RD Cells Research Articles

The actions of varenicline on alkaloids from Conium maculatum (poison hemlock), Lupinus sulphureus (sulphur lupine) and Nicotiana glauca (tree tobacco).

Evidence-based therapies to manage the clinical signs of intoxication caused by toxic plants in livestock are lacking. For that reason, the aim of this work was to develop a drug-based intervention for the management of clinical signs of piperidine alkaloid intoxication in livestock. The actions of anabasine, coniine, γ-coniceine, and two total alkaloid extracts from Lupinus sulphureus were compared in the presence and absence of the nicotinic acetylcholine receptor partial agonist varenicline in RD cells, mice and goats. Pretreatment of RD cells with 10.0μM varenicline significantly shifted the anabasine fifty percent effective concentration (EC50) value to a greater concentration and blocked the response of the cells to coniine. γ-coniceine did not have any effect on RD cells as measured by membrane potential sensing dye. Swiss Webster mice median lethal dose (LD50) values for anabasine, coniine, γ-coniceine were 1.5, 5.5, and 3.7mg/kg respectively, and pretreatment with 10.0mg/kg i. p. dosed varenicline shifted the LD50 values to 4.2, 9.1, and 4.3mg/kg respectively. The rodent LD50 value of the Pendelton, WA L. sulphureus quinolizidine alkaloid extract was shifted to a lesser concentration by varenicline while the Ritzville, WA L. sulphureus piperidine alkaloid extract was shifted to a greater concentration by varenicline. The clinical signs of intoxication in goats orally dosed with Conium maculatum were exacerbated by 0.5, 1.0 and 10.0mg/kg i. v. dosed varenicline. These results suggest that varenicline was effective at shifting piperidine alkaloid EC50 values in RD cells and increasing piperidine but not quinolizidine alkaloid LD50 values in mice and was not useful at managing the clinical signs of poison hemlock intoxication in goats.

The antiviral effect and potential mechanism of Houttuynia cordata thunb. (HC) against coxsackievirus A4

Ethnopharmacological relevanceHand, foot, and mouth disease (HFMD) is mainly caused by various of enteroviruses such as enterovirus 71 (EVA71), coxsackievirus A16 (CVA16), CVA6, and CVA10 in infants and children under 5 years old. During the past 5 years, CVA4 has become the dominant pathogen resulting in HFMD in China. However, there are no effective vaccines and antiviral drugs available. Houttuynia cordata Thunb (HC). is a Chinese herbal medicine eaten as vegetables for treating viral infection diseases, but whether HC has anti-CVA4 effect remains unclear. Aim of the studyIn this study, we want to investigate the antiviral activity of HC against CVA4 in vitro and in vivo and elucidate the potential mechanism of HC against CVA4. Materials and methodsMTT assay were used to evaluate the cytotoxicity of HC. Virus titers assay, CPE assay, violet staining and immunofluorescence were used to investigate the antiviral effect of HC against CVA4. A 13-day-old suckling mice model was established to evaluate the therapeutic efficacy of HC against CVA4 infection. Western blot, qRT-PCR and time-of-drug addition assay were performed to elucidate the potential mechanism of HC against CVA4 infection. ResultsMTT assay indicated the cytotoxicity concentration of HC on Vero cells and RD cells were more than 1 mg/ml, suggesting that the low cytotoxicity of HC. In vitro antiviral assay revealed that HC could dose-dependently prevent the CPE, suppress the release of newborn virus, and inhibit the replication of CVA4 by decreasing viral RNA transcription and protein expression with IC50 of 88.96 μg/mL. A time-of-addition assay showed that HC mainly exerted anti-CVA4 effect by inhibiting virus replication at the post-entry stage. In vivo results further demonstrated that HC could effectively prevent the lethal infection of CVA4 by promoting survival, improving clinical symptoms, prolonging the survival time, inhibiting excessive inflammatory responses, and reducing pathological injury in vivo. Mechanistic studies revealed inhibition of p38 MAPK and JNK pathway over-activation may be the primary mechanism of HC against CVA4 infection. ConclusionIn summary, our results for the first time demonstrated that HC not only effectively inhibited CVA4 replication, but also partially protected the lethal infection of CVA4 in vivo. Furthermore, pharmacological mechanism studies revealed that the primary mechanism of HC against CVA4 infection may be associated with its effect of inhibiting over-activation of p38 MAPK and JNK signaling pathways caused by enteroviruses. Our finding indicated that HC might be a potential innovative medicine for treating HFMD.

Evaluation of the antiviral activity of different extracts of Gliricidia sepium and Xylopia aethiopica on enteroviruses isolated in Côte d'Ivoire

Objective : The emergence of viral infections has highlighted the need for new antiviral molecules. This study is part of an exploration into finding new antiviral compounds from medicinal plants used in traditional pharmacopoeia in Côte d'Ivoire. The aim is to determine which of the extracts from Gliricidia sepium leaves and Xylopia aethiopica fruits (hydroalcoholic, hexane/water, ethyl acetate/water, aqueous) exhibits the best antiviral activity. Methodology: The cytotoxicity of each partition P1 (hydroalcoholic), P2 (hexane/water), P3 (ethyl acetate/water), P4 (aqueous) of Gliricidia sepium leaves and P1' (hydroalcoholic), P2' (hexane/water), P3' (ethyl acetate/water), P4' (aqueous) of Xylopia aethiopica fruits is determined on RD cells. The antiviral activity of these partitions was then evaluated against a type 1 enterovirus. Results: The hexane-water partition showed high antiviral activity against type 1 enterovirus, with an inhibition of 65.78±9.21% at a concentration of 1 μg/mL for Gliricidia sepium leaves, compared to a non-significant inhibition of 86.84±5.26% at 31.25 μg/mL for Xylopia aethiopica fruits. Additionally, the aqueous partition of Gliricidia sepium induced an inhibition of 50±0.01% at a concentration of 1.95 μg/mL. Conclusion: Hexane-water and aqueous partitions of Gliricidia sepium showed́ potent antiviral activitý against enterovirus 1 than those of Xylopia. aethiopica. Keywords: plant partition, RD cells, enterovirus type 1.

Comparative study of the propagation and plaque titration conditions for human coronavirus OC43 as a surrogate for SARS-CoV-2.

The ongoing COVID-19 pandemic is threatening human health globally. The development of effective drugs and vaccines against SARS-CoV-2 is hindered by the limited access to high-biosafety-level facilities. Although human coronavirus (HCoV) OC43, a low-pathogenic endemic human coronavirus, has been used as a surrogate virus for SARS-CoV-2 research, a standard technique for HCoV-OC43 culture and plaque titration has not been established. Our objective was to establish optimized culture and titration protocols for HCoV-OC43. The growth kinetics and permissibility to HCoV-OC43 infection of seven different cell lines were examined concurrently at two different temperatures, 33°C and 37°C. Cell lines exhibiting a cytopathic effect (CPE) were selected for plaque titration. No significant difference in the rate of cell growth was observed at the two temperatures tested. Interestingly, HCoV-OC43 was found not to be a high-temperature-sensitive virus, since it grew well at 37°C. Although RD, LLC-MK2, MRC-5, and HCT-8 cell lines supported virus growth with an obvious cytopathic effect and a high yield of virus after two days of infection, only RD cells were suitable for producing countable plaques. The incubation of the cells with 1.2% low-viscosity Avicel as an overlay medium at 37°C for 4 days appeared to promote clearer and sharper plaque morphology. However, further optimization of the plaque titration protocol is still required due to the continued observation of plaque size variation and hazy zones. We propose a cost-effective protocol for HCoV-OC43 culture and plaque titration that can be implemented at a standard conventional temperature without the need for additional special equipment.

Phospholipase Cδ-4 (PLCδ4) Acts as a Nuclear Player to Influence Cyclin B Expression in the Embryonal Rhabdomyosarcoma Cell Lines RD and A204.

Rhabdomyosarcoma (RMS), the most common form of sarcoma typical of pediatric age, arises from the malignant transformation of the mesenchymal precursors that fail to differentiate into skeletal muscle cells. Here, we investigated whether the protein phospholipase C δ4 (PLCδ4), a member of the PLC family involved in proliferation and senescence mechanisms of mesenchymal stromal stem cells, may play a role in RMS. Our molecular and morpho-functional data reveal that PLCδ4 is highly expressed in the fusion-negative, p53-positive, SMARCB1 heterozygous mutated embryonal RMS (ERMS) cell line A204, while it is poorly expressed in the ERMS cell lines RD (fusion-negative, MYC amplification, N-RAS (Q61H), homozygous mutated p53) and Hs729 (homozygous mutated p53) and the alveolar rhabdosarcoma (ARMS) cell line SJCRH30 (RH30; fusion positive, heterozygous mutated RARA, polyheterozygous mutated p53). To characterize the role of PLCδ4, the RD cell line was stably transfected with wild-type PLCδ4 (RD/PLCδ4). Overexpressed PLCδ4 mainly localized to the nucleus in RD cells and contributed to the phosphorylation of PRAS40 (T246), Chk2(T68), WNK1(T60), and Akt 1/273 (S473), as revealed by proteome profiler array analysis. Overexpression of PLCδ4 in RD cells enhanced cyclin B1 expression and resulted in G2/M-phase cell cycle arrest. In contrast, siRNA-mediated knockdown of PLCδ4 in A204 cells resulted in reduced cyclin B1 expression. Our study identifies a novel role for nuclear PLCδ4 as a regulator of cyclin B1 via Akt-dependent phosphorylation. The modulation of PLCδ4 expression and its downstream targets could represent a crucial signaling pathway to block embryonal RMS cell proliferation.

Characterization of cross-reactivity of coxsackievirus A2 VP1-specific polyclonal antibodies with enterovirus A71, coxsackievirus A16, and coxsackievirus A6

Coxsackievirus A2 (CVA2) is associated with multiple diseases in children. Currently, there is limited research on immunological detection methods for CVA2. Herein, the VP1 gene of CVA2 strain 201711, belonging to cluster 2 within genotype D, was analyzed. The structures of VP1 from CVA2 strains 201711, 7-1 and 12-1, enterovirus A71 (EV-A71) strain 201713, coxsackievirus A16 (CVA16) strain 201717, and coxsackievirus A6 (CVA6) strain JLS10 were compared. The Escherichia coli BL21(DE3)/pET vector system was employed to express the recombinant protein containing the entire VP1 of CVA2 strain 201711. Mice were immunized with the purified protein, and the sera were collected and used to specifically identify the VP1 in CVA2-infected RD cells by Western blot and immunofluorescence assay. There was no evident cross-reactivity of the sera with the VP1 of EV-A71, CVA16, and CVA6 strains mentioned above. Therefore, this study provided mouse-specific anti-CVA2 VP1 polyclonal antibodies for CVA2 detection.

Immunogenicity of trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against EV-A71 and CV-A16.

Aim: To develop a trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against hand foot and mouth disease (HFMD) and assess its immunogenicity in mice.Materials & methods: Trivalent plasmid carrying the VP1 and VP2 genes of EV-A71, VP1 gene of CV-A16 was encapsulated in Chitosan-TPP nanoparticles through ionic gelation. In vitro characterization and in vivo immunization studies of the CS-TPP-NPs (pIRES-VP121) were performed.Results: Mice administered with CS-TPP NPs (pIRES-VP121) intramuscularly were observed to have the highest IFN-γ response. Sera from mice immunized with the naked pDNA and CS-TPP-NPs (pIRES-VP121) demonstrated good viral clearance against wild-type EV-A71 and CV-A16 in RD cells.Conclusion: CS-TPP-NPs (pIRES-VP121) could serve as a prototype for future development of multivalent HFMD DNA vaccine candidates.

LC/MS-guided isolation and GNPS procedures to identify flavonoid of HCoV-OC43 inhibitors

The screening of based target compounds supported by LC/MS, MS/MS and Global Natural Products Social (GNPS) used to identify the compounds 1–10 of Butea monsperma. They were evaluated in human malignant embryonic rhabdomyoma cells (RD cells) infected with Human coronavirus OC43 (HCoV-OC43) and showed significant inhibitory activity. Target inhibition tests showed that compounds 6 and 8 inhibited the proteolytic enzyme 3CLpro, which is widely present in coronavirus and plays an important role in the replication process, with an effective IC50 value. The study confirmed that dioxymethylene of compound 8 may be a key active fragment in inhibiting coronavirus (EC50 7.2 μM, SI > 139.1). The results have led to identifying natural bioactive compounds for possible inhibiting HCoV-OC43 and developing drug for Traditional Chinese Medicine (TCM).

Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling

BackgroundAstragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms.MethodsThe GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman’s algorithm.ResultsThis study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis.ConclusionThis study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.Graphical

Abstract 4738: Overcoming therapy resistance in rhabdomyosarcoma by targeting the PI3K-MAPK pathway

Abstract Rhabdomyosarcoma (RMS) is a devastating pediatric cancer. There has not been any significant change in the treatment options for the last 4 decades, especially for relapsed or advanced disease. There is an urgent need to identify alternative treatment options to improve survival outcomes of RMS patients. RMS is comprised of two major subtypes as defined by the presence or absence of PAX3/PAX7-FOXO1 gene fusion. Fusion-negative RMS (FN RMS) is characterized by alterations in the RAS-PI3K-MAPK pathway in >90% of the cases. However, the role of the PI3K-MAPK pathway in therapy resistance and disease relapse remains poorly characterized. We have generated two FN-RMS lines, RD and SMS-CTR, with resistance to the standard-of-care chemotherapeutic agent, vincristine, through long-term culturing in incrementally increased concentrations of the drug. By RNA sequencing, vincristine-resistant RD and SMS-CTR cells demonstrated increased expression of genes in the RAS-PI3K-MAPK pathway compared to the parental lines. We hypothesize that increased activity of the PI3K-MAPK pathway contributes to therapy resistance in FN RMS. To test the hypothesis, we first showed that targeted disruption of PIK3CA by shRNA inhibited the cell growth of vincristine-resistant FN RMS cells. The same cells also showed increased sensitivity to the MEK inhibitor, trametinib, as well as the PIK3Cα inhibitor, alpelisib, compared to the parental cells. We then showed that zebrafish FN-RMS tumors treated with the combination of vincristine and alpelisib significantly inhibited tumor growth compared to the treatment with each agent alone. Through integrative analysis of RNA sequencing and ATAC-seq data, we identified MYOD1 and NFATC2 as among the top candidate upstream regulators. Targeted disruption of either MYOD1 or NFATC2 by CRISPR/Cas9 reduced cell growth of vincristine-resistant FN RMS cells and altered expression of the genes in the PI3K-MAPK pathway, implicating MYOD1 and NFATC2 in driving abnormal activity of this pathway. To assess the relevance of our findings in the clinical settings, we showed that selective components in the PI3K-MAPK pathways are up-regulated in 3 matched primary-recurrence/metastasis paraffinized tissue samples of human FN RMS by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Our findings indicate that targeting the PI3K-MAPK pathway is a promising targeted therapy option for overcoming therapy resistance in RMS. Citation Format: Yadong Wang, Eleanor Chen. Overcoming therapy resistance in rhabdomyosarcoma by targeting the PI3K-MAPK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4738.

Abstract 2863: CDKN1A prevents IR-induced apoptosis in rhabdomyosarcoma tumor via repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis

Abstract Rhabdomyosarcoma (RMS) is a tumor of the muscle and is the most common soft tissue cancer in children and teens, with approximately 400 to 500 new cases every year in the United States. While we have a good understanding of the interplay between pro- and anti-apoptotic regulators, much remains to be learned about how the decision to trigger apoptosis or not is controlled at the transcriptional level in RMS tumors. We strive to elucidate how pathways that drive apoptosis avoidance are epi-genetically pre-programmed in cells. We have discovered that SNAI2, a transcriptional repressor, acts as a master switch to either activate or dampen apoptosis in response to ionizing radiation (IR). While elevated SNAI2 results in transcriptional repression of the pro-apoptotic factor BIM and protection from apoptosis post-IR, a small number of SNAI2 null cells survive radiation to reproliferate in vivo, revealing the existence of hitherto unappreciated mechanisms that support the survival of irradiated cells. Hence, we hypothesized that there are additional genes or pathways in cells lacking SNAI2 that are co-opted to drive relapse disease. We have identified that CDKN1A or p21 expression is robustly increased in SNAI2 null cells. CDKN1A is thought to be a tumor suppressor. However, our preliminary data indicate that CDKN1A is an oncogene, and in RMS patients, high tumor expression is associated with poor outcomes. We found that CDKN1A expression in combination with SNAI2 can predict resistance to radiation across RMS cell lines. We next developed control RD, Rh30, and Rh18 cells, either CDKN1A or both CDKN1A and SNAI2 ablated using CRISPR/Cas9 reagents with control cells expressing control gRNAs. Ablation of CDKN1A, while not affecting proliferation, led to a significant increase in apoptosis post-radiation, and this effect was increased in SNAI2/CDKN1A double knockout cells. Further, in colony-forming assays, we find that double-ablated cells form significantly fewer colonies than control and single knockout cells. Loss of CDKN1A, SNAI2, or CDKN1A/SNAI2 led to a G2/M cell cycle block, indicating that the cell cycle effects of p21 might not be responsible for the phenotypes observed. Ongoing xenograft experiments have determined the effect we observe in vitro will also translate in vivo. Given that SNAI2 and CDKN1A are heterogeneously expressed, respectively, we will interrogate CDKN1A for its role in protecting differentiated cells from apoptosis. We will determine if CDKN1A prevents apoptosis by repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis specific to differentiated cells. Our study will show that critical primary and alternative apoptosis resistance mechanisms are epi-genetically pre-programmed in cells by SNAI2. Citation Format: Paulomi S. Modi, Myron Ignatius, Long Wang, Prethish Sreenivas. CDKN1A prevents IR-induced apoptosis in rhabdomyosarcoma tumor via repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2863.

Occurrence of poliovirus and non-polio enterovirus among children with acute flaccid paralysis in Cameroon from 2015 to 2020.

Poliovirus (PV) and non-polio enteroviruses (NPEV) belong to the Picornaviridae family. They are found worldwide and are responsible for a wide range of diseases such as acute flaccid paralysis (AFP). This study aimed to evaluate the detection rate of PV and NPEV in stool samples from children under fifteen years of age presenting with AFP in Cameroon and their distribution over time. Stool samples were collected as part of poliovirus surveillance throughout Cameroon from 2015 to 2020. Virus isolation was performed using RD and L20B cells maintained in culture. Molecular methods such as intratypic differentiation were used to identify PVs serotypes and analysis of the VP1 genome was performed. A total of 12,354 stool samples were analyzed. The EV detection rate by virus isolation was 11.42% (1411/12354). This rate varied from year to year with a mean distribution of 11.41 with a 95% confidence interval [11.37; 11.44]. Of the viruses detected, suspected poliovirus accounted for 31.3% (442/1411) and NPEV 68.67% (969/1411). No wild poliovirus (WPV) was isolated. Sabin types 1 and 3 were continuously isolated. Surprisingly, from February 2020, vaccine-derived PV type 2 (VDPV2) was detected in 19% of cases, indicating its resurgence. This study strongly supports the successful elimination of WPV in Cameroon and the resurgence of VDPV2. However, as long as VDPV outbreaks continue to be detected in Africa, it remains essential to monitor how they spread.

Emergence of Recombinant Subclade D3/Y in Coxsackievirus A6 Strains in Hand-Foot-and-Mouth Disease (HFMD) Outbreak in India, 2022.

Coxsackievirus-A6 (CV-A6) is responsible for more severe dermatological manifestations compared to other enteroviruses such as CV-A10, CV-A16, and EV-A71, causing HFMD in children and adults. Between 2005 and 2007, the recombinant subclade D3/RF-A started to expand globally, and a CV-A6 pandemic started. The study aimed to conduct whole-genome sequencing (WGS) of an isolated CV-A6 strain from currently circulating HFMD cases from India in 2022. Gene-specific RT-PCR and sequencing were used to perform molecular characterization of the isolated virus. Confirmation of these isolates was also performed by transmission electron microscopy and WGS. Among eleven positive clinical enterovirus specimens, eight CV-A6 strains were successfully isolated in the RD cell line. Isolates confirmed the presence of the CV-A6 strain based on VP1 and VP2 gene-specific RT-PCR. Sequences of isolates were clustered and identified as the novel CV-A6 strain of the D3/Y sub-genotype in India. The studies revealed that the D3/Y sub-genotype is being introduced into Indian circulation. The predicted putative functional loops found in VP1 of CV-A6 showed that the nucleotide sequences of the amino acid were a remarkably conserved loop prediction compatible with neutralizing linear epitopes. Therefore, this strain represents a potential candidate for vaccine development and antiviral studies.

Molecular characterization of ANKRD1 in rhabdomyosarcoma cell lines: expression, localization, and proteasomal degradation.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting.

Comprehensive Elucidation of the Role of L and 2A Security Proteins on Cell Death during EMCV Infection.

The EMCV L and 2A proteins are virulence factors that counteract host cell defense mechanisms. Both L and 2A exhibit antiapoptotic properties, but the available data were obtained in different cell lines and under incomparable conditions. This study is aimed at checking the role of these proteins in the choice of cell death type in three different cell lines using three mutants of EMCV lacking functional L, 2A, and both proteins together. We have found that both L and 2A are non-essential for viral replication in HeLa, BHK, and RD cell lines, as evidenced by the viability of the virus in the absence of both functional proteins. L-deficient infection led to the apoptotic death of HeLa and RD cells, and the necrotic death of BHK cells. 2A-deficient infection induced apoptosis in BHK and RD cells. Infection of HeLa cells with the 2A-deficient mutant was finalized with exclusive caspase-dependent death with membrane permeabilization, morphologically similar to pyroptosis. We also demonstrated that inactivation of both proteins, along with caspase inhibition, delayed cell death progression. The results obtained demonstrate that proteins L and 2A play a critical role in choosing the path of cell death during infection, but the result of their influence depends on the properties of the host cells.

Antibacterial, antioxidant, and anticancer potential of green fabricated silver nanoparticles made from Viburnum grandiflorum leaf extract

BackgroundRecently, researchers are focusing on creating new tools to combat the antibiotic resistant bacteria and malignancy issues, which pose significant threats to humanity. Biosynthesized silver nanoparticles (AgNPs) are thought to be a potential solution to these issues. The biosynthesis method, known for its environmentally friendly and cost-effective characteristics, can produce small-sized AgNPs with antimicrobial and anticancer properties. In this study, AgNPs were bio-fabricated from the distilled water and methanolic extracts of Viburnum grandiflorum leaves. Physio-chemical characterization of the bio-fabricated AgNPs was conducted using UV-visible spectroscopy, scanning electron microscopy, energy dispersive X-ray, and X-ray diffraction analysis.ResultsAgNPs produced from the methanol extract were smaller in size (12.28 nm) compared to those from the aqueous extract (17.77 nm). The bioengineered AgNPs exhibited a circular shape with a crystalline nature. These biosynthesized AgNPs demonstrated excellent bactericidal activity against both gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria. Highest antibacterial activity was observed with the methanol extract against P. aeruginosa (14.66 ± 0.74 mm). AgNPs from the methanol extract also displayed the highest antioxidant activity, with an IC50 value of 188.00 ± 2.67 μg/mL against 2,2-diphenyl-1-picrylhydrazyl (DPPH). Furthermore, AgNPs exhibited notable cytotoxic activity against Rhabdomyosarcoma cell line (RD cell) of human muscle cancer cell. The IC50 values calculated from the MTT assay were 26.28 ± 1.58 and 21.49 ± 1.44 μg/mL for AgNPs synthesized from aqueous and methanol extracts, respectively.ConclusionThe methanol extract of V. grandiflorum leaves demonstrates significant potential for synthesizing AgNPs with effective antibacterial, antioxidant, and anticancer actions, making them applicable in various biomedical applications.

Chemical Constituents and Anticancer Activities of the Extracts from Phlomis × commixta Rech. f. (P. cretica × P. lanata).

The present work is the first report on the ingredients of the P. × commixta hybrid, a plant of the genus Phlomis. So far, thirty substances have been isolated by various chromatographic techniques and identified by spectroscopic methods, such as UV/Vis, NMR, GC-MS and LC-MS. The compounds are classified as flavonoids: naringenin, eriodyctiol, eriodyctiol-7-O-β-D-glucoside, luteolin, luteolin-7-O-β-D-glucoside, apigenin, apigenin-7-O-β-D-glucoside, diosmetin-7-O-β-D-glucoside, quercetin, hesperetin and quercetin-3-O-β-D-glucoside; phenylpropanoids: martynoside, verbascoside, forsythoside B, echinacoside and allysonoside; chromene: 5,7-dihydroxychromone; phenolic acids: caffeic acid, p-hydroxybenzoic acid, chlorogenic acid, chlorogenic acid methyl ester, gallic acid, p-coumaric acid and vanillic acid; aliphatic hydrocarbon: docos-1-ene; steroids: brassicasterol and stigmasterol; a glucoside of allylic alcohol, 3-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranosyl-oct-1-ene-3-ol, was fully characterized as a natural product for the first time. Two tyrosol esters were also isolated: tyrosol lignocerate and tyrosol methyl ether palmitate, the latter one being isolated as a natural product for the first time. Moreover, the biological activities of the extracts from the different polarities of the roots, leaves and flowers were estimated for their cytotoxic potency. All root extracts tested showed a high cytotoxic activity against the Hep2c and RD cell lines.

TLR4 signalling: the key to controlling EV71 replication and inflammatory response.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity.

Indole Family and Neomycin Sulfate: Inductors of Differentiation in C2C12 and RD cell Lines

Rhabdomyosarcoma (RMS) is a highly aggressive tumor primarily affecting the pediatric population, that generally originates from a failure in the embryonic differentiation of myogenic precursor cells. Standard treatment involves surgery, chemotherapy and radiation therapy, with a poor prognosis, especially when it spreads to other parts of the body, highlighting the need for new treatment approaches. In this study, cellular differentiation was investigated as a potential therapeutic approach for an embryonal subtype of RMS (eRMS) employing a human eRMS cell line, RD; and comparing it with a normal murine myoblast cell line, C2C12. We observed that not only a serum free or a low serum concentration in the cultured medium induce morphological changes towards differentiations, but also staurosporine, bisindolylmaleimide, and neomycin sulfate (direct or indirect protein kinase inhibitors) induces differentiation in RD and C2C12 cell lines, suggesting a crucial role of PKC, PLC and its signaling pathways in a proliferation/differentiation switch. These findings underscore the importance of exploring cellular differentiation as a therapeutic strategy against RMS and highlight the need for further research into the underlying mechanisms of kinase inhibitor-induced cellular differentiation as a potential cancer treatment.

Epidemiology and molecular detection of human adenovirus and non-polio enterovirus in fecal samples of children with acute gastroenteritis: A five-year surveillance in northern Brazil.

Acute gastroenteritis (AGE) is a common pediatric infection that remains a significant cause of childhood morbidity and mortality worldwide, especially in low-income regions. Thus, the objective of this study was to detect human adenovirus (HAdV) and non-polio enterovirus (NPEV) in fecal samples from the Gastroenteritis Surveillance Network, and to identify circulating strains by nucleotide sequencing. A total of 801 fecal samples were tested using qPCR/RT-qPCR, and 657 (82.0%) were inoculated into HEp-2C and RD cell lines. The HAdV and NPEV positivity rates obtained using qPCR/RT-qPCR were 31.7% (254/801) and 10.5% (84/801), respectively, with 5.4% (43/801) co-detection. Cytopathic effect was observed in 9.6% (63/657) of patients, 2.7% (18/657) associated with HAdV, and 6.2% (41/657) associated with NPEV after testing by ICC-PCR. A comparison of the two methodologies demonstrated an agreement of 93.5% for EVNP and 64.4% for HAdV. These two viruses were detected throughout the study period, with HAdV positivity rates ranging from 41% in Amapá to 18% in Pará. The NEPV varied from 18% in Pará/Rondônia to 3% in Acre. The most affected age group was over 60 months for both HAdV and NPEV. Samples previously positive for rotavirus and norovirus, which did not show a major difference in the presence or absence of diarrhea, fever, and vomiting, were excluded from the clinical analyses of these two viruses. These viruses circulated over five years, with a few months of absence, mainly during the months corresponding to the waves of SARS-CoV-2 infection in Brazil. Five HAdV species were identified (A, B, C, D, and F), with a greater predominance of HAdV-F41 (56.5%) followed by HAdV-C (15.2%). Three NPEV species (A, B, and C) were detected, with serotypes E14 (19.3%) and CVA-24 (16.1%) being the most prevalent. The present study revealed a high diversity of NPEV and HAdV types circulating in children with AGE symptoms in the northern region of Brazil.