Efficacy and Safety of Long-Term Corticosteroid Monotherapy in 26 Cases of Nephrotic Syndrome with Biopsy-Proven Membranous Nephropathy Induced by Seronegative Hepatitis B Virus-Associated Glomerulonephritis.

BACKGROUND: Hepatitis B virus-associated glomerulonephritis (HBV-GN) can occur in patients with negative HBV serological antigens. Little is known about the treatment of seronegative HBV-GN (sn HBV-GN). The aim of this prospective study was to evaluate the efficacy and safety of corticosteroids in the treatment of sn HBV-GN. METHODS: Twenty-six patients with nephrotic syndrome induced by seronegative HBV-associated membranous nephropathy were enrolled. The patients were given methylprednisolone (0.8 mg/kg/day) for 12-24 weeks, tapered by a 2-mg reduction every 1-3 months. Patients were followed up for 6-36 months. Complete remission (CR) was defined as proteinuria <0.3 g/24 h. Partial remission (PR) was defined as proteinuria of 0.3-3.5 g/24 h that was reduced ≥50% of the baseline level. RESULTS: The effective remission (including CR and PR) rates of nephrotic syndrome were 23.1%, 61.5%, 73.1%, 76.2%, 90.5%, and 81.0%, respectively, after 1, 3, 6, 12, 24, and 36 months. Nineteen patients achieved effective remission after 11.68 ± 7.15 months. The level of serum albumin improved from 24.34 ± 6.71 g/L at baseline to 39.61 ± 7.45 g/L at the 36th month significantly. After treatment, the level of serum Cr was similar to the baseline. Only 2 patients relapsed. The primary adverse reaction was infection. None of the patients showed evidence of HBV replication. CONCLUSION: The long-term middle-dose corticosteroid therapy without antiviral drugs is effective and safe for membranous sn HBV-GN patients. For sn HBV-GN patients, the monitoring of HBV DNA and HBV markers in the serum is necessary during the corticosteroid monotherapy. TRIAL REGISTRATION: The Chinese Clinical Trial Registry (ChiCTR1900022518).

A case presentation of an IgA nephropathy patient with Vogt-Koyanagi-Harada syndrome.

BACKGROUND: Vogt-Koyanagi-Harada syndrome is a rare disease characterized by skin and eyelash bleaching, chronic granulomatous iridocyclitis and exudative retinal detachment, and aseptic meningitis and encephalopathy. IgA nephropathy complicated by Vogt-Koyanagi-Harada syndrome is very rare, even though they might have similar pathogeneses. Ocular lesions often are not examined when patients are diagnosed with IgA nephropathy, which affects the prognosis. CASE PRESENTATION: We describe a 55-year-old male IgA nephropathy patient who was admitted with high fever and hematuria. Physical examination revealed impaired binocular vision with blurred vision, impaired hearing, and a congestive rash on the chest and back. Renal ultrasound examination showed no abnormalities. Laboratory examination showed that glomerulonephritis was complicated by infection, and anti-infection therapy was ineffective. Bilateral fluorescein angiography showed Vogt-Koyanagi-Harada syndrome. Further renal biopsy confirmed IgA nephropathy. Hormone shock therapy and cyclophosphamide adjuvant therapy were administered, and the patient's symptoms improved. CONCLUSION: For the first time, we reported the case of simultaneous onset of IgA nephropathy and Vogt-Koyanagi-Harada syndrome, which is very rare. The onset of Vogt-Koyanagi-Harada syndrome is rapid and serious, while that of IgA nephropathy is relatively milder, making it easy for specialized doctors to neglect this condition. Doctors should be highly alert to the clinical concomitant occurrence of the two diseases with similar mechanisms, especially in the case of neurological defects and ocular symptoms in IgA nephropathy patients, since timely immunosuppressive treatment may improve the outcome of ocular diseases.

The relationships among hyperuricemia, body mass index and impaired renal function in type 2 diabetic patients.

Chronic kidney disease (CKD) is a common chronic microvascular complication and the major cause of death in diabetic patients. This study was conceived to explore the possible mechanisms of how hyperuricemia and obesity contribute to renal function impairment in type 2 diabetic (T2DM) patients. A cross-sectional study in 609 participants recruited from a T2DM population in North China was conducted. The multiplicative interaction between body mass index (BMI) and uric acid (UA) level was assessed using an interaction term in a logistic regression analysis. Our results indicate that male T2DM patients having higher BMI (OR 1.711, p = 0.038), blood urine nitrogen (BUN) (OR 1.100, p = 0.034), and 24-hour urinary micro-albumin levels (OR 1.004, p = 0.021) were much more likely to have high UA. Whereas, for female T2DM patients, the OR of BMI, BUN, and triglyceride were 1.169 (p = 0.001), 1.337 (p = 0.000), and 1.359 (p = 0.006), respectively. In this study population, obesity and elevated UA work together to increase the risk of renal injury. In vitro experiments indicate that reactive oxygen species (ROS) production increased with UA treatment in human renal glomerular endothelial cells (HRGECs), while endothelial nitric oxide synthase (eNOS) production level dropped. UA also increased monocyte chemotactic protein-1 (MCP-1) expression and nuclear factor kappa B (NF-κB) activation. Taken together, our results indicate that high concentrations of UA lead to endothelial dysfunction through the activation of the inflammatory response and induction of oxidative stress, even in non-obese T2DM patients.

Synthesis of Supported Ultrafine Non-noble Subnanometer-Scale Metal Particles Derived from Metal-Organic Frameworks as Highly Efficient Heterogeneous Catalysts.

The properties of supported non-noble metal particles with a size of less than 1 nm are unknown because their synthesis is a challenge. A strategy has now been created to immobilize ultrafine non-noble metal particles on supports using metal-organic frameworks (MOFs) as metal precursors. Ni/SiO2 and Co/SiO2 catalysts were synthesized with an average metal particle size of 0.9 nm. The metal nanoparticles were immobilized uniformly on the support with a metal loading of about 20 wt%. Interestingly, the ultrafine non-noble metal particles exhibited very high activity for liquid-phase hydrogenation of benzene to cyclohexane even at 80 °C, while Ni/SiO2 with larger Ni particles fabricated by a conventional method was not active under the same conditions.

TOX and CDKN2A/B Gene Polymorphisms Are Associated with Type 2 Diabetes in Han Chinese.

To study associations between type 2 diabetes (T2DM) candidate genes and microvascular complications of diabetes (MVCDs), we performed case-control association studies for both T2DM and MVCDs in Han Chinese subjects. We recruited 1,939 unrelated Han Chinese T2DM patients and 918 individuals with normal blood glucose levels as nondiabetic controls. Among T2DM patients, 1116 have MVCDs, 266 have a history of T2DM of >10 years but never developed MVCDs. Eighty-two single-nucleotide polymorphisms (SNPs) in 54 candidate genes were genotyped. Discrete association studies were performed by the PLINK program for T2DM and MVCDs. Significant associations were found among candidate gene SNPs and T2DM, including rs1526167 of the TOX gene (allele A, P = 2.85 × 10(-9), OR = 1.44). The SNP rs10811661 of the CDKN2A/B gene was also associated with T2DM (allele T, P = 4.09 × 10(-7), OR = 1.36). When we used control patients with >10 years of T2DM history without MVCD, we found that the G allele of SNP rs1526167 of the TOX gene was associated with MVCD (nominal P = 4.33 × 10(-4)). In our study, significant associations were found between TOX and CDKN2A/B gene SNPs and T2DM. The TOX polymorphism might account for the higher risk of T2DM and the lower risk of MVCDs in the Han Chinese population.

Cu and Boron Doped Carbon Nitride for Highly Selective Oxidation of Toluene to Benzaldehyde.

A novel Cu and boron doped graphitic carbon nitride catalyst (Cu-CNB) was synthesized using cheap precursors and systematically characterized. The selective oxidation of toluene proceeded very smoothly over the catalyst at 70 °C using tert-butyl hydroperoxide (TBHP) as the oxidant to exclusively afford benzaldehyde. The catalyst can be used for at least five cycles without decrease in activity and selectivity.

Heterogeneous copper-catalyzed hydroxylation of aryl iodides under air conditions.

In this work, the ligand-free heterogeneous copper Cu-g-C3N4 was synthesized and used for the hydroxylation of aryl iodides to synthesize phenols using cheap bases. The catalyst was conveniently prepared, air-tolerant, reusable and scalable, and is very efficient for a wide range of substrates. The synthesis of substituted phenols can be carried out under air conditions and has great potential for practical applications.

Synthesis of binuclear phenoxyimino organoaluminum complexes and their use as the catalyst precursors for efficient ring-opening polymerisation of ε-caprolactone.

A series of novel binuclear phenoxyimino organoaluminum complexes of the type [(RN=CH)C6H3OAlMe2]2 [R = C6H5 (2a), 2,6-(i)Pr2C6H3 (2b), 2,6-Ph2C6H3 (2c), adamantyl (2d), (t)Bu (2e)] have been prepared in high yields, and these complexes were identified by (1)H, (13)C NMR and elemental analysis. Structural analysis for 2a-e revealed that these complexes have a distorted tetrahedral geometry around Al and both the Al-O and the Al-N bond distances were considerably influenced by substituents in the imino groups. The complexes were tested as catalyst precursors for ring-opening polymerisation (ROP) of ε-caprolactone (CL) in the presence of BnOH, and their catalytic activities were strongly affected by the catalyst structures and polymerisation conditions. An efficient living ROP has been achieved using the 2b/BnOH system.

[Clinical analysis of drug-induced Pseudo-Bartter's syndrome: a report of five cases].

OBJECTIVE: To summarize the clinical characteristics and outcomes of Pseudo-Bartter's syndrome and explore its pathogenesis. METHODS: The clinical data of 5 cases of Pseudo-Bartter's syndrome at our ward from May 2008 to December 2010 was analyzed retrospectively. RESULTS: All patients were female. Long-term regimen of purgative or diuretics was prescribed. The clinical features included normotension, hypokalemic alkalosis and activation of renin-angiotensin-aldosterone. The pathological results of 3 cases of kidney biopsy showed the hyperplasia of juxtaglomerular apparatus, thickness of arteriole, infiltration of lymphocytes and monocytes and degeneration of renal tubule. Upon a definitive diagnosis, purgative or diuretics was discontinued and supplement therapy of potassium chloride initiated. The results of laboratory tests reverted to normal ranges within 4 weeks. CONCLUSION: Purgative or diuretics should be prescribed appropriately to avoid the occurrence of Pseudo-Bartter's syndrome.

Microchip electrophoresis-SDS methods with high-resolution and silver stain sensitivity for quality screening and quantitation of protein products.

Two microchip electrophoresis (ME)-SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate-coated proteins within 1 min. In the high-resolution ME-SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen-binding (Fab) assemblies can be readily resolved and individually quantified. A high-sensitivity ME-SDS method was also developed with sensitivity comparable to that of SDS-PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low-expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high-resolution and the high-sensitivity ME-SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.

[Identification of a mutation in exon 4 of apolipoprotein E gene in a family affected with lipoprotein glomerulopathy].

OBJECTIVE: To identify potential mutation of apolipoprotein E (apoE) gene in a male patient affected with lipoprotein glomerulopathy (LPG), his mother and his sister. METHODS: The patient and his mother both had histologically confirmed LPG. His sister and his father were asymptomatic. Genomic DNA was extracted from peripheral blood samples. PCR products of the coding region of exons 3 and 4 of the apoE gene were cloned into a pTA2 vector and sequenced. Genetic variations of the apoE gene were detected using PCR and restriction fragment length polymorphism (RFLP). RESULTS: An apoE gene mutation was identified in the patient's family. Sequence analysis confirmed a 9-bp deletion in the exon 4 of apoE gene from nt 484 to 492. The 9-bp deletion resulted in loss of 3 amino acids at positions 143-145. The sister of the propositus carried the same mutation, though she had neither proteinuria nor elevated plasma apoE. Sequence analysis of exon 3 showed no abnormality. No abnormalities were found in the father's apoE gene sequence. Analysis of genetic variations of the apoE gene by PCR and RFLP confirmed a 57 bp fragment consistent with the 9-bp deletion in exon 4. The father had a normal ε 3 ε 3 genotype. CONCLUSION: The 9 bp deletion of apoE may be associated with the pathogenesis of LPG.

High throughput profiling of charge heterogeneity in antibodies by microchip electrophoresis.

A high throughput microchip capillary zone electrophoresis (CZE) method was developed for the analysis of charge heterogeneity in antibodies. The method utilizes high speed microchip electrophoresis separation and is well-suited for high throughput charge profiling of antibodies during process and formulation development. The method involves derivatization of protein molecules with Cy5 N-hydroxysuccinimide ester (NHS-ester), which does not change the protein charge profile and enables fluorescence detection on a commercial microchip instrument. The sample preparation can be performed in 96-well microtiter plates within 1 h, and each sample analysis takes only 80 s. Protein charge variants with a pI difference of 0.1 can be readily resolved in the 12.5 mm microfluidic channel. Charge profiles similar to those obtained using conventional CZE technology were found for all antibodies tested (pIs in the range of 7.5-9.2). The separation efficiency corresponds to 1.2 × 10(4) theoretical plates (1.0 µm plate height). Assay performance is assessed by demonstrating specificity, carryover, linearity, limit of detection, and precision.

Selective covalent bond formation in polypeptide ions via gas-phase ion/ion reaction chemistry.

Primary amines present in protonated polypeptides can be covalently modified via gas-phase ion/ion reactions using bifunctional reagent ions. The use of reagent anions with a charge-bearing site that leads to strong interactions with the polypeptide, such as sulfonic acid, gives rise to the formation of a long-lived adduct. A distinct reactive functional group, an aldehyde in the present case, can then undergo reaction with the peptide. Collisional activation of the adduct ion formed from a reagent with an aldehyde group and a peptide ion with a primary amine gives rise to water loss in conjunction with imine (Schiff base) formation. The covalently bound modification is retained upon subsequent collisional activation. This work demonstrates the ability to selectively modify polypeptide ions in the gas phase within the context of a multistage mass spectrometry experiment.

Dissociation of disulfide-intact somatostatin ions: the roles of ion type and dissociation method.

The dissociation chemistry of somatostatin-14 was examined using various tandem mass spectrometry techniques including low-energy beam-type and ion trap collision-induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide-gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin-14 is present within a loop defined by the disulfide linkage between Cys-3 and Cys-14. The generation of readily interpretable sequence-related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH(2)-S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H](2+) species. The products were generated by a combination of S-S bond cleavage and amide bond cleavage. ETD of the [M+3H](3+) ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S-S bond and an N-C(alpha) bond can be cleaved following a single electron transfer reaction.

Tailored-waveform collisional activation of peptide ion electron transfer survivor ions in cation transmission mode ion/ion reaction experiments.

Broadband resonance excitation via a tailored waveform in a high pressure collision cell (Q2) on a hybrid quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been implemented for cation transmission mode electron transfer ion/ion reactions of tryptic polypeptides. The frequency components in the broadband waveform were defined to excite the first generation intact electron transfer products for relatively large tryptic peptides. The optimum amplitude of the arbitrary waveform applied has been determined empirically to be 3.0 V(p-p), which is effective for relatively high mass-to-charge (m/z) ratio precursor ions with little elimination of sequence information for low m/z ions. The application of broadband activation during the transmission mode ion/ion reaction obviates frequency and amplitude tuning normally associated with ion trap collision induced dissociation (CID). This approach has been demonstrated with triply and doubly charged tryptic peptides with and without post-translational modifications. Enhanced structural information was achieved by production of a larger number of informative c- and z-type fragments using the tailored waveform on unmodified and modified (phosphorylated and glycosylated) peptides when the first generation intact electron transfer products fell into the defined frequency range. This approach can be applied to a wide range of tryptic peptide ions, making it attractive as a rapid and general approach for ETD LC-MS/MS of tryptic peptides in a QqTOF instrument.

Electron transfer dissociation of iTRAQ labeled peptide ions.

Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.

Solid-phase synthesis of alpha-glucosamine sulfoforms with fragmentation analysis by tandem mass spectrometry.

Sulfated epitopes of alpha-glucosamine (GlcN sulfoforms) were prepared by solid-phase synthesis as models of internal glucosamines within heparan sulfate. An orthogonally protected 2'-hydroxyethyl GlcN derivative was immobilized on a trityl resin support and subjected to regioselective deprotection and sulfonation conditions, which were optimized with the aid of on-resin infrared or Raman analysis. The sulfoforms were cleaved from the resin under mild Lewis acid conditions without affecting the O- or N-sulfate groups and purified by reversed-phase high-performance liquid chromatography (HPLC). The alpha-GlcN sulfoforms and their 4- O-benzyl ethers were examined by electrospray ionization tandem mass spectrometry (ESI-MS/MS), with product ion spectra produced by collision-induced dissociation (CID). ESI-MS/MS revealed significant differences in parent ion stabilities and fragmentation rates as a function of sulfate position. Ion fragmentation by CID resulted in characteristic mass losses with strong correlation to the positions of both free hydroxyl groups and sulfate ions. Most of these fragmentation patterns are consonant with elimination pathways, and suggest possible strategies for elucidating the structures of glucosamine-derived sulfoforms with identical m/ z ratios. In particular, fragmentation analysis can easily distinguish GlcN sulfoforms bearing the relatively rare 3- O-sulfate from isomers with the more common 6- O-sulfate.

Rapidly alternating transmission mode electron-transfer dissociation and collisional activation for the characterization of polypeptide ions.

Cation transmission/electron-transfer reagent anion storage mode electron-transfer ion/ion reactions and beam-type collisional activation of the polypeptide ions are performed in rapid succession in the high-pressure collision cell (Q2) of a quadrupole/time-of-flight tandem mass spectrometer (QqTOF), where the electron-transfer reagent anions are accumulated. Duty cycles for both electron-transfer dissociation (ETD) and collision-induced dissociation (CID) experiments are improved relative to ion trapping approaches since there are no discrete ion storage and reaction steps for ETD experiments and no discrete ion storage step and frequency tuning for CID experiments. For this technique, moderately high resolution and mass accuracy are also obtained due to mass analysis via the TOF analyzer. This relatively simple approach has been demonstrated with a triply charged tryptic peptide, a triply charged tryptic phosphopeptide, and a triply charged tryptic N-linked glycopeptide. For the tryptic peptide, the sequence is identified with more certainty than would be available from a single method alone due to the complementary information provided by these two dissociation methods. Because of the complementary information derived from both ETD and CID dissociation methods, peptide sequence and post-translational modification (PTM) sites for the phosphopeptide are identified. This combined ETD and CID approach is particularly useful for characterizing glycopeptides because ETD generates information about both peptide sequence and locations of the glycosylation sites, whereas CID provides information about the glycan structure.

Electron transfer dissociation of peptides generated by microwave D-cleavage digestion of proteins.

The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.

Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions.

Cationic peptide electron-transfer products that do not fragment spontaneously are exposed to ion trap collisional activation immediately upon formation while they pass through a high-pressure collision cell (Q2), where the electron-transfer reagent anions are stored. Radial ion acceleration, which is normal to the ion flow, is implemented by applying an auxiliary dipolar alternating current to a pair of opposing rods of the Q2 quadrupole array at a frequency in resonance with the surviving electron-transfer products. Collisional cooling of cations in the pressurized Q2 ensures efficient overlap of the positive and negative ions for ion/ion reactions and also gives rise to relatively long residence times (milliseconds) for ions in Q2, making it possible to fragment ions via radial excitation during their axial transmission. The radial activation for transmission mode electron-transfer ion/ion reactions has been demonstrated with a doubly protonated tryptic peptide, a triply protonated phosphopeptide, and [M + 7H]7+ ions of ubiquitin. In all cases, significant increases in fragment ion yields and structural information from electron-transfer dissociation (ETD) were observed, suggesting the utility of this method for improving transmission mode ETD performance for relatively low charge states of peptides and proteins.