Outcome of tailored therapy in rheumatic heart disease with persistent atrial fibrillation (RHD-AF).

INTRODUCTION: Rheumatic heart disease with persistent atrial fibrillation (RHD-AF) is associated with increased morbidity. However, there is no standardized approach for the maintenance of sinus rhythm (SR) in them. We aimed to determine the utility of a stepwise approach to achieve SR in RHD-AF. METHODS: Consecutive patients with RHD-AF from July 2021 to August 2023 formed the study cohort. The stepwise approach included pharmacological rhythm control and/or electrical cardioversion (Central illustration). In patients with recurrence, additional options included AF ablation or pace and ablate strategy with conduction system pacing or biventricular pacing. Clinical improvement, NT-proBNP, 6-Minute Walk Test (6MWT), heart failure (HF) hospitalizations, and thromboembolic complications were documented during follow-up. RESULTS: Eighty-three patients with RHD-AF (mean age 56.13 ± 9.51 years, women 72.28%) were included. Utilizing this approach, 43 (51.81%) achieved and maintained SR during the study period of 11.04 ± 7.14 months. These patients had improved functional class, lower NT-proBNP, better distance covered for 6MWT, and reduced HF hospitalizations. The duration of AF was shorter in patients who achieved SR, compared to those who remained in AF (3.15 ± 1.29 vs 6.93 ± 5.23, p = 0.041). Thirty-five percent (29) maintained SR after a single cardioversion over the study period. Only one underwent AF ablation. Of the 24 who underwent pace and ablate strategy, atrial lead was implanted in 22 (hybrid approach), and 50% of these achieved and maintained SR. Among these 24, none had HF hospitalizations, but patients who maintained SR had further improvement in clinical and functional parameters. CONCLUSIONS: RHD-AF patients who could achieve SR with a stepwise approach, had better clinical outcomes and lower HF hospitalizations.

Multifunctional characteristics of biosynthesized CoFe2O4@Ag nanocomposite by photocatalytic, antibacterial and cytotoxic applications.

Carissa carandas, a traditional medicinal herb with a high concentration of antioxidant phytochemicals, has been used for thousands of years in the Ayurveda, Unani, and homoeopathic schools of medicine. By employing Carissa carandas bark extract as a reducing and capping agent in green biosynthesis, we extend this conventional application to produce CoFe2O4 and CoFe2O4@Ag nanocomposite. A variety of techniques have been used to characterize the synthesised nanocomposite, including UV-Vis, FTIR, XRD, FESEM, EDX, and BET. The CoFe2O4 and CoFe2O4@Ag nanocomposite demonstrated promising antibacterial action against human bacterial pathogens like B. subtilis and S. aureus as gram positive and P. aeruginosa and E. coli as gram negative with inhibition zones of 24.3 ± 0.57, 17.4 ± 0.75 and 20.5 ± 0.5, 19.8 ± 1.6 mm respectively, and the obtained results were superior to the nanocomposite without silver. Moreover, in-vitro cytotoxicity effects of biosynthesized CoFe2O4 and CoFe2O4@Ag were performed on the human breast cancer cell MCF-7. It was found that the MCF-7 cells' 50% inhibitory concentration (IC50) was 60 µg/mL. Additionally, biosynthesized CoFe2O4 and CoFe2O4@Ag nanocomposite was used to demonstrate the photocatalytic eradication of Rhodamine Blue (RhB). Due to the addition of Ag, which increases surface area, conductivity, and increased charge carrier separation, the CoFe2O4@Ag nanocomposite exhibits a high percentage of photocatalytic degradation of ⁓ 98% within 35 min under UV light irradiation. The photocatalytic performance of as-synthesised nanocomposite was evaluated using dye degradation-adsorption in both natural light and dark condition. Under dark conditions, it was found that 2 mg mL-1 CoFe2O4@Ag in RhB aqueous solution (5 ppm) causes dye adsorption in 30 min with an effectiveness of 72%. Consequently, it is anticipated that the CoFe2O4@Ag nanocomposite will be a promising photocatalyst and possibly a noble material for environmental remediation applications.

Analysis of chemical engineering curriculum to improve process safety competency.

Continuous process safety (PS) development is the key to maintaining a good PS system, and its competency plays a substantial role. However, PS incompetency can still be demonstrated in several process-related accidents, particularly major catastrophic incidents. To mitigate this gap, universities' PS education is analysed. Because PS is an important element of chemical engineering (CE), this study seeks to identify the most prevalent PS subjects taught in the top 300 Quacquarelli Symonds ranking (2019) universities. Findings indicate that PS education remains insufficiently addressed in undergraduate CE curricula over the years. Twelve common topics, i.e., human factors; management of hazards, incidents, and risk; design; fire and explosion; legislation and standards; sustainability; process control; economics; toxicology; and software are identified. Notably, sustainability is acknowledged to be a new common PS topic, depicting its demand for industrial evolution. Ultimately, strengthening the collaboration between universities and industries is required to develop graduates' PS competency.Abbreviations: ALARP: as low as reasonably practicable; CAD: computer-aided design; CE: chemical engineering; ETA: event tree analysis; FTA: fault tree analysis; FMEA: failure mode and effect analysis; HAZAN: hazard analysis; HAZID: hazard identification; HAZOP: hazard and operability; HSE: health, safety and environment; HYSYS: Hyprotech Systems; LCA: life cycle analysis; LOPA: layer of protection analysis; MS: Microsoft; ORP: occupational risk prevention; PC: personal computer; PHA: process hazard analysis; PS: process safety; PSM: process safety management; QS: Quacquarelli Symonds; SMS: safety management system.

The potential of polyethylene microplastics to transport copper in aquatic systems: Adsorption and desorption studies.

Heavy metals and microplastics are two types of general contaminants that can cause significant damage to water systems and organisms. However, the interaction of heavy metal ions with microplastic in aquatic systems received less attention compared with that of organic contaminants. This study aims to explore the interaction of copper (II) ions with microplastic (polyethylene) in aquatic systems. The adsorption experiments were performed by varying several operational parameters including equilibrium pH, initial Cu (II) concentrations, and contact times. The microplastic was characterized using X-ray diffraction, scanning electron microscopy, and Fourier transform infrared. The results confirmed the porous nature of the microplastic surface and the existence of various binding sites. The maximum Cu (II) uptake by microplastic was recorded as 1.23 mg/g at pH 5, according to the Langmuir adsorption isotherm. The experimental isotherm data exhibited a good fit to the Toth model, followed by the Langmuir and Freundlich equations, according to the correlation coefficient and %error values. The pseudo-first kinetics equation showed a better fit to copper (II) kinetics data compared with the pseudo-second kinetics equation. Elution of copper (II) ions from copper (II)-loaded microplastic was attempted using several elutants, and the results indicated that 0.01 M HNO3 performed well with elution efficiency over 99.5%. Thus, the elution experiments furnished proof that Cu-loaded microplastic may leach Cu (II) ions under rich acidic conditions, thereby aiding the transport of Cu (II) ions into the digestive tracts of aquatic organisms. PRACTITIONER POINTS: Polyethylene microplastics showed potential to sorb copper ions. The mechanism was electrostatic interaction between microplastics and metal ions. Maximum copper adsorption by microplastic was recorded as 1.23 mg/g. Once desorbed, Cu(II) transferred into the digestive tracts of aquatic organisms.

An ultra-sensitive laccase/polyaziridine-bismuth selenide nanoplates modified GCE for detection of atenolol in pharmaceuticals and urine samples.

The analysis of ß-blockers in pharmaceutical, biological and environmental samples has gained much interest due to their wide applications. The aim of this study was to develop an enzyme-based biosensor using hexagonal-shaped low-dimensional Bi2Se3 NPs decorated with laccase through polyaziridine (PAZ) modified glassy carbon electrode (Lac/PAZ-Bi2Se3 NPs/GCE). Surface properties were examined using SEM, TEM, EDX, XRD, XPS, FTIR, UV-Visible, and zeta potential. Electrochemical studies were performed with cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The enzymatic biosensor exhibited excellent catalytic activity towards the oxidation of ATN at +1.05 V (vs Ag/AgCl). Under the optimum experimental conditions, Ip (µA) was linearly related to the concentrations of ATN in the range of 3 to 130 µM (R2 = 0.9972) with an LOD of 0.15 µM and 0.21 µM with and without Lac enzyme. Additionally, the validation of the biosensor was tested to determine ATN on within a day and between-day basis. The biosensor was applied successfully to detect ATN in real samples. The obtained recoveries range from 98.5 % to 99.2 % with an RSD (n = 5) of 0.95 (±0.02). The findings of this study have potential biomedical applications in drug detection employing a promising nano electrode sensor of Lac/PAZ-Bi2Se3 NPs/GCE.

Fusing an insoluble protein to GroEL apical domain enhances soluble expression in Escherichia coli.

A protocol for increasing soluble protein expression by fusing the chaperone GroEL apical domain with a gene of interest is described herein. GroEL apical domain, the minichaperone that functions independently of GroES and ATP in protein folding, is cloned downstream of the lambda CII ribosome binding site in the parent pRE vector. The pRE vector has tightly controlled transcription suitable for expressing toxic proteins. The GroEL minichaperone is fused to a glycine-serine rich linker followed by the enterokinase protease recognition sequence. A number of genes that are recalcitrant to protein production in the parent pRE vector 5were cloned into the pRE:GroEL fusion vector and successfully expressed as fusion proteins in Escherichia coli.

Resection of Olfactory Groove Meningiomas Through Unilateral vs. Bilateral Approaches: A Systematic Review and Meta-Analysis.

Introduction: Consensus is limited regarding optimal transcranial approaches (TCAs) for the surgical resection of olfactory groove meningiomas (OGMs). This systematic review and meta-analysis aims to examine operative and peri-operative outcomes of unilateral compared to bilateral TCAs for OGMs. Methods: Electronic databases were searched from inception until December 2019 for studies delineating TCAs for OGM patients. Patient demographics, pre-operative symptoms, surgical outcomes, and complications were evaluated and analyzed with a meta-analysis of proportions. Results: A total of 27 observational case series comparing 554 unilateral vs. 451 bilateral TCA patients were eligible for review. The weighted pooled incidence of gross total resection is 94.6% (95% CI, 90.7-97.5%; I 2 = 59.0%; p = 0.001) for unilateral and 90.9% (95% CI, 85.6-95.4%; I 2 = 58.1%; p = 0.003) for bilateral cohorts. Similarly, the incidence of OGM recurrence is 2.6% (95% CI, 0.4-6.0%; I 2 = 53.1%; p = 0.012) and 4.7% (95% CI, 1.4-9.2%; I 2 = 55.3%; p = 0.006), respectively. Differences in oncologic outcomes were not found to be statistically significant (p = 0.21 and 0.35, respectively). Statistically significant differences in complication rates in bilateral vs. unilateral TCA cohorts include meningitis (1.0 vs. 0.0%; p = 0.022) and mortality (3.2 vs. 0.2%; p = 0.007). Conclusions: While both cohorts have similar oncologic outcomes, bilateral TCA patients exhibit higher post-operative complication rates. This may be explained by underlying tumor characteristics necessitating more radical resection but may also indicate increased morbidity with bilateral approaches. However, evidence from more controlled, comparative studies is warranted to further support these findings.

Risk of Distal Embolization From tPA (Tissue-Type Plasminogen Activator) Administration Prior to Endovascular Stroke Treatment.

BACKGROUND AND PURPOSE: In large artery occlusion stroke, both intravenous (IV) tPA (tissue-type plasminogen activator) and endovascular stroke treatment (EST) are standard-of-care. It is unknown how often tPA causes distal embolization, in which a procedurally accessible large artery occlusion is converted to a more distal and potentially inaccessible occlusion. METHODS: We analyzed data from a decentralized stroke telemedicine program in an integrated healthcare delivery system covering 21 hospitals, with 2 high-volume EST centers. We captured all cases sent for EST and examined the relationship between IV tPA administration and the rate of distal embolization, the rate of target recanalization (modified Treatment in Cerebral Infarction scale 2b/3), clinical improvement before EST, and short-term and long-term clinical outcomes. RESULTS: Distal embolization before EST was quite common (63/314 [20.1%]) and occurred more often after IV tPA before EST (57/229 [24.9%]) than among those not receiving IV tPA (6/85 [7.1%]; P<0.001). Distal embolization was associated with an inability to attempt EST: after distal embolization, 26/63 (41.3%) could not have attempted EST because of the new clot location, while in cases without distal embolization, only 8/249 (3.2%) were unable to have attempted EST (P<0.001). Among patients who received IV tPA, 13/242 (5.4%) had sufficient symptom improvement that a catheter angiogram was not performed; 6/342 (2.5%) had improvement to within 2 points of their baseline NIHSS. At catheter angiogram, 2/229 (0.9%) of patients who had received tPA had complete recanalization without distal embolization. Both IV tPA and EST recanalization were associated with improved long-term outcome. CONCLUSIONS: IV tPA administration before EST for large artery occlusion is associated with distal embolization, which in turn may reduce the chance that EST can be attempted and recanalization achieved. At the same time, some IV tPA-treated patients show symptomatic improvement and complete recanalization. Because IV tPA is associated with both distal embolization and improved long-term clinical outcome, there is a need for prospective clinical trials testing the net benefit or harm of IV tPA before EST.

Synthesis of green marine algal-based biochar for remediation of arsenic(V) from contaminated waters in batch and column mode of operation.

The sorption behavior of biochar derived from green seaweed (Ulva reticulata) toward arsenic(V) ions was explored in both batch and continuous modes. The pH edge experiments indicated optimum arsenic(V) sorption observed at pH 4, with maximum sorptional capacity of 7.67 mg/g through isotherm experiments. The kinetic experimental trials indicated that arsenic(V) sorption onto biochar was a fast electrostatic attraction process, with maximum removal occurred within 30 min. The sorption isotherms were modeled using the Toth, Redlich-Peterson, Langmuir and Freundlich isotherm models while the adsorption kinetics was modeled using the pseudo-first- and pseudo-second-order kinetic equations. The three-parameter models (Redlich-Peterson and Toth) better described the isotherm data, whereas pseudo-first-order model represented kinetic data well with low error and high correlation coefficient values. Among the different alkaline and acidic elutants investigated, the solution of 0.01 M NaOH effectively desorbed arsenic(V) from spent biochar. The feasibility of the biochar in continuous remediation of arsenic(V) from contaminated waters was explored in an up-flow fixed column. The biochar exhibited arsenic(V) removal efficiency and sorptional uptake of 59.5% and 8.12 mg/g, respectively. The biochar-loaded column was effectively desorbed using NaOH (0.01 M), with desorption efficiency of 99.5%.

Comparative Site-Specific N-Glycosylation Analysis of Lactoperoxidase from Buffalo and Goat Milk Using RP-UHPLC-MS/MS Reveals a Distinct Glycan Pattern.

The N-glycan pattern of lactoperoxidase (LPO) from buffalo and goat milk was analyzed with the corresponding site of attachment. The enzyme was purified from whey on cation exchange chromatography, proteolyzed using chymotrypsin, and the resulting (glyco)peptides were directly analyzed on reverse phase ultrahigh performance liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. N-Glycans such as high mannose, complex, and hybrid types were identified in buffalo and goat LPO. Among sialylated complex and hybrid types, the terminal Neu5Ac linked to either LacNAc/LacdiNAc found exclusively in buffalo, whereas Neu5Gc linked to LacdiNAc was predominant in goat LPO. N-Glycans at Asn6 and Asn349 in buffalo LPO were completely core fucosylated, while these sites in goat LPO showed differential fucosylation. Differential occupancy was observed at Asn112 with or without nonfucosylated complex and hybrid types, whereas mainly high mannose glycans were found in Asn222 in both of the LPOs. The presence of glycan isomers in buffalo and goat LPO was also observed. Despite the presence of distinct complex and hybrid glycans, the common glycosylation features in buffalo and goat LPO were identified and are comparable with those of bovine LPO. This finding could be useful in exploring the beneficial role of these glycans as functional ingredients for food products.

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli.

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.

Structural and luminescence properties of Sm3+ -doped bismuth phosphate glass for orange-red photonic applications.

In the present study, the effect of bismuth oxide (Bi2 O3 ) content on the structural and optical properties of 0.5Sm3+ -doped phosphate glass and the effect of concentration on structural and optical properties of Sm3+ -doped bismuth phosphate (BiP) glass were studied. Structural characterization was accomplished using X-ray diffraction (XRD), scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS), Fourier transform infrared (FTIR) spectroscopy and 31 P nuclear magnetic resonance (NMR) spectroscopy. Optical properties were studied using absorption, photoluminescence and decay measurements. Using optical absorption spectra, Judd-Ofelt parameters were derived to determine the local structure and bonding in the vicinity of Sm3+ ions. The emission spectra of Sm3+ -doped BiP glass showed two intense emission bands, 4 G5/2 →6 H7/2 (orange) and 4 G5/2 →6 H9/2 (red) for which the stimulated emission cross-sections (σe ) and branching ratios (ß) were found to be higher. The quantum efficiencies were also calculated from decay measurements recorded for the 4 G5/2 level of Sm3+ ions. The suitable combination of Bi2 O3 (10 mol%) and Sm3+ (0.5 mol%) ions in these glasses acted as an efficient lasing material and might be suitable for the development of visible orange-red photonic materials.

Spectral investigations on Eu3+ ,Sm3+ -doped and Sm3+ /Eu3+ co-doped potassium-fluoro-phosphate glass emitting intense orange-red for lighting applications.

Potassium fluoro-phosphate (KFP) glass singly doped with different concentrations of europium (Eu3+ ) or samarium (Sm3+ ) or co-doped (Sm3+ /Eu3+ ) was prepared, and their luminescence spectra were investigated. The phase composition of the product was verified by X-ray diffraction analysis. Optical transition properties of Eu3+ in the studied potassium phosphate glass were evaluated in the framework of the Judd-Ofelt theory. The radiative transition rates (AR ), fluorescence branching ratios (ß), stimulated emission cross-sections (σe ) and lifetimes (τexp ) for certain transitions or levels were evaluated. Red emission of Eu3+ was exhibited mainly by the 5 D0 →7 F2 transition located at 612 nm. Concentration quenching and energy transfer were observed from fluorescence spectra and decay curves, respectively. It was found that the lifetimes of the 5 D0 level increased with increase in concentration and then decreased. By co-doping with Sm3+ , energy transfer from Sm3+ to Eu3+ occurred and contributed to the enhancement in emission intensity. Intense orange-red light emission was obtained upon sensitizing with Sm3+ in KFP glass. This approach shows significant promise for use in reddish-orange lighting applications. The optimized properties of the Sm3+ /Eu3+ co-doped potassium phosphate glass might be promising for optical materials.

Impact of Bi2O3 on structural properties and lasing effects in Nd3+ doped bismuth phosphate glasses at 1.053µm emission.

Influence of addition of bismuth oxide (Bi2O3) on structural and optical properties of 0.5Nd3+doped different phosphate glasses prepared by melt quenching technique were reported. The structural properties were analysed by FT-IR and 31P MAS NMR techniques. 31P NMR results showed that, with the variation of Bi2O3 concentration in the prepared phosphate glass matrices there was no significant change in the structure. The optical properties have been analysed using Judd-Ofelt (J-O) theory. From absorption spectra, three phenomenological J-O intensity parameters (Ω2, Ω4, Ω6) have been calculated and these parameters were used to estimate the radiative properties. The J-O intensity parameters increased with increase in the concentration of Bi2O3, which confirmed the higher covalency, asymmetry and rigidity. The branching ratios (ß) and emission cross-sections (σ) were calculated from the emission spectra of Nd3+ doped bismuth phosphate (BiP) glasses. Among different glass matrices 15mol% of Bi2O3 glass matrix has higher ß and σ values which are useful for the laser applications. The improved emission cross-section value with the addition of Bi2O3 content results low threshold and high gain applications. The decay curves of all these BiP glasses showed single exponential behaviour.

Concentration-dependent studies of Nd3+ -doped zinc phosphate glasses for NIR photoluminescence at 1.05 µm.

Nd3+ -doped lead-free zinc phosphate glasses with the chemical compositions (60-x) NH4 H2 PO4  + 20ZnO + 10BaF2  + 10NaF + xNd2 O3 (where x = 0.5, 1.0, 1.5, 2.0 and 2.5 mol%) were prepared using a melt quenching technique. Vibrational bands were assigned and clearly elucidated by Raman spectral profiles for all the glass samples. Judd-Ofelt (J-O) intensity parameters (Ωλ : λ = 2, 4, 6) were obtained from the spectral intensities of different absorption bands of Nd3+ ions. Radiative properties such as radiative transition probabilities (AR ), radiative lifetimes (τR ) and branching ratios (ßR ) for different excited states were calculated using J-O parameters. The near infrared (NIR) photoluminescence spectra exhibited three emission bands (4 F3/2 level to 4 I13/2 , 4 I11/2 and 4 I9/2 states) for all the concentrations of Nd3+ ions. Various luminescence properties were studied by varying the Nd3+ concentration for the three spectral profiles. Fluorescence decay curves of the 4 F3/2 level were recorded. The energy transfer mechanism that leads to quenching of the 4 F3/2 state lifetimes was discussed at higher concentration of Nd3+ ions. These glasses are suggested as suitable hosts to produce efficient lasing action in NIR region at 1.05 µm.

Synthesis,isolation,identification and characterization of new process-related impurity in isoproterenol hydrochloride by HPLC,LC/ESI-MS and NMR / 药物分析学报

One unknown impurity(Imp-II)during the analysis of laboratory batches of isoproterenol hydrochloride was detected in the level ranging from 0.04% to 0.12% by high performance liquid chromatography with UV detection. The unknown impurity structure was proposed as 4-[2-(propan-2-ylamino)ethyl]benzene-1,2-diol (Imp-Ⅱ)using the liquid chromatography–mass spectrophotometry(LC–MS)analysis.Imp-Ⅱ was isolated by semi-preparative liquid chromatography from the impurity-enriched reaction crude sample. Its proposed structure was confirmed by nuclear magnetic spectroscopy such as 1H, 13C,DEPT(1D NMR),HSQC(2D NMR) and infrared spectroscopy(IR),and retention time and purity with HPLC followed by the chemical synthesis. Due to less removable nature of Imp-II during the purification,the synthetic process was optimized proficiently to control the formation of Imp-II below to the limit<0.12% in the course of reaction.The new chemical route was developed for the preparation of this impurity in required quantity with purity to use as reference standard. The most probable mechanism for the formation of Imp-II was discussed in details.

Production, Purification, and Characterization of ¹5N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements.

Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.

Protein labeling in Escherichia coli with (2)H, (13)C, and (15)N.

A number of structural biology techniques such as nuclear magnetic resonance spectroscopy and small-angle neutron scattering can be performed with proteins with nuclei at natural isotope abundance. However, the use of proteins labeled with stable isotopes ((2)H, (13)C, and (15)N) enables greater experimental flexibility. In this chapter, several methods for uniform and fractional protein labeling with stable isotopes using Escherichia coli in a defined media are described. The methods described can be used for labeling with single or multiple isotopes.

Extreme Expression of DNA Repair Protein Apurinic/Apyrimidinic Endonuclease 1 (APE1) in Human Breast Cancer As Measured by Liquid Chromatography and Isotope Dilution Tandem Mass Spectrometry.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. We previously developed an approach using liquid chromatography and tandem mass spectrometry with isotope dilution to accurately measure APE1 levels. Here, we applied this methodology to measure APE1 levels in normal and cancerous human breast tissues. Extreme expression of APE1 in malignant tumors was observed, suggesting that breast cancer cells may require APE1 for survival. Accurate measurement of APE1 may be essential for the development of novel treatment strategies and APE1 inhibitors as anticancer drugs.