Volume and function changes of left atrium and left ventricle in patients with ejection fraction preserved heart failure measured by a three dimensional dynamic heart model.

The accurate diagnosis of HFpEF is still challenging and controversial. In this study, we used 3D-DHM technology to compare the differences of cardiac structure and function between HFpEF patients and healthy controls, as well as the differences of two-dimensional and three-dimensional cardiac function in HFpEF patients. Echocardiography with 3D-DHM and conventional two-dimensional (2D) methods were applied to measure the volume and function parameters of left atrium and ventricle of patients with HFpEF and healthy controls. Significant differences of 3D cardiac function indexes including LVESV, 3D-LVEF, ESL, SV, CI, EDmass, LAVmax, LAVmin, LAEF, and LAVI were observed between patients with HFpEF and controls (P < 0.05). However, no significant difference of LVEDV and EDL were observed (P > 0.05). In addition, we found no significant between-group difference in 2D cardiac function indexes such as LVDD and 2D-LVEF (P > 0.05), but the LAD, LVSD, LVPW, IVS, E, E/A, and E/e ' were significantly different between groups (P < 0.05). There was no significant difference between 3D-LVEF and 2D-LVEF in the control group (P > 0.05), while 3D-LVEF in the HFpEF group was lower than 2D-LVEF(P < 0.05). Among the two-dimensional and three-dimensional parameters of HFpEF patients, the parameters related to diastolic function changed more significantly than those of the normal group, and the three-dimensional LVEF of HFpEF patients decreased. The three-dimensional cardiac function parameters analyzed by DHM can provide more information regarding myocardial mechanics.

Efficient production and characterization of soluble active human ß-1,2-N-acetylglucosaminyltransferase II in bacteria.

In humans, almost all the cell surface and secreted glycoproteins are modified with complex-type N-glycans. Thus, it is essential to obtain complex-type N-glycans to fully understand the biological properties of glycoproteins. Here, human ß-1,2-N-acetylglucosaminyltransferase II (hGnT-II), a Golgi-localized enzyme integral to complex-type N-glycan biosynthesis, was cloned as a truncated transmembrane form (GnT-II-ΔTM) and heterologously overexpressed in Escherichia coli. Our results showed that hGnT-II could be overexpressed in its soluble form by fusing the truncated enzyme with a thioredoxin (Trx)-tag in the Rosetta-Gami 2 strain. Using the optimized induction conditions, the expression level of recombinant protein was enhanced to yield approximately 4 mg per liter culture after affinity purification. The enzyme exhibited appropriate glycosyltransferase activity, and the calculated Km value was 52.4 µM, similar to the protein expressed in mammalian cells. Furthermore, the effect of MGAT2-CDG mutations on enzyme activity was also measured. These results suggested that the E. coli expression system was capable of the large-scale production of bioactive hGnT-II, which can be used for functional study and effective synthesis of complex-type N-glycans.

Nanomolar ß-glucosidase and ß-galactosidase inhibition by enantiomeric α-1-C-alkyl-1,4-dideoxy-1,4-imino-arabinitol derivatives.

A series of α-1-C-alkyl DAB (1,4-dideoxy-1,4-imino-d-arabinitol) and LAB (1,4-dideoxy-1,4-imino-l-arabinitol) derivatives with aryl substituents have been designed as analogues of broussonetine W (12), and assayed as glycosidase inhibitors. While the inhibition spectrum of α-1-C-alkyl DAB derivative 16 showed a good correlation to that of broussonetine W (12), introduction of substituents on the terminal aryl (17a-f) or hydroxyl groups at C-1' position of the alkyl chains (18a-e) decreased their α-glucosidase inhibitions but greatly improved their inhibitions of bovine liver ß-glucosidase and ß-galactosidase. Furthermore, epimerization of C-1' configurations of compounds 18a-e clearly lowered their inhibition potency of bovine liver ß-glucosidase and ß-galactosidase. Notably, some of the α-1-C-alkyl DAB derivatives were also found to have potent human lysosome ß-glucosidase inhibitions. In contrast, enantiomers of compounds 18a-e and 1'-epi-18a-e generally showed increased α-glucosidase inhibitions, but sharply decreased bovine liver ß-glucosidase and ß-galactosidase inhibitions. Molecular docking calculations unveiled the novel two set of binding modes for each series of compounds; introduction of C-1' hydroxyl altered the conformations of the pyrrolidine rings and orientation of their long chains, resulting in improved accommodation in the hydrophobic grooves. The compounds reported herein are very potent ß-glucosidase and ß-galactosidase inhibitions with novel binding mode; and the structure-activity relationship provides guidance for design and development of more pyrrolidine pharmacological chaperones for lysosomal storage diseases.

Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease.

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Measuring of strain parameters reflects changes of right ventricular function before and after thrombolytic therapy in patients with acute pulmonary embolism.

Strain parameters on speckle tracking echocardiography (STE) have been proposed as effective indexes for evaluating right ventricular (RV) function. This pilot study investigated the role of STE-derived strain parameters in assessing global and regional RV myocardial mechanical changes in patients with acute pulmonary embolism (PE) before and after thrombolytic therapy. In this case-control study, a total of 73 PE patients, 34 with pulmonary hypertension (PH) and 39 without PH, who underwent thrombolytic therapy were included. Healthy volunteers were included as controls. The peak longitudinal systolic strain (PLSS) and time to PLSS (TTP) for the global and regional RV were analyzed by STE software immediately before and 14 days after thrombolytic therapy. Changes in STE-derived strain parameters and conventional ultrasound parameters were compared. PLSS and TTP decreased before treatment in PE patients compared with measurements in the control group, particularly in those with PH. Also, the strain parameters decreased more significantly for the free wall than for the septum wall (P < 0.05). Moreover, the RV diastolic diameter (RVDD) and RV/left ventricular (LV) diameter ratio increased, while RV fraction shortening (RVFS), RV fractional area change (RVFAC), tricuspid regurgitation pressure gradient (TRPG), and tricuspid annular peak systolic excursion (TAPSE) decreased (P < 0.05). The global strain parameters for the RV were positively correlated with RVDD and RV/LV diameter ratio, but negatively correlated with RVFS, RVFAC, TRPG, and TAPSE (P < 0.05). After treatment, the strain parameters differed significantly between PE patients with PH and controls but did not differ between PE patients without PH and controls. STE-derived parameters are effective for detecting changes in global and regional RV function in PE patients with or without acute PH.

Two novel chiral tetranucleate copper-based complexes: syntheses, crystal structures, inhibition of angiogenesis and the growth of human breast cancer in vitro and in vivo.

The single crystals of two novel chiral tetranucleate copper(II)-based complexes (TNCu-A and TNCu-B) containing L-methioninol-derived Schiff-bases were obtained. Their single structures were characterized by X-ray single crystal diffraction, infrared (IR) rays, elemental analysis, and liquid chromatography-mass spectrometry analysis. TNCu-A can effectively inhibit human umbilical vein endothelial cells (HUVECs) to form a tubular structure and it induces apoptosis of human triple-negative breast cancer MDA-MB-231 cells and HUVECs in vitro in a mitochondria dependent manner. Moreover, in vivo TNCu-A can remarkably inhibit the growth of triple-negative breast cancer from which MDA-MB-231 cells were xenografted into severely immunodeficient nude mice by inhibiting proliferation, inducing apoptosis of MDA-MB-231 cells by dramatically inhibiting the expression of the anti-apoptotic protein Bcl-2 and up-regulating the expressions of proapoptotic proteins caspase-9 and Bax, and simultaneously inhibiting tumor angiogenesis by decreasing the density of vascular endothelial cells and suppressing migration and even partially inducing apoptosis.

Synthesis and glycosidase inhibition of 5-C-alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives.

5-C-Alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives have been designed and synthesized efficiently from an l-sorbose-derived cyclic nitrone. The DNJ and l-ido-DNJ derivatives with C-5 alkyl chains ranging from methyl to dodecyl were assayed against various glycosidases to study the effect of chain length on enzyme inhibition. Glycosidase inhibition study of DNJ derivatives showed potent and selective inhibitions of α-glucosidase; DNJ derivatives with methyl, pentyl to octyl, undecyl and dodecyl as C-5 branched chains showed significantly improved rat intestinal maltase inhibition. In contrast, most 5-C-alkyl-l-ido-DNJ derivatives were weak or moderate inhibitors of the enzymes tested, with only three compounds found to be potent α-glucosidase inhibitors. Docking studies showed different interaction modes of 5-C-ethyl-DNJ and 5-C-octyl-DNJ with ntMGAM and also different binding modes of 5-C-alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives; the importance of the degree of accommodation of the C-5 substituent in the hydrophobic groove and pocket may account for the variation of glycosidase inhibition in the two series of derivatives. The results reported herein are helpful in the design and development of α-glucosidase inhibitors; this may lead to novel agents for the treatment of viral infection and type II diabetes.

Chemo-enzymatic synthesis of the ALG1-CDG biomarker and evaluation of its immunogenicity.

Congenital disorders of glycosylation (CDG) are a growing group diseases that result from defects in genes involved in glycan biosynthesis pathways. One tetrasaccharide, i.e., Neu5Ac-α2, 6-Gal-ß1, 4-GlcNAc-ß1, 4-GlcNAc, was recently reported as the biomarker of ALG1-CDG, the disease caused by ALG1 deficiency. To develop a novel diagnostic method for ALG1-CDG, chemo-enzymatic synthesis of the tetrasaccharide biomarker linked to phytanyl phosphate and the biomarker's immune stimulation were investigated in this study. The immunization study using liposomes bearing phytanyl-linked tetrasaccharide revealed that they stimulated a moderate immune response. The induced antibody showed strong binding specificity for the ALG1-CDG biomarker, indicating its potential in medical applications.

Reconstitution of the lipid-linked oligosaccharide pathway for assembly of high-mannose N-glycans.

The asparagine (N)-linked Man9GlcNAc2 is required for glycoprotein folding and secretion. Understanding how its structure contributes to these functions has been stymied by our inability to produce this glycan as a homogenous structure of sufficient quantities for study. Here, we report the high yield chemoenzymatic synthesis of Man9GlcNAc2 and its biosynthetic intermediates by reconstituting the eukaryotic lipid-linked oligosaccharide (LLO) pathway. Endoplasmic reticulum mannosyltransferases (MTases) are expressed in E. coli and used for mannosylation of the dolichol mimic, phytanyl pyrophosphate GlcNAc2. These recombinant MTases recognize unique substrates and when combined, synthesize end products that precisely mimic those in vivo, demonstrating that ordered assembly of LLO is due to the strict enzyme substrate specificity. Indeed, non-physiological glycans are produced only when the luminal MTases are challenged with cytosolic substrates. Reconstitution of the LLO pathway to synthesize Man9GlcNAc2 in vitro provides an important tool for functional studies of the N-linked glycoprotein biosynthesis pathway.

Situational Analysis of Low-density Lipoprotein Cholesterol Control and the Use of Statin Therapy in Diabetes Patients Treated in Community Hospitals in Nanjing, China.

BACKGROUND: Comprehensive management of diabetes should include management of its comorbid conditions, especially cardiovascular complications, which are the leading cause of morbidity and mortality among patients with diabetes. Dyslipidemia is a comorbid condition of diabetes and a risk factor for cardiovascular complications. Therefore, lipid level management is a key of managing patients with diabetes successfully. However, it is not clear that how well dyslipidemia is managed in patients with diabetes in local Chinese health-care communities. This study aimed to assess how well low-density lipoprotein cholesterol (LDL-C) was managed in Nanjing community hospitals, China. METHODS: We reviewed clinical records of 7364 diabetic patients who were treated in eleven community hospitals in Nanjing from October 2005 to October 2014. Information regarding LDL-C level, cardiovascular risk factors, and use of lipid-lowering agents were collected. RESULTS: In patients without history of cardiovascular disease (CVD), 92.1% had one or more CVD risk factors, and the most common CVD risk factor was dyslipidemia. The overall average LDL-C level was 2.80 ± 0.88 mmol/L, which was 2.62 ± 0.90 mmol/L and 2.82 ± 0.87 mmol/L in patients with and without CVD history respectively. Only 38% of all patients met the target goal and 37.3% of patients who took lipid-lowering agents met target goal. Overall, 24.5% of all patients were on lipid-lowering medication, and 36.3% of patients with a CVD history and 20.9% of patients without CVD history took statins for LDL-C management. The mean statin dosage was 13.9 ± 8.9 mg. CONCLUSIONS: Only a small portion of patients achieved target LDL-C level, and the rate of using statins to control LDL-C was low. Managing LDL-C with statins in patients with diabetes should be promoted, especially in patients without a CVD history and with one or more CVD risk factors.

[Prokaryotic expression of the extracellular region of human CD1d and preparation of its polyclonal antibody].

AIM: To explore the prokaryotic expression of the extracellular region of human CD1d (hCD1d) and prepare its polyclonal antibody. METHODS: The gene encoding the extracellular region of hCD1d was amplified by PCR and cloned into prokaryotic expression vector pET28, then expressed in E.coli BL21 (DE3) with IPTG induction. The recombinant protein was purified by Ni2+-NTA agarose column and then used as immunogen to immunize the mouse. The generated polyclonal antibody was evaluated by ELISA, Western blot and immunohistochemical staining (IHC), respectively. RESULTS: The recombinant extracellular region of hCD1d was successfully expressed and purified. The polyclonal antibody with high titer and high specificity was obtained, which could recognize the native hCD1d in the human small intestinal tissues. CONCLUSION: The recombinant extracellular region of hCD1d has been obtained. The antibody with high titer and high specificity against the extracellular region of hCD1d from the mouse has been successfully prepared, which lays a foundation for further research into the detection and functional study of CD1d.

[Preparation and characterization of the rabbit polyclonal antibody against the alpha3 domain of human CD1d].

AIM: To prepare the rabbit antibody against the alpha3 domain of the human CD1d (hCD1d-alpha3). METHODS: The gene fragment coding for hCD1d-alpha3 was amplified by PCR and cloned into prokaryotic expression vector pET28, then expressed in E.coli BL21(DE3). The recombinant protein hCD1d-alpha3 was purified with Ni(2+)-NTA agarose column and used as immunogen to immunize the rabbit. The titer and specificity of the anti-hCD1d-alpha3 antibody from the rabbit were analyzed by ELISA, Western blot and immunohistochemistry, respectively. RESULTS: The recombinant hCD1d-alpha3 was successfully expressed and purified, and the polyclonal anit-hCD1d-alpha3 antibody was successfully prepared. The titer of the antiserum was 1:6 400 by ELISA. Western blot analysis showed this antiboday reacted specifically with hCD1d. Immunohistochemistry analysis showed the antibody could recognize the native hCD1d in the human intestinal tissue. CONCLUSION: The anti-CD1d-alpha3 antibody from the rabbit with high titer and specificity has been prepared with purified recombinant hCD1d-alpha3 as immunogen, which lays a foundation for further research into detection and functional study of CD1d.

[Prokaryotic expression of human mast cell chymase gene and preparation of its polyclonal antibody].

AIM: To express the human mast cell chymase cDNA in E.coli and prepare the antibody against human mast cell chymase with recombinant chymase. METHODS: The human mast cell chymase cDNA was cloned by RT-PCR. The recombinant chymase was expressed in E.coli with L-Arabinose induction and purified by Ni-NTA agarose column. Then the purified chymase was used as immunogen to immunize the rabbit. The titer and specificity of the anti-chymase antibody from the rabbit were analyzed by indirect ELISA and Western blot, respectively. RESULTS: The recombinant chymase was successfully expressed in E.coli, and the polyclonal anit-chymase antibody was prepared by immunizing the rabbit with the purified recombinant chymase. The titer of the generated antiserum was detected to be 1:12 800 by ELISA. Western blot analysis showed this antibody bound specifically with chymase. CONCLUSION: The anti-chymase antibody from the rabbit with high titer and specificity has been prepared with purified recombinant chymase as immunogen, which lays a foundation for further research into detection and function of chymase.

[Preparation and characterization of rabbit antibody against human mast cell carboxypeptidase].

AIM: To prepare antibody against human mast cell carboxypeptidase (hMC-CP) by using recombinant hMC-CP expressed in E.coli, and to characterize the antibody. METHODS: hMC-CP was expressed in E.coli with L-Arabinose induction and purified through Ni-NTA column. The purified hMC-CP as immunogen was used to immunize rabbit. The titer and the specificity of the rabbit anti-hMC-CP antibody was analyzed by indirect ELISA and Western blot respectively. RESULTS: The hMC-CP was successfully expressed and purified. The polyconal anit-hMC-CP antibody was prepared by immunizing rabbit using the purified recombinant protein, The titer of the generated antiserum was 1:6 400 by ELISA. Western blot analysis showed that this antibody could bind with hMC-CP specifically. CONCLUSION: The rabbit anti-hMC-CP antibody with high titer and high specificity has been prepared by using purified recombinant hMC-CP as immunogen, which lays the foundation for further research on detection and function of hMC-CP.