New free radical-initiated peptide sequencing (FRIPS) mass spectrometry reagent with high conjugation efficiency enabling single-step peptide sequencing.

A newly designed TEMPO-FRIPS reagent, 4-(2,2,6,6-tetramethylpiperidine-1-oxyl) methyl benzyl succinic acid N-hydroxysuccinimide ester or p-TEMPO-Bn-Sc-NHS, was synthesized to achieve single-step free radical-initiated peptide sequencing mass spectrometry (FRIPS MS) for a number of model peptides, including phosphopeptides. The p-TEMPO-Bn-Sc-NHS reagent was conjugated to target peptides, and the resulting peptides were subjected to collisional activation. The peptide backbone dissociation behaviors of the MS/MS and MS3 experiments were monitored in positive ion mode. Fragment ions were observed even at the single-step thermal activation of the p-TEMPO-Bn-Sc-peptides, showing mainly a-/x- and c-/z-type fragments and neutral loss ions. This confirms that radical-driven peptide backbone dissociations occurred with the p-TEMPO-Bn-Sc-peptides. Compared to the previous version of the TEMPO reagent, i.e., o-TEMPO-Bz-C(O)-NHS, the newly designed p-TEMPO-Bn-Sc-NHS has better conjugation efficiency for the target peptides owing to its improved structural flexibility and solubility in the experimental reagents. An energetic interpretation using the survival fraction as a function of applied normalized collision energy (NCE) ascertained the difference in the thermal activation between p-TEMPO-Bn-Sc- and o-TEMPO-Bz-C(O)- radical initiators. This study clearly demonstrates that the application of the p-TEMPO-Bn-Sc- radical initiator can improve the duty cycle, and this FRIPS MS approach has the potential to be implemented in proteomics studies, including phosphoproteomics.

Glaucoma Care of Incarcerated Patients at an Academic Institution: A Case-Control Study.

PURPOSE: To evaluate medication and follow-up adherence in incarcerated patients examined at an academic glaucoma clinic, in comparison to nonincarcerated controls. METHODS: Retrospective, case-control study. Consecutive prisoners presenting for initial visits in the Glaucoma Clinic at the Illinois Eye and Ear Infirmary between December 2015 and December 2017 were included in the study. Nonincarcerated patients seen in the same Glaucoma Clinic with similar initial visit dates, age, race, sex, and disease severity were selected as controls. Glaucoma Clinic visits from each patient were reviewed until December 2018. Examination information, surgical intervention, follow-up and treatment recommendations, and patient-reported medication usage were recorded for each visit. Number of visits, loss to follow-up, follow-up delays, and medication nonadherence were studied as primary outcome measures. RESULTS: Twenty-four prisoners and 24 nonincarcerated controls were included. Prisoners had an average of 2.46 ± 2.38 visits during the study period, compared to 5.04 ± 3.25 for controls (P = 0.001). Follow-up visits occurred more than 30 days after the recommended follow-up time in 57.4% (95% confidence interval [CI]: 44.2%-70.6%) of prisoners, compared to 17.9% (95% CI: 10.2%-25.6%) of controls (P < 0.00001). 70.8% of prisoners (95% CI: 66.3-74.5%) were lost to follow-up, compared to 29.2% of controls (95% CI: 25.5%-32.9%; P < 0.01). Medication nonadherence rates were similar between prisoners (13.6%; 95% CI: 12.1%-15.2%) and controls (12.0%; 95% CI: 11.4%-12.6%; P = 0.78). CONCLUSIONS: Glaucoma follow-up adherence was significantly worse in prisoners compared to a nonincarcerated control population. Further study into causative factors is needed.

Vaccination in pediatric cancer survivors: Vaccination rates, immune status, and knowledge regarding compliance.

BACKGROUND: Vaccination recommendations for childhood cancer survivors are ambiguous. Limited data exist on vaccination rates and patient/caregiver knowledge of vaccination postchemotherapy. PROCEDURE: A single-institution study of childhood cancer survivors treated from 1996 to 2018. Study included a retrospective chart review assessing patient's vaccination status, survey of patient's/caregiver's knowledge/beliefs regarding vaccination postchemotherapy, and assessment of immunoglobulin titers. RESULTS: A total of 120 patient charts were included. Vaccination records were available for 82% (98/120) of patients, 57% (56/98) were up to date with vaccinations before chemotherapy, and 83% (81/98) received vaccinations after chemotherapy. Children who resumed vaccination postchemotherapy were younger at cancer diagnosis compared to those who did not resume vaccination (2 vs 4 years, P < .02). Median time since chemotherapy was higher in vaccinated versus unvaccinated patients (107 vs 60 months, P < .02). Immunoglobulin titers were assessed in 27 patients, and 74% (20/27) were not immune to one or more infections tested. Lack of immunity to pneumococcal strains was the most common. There was no difference in median age at diagnosis or time since chemotherapy completion in immune versus nonimmune patients. In 33 surveyed patients/caregivers, 33% (11/33) were not advised about resuming vaccinations postchemotherapy. Over one-third (12/33) of respondents were concerned about vaccination safety after chemotherapy, although 88% (29/33) agreed they would vaccinate if recommended by their pediatrician/pediatric oncologist. CONCLUSIONS: Most childhood cancer survivors resume vaccinations postchemotherapy. Considerable variability exists in vaccination timing after chemotherapy. Pediatric oncologists play a central role in educating patients/pediatricians about vaccination recommendations postchemotherapy.

Botulinum toxin for the treatment of cyclic esotropia in a child with Chiari type I malformation.

We report the case of a 4-year-old boy who presented with cyclic esotropia in the setting of a Chiari type I malformation. He was treated with a single administration of botulinum toxin and remained orthotropic at 7-months. To our knowledge, this is the first reported case of cyclic esotropia with Chiari I malformation successfully treated using botulinum toxin.

The Relationship Between Optic Nerve Cup-to-Disc Ratio and Retinal Nerve Fiber Layer Thickness in Suspected Pediatric Glaucoma.

PURPOSE: To evaluate the relationship between optic nerve cup-to-disc ratio and peripapillary retinal nerve fiber layer (RNFL) thickness in suspected pediatric glaucoma with large cup-to-disc ratios. METHODS: This was a retrospective study undertaken at a single academic institution. Eighty-six eyes of 43 patients who presented with large (≥ 0.5) cup-to-disc ratios in both eyes and without elevated intraocular pressure were evaluated using spectral-domain optical coherence tomography. Global and sectoral peripapillary RNFL thickness measurements, Bruch's membrane opening size, refractive error in spherical equivalents, and intraocular pressure levels were recorded for all patients. Cup-to-disc ratios were manually derived using digital fundus images (D-cup-to-disc ratio). Parameters were compared between gender or race by t tests or analysis of variance. The differences in the relationship among the clinical parameters between two eyes were assessed using generalized estimation equation modeling followed by Pearson's correlation analysis. RESULTS: Forty-three patients (25 boys and 18 girls) with a mean age of 9.3 ± 2.7 years (range: 5 to 15 years) were included. The mean global peripapillary RNFL thickness and the D-cup-to-disc ratio of study eyes were 99.0 ± 9.2 µm and 0.66 ± 0.03, respectively. The peripapillary RNFL thickness was found to be correlated with refractive error (r = 0.404; P = .008) and Bruch's membrane opening size (r = 0.410; P = .008) but not with cup-to-disc ratios (r = 0.029; P = .858) or patient age (r = -0.044; P = .797). CONCLUSIONS: In patients with suspected pediatric glaucoma who present with large cup-to-disc ratios, RNFL thickness does not correlate with the degree of optic nerve cupping. Myopic refractive errors and Bruch's membrane opening size need to be taken into consideration to prevent misinterpretation of peripapillary RNFL measurements. [J Pediatr Ophthalmol Strabismus. 2020;57(2):90-96.].

Glaucoma Care of Prison Inmates at an Academic Hospital.

Importance: Glaucoma care for prison inmates is underrepresented in the literature even though managing the treatment of such patients may provide unique challenges. Objectives: To evaluate the glaucoma profile of prison inmates treated at an academic ophthalmology center and to report on the medical and surgical management and follow-up metrics. Design, Setting, and Participants: This retrospective cohort study assessed data from 82 incarcerated patients treated at the glaucoma clinic, an academic referral center at the University of Illinois at Chicago, between January 2013 and December 2017. Main Outcomes and Measures: Diagnosis, glaucoma severity, medical and surgical interventions, and patient-reported medication adherence were recorded for each visit. Recommended and actual follow-up times were recorded and compared. Data analyses were conducted from January 2013 to December 2018. Results: In total, 82 patients (161 eyes) had 375 visits during the study period. All patients were male and ranged from 20 to 75 years of age (mean [SD] age, 50.8 [11.9] years). Most participants were black patients (65 [79.3%]). The most common diagnoses were primary open-angle glaucoma (POAG; 53 eyes [32.9%]) and POAG suspect (52 eyes [32.3%]). Glaucoma severity ranged from mild (25 of 77 eyes [32.5%]) to advanced (41 of 77 eyes [53.2%]). Overall, 59 patients (73.2%) were treated medically with up to 4 topical agents (40.0%). Of those treated, 70.0% of patients (95% CI, 57.7%-81.2%) reported medication nonadherence during at least 1 visit. Medication nonadherence was more common among those taking 4 different topical medications (21 of 24 [87.5%]) compared with others taking fewer medications (20 of 35 [57.1%]), for a difference of 30.4% (95% CI, 7.0%-53.6%; P = .02), and among those with advanced disease (22 of 26 [84.6%]) compared with glaucoma suspect (6 of 13 [46.2%]), for a difference of 38.4% (95% CI, 9.3%-67.5%; P = .02). Nineteen office procedures, including laser peripheral iridotomy and laser trabeculoplasty, were performed on 14 eyes. Seventeen incisional glaucoma procedures were performed on 15 eyes, including glaucoma drainage device implant (11 procedures [64.7%]) and trabeculectomy (3 procedures [17.6%]). Only 26.6% of return office visits (95% CI, 21.3%-32.3%) occurred within the recommended follow-up time frame. Furthermore, 93 patients (34.8%; 95% CI, 28.2%-40.0%) were seen more than 1 month after the recommended follow-up. Conclusions and Relevance: Despite incarceration in prison, where medication administration and appointment attendance are theoretically controlled, the results of this study suggested that substantial medication and follow-up nonadherence exists among inmates.

LC-MS/MS Software for Screening Unknown Erectile Dysfunction Drugs and Analogues: Artificial Neural Network Classification, Peak-Count Scoring, Simple Similarity Search, and Hybrid Similarity Search Algorithms.

Screening and identifying unknown erectile dysfunction (ED) drugs and analogues, which are often illicitly added to health supplements, is a challenging analytical task. The analytical technique most commonly used for this purpose, liquid chromatography-tandem mass spectrometry (LC-MS/MS), is based on the strategy of searching the LC-MS/MS spectra of target compounds against database spectra. However, such a strategy cannot be applied to unknown ED drugs and analogues. To overcome this dilemma, we have constructed a standalone software named AI-SIDA (artificial intelligence screener of illicit drugs and analogues). AI-SIDA consists of three layers: LC-MS/MS viewer, AI classifier, and Identifier. In the second AI classifier layer, an artificial neural network (ANN) classification model, which was constructed by training 149 LC-MS/MS spectra (including 27 sildenafil-type, 6 vardenafil-type, 11 tadalafil-type ED drugs/analogues and other 105 compounds), is included to classify the LC-MS/MS spectra of the query compound into four categories: i.e., sildenafil, vardenafil, and tadalafil families and non-ED compounds. This ANN model was found to show 100% classification accuracy for the 187 LC-MS/MS modeling and test data sets. In the third Identifier layer, three search algorithms (pick-count scoring, simple similarity search, and hybrid similarity search) are implemented. In particular, the hybrid similarity search was found to be very powerful in identifying unknown ED drugs/analogues with a single modification from the library ED drugs/analogues. Altogether, the AI-SIDA software provides a very useful and powerful platform for screening unknown ED drugs and analogues.

Synergistic effects of urban tributary mixing on dissolved organic matter biodegradation in an impounded river system.

Dams and wastewater may greatly perturb riverine fluxes of dissolved organic matter (DOM) and CO2, yet little is known about the relationships between altered DOM quality and CO2 emission in eutrophic impounded river systems. A basin-wide field survey of surface water CO2 and dissolved organic carbon (DOC) was combined with laboratory incubations to examine how dams and urban tributaries delivering treated wastewater influence longitudinal patterns in DOM properties and CO2 along the impounded Han River traversing Seoul metropolitan area. Fluorescent DOM indices including parallel factor analysis (PARAFAC) components were used to characterize DOM in relation to biodegradable DOC (BDOC). Compared with distinct downstream increases in DOC and CO2, BDOC concentration and its proportion in DOC (%BDOC) were highly variable along the mainstem and peaked at urban tributaries. Longitudinal increases in fluorescence index (FI), biological index (BIX), and two PARAFAC components (C2 and C3) contrasted with general decreases in humification index (HIX) and C1, reflecting increasing downstream inputs of anthropogenic DOM. During a 5-day incubation employing continuous CO2 measurements, the cumulative production of CO2 in the mainstem water mixed with urban tributary water was significantly higher than the level expected for conservative mixing of the two samples, indicating a synergistic enhancement of DOM biodegradation. Molecular formulas identified by Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) revealed more consumed molecules in the mainstem water and more newly produced molecules in the tributary water over the 5-day incubation, implying abundant labile components in the mainstem water discharged from the upstream dam and highly processed tributary DOM limited in immediately biodegradable organic materials. Downstream increases in CO2 and DOC along the Han River, combined with the synergistic effect observed in the mixed water, suggest that mixing wastewater-derived DOM with labile autochthonous DOM can enhance CO2 production in the river system perturbed by impoundment and wastewater.

Free Radical-Initiated Peptide Sequencing Mass Spectrometry for Phosphopeptide Post-translational Modification Analysis.

Free radical-initiated peptide sequencing mass spectrometry (FRIPS MS) was employed to analyze a number of representative singly or doubly protonated phosphopeptides (phosphoserine and phosphotyrosine peptides) in positive ion mode. In contrast to collision-activated dissociation (CAD) results, a loss of a phosphate group occurred to a limited degree for both phosphoserine and phosphotyrosine peptides, and thus, localization of a phosphorylated site was readily achieved. Considering that FRIPS MS supplies a substantial amount of collisional energy to peptides, this result was quite unexpected because a labile phosphate group was conserved. Analysis of the resulting peptide fragments revealed the extensive production of a-, c-, x-, and z-type fragments (with some minor b- and y-type fragments), suggesting that radical-driven peptide fragmentation was the primary mechanism involved in the FRIPS MS of phosphopeptides. Results of this study clearly indicate that FRIPS MS is a promising tool for the characterization of post-translational modifications such as phosphorylation. Graphical Abstract.

Effects of Resveratrol on the Renin-Angiotensin System in the Aging Kidney.

The renin-angiotensin system (RAS), especially the angiotensin II (Ang II)/angiotensin II type 1 receptor (AT1R) axis, plays an important role in the aging process of the kidney, through increased tissue reactive oxygen species production and progressively increased oxidative stress. In contrast, the angiotensin 1-7 (Ang 1-7)/Mas receptor (MasR) axis, which counteracts the effects of Ang II, is protective for end-organ damage. To evaluate the ability of resveratrol (RSV) to modulate the RAS in aging kidneys, eighteen-month-old male C57BL/6 mice were divided into two groups that received either normal mouse chow or chow containing resveratrol, for six months. Renal expressions of RAS components, as well as pro- and antioxidant enzymes, were measured and mouse kidneys were isolated for histopathology. Resveratrol-treated mice demonstrated better renal function and reduced albuminuria, with improved renal histologic findings. Resveratrol suppressed the Ang II/AT1R axis and enhanced the AT2R/Ang 1-7/MasR axis. Additionally, the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, 8-hydroxy-2'-deoxyguanosine, 3-nitrotyrosine, collagen IV, and fibronectin was decreased, while the expression of endothelial nitric oxide synthase and superoxide dismutase 2 was increased by resveratrol treatment. These findings demonstrate that resveratrol exerts protective effects on aging kidneys by reducing oxidative stress, inflammation, and fibrosis, through Ang II suppression and MasR activation.

Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury.

BACKGROUND: Two important issues in the aging kidney are mitochondrial dysfunction and oxidative stress. An Nrf2 activator, resveratrol, is known to have various effects. Resveratrol may prevent inflammation and oxidative stress by activating Nrf2 and SIRT1 signaling. We examined whether resveratrol could potentially ameliorate the cellular condition, such as renal injury due to cellular oxidative stress and mitochondrial dysfunction caused by aging. METHODS: Male 18-month-old C57BL/6 mice were used. Resveratrol (40 mg/kg) was administered to aged mice for 6 months. We compared histological changes, oxidative stress, and aging-related protein expression in the kidney between the resveratrol-treated group (RSV) and the control group (cont). We performed experiments using small-interfering RNAs (siRNAs) for Nrf2 and SIRT1 in cultured HK2 cells. RESULTS: Resveratrol improved renal function, proteinuria, histological changes and inflammation in aging mice. Also, expression of Nrf2-HO-1-NOQ-1 signaling and SIRT1-AMPK-PGC-1α signaling was increased in the RSV group. Transfection with Nrf2 and SIRT1 siRNA prevented resveratrol-induced anti-oxidative effect in HK2 cells in media treated with H2O2. CONCLUSIONS: Activation of the Nrf2 and SIRT1 signaling pathways ameliorated oxidative stress and mitochondrial dysfunction. Pharmacological targeting of Nrf2 signaling molecules may reduce the pathologic changes of aging in the kidney.

TEMPO-Assisted Free Radical-Initiated Peptide Sequencing Mass Spectrometry (FRIPS MS) in Q-TOF and Orbitrap Mass Spectrometers: Single-Step Peptide Backbone Dissociations in Positive Ion Mode.

The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044-7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research. Graphical Abstract ᅟ.

Age-Associated Changes in the Vascular Renin-Angiotensin System in Mice.

Background. This study evaluated whether the change in the renin-angiotensin system (RAS) is associated with arterial aging in mice. Methods. Histologic changes and expressions of transforming growth factor-ß (TGF-ß), collagen IV, fibronectin, angiotensin II (Ang II), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), prorenin receptor (PRR), Mas receptor (MasR), endothelial nitric oxide synthase (eNOS), NADPH oxidase 2 and oxidase 4 (Nox2 and Nox4), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 3-nitrotyrosine, and superoxide dismutase 1 and dismutase 2 (SOD1 and SOD2) were measured in the thoracic aortas from 2-month-old, 12-month-old, and 24-month-old C57/BL6 mice. Results. Twenty-four-month-old mice showed significantly increased aortic media thickness and expressions of TGF-ß, collagen IV, and fibronectin, compared to 2-month-old and 12-month-old mice. The expressions of PRR, ACE, and Ang II, and AT1R-positive area significantly increased, whereas expressions of ACE2 and MasR and AT2R-positive area decreased with age. The expressions of phosphorylated serine(1177)-eNOS, SOD1, and SOD2 decreased, and the 8-OHdG-positive area and the 3-nitrotyrosine-positive area increased with age. The expression of Nox2 significantly increased with age, but that of Nox4 did not change. Conclusions. The enhanced PRR-ACE-Ang II-AT1R axis and reduced ACE2-MasR axis were associated with arterial aging in mice.

Role of Glyoxylate Shunt in Oxidative Stress Response.

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

Endogenous hydrogen peroxide increases biofilm formation by inducing exopolysaccharide production in Acinetobacter oleivorans DR1.

In this study, we investigated differentially expressed proteins in Acinetobacter oleivorans cells during planktonic and biofilm growth by using 2-dimensional gel electrophoresis combined with matrix-assisted laser desorption time-of-flight mass spectrometry. We focused on the role of oxidative stress resistance during biofilm formation using mutants defective in alkyl hydroperoxide reductase (AhpC) because its production in aged biofilms was enhanced compared to that in planktonic cells. Results obtained using an ahpC promoter-gfp reporter vector showed that aged biofilms expressed higher ahpC levels than planktonic cells at 48 h. However, at 24 h, ahpC expression was higher in planktonic cells than in biofilms. Deletion of ahpC led to a severe growth defect in rich media that was not observed in minimal media and promoted early biofilm formation through increased production of exopolysaccharide (EPS) and EPS gene expression. Increased endogenous H2O2 production in the ahpC mutant in rich media enhanced biofilm formation, and this enhancement was not observed in the presence of antioxidants. Exogenous addition of H2O2 promoted biofilm formation in wild type cells, which suggested that biofilm development is linked to defense against H2O2. Collectively, our data showed that EPS production caused by H2O2 stress enhances biofilm formation in A. oleivorans.

Risk factors in the progression of BK virus-associated nephropathy in renal transplant recipients.

BACKGROUND/AIMS: BK virus-associated nephropathy (BKVAN) is an important cause of allograft dysfunction in kidney transplant recipients. It has an unfavorable clinical course, and no definite treatment guidelines have yet been established. Here, we report our center's experience with biopsy-proven BKVAN and investigate factors associated with its progression. METHODS: From January 2004 to April 2013, 25 patients with BKVAN were diagnosed by biopsy at Seoul St. Mary's Hospital. Of the 25 patients, 10 were deceased-donor transplant recipients and 15 were living-donor transplant recipients. Three of the patients underwent retransplantation. The primary immunosuppressant used was tacrolimus in 17 patients and cyclosporine in eight patients. RESULTS: BKVAN was observed at a mean duration of 22.8 ± 29.1 months after transplantation. The mean serum creatinine level at biopsy was 2.2 ± 0.7 mg/dL. BKVAN occurred with acute rejection in eight patients (28%). Immunosuppression modification was performed in 21 patients (84%). Additionally, leflunomide and intravenous immunoglobulin were administered to 13 patients (52%) and two (8%), respectively. Allograft loss occurred in five patients (27.8%) during the follow- up period at 0.7, 17.1, 21.8, 39.8, and 41.5 months after the BKVAN diagnosis. Advanced stages of BKVAN, increased creatinine levels, and accompanying acute rejection at the time of BKVAN diagnosis increased the risk of allograft failure. CONCLUSIONS: The clinical outcomes in patients with biopsy-proven BKVAN were unfavorable in the present study, especially in patients with advanced-stage BKVAN, poor renal function, and acute allograft rejection.

Molecular mechanism involved in the response to hydrogen peroxide stress in Acinetobacter oleivorans DR1.

Two-dimensional gel electrophoresis was conducted to investigate the effect of H2O2 on whole protein expression in Acinetobacter oleivorans DR1. Functional classification of 13 upregulated proteins using MALDI-TOF mass spectrometry showed relationships with oxidative stress, energy production and conversion, nucleotide and amino acid metabolism, membrane-related, ion transport, and chaperone-related functions. Alignment of OxyR-binding regions from Pseudomonas aeruginosa and Escherichia coli with promoters of identified proteins revealed that only ahpC, ahpF, and trxB (thioredoxin-disulfide reductase) genes, along with a newly found oprC (putative outer membrane receptor protein) gene, have OxyR-binding sites. The oxyR and ahpC mutants were more sensitive to H2O2 and showed growth defects in both nutritional and n-hexadecane-amended media. Four catalases present in the genome of A. oleivorans DR1 were not detected, which led us to confirm the expression and activity of those catalases in the presence of H2O2. The expression patterns of the four catalase genes differed at different concentrations of H2O2. Interestingly, the promoters of both known OxyR-controlled katG gene (AOLE_17390) and putative small catalase gene (AOLE_09800) have OxyR-binding sites. Gel-shift assay confirmed OxyR binding to the promoter regions of newly identified OxyR-controlled genes encoding OprC and a putative catalase. Hierarchical expression and OxyR-binding of several OxyR-controlled genes suggested that concentration is an important factor in inducing the set of genes under H2O2 stress.

Molecular Mechanisms of Enhanced Bacterial Growth on Hexadecane with Red Clay.

Red clay was previously used to enhance bioremediation of diesel-contaminated soil. It was speculated that the enhanced degradation of diesel was due to increased bacterial growth. In this study, we selected Acinetobacter oleivorans DR1, a soil-borne degrader of diesel and alkanes, as a model bacterium and performed transcriptional analysis using RNA sequencing to investigate the cellular response during hexadecane utilization and the mechanism by which red clay promotes hexadecane degradation. We confirmed that red clay promotes the growth of A. oleivorans DR1 on hexadecane, a major component of diesel, as a sole carbon source. Addition of red clay to hexadecane-utilizing DR1 cells highly upregulated ß-oxidation, while genes related to alkane oxidation were highly expressed with and without red clay. Red clay also upregulated genes related to oxidative stress defense, such as superoxide dismutase, catalase, and glutaredoxin genes, suggesting that red clay supports the response of DR1 cells to oxidative stress generated during hexadecane utilization. Increased membrane fluidity in the presence of red clay was confirmed by fatty acid methyl ester analysis at different growth phases, suggesting that enhanced growth on hexadecane could be due to increased uptake of hexadecane coupled with upregulation of downstream metabolism and oxidative stress defense. The monitoring of the bacterial community in soil with red clay for a year revealed that red clay stabilized the community structure.

Bromine isotopic signature facilitates de novo sequencing of peptides in free-radical-initiated peptide sequencing (FRIPS) mass spectrometry.

We recently showed that free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) assisted by the remarkable thermochemical stability of (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) is another attractive radical-driven peptide fragmentation MS tool. Facile homolytic cleavage of the bond between the benzylic carbon and the oxygen of the TEMPO moiety in o-TEMPO-Bz-C(O)-peptide and the high reactivity of the benzylic radical species generated in •Bz-C(O)-peptide are key elements leading to extensive radical-driven peptide backbone fragmentation. In the present study, we demonstrate that the incorporation of bromine into the benzene ring, i.e. o-TEMPO-Bz(Br)-C(O)-peptide, allows unambiguous distinction of the N-terminal peptide fragments from the C-terminal fragments through the unique bromine doublet isotopic signature. Furthermore, bromine substitution does not alter the overall radical-driven peptide backbone dissociation pathways of o-TEMPO-Bz-C(O)-peptide. From a practical perspective, the presence of the bromine isotopic signature in the N-terminal peptide fragments in TEMPO-assisted FRIPS MS represents a useful and cost-effective opportunity for de novo peptide sequencing.