The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

[Effect of moxibustion on JAK2/STAT3 signal pathway in synovial tissues of rabbits with adjuvant arthritis under the overexpression of NLRP3 inflammatory bodies].

OBJECTIVE: To observe the effect of moxibustion on Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2-STAT3) signal pathway and interleukin (IL)-1ß and IL-18 in synovial fluid of adjuvant arthritis (AA) rabbits, so as to explore the mechanism of moxibustion in the treatment of rheumatoid arthritis. METHODS: Twenty-eight rabbits were randomized into control, model, moxibustion, and NLRP3 overexpression groups, with 7 rabbits in each group. AA rabbit model was established by injection of Freund's complete adjuvant (FCA, 0.5 mL/kg) into the rabbits' bilateral hind-knee joint cavities. On the third day after modeling, the NLRP3 lentiviral vector (40 µL) were injected into the bilateral hind-knee joint cavities of rabbits in NLRP3 overexpression group. Moxibustion was used to bilateral "Shenshu" (BL23) and "Zusanli" (ST36), 5 cones every time, once daily, 6 days a week for 3 weeks in the moxibustion and NLRP3 overexpression groups. The perimeters of rabbits' hind legs were measured after modeling and after the intervention. The contents of IL-1ß, IL-18 in synovial fluid were detected by ELISA and the expression levels of NLRP3, JAK2 and STAT3 mRNAs in synovial tissue were detected by real-time PCR. RESULTS: In comparison with the control group, the perimeters of bilateral knee joints were significantly increased at each time point (P<0.05),and the contents of IL-1ß, IL-18 in synovial fluid and the expression levels of NLRP3, JAK2, STAT3 mRNA in synovial tissue were significantly increased (P<0.05) in the model group. Compared with the model group, the perimeters of bilateral knee joints were significantly decreased (P<0.05), and the contents of IL-1ß, IL-18 in synovial fluid and the expression levels of NLRP3, JAK2, STAT3 mRNAs in synovial tissue were significantly decreased (P<0.05) in the moxibusion group. Compared with the moxibustion group, the above indexes were higher in the NLRP3 overexpression group (P<0.05). CONCLUSION: Moxibustion may play an anti-inflammatory and detumescent role in AA rabbits by regulating JAK2-STAT3 signal pathway, and its therapeutic effect may be closely related to the expression of NLRP3.

Case Report: Identification of Potential Prognosis-Related TP53 Mutation and BCL6-LPP Fusion in Primary Pituitary Lymphoma by Next Generation Sequencing: Two Cases.

Background: Primary pituitary lymphoma (PPL) is an extremely rare disease with poor prognosis. Although PPL has been shown to be different from classical primary central nervous system lymphoma because of the embryological origin of structures, individual and precise treatment of PPL remains unknown. Methods: A 61-year-old man and a 65-year-old woman both diagnosed with primary pituitary diffuse large B cell lymphoma underwent genetic analysis of cerebrospinal fluid and tumor tissue by next generation sequencing. Results: In the first case, partial remission was achieved following R²-MTX chemotherapy. In the other case with TP53 mutation and BCL6-LPP fusion, disease progressed although different chemotherapy regimens were given. Conclusion: The gene mutation of TP53 and BCL6 may be identified as a marker responsible for prognostic difference in patients with PPL. Genetic analysis may provide a novel approach for precise management and prognosis prediction.

[Mechanism on moxibustion for rheumatoid arthritis based on PD-1/PD-L1 signaling pathway].

OBJECTIVE: To explore the mechanism of moxibustion on the treatment of rheumatoid arthritis (RA) in the perspective of programmed cell death-1 and its ligand 1 (PD-1/PD-L1). METHODS: A total of 30 Japanese big ear white rabbits were randomly divided into a control group, a model group and a moxibustion group, 10 rabbits in each one. In the model group and the moxibustion group, RA model was prepared by the injection of Freund's complete adjuvant (FCA) into the hind knee joint cavities of each rabbit. In the control group, 0.9% sodium chloride solution of the same dose was injected. On the 8th day of experiment, in the moxibustion group, moxibustion was applied to "Shenshu" (BL 23) and "Zusanli" (ST 36), 5 cones at each acupoint, on the bilateral sides alternatively, once a day, 6 treatments as one course, with an interval of 1 days between the treatment courses. Totally, 3 courses of treatment were required. On the 1st, 7th, 14th, 21st and 28th days of experiment, successively, the circumference of the bilateral knee joints was measured with the tape. On the 28th day of experiment, H.E. staining was adopted to observe the histopathological morphology and to evaluate the score of knee synovial tissue. ELISA was used to determined the concentrations of soluble PD-1 (sPD-1) and its ligand 1 (sPD-L1), the interleukin 2 (IL-2) and IL-17 in knee synovial fluid and the concentrations of sPD-1 and sPD-L1 in serum. The histochemistry method was used to determine the expressions of membrane PD-1 (mPD-1) and its ligand 1 (mPD-L1) in spleen tissue. RESULTS: On the 14th, 21st and 28th days of experiment, the circumference of both knee joints was increased in each of the rabbits in the model group as compared with the control group (P<0.01), and it was reduced significantly in the moxibustion group as compared with the model group (P<0.01). Compared with the control group, the hyperplasia of synovial tissue and fibrous tissue, as well as inflammatory cell infiltration were increased obviously in the model group (P<0.01), and they were reduced significantly in the moxibustion group as compared with the model group (P<0.01, P<0.05). Compared with the control group, the concentrations of IL-2 and IL-17 in knee synovial fluid were increased in the rabbits of the model group (P<0.01). Compared with the model group, after the intervention with moxibustion, the concentrations of IL-2 and IL-17 in knee synovial fluid were reduced in the rabbits of the moxibustion group (P<0.05). Compared with the control group, the concentrations of sPD-1 and sPD-L1 in knee synovial fluid and serum in the rabbits of the model group were all increased (P<0.05). Compared with the model group, the concentration of sPD-1 in the knee synovial fluid and serum were reduced in the rabbits of moxibustion group (P<0.05). Compared with the control group, the expressions of mPD-1 and mPD-L1 in spleen tissue were increased obviously in the rabbits of the model group (P<0.05). Compared with the model group, the expression of mPD-L1 in spleen tissue was up-regulated in the rabbits of the moxibustion group (P<0.05). CONCLUSION: Moxibustion could inhibit the over-activation of T cells by enhancing the negative regulation of PD-1/PD-L1 signaling pathway so as to play its effect in treatment of RA.

Moxibustion benignantly regulates circadian rhythm of REV-ERBα in RA rats.

The key clinical symptoms and previous findings of RA show a circadian variation, with more prominent joint swelling, stiffness, and pain occurring in the early morning. Moxibustion is able to relieve RA in various pass ways, however, there is no verifying study results for the pathological rhythm of RA. Therefore, we conducted this work to verify whether moxibustion could adjust RA circadian rhythm according to regulate core clock genes. Based on these previous findings that circadian timekeeping is disturbed in RA at molecular level, the aim of this study was to observe the influence of moxibustion on expression level and circadian rhythm of REV-ERBα at different tissues of RA rats. Furthermore, the expression level of core clock genes closely related to RA were evaluated by RT-PCR. 96 SD rats were randomly assigned as 1:1:1:1 ratio to 4 groups for normal control group, RA model group, 5-7 am moxibustion group, and 5-7 pm moxibustion group. RT-PCR was used to measure the relatively expression quantity of REV-ERBα, CLOCK, BMAL1, and PER2 in hypothalamus, hippocampus, and adrenal gland. In RA rats, the expression level of REV-ERBα mRNA were up-regulated in different tissues, and moxibustion potentially up-regulated them in different degrees. In untreated RA rats, the circadian rhythm of REV-ERBα mRNA in hippocampus and adrenal gland both disappeared (P>0.05) and moxibustion was able to recover them (P<0.05). The expression level of CLOCK and PER2 mRNA in hippocampus and adrenal gland were down-regulated significantly (P<0.05) in RA model rats, while moxibustion up-regulated both of them in hippocampus (P<0.05). These results suggested together that moxibustion can benign regulate circadian rhythm of REV-ERBα in different tissues of RA rats. It was revealed that moxibustion not only recovered the losing diurnal oscillation of REV-ERBα in hippocampus and adrenal gland, but also adjusted the circadian rhythm of REV-ERBα in hypothalamus, hippocampus, and adrenal gland to close the normal circadian pattern.

Identifying Antigens Recognized by Cytolytic T Lymphocytes on Tumors.

Identification of antigenic peptides recognized by cytolytic T lymphocytes (CTL) is a prerequisite for the development of targeted cancer immunotherapy approaches. This chapter provides a global approach for the identification of peptides recognized by CTL. It implies the identification of the HLA molecule presenting the peptide as well as the design and screening of a cDNA library derived from the tumor cells. Methods used for the identification of spliced peptides on tumors are also described.

[Effects of moxibustion at different time points on foot swelling, serum level of TNF-α and circadian rhythm in rats with rheumatoid arthritis].

OBJECTIVE: To study the effects of moxibustion at different time points on serum level of tumor necrosis factor-α (TNF-α) in rats with rheumatoid arthritis (RA), and to explore its regulation mechanism on circadian rhythm. METHODS: A total of 96 Sprague-Dawley (SD) adult rats were randomly assigned into a blank group, a model group, a moxibustion at 5-7 AM group and a moxibustion at 5-7 PM group, 24 rats in each group, half male and half female. Each group was further divided into a 0 AM group, a 6 AM group, a 12 N group and a 6 PM group, 6 rats in each group. All rats were treated with the 12 h/12 h light-dark cycle in the whole process of experiment. Except for the blank group, all rats were treated with intracutaneous injection of freund's complete adjuvant (FCA) at right foot pad to establish the RA model. The rats at the two moxibustion groups were treated with grain-sized moxibustion at "Shenshu" (BL 23) and "Zusanli" (ST 36) at 5-7 AM and 5-7 PM, respectively, one side per treatment, once a day; six treatments were taken as one course and 3 courses were given with an interval of one day between courses. The rats in the remaining groups were treated with identical fixation but without moxibustion intervention. The right foot volume was measured before model establishment, after model establishment and after treatment. The blood samples were collected after treatment and the serum level of TNF-α was measured by ELISA. The SPSS 21.0 software and Halberg Cosinor were adopted to analyze the experiment data. RESULTS: After treatment, compared with the blank group, the foot swelling severity was significantly increased in the model group, moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (all P<0.01); compared with the model group, the foot swelling severity was significantly reduced in the moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (both P<0.01). Compared with the blank group, the serum level of TNF-α was increased significantly in the model group and moxibustion at 5-7 AM group (both P<0.05); compared with the model group, the serum level of TNF-α was reduced significantly in the moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (both P<0.05). The serum level of TNF-α showed circadian rhythm in all the groups (all P<0.05), and the peak appeared at night phase; compared with the blank group, the median value of TNF-α was increased significantly in the model group (P<0.05), the peak phase was delayed and the amplitude was increased (P<0.05); compared with the model group, the median value of TNF-α was significantly reduced in the moxibustion at 5-7 AM group and moxibustion at 5-7 PM group (P<0.01), the peak phase was advanced and the amplitude was reduced (P<0.05). CONCLUSION: Moxibustion could effectively reduce the serum level of TNF-α to relieve the foot swelling severity in RA rats. Moxibustion could regulate the circadian rhythm of TNF-α to play its effects on the inhibition of the synthesis of TNF-α. No efficacy is observed between the treatment at 5-7 AM and 5-7 PM.

[Effects of Chronological Moxibustion on Circadian Rhythm Activities of Hypothalamus-Pituitary-Axis in Rheumatoid Arthritis Rats].

OBJECTIVE: To observe the effect of chronological moxibustion on plasma corticosterone (CORT), corticotrophin releasing factor (CRF) and adrenocorticotropic hormone (ACTH) levels in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA. METHODS: A total of 144 SD rats were randomly divided into normal control, model, moxibustion, sham-adrenalectomy (ADX, sham-ADX), ADX and ADX+moxibustion groups which were further separately divided into 4 subgroups (4 time-points: 12 pm, 6 am, 12 am, 6 pm, n = 6/subgroup). The RA model was established by subcutaneous injection of Freund's complete adjuvant (FCA, 0. 1 mL) into the right footpad (for rats of the latter 5 groups). In rats of the ADX and ADX+ moxibustion groups, bilateral adrenal glands were removed under anesthesia. Moxibustion was applied to unilateral "Shenshu" (BL 23) and "Zusanli" (ST 36) for 10 min, once daily for 6 days in a week, and continuously for 3 weeks. The paw swelling volume was measured and plasma CORT, ACTH and CRF contents were assayed by ELISA and the circadian rhythm was analyzed by cosine curve fitting (cosinor) method. RESULTS: The paw swelling volume was significantly increased in the model group than in the normal control group (P<0.05), and decreased remarkably in the moxibustion group (P<0.05). The paw volume was markedly increased in the ADX +moxibustion group in comparison with the moxibustion group (P<0.05). In intact rats, plasma CRF and ACTH contents were significantly increased (P<0.05), and plasma CORT was obviously decreased in the model group (P<0.05). After moxibustion, the increased plasma CRF and ACTH contents and the decreased plasma CORT level Nere considerably reversed (P<0.05). In ADX rats, plasma ORE, ACTH and CORT contents of ADX and ADX + moxibustion groups had no significant.changes compared with the sham-ADX group (P>0.05). Results of the cosine curve fitting analysis showed that the peak phases of plasma CORT contents were -355.78 degrees at about 23:43 in the normal control group, -309.05 degrees at about 20:36 in the model group, -326.5 degrees at about 21:46 in the moxibustion group, -291.65 degrees at about 19:27 in the ADX group and -300.87 degrees at about 20:31 in the ADX + moxibustion group. The peak phases of plasma ACTH contents were -324.08 degrees at about 21:37 in the normal control group, -295.39 degrees at about 19:41 in the model group, -310.81 degrees at about 20:43 in the moxibustion group, -146.51 degrees at about 9:46 in the ADX group and -267.64 degrees at about 17:50 in the ADX+ moxibustion group. The peak phases of plasma CRF contents were -257.47 degrees at about 17:10 in the normal control group, -184.74 degrees at about 12:19 in the model group, -263.00 degrees at about 17:32 in the moxibustion group, -202.46 degrees at about 13:30 in the ADX group and -232.84 degrees at about 15:31 in the ADX+ moxibustion group. It suggests that in the intact RA rats, moxibustion intervention may inhibit modeling-induced decrease of the circadian rhythm of plasma CORT, and hyperactivity of ACTH and CRF circadian rhythm. CONCLUSION: VAoxibustion intervention can relieve paw swelling in RA rats and modulate the circadian rhythm activities of plasma CORT, ACTH and ORE levels (HPA axis activities) in intact animals.

[Effects of grain-sized moxibustion from 7 am to 9 am on circadian rhythm of inflammatory factor IL-6 in rats with rheumatoid arthritis].

OBJECTIVE: To explore the rhythm regulatory mechanism of interleukin-6 (IL-6) in the process of moxibustion for rheumatoid arthritis (RA). METHODS: A total of 144 Sprague-Dawley (SD) rats were randomly divided into a blank group, a model group, a moxibustion group, a sham operation group, an operation group, an operation+moxibustion group, 24 rats in each one. Each group was divided into 4 time points (0:00 am, 6:00' am, 12:00 am, 6:00 pm), 6 rats in each time point. The Light-Dark 12 : 12 was given in all rats for light-dark cycle. Except the blank group, rats in the remaining groups were treated with intracutaneous injection of freund's complete adjuvant at right-side foot to establish the model of RA. After the model establishment, bilateral adrenal, glands were removed in the operation group and operation + moxibustion group, while those in the sham operation group were not removed with identical operation procedure. Rats in the moxibustion group and operation + moxibustion group were treated with grain-sized moxibustion from 7:00 am to 9:00 am at "Shenshu" (BL 23) and "Zusanli" (ST 36) once everyday, 6 times were taken as one session and 3 sessions were required tatclly, while rats in the remaining groups received identical fixation without moxibustion. The general health state and foot volume of rats were measured before model establishment, after establishment and after treatment. After treatment, rats were sacrificed at each time point to collect the blood sample and measure the content of IL-6 by using enzymne-immunoassay method. RESULTS: Compared with the blank group, the foot swelling in the model group was obviously increased (P<0. 05); the IL-6 maintained circadian rhythm (P<0. 05), but the peak phase had a backward trend, famplitude had an increased trend and the median was significantly lifted (P<0. 05). Compared with model group, !the foot swelling in the moxibustion group was obviously decreased (P<0. 05); the IL-6 maintained circadian. rhythm (P<0. 05), and the peak phase had a forward trend, amplitude had a decreased trend and the median was significantly reduced (P<0. 05). Compared with the moxibustion group, the foot swelling in the operation--moxibustion group was obviously increased (P < 0.05); the IL-6 maintained circadian rhythm (P < 0.5), but the peak phase moved forwrd, and the median was significantly elevated (P < 0.05). CONCLUSION: The IL-6 in plasma maintains significant pathological circadian rhythm in RA rats; with the complete hypothalamic-pituitary-adrenal axis, moxibustion is likely to regulate the circadian rhythm of IL-6 to play an important role of anti-inflammatory effect in RA rats.

Preclinical safety evaluation of human mesenchymal stem cell transplantation in cerebrum of nonhuman primates.

The efficacy of stem cell transplantation for promoting recovery of patients with neurological diseases, such as stroke, has been reported in several studies. However, the safety of the intracerebral transplantation of human mesenchymal stem cells (hMSCs) remains unclear. The aim of the study was to evaluate the safety of hMSCs transplanted in cerebrum of Macaca fascicularis and to provide evidence for clinical application. A total of 24 M fascicularis were assigned to 3 groups randomly: low dose (3.0 × 10(5) cells/kg), high dose (2.5 × 10(6) cells/kg), and the control (normal saline [NS]). Human mesenchymal stem cells or NS were injected into each monkey for 2 times, with an interval of 3 weeks. The injection point was located outside of the right putamen, according to a stereotactic map and preoperative magnetic resonance imaging of the monkeys. Animal health, behavior, biophysical and biochemical parameters, and brain neurological function were routinely monitored over a 6-month period posttransplantation, and the histopathologic examinations were also performed. The results showed that local pathologic damage including local tissue necrosis and inflammation was induced after the injection. The damage of low-dose and high-dose groups was greater than that of the control group, yet over time, the damage could be repaired gradually. No major hMSCs-associated changes were induced from other indicators, and the transplantation of hMSCs in monkeys did not affect total immunoglobulin (Ig) M, total IgG, CD3, CD4, or CD8 values. We therefore conclude that transplantation of hMSCs to the cerebrum represents a safe alternative for clinical application of neurological disorders.

A novel KRAB zinc-finger protein, ZNF480, expresses in human heart and activates transcriptional activities of AP-1 and SRE.

The zinc-finger motif found in many transcription factors is thought to be important for human heart development and diseases. In this study, we have identified and characterized a novel zinc-finger gene named ZNF480 using degenerate primers from an early human embryo heart cDNA library. ZNF480 contains a KRAB-A box and 12 C2H2 zinc fingers. The cDNA sequence contains an open reading frame of 1551 bp, encoding a putative protein of 516 amino acid residues with a predicted molecular mass of 57 kDa. Northern blot analysis indicates that a 4.7kb transcript specific for ZNF480 is expressed only in embryonic heart. In the adult tissues, the expression of ZNF480 is restricted largely to heart, skeletal muscle, pancreas, and placenta. Overexpression of ZNF480 in cells activates the transcriptional activities of AP-1 and SRE. Therefore, our data suggest that ZNF480 may act as a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.