Metabolic reprogramming and biosensor-assisted mutagenesis screening for high-level production of L-arginine in Escherichia coli.

L-arginine is a value-added amino acid with promising applications in the pharmaceutical and nutraceutical industries. Further unleashing the potential of microbial cell factories to make L-arginine production more competitive remains challenging due to the sophisticated intracellular interaction networks and the insufficient knowledge of global metabolic regulation. Here, we combined multilevel rational metabolic engineering with biosensor-assisted mutagenesis screening to exploit the L-arginine production potential of Escherichia coli. First, multiple metabolic pathways were systematically reprogrammed to redirect the metabolic flux into L-arginine synthesis, including the L-arginine biosynthesis, TCA cycle, and L-arginine export. Specifically, a toggle switch responding to special cellular physiological conditions was designed to dynamically control the expression of sucA and pull more carbon flux from the TCA cycle toward L-arginine biosynthesis. Subsequently, a biosensor-assisted high-throughput screening platform was designed and applied to further exploit the L-arginine production potential. The best-engineered ARG28 strain produced 132 g/L L-arginine in a 5-L bioreactor with a yield of 0.51 g/g glucose and productivity of 2.75 g/(L ⋅ h), which were the highest values reported so far. Through whole genome sequencing and reverse engineering, Frc frameshift mutant, PqiB A78P mutant, and RpoB P564T mutant were revealed for enhancing the L-arginine biosynthesis. Our study exhibited the power of coupling rational metabolic reprogramming and biosensor-assisted mutagenesis screening to unleash the cellular potential for value-added metabolite production.

Sedation and analgesia requirements during venovenous extracorporeal membrane oxygenation in acute respiratory distress syndrome patients.

INTRODUCTION: The purpose of this study is to describe sedation and analgesia management, and identify the factors associated with increased demand for medication in acute respiratory distress syndrome (ARDS) patients receiving venovenous extracorporeal membrane oxygenation (VV-ECMO). METHODS: This retrospective, single-center study included consecutive adult ARDS patients who received VV-ECMO for at least 24 hours from January 2018 to December 2020 in a comprehensive intensive care unit. The electronic medical records were retrospectively reviewed to collect data. RESULTS: Forty-two adult patients meeting the inclusion criteria were included in the study. Midazolam, sufentanil, and remifentanil were main sedatives and analgesics used in the patient population. The morphine equivalents, representative of the demand for opioids, was 512.9 (IQR, 294.5, 798.2) mg/day. The midazolam equivalents, representative of benzodiazepine requirement, was 279.6 (IQR, 208.8, 384.5) mg/day. The levels of serum creatinine, total bilirubin, lactic acid, SOFA score, and APACHE Ⅱ score at cannulation were found to be associated with opiate or benzodiazepine requirements. Multiple linear regression analysis revealed a linear correlation between midazolam equivalents and morphine equivalents (p < 0.001). In addition, there was a negative linear correlation between Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) score and midazolam equivalents (p = 0.024). CONCLUSIONS: The sedation and analgesia requirements of ARDS patients receiving VV-ECMO often increase simultaneously. More large-scale studies are needed to confirm the risk factors for increased sedation and analgesia needs in patients supported on VV-ECMO.

Strategy for the design of a bioreactor for L-lysine immobilized fermentation using Corynebacterium glutamicum.

Biofilm-immobilized fermentation is a novel strategy that has been utilized in L-lysine fermentation. In this study, we describe a strategy for designing bioreactors for immobilized fermentation. We have constructed steel structures in which the carriers can be sewn, forming several star-like structures with different angles, and changing the ventilation robot to the aeration tray. In a 10-L bioreactor, this structure with 12 angles assisted the immobilized system to remedy the gap between free-cell and immobilized fermentation in the conversion rate. In a 50-L bioreactor, this enlarged structure with 16 angles illustrated a 4.61% higher conversion rate than the free-cell fermentation (67.75%) and increased the production by 28.56%. This successful case is the first step towards to industrial production of biofilm-based immobilized fermentation.Key points• The designed steel structure is useful for L-lysine immobilized fermentation in a 10-L bioreactor.• The conversion rate of immobilized fermentation increased from 13.99 to 60.07% and is 1.03% higher than that of the free-cell fermentation.• The conversion rate of the redesigned 50-L bioreactor is higher than that of free-cell fermentation.

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline.

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.

Application of a dissolved oxygen control strategy to increase the expression of Streptococcus suis glutamate dehydrogenase in Escherichia coli.

The accumulation of acetate in Escherichia coli inhibits cell growth and desired protein synthesis, and cell density and protein expression are increased by reduction of acetate excretion. Dissolved oxygen (DO) is an important parameter for acetate synthesis, and the accumulation of acetate is inversely correlated to DO level. In this study, the effect of DO levels on glutamate dehydrogenase (GDH) expression was investigated, and then different DO control strategies were tested for effects on GDH expression. DO control strategy IV (50% 0-9 h, 30% 9-18 h) provided the highest cell density (15.43 g/L) and GDH concentration (3.42 g/L), values 1.59- and 1.99-times higher than those achieved at 10% DO. The accumulation of acetate was 2.24 g/L with DO control strategy IV, a decrease of 40.74% relative to that achieved for growth at 10% DO. Additionally, under DO control strategy IV, there was lower expression of PoxB, a key enzyme for acetate synthesis, at both the transcriptional and translational level. At the same time, higher transcription and protein expression levels were observed for a glyoxylate shunt gene (aceA), an acetate uptake gene (acs), gluconeogensis and anaplerotic pathways genes (pckA, ppsA, ppc, and sfcA), and a TCA cycle gene (gltA). The flux of acetate with DO strategy IV was 8.4%, a decrease of 62.33% compared with the flux at 10% DO. This decrease represents both lower flux for acetate synthesis and increased flux of reused acetate.

[A school-aged boy with nephrotic syndrome with cough for one month and shortness of breath for half a month].

A boy, aged 6 years and 11 months, was admitted due to nephrotic syndrome for 2 years, cough for 1 month, and shortness of breath for 15 days. The boy had a history of treatment with hormone and immunosuppressant. Chest CT after the onset of cough and shortness of breath showed diffuse ground-glass opacities in both lungs. Serum (1, 3)-beta-D glucan was tested positive, and the nucleic acid of cytomegalovirus was detected in respiratory secretions. After the anti-fungal and anti-viral treatment, the child improved temporarily but worsened again within a short period of time. Pneumocystis jirovecii was identified by Gomori's methenamine silver staining in bronchoalveolar lavage fluid. The child was diagnosed with severe pneumonia (Pneumocystis jirovecii and cytomegalovirus infection), acute respiratory distress syndrome, and nephrotic syndrome. After anti-infective therapy with sulfamethoxazole/trimethoprim and ganciclovir and respiratory support, the child still experienced progressive aggravation of dyspnea and tension pneumothorax, and extracorporeal membrane oxygenation (ECMO) was given on day 13 of invasive ventilation. Anti-infective therapy with sulfamethoxazole/trimethoprim, ganciclovir, and linezolid, anticoagulation therapy, sedation therapy, nutrition, and comprehensive management of the respiratory tract were given during ECMO. The child was successfully weaned from ECMO after 72 days, resulting in a length of hospital stay of 134 days. The child was followed up for 6 months after discharge, and there was a significant improvement on lung CT, without organ dysfunction. It is concluded that Pneumocystis jirovecii pneumonia is a potential lifethreatening infection for children with low immunity, and that ECMO can effectively improve the prognosis of children with severe respiratory distress syndrome.

gDNA-Prot: Predict DNA-binding proteins by employing support vector machine and a novel numerical characterization of protein sequence.

DNA-binding proteins are the functional proteins in cells, which play an important role in various essential biological activities. An effective and fast computational method gDNA-Prot is proposed to predict DNA-binding proteins in this paper, which is a DNA-binding predictor that combines the support vector machine classifier and a novel kind of feature called graphical representation. The DNA-binding protein sequence information was described with the 20 probabilities of amino acids and the 23 new numerical graphical representation features of a protein sequence, based on 23 physicochemical properties of 20 amino acids. The Principal Components Analysis (PCA) was employed as feature selection method for removing the irrelevant features and reducing redundant features. The Sigmod function and Min-max normalization methods for PCA were applied to accelerate the training speed and obtain higher accuracy. Experiments demonstrated that the Principal Components Analysis with Sigmod function generated the best performance. The gDNA-Prot method was also compared with the DNAbinder, iDNA-Prot and DNA-Prot. The results suggested that gDNA-Prot outperformed the DNAbinder and iDNA-Prot. Although the DNA-Prot outperformed gDNA-Prot, gDNA-Prot was faster and convenient to predict the DNA-binding proteins. Additionally, the proposed gNDA-Prot method is available at http://sourceforge.net/projects/gdnaprot.

Predicting the Prognosis of Bladder Cancer Patients Through Integrated Multi-omics Exploration of Chemotherapy-Related Hypoxia Genes.

Bladder cancer is a prevalent malignancy with high mortality rates worldwide. Hypoxia is a critical factor in the development and progression of cancers. However, whether and how hypoxia-related genes (HRGs) could affect the development and the chemotherapy response of bladder cancer is still largely unexplored. This study comprehensively explored the complex molecular landscape associated with hypoxia in bladder cancer by analyzing 260 hypoxia genes based on transcriptomic and genomic data in 411 samples. Employing the 109 dysregulated hypoxia genes for consensus clustering, we delineated two distinct bladder cancer clusters characterized by disparate survival outcomes and distinct oncogenic roles. We defined a HPscore that was correlated with a variety of clinical features, including TNM stages and pathologic grades. Tumor immune landscape analysis identified three immune clusters and close interactions between hypoxia genes and the various immune cells. Utilizing a network-based method, we defined 129 HRGs exerting influence on apoptotic processes and critical signaling pathways in cancer. Further analysis of chemotherapy drug sensitivity identified potential drug-target HRGs. We developed a Risk Score model that was related to the overall survival of bladder cancer patients based on doxorubicin-target HRGs: ACTG2, MYC, PDGFRB, DHRS2, and KLRD1. This study not only enhanced our understanding of bladder cancer at the molecular level but also provided promising avenues for the development of targeted therapies, representing a significant step toward the identification of effective treatments and addressing the urgent need for advancements in bladder cancer management.

Diabetic conditions promote drug coating degradation but prevent endothelial coverage after stenting.

The endothelialization of drug-eluting stents is delayed after implantation in patients with diabetes. Although numerous factors were implicated in hyperglycemia-induced endothelial dysfunction, the effects of stent drug coating degradation on endothelial dysfunction remains unclear. We hypothesized that diabetic conditions promote drugcoating degradation and enhance antiproliferative agent release, but that the rapid release of these antiproliferative agents inhibits endothelial cell proliferation leading to poor reendothelialization post-stenting. To verify this hypothesis, a dynamic hyperglycemic circulation system was introduced to measure the profile of drugcoating degradation in vitro. Flow cytometry and RNA sequencing were performed to evaluate endothelial cell proliferation. Moreover, a Type 1 diabetic rabbit model was generated and a rescue experiment conducted to evaluate the effects of rapid drugcoating elution on endothelial coverage in vivo. The main findings were as follows: 1) diabetic conditions promoted drugcoating degradation and increased antiproliferative agent release; 2) this increase in antiproliferative agent release inhibited endothelial cell proliferation and delayed endothelial coverage; and 3) strict glycemic control attenuated drugcoating degradation and promoted endothelial coverage post-stenting. This is the first study to illustrate rapid drugcoating degradation and its potential effects on endothelial recovery under diabetic conditions, highlighting the importance of strict glycemic management in patients with diabetes after drug-eluting stent implantation. STATEMENT OF SIGNIFICANCE: Diabetic conditions promote drug coating degradation and increase the release of antiproliferative agents. Rapid drug coating degradation under diabetic conditions inhibits endothelial cell proliferation and delays endothelialization. Strict glycemic control attenuates drug coating degradation and promotes endothelialization.

Strategy for pH control and pH feedback-controlled substrate feeding for high-level production of L-tryptophan by Escherichia coli.

Optimum production of L-tryptophan by Escherichia coli depends on pH. Here, we established conditions for optimizing the production of L-tryptophan. The optimum pH range was 6.5-7.2, and pH was controlled using a three-stage strategy [pH 6.5 (0-12 h), pH 6.8 (12-24 h), and pH 7.2 (24-38 h)]. Specifically, ammonium hydroxide was used to adjust pH during the initial 24 h, and potassium hydroxide and ammonium hydroxide (1:2, v/v) were used to adjust pH during 24-38 h. Under these conditions, NH4 (+) and K(+) concentrations were kept below the threshold for inhibiting L-tryptophan production. Optimization was also accomplished using ratios (v/v) of glucose to alkali solutions equal to 4:1 (5-24 h) and 6:1 (24-38 h). The concentration of glucose and the pH were controlled by adjusting the pH automatically. Applying a pH-feedback feeding method, the steady-state concentration of glucose was maintained at approximately 0.2 ± 0.02 g/l, and acetic acid accumulated to a concentration of 1.15 ± 0.03 g/l, and the plasmid stability was 98 ± 0.5 %. The final, optimized concentration of L-tryptophan was 43.65 ± 0.29 g/l from 52.43 ± 0.38 g/l dry cell weight.

Earliest curry in Southeast Asia and the global spice trade 2000 years ago.

The global spice trade has played an essential role in world history. However, because of poor preservation conditions, archaeobotanical remains of spices have been limited in archaeological contexts until now. This study reports evidence for spice processing from the archaeological site of Oc Eo in southern Vietnam, an entrepôt of the state of Funan that was occupied during the early centuries CE. Analysis of plant microremains recovered from the surfaces of Oc Eo grinding stone tools thought to be of South Asian origin has identified culinary spices that include turmeric, ginger, fingerroot, sand ginger, galangal, clove, nutmeg, and cinnamon. These spices are indispensable ingredients used in the making of curry in South Asia today. We suggest that South Asian migrants or visitors introduced this culinary tradition into Southeast Asia during the period of early trade contact via the Indian Ocean, commencing about 2000 years ago.

Expression and characterization of α-L-arabinofuranosidase derived from Aspergillus awamori and its enzymatic degradation of corn byproducts with xylanase.

In this study, α-L-arabinofuranosidase (AF) from Aspergillus awamori was heterologously expressed in Pichia pastoris X33, with a 1-fold increase in AF activity after codon and vector optimization. AF remained stable at 60-65 °C and displayed a broad pH stability range of 2.5-8.0. It also demonstrated considerable resistance to pepsin and trypsin. Furthermore, compared with xylanase alone, AF with xylanase exhibited a marked synergistic effect in the degradation of expanded corn bran, corn bran, and corn distillers' dried grains with solubles, reducing sugars by 3.6-fold, 1.4-fold, and 6.5-fold, respectively, with the degree of synergy increasing to 4.61, 2.44, and 5.4, respectively, while in vitro dry matter digestibility values were 17.6%, 5.2%, and 8.8%, respectively. After enzymatic saccharification, corn byproducts were converted to prebiotic xylo-oligosaccharides and arabinoses, thereby demonstrating the favorable properties of AF in the degradation of corn biomass and its byproducts.

Long non-coding RNA BBOX1-antisense RNA 1 enhances cell proliferation and migration and suppresses apoptosis in oral squamous cell carcinoma via the miR-3940-3p/laminin subunit gamma 2 axis.

Long non-coding RNAs (lncRNAs) play an essential role in oral squamous cell carcinoma (OSCC). We aimed to demonstrate the effects of lncRNA gamma-butyrobetaine hydroxylase 1 (BBOX1)-antisense RNA 1 (AS1) in OSCC and its regulatory mechanisms. The levels of BBOX1-AS1, microRNA (miR)-3940-3p, and laminin subunit gamma 2 (LAMC2) in OSCC were determined using reverse transcription-quantitative polymerase chain reaction. The correlations among BBOX1-AS1, miR-3940-3p, and LAMC2 were validated using luciferase, pull-down, and RNA immunoprecipitation assays. Cell proliferation, migration, and apoptosis were examined. BBOX1-AS1 and LAMC2 were notably overexpressed in OSCC, while miR-3940-3p showed the opposite trend. BBOX-1-AS1 silencing reduced the cell proliferation and migration, while promoting apoptosis. Mechanistically, BBOX1-AS1 modulates LAMC2 expression by competitively binding to miR-3940-3p. miR-3940-3p inhibition alleviated the inhibitory effects of BBOX1-AS1 deficiency on OSCC development. LAMC2 knockdown reversed these changes. Our results revealed that BBOX1-AS1 promotes the malignant phenotype of OSCC cells via the upregulation of LAMC2 expression by targeting miR-3940-3p, indicating that BBOX1-AS1 may be a novel target for OSCC intervention.

Role of Quantitative EEG and EEG Reactivity in Traumatic Brain Injury.

OBJECTIVE: This study aimed to explore the effectiveness of quantitative electroencephalogram (EEG) and EEG reactivity (EEG-R) to predict the prognosis of patients with severe traumatic brain injury. METHODS: This was a prospective observational study on severe traumatic brain injury. Quantitative EEG monitoring was performed for 8 to 12 hours within 14 days of onset. The EEG-R was tested during the monitoring period. We then followed patients for 3 months to determine their level of consciousness. The Glasgow Outcome Scale (GOS) score was used. The score 3, 4, 5 of GOS were defined good prognosis, and score 1 and 2 as poor prognosis. Univariate and multivariate analyses were employed to assess the association of predictors with poor prognosis. RESULTS: A total of 56 patients were included in the study. Thirty-two patients (57.1%) awoke (good prognosis) in 3 months after the onset. Twenty-four patients (42.9%) did not awake (poor prognosis), including 11 cases deaths. Univariate analysis showed that Glasgow coma scale (GCS) score, the amplitude-integrated EEG (aEEG), the relative band power (RBP), the relative alpha variability (RAV), the spectral entropy (SE), and EEG-R reached significant difference between the poor-prognosis and good-prognosis groups. However, age, gender, and pupillary light reflex did not correlate significantly with poor prognosis. Furthermore, multivariate logistic regression analysis showed that only RAV and EEG-R were significant independent predictors of poor prognosis, and the prognostic model containing these 2 variables yielded a predictive performance with an area under the curve of 0.882. CONCLUSIONS: Quantitative EEG and EEG-R may be used to assess the prognosis of patients with severe traumatic brain injury early. RAV and EEG-R were the good predictive indicators of poor prognosis.

Rice and millet cultivated in Ha Long Bay of Northern Vietnam 4000 years ago.

Research has generally outlined that the Neolithic East Asian farmers expanded into Southeast Asia, leading to substantial social and cultural transformations. However, the associated archaeobotanical evidence until now has been insufficient to clarify the exact timing, dispersal route, and farming package of the emergence of agriculture in Mainland Southeast Asia. To clarify these issues, the micro-plant remains of phytolith and starch from three Neolithic sites in Ha Long Bay were extracted and analyzed. This study validates the earliest evidence of co-cropping in northern Vietnam, involving the cultivation of rice together with foxtail millet at 4000 years BP or slightly earlier. Moreover, the results indicate that at least two patterns of subsistence strategy were practiced simultaneously during the initial farming phase in the region. The Trang Kenh people, a regional variant of the Phung Nguyen cultural group often have been seen as the first farmers in northern Vietnam, and they mainly practiced a cereal-based subsistence strategy with more vital cultural characteristics of southern China origin. Meanwhile, the Ha Long people, mainly composed of indigenous hunter-gatherer descendants, continued to utilize a wide range of their preferred plant resources such as taros, yams, and acorns, while they absorbed and incorporated new elements such as millet and rice into their food system. This study provides solid information to understand the diverse economic systems among different cultural groups in Vietnam.

Molecular characterization of a novel GSTO2 of Fasciola hepatica and its roles in modulating murine macrophages.

Fascioliasis is an important zoonotic helminthic disease caused by Fasciola hepatica and poses a serious threat to global public health. To evade the immune response of its host (humans or animals), F. hepatica secretes various antioxidant enzymes such as glutathione transferase (GST) to facilitate its invasion, migration and parasitism in vivo. To investigate the biological functions of a novel omega-class GST (GSTO), the molecular features of GSTO2 of F. hepatica were analyzed by online software, and the biochemical properties in vitro of recombinant GSTO2 (rGSTO2) were dissected. Then, the regulatory roles of rGSTO2 protein in murine macrophages in vitro were further explored. The results revealed that the GSTO2 gene encodes 254 amino acids, which harbor the characteristic N-terminal domain (ßαßαßßα) and C-terminal domain (α-helical) of the cytoplasmic GST superfamily. GSTO2 was mainly expressed in F. hepatica vitelline follicles, intestinal tract, excretory pores and vitelline cells, with thioltransferase and dehydroascorbate reductase activities. Moreover, rGSTO2 protein could be taken up by murine macrophages and significantly inhibit the viability of macrophages. In addition, rGSTO2 protein could significantly promote apoptosis and modulate the expression of cytokines in macrophages. These findings suggested that F. hepatica GSTO2 plays an important role in modulating the physiological functions of macrophages, whereby this protein might be involved in immunomodulatory and anti-inflammatory roles during infection. This study provided new insights into the immune-evasion mechanism of F. hepatica and may contribute to the development of a potential anti-inflammatory agent.

Title: Caractérisation moléculaire d'une nouvelle GSTO2 de Fasciola hepatica et ses rôles dans la modulation des macrophages murins. Abstract: La fasciolase est une importante maladie helminthique zoonotique causée par Fasciola hepatica, qui constitue une menace sérieuse pour la santé publique mondiale. Pour échapper à la réponse immunitaire de son hôte (humain ou animal), F. hepatica sécrète diverses enzymes antioxydantes telles que la glutathion transférase (GST) pour faciliter son invasion, sa migration et son parasitisme in vivo. Pour étudier les fonctions biologiques d'une nouvelle GST de classe oméga (GSTO), les caractéristiques moléculaires de la GSTO2 de F. hepatica ont été analysées par un logiciel en ligne et les propriétés biochimiques in vitro de sa protéine recombinante (rGSTO2) ont été disséquées. Ensuite, les rôles régulateurs de la protéine rGSTO2 sur les macrophages murins in vitro ont été explorés plus avant. Les résultats ont révélé que le gène GSTO2 code pour 254 acides aminés, qui abritent le domaine N-terminal caractéristique (ßαßαßßα) et le domaine C-terminal (α-hélicoïdal) de la superfamille GST cytoplasmique. Chez F. hepatica, GSTO2 était principalement exprimée dans les follicules vitellins, le tractus intestinal, les pores excréteurs et les cellules vitellines, avec des activités de thioltransférase et de déhydroascorbate réductase. De plus, la protéine rGSTO2 a pu être absorbée par les macrophages murins et inhiber de manière significative la viabilité des macrophages. Enfin, la protéine rGSTO2 a pu favoriser de manière significative l'apoptose et moduler l'expression des cytokines dans les macrophages. Ces résultats suggèrent que la GSTO2 de F. hepatica joue un rôle important dans la modulation des fonctions physiologiques des macrophages, cette protéine pouvant être impliquée dans des rôles immunomodulateurs et anti-inflammatoires au cours de l'infection. Cette étude a fourni de nouvelles informations sur le mécanisme d'évasion immunitaire de F. hepatica et pourrait contribuer au développement d'un agent anti-inflammatoire potentiel.

MicroRNA 617 Targeting SERPINE1 Inhibited the Progression of Oral Squamous Cell Carcinoma.

Serpin family E member 1 (SERPINE1) is a serine proteinase inhibitor (serpin) upregulated in diverse types of cancer, including oral squamous cell carcinoma (OSCC), and it functions in an oncogenic role. Hence, exploring pathological mechanism underlying high expression of SERPINE1 is crucial to the targeted therapy of OSCC. Bioinformatics analysis was performed to identify the microRNA (miRNA) and the candidate gene contributing to OSCC progression. The viability, proliferation, and apoptosis of the OSCC cell were evaluated using Cell Counting Kit-8 (CCK-8) assay, BrdU assay, and cell apoptosis assay, respectively. The RNA pulldown assay and luciferase reporter assay were conducted to verify the relationship between SERPINE1 and miRNA 617 (miR-617). SERPINE1 was aberrantly upregulated in OSCC tissues and cell lines. Genetically inhibiting SERPINE1 led to reduction of OSCC cell viability and proliferation and elevation of OSCC cell apoptosis. According to bioinformatics analysis, miR-617 contained a response element for SERPINE1 overexpression, which is validated by the RNA pulldown and luciferase reporter assays. Furthermore, miR-617 was detected to be downregulated in OSCC tissues and cell lines, and it displayed a negative correlation with advanced stages. Besides, miR-617 mimic or inhibitor transfection could suppress or boost the SERPINE1 expression. More importantly, miR-617 mimic could block the effect of SERPINE1 overexpression on OSCC cell proliferation, viability, and apoptosis. SERPINE1 acted as a proproliferative oncogenic factor that is partly regulated by miR-167 downregulation in OSCC cells. Therefore, the miR-617/SERPINE1 axis is a potential therapeutic target against OSCC.

miR-532-3p inhibits the progression of tongue squamous cell carcinoma by targeting podoplanin.

BACKGROUND: The association between miR-532-3p and tongue squamous cell carcinoma (TSCC) has been examined in the literature to improve the survival rate of patients with this tumor. However, further studies are needed to confirm the regulatory roles of this microRNA (miRNA) in TSCC. The objective of this study was to investigate the roles played by and the underlying mechanism used by the miR-532-3p/podoplanin (PDPN) axis in TSCC development. METHODS: Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) were performed to evaluate the PDPN expression level in TSCC tissues and cells. The proliferative, adhesive, and migratory capabilities of TSCC cells (CAL-27 and CTSC-3) were examined using cell counting kit-8 (CCK-8), cell adhesion, and wound-healing assays, respectively. The dual-luciferase reporter (DLR) assay was later conducted to confirm the relationship between miR-532-3p and PDPN. RESULTS: The results indicated that PDPN expression was enriched in TSCC tissues and cells, and that the expression of PDPN was associated with some clinicopathological parameters of TSCC, including lymph node metastasis (P = 0.001), tumor-node-metastasis (TNM) staging (P = 0.010), and grading (P = 0.010). Further analysis also showed that PDPN knockdown inhibited the viability, adhesive ability, and migratory capacity of CAL-27 and CTSC-3 cells, effects that could be reversed by the application of a miR-532-3p inhibitor. Additionally, PDPN was found to be a direct target of miR-532-3p. CONCLUSIONS: This research suggested that by targeting PDPN, miR-532-3p could inhibit cell proliferation viability, adhesion, and migration in TSCC. Findings also revealed that the miR-532-3p/PDPN axis might provide more insights into the prognosis and treatment of TSCC.

Differences in clinical characteristics between younger and older patients with COVID-19 and their relationship with the length of hospital stay.

Background: The coronavirus disease 2019 (COVID-19) pandemic is currently threatening the health of individuals worldwide. We compared the clinical characteristics between younger patients (aged <60 years) and older patients (aged ≥60 years) with COVID-19, detected the risk factors associated with a prolonged hospital stay, and examined the treatments commonly used with a particular focus on antiviral therapies. Methods: This retrospective study was conducted at the West Campus, Union Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China). The sample consisted of 123 patients admitted to the hospital between 9th February, 2020, and 3rd March, 2020. The data related to the demographics, laboratory findings, and treatment were analyzed to identify discrepancies between younger and older patients and those with and without primary diseases. The risk factors that contribute to a prolonged hospital stay were subsequently identified. Results: Patients aged ≥60 years required longer hospital stay than younger patients (P=0.001). The percentage of lymphocytes was significantly lower in older patients and those with primary diseases (P=0.016 and P=0.042, respectively). The findings revealed that the risk factors that contributed to the length of hospital stay were age, the number of days of illness before hospitalization, white blood cell (WBC) count and albumin levels at admission, a neutrophil fraction at discharge, and antibiotic treatment. Analysis using a model that consisted of the above five risk factors for predicting prolonged hospital stay (>14 days) yielded an area under the ROC (AuROC) curve of 0.716. Antiviral and antibiotic treatments were administered to 97.6% and 39.0% of patients, respectively. The antiviral drugs most commonly administered were traditional Chinese medicine (83.7%) and arbidol (75.6%). Conclusions: In this study, older patients and those with primary diseases were at a higher risk of worse clinical manifestations. The physicians who treat the patients should pay close attention to the risk factors that contribute to the length of hospital stay, which could be used for predicting prolonged hospital stay. Traditional Chinese medicine and arbidol were the most frequently used antiviral drugs. Nevertheless, the extent to which these medications can effectively treat COVID-19 warrants further investigation.