A naturally occurring 4-bp deletion in the intron 4 of p53 creates a spectrum of novel p53 isoforms with anti-apoptosis function.

p53 functions as a tumor suppressor by transcriptionally regulating the expression of genes involved in controlling cell proliferation or apoptosis. p53 and its isoform Δ133p53/Δ113p53 form a negative regulation loop in that p53 activates the expression of Δ133p53/Δ113p53 while Δ133p53/Δ113p53 specifically antagonizes p53 apoptotic activity. This pathway is especially important to safeguard the process of embryogenesis because sudden activation of p53 by DNA damage signals or developmental stress is detrimental to a developing embryo. Here we report the identification of five novel p53 isoforms. p53ß is generated due to alternative splicing of the intron 8 of p53 while the other four, namely, TA2p53, TA3p53, TA4p53 and TA5p53, result from the combination of alternative splicing of intron 1 (within intron 4 of the p53 gene) of the Δ113p53 gene and a naturally occurring CATT 4 bp deletion within the alternative splicing product in zebrafish. The CATT 4 bp deletion creates four translation start codons which are in-frame to the open reading frame of Δ113p53. We also show that TAp53 shares the same promoter with Δ113p53 and functions to antagonize p53 apoptotic activity. The identification of Δ113p53/TA2/3/4/5p53 reveals a pro-survival mechanism which operates robustly during embryogenesis in response to the DNA-damage condition.

Comparison of Third-Generation Sequencing Technology and Traditional Microbiological Detection in Pathogen Diagnosis of Lower Respiratory Tract Infection.

BACKGROUND: It is common to obtain a low detection rate and unsatisfactory detection results in complex infection or rare pathogen detection. This retrospective study aimed to illustrate the application value and prospect of the third-generation sequencing technology in lower respiratory tract infection disease. METHODS: This study recruited 70 patients with lower respiratory tract infection (LRTI). Pathogen detection of bronchoalveolar lavage fluid (BALF) from all patients was performed using nanopore metagenomic sequencing technology and traditional culture. BALF culture combined with quantitiative PCR (qPCR) was used as a reference standard to analyze the sensitivity and specificity of nanopore sequencing technology. The current study also collected the examination results of enrolled samples using technical methods sputum culture, tuberculosis DNA (TB-DNA), and Xpert MTB/RIF and analyzed the detection efficiency of nanopore sequencing for Mycobacterium tuberculosis. RESULTS: The positive rates of pathogens in 70 BALF samples detected by conventional culture and nanopore sequencing were 25.71% and 84.29%, respectively. Among the 59 positive BALF cases using nanopore sequencing, a total of 31 pathogens were identified, of which the proportions of bacteria, fungi, viruses, and other pathogens were 50%, 17%, 32%, and 1%, respectively. Using the results combined with culture and qPCR detection methods as the standard, the pathogen detection of BALF using nanopore sequencing had a sensitivity of 70% and a specificity of 91.7%. Additionally, the positive rate of the detection of M. tuberculosis using nanopore sequencing was 33.3% (6/18). The clinical medication plans of 74.3% (52/70) of the patients were referred to the nanopore sequencing results, of which 31 cases changed their treatment strategy, 21 supported the previous treatment plans, and 90% (47/52) of the patients finally had clinical improvement. CONCLUSIONS: BALF detection using nanopore sequencing technology improves the process of detecting pathogens in patients with LRTI, especially for M. tuberculosis, fungi, and viruses, by reducing the report time from three days to six hours. The clinical application prospect of nanopore sequencing technology is promising in the pathogen diagnosis of LRTI.

[Spatial and temporal variations of landscape ecological risk in the dry and hot valley of the Jinsha River during 2000-2020]. / 2000­2020年金沙江干热河谷景观生态风险的时空变化.

Scientific assessment of landscape ecological risk in ecologically fragile areas of the upper reaches of the Yangtze River is of great significance to regional ecological regulation and construction of the Yangtze River ecological security barrier. With the dry-hot valley area of Jinsha River in Yunnan Province as the research area, we constructed a landscape ecological risk evaluation model, and analyzed the spatial and temporal variations of regional landscape ecological risk. The results showed that the average values of landscape ecological risk index (LER) in the study area were 0.414, 0.398, and 0.462 in 2000, 2010 and 2020, respectively. The LER value of the whole region had reached a higher risk level by 2020. In 2000 and 2010, the landscape ecological risk zones of each level were staggered, and the high-risk zones showed a centralized distribution in 2020. During the two decades, the average LER of each section in the study area was around 0.42, which was close to the high risk level, indicating high landscape ecological risk level. The area of middle and low risk zones had decreased, while the area of high risk zone had significantly increased. The area of high risk zone in the western and middle sections was much higher than that in the eastern section. The area with significant changes of landscape ecological risk accounted for about 55% of the total study area, with obvious spatial agglomeration characteristics of significant increase and decrease of risk. The competition between government-led ecological management policies and measures and market-led land use activities was the main cause of landscape ecological risk variations in this region. In the future, the driving mechanism of climate change coupled with human activities on global and local landscape ecological risk changes in the study area should be uncovered to effectively cope with regional ecological risks.

Using an untargeted metabolomics approach to analyze serum metabolites in COVID-19 patients with nucleic acid turning negative.

Background: The coronavirus disease of 2019 (COVID-19) is a severe public health issue that has infected millions of people. The effective prevention and control of COVID-19 has resulted in a considerable increase in the number of cured cases. However, little research has been done on a complete metabonomic examination of metabolic alterations in COVID-19 patients following treatment. The current project pursues rigorously to characterize the variation of serum metabolites between healthy controls and COVID-19 patients with nucleic acid turning negative via untargeted metabolomics. Methods: The metabolic difference between 20 COVID-19 patients (CT ≥ 35) and 20 healthy controls were investigated utilizing untargeted metabolomics analysis employing High-resolution UHPLC-MS/MS. COVID-19 patients' fundamental clinical indicators, as well as health controls, were also collected. Results: Out of the 714 metabolites identified, 203 still significantly differed between COVID-19 patients and healthy controls, including multiple amino acids, fatty acids, and glycerophospholipids. The clinical indexes including monocytes, lymphocytes, albumin concentration, total bilirubin and direct bilirubin have also differed between our two groups of participators. Conclusion: Our results clearly showed that in COVID-19 patients with nucleic acid turning negative, their metabolism was still dysregulated in amino acid metabolism and lipid metabolism, which could be the mechanism of long-COVID and calls for specific post-treatment care to help COVID-19 patients recover.

Ribonuclease like 5 regulates zebrafish yolk extension by suppressing a p53-dependent DNA damage response pathway.

Ribonuclease like 5 (Rnasel5) is a novel member of the zebrafish ribonuclease A family and its expression is increased during early embryogenesis. However, the in vivo biological function of Rnasel5 remains to be elucidated. Here, we report that knockdown of Rnasel5 by morhpolinos caused shrunken yolk extension as well as increased DNA damage at yolk syncytial layer and external tissue layers via the activation of p53 pathway. In addition, the morphological defects caused by Rnasel5 knockdown can be partially rescued by mRNA injection. Our findings provide the first functional characterization of Rnasel5 in zebrafish development and reveal its critical role in yolk extension by modulation of the p53 pathway.

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

Protein interaction between p53 and Δ113p53 is required for the anti-apoptotic function of Δ113p53.

Zebrafish Δ113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, Δ113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed Δ113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and Δ113p53 proteins also interact with each other, and if this interaction is required for Δ113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and Δ113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C10 for detection, we demonstrated that endogenous Δ113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six Δ113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those Δ113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein-protein interaction between Δ113p53 and p53 is essential for the anti-apoptotic function of Δ113p53. In addition, the two Δ113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of Δ113p53.

Expression of CCR7 and Tim-3 in Childhood Patients with Acute Lymphoblastic Leukemia and Their Predictive Value for Prognosis / 中国实验血液学杂志

OBJECTIVE@#To analyze the expression of CCR7 and Tim-3 in childhood patients with acute lymphoblastic leukemia (ALL) and their predictive value for prognosis.@*METHODS@#Eighty-six newly diagnosed ALL childhood patients from January 2007 to January 2017 treated in our hospital were selected. The expression level of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients were detected by flow cytometry, all the patients were divided into the recurrence group and non-recurrence group according to the follow-up results, the differences in the expressions of CCR7, Tim-3 between the two groups were compared. The correlation between the expression of CCR7 , Tim-3 and the clinicopathologic features of ALL patients were analyzed, the predictive value of CCR7 and Tim-3 for the prognosis of newly ALL patients were evaluated by ROC curve, and the relationship between serum CCR7, Tim-3 and prognosis were analyzed.@*RESULTS@#The expression levels of CCR7 and Tim-3 in recurrence group were significantly higher than those in non-recurrence group(P0.05). The exogenous infiltration rate of patients with high expression of CCR7 and Tim-3 was significantly higher than those in low expression group (P<005). The high expression rate 76.9% of Tim-3 in patients with T-ALL was significantly higher than that of B-ALL patients with Tim-3 high expression rate 45.2% (P<0.05). The median OS of patients with CCR7 level ≥45.97% and <45.97% were 9.3 months and 13.6 months respectively(P=0.004), and the Tim-3≥53.54% and Tim-3<53.54% were 9.1 months and 13.6 months respectively(P=0.001). The results of Cox's multi-factor regression analysis showed that CCR7 level(HR=1.024, 95 CI 1.001-1.049) and Tim-3 level (HR=1.879, 95 CI 1.183- 2.985) were the independent risk factors that affect the OS in ALL patients(P<0.05).@*CONCLUSION@#The expression of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients shows good predictive value for prognosis, and the combination of CCR7 and Tim-3 can improve the sensitivity of the detection, the higher expression of CCR7 and Tim-3 can be used as potential indexes in prognosis evaluate.

Expression of Wif-1 and β-catenin in the Wnt pathway in childhood acute lympho-blastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression and possible roles of Wnt inhibitory factor-1 (Wif-1) and β-catenin in the Wnt pathway in childhood acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>The clinical data of 35 children who had newly-diagnosed ALL and achieved complete remission on day 33 of remission induction therapy were retrospectively reviewed. The children before treatment were considered as the incipient group, and those who achieved complete remission on day 33 were considered as the remission group. Fifteen children with non-malignant hematologic diseases were enrolled as the control group. RT-PCR was used to measure the mRNA expression of Wif-1 and β-catenin. ELISA was used to measure the protein expression of Wif-1.</p><p><b>RESULTS</b>Compared with the control and remission groups, the incipient group had significantly lower mRNA and protein expression of Wif-1 and significantly higher mRNA expression of β-catenin (P<0.05). In the incipient and remission groups, high-risk children showed significantly higher mRNA expression of β-catenin and significantly lower mRNA and protein expression of Wif-1 than the medium- and low-risk children (P<0.05). In the incipient and remission group, the children with T-cell acute lymphoblastic leukemia showed significantly higher mRNA expression of β-catenin and significantly lower mRNA and protein expression of Wif-1 compared with those with B-lineage acute lymphoblastic leukemia (P<0.05). In each group, there was a negative correlation between the mRNA expression of Wif-1 and β-catenin (P<0.05).</p><p><b>CONCLUSIONS</b>Reduced expression of Wif-1 and increased expression of β-catenin may be involved in the pathogenesis of childhood ALL, and the degree of reduction in Wif-1 and/or increase in β-catenin may be related to prognosis.</p>