Integrated single-cell RNA-seq analysis revealed podocyte injury through activation of the BMP7/AMPK/mTOR mediated autophagy pathway.

BACKGROUND: Nephrotic syndrome (NS) is a chronic kidney disease mainly caused by impaired podocytes, ultimately resulting in massive proteinuria or even end-stage renal disease (ESRD). METHODS: The objective of this study was to explore the potential pathogenesis of NS caused by podocyte injury, and further explore the underlying mechanism through data mining, bioinformatics analysis, and experimental verification. The integrated analyses including Seurat, CellChat, gene ontology (GO), and molecular docking were performed based on the single-cell RNA-seq data (scRNA-seq). The adriamycin (ADR)-induced podocyte injury model in vitro was established to conduct the experimental verification for bioinformatics analysis results through western blot and real-time quantitative PCR (RT-qPCR). RESULTS: The results of bioinformatics analysis revealed that the bone morphogenetic protein (BMP) signaling pathway was involved in the podocyte-to-podocyte communication, which plays a crucial role in podocyte injury. The expression of BMP7 was significantly increased in ADR-induced podocytes through activating the Adenosine-monophosphate activated-protein kinase/Mammalian target of rapamycin (AMPK/mTOR) mediated autophagy pathway, and these findings were confirmed by in vitro experiments. CONCLUSION: This study first demonstrated that BMP7 participated in ADR-induced podocyte injury. The BMP7/AMPK/mTOR mediated autophagy pathway may play a crucial role in podocyte injury, which may be the potential therapeutic target for NS patients.

The protective roles of liraglutide on Kawasaki disease via AMPK/mTOR/NF-κB pathway.

Kawasaki disease (KD) is an acute febrile rash illness among children of unknown etiology, with coronary artery injury. The main purpose of this study was to investigate the protective effects of liraglutide on KD, and elucidate the underlying mechanisms. The candida albicans water-soluble fraction (CAWS)-induced coronary arteritis of mouse KD model in vivo and tumor necrosis factor α (TNF-α) induced endothelial cell injury of human umbilical vein endothelial cell (HUVEC) model in vitro were used to explore the anti-inflammation and anti-apoptosis effects of liraglutide on KD. In vivo results showed that liraglutide could significantly alleviate the coronary artery injury of KD mice, as evidenced by the reduction of inflammatory infiltration around the coronary arteries, downregulation of inflammatory cytokines and chemokines expressions, and decrease of TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) positive cell rates. The results in vitro also displayed that liraglutide could markedly relieve the inflammatory of TNF-α induced HUVECs through downregulating the expressions of inflammatory and chemokine indicators as well as inhibit TNF-α induced HUVEC apoptosis by the less ratio of apoptotic cells, the more loss of mitochondrial membrane potential (△Ψm), the lower level of intracellular reactive oxygen species (ROS), and the more ratio of BCL-2/BAX. Further in vivo and in vitro studies demonstrated that liraglutide could rescue endothelial cell injury through AMPK/mTOR/NF-κB pathway. In conclusion, liraglutide could play protective roles on KD through inhibiting endothelial cell inflammation and apoptosis via the activation of AMPK/mTOR/NF-κB pathway.

The protective roles of allicin on type 1 diabetes mellitus through AMPK/mTOR mediated autophagy pathway.

Background: Type 1 diabetes mellitus (T1DM) is one of the most common endocrine and metabolic diseases in children. Pancreatic ß cells are thought to be critical cells involved in the progression of T1DM, and their injury would directly lead to impaired insulin secretion. Purpose: To investigate the protective effects of allicin on pancreatic ß cell injury and elucidate the underlying mechanism. Methods: The streptozotocin (STZ)-induced mouse T1DM model in vivo and STZ-induced pancreatic ß cell Min6 model in vitro were used to explore the effects of allicin on T1DM. The experiments include fasting blood glucose test, oral glucose tolerance detection, HE staining, immunohistochemistry, immunofluorescence, TUNEL staining, western blot, real-time quantitative PCR (RT-qPCR), and flow cytometry. Results: Allicin could significantly decrease blood glucose level, improve islet structure and insulin expression, and inhibit apoptosis to reduce STZ-induced pancreatic ß cell injury and loss through activating AMPK/mTOR mediated autophagy pathway. Conclusion: Allicin treatment significantly reduced STZ-induced T1DM progression, suggesting that allicin may be a potential therapy option for T1DM patients.

Establishment of an induced pluripotent stem cell line from a Noonan syndrome patient with the heterozygote mutation p.S257L (c.770C > T) in RAF1 gene.

Noonan Syndrome (NS) is an inherited autosome dominant disorder syndrome, which can be caused by the mutations of serine/threonine kinase rapidly accelerated fibrosarcoma 1 (RAF1) gene. Here, an induced pluripotent stem cell (iPSC) line named WMUi022-A derived from urine cells (UCs) of a 9-year-old male NS patient with the heterozygote RAF1 gene mutation p.S257L (c.770C > T) was established through the commercial Sendai virus reprogramming kit. The pluripotent markers like OCT4 and SOX2 can be expressed positively in WMUi022-A, which can be induced into three germ layers in vitro as well as maintain a normal karyotype (46, XY).

Generation of a human induced pluripotent stem cell line (WMUi021-A) from a Gitelman syndrome patient carrying a SLC12A3 gene mutation (c.179C > T).

Gitelman Syndrome (GS) is an inherited autosome recessive disorder syndrome, which can be caused by the gene mutations of solute carrier family 12 member 3 gene (SLC12A3). In present study, the urine cells (UCs) of a 7-year-old male GS patient with the homozygote SLC12A3 gene mutation p.T60M (c.179C > T) were reprogrammed into induced pluripotent stem cells (iPSCs) named WMUi021-A through the commercial Sendai virus reprogramming kit. The pluripotent markers OCT4 and SOX2 can be expressed positively in WMUi021-A, which can be differentiated into three germ layers in vitro as well as maintain a stable karyotype (46, XY).

Establishment of a human induced pluripotent stem cell line (WMUi031-A) from a Lowe syndrome patient carrying a OCRL gene mutation (c.2626dupA).

Lowe Syndrome (LS) is a rare X-linked multisystemic disorder syndrome, which can be caused by the gene mutations of OCRL. In present study, the urine cells (UCs) derived from a 12-year-old male LS patient with the hemizygote OCRL gene mutation p.M876N (c.2626dupA) were reprogrammed into induced pluripotent stem cells (iPSCs) named WMUi031-A through the commercial Sendai virus reprogramming kit. The pluripotent markers OCT4 and SOX2 can be expressed positively in WMUi031-A, which can be differentiated into three germ layers in vitro as well as maintain a stable karyotype (46, XY).

Establishment of an induced pluripotent stem cell line from a Antley-Bixler Syndrome (ABS) patient with the homozygote mutation p.R457H (c.1370G>A) in POR gene.

Antley-Bixler syndrome (ABS) is a rare inherited autosome recessive malformation syndrome, which can be caused by the gene mutations of cytochrome P450 oxidoreductase (POR). In this study, the urine cells (UCs) derived from a 5-year-old female ABS patient with the homozygote POR gene mutation p.R457H (c.1825C>G) were reprogramming into induced pluripotent stem cells (iPSCs) named WMUi018-A using a commercial Sendai virus reprogramming kit. The pluripotent markers of stem cells like OCT4 and SOX2 can be positively expressed in this iPSC line, which can be induced to differentiate into three germ layers in vitro and maintain a stable karyotype (46, XX).

Generation of an induced pluripotent stem cell line from a Bartter syndrome patient with the homozygote mutation p.A244D (c.731C>A) in SLC12A1 gene.

Bartter Syndrome (BS) is a group of rare inherited autosome-recessive disease, which can be caused by the gene mutations of sodium-potassium-chloride cotransporter gene (SLC12A1). Here, the urine cells (UCs) derived from a 4-year-old female BS patient with the homozygote SLC12A1 gene mutation p.A244D (c.731C>A) were reprogramming into induced pluripotent stem cells (iPSCs) named WMUi019-A using a commercial Sendai virus reprogramming kit. The pluripotent stem cell markers like OCT4 and SSEA4 can be positively expressed in this iPSC line, which can also be induced to differentiate into three germ layers in vitro and maintain a stable karyotype (46, XY).

Establishment of an induced pluripotent stem cell line (WMUi016-A) from a patient with X-linked Dent disease (X-Dent) carrying the hemizygote mutation p.R718* (c.2152C > T) in the CLCN5 gene.

The gene mutations of the chloride channel gene (CLCN5) can lead to the inherited X-linked Dent disease (X-Dent). The urine cells of a 4-year-old male X-Dent patient with the hemizygous CLCN5 gene mutation p.R718* (c.2152C > T) were reprogrammed into induced pluripotent stem cells (iPSCs) using integration free Sendai virus reprogramming system. The generated iPSCs stably expressed pluripotent stem cell markers and can be induced to differentiate into three germ layers in vitro. The karyotype of the generated iPSCs was normal (46, XY).

Generation of an urine-derived induced pluripotent stem cell line WMUi017-A from a X-linked Renpenning syndrome (X-RSY) patient with the hemizygous PQBP1 gene mutation p.P609A (c.1825C>G).

The mutations of polyglutamine binding protein 1 gene (PQBP1) can lead to the rare inherited X-linked Renpenning syndrome (X-RSY). Here, an induced pluripotent stem cell (iPSC) line WMUi017-A was generated through reprogramming the urine cells of a 5-year-old male X-RSY patient with the hemizygous PQBP1 gene mutation p.P609A (c.1825C>G) using the commercial Sendai virus reprogramming system. The established iPSCs can stably express pluripotent stem cell markers OCT4 and NANOG, and can be induced into three germ layers and maintain a normal karyotype (46, XY) in vitro.

Neutrophil-to-lymphocyte ratio, a critical predictor for assessment of disease severity in patients with COVID-19.

INTRODUCTION: Monitoring of laboratory indicators is important for predicting changes in disease severity and clinical outcomes. We aimed to identify the critical predictors that can effectively assess the disease conditions of patients with COVID-19 by analyzing the clinical characteristics and laboratory findings of patients with SARS-CoV-2 infection. METHODS: All consecutive patients (n = 294) with confirmed SARS-CoV-2 infection admitted to the General Hospital of Central Theater Command of the PLA from February 6 to February 21, 2020, were enrolled. These patients were divided into the severe group and the nonsevere group according to disease severity during hospitalization. RESULTS: The median neutrophil-to-lymphocyte ratio (NLR) value of the severe patients was dramatically higher than that of the nonsevere patients (10.4 vs 2.6; P < .001). The NLR value equal to 5 was a boundary value worthy of reference, because more than 80% severe patients had an NLR value greater than 5 and over 80% nonsevere patients had an NLR value less than 5. The NLR value of these COVID-19 patients was positively and respectively correlated with the values of C-reactive protein (R = .5921, P < .001), lactate dehydrogenase (R = .4509, P < .001), procalcitonin (R = .5504, P < .001), fibrinogen (R = .4710, P < .001), and D-dimers (R = .4425, P < .001). However, the NLR value was merely and positively correlated with the interleukin-6 value (R = .3594, P < .05), but had no correlations with the values of interleukin-10, interleukin-4, interleukin-17, interferon-γ, and tumor necrosis factor-α (P > .05). DISCUSSION: Neutrophil-to-lymphocyte ratio is a critical predictor for assessment of disease severity in patients with COVID-19, and it has a close relation with the laboratory indicators related to disease conditions.

Generation of a human induced pluripotent stem cell line with Cas9 driven by Tet-on operator via AAVS1 safe harbor gene-editing.

Human induced pluripotent stem (iPS) cells expressing Cas9 protein are valuable for the pathogenic mechanism study and drug discovery. These cells can be efficiently induced to differentiate into disease cell models with specific mutations through adding designed sgRNAs. Here, we generated a human gene-editable iPS cell line by gene editing method that Cas9 gene driven by Tet-on operator was perfectly integrated into the human AAVS1 safe harbor locus. The established Cas9 expression iPS cell line named as WMUi013-A can express endogenous pluripotent markers, has the ability to differentiate into the three germ layers, and possesses a normal karyotype.

Generation of an urine-derived induced pluripotent stem cell line from a 5-year old X-linked Alport syndrome (X-LAS) patient.

The gene mutations of the collagen type IV alpha 5 chain (COL4A5) can lead to the inherited haematuria to end-stage renal disease X-linked Alport syndrome (X-LAS). The urine cells of a 5-year-old male X-LAS patient carrying a hemizygous COL4A5 gene mutation p.G1433V (c.4298G>T) were reprogrammed to induced pluripotent stem cells (iPSCs) with Sendai virus reprogramming kit containing OCT4, SOX2, c-MYC, and KLF4 Yamanaka factors. The generated iPSC line WMUi015-A stably expressed pluripotent markers, maintained a normal karyotype (46, XY), and had differentiation potential into three germ layers in vitro.

Clinical application of Chemiluminescence Microparticle Immunoassay for SARS-CoV-2 infection diagnosis.

BACKGROUND: The unsatisfactory accuracy and capacity of real time RT-PCR depends on several unavoidable reasons, which cannot meet the demands for COVID-19 diagnosis. METHODS: 206 serum samples were collected from patients who were treated in the General Hospital of the Central Theater Command of the PLA between January 18 and April 4, 2020. 270 serum samples from healthy blood donors were used as control. IgM and total antibodies (Ab) against SARS-CoV-2 were detected by Chemiluminescence Microparticle Immunoassay (CMIA). RESULTS: Among the 206 patients, the positive rate of IgM and Ab were 149/206 (72.3 %) and 187/206 (90.8 %), respectively. And the specificity of IgM and Ab detection were 99.3 % and 98.9 %, respectively. The sensitivity of CMIA for Ab detection was significantly higher than that of IgM. An increase of the positive rate and S/CO value for detecting IgM and Ab accompanied with the increasing of days post-disease onset (d.p.o.) were observed. The positive rate of Ab detected by CMIA increased rapidly after 7 d.p.o., while that of IgM was obviously increased after 14 d.p.o.. In addition, the age and gender of these patients did not affect the seroconversion and titer of antibodies during the whole course. The disease-severity of patients had no effect on the seroconversion of antibodies. However, the critical patients possessed a much higher antibody titers than the no-critical cases after 14 d.p.o.. CONCLUSIONS: The CMIA can provide important complementation to nucleic acid assay and help to enhance the accuracy and capacity of diagnosis of SARS-CoV-2 infection.

A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients.

In this study, we aimed to evaluate the diagnostic value of serological assay for SARS-CoV-2. A newly-developed ELISA assay for IgM and IgG antibodies against N protein of SARS-CoV-2 was used to screen the serums of 238 admitted hospital patients between February 6 and February 14, 2020 with confirmed or suspected SARS-CoV-2. SARS-CoV-2 RNA was detected on pharyngeal swab specimens using real time RT-PCR. 194 (81.5%) of the serums were detected to be antibody (IgM and/or IgG) positive, significantly higher than the positive rate of viral RNA (64.3%). There was no difference in the positive rate of antibodies between the confirmed patients (83.0%, 127/153) and the suspected patients (78.8%, 67/85), whose nucleic acid tests were negative. The antibody positive rates were very low in the first five days after initial onset of symptoms, and then rapidly increased as the disease progressed. After 10 days, the antibody positive rates jumped from below 50% to over 80%. However, the positive rates of viral RNA maintained above 60% in the first 11 days after initial onset of symptoms, and then rapidly decreased. Overall, the suspected patients were most likely infected by SARS-CoV-2. Before the 11th day after initial onset of symptoms, nucleic acid test is key for confirmation of viral infection. The combination of serological assay can greatly improve the diagnostic efficacy. After the 11th day post-disease onset, the diagnosis for viral infection should be majorly dependent on serological assay.

Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for Detecting Antibodies against SARS-CoV-2.

At present, PCR-based nucleic acid detection cannot meet the demands for coronavirus infectious disease (COVID-19) diagnosis. Two hundred fourteen confirmed COVID-19 patients who were hospitalized in the General Hospital of Central Theater Command of the People's Liberation Army between 18 January and 26 February 2020 were recruited. Two enzyme-linked immunosorbent assay (ELISA) kits based on recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (rN) and spike protein (rS) were used for detecting IgM and IgG antibodies, and their diagnostic feasibility was evaluated. Among the 214 patients, 146 (68.2%) and 150 (70.1%) were successfully diagnosed with the rN-based IgM and IgG ELISAs, respectively; 165 (77.1%) and 159 (74.3%) were successfully diagnosed with the rS-based IgM and IgG ELISAs, respectively. The positive rates of the rN-based and rS-based ELISAs for antibody (IgM and/or IgG) detection were 80.4% and 82.2%, respectively. The sensitivity of the rS-based ELISA for IgM detection was significantly higher than that of the rN-based ELISA. We observed an increase in the positive rate for IgM and IgG with an increasing number of days post-disease onset (d.p.o.), but the positive rate of IgM dropped after 35 d.p.o. The positive rate of rN-based and rS-based IgM and IgG ELISAs was less than 60% during the early stage of the illness, 0 to 10 d.p.o., and that of IgM and IgG was obviously increased after 10 d.p.o. ELISA has a high sensitivity, especially for the detection of serum samples from patients after 10 d.p.o., so it could be an important supplementary method for COVID-19 diagnosis.

Transcriptional Regulation Between the Two Global Regulators RovA and CRP in Yersinia pestis biovar Microtus.

Yersinia pestis is a dangerous bacterial pathogen that can cause plague. Both RovA and cyclic AMP receptor protein (cAMP-CRP) are required for regulating biofilm- and virulence-related genes in Y. pestis. In this study, the transcriptional regulation between RovA and cAMP-CRP were analyzed by using primer extension, quantitative RT-PCR, LacZ fusion, and electrophoretic mobility shift assay. The results indicated that RovA repressed crp transcription in an indirect manner, while that RovA had no regulatory action on cyaA at the transcriptional level. In addition, cAMP-CRP did not regulate the transcription of rovA. Taken together with our previous results, complex regulatory interactions of RovA, cAMP-CRP, and PhoP/PhoQ in Y. pestis were revealed, which would promote us gain deeper understanding about coordinative modulation of biofilm- and virulence-related regulator genes.