Validated spectrofluorimetric method for the determination of netilmicin based on its interaction with o-phthalaldehyde/mercaptoethanol: Evaluation of the method's greenness.

In this study, netilmicin (NTM) was selectively assessed in its dosage forms after a facile derivatization reaction. The proposed approach was based on the interaction between NTM and o-phthalaldehyde/2-mercaptoethanol (Roth's reagent). The reaction product was fluorometrically measured at λemission of 434 nm after λexcitation of 338 nm. All reaction conditions for achieving the optimum fluorescence switch-on activity were visualized and monitored. Moreover, the method was validated under ICH guidelines, and was linear over the range 30-210 ng/ml after plotting netilmicin concentrations against the corresponding fluorescence intensity values. In addition, the selectivity of the developed method was investigated against either the co-formulated drug (dexamethasone) or a common ophthalmic drop excipient (benzalkonium chloride) without interference from either of them. Furthermore, the developed method was applied to assay netilmicin in various samples of pharmaceutical eye drops with good recovery. Finally, multicriteria greenness and whiteness metrics were used to evaluate the sustainability, greenness, and whiteness of the approach. The applied tools were the AGREE algorithm, the RGB 12 algorithm, and HEXAGON.

Use of thiol reagents to disperse cold autoagglutination.

Thiol reagents dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) are sulfhydryl reagents that can be used to disperse cold autoagglutinins coating red blood cells (RBCs). DTT and 2-ME are primarily used when warm washing of the coated RBCs fails to successfully disperse the cold autoantibody. Using a weak concentration of DTT or 2-ME, the cold IgM agglutinin can be removed from the coated RBCs without disrupting the IgG or complement coating the RBCs. The treated RBCs can be used for ABO typing, antigen typing, or the direct antiglobulin test.

Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae.

2-mercaptoethanol (2-ME), alongside polyvinylpyrrolidone is commonly used in plant DNA extractions to deal with polyphenols, which could interfere with extraction and downstream applications. 2-ME is also commonly used to denature proteins and nucleases, especially RNAses. On the contrary, we found that the presence of 2-ME in lysis buffer interfered with DNA extraction from 12 strains of freshwater microalgae, resulting in DNA with poor integrity. We also found that the TNES-urea buffer, commonly used for preservation and DNA extraction from fish, appears as effective as the SDS and CTAB buffer for some microalgae strains. Results from our study suggests that the inclusion of 2-ME in DNA extraction protocols may be detrimental for isolation of good quality DNA from freshwater microalgae, and therefore recommend eliminating it or testing varying concentrations of 2-ME when developing species-specific extraction protocols for microalgae.

Optimization of o-phtaldialdehyde/2-mercaptoethanol postcolumn reaction for the hydrophilic interaction liquid chromatography determination of memantine utilizing a silica hydride stationary phase.

A rapid procedure for the determination of memantine based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o-phtaldialdehyde/2-mercaptoethanol was performed at excitation and emission wavelengths of 345 and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, and reagents concentration were studied due to steric hindrance of amino group of memantine. The derivatization reaction was applied for the hydrophilic interaction liquid chromatography method which was based on Cogent Silica-C stationary phase with a mobile phase consisting of a mixture of 10 mmol/L citric acid and 10 mmol/L o-phosphoric acid (pH 6.0) with acetonitrile using an isocratic composition of 2:8 v/v. The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, accuracy, precision, and selectivity according to the International Conference on Harmonisation guidelines. The developed method was successfully applied for the analysis of commercial memantine tablets.

Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR.

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4-1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4-1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40-50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.

Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida.

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteins gave the characteristic maximum at 450nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with TH2-dominant inflammation. Thymic stromal lymphopoietin (TSLP) is a cytokine that triggers dendritic cell-mediated TH2 inflammatory responses and that enhances IL-1-dependent TH2 cytokine production in mast cells. Although increased TSLP mRNA levels have been found in nasal polyps (NPs), expression of TSLP protein and its function in patients with chronic rhinosinusitis (CRS) have not been fully explored. OBJECTIVES: The objective of this study was to investigate the role of TSLP in patients with CRS. METHODS: We investigated the presence and stability of TSLP protein in NPs using ELISA and Western blotting and investigated the function of TSLP in nasal tissue extracts with a bioassay based on activation of human mast cells. RESULTS: Although TSLP mRNA levels were significantly increased in NP tissue from patients with CRSwNP compared with uncinate tissue from patients with CRS or control subjects, TSLP protein was significantly decreased in NP tissue, as detected by using the commercial ELISA kit. We found that recombinant TSLP was time-dependently degraded by NP extracts, and this degradation was completely inhibited by a protease inhibitor cocktail, suggesting that TSLP is sensitive to tissue proteases. Interestingly, NP extract-treated TSLP had higher activity in mast cells, although the amount of full-length TSLP was reduced up to 85%. NP extracts significantly enhanced IL-1ß-dependent IL-5 production in mast cells compared with uncinate tissue homogenates, and responses were significantly inhibited by anti-TSLP, suggesting that NPs contain biologically relevant levels of TSLP activity. CONCLUSION: TSLP and its metabolic products might play an important role in the inflammation seen in patients with CRSwNP.

Seroprevalence of brucellosis antibodies and associated risk factors among the hospitalized patient, Aleppo, Syria: a hospital-based cross-sectional study.

Introduction and importance: Brucellosis is one of the most common infectious diseases in the world, especially in developing countries. Recent reports show that Syria is among the top ten countries where brucellosis is most prevalent. The purpose of this study is to estimate the seroprevalence of brucellosis antibodies among the hospitalized patients, in one of the largest hospitals in northern Syria. Materials and methods: A cross-sectional study was conducted among the hospitalized patients. The authors used a questionnaire to collect sociodemographic and brucellosis-related data from the patients. The authors also collected blood samples from these patients to be screened for brucellosis antibodies using Wright Coombs Agglutination and 2-mercaptoethanol tests, during the period from November 2021 and March 2022. Results: Among the 776 patients who were recruited in the study, the seroprevalence of brucellosis antibodies was 13.1% (n=776). The highest prevalence was among the female sex (16.7%, n=298), middle aged group 12-40 years (24.1%, n=116), and patients with history of brucellosis (30.1%, n=53). Among the positive samples, the findings of 2-mercaptoethanol tests show that (14.7%, n=102) were positive (presence of IgG Antibodies), and (75.5%, n=102) were negative. Conclusion: This study is the first to describe the epidemiology of brucellosis in northern Syria. It clearly shows high rates of positivity, which reflects immense challenges facing the public health sector in Syria. The best next step in light of this crisis is to raise awareness among population about brucellosis and its risk factor.

Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis.

Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.

First seroprevalence and molecular identification report of Brucella canis among dogs in Greater Cairo region and Damietta Governorate of Egypt.

Background and Aim: Given the rise in stray and imported dogs in Egypt over the past 5 years, it is surprising that no report of Brucella canis infection in dogs or humans has been documented in Egypt's published papers. This study aimed to detect the presence of antibodies against the rough (B. canis) and smooth Brucellae among dogs in Egypt and to characterize the Brucella species circulating in dogs. Materials and Methods: Blood samples (n = 449) were collected from owned and stray dogs in the Greater Cairo region (n = 309) and Damietta governorate (n = 140). The apparent, true, and total seroprevalence of canine brucellosis caused by B. canis infection were calculated using the 2-mercaptoethanol tube agglutination test (2-ME TAT) and rapid slide agglutination test (RSAT). We used the rose Bengal test (RBT) and the buffered acidified plate antigen test (BAPAT) to check the serum samples from dogs for the presence of antibodies against smooth Brucellae. Three polymerase chain reaction (PCR) assays - Bruce-ladder PCR, B. canis species-specific PCR (BcSS-PCR), and Abortus Melitensis Ovis Suis (AMOS)-PCR - were used to determine the Brucella species in the buffy coats of the serologically positive dogs. Results: The overall apparent and true prevalence of B. canis infection in dogs were estimated to be 3.8% and 13.2%. The estimated true prevalence in stray dogs (15%) was higher than in owned dogs (12.5%). The BAPAT and the RBT using smooth antigens revealed that 11 (2.4%) and 9 (2%) were positive. Bruce-ladder PCR targeting eryC, ABC, and Polysaccharide deacetylase genes was able to identify B. canis in nine out of 17 buffy coat samples. AMOS-PCR identified the eight undetermined Brucella species by Bruce-ladder PCR as Brucella abortus (n = 4) and Brucella melitensis (n = 4). To exclude the presence of Brucella suis, a one-step species-specific BcSS-PCR was performed and specifically amplified all B. canis DNA (n = 9) the same as did the Bruce-ladder PCR. Conclusion: To the best of our knowledge, this is the first report of B. canis detection in dogs in Egypt. Molecular identification of B. abortus and B. melitensis in the Egyptian canines highlights the role of stray dogs in brucellosis remerging in Brucellosis-free dairy farms. Brucella canis infection can be diagnosed specifically with the one-step BcSS-PCR. The obtained results set-an-alarm to the veterinary authorities to launch plans to control this disease in dogs.

Brucellar epididymo-orchitis: Symptoms, diagnosis, treatment and follow-up of 50 patients in Iran.

Introduction: Epididymo-orchitis (EO) is a disease of both the epididymis and ipsilateral testis. Brucellar epididymo-orchitis (BEO) is an uncommon localized infection of the testis and epididymis which occurs in about 2-14 % of all patients with brucellosis as a result of urine Brucella removal or due to blood-borne septic metastasis. Methods: Between January 2018 and June 2021, 50 patients with fever, chills, swelling, and pain of the testicle (testicles) were referred to our center. Two approaches were used for the treatment of brucellarepididymo-orchitis among these individuals. Intravenous Gentamicin and Doxycycline were used in seven cases, while Rifampicin was added to this combination for the remaining 43 patients. Intravenous Gentamicin was administered for 7 days and the other drugs were used for 45 days. All patients were followed up for six months by monitoring the symptoms and signs of the disease. Results: None of the patients had been diagnosed with brucellosis before referral to our clinic. 43 patients were successfully treated by. Intravenous Gentamicin, Doxycycline and Rifampicin, whereas seven patients were fully treated using. Intravenous Gentamicin and Doxycycline. The two therapeutic groups were hospitalized for 7.56 ± 3.45 (3-23) and 10.14 ± 1.77 (8-13) days, respectively. Treatment failure, drug side effects, and disease complications were not observed in any of the cases over a 6-month follow-up period. Conclusions: Physicians should be alert regarding Brucellarepididymo-orchitis (BEO) within the differential diagnosis of nonspecific epididymo-orchitis, especially in regions where the disease is endemic. Delay in diagnosis or inappropriate management of BEO may result in complications.

Water-soluble protein from walleye pollock (Gadus chalcogrammus) suppresses lipopolysaccharide-induced inflammation by attenuating TLR4-MyD88 expression in macrophages.

Water-soluble protein (WSP) from fish meat is abundant in the waste effluent generated via the surimi manufacturing process. This study investigated the anti-inflammatory effects and mechanisms of fish WSP using primary macrophages (MΦ) and animal ingestion. MΦ were treated with digested-WSP (d-WSP, 500 µg/mL) with or without lipopolysaccharide (LPS) stimulation. For the ingestion study, male ICR mice (5 weeks old) were fed 4% WSP for 14 days following LPS administration (4 mg/kg body weight). d-WSP decreased the expression of Tlr4, an LPS receptor. Additionally, d-WSP significantly suppressed the secretion of inflammatory cytokines, phagocytic ability, and Myd88 and Il1b expressions of LPS-stimulated macrophages. Furthermore, the ingestion of 4% WSP attenuated not only LPS-induced IL-1ß secretion in the blood but also Myd88 and Il1b expressions in the liver. Thus, fish WSP decreases the expressions of the genes involved in the TLR4-MyD88 pathway in MΦ and the liver, thereby suppressing inflammation.

Ferroptosis model system by the re-expression of BACH1.

Ferroptosis is a regulated cell death induced by iron-dependent lipid peroxidation. The heme-responsive transcription factor BTB and CNC homology 1 (BACH1) promotes ferroptosis by repressing the transcription of genes involved in glutathione (GSH) synthesis and intracellular labile iron metabolism, which are key regulatory pathways in ferroptosis. We found that BACH1 re-expression in Bach1-/- immortalized mouse embryonic fibroblasts (iMEFs) can induce ferroptosis upon 2-mercaptoethanol removal, without any ferroptosis inducers. In these iMEFs, GSH synthesis was reduced, and intracellular labile iron levels were increased upon BACH1 re-expression. We used this system to investigate whether the major ferroptosis regulators glutathione peroxidase 4 (Gpx4) and apoptosis-inducing factor mitochondria-associated 2 (Aifm2), the gene for ferroptosis suppressor protein 1, are target genes of BACH1. Neither Gpx4 nor Aifm2 was regulated by BACH1 in the iMEFs. However, we found that BACH1 represses AIFM2 transcription in human pancreatic cancer cells. These results suggest that the ferroptosis regulators targeted by BACH1 may vary across different cell types and animal species. Furthermore, we confirmed that the ferroptosis induced by BACH1 re-expression exhibited a propagating effect. BACH1 re-expression represents a new strategy for inducing ferroptosis after GPX4 or system Xc- suppression and is expected to contribute to future ferroptosis research.

The Impact of Sodium Dodecyl Sulfate and 2-Mercaptoethanol on Antibody and Antigen Binding.

OBJECTIVE: To evaluate the effect of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) on antigen-antibody binding when incubated at 100°C, which is the pretreatment temperature required for western blots. METHODS: Serum that tested positive for hepatitis B surface antigen (HBsAg) plus loading buffer were mixed at a ratio of 4:1 and incubated in a water bath. We then detected HBsAg using double immunodiffusion and ELISA. RESULTS: The HBsAg titer was 1:512 in the control group when incubated at 37°C. Incubation with SDS at 100°C reduced the antigen titer to 1:32. The inhibitory effect on HBsAg titer reached 96.9% after incubation at 100°C with SDS and 2-ME. CONCLUSION: We detected strong inhibition of antigens in western blots via SDS and 2-ME. It is likely that false-negative results will be obtained from western blots of antigens with weak resistance to these reagents.

2-Mercaptoethanol promotes porcine oocyte maturation in vitro by maintaining autophagy homeostasis.

2-Mercaptoethanol (2-ME) is often used as an antioxidant to optimize culture systems for in vitro oocyte maturation in livestock. However, the relationship between 2-ME and autophagy has not yet been elucidated. In this study, we hypothesized that 2-ME can promote porcine oocyte maturation in vitro by maintaining autophagy homeostasis. To test this hypothesis, we explored the effects of 2-ME on the maturation of porcine oocytes exposed to an autophagy activator (rapamycin) or an autophagy inhibitor (3-methyladenine, i.e., 3-MA) in vitro. Rapamycin-induced autophagy over-activation significantly increased autophagy- and apoptosis-related gene expression, oxidative stress, apoptosis rates, abnormal mitochondrial redistribution, and significantly decreased oocyte first polar body extrusion (PBE) rates, spindle/chromosome integrity and developmental competence. 3-MA-mediated autophagy inhibition exerted similar effects on all these parameters except the expression of genes that promote autophagy and inhibit apoptosis. Importantly, 2-ME supplementation significantly attenuated the detrimental effects of rapamycin and 3-MA. Interestingly, we observed that 44 h of coincubation with rapamycin/3-MA and 2-ME restored autophagy homeostasis in vitro. In conclusion, our study confirmed that 2-ME promotes porcine oocyte maturation and embryo development in vitro by maintaining autophagy homeostasis and lays a foundation for further research on the underlying mechanism.

2-Mercaptoethanol protects against DNA double-strand breaks after kidney ischemia and reperfusion injury through GPX4 upregulation.

BACKGROUND: Kidney ischemia reperfusion injury (IRI) is characterized by tubular cell death. DNA double-strand breaks is one of the major sources of tubular cell death induced by IRI. 2-Mercaptoethanol (2-ME) is protective against DNA double-strand breaks derived from calf thymus and bovine embryo. Here, we sought to determine whether treatment with 2-ME attenuated DNA double-strand breaks, resulting in reduced kidney dysfunction and structural damage in IRI. METHODS: Kidney IRI or sham-operation in mice was carried out. The mice were treated with 2-ME, Ras-selective lethal 3, or vehicle. Kidney function, tubular injury, DNA damage, antioxidant enzyme expression, and DNA damage response (DDR) kinases activation were assessed. RESULTS: Treatment with 2-ME significantly attenuated kidney dysfunction, tubular injury, and DNA double-strand breaks after IRI. Among DDR kinases, IRI induced phosphorylation of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR), but IRI reduced phosphorylation of other DDR kinases including ataxia telangiectasia and Rad3 related, checkpoint kinase 1 (Chk1), Chk2, and Chinese hamster cells 1 (XRCC1). Treatment with 2-ME enhanced phosphorylation of ATM and ATM-mediated effector kinases in IRI-subjected kidneys, suggesting that 2-ME activates ATM-mediated DDR signaling pathway. Furthermore, 2-ME dramatically upregulated glutathione peroxidase 4 (GPX4) in IRI-subjected kidneys. Inhibition of GPX4 augmented adverse IRI consequences including kidney dysfunction, tubular injury, DNA double-strand breaks, and inactivation of ATM-mediated DDR signaling pathway after IRI in 2-ME-treated kidneys. CONCLUSIONS: We have demonstrated that exogenous 2-ME protects against DNA double-strand breaks after kidney IRI through GPX4 upregulation and ATM activation.

Micellar sensitized Resonance Rayleigh Scattering and spectrofluorometric methods based on isoindole formation for determination of Eflornithine in cream and biological samples.

α-Difluoromethylornithine or Eflornithine is an FDA-approved drug used for the treatment of Sleeping Sickness (as vials dosage form) and also used for diminishing the unwanted excess facial hair in the hirsutism (as creams dosage form). The proposed work is based on the condensation interaction between the amino moiety of Eflornithine and O-phthalaldehyde/2-mercaptoethanol to form a highly fluorescent isoindole derivative. The fluorescence and the Resonance Rayleigh Scattering (RRS) intensities of the reaction product were greatly augmented upon the addition of hexadecyl-trimethyl ammonium bromide by 153% and 250%, respectively. After optimization of the reaction conditions, the formed isoindole derivative was measured fluorometrically at λemission= 429 nm after λexcitation= 337 nm. Moreover, the significant augmentation in the RRS intensity of the formed product was measured at λmax= 422 nm. In regards to accuracy, sensitivity, robustness and precision, the proposed methods were validated according to ICH guidelines. Furthermore, the proposed methods were successfully applied for the assay of Eflornithine in various commercial brands of the pharmaceutical cream samples with good recovery. In addition to the current fluorometric method was confirmed to be effective in the assaying of Eflornithine in spiked plasma and urine specimens with good recovery.

2-Mercaptoethanol (2-ME)-based IATs or Polybrene method mitigates the interference of daratumumab on blood compatibility tests.

OBJECTIVES: Treating red blood cells (RBCs) with dithiothreitol (DTT) is a wildly-recommended to overcome the interference of the daratumumab (DARA) with blood compatibility testing. Nevertheless, DTT can be hard to obtain in the clinical laboratory, while its use in routine practice may be time-consuming. In the following study, we explored the feasibility of using a commercial 2-mercaptoethanol (2-ME) working solution or the time-saving Polybrene method to mitigate DARA interference. METHODS: Antibody screening and cross-matching were performed using 2-ME or DTT-based indirect antiglobulin tests (IATs) and Polybrene method (with human IgG anti-E same IATs titer as DARA as positive control) on 37 samples. Most clinically important blood group antigens on RBCs were detected after treatment with 2-ME or DTT. RESULTS: Treating RBCs with 2-ME eliminates the DARA interference with the antibody screening or cross-matching; yet, K antigen is denatured during treatment. DARA does not interfere with antibody screening and cross-matching via Polybrene method, while 2+ agglutinations of anti-E antibody with the same titer (IATs method) as DARA could be observed in the positive controls via this method. CONCLUSION: 2-ME-based IATs or Polybrene method could replace DTT-based IATs to mitigate DARA interference.

Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13.

A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 µM and 7.1 µmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab.

BACKGROUND: Monoclonal antibodies (mAbs) and their derivatives have become one of the most important classes of therapeutic drugs. Their multiple applications increased the interest for understanding their complex structure. In vivo, animal cells are able to fold mAbs correctly (Song et al, J Biosci Bioeng 110:135-40, 2010), whereas previous in vitro approaches were scarce and mostly unsuccessful. RESULTS: In this work, we compared in vitro assembly characteristics of trastuzumab, produced either by A) physical separation and refolding of its sub-units or B) direct joining of individually produced heavy and light chains. Native and denatured structures of trastuzumab were determined by SEC-HPLC, HIC-HPLC and SDS-PAGE. CONCLUSIONS: Our results demonstrate the requirement of correctly folded HC, forming disulfide-bonded dimers, in order to form a fully functional mAb. Otherwise, the unfolded HC tend to precipitate. We were able to assemble trastuzumab in this fashion by only mixing them to LC in pH-buffered conditions, while monomeric HC structure was too unstable to render a functional mAb. This approach has been used in the generation of homogeneous ADC, with results pending to be published.