Serum dithiothreitol-oxidizing capacity (DOC) is a promising biomarker for excluding significant liver fibrosis: a proof-of-concept study.

BACKGROUND: APRI and FIB-4 scores are used to exclude clinically significant fibrosis (defined as stage ≥ F2) in patients with chronic viral hepatitis. However, the cut-offs for these scores (generated by Youden indices) vary between different patient cohorts. This study aimed to evaluate whether serum dithiothreitol-oxidizing capacity (DOC), i.e., a surrogate test of quiescin sulfhydryl oxidase-1, which is a matrix remodeling enzyme, could be used to non-invasively identify significant fibrosis in patients with various chronic liver diseases (CLDs). METHODS: Diagnostic performance of DOC was compared with APRI and FIB-4 for identifying significant fibrosis. ROC curve analyses were undertaken in: a) two chronic hepatitis B (CHB) cohorts, independently established from hospitals in Wenzhou (n = 208) and Hefei (n = 120); b) a MASLD cohort from Wenzhou hospital (n = 122); and c) a cohort with multiple CLD etiologies (except CHB and MASLD; n = 102), which was identified from patients in both hospitals. Cut-offs were calculated using the Youden index. All CLD patients (n = 552) were then stratified by age for ROC curve analyses and cut-off calculations. RESULTS: Stratified by CLD etiology or age, ROC curve analyses consistently showed that the DOC test was superior to APRI and FIB-4 for discriminating between clinically significant fibrosis and no fibrosis, when APRI and FIB-4 showed poor/modest diagnostic performance (P < 0.05, P < 0.01 and P < 0.001 in 3, 1 and 3 cohort comparisons, respectively). Conversely, the DOC test was equivalent to APRI and FIB-4 when all tests showed moderate/adequate diagnostic performances (P > 0.05 in 11 cohort comparisons). DOC had a significant advantage over APRI or FIB-4 scores for establishing a uniform cut-off independently of age and CLD etiology (coefficients of variation of DOC, APRI and FIB-4 cut-offs were 1.7%, 22.9% and 47.6% in cohorts stratified by CLD etiology, 2.0%, 26.7% and 29.5% in cohorts stratified by age, respectively). The uniform cut-off was 2.13, yielded from all patients examined. Surprisingly, the uniform cut-off was the same as the DOC upper limit of normal with a specificity of 99%, estimated from 275 healthy control individuals. Hence, the uniform cut-off should possess a high negative predictive value for excluding significant fibrosis in primary care settings. A high DOC cut-off with 97.5% specificity could be used for detecting significant fibrosis (≥ F2) with an acceptable positive predictive value (87.1%). CONCLUSIONS: This proof-of-concept study suggests that the DOC test may efficiently rule out and rule in significant liver fibrosis, thereby reducing the numbers of unnecessary liver biopsies. Moreover, the DOC test may be helpful for clinicians to exclude significant liver fibrosis in the general population.

Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

ER stress-induced transcriptional response reveals tolerance genes in yeast.

Saccharomyces cerevisiae is important for protein secretion studies, yet the complexities of protein synthesis and secretion under endoplasmic reticulum (ER) stress conditions remain not fully understood. ER stress, triggered by alterations in the ER protein folding environment, poses substantial challenges to cells, especially during heterologous protein production. In this study, we used RNA-seq to analyze the transcriptional responses of yeast strains to ER stress induced by reagents such as tunicamycin (Tm) or dithiothreitol (DTT). Our gene expression analysis revealed several crucial genes, such as HMO1 and BIO5, that are involved in ER-stress tolerance. Through metabolic engineering, the best engineered strain R23 with HMO1 overexpression and BIO5 deletion, showed enhanced ER stress tolerance and improved protein folding efficiency, leading to a 2.14-fold increase in α-amylase production under Tm treatment and a 2.04-fold increase in cell density under DTT treatment. Our findings contribute to the understanding of cellular responses to ER stress and provide a basis for further investigations into the mechanisms of ER stress at the cellular level.

Kinetics of ascorbate and dithiothreitol oxidation by soluble copper, iron, and manganese, and 1,4-naphthoquinone: Influence of the species concentration and the type of fluid.

An alternative metric to account for particulate matter (PM) composition-based toxicity is the ability of PM-species to generate reactive oxygen species (ROS) and deplete antioxidants, the so-called oxidative potential (OP). Acellular OP assays are the most used worldwide, mainly those based on ascorbic acid (AA) and dithiothreitol (DTT) depletion; OP values are calculated from AA/DTT concentration over time kinetic curves. Since a great variability in OP-DTT and OP-AA values can be found in the literature, the understanding of those factors affecting the kinetic rate of AA and DTT oxidation in the presence of PM-bound species will improve the interpretation of OP values. In this work, a kinetic study of the oxidation rate of AA and DTT driven by species usually found in PM (transition metals and naphthoquinone (NQ)) was carried out. In particular, the influence of the concentration of Cu(II), Fe(II), Fe(III), Mn(II), Mn(III), and 1,4-NQ, and the type of fluid used in the assay (phosphate buffer (PB), phosphate buffer saline (PBS) and artificial lysosomal fluid (ALF)) is analysed and discussed. The reaction orders with respect to the AA/DTT and the active compound, and the kinetic rate constants were also determined. The results show great variability in OP values among the studied species depending on the fluid used; the OP values were mostly higher in PB0.05 M, followed by PBS1x and ALF. Moreover, different species concentration-responses for OP-DTT/OP-AA were obtained. These differences were explained by the different reaction orders and kinetic rate constants obtained for each active compound in each fluid.

Redox-dependent Cd2+ inhibition of BK-type Ca2+-activated K+ channels.

Large-conductance Ca2+-activated K+ channels (BK channels) are formed by Slo1 subunits as a homotetramer. Besides Ca2+, other divalent cations, such as Cd2+, also activate BK channels when applied intracellularly by shifting the conductance-voltage relation to more negative voltages. However, we found that if the inside-out patch containing BK channels was treated with solution containing reducing agents such as dithiothreitol (DTT), then subsequent Cd2+ application completely inhibited BK currents. The DTT-dependent Cd2+ inhibition could be reversed by treating the patch with solutions containing H2O2, suggesting that a redox reaction regulates the Cd2+ inhibition of BK channels. Similar DTT-dependent Cd2+ inhibition was also observed in a mutant BK channel, Core-MT, in which the cytosolic domain of the channel is deleted, and in the proton-activated Slo3 channels but not observed in the voltage-gated Shaker K+ channels. A possible mechanism for the DTT-dependent Cd2+ inhibition is that DTT treatment breaks one or more disulfide bonds between cysteine pairs in the BK channel protein and the freed thiol groups coordinate with Cd2+ to form an ion bridge that blocks the channel or locks the channel at the closed state. However, surprisingly, none of the mutations of all cysteine residues in Slo1 affect the DTT-dependent Cd2+ inhibition. These results are puzzling, with an apparent contradiction: on one hand, a redox reaction seems to regulate Cd2+ inhibition of the channel, but on the other hand, no cysteine residue in the Slo1 subunit seems to be involved in such inhibition.

A Facile and Green Approach for the Preparation of Amine-Functionalized Poly(ethylene glycol) by Reducing Poly(ethylene glycol) Azide with Dithiothreitol.

A facile and green approach for the preparation of PEGn-NH2s from PEGn-N3s in water with DTT as the reduction reagent has been developed, avoiding the introduction of metal ions and difficulties in purification compared to the traditional synthesis process of PEGn-NH2s. A series of high-purity linear and multiarm PEGn-NH2s with different molecular weights were synthesized, demonstrating the versatility of this method. Additionally, HS-PEG45-NH2 with high fidelity of thiol and amine was easily prepared through the one-step two functional group conversion of N3-PEG45-S-S-PEG45-N3, and the PEG-based NH2-PEG@AuNPs were also prepared. This technology will promote the application of PEGn-NH2s in the fields of medicine and biomaterials.

Dithiothreitol reduces oxidative stress and necrosis caused by ultraviolet A radiation in L929 fibroblasts.

Ultraviolet A (UVA) radiation, present in sunlight, can induce cell redox imbalance leading to cellular damage and even cell death, compromising skin health. Here, we evaluated the in vitro antioxidant and photochemoprotective effect of dithiothreitol (DTT). DTT neutralized the free radicals 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+), 2,2-diphenyl-1-picrylhydrazyl (DPPH·), and superoxide anion (O2·-) in in vitro assays, as well as the ferric ion (Fe3+) in the ferric reducing antioxidant power (FRAP) assay. We also evaluated the effect of DTT pre-treatment in L929 dermal fibroblasts and DTT (50 and 100 µM) led to greater cell viability following UVA-irradiation compared to cells that were untreated. Furthermore, the pre-treatment of cells with DTT prevented the increase of intracellular reactive oxygen species (ROS) production, including hydrogen peroxide (H2O2), lipid peroxidation, and DNA condensation, as well as the decrease in mitochondrial membrane potential (Δψm), that occurred following irradiation in untreated cells. The endogenous antioxidant system of cells was also improved in irradiated cells that were DTT pre-treated compared to the untreated cells, as the activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes remained as high as non-irradiated cells, while the activity levels were depleted in the untreated irradiated cells. Furthermore, DTT reduced necrosis in UVA-irradiated fibroblasts. Together, these results showed that DTT may have promising use in the prevention of skin photoaging and photodamage induced by UVA, as it provided photochemoprotection against the harmful effects of this radiation, reducing oxidative stress and cell death, due mainly to its antioxidant capacity.

Effects of long-range transport on carboxylic acids, chlorinated VOCs, and oxidative potential in air pollution events.

In the context of air quality research, the collection and analysis of fine particulate matter (PM2.5, with a diameter less than 2.5 µm) and volatile organic compound (VOCs) play a pivotal role in understanding and addressing environmental issues across the Korean Peninsula. PM2.5 and VOCs were collected over 4-hr intervals from October 17 to November 26, 2021 during the 2021 Satellite Integrated Joint Monitoring of Air Quality campaign at Olympic Park in the Republic of Korea to understand the factors controlling air quality over the Seoul Metropolitan Area. Source apportionment was performed using the positive matrix factorization (PMF) model incorporating PM2.5 and VOCs. The factor identified by chlorinated VOCs as a major component was presumed to be due to transboundary influx and was referred to as the long-range transport factor. The long-range transport factor of PM2.5 was composed of NO3-, SO42-, NH4+, and di-carboxylic acids. Back trajectory analysis showed that the airflows originated from China and passed through the west coast of Korea to the Korean Peninsula. In the PMF results using PM2.5 and VOCs, long-range transport factors were identified in both analyses, and the high correlation observed between these factors confirms that they were transported from abroad. The dithiothreitol oxidation potential normalized to quinine showed the highest oxidation potential during the same period as the long-range transport factors increased. In conclusion, PM2.5 from external sources significantly contribute to elevated levels of dithiothreitol assay-oxidative potential (DTT-OP) in Korea. The toxic concentration, expressed as the mean ± standard deviation, was determined to be 0.29 ± 0.05 µM/m³, peaking at 0.39 µM/m³. This level is 1.8 times higher than that observed outside the event period. A notable increase in secondary pollutants was observed during these periods. These pollutants are known to enhance oxidative potential, thereby potentially impacting human health.

Size-distribution and driving factors of aerosol oxidative potential in rural kitchen microenvironments of northeastern India.

This study reports size-resolved dithiothreitol (DTT)-based oxidative potential (OP: total and water-soluble) in rural kitchens using liquefied petroleum gas (LPG), firewood (FW), and mixed biomass (MB) fuels in northeastern (NE) India. In comparison to LPG, volume-normalized total OP (OPtotal(v)DTT) was enhanced by a factor of ∼5 in biomass-using kitchens (74 ± 35 to 78 ± 42 nmol min-1 m-3); however, mass-normalized total OP (OPtotal(m)DTT) was similar between LPG and FW users and higher by a factor of 2 in MB-using kitchens. The water-insoluble OP (OPwi(v, m)DTT) fraction in OPtotal(v, m)DTT was greater than 50% across kitchens. Size distributions across kitchens and OPDTT categories ranged from unimodal to trimodal. OPws(v)DTT was driven by metals as well as organics across size fractions while OPwi(v)DTT was majorly constrained by metals with an increasing importance of organics in fine particles of biomass-using kitchens. Multiple linear regression analysis revealed that Cu and Ba explained 71% of the OPtotal(v)DTT variability in LPG-using kitchens, while water-soluble organic carbon (WSOC) and Ba were responsible for 44% variability in FW-using kitchens. Finally, the high internal dose of OPtotal(v)DTT (28-31 nmol min-1 m-3) in biomass-using kitchens established the severity of oxidative stress on the exposed population.

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Adoption of nanoparticle micro-infusion valve in venous port access for tumor patients and self-care analysis.

It aimed to discuss the adoption of nanoparticle micro-infusion valves in the totally implantable venous access port (TIVAP) of tumor patients and analyze the self-care of patients after surgery. A total of 189 tumor patients who received TIVAP chemotherapy were selected from the day chemotherapy outpatient department of the Second People's Hospital of Lianyungang City from June to October 2021. The questionnaire survey was conducted to collect and analyze the general information about the patients and the patient's self-care ability. All the surveys and analyses were performed using a general information questionnaire, self-care competence scale, self-management efficacy scale, social support scale, and self-rating anxiety scale. Moreover, the nanoparticle micro-infusion valve was prepared, and its characterization, in vitro drug release, and cytotoxicity were studied. The self-management ability of patients was concentrated in the middle and high levels. Marital status, education level, and economic status all had a great impact on the patient's self-care ability. The infusion of dithiothreitol (DTT) increased the drug release of the nanoparticle micro-infusion valve. When pH=5.0, the drug-loaded nanoparticle micro-infusion valve release amount was greater than when pH=7.4. When pH=5.0 and pH=7.4, the cell survival rates under the drug-loaded nanoparticle micro-infusion valve were both lower than that without drug loading. Nanoparticle micro-infusion valves can specifically identify tumor cells, and have good adoption prospects in TIVAP implantation in tumor patients. Gender, marital status, education level, etc. all affected the self-care ability of patients.

Staphylococcus aureus Detection in Milk Using a Thickness Shear Mode Acoustic Aptasensor with an Antifouling Probe Linker.

Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination of food. In this work, an aptasensor based on a thickness shear mode acoustic method (TSM) with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow's milk. The frequency variation and dissipation data demonstrated the correct immobilization of the components. The analysis of viscoelastic properties suggests that DNA aptamers bind to the surface in a non-dense manner, which favors the binding with bacteria. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the sensor's antifouling properties, which is based on 3-dithiothreitol propanoic acid (DTTCOOH) antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) quartz crystals, the sensitivity of the sensor's antifouling in milk improved by about 82-96%. The excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow's milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.

Measurement artifacts in the dithiothreitol (DTT) oxidative potential assay caused by interactions between aqueous metals and phosphate buffer.

Metals in particulate matter (PM) are hypothesized to have enhanced toxicity based on their ability to catalyze reactive oxygen species (ROS) formation. Acellular assays are used to measure the oxidative potential (OP) of PM and its individual components. Many OP assays, including the dithiothreitol (DTT) assay, use a phosphate buffer matrix to simulate biological conditions (pH 7.4 and 37 °C). Prior work from our group observed transition metal precipitation in the DTT assay, consistent with thermodynamic equilibria. In this study, we characterized the effects of metal precipitation on OP measured by the DTT assay. Metal precipitation was affected by aqueous metal concentrations, ionic strength, and phosphate concentrations in ambient PM sampled in Baltimore, MD and a standard PM sample (NIST SRM-1648a, Urban Particulate Matter). Critically, differences in metal precipitation induced differing OP responses of the DTT assay as a function of phosphate concentration in all PM samples analyzed. These results indicate that comparison of DTT assay results obtained at differing phosphate buffer concentrations is highly problematic. Further, these results have implications for other chemical and biological assays that use phosphate buffer for pH control and their use to infer PM toxicity.

Better resolving of anti-CD38 antibody interference with blood compatibility testing by using manual polybrene method compared with dithiothreitol-pretreatment indirect antiglobulin test.

BACKGROUND: It is advised to pretreat the reagent erythrocytes with Dithiothreitol (DTT) to denature the surface CD38 to prevent anti-CD38 monoclonal antibodies (MoAb) from interfering with the blood compatibility test. Anti-CD38 has little impact on the Polybrene test, but it is still unknown how sensitive it is to detect irregular antibodies and how effective it is when compared to the standard DTT-based method. METHODS: Twenty-one patients receiving daratumumab (N = 13) and isatuximab (N = 8) had their serum collected. Standard anti-sera (anti-c, D, E, Fyb , Jka , M, Mia ) with serial dilution were added to patients' serum. Antibody screening tests were performed simultaneously using the manual polybrene method (MP) and DTT-pretreated, automatic indirect antiglobulin test (DTT-IAT) to compare the detection sensitivity. These two methods' operating times and costs were also analyzed. RESULTS: Both MP and DTT-IAT can overcome the interference caused by anti-CD38 MoAb. However, MP is more sensitive in detecting anti-M and anti-Mia and is comparable to DTT-IAT in detecting other antibodies. In terms of cost and operating time, MP is also far superior to DTT-IAT. CONCLUSION: MP is a cost-effective alternative to DTT-IAT in resolving anti-CD38 interference and is especially suitable for populations with a high prevalence of anti-M and anti-Mia . However, both methods have a well-known drawback of low detection sensitivity for anti-K, and K-units should be provided to patients to prevent hemolytic transfusion reactions.

Inactivation of Human Aldehyde Oxidase by Small Sulfhydryl-Containing Reducing Agents.

Human aldehyde oxidase (hAOX1) is a molybdoflavoenzyme that belongs to the xanthine oxidase (XO) family. hAOX1 is involved in phase I drug metabolism, but its physiologic role is not fully understood to date, and preclinical studies consistently underestimated hAOX1 clearance. In the present work, we report an unexpected effect of the common sulfhydryl-containing reducing agents, e.g., dithiothreitol (DTT), on the activity of hAOX1 and mouse aldehyde oxidases. We demonstrate that this effect is due to the reactivity of the sulfido ligand bound at the molybdenum cofactor with the sulfhydryl groups. The sulfido ligand coordinated to the Mo atom in the XO family of enzymes plays a crucial role in the catalytic cycle and its removal results in the total inactivation of these enzymes. Because liver cytosols, S9 fractions, and hepatocytes are commonly used to screen the drug candidates for hAOX1, our study suggests that DTT treatment of these samples should be avoided, otherwise false negative results by an inactivated hAOX1 might be obtained. SIGNIFICANCE STATEMENT: This work characterizes the inactivation of human aldehyde oxidase (hAOX1) by sulfhydryl-containing agents and identifies the site of inactivation. The role of dithiothreitol in the inhibition of hAOX1 should be considered for the preparation of hAOX1-containing fractions for pharmacological studies on drug metabolism and drug clearance.

Direct inhibition of human and rat 11ß-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis.

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11ß-HSD2. C8-C14 PFAS at 100 µM significantly inhibited human 11ß-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 µM) > C11 (15.09 µM) > C12 (18.43 µM) > C9 (20.93 µM) > C13 (124 µM) > C14 (147.3 µM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11ß-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11ß-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11ß-HSD2 but not rat 11ß-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11ß-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11ß-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11ß-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11ß-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11ß-HSD2.

Long-term storage protocol of reagent red blood cells treated with 0.01M dithiothreitol (DTT) for pre-transfusion testing of patients receiving anti-CD38 therapy, daratumumab.

OBJECTIVE: Use red blood cell stabilizer to store the antibody screening and antibody identification reagent red blood cells (RBCs) treated with 0.01 mol/L DTT and investigate its value in the pre-transfusion examinations of patients treated with daratumumab. METHOD: Determined the optimal incubation time for the 0.01 mol/L DTT-treated RBCs method by evaluating the effect of treatment at different time points. Added ID-CellStab to store DTT-treated RBCs, determined the maximum shelf life of reagent RBCs by monitoring the hemolysis index, and assessed changes in the antigenicity of blood group antigens on the surface of RBCs during storage with antibody reagents. RESULT: A protocol for long-term storage of reagent red blood cells treated with the 0.01 mol/L DTT method was established. The optimal incubation time was 40-50 min. RBCs could be stored stably for 18 days after adding ID-CellStab. The protocol was able to eliminate pan-agglutination caused by daratumumab, with no significant changes in the antigens of most blood group systems, except for some attenuation of K antigen and Duffy blood group system antigens during the storage period. CONCLUSION: The storage protocol of reagent RBCs based on the 0.01 mol/L DTT method does not affect the detection of most blood group antibodies and retains a certain degree of detection ability for anti-K antibodies, allowing patients treated with daratumumab to quickly perform pre-transfusion examinations, making up for the shortcomings of currently commercial reagent RBCs.

Synthesis of 4-thio-d-glucopyranose and interconversion to 4-thio-d-glucofuranose.

Sulfur containing glycosides offer an exciting prospect for inclusion within noncanonical glycan sequences, particularly as enabling probes for chemical glycobiology and for carbohydrate-based therapeutic development. In this context, we required access to 4-thio-d-glucopyranose and sought its chemical synthesis. Unable to isolate this material in homogenous form, we observed instead a thermodynamic preference for interconversion of the pyranose to 4-thio-d-glucofuranose. Accordingly, we present an improved method to access both bis(4-thio-d-glucopyranoside)-4,4'-disulfide and 4-thio-d-glucofuranose from a single precursor, demonstrating that the latter compound can be accessed from the former using a dithiothreitol controlled reduction of the disulfide. The dithiothreitol-mediated interconversion between pyranose (monomer and disulfide) and furanose forms for this thiosugar is monitored by 1H NMR spectroscopy over a 24-h period. Access to these materials will support accessing sulfur-containing mimetics of glucose and derivatives therefrom, such as sugar nucleotides.

Dithiotreitol pre-treatment of synovial fluid samples improves microbiological counts in peri-prosthetic joint infection.

PURPOSE: Synovial fluid cultures of periprosthetic joint infections (PJI) may be limited by bacteria living in the fluids as biofilm-aggregates. The antibiofilm pre-treatment of synovial fluids with dithiotreitol (DTT) could improve bacterial counts and microbiological early stage diagnosis in patients with suspected PJI. METHODS: Synovial fluids collected from 57 subjects, affected by painful total hip or knee replacement, were divided into two aliquots, one pre-treated with DTT and one with normal saline. All samples were plated for microbial counts. Sensitivity of cultural examination and bacterial counts of pre-treated and control samples were then calculated and statistically compared. RESULTS: Dithiothreitol pre-treatment led to a higher number of positive samples, compared to controls (27 vs 19), leading to a statistically significant increase in the sensitivity of the microbiological count examination from 54.3 to 77.1% and in colony-forming units count from 1884 ± 2.129 CFU/mL with saline pre-treatment to 20.442 ± 19.270 with DTT pre-treatment (P = 0.02). CONCLUSIONS: To our knowledge, this is the first report showing the ability of a chemical antibiofilm pre-treatment to increase the sensitivity of microbiological examination in the synovial fluid of patients with peri-prosthetic joint infection. If confirmed by larger studies, this finding may have a significant impact on routine microbiological procedures applied to synovial fluids and brings further support to the key role of bacteria living in biofilm-formed aggregates in joint infections.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH.

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross ß sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.