Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

BACKGROUND AND OBJECTIVES: The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system. MATERIALS AND METHODS: Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins. RESULTS: Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins. CONCLUSIONS: Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

RNA Modifications and Epigenetics in Modulation of Lung Cancer and Pulmonary Diseases.

Lung cancer is the leading cause of cancer-related mortality worldwide, and its tumorigenesis involves the accumulation of genetic and epigenetic events in the respiratory epithelium. Epigenetic modifications, such as DNA methylation, RNA modification, and histone modifications, have been widely reported to play an important role in lung cancer development and in other pulmonary diseases. Whereas the functionality of DNA and chromatin modifications referred to as epigenetics is widely characterized, various modifications of RNA nucleotides have recently come into prominence as functionally important. N6-methyladosine (m6A) is the most prevalent internal modification in mRNAs, and its machinery of writers, erasers, and readers is well-characterized. However, several other nucleotide modifications of mRNAs and various noncoding RNAs have also been shown to play an important role in the regulation of biological processes and pathology. Such epitranscriptomic modifications play an important role in regulating various aspects of RNA metabolism, including transcription, translation, splicing, and stability. The dysregulation of epitranscriptomic machinery has been implicated in the pathological processes associated with carcinogenesis including uncontrolled cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In recent years, with the advancement of RNA sequencing technology, high-resolution maps of different modifications in various tissues, organs, or disease models are being constantly reported at a dramatic speed. This facilitates further understanding of the relationship between disease development and epitranscriptomics, shedding light on new therapeutic possibilities. In this review, we summarize the basic information on RNA modifications, including m6A, m1A, m5C, m7G, pseudouridine, and A-to-I editing. We then demonstrate their relation to different kinds of lung diseases, especially lung cancer. By comparing the different roles RNA modifications play in the development processes of different diseases, this review may provide some new insights and offer a better understanding of RNA epigenetics and its involvement in pulmonary diseases.

Tumor Necrosis Factor-Alpha Exacerbates Viral Entry in SARS-CoV2-Infected iPSC-Derived Cardiomyocytes.

The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.

Improvement of Left Ventricular Remodelling by Inhibition of NF-κB in a Rat Model of Myocardial Infarction.

BACKGROUND: To investigate the effects of inhibition of NF-κB activation on left ventricular (LV) remodelling in a rat model of myocardial infarction (MI). METHODS: The acute MI model was established by ligation of left anterior descending coronary artery. Pyrrolidine dithiocarbamate (PDTC) (20mg/kg, Qd) was administered intraperitoneally to inhibit NF-κB activation. Eight weeks later, the cardiac structure and LV ejection fraction were assessed with echocardiography. The rat body, heart, and LV weights were measured to calculate LV mass indices. Activation of NF-κB in non-infarcted myocardium was detected by a TransAM NF-κB p65 Transcription Factor Assay Kit. Cardiac collagen volume fraction was evaluated by Masson staining. RESULTS: Eight weeks after the MI model was established, the LV posterior wall thickness in PDTC and MI group was 1.75±0.07mm and 1.85±0.07mm respectively (p<0.05). The LV mass index in the PDTC group (2.53±0.09) was lower than in the MI group (2.65±0.08, p<0.05). The LVEF in the PDTC group (63.89%±4.21%) was higher than in the MI group (42.73%±8.94%, p<0.05). The interstitial collagen deposition in the non-infarcted myocardium in the PDTC group was less than in the MI group (7.25%±1.88% vs. 10.09%±2.19%, p<0.05). CONCLUSION: Inhibition of activation of NF-κB may result in improvement of myocardial remodelling after myocardial infarction, which is possibly attributable to reduced collagen deposition in non-infarcted areas.

Integration of single-cell sequencing and bulk RNA-seq to identify and develop a prognostic signature related to colorectal cancer stem cells.

The prognosis for patients with colorectal cancer (CRC) remains worse than expected due to metastasis, recurrence, and resistance to chemotherapy. Colorectal cancer stem cells (CRCSCs) play a vital role in tumor metastasis, recurrence, and chemotherapy resistance. However, there are currently no prognostic markers based on CRCSCs-related genes available for clinical use. In this study, single-cell transcriptome sequencing was employed to distinguish cancer stem cells (CSCs) in the CRC microenvironment and analyze their properties at the single-cell level. Subsequently, data from TCGA and GEO databases were utilized to develop a prognostic risk model for CRCSCs-related genes and validate its diagnostic performance. Additionally, functional enrichment, immune response, and chemotherapeutic drug sensitivity of the relevant genes in the risk model were investigated. Lastly, the key gene RPS17 in the risk model was identified as a potential prognostic marker and therapeutic target for further comprehensive studies. Our findings provide new insights into the prognostic treatment of CRC and offer novel perspectives for a systematic and comprehensive understanding of CRC development.

Ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate attenuates the malignant biological behaviors of non-small cell lung cancer via suppressing EGFR/PI3K/AKT/mTOR signaling pathway.

OBJECTIVE: The goal of the study is to examine the impact on the malignant biological behaviors of non-small cell lung cancer (NSCLC) of a novel coumarin derivative, ethyl 2,2-difluoro-2-(2-oxo-2H-chromen-3-yl) acetate (C2F). It also aims to define its underlying mechanism. METHODS: NSCLC cell lines and xenograft nude mice model were conducted to explore the anti-NSCLC effects of C2F in vitro and in vivo. Then, network pharmacology analysis and molecular docking were applied to estimate the possible targets of C2F in NSCLC. Finally, the underlying mechanism of C2F against NSCLC cellular proliferation and tumor development was confirmed using inhibitors or activators of the PI3K/AKT signaling pathway. RESULTS: Our results showed that C2F was able to inhibit proliferation, migration, and invasion of NSCLC cell lines, induce cell cycle arrest and apoptosis in vitro, and prevent tumor growth in vivo. In addition, the estimated glomerular filtration rate and its downstream pathway (PI3K/AKT/mTOR) were found to be critical for the anti-NSCLC activity of C2F. CONCLUSIONS: C2F inhibits malignant biological behaviors of NSCLC by suppressing EGFR/PI3K/AKT/mTOR signaling pathway.

Maternal Cold-Reacting Immunoglobulin G Anti-M of MNS Blood Group System Causing Hemolytic Disease of the Fetus.

Several cases of the hemolytic disease of the fetus and newborn (HDFN) caused by immunoglobulin G (IgG) anti-M antibodies have been reported, in which almost all the HDFN-associated anti-M were warmly reacting. Here we report two cases of severe HDFN associated with cold-reacting IgG anti-M. In both cases, pregnancy was terminated, in weeks 33 and 23 respectively, due to a diagnosis of fetal growth retardation (FGR). To our knowledge, these are the most severe HDFN cases caused by cold-reacting IgG anti-M.

The Risk Factors-Based Nursing Case Management Could Effectively Reduce the Incidence of Pressure Sores in Hospitalized Patients.

BACKGROUND: To explore the effects of risk factors-based nursing management on the occurrence of pressure sores in hospitalized patients. METHODS: From Jan 2018 to Jun 2018, 289 hospitalized patients were divided into pressure sores group [100] and control group [189] for retrospective analysis. Overall, 260 hospitalized patients from Jun 2018 to Dec 2018 were followed up for nursing intervention. Overall 130 patients received risk factors-based nursing case management were in the intervention group, whereas 130 patients who received routine nursing care were in the control group. The chi-square test and t-test were used to compare the count data and the measurement data between groups, respectively. RESULTS: Age, body weight and proportions of patients with impaired nutritional intake, diabetes or stroke in pressure sores group were higher than those in normal group (P<0.05). Hospital stay and operative time in pressure sores group was longer than those in normal group (P<0.05). The frequency of assistant activity in pressure sores group was significantly lower than that in control group (P<0.05).In addition, the score of uroclepsia in pressure sores group was lower than that in normal group (P<0.05). Patients in the intervention group showed lower risk for pressure sores and more satisfied than patients in control group (P<0.001). CONCLUSION: Advanced age, high body weight, diabetes and stroke, long hospital stay, long operative time, poor nutritional status and severe uroclepsia were independent risk factors of pressure sores. Risk factors-based nursing case management can effectively reduce the occurrence and risk of pressure sores for hospitalized patients.

Incidence, prognosis, and risk factors of sepsis-induced cardiomyopathy.

BACKGROUND: At present, large-scale studies on the clinical characteristics of sepsis-induced cardiomyopathy (SIC) are lacking. AIM: To investigate the clinical characteristics of SIC. METHODS: Based on the analysis of the MIMIC-III public database, we performed a large-scale retrospective study involving sepsis patients who were admitted to the intensive care unit (ICU) and had no concomitant cardiac disease. We used propensity score matching analysis and multivariate logistic regression to ensure the robustness of the results. The primary outcome was hospital mortality, and the secondary outcomes included the number of patients who received mechanical ventilation or renal replacement therapy during their hospital stay, the number of patients administered with vasopressors, the length of ICU stay, and the length of hospital stay. RESULTS: In the present study, after screening 38605 patients, 3530 patients with sepsis were included. A total of 997 patients met the SIC diagnostic criteria, and the incidence of SIC was 28.20% (95% confidence interval [CI]: 26.80%-29.70%). Compared to patients in the non-SIC group, patients in the SIC group were of older age and had a higher Simplified Acute Physiology Score (SAPS)-I score, SAPS-II score, and Elixhauser comorbidity index (ECI). A total of 367 (36.8%) of 997 patients in the SIC group and 818 (32.3%) of 2533 patients in the non-SIC group died in the hospital, which resulted in a significant between-group difference (odds ratios = 1.22, 95%CI: 1.05-1.42; P = 0.011). For the secondary outcomes, more patients in the SIC group received mechanical ventilation and vasopressors. Multivariate logistic regression analysis showed that age, male sex, ECI, hemoglobin level, diabetes, and mechanical ventilation use on the first day of ICU admission were risk factors for SIC. CONCLUSION: Compared with non-SIC patients, hospital mortality is higher in SIC patients.

The roles of m6A RNA modifiers in human cancer.

Like DNA and proteins, RNA is subject to numerous (over 160) covalent modifications which play critical roles to regulate RNA metabolism. Among these modifications, N-methyladenosine (mA) is the most prevalent RNA methylation on mRNA which occurs on around 25% of transcripts. The recent studies demonstrated that mA participates in many aspects of RNA processing, including splicing, nuclear exporting, translation, stabilization, etc. Therefore, it revealed a new layer of regulatory mechanism for gene expression and has been termed "RNA Epigenetics" or "Epitranscriptomics". RNA mA is regulated and exerts its functions by three groups of "mA RNA modifiers" including mA methyltransferases (writers), mA demethylases (erasers), and mA binding proteins (readers). In this review, we would summarize and discuss the current understandings of the roles of the conventional mA RNA modifiers in human cancers.

Highlight of Immune Pathogenic Response and Hematopathologic Effect in SARS-CoV, MERS-CoV, and SARS-Cov-2 Infection.

A sudden outbreak of COVID-19 caused by a novel coronavirus, SARS-CoV-2, in Wuhan, China in December 2019 quickly grew into a global pandemic, putting at risk not only the global healthcare system, but also the world economy. As the disease continues to spread rapidly, the development of prophylactic and therapeutic approaches is urgently required. Although some progress has been made in understanding the viral structure and invasion mechanism of coronaviruses that may cause severe cases of the syndrome, due to the limited understanding of the immune effects caused by SARS-CoV-2, it is difficult for us to prevent patients from developing acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF), the major complications of coronavirus infection. Therefore, any potential treatments should focus not only on direct killing of coronaviruses and prevention strategies by vaccine development, but also on keeping in check the acute immune/inflammatory responses, resulting in ARDS and PF. In addition, potential treatments currently under clinical trials focusing on killing coronaviruses or on developing vaccines preventing coronavirus infection largely ignore the host immune response. However, taking care of SARS-CoV-2 infected patients with ARDS and PF is considered to be the major difficulty. Therefore, further understanding of the host immune response to SARS-CoV-2 is extremely important for clinical resolution and saving medication cost. In addition to a breif overview of the structure, infection mechanism, and possible therapeutic approaches, we summarized and compared the hematopathologic effect and immune responses to SARS-CoV, MERS-CoV, and SARS-CoV-2. We also discussed the indirect immune response caused by SARS and direct infection, replication, and destroying of immune cells by MERS-CoV. The molecular mechanisms of SARS-CoV and MERS-CoV infection-induced lymphopenia or cytokine storm may provide some hint toward fight against SARS-CoV-2, the novel coronavirus. This may provide guidance over using immune therapy as a combined treatment to prevent patients developing severe respiratory syndrome and largely reduce complications.

[Protective effect of curcumin on hepatocytes in rats with sepsis].

OBJECTIVE: To investigate the protective effect of curcumin on hepatocytes in rats with sepsis. METHODS: Eighty healthy male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, Xuebijing group and curcumin group (20 rats in each group) according to the random number table method. The animal model of sepsis was established by cecal ligation and puncture (CLP). In the sham operation group, the cecum was removed only after the operation. The rats in Xuebijing group and curcumin group were injected with 4 mL/kg Xuebijing, 100 mg/kg curcumin intraperitoneally at 0, 8 and 16 hours after operation (diluted with normal saline to 4 mL/kg) respectively; Sham operation group and sepsis group were injected with the same volume of normal saline. Five rats in each group were sacrificed at 2, 6, 12 and 24 hours after operation, the blood sample was collected, and liver tissues were harvested. The levels of serum procalcitonin (PCT), tumor necrosis factor-α (TNF-α) and interleukin (IL-6, IL-1ß) were measured by enzyme linked immunosorbent assay (ELISA), the pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining, and apoptosis index (AI) was measured by TdT-mediated dUTP nick end labeling (TUNEL) method. RESULTS: The degree of hepatocyte injury in sepsis group increased gradually with time, the apoptotic cells gradually increased, and the AI of liver cells increased to 24 hours; serum levels of PCT, TNF-α, IL-6 and IL-1ß were significantly higher than those in the sham operated group at 2 hours after operation and gradually increased to peak at 12 hours. The injury degree of liver tissue in Xuebijing group and curcumin group was significantly lighter than that in sepsis group, and the number of apoptotic cells were significantly decreased; the AI of hepatocytes and serum levels of PCT, TNF-α, IL-6 and IL-1ß were significantly lower than those of sepsis group from 2 hours [AI: (11.89±1.34)%, (11.56±0.96)% vs. (23.59±2.00)% at 2 hours, (28.95±1.40)%, (30.35±1.20)% vs. (52.05±1.31)% at 24 hours; PCT (µg/L): 1.27±0.18, 1.13±0.19 vs. 2.41±0.21 at 2 hours, 5.07±0.45, 5.09±0.42 vs. 8.68±0.58 at 12 hours; TNF-α (ng/L): 127.93±9.53, 124.73±7.47 vs. 217.28±14.24 at 2 hours, 171.03±8.58, 168.68±6.95 vs. 314.13±14.39 at 12 hours; IL-6 (ng/L): 132.15±9.27, 136.14±8.42 vs. 153.35±12.64 at 2 hours, 211.65±8.52, 213.37±8.96 vs. 298.11±12.35 at 12 hours; IL-1ß (ng/L): 33.59±1.49, 35.05±1.00 vs. 61.84±3.21 at 2 hours; 81.76±2.80, 84.06±3.42 vs. 132.24±2.58 at 12 hours, all P < 0.05]. There was no significant difference in the above indexes between Xuebijing group and curcumin group. CONCLUSIONS: Curcumin can inhibit the inflammatory response of hepatocytes in sepsis rats and reduce the apoptosis of hepatocytes, which can protect hepatocytes from sepsis.

[The dose-dependent protective effect of curcumin on hepatocyte of rats with sepsis].

Objective: To observe the protective effect of different doses of curcumin on hepatocytes of rats with sepsis. Methods: 100 healthy male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, and low, medium, high dose curcumin intervention groups (L-cur, M-cur, H-cur groups), with 20 rats in each group. The animal model of sepsis was reproduced by cecal ligation and puncture (CLP) method, and in the sham operation group the cecum was just taken out and returned. In the L-cur, M-cur, H-cur groups curcumin was immediately injected after CLP with a dose of 50, 100, 150 mg/kg, respectively, and the rats in sham operation group and sepsis group were given the same amount of normal saline. Five rats in each group were sacrificed at 2, 6, 12, 24 hours after operation, and the hepatic tissues and blood samples were obtained. The pathological changes in hepatic tissues were observed under a microscope, and hepatocytes apoptosis and apoptosis index (AI) of hepatocytes were determined with transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method, and the levels of serum procalcitonin (PCT), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were determined with enzyme linked immunosorbent assay (ELISA) method. Results: Microscopic examination showed that the damage degree of hepatic tissues was significantly increased in sepsis group; the number of apoptotic cells and damage degree of hepatic tissues were increased gradually over time. The damage degree of hepatic tissues in curcumin groups was lessened as compared with sepsis group, especially in M-cur group. There were no significant changes in AI and serum PCT, TNF-α, and IL-1ß levels at any of the time points tested in the sham operation group. The AI, serum PCT, TNF-α, and IL-1ß levels in the sepsis group were significantly higher than those in the sham operation group from 2 hours after operation on [AI: (23.59±2.00)% vs. (2.02±0.13)%, PCT (µg/L): 2.41±0.21 vs. 0.81±0.01, TNF-α (ng/L): 217.28±14.24 vs. 80.02±2.26, IL-1ß (ng/L): 61.84±3.21 vs. 25.78±1.29, all P < 0.05], and they showed a gradually increasing tendency. AI reached peak value at 24 hours after operation [(52.05±1.31)%]; PCT, TNF-α and IL-1ß reached the peak values at 12 hours after operation [(8.68±0.58) µg/L, (314.13±14.39) ng/L, (132.24±2.58) ng/L, respectively]. Curcumin intervention significantly reduced the levels of AI, TNF-α, PCT and IL-1ß in hepatocytes of septic rats, especially in M-cur group [AI: (11.56±0.96)% vs. (23.59±2.00)% at 2 hours, (30.35±1.20)% vs. (52.05±1.31)% at 24 hours; PCT (µg/L): 1.13±0.19 vs. 2.41±0.21 at 2 hours, 5.09±0.42 vs. 8.68±0.58 at 12 hours; TNF-α (ng/L): 124.73±7.47 vs. 217.28±14.24 at 2 hours, 168.68±6.95 vs. 314.13±14.39 at 12 hours; IL-1ß (ng/L): 35.05±1.00 vs. 61.84±3.21 at 2 hours, 84.06±3.42 vs. 132.24±2.58 at 12 hours; all P < 0.05]. Conclusions: Curcumin can inhibit the inflammatory reaction of hepatocytes of rats, prevent apoptosis, and protect the hepatocytes of rats with sepsis. The concentration of curcumin with the most significant effect is 100 mg/kg, which is the medium dosage.

CpG oligodeoxynucleotides are a potent adjuvant for an inactivated polio vaccine produced from Sabin strains of poliovirus.

Poliovirus transmission is controlled globally through world-wide use of a live attenuated oral polio vaccine (OPV). However, the imminence of global poliovirus eradication calls for a switch to the inactivated polio vaccine (IPV). Given the limited manufacturing capacity and high cost of IPV, this switch is unlikely in most developing and undeveloped countries. Adjuvantation is an effective strategy for antigen sparing. In this study, we evaluated the adjuvanticity of CpG oligodeoxynucleotides (CpG-ODN) for an experimental IPV produced from Sabin strains of poliovirus. Our results showed that CpG-ODN, alone or in combination with alum, can significantly enhance both the humoral and cellular immune responses to IPV in mice, and, consequently, the antigen dose could be reduced substantially. Therefore, our study suggests that the global use of IPV could be facilitated by using CpG-ODN or other feasible adjuvants.