Background: Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear. Methods: We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP). Results: We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. Conclusions PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.

Background: Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear. Methods: We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP). Results: We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. Conclusions PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.

Background: Nasopharyngeal carcinoma (NPC) is characterized by high programmed death-ligand 1 (PD-L1) expression and abundant infiltration of non-malignant lymphocytes, which renders patients potentially suitable candidates for immune checkpoint blockade therapies. Palate, lung, and nasal epithelium clone (PLUNC) inhibit the growth of NPC cells and enhance cellular apoptosis and differentiation. Currently, the relationship between PLUNC (as a tumor-suppressor) and PD-L1 in NPC is unclear. Methods: We collected clinical samples of NPC to verify the relationship between PLUNC and PD-L1. PLUNC plasmid was transfected into NPC cells, and the variation of PD-L1 was verified by western blot and immunofluorescence. In NPC cells, we verified the relationship of PD-L1, activating transcription factor 3 (ATF3), and β-catenin by western blot and immunofluorescence. Later, we further verified that PLUNC regulates PD-L1 through β-catenin. Finally, the effect of PLUNC on β-catenin was verified by co-immunoprecipitation (Co-IP). Results: We found that PLUNC expression was lower in NPC tissues than in paracancer tissues. PD-L1 expression was opposite to that of PLUNC. Western blot and immunofluorescence showed that β-catenin could upregulate ATF3 and PD-L1, while PLUNC could downregulate ATF3/PD-L1 by inhibiting the expression of β-catenin. PLUNC inhibits the entry of β-catenin into the nucleus. Co-IP experiments demonstrated that PLUNC inhibited the interaction of DEAD-box helicase 17 (DDX17) and β-catenin. Conclusions PLUNC downregulates the expression of PD-L1 by inhibiting the interaction of DDX17/β-catenin in NPC.

Transcriptional regulation based on transcription factors is an effective regulatory method widely used in microbial cell factories. Currently, few naturally transcriptional regulatory elements have been discovered from Saccharomyces cerevisiae and applied. Moreover, the discovered elements cannot meet the demand for specific metabolic regulation of exogenous compounds due to the high background expression or narrow dynamic ranges. There are abundant transcriptional regulatory elements in plants. However, the sequences and functions of most elements have not been fully characterized and optimized. Particularly, the applications of these elements in microbial cell factories are still in the infancy stage. In this study, natural regulatory elements from Medicago truncatula were selected, including the transcription factors MtTASR2 and MtTASR3, along with their associated promoter P_roHMGR1, for functional characterization and engineering modification. We constructed an inducible transcriptional regulation tool and applied it in the regulation of heterologous β-carotene synthesis in S. cerevisiae, which increased the β-carotene production by 7.31 folds compared with the original strain. This study demonstrates that plant-derived transcriptional regulatory elements can be used to regulate the expression of multiple genes in S. cerevisiae, providing new strategies and ideas for the specific regulation and application of these elements in microbial cell factories.
Medicago truncatula/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Transcription Factors/genetics* ; beta Carotene/biosynthesis* ; Promoter Regions, Genetic/genetics* ; Gene Expression Regulation, Plant ; Metabolic Engineering/methods* ; Regulatory Elements, Transcriptional/genetics* ; Plant Proteins/genetics*

Objective:To discuss the effects of 5-aza-2'-deoxycytidine(5-Aza-CdR)on autophagy and apoptosis of the TPC-1 cells in subcutaneous xenograft tumor tissue of the nude mice,and to clarify its mechanism.Methods:Sixteen female BALB/c nude mice were inoculated with human papillary thyroid carcinoma(PTC)TPC-1 cells in the right axilla to establish the xenograft tumor model.After tumor formation,the mice were randomly divided into control group and experiment group(n=8).The nude mice in control group were given an intraperitoneal injection of saline,while the nude mice in experiment group were given the intraperitoneal injection of 5-Aza-CdR,administered once every other day for four weeks.The growth status of xenograft tumor of the mice in both groups was observed,and the mice were sacrificed after the final administration and the tumor weights of the nude mice in two groups were detected.HE staining was used to observe the pathomorphology of xenograft tumor tissue of the nude mice in both groups;immunohistochemistry was used to detect the expression levels of microtubule-associated protein light chain 3(LC3),B-cell lymphoma 2(Bcl-2),and Bcl-2-associated X protein(Bax)in xenograft tumor tissue of the nude mice in two groups;real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of LC3,Beclin-1,Bcl-2,Bax,mitogen-activated protein kinase kinase(MEK),extracellular signal-regulated kinase 1(ERK1),extracellular signal-regulated kinase 2(ERK2),phosphorylated EPK1(p-ERK1),and phosphorylated EPK2(p-ERK2)mRNA and proteins in xenograft tumor tissue of the nude mice in two groups.Results:Compared with control group,the tumor volume and weight of the nude mice in experiment group were significantly decreased(P＜0.01).The number of cancer cells of the nude mice in control group was high,and the cells were densely arranged,with irregular shapes,clear nuclear staining,large overlapping nuclei,and lobulation,showing significant pathological mitotic figures consistent with PTC pathological characteristics.The number of cancer cells of the nude mice in experiment group showed a significant decresing trend,and the cells were sparse arrangement,nuclear shrinkage,and less distinct nuclei,with a significant increase in connective tissue.Compared with control group,the expression levels of LC3B and Beclin-1 mRNA and proteins in xenograft tumor tissue of the nude mice in experiment group were significantly increased(P＜0.05)and the ratio of and Bax/Bcl-2 was increased(P＜0.05 or P＜0.01),while the expression levels of MEK,ERK1/2,and p-ERK1/2 mRNA and proteins were significantly decreased(P＜0.05 or P＜0.01).Conclusion:5-Aza-CdR can inhibit the growth of TPC-1 cells in subcutaneous xenograft tumor tissue of the nude mice,induce the autophagy,and promote the apoptosis of the tumor cells.The mechanism may be related to the inhibition of the mitogen-activated protein kinase(MAPK)/MEK/extracellular signal-regulated kinase(ERK)signaling pathways.

Objective:To investigate the expression and significance of programmed death ligand 1 (PD-L1) and programmed death 1 (PD-1) in colorectal cancer complicated by schistosomiasis.Methods:A total of 134 patients with colorectal cancer who received treatment in Xuancheng People's Hospital during 2014-2021 were included in this study. These patients consisted of 74 patients with colorectal cancer combined with schistosomiasis (patient group) and 60 patients with only colorectal cancer (control group). The expression of PD-L1 and PD-1 in colorectal cancer tissue was detected by an immunohistochemical method. The differences in PD-L1 and PD-1 expression were compared between the two groups. The relationships between PD-L1 and PD-1 expression and clinical pathological characteristics were determined.Results:The positive expression rates of PD-L1 and PD-1 in cancer cells and interstitial lymphocytes were 55.4% and 60.8% respectively in the patient group and they were 35.0% and 40.0% respectively in the control group. The positive expression rates of PD-L1 and PD-1 were significantly higher in the patient group than the control group ( χ2 = 5.55, 5.74, both P < 0.05). The expressions of PD-L1 and PD-1 in the patient group were correlated with lymph node metastasis and high tumor-node-metastasis stage ( P < 0.05). Conclusion:PD-L1 and PD-1 are highly expressed in colorectal cancer complicated by schistosomiasis and are related to their invasive behavior. PD-1/PD-L1 singaling pathway may be involved in the molecular mechanism underlying the occurrence and development of colorectal cancer complicated by schistosomiasis. Blocking PD-1/PD-L1 signaling pathway may be a new strategy for immunotherapy of colorectal cancer complicated by schistosomiasis.

Objective:To investigate the effects of sodium fluoride on growth and development and serum oxidative stress of offspring rats.Methods:Twenty-four clean female SD rats and 24 clean male SD rats were selected, weighting 180 - 220 g, and mating in the same cage for 10 d according to 1 ∶ 1 for male and female. According to body weight by random number table method, the female rats were divided into control group, low-dose fluoride group, and high-dose fluoride group, 8 rats in each group. They were drunk 0, 100 and 200 mg/L sodium fluoride solution prepared with purified water, respectively, and they all ate standard feed. The female rats were exposed to fluoride from the 0th day of pregnancy to the 3rd week after the offspring rats were born (before weaning). After weaning, 10 female offspring rats were selected from each group and continued to be exposed to fluoride in the same amount and manner until the 12th week after birth. The body weight, body length and hind limb length of the offspring rats were measured every week before weaning and every two weeks after weaning. After 12th week of exposure to fluoride, blood samples were taken from abdominal aortas to detect the levels of serum superoxide dismutase (SOD), malondialdehyde (MDA), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC).Results:At the 2nd week after birth, the body weight [(24.87 ± 3.36) g], body length [(6.37 ± 0.52) cm] and hind limb length [(2.27 ± 0.13) cm] of the offspring rats in high-dose fluoride group were lower than those in control group [(29.23 ± 4.19) g, (6.92 ± 0.47), (2.44 ± 0.16) cm, P < 0.05], but there was no statistically significant difference between low-dose fluoride group and control group and high-dose fluoride group ( P > 0.05). At 3rd to 12th weeks after birth, the body weight, body length and hind limb length of the offspring rats in high-dose fluoride group were lower than those in low-dose fluoride group and control group ( P < 0.05), the low-dose fluoride group were lower than those in control group ( P < 0.05). Serum SOD, GSH-Px and T-AOC levels in control group [(176.51 ± 29.55), (985.23 ± 164.80) U/ml, (0.864 ± 0.167) mmol/L] were higher than those in low-dose fluoride group [(127.98 ± 24.41), (776.53 ± 107.85) U/ml, (0.639 ± 0.110) mmol/L] and high-dose fluoride group [(99.75 ± 14.56), (425.14 ± 78.67) U/ml, (0.441 ± 0.072) mmol/L], the levels of MDA and iNOS [(3.37 ± 0.73) nmol/ml, (189.00 ± 44.67) pg/ml] were lower than those in low-dose fluoride group [(8.22 ± 1.38) nmol/ml, (305.60 ± 73.41) pg/ml] and high-dose fluoride group [(14.81 ± 1.81) nmol/ml, (431.00 ± 91.19) pg/ml], the differences were statistically significant ( P < 0.05); the levels of serum SOD, GSH-Px and T-AOC in high-dose fluoride group were lower than those in low-dose fluoride group, and the levels of MDA and iNOS were higher than those in low-dose fluoride group ( P < 0.05). Conclusion:Excessive fluoride can increase the serum oxidative stress level of offspring rats, which may affect the growth and development of offspring rats.

Objectives:To study the feasibility of using ultrasound to evaluate diaphragm function in patients with invasive mechanical ventilation.Methods:From March to December 2017, 40 adult patients with acute respiratory distress syndrome who were admitted to the Department of Critical Care Medicine, Xiangya Hospital, Central South University for more than 48 hours were included. Diaphragmatic excursion and thickness of bilateral anterior, middle and posterior parts were measured by ultrasound for 5 consecutive days.Results:(1) Compared with the diaphragmatic excursion of the right [anterior: (11.05±3.04) mm; middle: (12.08±2.71) mm; posterior: (11.51±3.33) mm] and left [anterior: (13.63±7.52) mm; middle: (15.44±7.52) mm; posterior: (14.76±6.93) mm] sides on day 1, the diaphragmatic excursion of the right [anterior: (8.90±3.65) mm; middle: (10.02±4.24) mm; posterior: (10.25±4.38) mm] and left [anterior: (9.82±1.96) mm; middle: (11.60±1.13) mm; posterior: (11.52±1.98) mm] sides decreased significantly on day 3 ( P<0.05). Bilateral anterior, middle and posterior diaphragmatic excursion recovered on day 5, and was higher than the baseline levels on day 1, with the left middle and posterior diaphragmatic excursion changing most significantly. (2) Compared with day 1, 2, 3, the thickening fraction of bilateral anterior, middle and posterior diaphragm were significantly decreased on day 4, with the left middle part [day 1: (33.87±14.34)%; day 2: (37.26±13.91)%; day 3: (30.56±14.27)%; day 4: (15.53±5.68)%] and the left posterior part [day 1: (35.50±15.69)%; day 2: (39.84±15.32)%; day 3: (29.06±14.96)%; day 4: (13.30±5.79)%] changing most significantly ( P<0.05). The thickening fractions of left anterior, middle and posterior diaphragm recovered on day 5 compared with that on day 4, but still lower than those on day 1 ( P<0.05). Conclusions:It is feasible to evaluate the diaphragm function in patients with invasive mechanical ventilation by ultrasound, which can provide guidance for preventing diaphragmatic atrophy and withdrawing from mechanical ventilation.

Objective: To investigate the clinical significance of transcranial Doppler (TCD) in early diagnosis of sepsis-associated encephalopathy(SAE). Methods: Septic patients admitted to the intensive care unit(ICU) were recruited at Xiangya Hospital, Central South University from July 2015 to March 2016. Clinical data and TCD parameters during 24 hours after admission were collected. All patients were screened for delirium using the confusion assessment method for the intensive care unit (CAM-ICU) twice a day. The gold standard of the diagnosis of SAE was positive CAM-ICU evaluation. Patients were divided into SAE group and the non-SAE group. TCD data including systolic velocity (Vs), diastolic velocity (Vd), mean velocity (Vm), pulsatility index (PI) and resistant index (RI) were analyzed to determine the optimal diagnostic cut-off value. Results: A total of 43 patients were enrolled including 12 in SAE group and 31 in non-SAE group. Vm and Vd were lower in SAE group [Vm: (53.50±12.22) cm/s vs. (61.68±9.63) cm/s, P<0.05; Vd: (33.42±10.87) cm/s vs. (43.16±7.84) cm/s, P<0.01] but PI and RI were significant higher in SAE group[PI:(1.16±0.2) vs. (0.90±0.15), P<0.01;RI:(0.65±0.08) vs. (0.56±0.06), P<0.01] than in non-SAE group. The cut-off values of Vs, Vm, Vd, PI and RI for the diagnosis of SAE were 112cm/s, 55.50cm/s, 34.50cm/s, 1.16, 0.65, respectively, with the relevant sensitivities of 19.4%, 83.9%, 93.5%, 58.3%, 58.3% and the specificities of 100.0%, 50.0%, 58.3%, 96.8%, 96.8%, respectively. The diagnostic AUC of Vd, PI and RI were 0.741, 0.808 and 0.808 respectively. Conclusions The parameter changes of TCD suggest that the pathogenesis of SAE is related to cerebral hypoperfusion, TCD is a helpful method for the early diagnosis of SAE.

To explore the present status of fluid therapy and clinical outcome in critically ill patients in intensive care units (ICU).ICU patients consecutively admitted to our ICU were prospectively enrolled.Patients' demographics,laboratory data,fluid record and clinical outcome were collected.Fluid intake quantity of all patients was at peak on the fifth day which was 2 806 (1 997,3 582)ml.From the fourth day in ICU,fluid balance started to benegative as-84 (-1 127,612)ml and gradually increased.Crystalloid solution was the main components.For treatment purposes,medication injections and nutrients were major fluids.Positive correlations were found between total fluid intake quantity,total crystalloid volume,total colloidal volume and hospital stay,ICU stay,duration of intubation (r values as 0.211,0.686,0.282,0.155,0.506,0.174,0.209,0.072,0.292,respectively P＜0.05).Moreover,positive correlations were also demonstrated between total colloidal volume and total bilirubin,direct bilirubin,alanine transaminase,aspartate transaminase,blood urea nitrogen,serum creatinine (r values as 0.196,0.242,0.190,0.335,0.284,0.223,respectively P＜0.05).