Long noncoding RNA KCNMA1-AS2 regulates the function of colorectal cancer cells and sponges miR-1227-5p.

BACKGROUND: Many long noncoding RNAs (lncRNAs) with altered expression significantly influence colorectal cancer (CRC) progression and behavior. The functions of many lncRNAs in CRC are not clear yet. This study aimed to discover novel lncRNA entities and comprehensively examine and validate their roles and underlying molecular mechanisms in CRC. METHODS: Tissue samples, both tumourous and non-tumourous, from three CRC patients were submitted for sequencing. Following expression validation in samples from ten patients and four CRC cell lines. The lncRNA KCNMA1-AS2 was synthesized by In-vitro transcription RNA synthesis and the lncRNA was directly transfected into CRC cell lines to overexpress. Functional assays including MTT proliferation assay, Annexin-V/propidium iodide apoptosis assay, wound healing migration assay and cell cycle assays were performed to evaluate the effect of overexpression of KCNMA1-AS2. Furthermore, the binding of KCNMA1-AS2 to miR-1227-5p was confirmed using dual luciferase reporter assays and qPCR analyses. Subsequent bioinformatics analyses identified 58 potential downstream targets of miR-1227-5p across three databases. RESULTS: In this study, we identified the lncRNA KCNMA1-AS2, the expression of which was down-regulated consistently in cancer tissues and CRC cell lines compared to non-cancerous tissues. The overexpression of lncRNA KCNMA1-AS2 led to significant reduction in CRC cell proliferation and migration, increase in cell apoptosis, and more cells arrested in S phase. Additionally, the interaction between KCNMA1-AS2 and miR-1227-5p was confirmed through dual luciferase reporter assay and qPCR analysis. It is also putatively predicted that MTHFR and ST8SIA2 may be linked to CRC based on bioinformatics analyses. CONCLUSIONS: LncRNA KCNMA1-AS2 exhibited distinct gene expression patterns in both CRC tissue and cell lines, impacting various cellular functions while also acting as a sponge for miR-1227-5p.The findings spotlight lncRNA KCNMA1-AS2 as a potential marker for diagnosis and treatment of CRC.

Independent and joint associations of multiple metals exposure with vital capacity index: a cross-sectional study in Chinese children and adolescents.

OBJECTIVE: The current study aimed to explore the relationships between urinary metals and vital capacity index (VCI) in 380 children and adolescents in Northeast China using a variety of statistical methods. METHODS: A cross-sectional survey was conducted among 380 children and adolescents in Liaoning Province, China. To assess the relationships between urinary metals and VCI, Elastic-net (ENET) regression, multivariate linear regression, weighted quantile sum (WQS), bayesian kernel machine regression (BKMR) and quantile-based g computation (qgcomp) were adopted. RESULTS: The ENET model selected magnesium (Mg), vanadium (V), manganese (Mn), arsenic (As), tin (Sn) and lead (Pb) as crucial elements. In multiple linear regression, we observed urinary Pb, Mn was negatively correlated with VCI individually in both total study population and adolescents (all p values < 0.05) in the adjustment model. The WQS indices were negatively related with VCI in total study population (ß=-3.19, 95%CI: -6.07, -0.30) and adolescents (ß=-3.46, 95%CI: -6.58, -0.35). The highest weight in total study population was Pb (38.80%), in adolescents was Mn (35.10%). In the qgcomp, Pb (31.90%), Mn (27.20%) were the major negative contributors to the association in the total population (ß=-3.51, 95%CI: -6.29, -0.74). As (42.50%), Mn (39.90%) were the main negative contributors (ß=-3.95, 95% CI: -6.68, -1.22) among adolescents. The results of BKMR were basically consistent with WQS and qgcomp analyses. CONCLUSIONS: Our results indicated that Pb and Mn were priority toxic materials on VCI. The cumulative effect of metals was negatively related to VCI, and this relationship was more pronounced in adolescents.

Global, regional, and national burden of cancers attributable to particulate matter pollution from 1990 to 2019 and projection to 2050: Worsening or improving?

Particulate matter pollution (PMP) has been identified as a substantial contributor to cancer. However, accurately delineating the evolving trends in cancer burden attributable to PMP remains an ongoing challenge. The 1990-2019 disability-adjusted life years (DALYs) were used for cancers attributable to PMP from the Global Burden and Disease Study (GBD) 2019, including ambient particulate matter pollution (APMP) and household air pollution from solid fuels (HAP). The joinpoint regression and the Bayesian age-period-cohort (BAPC) model were employed to assess the corresponding trends over the periods 1990-2019 and 2020-2050, respectively. Additionally, statistical models such as frontier analysis and health inequality analysis were also utilized. During the 30-year period, cancer DALYs attributable to APMP increased globally, while those attributable to HAP and PMP decreased. Cancer DALYs attributable to APMP were positively correlated with socio-demographic index (SDI), while those attributable to PMP and HAP were negatively correlated with SDI. Frontier analysis identified the countries and regions requiring urgent action to mitigate PMP-attributable cancer. Finally, it was anticipated that the cancer burden attributable to APMP would increase during 2020 to 2050, while the burden attributable to HAP and PMP would decrease. This study conducted an epidemiological investigation of the burden of cancer attributable to APMP, HAP and PMP in various regions and populations worldwide, providing epidemiological insights into the global burden of cancer attributable to PMP and guiding policy and research directions.

Genistein-3'-sodium sulfonate promotes brain functional rehabilitation in ischemic stroke rats by regulating astrocytes polarization through NF-κB signaling pathway.

The activation and polarization of astrocytes are involved in neuroinflammation and brain functional rehabilitation after ischemic stroke. Our previous studies display the neuroprotective effect of genistein-3'-sodium sulfonate (GSS) in the acute phase of cerebral ischemia-reperfusion injury (CI/RI). This study aimed to investigate the brain function improvement of GSS during the recovery period after CI/RI in rats and to explore the potential mechanism from the perspective of astrocyte activation and polarization. The transient middle cerebral artery occlusion (tMCAO) rats were treated with GSS (1 mg/kg) continuously for 28 days. The behavior tests were measured to assess neurological function. The mRNA and protein expression in affected cerebral cortex were detected on day 29 after tMCAO. Our results demonstrated that GSS treatment significantly improved the spatial and temporal gait parameters in the Catwalk gait test, prolonged the time on the stick and increased the rotation speed in the rotarod test, and decreased the time to find the hidden platform and increased the time in the target quadrant in the Morris water maze test. In addition, GFAP, GBP2, C3, IL-1ß protein expressions and Nos2A mRNA level were decreased, while Nrf2, BDNF, IL-10 protein expressions and Sphk1 and Nef2l2 mRNA levels increased after GSS treatment. Interestingly, GSS presented a strong binding affinity to TLR4 and suppressed the activation of NF-κB signaling. In conclusion, GSS can promote brain function recovery by inhibiting astrocyte activation and polarization to A1 phenotype, and enhancing astrocyte polarization to A2 phenotype via inactivating TLR4/NF-κB signaling, which provide a candidate compound for clinical rehabilitation therapy in the recovery period after ischemic stroke.

Optimization of extended Kozak elements enhances recombinant proteins expression in CHO cells.

In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.

Selective Detection of Dynamics-Complete Set of Correlations via Quantum Channels.

Correlations of fluctuations are essential to understanding many-body systems and key information for advancing quantum technologies. To fully describe the dynamics of a physical system, all time-ordered correlations (TOCs), i.e., the dynamics-complete set of correlations are needed. The current measurement techniques can only access a limited set of TOCs, and there has been no systematic and feasible solution for extracting the dynamic-complete set of correlations hitherto. Here we propose a platform-universal protocol to selectively detect arbitrary types of TOCs via quantum channels. In our method, the quantum channels are synthesized with various controls, and engineer the evolution of a sensor-target system along a specific path that corresponds to a desired correlation. Using nuclear magnetic resonance, we experimentally demonstrate this protocol by detecting a specific type of fourth-order TOC that has never been accessed previously. We also show that the knowledge of the TOCs can be used to significantly improve the precision of quantum optimal control. Our method provides a new toolbox for characterizing the quantum many-body states and quantum noise, and hence for advancing the fields of quantum sensing and quantum computing.

Embryonic origin of two ASD subtypes of social symptom severity: the larger the brain cortical organoid size, the more severe the social symptoms.

BACKGROUND: Social affective and communication symptoms are central to autism spectrum disorder (ASD), yet their severity differs across toddlers: Some toddlers with ASD display improving abilities across early ages and develop good social and language skills, while others with "profound" autism have persistently low social, language and cognitive skills and require lifelong care. The biological origins of these opposite ASD social severity subtypes and developmental trajectories are not known. METHODS: Because ASD involves early brain overgrowth and excess neurons, we measured size and growth in 4910 embryonic-stage brain cortical organoids (BCOs) from a total of 10 toddlers with ASD and 6 controls (averaging 196 individual BCOs measured/subject). In a 2021 batch, we measured BCOs from 10 ASD and 5 controls. In a 2022 batch, we  tested replicability of BCO size and growth effects by generating and measuring an independent batch of BCOs from 6 ASD and 4 control subjects. BCO size was analyzed within the context of our large, one-of-a-kind social symptom, social attention, social brain and social and language psychometric normative datasets ranging from N = 266 to N = 1902 toddlers. BCO growth rates were examined by measuring size changes between 1- and 2-months of organoid development. Neurogenesis markers at 2-months were examined at the cellular level. At the molecular level, we measured activity and expression of Ndel1; Ndel1 is a prime target for cell cycle-activated kinases; known to regulate cell cycle, proliferation, neurogenesis, and growth; and known to be involved in neuropsychiatric conditions. RESULTS: At the BCO level, analyses showed BCO size was significantly enlarged by 39% and 41% in ASD in the 2021 and 2022 batches. The larger the embryonic BCO size, the more severe the ASD social symptoms. Correlations between BCO size and social symptoms were r = 0.719 in the 2021 batch and r = 0. 873 in the replication 2022 batch. ASD BCOs grew at an accelerated rate nearly 3 times faster than controls. At the cell level, the two largest ASD BCOs had accelerated neurogenesis. At the molecular level, Ndel1 activity was highly correlated with the growth rate and size of BCOs. Two BCO subtypes were found in ASD toddlers: Those in one subtype had very enlarged BCO size with accelerated rate of growth and neurogenesis; a profound autism clinical phenotype displaying severe social symptoms, reduced social attention, reduced cognitive, very low language and social IQ; and substantially altered growth in specific cortical social, language and sensory regions. Those in a second subtype had milder BCO enlargement and milder social, attention, cognitive, language and cortical differences. LIMITATIONS: Larger samples of ASD toddler-derived BCO and clinical phenotypes may reveal additional ASD embryonic subtypes. CONCLUSIONS: By embryogenesis, the biological bases of two subtypes of ASD social and brain development-profound autism and mild autism-are already present and measurable and involve dysregulated cell proliferation and accelerated neurogenesis and growth. The larger the embryonic BCO size in ASD, the more severe the toddler's social symptoms and the more reduced the social attention, language ability, and IQ, and the more atypical the growth of social and language brain regions.

Acevaltrate promotes apoptosis and inhibits proliferation by suppressing HIF-1α accumulation in cancer cells.

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.

Overexpression of YTHDF3 increases the specific productivity of the recombinant protein in CHO cells by promoting the translation process.

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A.

Synthetic enhancers including TFREs improve transgene expression in CHO cells.

The human cytomegalovirus major immediate early gene (CMV) promoter is currently the most preferred promoter for recombinant therapeutic proteins (RTPs) production in CHO cells. To enhance the production of RTPs, five synthetic enhancers including multiple transcription factor regulatory elements (TFREs) were evaluated to enhance recombinant protein level in transient and stably transfected CHO cells. Compared with the control, four elements can enhance the report genes expression under both two transfected states. Further, the function of these four enhancers on human serum albumin (HSA) were investigated. We found that the transient expression can increase by up to 1.5 times, and the stably expression can maximum increase by up to 2.14 times. The enhancement of transgene expression was caused by the boost of their corresponding mRNA levels. Transcriptomics analysis was performed and found that transcriptional activation and cell cycle regulation genes were involved. In conclusion, optimization of enhancers in the CMV promoter could increase the production yield of transgene in transfected CHO cells, which has significance for developing high-yield CHO cell expression system.

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37 °C and PDI/XBP-1s/37 °C, the Adalimumab volume yield of PDI/XBP-1s/33 °C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.

Neurodevelopmental proteasomopathies represent a distinctive category of neurodevelopmental disorders (NDD) characterized by genetic variations within the 26S proteasome, a protein complex governing eukaryotic cellular protein homeostasis. In our comprehensive study, we identified 23 unique variants in PSMC5 , which encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, causing syndromic NDD in 38 unrelated individuals. Overexpression of PSMC5 variants altered human hippocampal neuron morphology, while PSMC5 knockdown led to impaired reversal learning in flies and loss of excitatory synapses in rat hippocampal neurons. PSMC5 loss-of-function resulted in abnormal protein aggregation, profoundly impacting innate immune signaling, mitophagy rates, and lipid metabolism in affected individuals. Importantly, targeting key components of the integrated stress response, such as PKR and GCN2 kinases, ameliorated immune dysregulations in cells from affected individuals. These findings significantly advance our understanding of the molecular mechanisms underlying neurodevelopmental proteasomopathies, provide links to research in neurodegenerative diseases, and open up potential therapeutic avenues.

Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

Dual blockade of CD47 and CD24 signaling using a novel bispecific antibody fusion protein enhances macrophage immunotherapy.

CD47 and its receptor signal regulatory protein α (SIRPα) act as a dominant antiphagocytic, "don't eat me" signal. Recent studies reveal CD24 as a novel target for cancer immunotherapy by macrophages in ovarian cancer and breast cancer. However, whether simultaneous blockade of CD47 and CD24 by a bispecific antibody may result in a potential synergy is still unclear. In the present study, we for the first time designed and developed a bispecific antibody fusion protein, PPAB001 for cotargeting CD47 and CD24. Data demonstrate that simultaneous blockade of CD47/SIRPα and CD24/Siglec-10 signaling by PPAB001 potently promoted macrophage phagocytosis of tumor cells. Compared to single CD47 or CD24 targeting agents, PPAB001 was more effective in inhibiting tumor growth in both mouse 4T-1 syngeneic and human SK-OV-3 xenogeneic tumor models. Mechanistically, we found that PPAB001 therapy markedly increased the proportion of tumor-infiltrating macrophages and upregulated interleukin-6 and tumor necrosis factor-α levels that were representative macrophage inflammatory cytokines. Notably, an increased ratio of M1/M2 in tumor-infiltrating macrophages in the mice treated with PPAB001 suggested that the dual blockade may promote the transition of macrophages from M2 to M1. Taken together, our data supported the development of PPAB001 as a novel immunotherapeutic in the treatment of CD47 and CD24 double-positive cancers.

Effects of Ambient O3 on Respiratory Mortality, Especially the Combined Effects of PM2.5 and O3.

BACKGROUND: In China, the increasing concentration of ozone (O3) has emerged as a significant air pollution issue, leading to adverse effects on public health, particularly the respiratory system. Despite the progress made in managing air pollution in China, it is crucial to address the problem of environmental O3 pollution at present. METHODS: The connection between O3 exposure and respiratory mortality in Shenyang, China, from 2014 to 2018 was analyzed by a time-series generalized additive regression model (GAM) with quasi-Poisson regression. Additionally, the potential combined effects of fine particulate matter (PM2.5) and O3 were investigated using the synergy index (SI). RESULTS: Our findings indicate that each 10 µg/m3 increase in O3 at lag 2 days was associated with a maximum relative risk (RR) of 1.0150 (95% CI: 1.0098-1.0202) for respiratory mortality in the total population. For individuals aged ≥55 years, unmarried individuals, those engaged in indoor occupations, and those with low educational attainment, each 10 µg/m3 increase in O3 at lag 07 days was linked to RR values of 1.0301 (95% CI: 1.0187-1.0417), 1.0437 (95% CI: 1.0266-1.0610), 1.0317 (95% CI: 1.0186-1.0450), and 1.0346 (95% CI: 1.0222-1.0471), respectively. Importantly, we discovered a synergistic effect of PM2.5 and O3, resulting in an SI of 2.372 on the occurrence of respiratory mortality. CONCLUSIONS: This study confirmed a positive association between O3 exposure and respiratory mortality. Furthermore, it highlighted the interaction between O3 and PM2.5 in exacerbating respiratory deaths.

CMV/AAT promoter of MAR-based episomal vector enhanced transgene expression in human hepatic cells.

We have previously developed a non-viral episomal vector based on matrix attachment region (MAR) that can facilitate plasmid replication episomally in mammal cells. In this study, we have focused on the development of an alternative tissue specific episomal vector by incorporating into cis-acting elements. We found that AAT promoter demonstrated the highest eGFP expression level in HepG2, Huh-7 and HL-7702 hepatic cells. Furthermore, hCMV enhancer when combined with AAT promoter significantly improved the eGFP expression level in the transfected HepG2 cells. The mean fluorescence intensity of eGFP in hCMV2 group was 1.33 fold, which was higher than that of the control (p < 0.01), followed by the hCMV1 group (1.21 fold). In addition, the percentages of eGFP-expressing cells in hCMV1 and hCMV2 groups were observed to be 49.3% and 57.2%, which were significantly higher than that of the enhancer-devoid control vector (44.3%) (p < 0.05). Moreover, the eGFP protein were up to 3.5 fold and 5.1 fold (p < 0.05), respectively. This observation could be related with the activities of some specific transcription factors (TFs) during the transcriptional process, such as SRF, REL and CREB1. The composite CMV/AAT promoter can be thus used for efficient transgene expression of MAR-based episomal vector in liver cells and as a potential gene transfer tools for the management of liver diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03774-x.

Case report: A novel frameshift mutation in BRSK2 causes autism in a 16-year old Chinese boy.

Serine/threonine protein kinases are involved in axon formation and neuronal polarization and have recently been implicated in autism spectrum disorder (ASD) and neurodevelopmental disorders (NDD). Here, we focus on BRSK2, which encodes brain-specific serine/threonine protein kinase 2. Although previous studies have reported 19 unrelated patients with BRSK2 pathogenic variation, only 15 of 19 patients have detailed clinical data. Therefore, more case reports are needed to enrich the phenotype associated with BRSK2 mutations. In this study, we report a novel de novo frameshift variant (c.442del, p.L148Cfs*39) identified by exome sequencing in a 16 year-old Chinese boy with ASD. The proband presented with attention-deficit, auditory hallucinations, limb tremor, and abnormal brain electrical activity mapping. This study expands the phenotypic spectrum of BRSK2-related cases and reveals the highly variable severity of disorders associated with BRSK2.

Effect of Host Cell Protein on Chinese Hamster Ovary Recombinant Protein Production and its Removal Strategies: A Mini Review.

Chinese hamster ovary cells are the main expression system for recombinant therapeutic proteins. During the production of these proteins, certain host cell proteins are secreted, broken down, and released by host cells in the culture along with the proteins of interest. These host cell proteins are often difficult to remove during the downstream purification process, and thus affect the quality, safety, and effectiveness of recombinant protein biopharmaceutical products and increase the production cost of recombinant therapeutic proteins. Therefore, host cell protein production must be reduced as much as possible during the production process and eliminated during purification. This article reviews the harm caused by host cell proteins in the production of recombinant protein drugs using Chinese hamster ovary cell, factors affecting host cell proteins, the monitoring and identification of these proteins, and methods to reduce their type and quantity in the final product.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.

Multifunctional calcium polyphenol networks reverse the hostile microenvironment of trauma for preventing postoperative peritoneal adhesions.

Abdominal adhesions, a commonly observed complication of abdominal surgery, have a high incidence and adversely affect patients' physical and mental health. The primary causes of abdominal adhesions are intraoperative trauma, acute inflammatory response, bleeding, and foreign body infection. Because most current treatment approaches for abdominal adhesions are limited, improved and novel postoperative anti-adhesion regimens are urgently needed. In this study, we developed calcium polyphenol network (CaPN) microspheres based on the self-assembly of the natural triphenolic compound gallic acid and Ca2+ in solution. The physicochemical properties of CaPNs, including their hemostatic, antibacterial, antioxidant, and anti-inflammatory activities, were investigated in vitro. Bleeding and cecal-abdominal wall adhesion models were established to observe the hemostatic activity of CaPNs and their preventive effect on postoperative abdominal wall adhesion in vivo. The results showed that CaPNs significantly reduced inflammation, oxidative stress, fibrosis, and abdominal adhesion formation and had good hemostatic and antibacterial properties. Our findings suggest a novel strategy for the prevention of postoperative adhesions.