Constitutive Expression Levels Research Articles

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

Does Constitutive Expression of Defense-Related Genes and Salicylic Acid Concentrations Correlate with Field Resistance of Potato to Black Scurf Disease?

Black scurf disease on potato caused by Rhizoctonia solani AG3 occurs worldwide and is difficult to control. The use of potato cultivars resistant to black scurf disease could be part of an integrated control strategy. Currently, the degree of resistance is based on symptom assessment in the field, but molecular measures could provide a more efficient screening method. We hypothesized that the degree of field resistance to black scurf disease in potato cultivars is associated with defense-related gene expression levels and salicylic acid (SA) concentration. Cultivars with a moderate and severe appearance of disease symptoms on tubers were selected and cultivated in the same field. In addition, experiments were conducted under controlled conditions in an axenic in vitro culture and in a sand culture to analyze the constitutive expression of defense-related genes and SA concentration. The more resistant cultivars did not show significantly higher constitutive expression levels of defense-related genes. Moreover, the level of free SA was increased in the more resistant cultivars only in the roots of the plantlets grown in the sand culture. These results indicate that neither expression levels of defense-related genes nor the amount of SA in potato plants can be used as reliable predictors of the field resistance of potato genotypes to black scurf disease.

DMD-Associated Dilated Cardiomyopathy: Genotypes, Phenotypes, and Phenocopies.

Variants in the DMD gene, that encodes the cytoskeletal protein, dystrophin, cause a severe form of dilated cardiomyopathy (DCM) associated with high rates of heart failure, heart transplantation, and ventricular arrhythmias. Improved early detection of individuals at risk is needed. Genetic testing of 40 male probands with a potential X-linked genetic cause of primary DCM was undertaken using multi-gene panel sequencing, multiplex polymerase chain reaction, and array comparative genomic hybridization. Variant location was assessed with respect to dystrophin isoform patterns and exon usage. Telomere length was evaluated as a marker of myocardial dysfunction in left ventricular tissue and blood. Four pathogenic/likely pathogenic DMD variants were found in 5 probands (5/40: 12.5%). Only one rare variant was identified by gene panel testing with 3 additional multi-exon deletion/duplications found following targeted assays for structural variants. All of the pathogenic/likely pathogenic DMD variants involved dystrophin exons that had percent spliced-in scores >90, indicating high levels of constitutive expression in the human adult heart. Fifteen DMD variant-negative probands (15/40: 37.5%) had variants in autosomal genes including TTN, BAG3, LMNA, and RBM20. Myocardial telomere length was reduced in patients with DCM irrespective of genotype. No differences in blood telomere length were observed between genotype-positive family members with/without DCM and controls. Primary genetic testing using multi-gene panels has a low yield and specific assays for structural variants are required if DMD-associated cardiomyopathy is suspected. Distinguishing X-linked causes of DCM from autosomal genes that show sex differences in clinical presentation is crucial for informed family management.

HSP90β prevents aging-related cataract formation through regulation of the charged multivesicular body protein (CHMP4B) and p53

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90β (HSP90β) plays a fundamental role in suppressing cataractogenesis. HSP90β is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90β is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90β silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90β can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90β silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90β and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90β is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

TUMOR CELL PANEL WITH CHARACTERIZED EXPRESSION OF PD-L1 FOR PRECLINICAL STUDIES OF ANTICANCER DRUGS AND IMMUNE CHECKPOINT INHIBITORS INTERACTION

PD-L1 (Programmed death-ligand 1), a membrane protein of the immunoglobulin superfamily, is one of the targets for cancer immunotherapy. A panel of 14 cancer cell cultures with different constitutive PD-L1 expression level was formed and characterized. The panel is recommended for preclinical studies of a cytostatic drug effect on PD-L1 expression and for predicting the ef cacy of their combination with immune checkpoint inhibitors.

Evolution and expression analysis of the caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene family in jute (Corchorus L.)

BackgroundJute is considered one of the most important crops for fiber production and multipurpose usages. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a crucial enzyme involved in lignin biosynthesis in plants. The potential functions of CCoAOMT in lignin biosynthesis of jute have been reported in several studies. However, little is known about the evolution of the CCoAOMT gene family, and either their expression level at different developing stages in different jute cultivars, as well as under abiotic stresses including salt and drought stress.ResultsIn the present study, 66 CCoAOMT genes from 12 species including 12 and eight CCoAOMTs in Corchorus olitorius and C. capsularis were identified. Phylogenetic analysis revealed that CCoAOMTs could be divided into six groups, and gene expansion was observed in C. olitorius. Furthermore, gene expression analysis of developing jute fibers was conducted at different developmental stages (15, 30, 45, 60, and 90 days after sowing [DAS]) in six varieties (Jute-179 [J179], Lubinyuanguo [LB], and Qiongyueqing [QY] for C. capsularis; Funong No.5 [F5], Kuanyechangguo [KY], and Cvlv [CL] for C. olitorius). The results showed that CCoAOMT1 and CCoAOMT2 were the dominant genes in the CCoAOMT family. Of these two dominant CCoAOMTs, CCoAOMT2 showed a constitutive expression level during the entire growth stages, while CCoAOMT1 exhibited differential expression patterns. These two genes showed higher expression levels in C. olitorius than in C. capsularis. The correlation between lignin content and CCoAOMT gene expression levels indicated that this gene family influences the lignin content of jute. Using real-time quantitative reverse transcription PCR (qRT-PCR), a substantial up-regulation of CCoAOMTs was detected in stem tissues of jute 24 h after drought treatment, with an up to 17-fold increase in expression compared to that of untreated plants.ConclusionsThis study provides a basis for comprehensive genomic studies of the entire CCoAOMT gene family in C. capsularis and C. olitorius. Comparative genomics analysis among the CCoAOMT gene families of 12 species revealed the close evolutionary relationship among Corchorus, Theobroma cacao and Gossypium raimondii. This study also shows that CCoAOMTs are not only involved in lignin biosynthesis, but also are associated with the abiotic stress response in jute, and suggests the potential use of these lignin-related genes to genetically improve the fiber quality of jute.

Resistance risk assessment of Fusarium pseudograminearum from wheat to prothioconazole

Fusarium crown rot (FCR), primarily caused by Fusarium pseudograminearum, poses significant threats to cereal crops worldwide. Prothioconazole is a demethylation inhibitor (DMI) fungicide used to control FCR. However, the risk of resistance in F. pseudograminearum to prothioconazole has not yet been evaluated. In this study, the sensitivity of a total of 255 F. pseudograminearum strains obtained from Henan Province, China to prothioconazole were determined by the mycelial growth inhibition. The results showed that the effective concentration to 50% growth inhibition (EC50) of these strains ranged from 0.4228 μg/mL to 2.5284 μg/mL, with a mean EC50 value of 1.0692 ± 0.4527 μg/mL (mean ± SD). Thirty prothioconazole-resistant mutants were obtained out of six selected sensitive parental strains by means of fungicide taming. The resistant mutants exhibited defects in vegetative growth, conidia production, and pathogenicity on wheat seedlings compared to their parental strains. Under ion, cell wall, and temperature stress conditions but not osmotic stress, all the mutants exhibited decreased growth rates compared with their parental strains, which was consistent with the control treatment. Cross-resistance test showed that there was a cross-resistance relationship between prothioconazole and four DMI fungicides, including prochloraz, metconazole, tebuconazole and hexaconazole, but no cross-resistance was observed between prothioconazole and carbendazim, phenamacril, fludioxonil, or azoxystrobin. Although no site mutation occurred on Cyp51a and Cyp51b genes, the constitutive expression level of the Cyp51a gene was significantly increased in all mutants. After being treated with prothioconazole, the Cyp51a and Cyp51b genes were significantly increased in both the resistant mutants and their parents. These results suggested that the resistance to prothioconazole of the mutants may be attributed to the changes of the relative expression level of Cyp51a and Cyp51b genes. Taken together, these results could provide a theoretical basis for the scientific use of prothioconazole in the field and fungicide resistance management strategies.

VanG- and D-Ala-D-Ser-dependent peptidoglycan synthesis and vancomycin resistance in Clostridioides difficile.

A Clostridioides difficile strain deficient in the ddl gene is unable to synthesize the dipeptide D-Ala-D-Ala, an essential component of peptidoglycan and the target of vancomycin. We isolated spontaneous suppressors of a ∆ddl mutation that allowed cell growth in the absence of D-Ala-D-Ala. The mutations caused constitutive or partly constitutive expression of the vancomycin-inducible vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The mutations mapped to the vanS or vanR genes, which regulate expression of the vanG operon. The constitutive level of vanG expression was about 10-fold above that obtained by vancomycin induction. The incorporation of D-Ala-D-Ser into peptidoglycan due to high expression of the vanG operon conferred only low-level resistance to vancomycin, but VanG was found to synthesize D-Ala-D-Ala in addition to D-Ala-D-Ser. However, the same, low resistance to vancomycin was also observed in cells completely unable to synthesize D-Ala-D-Ala and grown in the presence of D-Ala-D-Ser. D-Ala-D-Ala presence was required for efficient vancomycin induction of the vanG operon showing that vancomycin is not by itself able to activate VanS. D-Ala-D-Ser, similar to D-Ala-D-Ala, served as an anti-activator of DdlR, the positive regulator of the ddl gene, thereby coupling vanG and ddl expression.

Effect of Different Tolerable Levels of Constitutive mcr-1 Expression on Escherichia coli.

ABSTRACTTo study the effect of different tolerable levels of constitutive mcr-1 expression on Escherichia coli, and to provide direct evidence for moderate resistance mediated by mcr-1, construction of E. coli strains carrying mcr-1 on the chromosome with promoters of different strengths was conducted using λ-red recombination. Our results demonstrated that over-high expression of mcr-1 cannot be tolerated, and seven constructs with more than 200-fold mcr-1 transcriptional expression differences were obtained. The colistin MICs of the seven strains increased with the increase of MCR-1 levels, and the highest MIC was 8 μg/mL. Lower expression of mcr-1 didn’t demonstrate many effects on bacteria, while higher tolerable expression of mcr-1 tended to show fitness costs in growth rate, competitive ability, and cell structures, but no obvious change of virulence was observed in mice. Bacteria demonstrated colistin MICs of 4–8 μg/mL at mcr-1 expression levels similar to clinical isolates, which were the mcr-1 expression levels with relatively lower fitness costs.IMPORTANCE The effects of relatively lower tolerable levels of mcr-1 were not evaluated thoroughly, and direct evidence for moderate resistance mediated by mcr-1 was lacking. In the present study, we made constructs carrying mcr-1 on the E. coli K12 chromosome under the control of serial constitutive promoters of different strengths and studied the effects of different tolerable levels of mcr-1 expression in vitro and in vivo. The results demonstrated that generally, except QH0007 (the construct with the highest mcr-1 expression that showed some extent of cell death), the fitness costs of tolerable mcr-1 expression on bacteria were not apparent or low. Bacteria demonstrated colistin MICs of 4–8 μg/mL at mcr-1 expression levels similar to clinical isolates, which corresponded to the lower levels of mcr-1 expression that can lead to colistin resistance, indicating the cleverness of bacteria to balance the benefit and cost of MCR-1-mediated colistin resistance.

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.

Molecular characterization of thioester-containing proteins in Biomphalaria glabrata and their differential gene expression upon Schistosoma mansoni exposure.

Schistosomiasis is a disease caused by trematode parasites of the genus Schistosoma that affects approximately 200 million people worldwide. Schistosomiasis has been a persistent problem in endemic areas as there is no vaccine available, currently used anti-helmintic medications do not prevent reinfection, and most concerning, drug resistance has been documented in laboratory and field isolates. Thus, alternative approaches to curtail this human disease are warranted. Understanding the immunobiology of the obligate intermediate host of these parasites, which include the freshwater snail Biomphalaria glabrata, may facilitate the development of novel methods to stop or reduce transmission to humans. Molecules from the thioester-containing protein (TEP) superfamily have been shown to be involved in immunological functions in many animals including corals and humans. In this study we identified, characterized, and compared TEP transcripts and their expression upon S. mansoni exposure in resistant and susceptible strains of B. glabrata snails. Results showed the expression of 11 unique TEPs in B. glabrata snails. These transcripts present high sequence identity at the nucleotide and putative amino acid levels between susceptible and resistant strains. Further analysis revealed differences in several TEPs’ constitutive expression levels between resistant and susceptible snail strains, with C3-1, C3-3, and CD109 having higher constitutive expression levels in the resistant (BS90) strain, whereas C3-2 and TEP-1 showed higher constitutive expression levels in the susceptible (NMRI) strain. Furthermore, TEP-specific response to S. mansoni miracidia exposure reiterated their differential expression, with resistant snails upregulating the expression of both TEP-4 and TEP-3 at 2 h and 48 h post-exposure, respectively. Further understanding the diverse TEP genes and their functions in invertebrate animal vectors will not only expand our knowledge in regard to this ancient family of immune proteins, but also offer the opportunity to identify novel molecular targets that could aid in the efforts to develop control methods to reduce schistosomiasis transmission.

Predicting bacterial promoter function and evolution from random sequences.

Predicting function from sequence is a central problem of biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wildtype sequence rather than globally from any sequence. Using random mutant libraries, we developed a biophysical model that accounts for multiple features of σ70 binding bacterial promoters to predict constitutive gene expression levels from any sequence. We experimentally and theoretically estimated that 10-20% of random sequences lead to expression and ~80% of non-expressing sequences are one mutation away from a functional promoter. The potential for generating expression from random sequences is so pervasive that selection acts against σ70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. This pervasiveness of σ70-binding sites implies that emergence of promoters is not the limiting step in gene regulatory evolution. Ultimately, the inclusion of novel features of promoter function into a mechanistic model enabled not only more accurate predictions of gene expression levels, but also identified that promoters evolve more rapidly than previously thought.

Evaluation of cancer immunotherapy using mini-tumor chips.

Rationale: Predicting tumor responses to adjuvant therapies can potentially help guide treatment decisions and improve patient survival. Currently, tumor pathology, histology, and molecular profiles are being integrated into personalized profiles to guide therapeutic decisions. However, it remains a grand challenge to evaluate tumor responses to immunotherapy for personalized medicine.Methods: We present a microfluidics-based mini-tumor chip approach to predict tumor responses to cancer immunotherapy in a preclinical model. By uniformly infusing dissociated tumor cells into isolated microfluidic well-arrays, 960 mini-tumors could be uniformly generated on-chip, with each well representing the ex vivo tumor niche that preserves the original tumor cell composition and dynamic cell-cell interactions and autocrine/paracrine cytokines.Results: By incorporating time-lapse live-cell imaging, our mini-tumor chip allows the investigation of dynamic immune-tumor interactions as well as their responses to cancer immunotherapy (e.g., anti-PD1 treatment) in parallel within 36 hours. Additionally, by establishing orthotopic breast tumor models with constitutive differential PD-L1 expression levels, we showed that the on-chip interrogation of the primary tumor's responses to anti-PD1 as early as 10 days post tumor inoculation could predict the in vivo tumors' responses to anti-PD1 at the endpoint of day 24. We also demonstrated the application of this mini-tumor chip to interrogate on-chip responses of primary tumor cells isolated from primary human breast and renal tumor tissues.Conclusions: Our approach provides a simple, quick-turnaround solution to measure tumor responses to cancer immunotherapy.

PD-L1 sustains chronic, cancer cell–intrinsic responses to type I interferon, enhancing resistance to DNA damage

Programmed death ligand 1 (PD-L1), an immune-checkpoint protein expressed on cancer cells, also functions independently of the immune system. We found that PD-L1 inhibits the killing of cancer cells in response to DNA damage in an immune-independent manner by suppressing their acute response to type I interferon (IFN; IFN-I). In addition, PD-L1 plays a critical role in sustaining high levels of constitutive expression in cancer cells of a subset of IFN-induced genes, the IFN-related DNA damage resistance signature (IRDS) which, paradoxically, protects cancer cells. The cyclic GMP-AMP synthase-stimulator of the IFN genes (cGAS-STING) pathway is constitutively activated in a subset of cancer cells in the presence of high levels of PD-L1, thus leading to a constitutive, low level of IFN-β expression, which in turn increases IRDS expression. The constitutive low level of IFN-β expression is critical for the survival of cancer cells addicted to self-produced IFN-β. Our study reveals immune-independent functions of PD-L1 that inhibit cytotoxic acute responses to IFN-I and promote protective IRDS expression by supporting protective chronic IFN-I responses, both of which enhance the resistance of cancer cells to DNA damage.

Differential Yet Integral Contributions of Nrf1 and Nrf2 in the Human HepG2 Cells on Antioxidant Cytoprotective Response against Tert-Butylhydroquinone as a Pro-Oxidative Stressor.

In the past 25 years, Nrf2 (nuclear factor erythroid 2-related factor 2, also called NFE2L2) had been preferentially parsed as a master hub of regulating antioxidant, detoxification, and cytoprotective genes; albeit as a matter of fact that Nrf1 (nuclear factor erythroid 2-related factor 1, also called NFE2L1)—rather than Nrf2—is indispensable for cell homeostasis and organ integrity during normal growth and development. Herein, distinct genotypic cell lines (i.e., Nrf1α−/−, Nrf2−/−ΔTA, and caNrf2ΔN) are employed to determine differential yet integral roles of Nrf1 and Nrf2 in mediating antioxidant responsive genes to tert-butylhydroquinone (tBHQ) serving as a pro-oxidative stressor. In Nrf1α−/− cells, Nrf2 was highly accumulated but also could not fully compensate specific loss of Nrf1α’s function in its basal cytoprotective response against endogenous oxidative stress, though it exerted partially inducible antioxidant response, as the hormetic effect of tBHQ, against apoptotic damages. By contrast, Nrf2−/−ΔTA cells gave rise to a substantial reduction of Nrf1 in both basal and tBHQ-stimulated expression levels and hence resulted in obvious oxidative stress, but it can still be allowed to mediate a potent antioxidant response, as accompanied by a significantly decreased ratio of GSSG (oxidized glutathione) to GSH (reduced glutathione). Conversely, a remarkable increase of Nrf1 expression resulted from the constitutive active caNrf2ΔN cells, which were not manifested with oxidative stress, whether or not it was intervened with tBHQ. Such inter-regulatory effects of Nrf1 and Nrf2 on the antioxidant and detoxification genes (encoding HO-1, NQO1, GCLC, GCLM, GSR, GPX1, TALDO, MT1E, and MT2), as well on the ROS (reactive oxygen species)-scavenging activities of SOD (superoxide dismutase) and CAT (catalase), were further investigated. The collective results unraveled that Nrf1 and Nrf2 make distinctive yet cooperative contributions to finely tuning basal constitutive and/or tBHQ-inducible expression levels of antioxidant cytoprotective genes in the inter-regulatory networks. Overall, Nrf1 acts as a brake control for Nrf2’s functionality to be confined within a certain extent, whilst its transcription is regulated by Nrf2.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

Traces of SARS-CoV-2 RNA in Peripheral Blood Cells of Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus that caused coronavirus-related outbreaks over the past 20 years. The outbreak was first reported in December 2019 in Wuhan, China, but rapidly progressed into a pandemic of an unprecedented scale since the 1918 flu pandemic. Besides respiratory complications in patients with COVID-19, clinical characterization of severe infection cases showed several other comorbidities, including multiple organ failure, and septic shock. To better understand the systemic pathogenesis of COVID-19, we interrogated the virus's presence in the peripheral blood cells, which might provide a form of trafficking or hiding to the virus. By analyzing >2 billion sequence reads of high-throughput transcriptome sequence data from 180 samples of patients with active SARS-CoV-2 infection or healthy controls collected from 6 studies, we found evidence of traces of SARS-CoV-2 RNA in peripheral blood mononuclear cells in two samples from two independent studies. In contrast, the viral RNA was abundant in bronchoalveolar lavage specimens from the same patients. We also devised a “viral spike-to-actin” RNA normalization as a metric to compare across various samples and minimize errors caused by intersample variability in total human RNA abundance. Our observation suggests immune presentation and discounts the possibility of extensive viral infection of lymphocytes or monocytes.

ACE2 interaction with cytoplasmic PDZ protein enhances SARS-CoV-2 invasion

SummarySARS-CoV-2 is responsible for the global COVID-19 pandemic. Angiotensin converting enzyme 2 (ACE2) is the membrane-delimited receptor for SARS-CoV-2. Lung, intestine, and kidney, major sites of viral infection, express ACE2 that harbors an intracellular, carboxy-terminal PDZ-recognition motif. These organs prominently express the PDZ protein Na+/H+ exchanger regulatory factor-1 (NHERF1). Here, we report NHERF1 tethers ACE2 and augments SARS-CoV-2 cell entry. ACE2 directly binds both NHERF1 PDZ domains. Disruption of either NHERF1 PDZ core-binding motif or the ACE2 PDZ recognition sequence eliminates interaction. Proximity ligation assays establish that ACE2 and NHERF1 interact at constitutive expression levels in human lung and intestine cells. Ablating ACE2 interaction with NHERF1 accelerated SARS-CoV-2 cell entry. Conversely, elimination of the ACE2 C-terminal PDZ-binding motif decreased ACE2 membrane residence and reduced pseudotyped virus entry. We conclude that the PDZ interaction of ACE2 with NHERF1 facilitates SARS-CoV-2 internalization. β-Arrestin is likely indispensable, as with G protein-coupled receptors.

Herbivore‐induced plant volatiles mediate defense regulation in maize leaves but not in maize roots

Plant leaves that are exposed to herbivore‐induced plant volatiles (HIPVs) respond by increasing their defenses, a phenomenon referred to as priming. Whether this phenomenon also occurs in the roots is unknown. Using maize plants, Zea mays, whose leaves respond strongly to leaf HIPVs, we measured the impact of belowground HIPVs, emanating from roots infested by the banded cucumber beetle, Diabrotica balteata, on constitutive and herbivore‐induced levels of defense‐related gene expression, phytohormones, volatile and non‐volatile primary and secondary metabolites, growth and herbivore resistance in roots of neighbouring plants. HIPV exposure did not increase constitutive or induced levels of any of the measured root traits. Furthermore, HIPV exposure did not reduce the performance or survival of D. balteata on maize or its ancestor teosinte. Cross‐exposure experiments between HIPVs from roots and leaves revealed that maize roots, in contrast to maize leaves, neither emit nor respond strongly to defense‐regulating HIPVs. Together, these results demonstrate that volatile‐mediated defense regulation is restricted to the leaves of maize. This finding is in line with the lower diffusibility of volatiles in the soil and the availability of other, potentially more efficient, information conduits below ground.

Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically β1, β2, and β5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits β1, β2, and β5 can be substituted by the immune βli, β2i, and β5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.