[Application of transbronchial cryobiopsy in the diagnosis of postoperative complications after lung transplantation: a report of 6 cases].

Objective: To investigate the efficacy and safety of transbronchial cryobiopsy (TBCB) after lung transplantation. Methods: The clinical characteristics, TBCB procedure, diagnosis and treatment, and outcomes of lung transplant recipients of 6 patients (all male, aged 33-67 years) with TBCB in China-Japan Friendship Hospital from May to November 2021 were retrospectively analyzed. Results: Among the 6 patients diagnosed by TBCB, there were 2 cases of organizing pneumonia, 1 acute cellular rejection, 1 antibody-mediated rejection, and 1 bronchiolitis obliterans, and 1 diffuse alveolar damage. After the clinical diagnosis was confirmed, the condition improved after adjustment of the treatments followed. There were no serious complications related to the TBCB procedure. Conclusion: TBCB is valuable and relatively safe in the diagnosis of complications after lung transplantation, but the indications need to be strictly controlled.

[Safety and efficacy of transcatheter tricuspid valve replacement with LuX-Valve in patients with severe tricuspid regurgitation].

Objectives: To evaluate the safety and efficacy of LuX-Valve on the treatment of severe tricuspid regurgitation (TR). Methods: This is a prospective observational study. From September 2018 to March 2019, 12 patients with severe TR, who were not suitable for surgery, received LuX-Valve implantation in Changhai Hospital. LuX-Valve was implanted under general anesthesia and the guidance of transesophageal echocardiography and X-ray fluoroscopy. Access to the tricuspid valve was achieved via a minimally invasive thoracotomy and transatrial approach. Main endpoints were surgery success and device success. Surgery success was defined as successful implanting the device and withdrawing the delivery system, positioning the valve correctly and stably without severe or life-threatening adverse events. Device success was defined as satisfied valve function (TR severity reduction ≥ 2 grades, tricuspid gradient ≤ 6 mmHg (1 mmHg=0.133 kPa)), absence of malposition, valve failure and reintervention, major adverse events including device related mortality, embolization, conduction system disturbances and new onset shunt across ventricular septum at day 30 post implantation. Results: A total of 12 patients with severe to torrential TR were included in this study. The age was (68.5±6.9) years and 7 were female. All patients had typical right heart failure symptoms. Procedural success was achieved in all cases, there was no intraprocedural mortality or transfer to open surgery. TR significantly improved after LuX-Valve implantation (none/trivial in 8 patients, mild in 3 patients and moderate in 1 patient). The average device time was (9.2±4.2) minutes. Intensive care unit duration was 3.0 (2.0, 4.8) days. One patient died at postoperative day 18 due to non-surgery and device reasons. Transthoracic echocardiography at 30 days after operation showed that TR was significantly reduced (none/trivial in 8 patients, mild in 2 patients and moderate in 1 patient) and device success was achieved in 11 cases. All survived patients experienced a significant improvement in life quality with significantly improvement in New York Heart Association (NYHA) classification (Ⅰ and Ⅱ: 6/11 post operation vs. 0/11 before operation, P=0.012) and there were no device related complications in this patient cohort. Conclusions: LuX-Valve implantation is feasible, safe and effective for the treatment of patients with severe TR.

[Incidence and trend of occupational diseases in a district of Beijing from 2004 to 2017].

Objective: To investigate the incidence of occupational diseases in a District of Beijing, from 2004 to 2017 and to analyze the distribution characteristics and incidence trends of occupational diseases. Methods: The data of confirmed occupational disease cases data in the occupational disease and occupational health information monitoring system in a district of Beijing from 2004~2017 were collected to analyze the incidence and trends of occupational diseases. Results: In 2004~2017, a total of 161cases of occupational diseases were reported in a district of Beijing, mainly pneumoconiosis (113 cases, 70.19%) . The average age of onset of pneumoconiosis was (51.65 ±11.10) years old, and the average age of dust exposure was (13.14±8.07) years, mainly including silicosis accounting for 85.84%, concentrated in small collective enterprises. Pneumoconiosis was mainly female, with 80 cases accounting for 70.80% of the disease; most of the working years were 10-20 years, the age of onset of dust pneumoconiosis and the duration of dust exposure were statistically different (P<0.05) ; The distribution of pneumoconiosis industry was concentrated on the manufacture of jewellery and related articles in 91cases (80.53%) , Compared with the non-jewellery and related articles manufacturing industry, the average age of onset were statistically significant (P<0.05) . Occupational diseases other than pneumoconiosis were mainly male; occupational ear, nose and throat (ENT) and oral disease male accounted for 86.96% of the disease, mainly concentrated in small enterprises, state-owned enterprises, the majority of working years wereconcentrated in 20~30 years; occupational infectious diseases accountded for 93.33% of the disease, mainly concentrated in small collective enterprises, most of the working years were less than 10 years. Conclusion: Occupational diseases in a district of Beijing are mainly pneumoconiosis, mainly in small collective enterprises, mostly historical issues, the number of reports of occupational ENT and oral disease and occupational infectious diseases are increasing, it is important to strengthen supervision and protect the health of workers.

Comparison of EBSD patterns simulated by two multislice methods.

The extraction of crystallography information from electron backscatter diffraction (EBSD) patterns can be facilitated by diffraction simulations based on the dynamical electron diffraction theory. In this work, the EBSD patterns are successfully simulated by two multislice methods, that is, the real space (RS) method and the revised real space (RRS) method. The calculation results by the two multislice methods are compared and analyzed in detail with respect to different accelerating voltages, Debye-Waller factors and aperture radii. It is found that the RRS method provides a larger view field of the EBSD patterns than that by the RS method under the same calculation conditions. Moreover, the Kikuchi bands of the EBSD patterns obtained by the RRS method have a better match with the experimental patterns than those by the RS method. Especially, the lattice parameters obtained by the RRS method are more accurate than those by the RS method. These results demonstrate that the RRS method is more accurate for simulating the EBSD patterns than the RS method within the accepted computation time.

Dynamical electron diffraction simulation for non-orthogonal crystal system by a revised real space method.

In the transmission electron microscopy, a revised real space (RRS) method has been confirmed to be a more accurate dynamical electron diffraction simulation method for low-energy electron diffraction than the conventional multislice method (CMS). However, the RRS method can be only used to calculate the dynamical electron diffraction of orthogonal crystal system. In this work, the expression of the RRS method for non-orthogonal crystal system is derived. By taking Na2 Ti3 O7 and Si as examples, the correctness of the derived RRS formula for non-orthogonal crystal system is confirmed by testing the coincidence of numerical results of both sides of Schrödinger equation; moreover, the difference between the RRS method and the CMS for non-orthogonal crystal system is compared at the accelerating voltage range from 40 to 10 kV. Our results show that the CMS method is almost the same as the RRS method for the accelerating voltage above 40 kV. However, when the accelerating voltage is further lowered to 20 kV or below, the CMS method introduces significant errors, not only for the higher-order Laue zone diffractions, but also for zero-order Laue zone. These indicate that the RRS method for non-orthogonal crystal system is necessary to be used for more accurate dynamical simulation when the accelerating voltage is low. Furthermore, the reason for the increase of differences between those diffraction patterns calculated by the RRS method and the CMS method with the decrease of the accelerating voltage is discussed.

Mesenchymal Stem Cell Modulates T Follicular Helper Cell to Induce Immunotolerance of Islet Allograft.

OBJECTIVE: To verify whether mesenchymal stem cells protected the islet allograft via modulating follicular B helper T cells (Tfh) cells. METHODS: The recipient mice were divided into 5 groups. Group A: the diabetic group (n = 12); Group B: islets alone group (n = 12); Group C: MSCs and islets co-transplanted group (n = 12, MSCs = 0.5 × 10(6)); Group D: MSCs and islets co-transplanted group (n = 12, MSCs = 1 × 10(6)); Group E: MSCs and islets co-transplanted group (n = 12, MSCs = 2 × 10(6)); One control group of normal NOD mice was set as well. ELISA was used to examine the autoantibody level of GAD65 Ab, insulin autoantibodies, and insulin. The Tfh count was determined by fluorescence-activated cell sorting. The insulin expression of islet grafts, the infiltration of lymphocytes, and the Tfh cells were observed via hematoxylin and eosin staining and immunohistochemical staining. RESULTS: There was significant prolonged graft survival and more insulin expression of islet grafts observed in the co-transplant group. A lower level of the Tfh cells and autoantibody GAD65 Ab, insulin autoantibodies were also presented in the co-transplant group (P < .01). The infiltration of lymphocytes in the co-transplant group was notably less than in the islet-alone group (P < .01). CONCLUSIONS: Mesenchymal stem cells were able to protect the islet allograft by regulating the follicular helper T cells.

Hepatic stellate cells induce immunotolerance of islet allografts.

Activated hepatic stellate cells (HSCs) possess strong immune inhibitory activity. The present study highlighted the protective role of HSCs in islet transplantation. Recipients were randomly divided into 4 groups: a diabetic group, an HSC-alone group, an islet-alone transplant group, and a cotransplant group. Graft survival was compared among the 4 groups. Serum transforming growth factor ß (TGFß), tumor necrosis factor α, interleukin-1ß, and interferon gamma expression levels were measured. The infiltration of lymphocytes was observed via hematoxylin and eosin staining, and immunohistochemical examinations were performed. Results showed that allogeneic HSCs protect islet allografts better than syngeneic HSCs. There was significant prolonged graft survival and a higher level of TGFß in the cotransplant group (P < .01). The infiltration of lymphocytes in the cotransplant group was notably less than in the islet-alone group (P < .01). The formation of desmin-positive HSC packages was detected in the cotransplant group. In conclusion, allogeneic HSCs can better prolong the survival of islet allografts by stimulating TGFß expression and forming a biological capsule around the graft.

Cu2O island shape transition during Cu-Au alloy oxidation.

In situ transmission electron microscopy observations of the oxidation of (001) Cu-Au alloys indicate that the Cu2O islands that form undergo a remarkable transformation from an initially compact morphology to a dendritic structure as growth proceeds. Correspondingly, the surface composition becomes nonuniform and the fractal dimension associated with the islands evolves from 2.0 to a stable value of 1.87, indicating a transition in the rate-limiting mechanism of oxidation from oxygen surface diffusion to diffusion of copper through the increasingly gold-rich regions adjacent to the islands.

Structural analysis of regulatory protein domains using GST-fusion proteins.

The glutathione S-transferase (GST) fusion protein expression system has been used extensively to generate a large quantity of proteins for structural studies. To avoid the inter-domain flexibility introduced by the GST segment, GST-fusion proteins are normally cleaved with proteases to release the GST moiety prior to crystallization. Recently, several reports have shown that GST-fusion proteins can also be used as a vehicle to determine the crystal structures of the attached small peptides and biological regulatory domains. In comparison with the standard method, GST-fusion proteins are more easily crystallized under similar conditions. In addition, the structure of the desired protein or peptide can be determined using the molecular replacement method with the help of the GST structure. Thus, GST-fusion proteins can be used as a new technique for structural determination of small regulatory domains, especially of small peptides. Here, we review the recent progress on this technique, known as GST-driven crystallization. We have summarized and compared different methods of protein preparation and crystallization used by different groups. We have also compared the three-dimensional structures, especially those of the fused peptide segments. Finally, we have discussed the potential effects of the crystal packing on the crystal structure.

Relation between the flexibility of the WPD loop and the activity of the catalytic domain of protein tyrosine phosphatase SHP-1.

The conserved WPD loop of protein tyrosine phosphatases play an important role in the catalytic activity and the invariant aspartate residue acts as a general acid/base catalyst in the dephosphorylation reaction. In our previous report, we have demonstrated that the catalytic activities of the PTPs are influenced by the flexibility and stability of the WPD loop in its active "open" conformation [Yang et al., 1998]. Phosphatases with a more flexible WPD loop generally have higher specific activity. In this report, we modify the WPD loop of SHP-1 by alanine-scan mutation of the residues flanking the loop and measure their effects on the catalytic activity of the phosphatase. We show that the S418A, V424A, S426A, E427A, and P428A mutants increase the phosphatase activity, possibly due to the increased flexibility of the WPD loop, whereas the L417A, L417G and P425A mutants decrease its phosphatase activity. In addition, we propose that the two-proline residues in the WPD loop (Pro(420) and Pro(425) in SHP-1) work as pivotal points through a conserved hydrophobic network and allows residues between the pivotal points to have maximum flexibility in enhancing the phosphatase activity. Furthermore, our data suggest that the hydrolysis of the phosphoryl-cysteine intermediate, not its formation, is the rate-limiting step with p-nitrophenyl phosphate as the substrate while both the steps are rate-limiting with phosphotyrosine as the substrate.

Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate.

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

Protein tyrosine phosphatase SHP-1 specifically recognizes C-terminal residues of its substrates via helix alpha0.

The catalytic domain of protein tyrosine phosphatase SHP-1 possesses distinct substrate specificity. It recognizes the P-3 to P-5 residues of its substrates via the beta5-loop-beta6 region. To study the substrate specificity further, we determined the structure of the catalytic domain of SHP-1 (C455S) complexed with a less-favorable-substrate peptide originated from SIRPalpha. The complex has disordered N-terminal peptide structure and reduced interactions between the N-terminal peptide and the beta5-loop-beta6 region. This could be the basis for the lower affinity of peptide pY(427) for the catalytic domain of SHP-1. In addition, by comparing the SHP-1/less-favorable peptide complex structure with the SHP-1/substrate complex structures, we identified a novel substrate-recognition site in the catalytic domain of SHP-1. This site was formed by helix alpha0 and the alpha5-loop-alpha6 motif of SHP-1, and specifically bound residues at the P + 4 and further C-terminal positions of peptide substrates.

Phox homology domains specifically bind phosphatidylinositol phosphates.

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

[Esterification of octanoic acid with 1-octanol catalyzed by lipase in W/O microemulsions and in microemulsion-based organogels].

The esterification reactions of octanoic acid with 1-octanol catalyzed by Candida lypolytical (CL) lipase was studied in water-in-oil microemulsions formed by water/bis-(2-ethylhexyl)sulfosuccinate sodium (AOT)/isooctane. The results of kinetic study showed that the reaction follows a Ping-Pong Bi-Bi mechanism. The values of apparent kinetic parameters were determined. Lipase has also been immobilized in gelatin-containing AOT microemulsion-based organogels (MBGs) for retention of catalytic activity. These lipase-containing MBGs proved to be a solid-phase catalysts for use in apolar organic solvents, retaining its higher activity after many runs of esterification reactions.

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.

Structural basis for substrate specificity of protein-tyrosine phosphatase SHP-1.

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRPalpha, a member of the signal-regulatory proteins. We show that the variable beta5-loop-beta6 motif confers SHP-1 substrate specificity at the P-4 and further N-terminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in protein- tyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1.

Crystal structure of the nuclear matrix targeting signal of the transcription factor acute myelogenous leukemia-1/polyoma enhancer-binding protein 2alphaB/core binding factor alpha2.

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2alpha)/core-binding factor alpha (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2alphaB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

Kinetic comparison of the catalytic domains of SHP-1 and SHP-2.

The phosphatase activity of SH2-containing protein tyrosine phosphatase (SHP) is inhibited by its SH2 domains and C-terminal tail. In order to determine the inhibitory effects of the SH2 domains and C-terminal tail, we have expressed and purified the catalytic domains of SHP-1 and SHP-2, and the SH2 domain truncated SHP-1 and SHP-2. We have then measured their kinetic parameters using p-nitrophenyl phosphate (p-NPP) and phosphotyrosine (pY) as substrates under the same experimental conditions. The results indicate that the pH-dependent profiles of SHP-1 and SHP-2 are mainly determined by their catalytic domains. Both enzymes have maximum activity at pH 5.0. In addition, the phosphatase activity of different forms of SHP-1 and SHP-2 decreases as the salt concentration increases. Without SH2 domains, both SHP-1 and SHP-2 are no longer inhibited by their C-terminal tails. However, the C-terminal tail of SHP-1 can further prevent the salt inhibition of the phosphatase activity. Under the same experimental conditions, the catalytic domain of SHP-1 is two times more active than the catalytic domain of SHP-2.

Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1.

The crystal structures of the protein-tyrosine phosphatase SHP-1 catalytic domain and the complex it forms with the substrate analogue tungstate have been determined and refined to crystallographic R values of 0.209 at 2.5 A resolution and 0.207 at 2.8 A resolution, respectively. Despite low sequence similarity, the catalytic domain of SHP-1 shows high similarity in secondary and tertiary structures with other protein-tyrosine phosphatases (PTPs). In contrast to the conformational changes observed in the crystal structures of PTP1B and Yersinia PTP, the WPD loop (Trp419-Pro428) in the catalytic domain of SHP-1 moves away from the substrate binding pocket after binding the tungstate ion. Sequence alignment and structural analysis suggest that the residues in the WPD loop, especially the amino acid following Asp421, are critical for the movement of WPD loop on binding substrates and the specific activity of protein-tyrosine phosphatases. Our mutagenesis and kinetic measurements have supported this hypothesis.

Preliminary crystallographic study of glutathione S-transferase fused with the nuclear matrix targeting signal of the transcription factor AML-1/CBF-alpha2.

A glutathione S-transferase fused with the nuclear matrix targeting signal (GST-NMTS) of AML-1/CBF-alpha2 has been crystallized by the vapor diffusion method using polyethylene glycol (PEG) as the precipitant. The NMTS is a 31-amino-acid signal peptide that can target the AML-1/CBF-alpha2 protein to the nuclear matrix. The crystal belongs to tetragonal space group P43212 with unit cell dimensions a = b = 93.4 A, c = 57.6 A. There is one GST-fusion protein per asymmetric unit. Crystals diffracted to at least 2.7 A and are appropriate for structure determination.