Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

Effects of electroacupuncture on the PI3K/Akt/CREB signaling pathway and hippocampal neuronal apoptosis in diabetic cognitive impairment rats. / ç”µé’ˆå¯¹ç³–å°¿ç— è®¤çŸ¥åŠŸèƒ½éšœç¢å¤§é¼ PI3K/Akt/CREBä¿¡å·é€šè·¯å’Œæµ·é©¬ç¥žç»å ƒå‡‹äº¡çš„å½±å“.

OBJECTIVES: To observe the effects of electroacupuncture (EA) on the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/cAMP response element binding protein (CREB) signaling pathway-related proteins and hippocampal neuron apoptosis in diabetic cognitive impairment (DCI) rats, and to explore the mechanisms of EA in treating DCI. METHODS: Adult male SD rats were randomly divided into normal, model, and EA groups, with 12 rats in each group. The animal model of DCI was replicated using a high-fat, high-sugar diet combined with low-dose streptozotocin. The EA group received EA stimulation at "Yishu" (EX-B6), "Zusanli" (ST36), "Baihui" (GV20), and "Dazhui" (GV14). Blood glucose contents of the rats in each group were measured. The Morris water maze test was used to assess the learning and memory abilities of rats. Transmission electron microscopy was used to observe the ultrastructure of hippocampal CA1 neurons. Nissl staining was used to observe the pathological changes in hippocampal CA1 neurons. TUNEL staining was used to detect the apoptosis in hippocampal CA1 neurons. Western blot was used to detect the protein expression levels of p-PI3K/PI3K and p-Akt/Akt, as well as CREB, p-CREB, cysteine aspartate pro-tease (Caspase)-3, B-cell lymphoma-2 (Bcl-2), and Bcl-2 related X protein (Bax) in the hippocampal tissue of rats. RESULTS: Compared with the normal group, the rats' random blood glucose contents were significantly increased (P<0.01), the escape latency prolonged (P<0.01), and the original platform crossing counts reduced (P<0.01) in the model group. Significant damage to hippocampal CA1 neurons, a significantly increased neuronal apoptosis index (P<0.01), decreased ratio of p-PI3K/PI3K and p-Akt/Akt and expression of CREB, p-CREB and Bcl-2 proteins, increased expression of Caspase-3 and Bax proteins (P<0.01) were observed in the hippocampal tissue of rats in the model group. Compared with the model group, the rats in the EA group showed decreased random blood glucose content (P<0.01), shortened escape latency (P<0.01), increased original platform crossing counts (P<0.01), improved quantity and pathological morphology and ultrastructure of hippocampal CA1 neurons, reduced neuronal apoptosis index (P<0.01), increased ratio of p-PI3K/PI3K and p-Akt/Akt, and expression of CREB, p-CREB and Bcl-2 proteins (P<0.05, P<0.01) in the hippocampal tissue, and decreased expression of Caspase-3 and Bax proteins (P<0.01). CONCLUSIONS: EA can improve the learning and memory abilities of rats with DCI, and the mechanism may be related to the regulation of the expression of PI3K/Akt/CREB signaling pathway-related proteins, which attenuates the neuronal apoptosis in the hippocampus of rats, and improves the neural function.

Highly in vitro anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer cells.

BACKGROUND: Development of promising medicines from natural sources, specially venom, is of highly necessitated to combat against life-threatening cancers. Non-small cell lung cancer (NSCLC) has a significant percentage of mortalities. Melittin, from bee venom, is a potent anticancer peptide but its toxicity has limited its therapeutic applications. Accordingly, this study aims to synthesize niosomes with suitable stability and capacity for carrying melittin as a drug. Additionally, it seeks to evaluate the anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer. METHODS: The niosome was prepared by thin film hydration method. Cytotoxicity and apoptosis were assessed on A549, Calu-3, and MRC5 cells. Real-time PCR was used to determine expression of apoptotic and pro-apoptotic Bax, Bcl2, and Casp3 genes. Immunocytochemistry (ICC) was also used to confirm expression of the abovementioned genes. Furthermore, wound healing assay was performed to compare inhibition effects of melittin-loaded niosomes with free melittin on migration of cancer cells. RESULTS: IC50 values of melittin-loaded niosomes for A549, Calu-3, and MRC5 cells were respectively 0.69 µg/mL, 1.02 µg/mL, and 2.56 µg/mL after 72 h. Expression level of Bax and Casp3 increased '10 and 8' and '9 and 10.5' fold in A549 and Calu-3, whereas Bcl2 gene expression decreased 0.19 and 0.18 fold in the mentioned cell lines. The cell migration inhibited by melittin-loaded niosomes. CONCLUSIONS: Melittin-loaded niosomes had more anti-cancer effects and less toxicity on normal cells than free melittin. Furthermore, it induced apoptosis and inhibited cancer cells migration. Our results showed that melittin-loaded niosomes may be a drug lead and it has the potential to be future developed for lung cancer treatment.

Fish oil ameliorates ethanol-induced gastric injury in rat by modulating gene related to apoptosis.

Gastric ulcers are a type of digestive disease that can severely affect a person's quality of life. Our study aimed to investigate the effects of fish oil on ethanol-induced gastric ulcers in rats, with the purpose of providing more comprehensive information on the topic. The study looked at various factors such as gastric ulcer index, and nitric oxide (NO) levels in stomach tissue. To investigate apoptosis, the mRNA levels of Bax, Bcl-2, and Caspase 3 were analyzed. The results showed that fish oil can reduce gastric acidity and the gastric ulcer index in cases of ethanol-induced gastric ulcers. It was found that fish oil can increase NO levels and improve the anti-apoptotic system by increasing the expression of Bcl-2 while decreasing the expression of Bax and Caspase 3. In general, the study demonstrates that fish oil can protect the stomach from ethanol-induced damage by reducing the apoptosis pathway via nitric oxide.

Thymoquinone potentiates anti-cancer effects of cisplatin in oral squamous cell carcinoma via targeting oxidative stress.

Recent evidence has proved that thymoquinone as a natural polyphenol has great anticancer and anti-proliferative effects in cancer cells. In this study, we aimed to examine the effects of thymoquinone on increasing cisplatin-induced apoptosis human oral squamous cell carcinoma cells and its underlying molecular mechanisms. SCC-25 cancer cells treated by thymoquinone and cisplatin with different concentrations. Cell viability will determine by using MTT assay. The concentrations of reactive oxygen species (ROS) and antioxidant activities were determined using specific related kits. DNA damage, lipid, and protein oxidation were assessed. Real-time PCR and Western blot analysis will be used to determine the expression of apoptosis-related proteins including Bax, Bcl-2, and caspase-3. Combination of thymoquinone and cisplatin suppressed synergistically SCC-25 cancer cell viability and induced apoptosis in dose-depended manner. Cell treatment with combination of thymoquinone and cisplatin led to accumulation of ROS within cells and increase in the intracellular levels of DNA damage, protein and lipid peroxidation. In addition, the combination of thymoquinone and cisplatin modulated the mRNA and protein expression levels of apoptosis-related proteins including Bax, Bcl-2, and caspase-3. Thymoquinone potentiated cisplatin anti-cancer effect on OSCC by inducing oxidative stress in cells.

Generation of two human induced pluripotent stem cell lines with BAX and BAK1 double knock-out using CRISPR/Cas9.

Bcl-2-associated X protein (BAX) and Blc-2 homologous antagonist killer 1 (BAK) are two pro-apoptotic members of BCL2 family. Here, two BAX/BAK double knock-out human induced pluripotent stem cell lines (iPSC) we generated using CRISPR-Cas9 to generate apoptosis incompetent cell lines. The resulting cell lines were karyotypically normal, had typical morphology and expressed typical markers for the undifferentiated state.

Fertilization with follicular fluid reduces HSP70 and BAX expression on bovine in vitro embryos.

The in vivo fertilization process occurs in the presence of follicular fluid (FF). The aim of this study was to evaluate the effect of in vitro fertilization medium supplementation with 5% or 10% bovine follicular fluid (BFF) on the production of in vitro bovine embryos. FF was collected from ovarian follicles with a diameter of 8-10 mm, and cumulus-oocyte complexes (COCs) were co-incubated with sperm for 24 h in the commercial medium BotuFIV® (BotuPharma©), being distributed among the experimental groups: oocytes fertilized in a control medium; oocytes fertilized in a medium supplemented with 5% BFF; and oocytes fertilized in a medium supplemented with 10% BFF. After fertilization, the zygotes were cultured in vitro for 8 days. Embryo development was assessed through cleavage rates (day 2) and blastocyst formation rates (day 8). The relative expression of the genes OCT4, IFNT2, BAX, HSP70 and SOD2 was measured using the real-time polymerase chain reaction method. There was no difference (p > .05) among the different experimental groups in terms of cleavage rates and blastocyst formation rates. Regarding the gene expression results, only the blastocysts from oocytes fertilized with 10% BFF showed significantly lower expression of IFNT2 (p = .003) and SOD2 (p = .01) genes compared to blastocysts from oocytes fertilized in control medium alone, while there was no difference between blastocyst from oocytes fertilized in control medium and the ones from oocytes fertilized with 5% BFF. In addition to this, the blastocysts from oocytes fertilized with 5% BFF showed significantly reduced levels of expression of the heat shock protein HSP70 (p < .001) and the pro-apoptotic protein BAX (p = .015) compared to blastocysts from oocytes fertilized with control medium. This may indicate that lower supplementation of BFF to the IVF medium creates a more suitable environment for fertilization and is less stressful for the zygote.

Endogenous BAX and BAK form mosaic rings of variable size and composition on apoptotic mitochondria.

One hallmark of apoptosis is the oligomerization of BAX and BAK to form a pore in the mitochondrial outer membrane, which mediates the release of pro-apoptotic intermembrane space proteins into the cytosol. Cells overexpressing BAX or BAK fusion proteins are a powerful model system to study the dynamics and localization of these proteins in cells. However, it is unclear whether overexpressed BAX and BAK form the same ultrastructural assemblies following the same spatiotemporal hierarchy as endogenously expressed proteins. Combining live- and fixed-cell STED super-resolution microscopy, we show that overexpression of BAK results in novel BAK structures, which are virtually absent in non-overexpressing apoptotic cells. We further demonstrate that in wild type cells, BAK is recruited to apoptotic pores before BAX. Both proteins together form unordered, mosaic rings on apoptotic mitochondria in immortalized cell culture models as well as in human primary cells. In BAX- or BAK- single-knockout cells, the remaining protein is able to form rings independently. The heterogeneous nature of these rings in both wild type as well as single-knockout cells corroborates the toroidal apoptotic pore model.

Long non-coding RNA TINCR suppresses growth and epithelial-mesenchymal transition by inhibiting Wnt/ß-catenin signaling pathway in human pancreatic cancer PANC-1 cells: Insights from in vitro and in vivo studies.

There is increasing evidence that long non-coding RNAs (lncRNAs) play a crucial role in the development and progression of malignant tumors, particularly pancreatic cancer. In this study, the influence of the lncRNA TINCR on the behavior of human pancreatic cancer cells was investigated with the aim of deciphering its role in growth, migration, and invasion. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate TINCR expression in pancreatic cancer cells. Ectopic expression of TINCR in PANC-1 cells was induced to evaluate the effects on cell viability and apoptosis, examining the apoptotic genes Bax and Bcl-2. Migration and invasion assays were used to measure the impact of TINCR on these cellular processes. In vivo studies using a xenograft mouse model examined the effects of TINCR on tumor growth, epithelial-to-mesenchymal transition (EMT) markers, and the Wnt/ß-catenin signaling pathway. PANC-1 cells showed strikingly low TINCR expression compared to other pancreatic cancer cell lines. Ectopic TINCR expression reduced the viability of PANC-1 cells primarily by inducing apoptosis, as evidenced by increased Bax and decreased Bcl-2 expression. Overexpression of TINCR significantly increased the percentage of apoptotic cells. It also decreased the migration and invasion ability of PANC-1 cells, as demonstrated in wound healing and transwell assays. In addition, overexpression of TINCR-suppressed proteins is associated with the Wnt/ß-catenin signaling pathway in PANC-1 cells. In the xenograft mouse model, overexpression of TINCR inhibited tumor growth, EMT markers, and proteins associated with the Wnt/ß-catenin pathway. This study sheds light on the tumour-suppressive role of TINCR in PANC-1 cells and suggests its potential as a therapeutic target. These results shed light on the molecular mechanisms underlying the impact of TINCR on pancreatic cancer and offer promising opportunities for innovative therapeutic strategies to improve outcomes in this serious malignancy.

Detection and Characterization of Apoptosis-Related Proteins in Hippocampal Neurodegeneration: From mRNA Expression to Protein Quantification.

The involvement of apoptosis in neurodegeneration can be detected by quantifying the apoptotic proteins in hippocampal lysate. Apoptosis can occur due to the overproduction of apoptotic proteins under the influence of external trigger or due to the overexpression of the apoptotic genes. Thus, the imbalance in the production of apoptotic proteins can be quantified using the Western blotting technique and the overexpression of apoptotic genes in hippocampal DNA can be quantified using the real-time quantification of mRNA expression of the apoptotic proteins. Here we provide the methodology of detecting the apoptosis-related proteins like Bax and Bcl-2 and their mRNA expression in hippocampal neurodegeneration. In this chapter, we have described the methodology for quantification of mRNA expression of these apoptosis-related proteins in the hippocampal lysate using the real-time quantitative polymerase chain reaction (qPCR) technique and the methodology of detection and characterization of respective protein expression in the hippocampal lysate using the Western blotting technique.

STING dependent BAX-IRF3 signaling results in apoptosis during late-stage Coxiella burnetii infection.

STING (STimulator of Interferon Genes) is a cytosolic sensor for cyclic dinucleotides (CDNs) and initiates an innate immune response upon binding to CDNs. Coxiella burnetii is a Gram-negative obligate intracellular bacterium and the causative agent of the zoonotic disease Q fever. The ability of C. burnetii to inhibit host cell death is a critical factor in disease development. Previous studies have shown that C. burnetii inhibits host cell apoptosis at early stages of infection. However, during the late-stages of infection, there is host cell lysis resulting in the release of bacteria to infect bystander cells. Thus, we investigated the role of STING during late-stages of C. burnetii infection and examined STING's impact on host cell death. We show that the loss of STING results in higher bacterial loads and abrogates IFNß and IL6 induction at 12 days post-infection. The absence of STING during C. burnetii infection significantly reduces apoptosis through decreased caspase-8 and -3 activation. During infection, STING activates IRF3 which interacts with BAX. BAX then translocates to the mitochondria, which is followed by mitochondrial membrane depolarization. This results in increased cytosolic mtDNA in a STING-dependent manner. The presence of increased cytosolic mtDNA results in greater cytosolic 2'-3' cGAMP, creating a positive feedback loop and leading to further increases in STING activation and its downstream signaling. Taken together, we show that STING signaling is critical for BAX-IRF3-mediated mitochondria-induced apoptosis during late-stage C. burnetii infection.

Isolation and Identification of a Tibetan Pig Porcine Epidemic Diarrhoea Virus Strain and Its Biological Effects on IPEC-J2 Cells.

Porcine epidemic diarrhoea virus (PEDV) is a coronavirus that can cause severe watery diarrhoea in piglets, with high morbidity and mortality rates, seriously hindering the healthy development of the global swine industry. In this study, we isolated a strain of PEDV from Tibetan pigs and named it CH/GS/2022. Subsequently, we screened the apoptosis signals of PEDV-infected IPEC-J2 cells and studied the correlation between apoptosis signals and cell apoptosis. The results showed that different infections of PEDV induced different degrees of apoptosis in cells, and PEDV-induced cell apoptosis was dose-dependent. We then detected the expression of the p53, p38, JNK, Bax, and Bcl-2 genes in the apoptosis signal pathway. The results showed that 24 h after PEDV infection, the expression of the p53, p38, JNK, and Bax genes in IPEC-J2 cells increased significantly, while the expression of the Bcl-2 gene decreased significantly (p < 0.05). Subsequently, we used Western blot to detect the protein levels of these five genes, and the results showed that PEDV infection upregulated the expression of p53, p38, JNK, and Bax proteins (p < 0.05) while downregulating the expression of Bcl-2 protein (p < 0.05). Thus, it was initially inferred that PEDV infection could regulate cell apoptosis by activating the p53, p38, and JNK signalling pathways. Finally, we further investigated the apoptosis of the cells through the use of inhibitors. The results indicated that the p53 inhibitor Pifithrin-α has a significant inhibitory effect on the expression of the p53 protein after PEDV infection and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p53 is involved in PEDV-induced cell apoptosis. Similarly, the p38 MAPK inhibitor SB203580 has an inhibitory effect on the expression of the p38 protein and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p38 is also involved in PEDV-induced cell apoptosis. On the other hand, the JNK inhibitor SP600125 has no inhibitory effect on the expression of the JNK protein after PEDV infection, but the expression levels of Bax and Bcl-2 proteins have changed. Furthermore, it is noteworthy that SP600125 can inhibit the activity of apoptotic proteins but not their levels, resulting in reduced cell apoptosis. These preliminary results indicated that JNK may be involved in PEDV-induced IPEC-J2 cell apoptosis.

Quantitative analysis of biochemical characteristics and anti-cancer properties in MCF-7 breast cancer cell line: a comparative study between Ziziphus jujube honey and commercial honey.

BACKGROUND: There is increasing evidence that honey has anti-inflammatory, antioxidant, and anti-cancer effects. This study aims to assess and contrast the cytotoxic, anti-metastatic, and apoptotic effects of Ziziphus jujube honey and commercial honey on MCF7 cells. METHODS AND RESULTS: Two honey samples, Ziziphus jujube (JH) and commercial honey (CH), were categorized into high and low groups based on their phenolic content, antioxidant capacity, and diastase activity (PAD score). The viability and migration ability of MCF-7 cells treated with JH and CH were evaluated. Also, quantitative polymerase chain reaction (Q-PCR) was performed to assess the effect of the two honey samples on the expression of Bax, p53, p21 and Bcl-2 genes. JH had a total phenolic content of 606.4 ± 0.1 µg gallic acid equivalent/mg, while CH had a value of 112.1 ± 0.09 µg gallic acid equivalent/mg. The total antioxidant capacity of the two samples was compared. It was 203.5 ± 10.5µM/l in JH and 4.6 ± 10.5 µM/l in CH. In addition, JH had a diastatic activity of 524.1 ± 0.25 U/l, while CH had a value of 209.7 ± 0.56 U/l. According to the results, JH had a high PAD value, while CH had a low PAD value. Cell viability was measured using the results of the MTT assay. The results showed that JH inhibited the growth of MCF-7 cells more strongly (IC50 of 170 ± 4.2 µg/ml) than CH (IC50 of 385.3 ± 4.5 µg/l). The scratch assay showed that treatment with JH decreased the migration rate of MCF-7 cells in a dose-dependent manner compared to the CH and control groups. In addition, the results of q-PCR analysis showed significant upregulation of Bax, p53 and p21 genes and downregulation of Bcl-2 gene in the JH-treated group compared to the CH and control groups. CONCLUSION: These results showed that honey with an increased content of phenolic compounds, antioxidant capacity, and diastatic activity has anticancer properties by effectively suppressing tumor development. This suppression occurs via several mechanisms, including suppression of proliferation and metastasis, and promotion of apoptosis.

The potential therapeutic effect of melatonin in oxaliplatin combination therapy against chemoresistant colorectal cancer cells.

BACKGROUND: Oxaliplatin is one of the main therapeutics in colorectal cancer (CRC) chemotherapy. However, in light of multidrug resistance (MDR) phenotype development, the efficacy of oxaliplatin has decreased. This study aimed to assess the potential therapeutic effect of melatonin in oxaliplatin combination therapy for drug-resistant colorectal cancer cells. METHODS AND RESULTS: Initially, the oxaliplatin-resistant cell line was created of LS174T (LS174T/DR) by using the oxaliplatin IC50 concentration and resting cycles. MTT assays and flow cytometry were applied for assessing cell viability and apoptotic cells. The mRNA expression level of Bax, Bcl2, MT1, MT2, and ABCB1 as well as protein levels of ABCB1, Bcl2, BAX were measured by the qRT-PCR and western blot techniques respectively. P-gp activity was assessed by Rho123 staining. The IC50 concentration of oxaliplatin in resistant cells was increased from 500.7 ± 0.2 nM to 7119 ± 0.1 nM. Bcl2, MT1, MT2, and ABCB1 mRNA plus protein expression levels of Bcl2 and ABCB1 were significantly reduced in resistant cells, along with a marked increase in Bax mRNA and protein levels compared to parental cells. Rho 123 staining revealed a marked reduction in P-gp activities in the combination-treated group compared to the oxaliplatin-treated group. CONCLUSIONS: The results of cytotoxicity assays, MTT, and flow cytometry revealed that the combination of melatonin and oxaliplatin exerts synergistic effects on induction of oxaliplatin's cytotoxicity in CRC. Our research suggests that combining the treatments of melatonin and oxaliplatin may be considered as a new approach to overcoming oxaliplatin resistance in CRC patients.

Urolithin B protects PC12 cells against glutamate-induced toxicity.

BACKGROUND: The involvement of malfunctioning glutamate systems in various central nervous system (CNS) disorders is widely acknowledged. Urolithin B, known for its neuroprotective and antioxidant properties, has shown potential as a therapeutic agent for these disorders. However, little is known about its protective effects against glutamate-induced toxicity in PC12 cells. Therefore, in this study, for the first time we aimed to investigate the ability of Urolithin B to reduce the cytotoxic effects of glutamate on PC12 cells. METHODS: Different non-toxic concentrations of urolithin B were applied to PC12 cells for 24 h before exposure to glutamate (10 mM). The cells were then analyzed for cell viability, intracellular reactive oxygen species (ROS), cell cycle arrest, apoptosis, and the expression of Bax and Bcl-2 genes. RESULTS: The results of MTT assay showed that glutamate at a concentration of 10 mM and urolithin B at a concentration of 114 µM can reduce PC12 cell viability by 50%. However, urolithin B at non-toxic concentrations of 4 and 8 µM significantly reduced glutamate-induced cytotoxicity (p < 0.01). Interestingly, treatment with glutamate significantly enhanced the intracellular ROS levels and apoptosis rate in PC12 cells, while pre-treatment with non-toxic concentrations of urolithin B significantly reduced these cytotoxic effects. The results also showed that pre-treatment with urolithin B can decrease the Bax (p < 0.05) and increase the Bcl-2 (p < 0.01) gene expression, which was dysregulated by glutamate. CONCLUSIONS: Taken together, urolithin B may play a protective role through reducing oxidative stress and apoptosis against glutamate-induced toxicity in PC12 cells, which merits further investigations.

Immunological mechanisms of the nucleocapsid protein in COVID-19.

The emergence of corona virus disease 2019 (COVID-19), resulting from Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has left an indelible mark on a global scale, causing countless infections and fatalities. This investigation delves into the role of the SARS-CoV-2 nucleocapsid (N) protein within the HEK293 cells, shedding light on its influence over apoptosis, interferon signaling, and cytokines production. The N gene was amplified, inserted into the pAdTrack-CMV vector, and then transfected to the HEK293 cells. Changes in the expression of IRF3, IRF7, IFN-ß, BAK, BAX, and BCL-2 genes were evaluated. The levels of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α were also determined. The N protein exhibited an anti-apoptotic effect by modulating critical genes associated with apoptosis, including BAK, BAX, and BCL-2. This effect potentially prolonged the survival of infected cells. The N protein also played a role in immune evasion by suppressing the interferon pathway, evidenced by the downregulation of essential interferon regulatory factors of IRF3 and IRF7, and IFN-ß expression. The N protein expression led to a substantial increase in the production of proinflammatory cytokines of IL-6, IL-12, IL-1ß, and TNF-α. The N protein emerged as a versatile factor and was exerted over apoptosis, interferon signaling, and cytokine production. These findings carry potential implications for the development of targeted therapies to combat COVID-19 and mitigate its global health impact.

Inhibition of vinculin activity has an adverse effect on porcine ovarian cells.

The existing knowledge of the involvement of vinculin (VCL) in the control of ovarian cell functions is insufficient. To understand the role of VCL in the control of basic porcine ovarian granulosa cell functions, we decreased VCL activity by small interfering RNA (VCL siRNA). The expression of VCL, accumulation of VCL protein, cell viability, proliferation (accumulation of PCNA and cyclin B1), proportion of proliferative active cells, apoptosis (accumulation of bax, caspase 3, p53, antiapoptotic marker bcl2, and bax/bcl-2 ratio), DNA fragmentation, and release of steroid hormones and IGF-I were analyzed by RT‒qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT assay, TUNEL assay, and ELISA. The suppression of VCL activity inhibited cell viability, the accumulation of the proliferation-related proteins PCNA and cyclin B1, the antiapoptotic protein bcl2, and the proportion of proliferative active cells. Moreover, VCL siRNA inhibited the release of progesterone, estradiol, and IGF-1. VCL siRNA increased the proportion of the proapoptotic proteins bax, caspase 3, p53, the proportion of DNA fragmented cells, and stimulated testosterone release. Taken together, the present study is the first evidence that inhibition of VCL suppresses porcine granulosa cell functions. Moreover, the results suggest that VCL can be a potent physiological stimulator of ovarian functions.

Splicing Factor PQBP1 Curtails BAX Expression to Promote Ovarian Cancer Progression.

Splicing factor polyglutamine binding protein-1 (PQBP1) is abundantly expressed in the central nervous system during development, and mutations in the gene cause intellectual disability. However, the roles of PQBP1 in cancer progression remain largely unknown. Here, it is shown that PQBP1 overexpression promotes tumor progression and indicates worse prognosis in ovarian cancer. Integrative analysis of spyCLIP-seq and RNA-seq data reveals that PQBP1 preferentially binds to exon regions and modulates exon skipping. Mechanistically, it is shown that PQBP1 regulates the splicing of genes related to the apoptotic signaling pathway, including BAX. PQBP1 promotes BAX exon 2 skipping to generate a truncated isoform that undergoes degradation by nonsense-mediated mRNA decay, thus making cancer cells resistant to apoptosis. In contrast, PQBP1 depletion or splice-switching antisense oligonucleotides promote exon 2 inclusion and thus increase BAX expression, leading to inhibition of tumor growth. Together, the results demonstrate an oncogenic role of PQBP1 in ovarian cancer and suggest that targeting the aberrant splicing mediated by PQBP1 has therapeutic potential in cancer treatment.

Effect of photodynamic therapy mediated by hematoporphyrin derivatives on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells.

To investigate the effects of photodynamic therapy (PDT) mediated by hematoporphyrin derivatives (HPD) on the proliferation of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells. H446 cells and BEAS-2B cells were cultured in vitro with different concentrations of HPD(0, 5, 10, 12, 15, 20 µg/mL) for 4 h, and then irradiated with 630 nm laser with different energy densities (0, 25, 50, 75, 100 mW/cm2). Cell viability of H446 cells and BEAS-2B cells were detected by CCK8 assay. The cell apoptosis was observed with Annexin V-FTTC/PI double staining and Hoechst 33258. The RT-PCR examination was applied to detect the transcriptional changes of the mRNA of Bax、Bcl-2, and Caspase-9. The results of CCK8 showed that when the HPD was 15 µg/mL and the laser power density reached 50 mW/cm2, the cell viability was significantly decreased compared with the black control group. Hoechst 33258 staining showed that with the increase of HPD concentration, the cell density was reduced, and apoptotic cells increased. Flow cytometry assay revealed that the apoptotic rates of the HPD-PDT group of H446 cells and BEAS-2B cells were significantly different from those of the blank control group. The RT-PCR examination showed that the expression levels of Bax and Caspase-9 mRNA in the HPD-PDT group were up-regulated, while the expression levels of Bcl-2 mRNA were down-regulated significantly. HPD-PDT can inhibit H446 cells and BEAS-2B cells growth. The mechanism may be related to up-regulating the expression levels of Bax and Caspase-9 mRNA and down-regulating the expression levels of Bcl-2 mRNA.

Gold nanoparticles (AuNPs) decrease the viability of cervical cancer cells by inducing the BAX gene and activating antioxidant enzymes.

BACKGROUND: Cervical Cancer (CC), a leading cause of female mortality worldwide, demonstrates a direct association with high-risk human papillomavirus (HPV) infections. However, not all CC patients exhibit HPV infection, suggesting additional predisposing factors. Recently, disturbances in the oxidant-antioxidant balance have been implicated in CC development. This study explores the impact of gold nanoparticles (AuNPs) on the survival and antioxidant capacity of HeLa cells, aiming to contribute to novel CC therapy approaches. METHODS AND RESULTS: Synthesized and characterized AuNPs (25.5 nm, uniform distribution according to the DLS analysis) were administered to HeLa cells at varying concentrations. After 24 h, cell viability was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) (MTT) assay. Real-time PCR measured expression levels of apoptosis-related genes (BCL2 associated X (BAX) and p53). Catalase and superoxide dismutase (SOD) activities, key antioxidant enzymes, were also evaluated post-AuNP treatment. AuNPs dose-dependently reduced HeLa cell viability, with an IC50 value of 113 µg/ml. BAX gene expression significantly increased, indicating pro-apoptotic effects. Moreover, enzyme activities significantly rose under AuNP influence. CONCLUSIONS: AuNPs demonstrated the potential to induce HeLa cell death by upregulating pro-apoptotic BAX gene expression and altering antioxidant system enzyme activities. These findings underscore the promise of AuNPs as a therapeutic avenue for CC, emphasizing their impact on crucial cellular processes involved in cancer progression.