and content of IGF-1 in islet tissue were analyzed by flow analysis. The protein levels of SHP2, Rheb, mTORC1, Rictor and Raptor in islet tissue were analyzed by Western blot. The results showed that hsa-miR-424-5p mimic group had the highest expression of hsa-miR-424-5p in lymphocytes. The expression of PD-1 was in hsa-miR-424-5p inhibitor > miRNA control > hsa-miR-424-5p mimic, while the expression of T-bet, CXCR3 and STING was in hsa-miR-424-5p mimic > miRNA control > hsa-miR-424-5p inhibitor. The expression of IGF-1 protein in hsa-miR-424-5p inhibitor group was the highest (32.08%) and hardly expressed in hsa-miR-424-5p mimic group (2.36%). The expression of SHP2, Rheb, mTORC1, Rictor and Raptor of insulin histoproteins were in hsa-miR-424-5p mimic group > miRNA control of > hsa-miR-424-5p inhibitor group, with statistical differences. It indicates that hsa-miR-424-5p binding PD-1 signaling molecules can stimulate the immune effect through the mTORC signaling pathway and participates in the pathogenesis of T1D.

Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer

Background: The molecular mechanisms of ovarian cancer (OC) remain unclear. We sought to comprehensively identify miRNAs that are aberrantly expressed in OC. Methods: Differentially expressed miRNAs were screened from six pairs of primary and metastatic OC tissues; their possible functions were then analyzed and target genes were predicted by bioinformatics. Then gene expression profiling results were established by reverse transcription quantitative polymerase chain reaction and western blot. Target binding between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Results: Fifteen miRNAs and 10 target mRNAs were differentially expressed in primary and metastatic OC tissues. ITGB3, TGFβ2 and TNC correlated to miRNA function in metastatic OC. Compared with primary OC, RNA levels of hsa-miR-141-3p, hsa-miR-7-5p and hsa-miR-187-5p in metastatic OC tissues were potently decreased ( p < 0.05). However, a statistically prominent difference in hsa-miR-584-5p level between the two groups ( p > 0.05) was not observed. Comparing to primary OC, TGFβ2 and TNC were markedly increased ( p < 0.05). Luciferase activity was remarkably decreased after co-transfection of a wild-type TGFβ2 3’-UTR plasmid and miR-7-5p compared with a control plasmid, but no remarkable change after co-transfection of mutant TGFβ2 3’-UTR and miR-7-5p was demonstrated. Conclusions: Fifteen miRNAs and 10 mRNAs were differentially expressed in metastatic OC tissues compared with primary OC tissues, which suggested that they may participate in invasive and metastatic processes. Hsa-miR-141-3p, hsa-miR-187-5p and hsa-miR-7-5p expression was prominently lower in metastatic OC than in primary OC, while TGFβ2 and TNC expression was markedly higher in metastatic OC tissues. Hsa-miR-7-5p may bind to the TGFβ2 3’-UTR to inhibit its expression.

USP22 promotes pro-inflammatory responses in Pseudomonas aeruginosa-induced keratitis by targeting TRAF6

Background Pseudomonas aeruginosa (PA)-induced keratitis is characterized by inflammatory epithelial edema, stromal infiltration, corneal ulceration, and can lead to vision loss. In the present study, we aim to study the effect of ubiquitin-specific protease 22 (USP22) on PA-induced keratitis. Methods Lentivirus containing control plasmid or shRNA targeting USP22 were used to silence the expression of USP22 in mouse corneas and cultured RAW264.7 cells, the inflammatory processes were detected. Results We found the expression level of USP22 was significantly increased in both mouse corneas and cultured RAW264.7 cells after PA stimulation. In addition, we observed that silencing of USP22 attenuate disease progression, downregulate NF-κB pathway and suppressed the expression of pro-inflammatory cytokines after PA stimulation. Most importantly, we found the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was decreased by silencing of USP22, and USP22 was found to remove K48-linked poly-ubiquitination chains from TRAF6 to stabilize TRAF6 expression after PA infection. Conclusion This data indicated USP22 as a positive regulator of pro-inflammatory responses in PA induced keratitis.