A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells.

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 µM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells. KEY POINTS: • HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells. • By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased. • It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Microbial-mediated metal(loid) immobilization in mulch-covered tailings.

Mulch coverage of mining tailings can create anaerobic conditions and consequently establish an anoxic environment that promotes the metabolic processes of anaerobic microorganisms. This anoxic environment has the potential to decrease heavy metal mobility and bioavailability. While tailings exposed to sunlight have been extensively studied, research on the effects of microbial-mediated geochemical cycling of heavy metals in mulch-covered tailings is scarce. This study aimed to examine the effects of mulch coverage-induced alterations in the structures of tailing microbial communities on the biogeochemical processes associated with heavy metals. Mulch coverage significantly reduced the pH of the tailings and the tailings exhibited heavy metal bioavailability. Random forest analysis demonstrated that mulch coverage-induced changes in the As/Cd-contaminated fractions and nutrients (total organic carbon and total nitrogen) were the most crucial predictors of microbial diversity and ecological clusters in the tailings. Notably, different from direct metal(loid) immobilization, mulch coverage can facilitate heavy metal immobilization in tailings by promoting microbial-mediated Fe, S, and As reduction. Overall, this study demonstrated that mulch coverage of tailings contributed to a reduction in heavy metal mobilization, which can be attributed to shifts in microbial-mediated Fe, S, and As reduction processes.The study provides valuable insights into the potential of mulch coverage as a remediation strategy and underscores the importance of microbial-mediated processes in managing heavy metal pollution in tailing systems.

Preparation of Hematoporphyrin-Poly(Lactic Acid) Nanoparticles Encapsulated Perfluoropentane/Salicylic Acid for Enhanced US/CEST MR Bimodal Imaging.

Background: Medical imaging modalities, such as magnetic resonance imaging (MRI), ultrasound, and fluorescence imaging, have gained widespread acceptance in clinical practice for tumor diagnosis. Each imaging modality has its own unique principles, advantages, and limitations, thus necessitating a multimodal approach for a comprehensive disease understanding of the disease process. To enhance diagnostic precision, physicians frequently integrate data from multiple imaging modalities, driving research advancements in multimodal imaging technology research. Methods: In this study, hematoporphyrin-poly (lactic acid) (HP-PLLA) polymer was prepared via ring-opening polymerization and thoroughly characterized using FT-IR, 1H-NMR, XRD, and TGA. HP-PLLA based nanoparticles encapsulating perfluoropentane (PFP) and salicylic acid were prepared via emulsion-solvent evaporation. Zeta potential and mean diameter were assessed using DLS and TEM. Biocompatibility was evaluated via cell migration, hemolysis, and cytotoxicity assays. Ultrasonic imaging was performed with a dedicated apparatus, while CEST MRI was conducted using a 7.0 T animal scanner. Results: We designed and prepared a novel dual-mode nanoimaging probe SA/PFP@HP-PLLA NPs. PFP enhanced US imaging, while salicylic acid bolstered CEST imaging. With an average size of 74.43 ± 1.12 nm, a polydispersity index of 0.175 ± 0.015, and a surface zeta potential of -64.1 ± 2.11 mV. These NPs exhibit excellent biocompatibility and stability. Both in vitro and in vivo experiments confirmed the SA/PFP@HP-PLLA NP's ability to improve tumor characterization and diagnostic precision. Conclusion: The SA/PFP@HP-PLLA NPs demonstrate promising dual-modality imaging capabilities, indicating their potential for preclinical and clinical use as a contrast agent.

Spatio-seasonal patterns and sources of major ions in the Longjiang River catchment, Southern China.

River water quality is closely related to the major ion sources and hydrological conditions. However, there is a limited cognition about the geochemical sources and the seasonal variations of major ions. Thus, in this study, a total of 90 water samples were collected from the Longjiang River and its three tributaries in the dry and wet seasons. The samples were analyzed, including major ion concentrations and physicochemical parameters. Statistical analysis, such as correlation analysis and principal component analysis (PCA), was employed to investigate the spatial and seasonal variations in major ion composition and their respective sources. Our study revealed that the predominant major ions in the studied samples are Ca2+, Mg2+, HCO - 3, and SO2 - 4. Most of ions exhibited notable spatial disparities attributable to variations in geological settings and human activities. Regions characterized by igneous rock outcrops tend to exhibit higher levels of K+ and Na+, while areas with higher population densities in the middle and downstream segments show elevated concentrations of Cl-, NO - 3, SO2 - 4, Na+, and K+. The observed peak SO2 - 4 levels may be attributed to active mining operations. Most parameters displayed higher values in flood season than those in dry season due to dilution effects. Stoichiometric analysis indicated that carbonate weathering inputs contribute to over 85% of the mean total cation concentrations in the water, followed by contributions from silicates, atmospheric deposition, and anthropogenic inputs. On the whole, although the water quality remains non-polluted and is suitable for drinking and irrigation purposes, the enrichment of SO2 - 4 and NO - 3 may contribute to water eutrophication. Caution is warranted during the dry season due to reduced water flow resulting from dam interceptions and limited dilution capacity, potentially leading to elevated pollutant concentrations. Taken together, our results provided a scientific basis for water quality managements of monsoon rivers.

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.

The rhizosphere microbiome reduces the uptake of arsenic and tungsten by Blechnum orientale by increasing nutrient cycling in historical tungsten mining area soils.

The growth of pioneer plants in metal mining area soil is closely related to their minimal uptake of toxic elements. Pioneer plants can inhibit the uptake of toxic elements by increasing nutrient uptake. However, few studies have focused on the mechanisms by which the rhizosphere microbiome affect nutrient cycling and their impact on the uptake of toxic elements by pioneer plants. In this study, we selected Blechnum orientale to investigate the potential roles of the rhizosphere microbiome in nutrient cycling and plant growth in a historical tungsten (W) mining area. Our results showed that while the arsenic (As) and W contents in the soil were relatively high, the enrichment levels of As and W in the B. orientale were relatively low. Furthermore, we found that the As and W contents in plants were significantly negatively correlated with soil nutrients (S, P and Mo), suggesting that elevated levels of these soil nutrients could inhibit As and W uptake by B. orientale. Importantly, we found that these nutrients were also identified as the most important factors shaping rhizosphere microbial attributes, including microbial diversity, ecological clusters, and keystone OTUs. Moreover, the genera, keystone taxa and microbial functional genes enriched in the rhizosphere soils from mining areas played a key role in nutrient (S, P and Mo) bioavailability, which could further increase the nutrient uptake by B. orientale. Taken together, our results suggest that rhizosphere microorganisms can improve pioneer plant growth by inhibiting toxic element accumulation via the increase in nutrient cycling in former W mining areas.

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.

Novel Nanoprobe with Combined Ultrasonography/Chemical Exchange Saturation Transfer Magnetic Resonance Imaging for Precise Diagnosis of Tumors.

Given that cancer mortality is usually due to a late diagnosis, early detection is crucial to improve the patient's results and prevent cancer-related death. Imaging technology based on novel nanomaterials has attracted much attention for early-stage cancer diagnosis. In this study, a new block copolymer, poly(ethylene glycol)-poly(l-lactide) diblock copolymer (PEG-PLLA), was synthesized by the ring-opening polymerization method and thoroughly characterized using Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy (H-NMR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The obtained PEG-PLLA was used to prepare nanoparticles encapsulated with perfluoropentane and salicylic acid by the emulsion-solvent evaporation method, resulting in a new dual-mode nano-image probe (PEG-PLLA@SA·PFP). The zeta potential and mean diameter of the obtained nanoparticles were measured using dynamic light scattering (DLS) with a Malvern Zetersizer Nano. The in vitro biocompatibility of the PEG-PLLA nanoparticles was evaluated with cell migration, hemolysis, and cytotoxicity assays. Ultrasonic imaging was performed using an ultrasonic imaging apparatus, and chemical exchange saturation transfer (CEST) MRI was conducted on a 7.0 T animal scanner. The results of IR and NMR confirmed that the PEG-PLLA was successfully synthesized. The particle size and negative charge of the nanoparticles were 223.8 ± 2.5 nm and -39.6 ± 1.9 mV, respectively. The polydispersity of the diameter was 0.153 ± 0.020. These nanoparticles possessed good stability at 4 °C for about one month. The results of cytotoxicity, cell migration, and hemolysis assays showed that the carrier material was biocompatible. Finally, PEG-PLLA nanoparticles were able to significantly enhance the imaging effect of tumors by the irradiation of ultrasound and saturation by a radiofrequency pulse, respectively. In conclusion, these nanoparticles exhibit promising dual-mode capabilities for US/CEST MR imaging.

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.

Danggui Buxue decoction alleviates cyclophosphamide-induced myelosuppression by regulating ß-hydroxybutyric acid metabolism and suppressing oxidative stress.

CONTEXT: Danggui Buxue Decoction (DBD) is an effective complementary medicine in alleviating myelosuppression after chemotherapy (MAC). However, its mechanism of action is elusive. OBJECTIVE: To illustrate that regulating ß-hydroxybutyric acid (ß-OHB) metabolism and suppressing oxidative stress could be a potential mechanism of action for DBD in alleviating MAC. MATERIALS AND METHODS: After HPLC quantification and dose testing (3, 6 and 10 g/kg, gavage) of DBD, Sprague-Dawley rats were divided into control, cyclophosphamide (CTX) (30 mg/kg CTX for 5 days, intraperitoneal administration) and CTX + DBD groups (6 g/kg DBD for 14 days, gavage). Blood cell counts, thigh bone histological examination, ß-OHB levels, oxidative stress indices and HDAC1 activity were tested. The biological function of ß-OHB was verified in vitro (hBMSC cells were incubated in culture mediums that contained 40 µM CTX and ß-OHB in 0, 1, 2.5, 5, 10 mM) and in vivo (MAC rat model, 3 g/kg ß-OHB for 14 days, gavage). RESULTS: Rats in the CTX + DBD group showed upregulated blood cell counts (118-243%), ß-OHB levels (495 nmol/mL in blood, 122 nmol/mg in marrow supernatant) and downregulated HDAC1 activity (59%), and oxidative stress indices (60-85%). In vitro, 5 mM ß-OHB improved hBMSC cell migration (123%) and proliferation (131%). In vivo, rats treated with 3 g/kg ß-OHB showed upregulated blood cell counts (121-182%) and downregulated HDAC1 activity (64%) and oxidative stress indices (65-83%). DISCUSSION AND CONCLUSIONS: DBD, a traditional Chinese medicine, alleviates MAC by intervening in ß-OHB metabolism and oxidative stress.

Deep learning to reconstruct quasi-steady-state chemical exchange saturation transfer from a non-steady-state experiment.

The insufficiently long RF saturation duration and relaxation delay in chemical exchange saturation transfer (CEST)-MRI experiments may result in underestimation of CEST measurements. To maintain the CEST effect without prolonging the saturation duration and reach quasi-steady state (QUASS), a deep learning method was developed to reconstruct a QUASS CEST image pixel by pixel from non-steady-state CEST acquired in experiments. In this work, we established a tumor-bearing rat model on a 7 T horizontal bore small-animal MRI scanner, allowing ground-truth generation, after which a bidirectional long short-term memory network was formulated and trained on simulated CEST Z-spectra to reconstruct the QUASS CEST; finally, the ground truth yielded by experiments was used to evaluate the performance of the reconstruction model by comparing the estimates with the ground truth. For quantitation evaluation, linear regression analysis, structural similarity index (SSIM) and peak signal-to-noise ratio (peak SNR) were used to assess the proposed model in the QUASS CEST reconstruction. In the linear regression analysis of in vivo data, the coefficient of determination for six different representative frequency offsets was at least R2 = 0.9521. Using the SSIM and peak SNR as evaluation metrics, the reconstruction accuracies of in vivo QUASS CEST were found to be 0.9991 and 46.7076, respectively. Experimental results demonstrate that the proposed model provides a robust and accurate solution for QUASS CEST reconstruction using a deep learning mechanism.

[Advances in epigenetic regulation of Chinese hamster ovary cells].

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

Quantitative morphometric changes in vascular mild cognitive impairment patients: early diagnosis of dementia.

Vascular mild cognitive impairment (VMCI) is an early and reversible stage of dementia. Volume differences in regional gray matter may reveal the development and prognosis of VMCI. This study selected 2 of the most common types of VMCI, namely, periventricular white matter hyperintensities (PWMH, n = 14) and strategic single infarctions (SSI, n = 10), and used the voxel-based morphometry method to quantify their morphological characteristics. Meanwhile, age- and sex-matched healthy volunteers were included (n = 16). All the participants were neuropsychologically tested to characterize their cognitive function and underwent whole-brain magnetic resonance imaging scanning. Our results showed that the volumes of the bilateral temporal lobes and bilateral frontal gray matter were obviously diminished in the PWMH group. The atrophy volume difference was 4,086 voxels in the left temporal lobe, 4,154 voxels in the right temporal lobe, 1,718 voxels in the left frontal lobe, and 1,141 voxels in the right frontal lobe (P ≤ 0.001). Moreover, the characteristics of the gray matter atrophy associated with the PWMH were more similar to those associated with Alzheimer's disease than SSI, which further revealed the susceptibility for escalation from PWMH to dementia. In conclusion, PWMH patients and SSI patients have different morphological characteristics, which explain the different prognoses of VMCI.

Synthetic phase-sensitive inversion-recovery vessel for assessing extramural venous invasion in patients with rectal cancer: imaging quality and added value to T2-wighted imaging.

OBJECTIVES: To evaluate the imaging quality of a synthetic phase-sensitive inversion recovery (SyPSIR) vessel and to add value to T2-weighted imaging (T2WI) for extramural venous invasion (EMVI) detection in patients with rectal cancer. METHODS: Participants in this retrospective study underwent preoperative synthetic MRI between October 2020 and April 2022. SyPSIR image reconstruction was performed with a single inversion time of 10 ms. A junior and a senior radiologist evaluated the imaging quality, including overall imaging quality scores, motion artifact scores, and relative image signal intensity contrast between the tumor and peritumoral vessels (SItumor-vessel), of both T2WI and SyPSIR vessels. Differences in imaging quality between the two methods were assessed using the Wilcoxon signed-rank test and two-sample t-test. EMVI scores were recorded for T2WI and T2WI+SyPSIR vessel. The area under the receiver operating characteristic curve (AUC) was calculated to evaluate the diagnostic performance. RESULTS: A total of 106 patients (35 EMVI+ and 71 EMVI-) were evaluated. There were no statistically significant differences in the overall image quality scores, motion artifacts, or SItumor-vessel (p = 0.08-0.93) between the T2WI and SyPSIR vessels. On combining T2WI and SyPSIR vessels, the AUC for pathological EMVI+ diagnoses increased from 0.65 to 0.88 for the junior radiologist and from 0.86 to 0.96 for the senior radiologist. Furthermore, the sensitivity of the analyses by junior and senior radiologists increased from 0.40 to 0.77 and 0.49 to 0.86, respectively. CONCLUSION: A SyPSIR vessel can provide additional information to improve the diagnostic efficiency of pathological EMVI in rectal cancer, which may be beneficial for individualized clinical treatment. KEY POINTS: • SyPSIR vessel and T2WI had similar imaging quality. • EMVI evaluation in SyPSIR vessel has a high inter-observer agreement. • The SyPSIR vessel has the potential to improve the diagnostic efficiency of EMVI detection in rectal cancer.

Apilimod enhances specific productivity in recombinant CHO cells through cell cycle arrest and mediation of autophagy.

Chinese hamster ovary (CHO) cells are expected to acquire the ability to produce higher recombinant therapeutic protein levels using various strategies. Genetic engineering targeting the cell cycle and autophagy pathways in the regulation of cell death in CHO cell cultures has received attention for enhancing the production of therapeutic proteins. In this study, we examined the small-molecule compound apilimod, which was found to have a positive influence on recombinant protein expression in CHO cells. This was confirmed by selective blocking of the cell cycle at the G0/G1 phase. Apilimod treatment resulted in decreased expression of cyclin-dependent kinase 3 (CDK3) and Cyclin C and increased expression of cyclin-dependent kinase suppressor p27Kip1, which are critical regulators of G1 cell cycle progression and important targets controlling cell proliferation. Furthermore, total transcription factor EB (TFEB) was lower in apilimod-treated CHO cells than in control cells, resulting in decreased lysosome biogenesis and autophagy with apilimod treatment. These multiple effects demonstrate the potential of apilimod for development as a novel enhancer for the production of recombinant proteins in CHO cell engineering.

Arsenic shapes the microbial community structures in tungsten mine waste rocks.

Tungsten (W) is a critical material that is widely used in military applications, electronics, lighting technology, power engineering and the automotive and aerospace industries. In recent decades, overexploitation of W has generated large amounts of mine waste rocks, which generate elevated content of toxic elements and cause serious adverse effects on ecosystems and public health. Microorganisms are considered important players in toxic element migrations from waste rocks. However, the understanding of how the microbial community structure varies in W mine waste rocks and its key driving factors is still unknown. In this study, high-throughput sequencing methods were used to determine the microbial community profiles along a W content gradient in W mine waste rocks. We found that the microbial community structures showed clear differences across the different W levels in waste rocks. Notably, arsenic (As), instead of W and nutrients, was identified as the most important predictor influencing microbial diversity. Furthermore, our results also showed that As is the most important environmental factor that regulates the distribution patterns of ecological clusters and keystone ASVs. Importantly, we found that the dominant genera have been regulated by As and were widely involved in As biogeochemical cycling in waste rocks. Taken together, our results have provided useful information about the response of microbial communities to W mine waste rocks.

Deaminative Cyclization of Tertiary Amines for the Synthesis of 2-Arylquinoline Derivatives with a Nonsubstituted Vinylene Fragment.

With triethylamine as a vinylene source, a convenient protocol for the regioselective synthesis of ß,γ-nonsubstituted 2-arylquinolines from aldehydes and arylamines has been accomplished. The deaminative cyclization is also extended to long-chain tertiary alkylamines, enabling diverse alkyl groups to be concurrently installed into the pyridine rings. This process demonstrates a new conversion pathway for the simultaneous dual C(sp3)-H bond functionalization of tertiary amines, wherein the transient acyclic enamines generated in situ undergo the Povarov reaction.

[Development of an APRT-deficient CHO cell line and its ability of expressing recombinant protein].

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.

Automated detection of intracranial artery stenosis and occlusion in magnetic resonance angiography: A preliminary study based on deep learning.

BACKGROUND AND OBJECTIVES: Intracranial atherosclerotic stenosis of a major intracranial artery is the common cause of ischemic stroke. We evaluate the feasibility of using deep learning to automatically detect intracranial arterial steno-occlusive lesions from time-of-flight magnetic resonance angiography. METHODS: In a retrospective study, magnetic resonance images with radiological reports of intracranial arterial stenosis and occlusion were extracted. The images were randomly divided into a training set and a test set. The manual annotation of lesions with a bounding box labeled "moderate stenosis," "severe stenosis," "occlusion," and "absence of signal" was considered as ground truth. A deep learning algorithm based on you only look once version 5 (YOLOv5) detection model was developed with the training set, and its sensitivity and positive predictive values to detect lesions were evaluated in the test set. RESULTS: A dataset of 200 examinations consisted of a total of 411 lesions-242 moderate stenoses, 84 severe stenoses, 70 occlusions, and 15 absence of signal. The magnetic resonance images contained 291 lesions in the training set and 120 lesions in the test set. The sensitivity and positive predictive values were 64.2 and 83.7%, respectively. The detection sensitivity in relation to the location was greatest in the internal carotid artery (86.2%). CONCLUSIONS: Applying deep learning algorithms in the automated detection of intracranial arterial steno-occlusive lesions from time-of-flight magnetic resonance angiography is feasible and has great potential.

T2 relaxation time for the early prediction of treatment response to chemoradiation in locally advanced rectal cancer.

OBJECTIVES: Poor responders to chemoradiotherapy (CRT) for locally advanced rectal cancer (LARC) can still have a good prognosis if the treatment strategy is changed in time. However, no reliable predictor of early-treatment response has been identified. The purpose of this study was to investigate the role of T2 relaxation time in magnetic resonance imaging (MRI) for the early prediction of a pathological response to CRT in LARC. METHODS: A total of 123 MRIs were performed on 41 LARC patients immediately before, during, and after CRT. The corresponding tumor volume, T2 relaxation time, and apparent diffusion coefficient (ADC) values at different scan time points were obtained. The Mann-Whitney U test was used to compare the T2 relaxation time between pathological good responders (GR) and non-good responders (non-GR). The area under the curve (AUC) value was used to quantify the diagnostic ability of each parameter in predicting tumor response to CRT. RESULTS: Twenty-one (51%) and 20 (49%) were GRs and non-GRs, respectively. T2 relaxation time showed an excellent intraclass correlation coefficient (ICC) of > 0.85 at three-time points. It was significantly lower in the GR group than in the non-GR group during and after CRT. The early T2 decrease had a high AUC of 0.91 in differentiating non-GRs and GRs, similar to 0.90 of the T2 value after CRT. CONCLUSIONS: T2 relaxation time may help predict treatment response to CRT for LARC earlier, rather than having to wait until the end of CRT, thereby alleviating the physical burden for patients with no good response.