Silencing of tropomodulin 1 inhibits acute myeloid leukemia cell proliferation and tumor growth by elevating karyopherin alpha 2-mediated autophagy.

Evidence shows that tropomodulin 1 (TMOD1) is a powerful diagnostic marker in the progression of several cancer types. However, the regulatory mechanism of TMOD1 in tumor progression is still unclear. Here, we showed that TMOD1 was highly expressed in acute myeloid leukemia (AML) specimens, and TMOD1-silencing inhibited cell proliferation by inducing autophagy in AML THP-1 and MOLM-13 cells. Mechanistically, the C-terminal region of TMOD1 directly bound to KPNA2, and TMOD1-overexpression promoted KPNA2 ubiquitylation and reduced KPNA2 levels. In contrast, TMOD1-silencing increased KPNA2 levels and facilitated the nuclear transfer of KPNA2, then subsequently induced autophagy and inhibited cell proliferation by increasing the nucleocytoplasmic transport of p53 and AMPK activation. KPNA2/p53 inhibitors attenuated autophagy induced by silencing TMOD1 in AML cells. Silencing TMOD1 also inhibited tumor growth by elevating KPNA2-mediated autophagy in nude mice bearing MOLM-13 xenografts. Collectively, our data demonstrated that TMOD1 could be a novel therapeutic target for AML treatment.

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.

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.

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.

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.

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.

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.

Insights into the interaction mechanism of acid phosphatase from Lateolabrax japonicus livers and rosmarinic acid using multispectroscopy and molecular docking.

Acid phosphatase (ACP) is a key enzyme that hydrolyzes inosinic acid. The mechanisms underlying the interaction between rosmarinic acid (RA) and ACP and the inhibition of the enzyme were investigated using inhibition kinetics, UV-visible and fluorescence spectroscopy, circular dichroism, and molecular docking. The results showed that RA was a reversible inhibitor of ACP and that the inhibition mechanism was uncompetitive. The ACP fluorescence was quenched by RA, and the quenching mode was static. The interaction of ACP with RA was driven by H bonds and van der Waals forces. The addition of RA increased the α-helix content and decreased the ß-sheet, ß-turn, and random coil contents in ACP, thereby altering the secondary structure of the enzyme. This study enriched our understanding of inhibitory and interaction mechanisms involving ACP and RA.

Effects of tetrasodium pyrophosphate coupled with soy protein isolate on the emulsion gel properties of oxidative myofibrillar protein.

The effects of protein oxidation on the emulsion gel properties of myofibrillar protein (MP) in the presence of tetrasodium pyrophosphate (TSPP) and soybean protein isolate (SPI) were investigated from the perspective of interfacial protein interactions. The results showed that the emulsifying activity and emulsion stability of MP increased by 35.2 %-181.6 % with elevated H2O2 concentrations (1-20 mM), while the gel strength and water holding capacity of MP emulsions first increased to a maximum at 5 mM H2O2 and then decreased. TSPP and SPI further reinforced the effects caused by oxidation. The emulsifying properties of MP and its emulsion gel properties were closely related to surface hydrophobicity/hydrogen bonds/hydrophobic interactions and disulfide bonds among interfacial proteins, respectively. However, these correlations became difficult to define when TSPP and SPI were introduced. The study provides a theoretical basis for the strategy development to reduce protein oxidation damage on meat product quality.

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.

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.

High-pressure homogenization: A potential technique for transforming insoluble pea protein isolates into soluble aggregates.

High-pressure homogenization (HPH) is a technique that impacts the aggregation of globular proteins. In this study, the effect of HPH (at a pressure of 30/50 MPa for three cycles) was investigated on the aggregation states and functional properties of insoluble commercial pea protein isolates (CPPI). Results showed that HPH significantly improved the solubility, foaming and emulsifying capacity of CPPI. Samples treated at 50 MPa demonstrated better foaming and emulsifying capacity than that at 30 MPa. Surface hydrophobicity, intrinsic fluorescence, SDS-PAGE and FTIR analysis revealed that insoluble precipitates/aggregates (most legumins included) of CPPI were broken down and converted into soluble aggregates. Low-pressure HPH (30 MPa) can break non-covalent bonds (hydrophobic interactions), whereas higher pressure (50 MPa) can further break covalent bonds (SS). The study sheds light on the mechanism of disruption of insoluble CPPI under HPH and proposes a method to enhance their techno-functional properties for application in food formulations.

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.

Profiling of naturally occurring folates in a diverse soybean germplasm by HPLC-MS/MS.

Soybean is a rich source of folates. We optimised the extraction and detection of folates from soybean seeds by HPLC-MS/MS and analysed the folate content and composition of 1074 accessions. Total folate content ranged from 64.51 to 691.24 µg/100 g fresh weight, with 10-fold variation, and 60 elite accessions with over 400 µg/100 g of total folate were identified. The most abundant component was 5-CHO-H4folate, which accounted for an average of 60% of total folate content. Seed-coat colour, seed weight, ecoregion, and accession type significantly affected soybean folate content. Furthermore, 5-CH3-H4folate correlated positively with seed protein (r = 0.24***) and negatively with oil (r = -0.26***). The geographical distribution of folate according to accession origin revealed that accessions from Northeast China contain higher amounts of total folate and 5-CHO-H4folate. This study provides comprehensive and novel insights into the folate profile of soybean, which will benefit soybean breeding for folate enhancement.

Tetrasodium pyrophosphate ameliorates oxidative damage to the TGase-catalyzed gelation of actomyosins.

The present study attempted to investigate the interactive roles of protein oxidation (0-20 mM H2O2) and tetrasodium pyrophosphate (TSPP) on the crosslinking efficiency of actomyosin mediated by transglutaminase (TGase). Oxidation at 0-20 mM H2O2 was not conducive to TGase-mediated crosslinking as indicated by the relative reduction of free amine consumption from 35.3% to 11.7%, and caused the principle crosslinking sites to progressively convert from myosin subfragment-1 (S1) to subfragment-1 (S2) as evidenced by electrophoresis. However, the binding of TSPP to myosin alleviated oxidation suppression to TGase-catalyzed crosslinking in varying degrees and retarded the migration of crosslinking site from S1 to S2. Moreover, oxidation (especially 20 mM H2O2) decreased the final (90 °C) elasticity index (EI) and water holding capacity of TGase-treated actomyosin gel, while TSPP intensified those of TGase-catalyzed actomyosin gel, indicating that TSPP had a positive effect on ameliorating the oxidative stress to TGase-catalyzed gelation of actomyosin.

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.

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.

Umbilical artery tissue contains p75 neurotrophin receptor-positive pericyte-like cells that possess neurosphere formation capacity and neurogenic differentiation potential.

INTRODUCTION: The p75 neurotrophin receptor (p75NTR) is known as an efficient marker for the prospective isolation of mesenchymal stem cells (MSCs) and neural crest-derived stem cells (NCSCs). To date, there is quite limited information concerning p75NTR-expressing cells in umbilical cord (UC), although UC is known as a rich source of MSCs. We show for the first time the localization, phenotype, and functional properties of p75NTR+ cells in UC. METHODS: Human UC tissue sections were subjected to immunohistochemistry for MSC markers including p75NTR. Enzymatically isolated umbilical artery (UA) cells containing p75NTR+ cells were assessed for immunophenotype, clonogenic capacity, and differentiation potential. To identify the presence of neural crest-derived cells in the UA, P0-Cre/Floxed-EGFP reporter mouse embryos were used, and immunohistochemical analysis of UC tissue was performed. RESULTS: Immunohistochemical analysis revealed that p75NTR+ cells were specifically localized to the subendothelial area of the UA and umbilical vein. The p75NTR+ cells co-expressed PDGFRß, CD90, CD146, and NG2, phenotypic markers of MSCs and pericytes. Isolated UA cells possessed the potential to form neurospheres that further differentiated into neuronal and glial cell lineages. Genetic lineage tracing analysis showed that EGFP+ neural crest-derived cells were detected in the subendothelial area of UA with p75NTR immunoreactivity. CONCLUSIONS: These results show that UA tissue harbors p75NTR+ pericyte-like cells in the subendothelial area that have the capacity to form neurospheres and the potential for neurogenic differentiation. The lineage tracing data suggests the p75NTR+ cells are putatively derived from the neural crest.