Spurious High Platelet Count without PLT Flag(S) in a Patient with Severe Aplastic Anaemia.

BACKGROUND: Platelet (PLT) count is one of the most important parameters of automated hematology, as spurious PLT reports could affect medical judgement and bring significant risks. In most cases, spurious PLT will not be reported for review criteria, which will be triggered by abnormal PLT histograms and PLT flag(s). Here, we present a case of severe aplastic anemia after hematopoietic stem cell transplantation with spurious high platelet count with normal histogram and no PLT flag(s). METHODS: The electrical impedance channel (PLT-I) and the fluorescence channel (PLT-F) of Sysmex XN-series hematology analyzer was used to obtain PLT results. Then, the sample was retested by another hematology analyzer MINDRAY BC-7500 [NR] CRP, and incubation was performed to rule out cryoglobulin interference. Furthermore, a microscope was used to estimate the PLT count by the ratio of platelets to red blood cells and observe the morphology of cells. RESULTS: Both PLT-I and PLT-F test results were spuriously high, and microscopically assessed platelet counts were relatively reliable. The observed spiny cells and ghost cells caused by hemolysis may have contributed to the inaccuracy of instrumental counting in this case. CONCLUSIONS: For special hematologic patients, PLT-I with flags may not be sufficient for screening purposes and PLT-F is not always accurate. Multiple testing methods including manual microscopy are needed.

Platelet indices and angiogenesis markers in hypertensive disorders of pregnancy.

INTRODUCTION: Activated platelets exert a key role in the pathogenesis of preeclampsia (PE). There is evidence of distinctive patterns of platelet indices in PE in comparison to healthy pregnancies, therefore these indices can be potential tools for PE detection, risk stratification, and management. Considering the vascular aspects of its pathophysiology, PE is characterized by the increased levels of soluble FMS-like tyrosine kinase-1 (sFlt-1) an antiangiogenic factor, and reduced placental growth factor (PlGF), a proangiogenic factor. This study aimed to assess the platelet indices in hypertensive disorders of pregnancy (HDP) and its correlation with angiogenesis-related biomarkers. METHODS: The groups for the study were: control (n = 114); gestational hypertension; (n = 112), and PE (n = 42). The platelet indices included were platelet counts (PLT-I and PLT-F), mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), platelet large cell ratio (P-LCR), and immature platelet fraction (IPF# and IPF%). Serum levels of sFlt-1 and PlGF were assessed. RESULTS: PLT-I, PLT-F, and PCT% were lower in PE, while MPV, PDW, P-LCR, IPF%, and IPF# were increased. The parameter MPV presented the best performance for the discrimination of PE. There was a moderate positive correlation between sFlt-1 levels and MPV, PDW, and P-LCR. CONCLUSION: Platelet indices can be potentially applied as additional tools for the diagnosis and management of HDP. Activated platelets may act as an extra source of sFlt-1 in PE.

Performance evaluation of a novel platelet count parameter, hybrid platelet count, on the BC-780 automated hematology analyzer.

OBJECTIVES: Automated hematology analysis is expected to improve the performance of platelet counting. We evaluated the performance of a new platelet counting, hybrid (PLT-H) and also impedance (PLT-I) and optical (PLT-O) on the BC-780 automated hematology analyzer compared to the international reference method (IRM) in blood samples with thrombocytopenic and platelet interference. METHODS: The basic platelet count performance of the BC-780 automated hematology analyzer was evaluated according to the requirements of the Clinical Laboratory and Standards Institute (CLSI) Document H26-A2. Additionally, the thrombocytopenic (low PLT count) blood samples and the platelet interference blood samples including fragmented red blood cells (RBCs), microcytes or small RBCs, and giant platelets were determined with the BC-780 hematology analyzer compared to the IRM. RESULTS: Blank counting and the carry-over contamination rate of platelet count using the BC-780 both met the manufacturers' claim. For both 123 thrombocytopenic and 232 platelet interference blood samples (72 fragmented RBCs, 91 microcytes and 51 giant platelets), all three platelet counting methods exhibited high comparability with the IRM (the lowest correlation (r)=0.916). Interestingly, the comparability of PLT-H (r=0.928-0.986) with the IRM was better than that of PLT-I (r=0.916-0.979). CONCLUSIONS: The performance of PLT-H in the BC-780 met the manufacturer's specifications. PLT-H exhibits better reproducibility than did PLT-I, correlates well with the PLT-O for thrombocytopenic samples and demonstrates good anti-interference ability. PLT-H counting is therefore recommended as a zero-cost alternative platelet counting method for platelet interference samples in clinical settings.

Synthesis, lipoxygenase inhibition activity and molecular docking of oxamide derivative.

In this study, a range of oxamide ligands were synthesized by the reaction of amines with oxalyl chloride in basic medium. Spectroscopic and analytical techniques such as IR, 1H-NMR and ESI-MS techniques were used for characterization of the synthesized oxamides. The synthesized oxamides were screened for Lipoxygenase inhibition. Biological screening revealed that the oxamides possessed good lipoxygenase inhibition activities, whereas, the unsubstituted oxamide did not show any distinct lipoxygenase inhibition activity. Molecular docking studies of the oxamides were also carried out for lipoxygenase inhibition. The results obtained from molecular docking were well correlated with the empirical data.

Protective effect of lodoxamide on hepatic steatosis through GPR35.

Although GPR35 is an orphan G protein-coupled receptor, synthetic agonists and antagonists have been developed. Recently, cromolyn, a mast cell stabilizer, was reported as an agonist of GPR35 and was shown to exhibit antifibrotic effects through its actions on hepatocytes and stellate cells. In this study, the role of GPR35 in hepatic steatosis was investigated using an in vitro model of liver X receptor (LXR)-mediated hepatocellular steatosis and an in vivo model of high fat diet-induced liver steatosis. GPR35 was expressed in Hep3B human hepatoma cells and mouse primary hepatocytes. A specific LXR activator, T0901317, induced lipid accumulation in Hep3B cells. Lodoxamide, the most potent agonist of GPR35, inhibited lipid accumulation in a concentration-dependent manner. The protective effect of lodoxamide was inhibited by a specific GPR35 antagonist, CID2745687, and by siRNA-mediated knockdown of GPR35. The expression of SREBP-1c, a key transcription factor for lipid synthesis, was induced by T0901317 and the induction was inhibited by lodoxamide. Through the use of specific inhibitors of cellular signaling components, the lodoxamide-induced inhibition of lipid accumulation was found to be mediated through p38 MAPKs and JNK, but not through Gi/o proteins and ERKs. Furthermore, the protective effect of lodoxamide was confirmed in mouse primary hepatocytes. Lodoxamide suppressed high fat diet-induced fatty liver development, which suggested the protective function of GPR35 in liver steatosis. Therefore, the present data suggest that GPR35 may function to protect against fatty liver development.

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin.

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics.IMPORTANCE POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

Formation of Fluorinated Amido Esters through Unexpected C3-C4 Bond Fission in 4-Trifluoromethyl-3-oxo-ß-lactams.

4-Trifluoromethyl-3-oxo-ß-lactams were unexpectedly transformed into 2-[(2,2-difluorovinyl)amino]-2-oxoacetates as major products, accompanied by minor amounts of 2-oxo-2-[(2,2,2-trifluoroethyl)amino]acetates, upon treatment with alkyl halides and triethylamine in DMSO. This peculiar C3-C4 bond fission reactivity was investigated in-depth, from both an experimental and a computational point of view, in order to shed light on the underlying reaction mechanism.

Efficacy and safety of 0.1% lodoxamide for the long-term treatment of superior limbic keratoconjunctivitis.

PURPOSE: To report the therapeutic efficacy and safety of topical 0.1% lodoxamide in the long-term treatment of superior limbic keratoconjunctivitis. METHODS: Sixty-seven eyes of 34 patients with active SLK were studied. Therapeutic response was analyzed according to modified-Ohashi parameters. All eyes were treated with 0.1% lodoxamide twice daily, and those with moderate or severe inflammation received a short course (7-14 days) of 0.1% fluorometholone acetate at presentation and during a relapse. Patients were evaluated at regular intervals and followed up for ≥3 months on continuous therapy. Primary endpoints included inflammatory response; rates of inflammatory control and remission; relapses while on therapy or on remission; and therapeutic failure rate. RESULTS: The mean follow-up time on lodoxamide therapy was 15.3 months. The majority of eyes (82.0%) achieved control of inflammation in a mean time of 2.2 months. Of these, 42 (76.3%) eyes remained under control while on therapy for 13.8 months. There was a significant improvement of SLK-related signs by the third month on therapy (p < 0.05). A total of 24 (35.8%) eyes achieved remission. Relapses presented in 12 (18.0%) treated eyes and in 4 (16.6%) eyes on remission. Only 5 (7.4%) eyes failed to respond to therapy. In the majority of cases (95.3%), lodoxamide 0.1% was well tolerated and minor adverse effects not requiring stopping the medication were reported in only 4.7% of patients. CONCLUSIONS: Lodoxamide 0.1% is an efficacious therapeutic alternative for the treatment of active and chronic SLK. This medication has proved to be safe and well tolerated.

GPR35 mediates lodoxamide-induced migration inhibitory response but not CXCL17-induced migration stimulatory response in THP-1 cells; is GPR35 a receptor for CXCL17?

BACKGROUND AND PURPOSE: GPR35 has long been considered an orphan GPCR, because no endogenous ligand of GPR35 has been discovered. CXCL17 (a chemokine) has been reported to be an endogenous ligand of GPR35, and it has even been suggested that it be called CXCR8. However, at present there is no supporting evidence that CXCL17 does interact with GPR35. EXPERIMENTAL APPROACH: We applied two assay systems to explore the relationship between CXCL17 and GPR35. An AP-TGF-α shedding assay in GPR35 over-expressing HEK293 cells was used as a gain-of-function assay. GPR35 knock-down by siRNA transfection was performed in endogenously GPR35-expressing THP-1 cells. KEY RESULTS: In the AP-TGF-α shedding assay, lodoxamide, a well-known synthetic GPR35 agonist, was confirmed to be the most potent agonist among other reported agonists. However, neither human nor mouse CXCL17 had an effect on GPR35. Consistent with previous findings, G proteins Gαi/o and Gα12/13 were found to couple with GPR35. Furthermore, lodoxamide-induced activation of GPR35 was concentration-dependently inhibited by CID2745687 (a selective GPR35 antagonist). In endogenously GPR35-expressing THP-1 cells, lodoxamide concentration-dependently inhibited migration and this inhibitory effect was blocked by CID2745687 treatment or GPR35 siRNA transfection. However, even though CXCL17 stimulated the migration of THP-1 cells, which is consistent with a previous report, this stimulatory effect of CXCL17 was not blocked by CID2745687 or GPR35 siRNA. CONCLUSIONS AND IMPLICATIONS: The present findings suggest that GPR35 functions as a migration inhibitory receptor, but CXCL17-stimulated migration of THP-1 cells is not dependent on GPR35.

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments.

Genetic modification of large DNA fragments (gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity. In this study, we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction (PCR) targeting combined with Gibson assembly. In this strategy, the biosynthetic genes for peptidyl moieties (HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid (CPOAA) from the polyoxin biosynthetic gene cluster to generate a ~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette. The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic, polynik A, was obtained and verified. This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.

Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation.

Polyoxin, produced by Streptomcyes cacaoi var. asoensis and Streptomyces aureochromogenes, contains two non-proteinogenic amino acids, carbamoylpolyoxamic acid (CPOAA) and polyoximic acid. Although the CPOAA moiety is highly unusual, its biosynthetic logic has remained enigmatic for decades. Here, we address CPOAA biosynthesis by reconstitution of its pathway. We demonstrated that its biosynthesis is initiated by a versatile N-acetyltransferase, PolN, catalyzing L-glutamate (1) to N-acetyl glutamate (2). Remarkably, we verified that PolM, a previously annotated dehydrogenase, catalyzes an unprecedented tandem reduction of acyl-phosphate to aldehyde, and subsequently to alcohol. We also unveiled a distinctive acetylation cycle catalyzed by PolN to synthesize α-amino-δ-hydroxyvaleric acid (6). Finally, we report that PolL is capable of converting a rare sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid (7) to CPOAA. PolL represents an intriguing family of Fe(II)-dependent α-ketoglutarate dioxygenase with a cupin fold. These data illustrate several novel enzymatic reactions, and also set a foundation for rational pathway engineering for polyoxin production.

Fermentative production of high titer gluconic and xylonic acids from corn stover feedstock by Gluconobacter oxydans and techno-economic analysis.

High titer gluconic acid and xylonic acid were simultaneously fermented by Gluconobacter oxydans DSM 2003 using corn stover feedstock after dry dilute sulfuric acid pretreatment, biodetoxification and high solids content hydrolysis. Maximum sodium gluconate and xylonate were produced at the titer of 132.46g/L and 38.86g/L with the overall yield of 97.12% from glucose and 90.02% from xylose, respectively. The drawbacks of filamentous fungus Aspergillus niger including weak inhibitor tolerance, large pellet formation and no xylose utilization were solved by using the bacterium strain G. oxydans. The obtained sodium gluconate/xylonate product was highly competitive as cement retarder additive to the commercial product from corn feedstock. The techno-economic analysis (TEA) based on the Aspen Plus modeling was performed and the minimum sodium gluconate/xylonate product selling price (MGSP) was calculated as $0.404/kg. This study provided a practical and economic competitive process of lignocellulose utilization for production of value-added biobased chemicals.

Biodegradable Oxamide-Phenylene-Based Mesoporous Organosilica Nanoparticles with Unprecedented Drug Payloads for Delivery in Cells.

We describe biodegradable mesoporous hybrid nanoparticles (NPs) in the presence of proteins and their applications for drug delivery. We synthesized oxamide phenylene-based mesoporous organosilica nanoparticles (MON) in the absence of a silica source which had remarkably high organic content and high surface areas. Oxamide functions provided biodegradability in the presence of trypsin model proteins. MON displayed exceptionally high payloads of hydrophilic and hydrophobic drugs (up to 84 wt %), and a unique zero premature leakage without the pore capping, unlike mesoporous silica. MON were biocompatible and internalized into cancer cells for drug delivery.

Synthesis and structure of new tetracopper(II) complexes with N-benzoate-N'-[3-(diethylamino)propyl]oxamide as a bridging ligand: The influence of hydrophobicity on enhanced DNA/BSA-binding and anticancer activity.

Two new tetracopper(II) complexes bridged by N-benzoate-N'-[3-(diethylamino)propyl]oxamide (H3bdpox), and ended with 4,4'-dimethyl-2,2'-bipyridine (Me2bpy) or 2,2'-bipyridine (bpy), namely [Cu4(bdpox)2(Me2bpy)2](pic)2 (1) and [Cu4(bdpox)2(bpy)2](pic)2·2H2O (2) (where pic denotes the picrate anion) have been synthesized and characterized by X-ray single-crystal diffraction and other methods. In both complexes, four copper(II) ions are bridged alternately by the cis-oxamido and the carboxylato groups of two bdpox(3-) ligands to form a centrosymmetric cyclic tetranuclear cation, in which, the copper(II) ions at the endo- and exo-sites of cis-bdpox(3-) ligand have square-planar and square-pyramidal coordination geometries, respectively. The reactivity towards DNA/BSA suggests that these complexes can interact with HS-DNA through the intercalation mode and the binding affinity varies as 1>2 depending on the hydrophobicity, and effectively quench the fluorescence of protein BSA via a static mechanism. In vitro anticancer activities showed that the two complexes are active against the selected tumor cell lines, and the anticancer activities are consistent with their DNA-binding affinity.

Characterization and Inhibitor Screening of Plateau Zokor Lactate Dehydrogenase C4.

Lactate dehydrogenase C4 (LDH-C4) is considered to be a target protein for the development of contraceptives. In this work, the characterization of plateau zokor LDH-C4 and the screening of a series of N-substituted oxamic acids as inhibitors against zokor LDH-C4 were reported. The cDNA of zokor LDH-C gene was cloned and expressed in Escherichia coli, from which the protein was purified and further characterized. The protein was a tetramer (LDH-C4) and thermally stable up to 62 °C with a K m of 63.9 µM for pyruvate and with optimal pH values of 7.95 and 10.1 for the forward and backward reactions respectively. Virtual and in vitro screening against zokor LDH-C4 revealed eight N-substituted oxamic acids with IC50s ranging from 198 to 2513 µM, higher than that of oxamic acid (150 µM) and (ethylamino)(oxo)acetic acid (59 µM). The inhibition potencies of N-substituted oxamic acids tested are in the micromolar range, and the increase in the length of substituting chain seems not to increase inhibition potency.

Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase.

A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes did not activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors.

Synthesis and structure elucidation of new µ-oxamido-bridged dicopper(II) complex with in vitro anticancer activity: A combined study from experiment verification and docking calculation on DNA/protein-binding property.

A new oxamido-bridged dicopper(II) complex with formula of [Cu2(deap)(pic)2], where H2deap and pic represent N,N'-bis[3-(diethylamino)propyl]oxamide and picrate, respectively, was synthesized and characterized by elemental analyses, molar conductance measurements, IR and electronic spectral study, and single-crystal X-ray diffraction. The crystal structure analyses revealed that the two copper(II) atoms in the dicopper(II) complex are bridged by the trans-deap(2-) ligand with the distances of 5.2116(17)Å, and the coordination environment around the copper(II) atoms can be described as a square-planar geometry. Hydrogen bonding and π-π stacking interactions link the dicopper(II) complex into a three-dimensional infinite network. The DNA/protein-binding properties of the complex are investigated by molecular docking and experimental assays. The results indicate that the dicopper(II) complex can interact with HS-DNA in the mode of intercalation and effectively quench the intrinsic fluorescence of protein BSA by 1:1 binding with the most possible binding site in the proximity of Trp134. The in vitro anticancer activities suggest that the complex is active against the selected tumor cell lines, and IC50 values for SMMC-7721 and HepG2 are lower than cisplatin. The effects of the electron density distribution of the terminal ligand and the chelate ring arrangement around copper(II) ions bridged by symmetric N,N'-bis(substituted)oxamides on DNA/BSA-binding ability and in vitro anticancer activity are preliminarily discussed.

A minimally cytotoxic CD4 mimic as an HIV entry inhibitor.

Several CD4 mimics have been reported as HIV-1 entry inhibitors which can block the interaction between the viral envelope glycoprotein gp120 and the cell surface protein CD4. We previously found a lead compound 2 (YYA-021) with high anti-HIV activity and low cytotoxicity. Pharmacokinetic analysis however showed compound 2 to have wide tissue distribution and relatively high distribution volumes in rats and rhesus macaques. In the present study we searched for more hydrophilic CD4 mimics with a view to reducing tissue distribution. A new compound (5) with a 1,3-benzodioxolyl moiety was found to have relatively high anti-HIV activity and no significant cytotoxicity. Compound 5 is more hydrophilic than compound 2 and the pharmacokinetics of the intravenous administration of compound 5 in a rhesus macaque showed that compound 5 has lower tissue distribution than compound 2, suggesting that compound 5 possesses a better profile.

Synthesis of glycoaminooxy acid and N-oxyamide-linked glycolipids.

Aminooxyl sugar derivatives are versatile building blocks for the generation of various glycoconjugates with interesting bioactivities. We report herein a synthetic method for the preparation of orthogonally protected glycoaminooxy acid from methyl α-d-glycopyranoside in 7 steps. The key steps involve the selective protection, O-alkylation and Mitsunobu reaction. Fully deprotected N-oxyamide-linked novel glycolipids can be easily generated from the glycoaminooxy ester or from the 2-hydroxy free sugar in 5 or 6 steps.

Self-assembly of oxamidato bridged ester functionalised dirhenium metallastirrups: synthesis, characterisation and cytotoxicity studies.

A new set of ester functionalised Re(i)-based oxamidato bridged neutral dinuclear metallacycles were synthesised by self-assembly of four components from three building blocks in a facile one-pot reaction via an orthogonal bonding approach. Oxidative addition of oxamide ligands (H2L = N,N'-diphenyloxamide, and N,N'-dibenzyloxamide) to rhenium carbonyl (Re2(CO)10) in the presence of semi-rigid and flexible ditopic pyridyl ligands (L' = o-phenylene diisonicotinate (pdi), ethane diyl di-4-pyridine carboxylate (etdp) and 1,4-butane diyl di-4-pyridine carboxylate (budp)) having ester functionality afforded neutral dirhenium metallacycles of the general formula [(CO)3Re(µ-L)(µ-L')Re(CO)3] (1-5) under solvothermal reaction conditions. The metallacyclic compounds were characterised using elemental analyses, IR, UV-vis and NMR spectroscopic techniques. Structural analyses of 2-5 by single crystal X-ray diffraction methods revealed a stirrup like molecular framework in which two fac-Re(CO)3 units are bridged together by dissymmetrical NO∩ON bis-chelation of oxamide ligands (as a pedestal of stirrups) and further connected by a flexible ditopic tecton (as an arched anchor of stirrups) in an orthogonal fashion. The cytotoxicity activities of dirhenium metallacycles 1-5 were studied in vitro against three different cancer cell lines and normal cells.