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Heliyon Jun 2024Anti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and...
BACKGROUND
Anti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CL) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors .
OBJECTIVE
We aimed to evaluate two active compounds, AM and GM, from for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CL and ACE2 neutralization were major targets and explored whether any additional targets existed.
METHODS
We tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CL and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, 3CL activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA).
RESULTS
AM and GM reduced SARS-CoV-2 3CL and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC of 0.70-3.05 μM, in which wild-type B was the most susceptible strain (EC 0.70-0.79 μM). AM was slightly more efficient in the variants (EC 0.88-2.41 μM), resulting in higher selectivity indices (SI 3.65-10.05), compared to the GM (EC 0.94-3.05 μM, SI 1.66-5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CL activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM.
CONCLUSION
AM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system.
PubMed: 38867992
DOI: 10.1016/j.heliyon.2024.e31987 -
Translational Psychiatry Jun 2024Depression is a prevalent and incapacitating condition with a significant impact on global morbidity and mortality. Although the immune system's role in its pathogenesis...
Depression is a prevalent and incapacitating condition with a significant impact on global morbidity and mortality. Although the immune system's role in its pathogenesis is increasingly recognized, there is a lack of comprehensive understanding regarding the involvement of innate and adaptive immune cells. To address this gap, we conducted a multicenter case-control study involving 121 participants matched for sex and age. These participants had either an active (or current) major depressive episode (MDE) (39 cases) or a remitted MDE (40 cases), including individuals with major depressive disorder or bipolar disorder. We compared these 79 patients to 42 healthy controls (HC), analyzing their immunological profiles. In blood samples, we determined the complete cell count and the monocyte subtypes and lymphocyte T-cell populations using flow cytometry. Additionally, we measured a panel of cytokines, chemokines, and neurotrophic factors in the plasma. Compared with HC, people endorsing a current MDE showed monocytosis (p = 0.001), increased high-sensitivity C-reactive protein (p = 0.002), and erythrocyte sedimentation rate (p = 0.003), and an altered proportion of specific monocyte subsets. CD4 lymphocytes presented increased median percentages of activation markers CD69 (p = 0.007) and exhaustion markers PD1 (p = 0.013) and LAG3 (p = 0.014), as well as a higher frequency of CD4CD25FOXP3 regulatory T cells (p = 0.003). Additionally, patients showed increased plasma levels of sTREM2 (p = 0.0089). These changes are more likely state markers, indicating the presence of an ongoing inflammatory response during an active MDE. The Random Forest model achieved remarkable classification accuracies of 83.8% for MDE vs. HC and 70% for differentiating active and remitted MDE. Interestingly, the cluster analysis identified three distinct immunological profiles among MDE patients. Cluster 1 has the highest number of leukocytes, mainly given by the increment in lymphocyte count and the lowest proinflammatory cytokine levels. Cluster 3 displayed the most robust inflammatory pattern, with high levels of TNFα, CX3CL1, IL-12p70, IL-17A, IL-23, and IL-33, associated with the highest level of IL-10, as well as β-NGF and the lowest level for BDNF. This profile is also associated with the highest absolute number and percentage of circulating monocytes and the lowest absolute number and percentage of circulating lymphocytes, denoting an active inflammatory process. Cluster 2 has some cardinal signs of more acute inflammation, such as elevated levels of CCL2 and increased levels of proinflammatory cytokines such as IL-1β, IFNγ, and CXCL8. Similarly, the absolute number of monocytes is closer to a HC value, as well as the percentage of lymphocytes, suggesting a possible initiation of the inflammatory process. The study provides new insights into the immune system's role in MDE, paving the ground for replication prospective studies targeting the development of diagnostic and prognostic tools and new therapeutic targets.
Topics: Humans; Female; Male; Case-Control Studies; Depressive Disorder, Major; Adult; Immunophenotyping; Middle Aged; Cytokines; Monocytes; Bipolar Disorder; Inflammation; Antigens, CD; Flow Cytometry
PubMed: 38866753
DOI: 10.1038/s41398-024-02902-2 -
Cell Death & Disease Jun 2024Full-length p53 (p53α) plays a pivotal role in maintaining genomic integrity and preventing tumor development. Over the years, p53 was found to exist in various... (Review)
Review
Full-length p53 (p53α) plays a pivotal role in maintaining genomic integrity and preventing tumor development. Over the years, p53 was found to exist in various isoforms, which are generated through alternative splicing, alternative initiation of translation, and internal ribosome entry site. p53 isoforms, either C-terminally altered or N-terminally truncated, exhibit distinct biological roles compared to p53α, and have significant implications for tumor development and therapy resistance. Due to a lack of part and/or complete C- or N-terminal domains, ectopic expression of some p53 isoforms failed to induce expression of canonical transcriptional targets of p53α like CDKN1A or MDM2, even though they may bind their promoters. Yet, p53 isoforms like Δ40p53α still activate subsets of targets including MDM2 and BAX. Furthermore, certain p53 isoforms transactivate even novel targets compared to p53α. More recently, non-canonical functions of p53α in DNA repair and of different isoforms in DNA replication unrelated to transcriptional activities were discovered, amplifying the potential of p53 as a master regulator of physiological and tumor suppressor functions in human cells. Both regarding canonical and non-canonical functions, alternative p53 isoforms frequently exert dominant negative effects on p53α and its partners, which is modified by the relative isoform levels. Underlying mechanisms include hetero-oligomerization, changes in subcellular localization, and aggregation. These processes ultimately influence the net activities of p53α and give rise to diverse cellular outcomes. Biological roles of p53 isoforms have implications for tumor development and cancer therapy resistance. Dysregulated expression of isoforms has been observed in various cancer types and is associated with different clinical outcomes. In conclusion, p53 isoforms have expanded our understanding of the complex regulatory network involving p53 in tumors. Unraveling the mechanisms underlying the biological roles of p53 isoforms provides new avenues for studies aiming at a better understanding of tumor development and developing therapeutic interventions to overcome resistance.
Topics: Humans; Tumor Suppressor Protein p53; Protein Isoforms; Neoplasms; Animals; Drug Resistance, Neoplasm; Proto-Oncogene Proteins c-mdm2
PubMed: 38866752
DOI: 10.1038/s41419-024-06783-7 -
The Journal of Cell Biology Aug 2024CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4...
CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4 or DRF1 for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated isogenic cell lines deficient in either DBF4 or DRF1: these cells are viable but present signs of genomic instability, indicating that both can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, partial deficiency in MCM helicase phosphorylation, and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not on DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, mediating most of its functions in unperturbed DNA replication and upon replication interference.
Topics: DNA Replication; Humans; Cell Cycle Proteins; Protein Serine-Threonine Kinases; Phosphorylation; Genomic Instability; Adaptor Proteins, Signal Transducing; DNA-Binding Proteins
PubMed: 38865090
DOI: 10.1083/jcb.202402144 -
Antimicrobial Agents and Chemotherapy Jun 2024Islatravir (ISL) is a deoxyadenosine analog that inhibits HIV-1 reverse transcription by multiple mechanisms. Lenacapavir (LEN) is a novel capsid inhibitor that inhibits...
Islatravir (ISL) is a deoxyadenosine analog that inhibits HIV-1 reverse transcription by multiple mechanisms. Lenacapavir (LEN) is a novel capsid inhibitor that inhibits HIV-1 at multiple stages throughout the viral life cycle. ISL and LEN are being investigated as once-weekly combination oral therapy for the treatment of HIV-1. Here, we characterized ISL and LEN to assess combinatorial antiviral activity, cytotoxicity, and the potential for interactions between the two compounds. Bliss analysis revealed ISL with LEN demonstrated additive inhibition of HIV-1 replication, with no evidence of antagonism across the range of concentrations tested. ISL exhibited potent antiviral activity against variants encoding known LEN resistance-associated mutations (RAMs) with or without the presence of M184V, an ISL RAM in reverse transcriptase (RT) . Static resistance selection experiments were conducted with ISL and LEN alone and in combination, initiating with either wild-type virus or virus containing the M184I RAM in RT to further assess their barrier to the emergence of resistance. The combination of ISL with LEN more effectively suppressed viral breakthrough at lower multiples of the compounds' IC (half-maximal inhibitory concentration) values and fewer mutations emerged with the combination compared to either compound on its own. The known pathways for development of resistance with ISL and LEN were not altered, and no novel single mutations emerged that substantially reduced susceptibility to either compound. The lack of antagonism and cross-resistance between ISL and LEN support the ongoing evaluation of the combination for treatment of HIV-1.
PubMed: 38864613
DOI: 10.1128/aac.00334-24 -
Frontiers in Microbiology 2024spp. are known for their radiation resistance, toxic compound removal, and production of valuable substances. Therefore, developing gene expression systems for spp. is...
spp. are known for their radiation resistance, toxic compound removal, and production of valuable substances. Therefore, developing gene expression systems for spp. is crucial in advancing genetic engineering applications. To date, plasmid vectors that express foreign genes in and have been limited to plasmid pI3 and its derivatives. In contrast, plasmid vectors that express foreign genes in include plasmid pZT23 and its derivatives. In this study, we developed a new system for the stable introduction and retention of expression plasmids for . Two cryptic plasmids were removed from the wild-type strain to generate the TY3 strain. We then constructed a shuttle vector plasmid, pGRC5, containing the replication initiation region of the smallest cryptic plasmid, pDEGR-3, replication initiation region of the vector, pACYC184, and an antibiotic resistance gene. We introduced pGRC5, pZT23-derived plasmid pZT29H, and pI3-derived plasmid pRADN8 into strain TY3, and found their coexistence in cells. The quantitative PCR assay results found that pGRC5, pZT29H, and pRADN8 had relative copy numbers of 11, 26, and 5 per genome, respectively. Furthermore, we developed a new plasmid in which the luciferase gene was controlled by the promoter region, which contained radiation-desiccation response operator sequences for DdrO, a stress response regulon repressor in , hence inducing gene expression via ultraviolet-C light irradiation. These plasmids are expected to facilitate the removal and production of toxic and valuable substances, in , respectively, particularly of those involving multiple genes.
PubMed: 38863757
DOI: 10.3389/fmicb.2024.1387296 -
Frontiers in Immunology 2024Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent...
Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.
Topics: Humans; Dengue Virus; Dendritic Cells; Autophagy; Dengue; Autophagosomes; Secretory Pathway; Virus Replication; Extracellular Vesicles; Cells, Cultured
PubMed: 38863700
DOI: 10.3389/fimmu.2024.1260439 -
Veterinary Research Jun 2024Bovine coronavirus (BCoV) is a pneumoenteric virus that can infect the digestive and respiratory tracts of cattle, resulting in economic losses. Despite its...
Bovine coronavirus (BCoV) is a pneumoenteric virus that can infect the digestive and respiratory tracts of cattle, resulting in economic losses. Despite its significance, information regarding BCoV pathogenesis is limited. Hence, we investigated clinical signs, patterns of viral shedding, changes in antibody abundance, and cytokine/chemokine production in calves inoculated with BCoV via intranasal and oral. Six clinically healthy Korean native calves (< 30 days old), initially negative for BCoV, were divided into intranasal and oral groups and monitored for 15 days post-infection (dpi). BCoV-infected calves exhibited clinical signs such as nasal discharge and diarrhea, starting at 3 dpi and recovering by 12 dpi, with nasal discharge being the most common symptoms. Viral RNA was detected in nasal and fecal samples from all infected calves. Nasal shedding occurred before fecal shedding regardless of the inoculation route; however, fecal shedding persisted longer. Although the number of partitions was very few, viral RNA was identified in the blood of two calves in the oral group at 7 dpi and 9 dpi using digital RT-PCR analysis. The effectiveness of maternal antibodies in preventing viral replication and shedding appeared limited. Our results showed interleukin (IL)-8 as the most common and highly induced chemokine. During BCoV infection, the levels of IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1β were significantly affected, suggesting that these emerge as potential and reliable biomarkers for predicting BCoV infection. This study underscores the importance of BCoV as a major pathogen causing diarrhea and respiratory disease.
Topics: Animals; Cattle; Coronavirus, Bovine; Cattle Diseases; Virus Shedding; Coronavirus Infections; Republic of Korea; Feces; RNA, Viral; Antibodies, Viral; Cytokines; Male
PubMed: 38863015
DOI: 10.1186/s13567-024-01331-9 -
Scientific Reports Jun 2024Individuals with type 1 diabetes (T1D) carry a markedly increased risk of stroke, with distinct clinical and neuroimaging characteristics as compared to those without...
Individuals with type 1 diabetes (T1D) carry a markedly increased risk of stroke, with distinct clinical and neuroimaging characteristics as compared to those without diabetes. Using whole-exome or whole-genome sequencing of 1,051 individuals with T1D, we aimed to find rare and low-frequency genomic variants associated with stroke in T1D. We analysed the genome comprehensively with single-variant analyses, gene aggregate analyses, and aggregate analyses on genomic windows, enhancers and promoters. In addition, we attempted replication in T1D using a genome-wide association study (N = 3,945) and direct genotyping (N = 3,263), and in the general population from the large-scale population-wide FinnGen project and UK Biobank summary statistics. We identified a rare missense variant on SREBF1 exome-wide significantly associated with stroke (rs114001633, p.Pro227Leu, p-value = 7.30 × 10), which replicated for hemorrhagic stroke in T1D. Using gene aggregate analysis, we identified exome-wide significant genes: ANK1 and LRRN1 displayed replication evidence in T1D, and LRRN1, HAS1 and UACA in the general population (UK Biobank). Furthermore, we performed sliding-window analyses and identified 14 genome-wide significant windows for stroke on 4q33-34.1, of which two replicated in T1D, and a suggestive genomic window on LINC01500, which replicated in T1D. Finally, we identified a suggestively stroke-associated TRPM2-AS promoter (p-value = 5.78 × 10) with borderline significant replication in T1D, which we validated with an in vitro cell-based assay. Due to the rarity of the identified genetic variants, future replication of the genomic regions represented here is required with sequencing of individuals with T1D. Nevertheless, we here report the first genome-wide analysis on stroke in individuals with diabetes.
Topics: Adult; Female; Humans; Male; Middle Aged; Ankyrins; Diabetes Mellitus, Type 1; Genetic Predisposition to Disease; Genome-Wide Association Study; Membrane Proteins; Polymorphism, Single Nucleotide; Regulatory Sequences, Nucleic Acid; Stroke; Whole Genome Sequencing
PubMed: 38862513
DOI: 10.1038/s41598-024-61840-7 -
MBio Jun 2024Mammalian AIM-2-like receptor (ALR) proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host...
Mammalian AIM-2-like receptor (ALR) proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host innate immunity. In mice, the locus is highly polymorphic at the sequence and copy number level, and we show here that it is one of the most dynamic regions of the genome. One rapidly evolving gene within this region, , was introduced to the genome by gene conversion or an unequal recombination event a few million years ago. has a large, distinctive repeat region that differs in sequence and length among species and even closely related inbred strains. We show that IFI207 controls murine leukemia virus (MLV) infection and that it plays a role in the STING-mediated response to cGAMP, dsDNA, DMXXA, and MLV. IFI207 binds to STING, and inclusion of its repeat region appears to stabilize STING protein. The locus and provide a clear example of the evolutionary innovation of gene function, possibly as a result of host-pathogen co-evolution.IMPORTANCEThe Red Queen hypothesis predicts that the arms race between pathogens and the host may accelerate evolution of both sides, and therefore causes higher diversity in virulence factors and immune-related proteins, respectively . The gene family in mice has undergone rapid evolution in the last few million years and includes the creation of two novel members, and , in particular, became highly divergent, with significant genetic changes between highly related inbred mice. IFI207 protein acts in the STING pathway and contributes to anti-retroviral resistance via a novel mechanism. The data show that under the pressure of host-pathogen coevolution in a dynamic locus, gene conversion and recombination between gene family members creates new genes with novel and essential functions that play diverse roles in biological processes.
PubMed: 38860764
DOI: 10.1128/mbio.01209-24