Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles.

Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations - the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet-derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.

Predesigned Metal-Anchored Building Block for In Situ Generation of Pd Nanoparticles in Porous Covalent Organic Framework: Application in Heterogeneous Tandem Catalysis.

The development of nanoparticle-polymer-hybrid-based heterogeneous catalysts with high reactivity and good recyclability is highly desired for their applications in the chemical and pharmaceutical industries. Herein, we have developed a novel synthetic strategy by choosing a predesigned metal-anchored building block for in situ generation of metal (Pd) nanoparticles in the stable, porous, and crystalline covalent organic framework (COF), without using conventional reducing agents. In situ generation of Pd nanoparticles in the COF skeleton is explicitly confirmed from PXRD, XPS, TEM images, and 15N NMR spectral analysis. This hybrid material is found to be an excellent reusable heterogeneous catalyst for the synthesis of biologically and pharmaceutically important 2-substituted benzofurans from 2-bromophenols and terminal alkynes via a tandem process with the turnover number up to 1101. The heterogeneity of the catalytic process is unambiguously verified by a mercury poisoning experiment and leaching test. This hybrid material shows superior catalytic performance compared to commercially available homogeneous as well as heterogeneous Pd catalysts.

Odd-Even Alternation in Tautomeric Porous Organic Cages with Exceptional Chemical Stability.

Amine-linked (C-NH) porous organic cages (POCs) are preferred over the imine-linked (C=N) POCs owing to their enhanced chemical stability. In general, amine-linked cages, obtained by the reduction of corresponding imines, are not shape-persistent in the crystalline form. Moreover, they require multistep synthesis. Herein, a one-pot synthesis of four new amine-linked organic cages by the reaction of 1,3,5-triformylphloroglucinol (Tp) with different analogues of alkanediamine is reported. The POCs resulting from the odd diamine (having an odd number of -CH2 groups) is conformationally eclipsed, while the POCs constructed from even diamines adopt a gauche conformation. This odd-even alternation in the conformation of POCs has been supported by computational calculations. The synthetic strategy hinges on the concept of Schiff base condensation reaction followed by keto-enol tautomerization. This mechanism is the key for the exceptional chemical stability of cages and facilitates their resistance towards acids and bases.

Counteranion Driven Homochiral Assembly of a Cationic C3-Symmetric Gelator through Ion-Pair Assisted Hydrogen Bond.

The helical handedness in achiral self-assemblies is mostly complex due to spontaneous symmetry breaking or kinetically controlled random assembly formation. Here an attempt has been made to address this issue through chiral anion exchange. A new class of cationic achiral C3-symmetric gelator devoid of any conventional gelation assisting functional units is found to form both right- and left-handed helical structures. A chiral counteranion exchange-assisted approach is successfully introduced to control the chirality sign and thereby to obtain preferred homochiral assemblies. Formation of anion-assisted chiral assembly was confirmed by circular dichroism (CD) spectroscopy, microscopic images, and crystal structure. The X-ray crystal structure reveals the construction of helical assemblies with opposite handedness for (+)- and (-)-chiral anion reformed gelators. The appropriate counteranion driven ion-pair-assisted hydrogen-bonding interactions are found responsible for the helical bias control in this C3-symmetric gelator.

Topological analysis of the Na+/H+ exchanger.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed integral membrane protein present in mammalian cells. It is made up of a hydrophobic 500 amino acid membrane domain that transports and removes protons from within cells, and a regulatory intracellular cytosolic domain made of approximately 315 amino acids. Determining the structure of NHE1 is critical for both an understanding of the Na+/H+ exchange mechanism of transport, and in the design of new improved inhibitors for use in treatment of several diseases in which it is involved. Differing models of the NHE1 protein have been proposed. The first model suggested by two groups proposes that amino acids 1-500 form a 12 transmembrane segment spanning region in which amino acids 1-127 form two transmembrane segments, and amino acids 315-411 form a single transmembrane segment with membrane associated segments. A second model based on the structure of the Escherichia coli Na+/H+ exchanger protein proposes an overall similar topology, but suggests amino acids 1-127 are removed as a signal sequence and are not present in the mature protein. It also suggests a different topology of amino acids 315-411 to form three transmembrane segments. We used cysteine scanning accessibility and examination of glycosylation of the mature protein to characterize the NHE1 protein. Our results demonstrate that the model of NHE1 is correct which suggests that amino acids 1-127 form two transmembrane segments that remain connected to the mature protein, and the segment between amino acids 315-411 is one transmembrane segment.

Differential roles of tryptophan residues in the functional expression of human anion exchanger 1 (AE1, Band 3, SLC4A1).

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl(-)=HCO(-)(3) exchange across the erythrocyte plasma membrane. This transport activity resides in the 52 kDa C-terminal membrane domain (Gly(361)-Val(911)) predicted to span the membrane 14 times. To explore the role of tryptophan (Trp) residues in AE1 function, the seven endogenous Trp residues in the membrane domain were mutated individually to alanine (Ala) and phenylalanine (Phe). Expression levels, cell surface abundance, inhibitor binding and transport activities of the mutants were measured upon expression in HEK-293 cells. The seven Trp residues divided into three classes according the impact of mutations on the functional expression of AE1: Class 1, dramatically decreased expression (Trp(492) and Trp(496)); Class 2, decreased expression by Ala substitution but not Phe (Trp(648), Trp(662) and Trp(723)); and Class 3, normal expression (Trp(831) and Trp(848)). The results indicate that Trp residues play differential roles in AE1 expression and function depending on their location in the protein and that Trp mutants with low expression are misfolded and retained in the endoplasmic reticulum.

A C(3v)-symmetric tripodal urea receptor for anions and ion pairs: formation of dimeric capsular assemblies of the receptor during anion and ion pair coordination.

A new C(3v)-symmetric urea-based heteroditopic tripodal receptor capable of recognizing both anions and ion pairs was designed, synthesized, and characterized. The protonated receptor forms a sulfate complex which encapsulates a single DMF in the tripodal cavity of the receptor. However, the SO4(2-) anion is located outside the tripodal cavity and is stabilized by N-H···O hydrogen bonds from the urea functions of four receptor cations. With TBAHSO4 the receptor forms a contact ion pair complex, where both the TBA(+) and SO4(2-) groups are pseudoencapsulated in the tripodal cavity of the protonated receptor. Significantly, the receptor forms a charge-separated polymeric ion pair complex with K(+) and HPO4(2-) via formation of a dimeric capsular assembly of the receptor, in which three K(+) encapsulated dimeric capsular assemblies interdigitate to form a precise cavity that further encapsulates HPO4(2-). The receptor also forms an anion complex with CO3(2-) via formation of dimeric capsular self-assembly of the receptor. Solution-state binding studies of the receptor with oxyanions have also been carried out by (1)H NMR titration experiments, which show the oxyanion binding trend HCO3(-) > H2PO4(-) > HSO4(-), whereas no binding with NO3(-) and ClO4(-) anions is observed.

Comparative Study of Recurrence and Complications Using Various Sclerosants by Single Dart Technique in Treatment of Ganglion Cysts.

Ganglion cysts are tense,smooth,fluctuant,cystic and transilluminant swellings. They are commonly found on the dorsum of the wrist, at the scapholunate articulation and may involve volar wrist, tendon sheaths and even inter phalangeal joints. This study aims to compare the efficacy and the recurrence rates with triamcinolone, hyaluronidase and sodium tetradecyl sulphate,using the single dart technique. This prospective observational study was conducted on patients who presented to the general surgery outpatient department of our institute with ganglion cysts of wrist between January 2010 and August 2011 (20 months). A total of 180 patients were included in this study. The difference in the recurrence rates after sclerotherapy for ganglion cysts is statistically not significant between triamcinolone and hyaluronidase regimens as Z (P1-P2) = 1.70, p > 0.05 but the difference in the recurrence rates after sclerotherapy for ganglion cysts is statistically significant between triamcinolone and sodium tetradecyl sulphate regimens as Z (P1-P2) = 3.34, p < 0.05 . Chi-square value -10.33 (2 ° of freedom), p = 0.00571987 (significant at 5 % level). Intralesional injection of triamcinolone by single dart technique, therefore, may be considered as a simple, safe, cost effective, convenient, less invasive alternative to surgical excision of wrist ganglion cysts.

Fluorescence Turn on Sensor for Sulfate Ion in Aqueous Medium Using Tripodal-Cu(2+) Ensemble.

We report the selective recognition of sulfate anion in aqueous medium at biological pH 7.2 over the other interfering anions based on naphthoic acid bearing tripodal ligand by applying fluorescence turn off -on mechanism. The carboxylic acid groups in the ligand enhance the solubility in water and enable it to form complex with copper salt. Thus formed L-Cu2+ ensemble quench the fluorescence of the parent ligand and in turn recognize sulfate anion via revival of fluorescence intensity. The 1:2 stoichiometry was confirmed by ESI mass spectral data and Job's plot. The average binding constant was found to be 6.2×10(8) M(-2).

A substrate access tunnel in the cytosolic domain is not an essential feature of the solute carrier 4 (SLC4) family of bicarbonate transporters.

Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na(+)-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation. Introduction of single and double mutations at the equivalent arginine (Arg(283)) and at an interacting glutamate (Glu(85)) in the cytosolic domain of human AE1 (cdAE1) had no effect on the cell surface expression or the transport activity of AE1 expressed in HEK-293 cells. In addition, the membrane domain of AE1 (mdAE1) efficiently mediated anion transport. A 2.1-Å resolution crystal structure of cdΔ54AE1 (residues 55-356 of cdAE1) lacking the amino-terminal and carboxyl-terminal disordered regions, produced at physiological pH, revealed an extensive hydrogen-bonded network involving Arg(283) and Glu(85). Mutations at these residues affected the pH-dependent conformational changes and stability of cdΔ54AE1. As these structural alterations did not impair functional expression of AE1, the cytosolic and membrane domains operate independently. A substrate access tunnel within the cytosolic domain is not present in AE1 and therefore is not an essential feature of the SLC4 family of bicarbonate transporters.

Concurrent parathyroid carcinoma and adenoma: A rare presentation of a rarer disease entity.

Parathyroid carcinoma is a rare disease. But multiglandular parathyroid neoplasm is even rarer. A high level of suspicion, on the basis of clinical, hematological tests and intraoperative findings is necessary to treat this disease entity, particularly in the absence of palpable neck masses. Preoperative localization is important. Bilateral neck exploration should be done routinely and all 4 glands seen to avoid missing out other pathological glands.

Encapsulation of a discrete cyclic halide water tetramer [X2(H2O)2]2-, X = Cl-/Br- within a dimeric capsular assembly of a tripodal amide receptor.

A conformationally flexible C3v symmetric N-bridged tripodal amide receptor encapsulates a tetrameric halide water cluster [X2(H2O)2](2-) (X = Cl(-)/Br(-)) within its dimeric capsular assembly and forms a non-capsular 1D polymeric assembly with higher homologous iodide anions upon protonation.

Methods of retaining the suture in a sub-cuticular stitch using non absorbable suture - innovations and diversity of techniques.

Subcuticular sutures have been extensively used for closure of wounds, particularly when good cosmesis is required. Both absorbable and nonabsorbable sutures are used in subcuticular sutures. Nonabsorbable sutures have some distinct advantages over absorbable sutures from cosmetic point of view, when used in subcuticular sutures. The challenge remains in keeping the nonabsorbable suture secure and in situ. We present six different methods of securing the suture in position while closing the wound with a nonabsorbable suture (2-0/3-0 polypropylene, polyamide, or polyethylene).

Encapsulation of divalent tetrahedral oxyanions of sulfur within the rigidified dimeric capsular assembly of a tripodal receptor: first crystallographic evidence of thiosulfate encapsulation within neutral receptor capsule.

A simple tris(2-aminoethyl)amine based meta-chloro substituted tripodal thiourea receptor L has been extensively studied with two divalent oxyanions of sulfur, such as sulfate and thiosulfate, with identical dimensionality. The solid state crystal structure of the anion complexes with L reveal that the anions are encapsulated within the dimeric rigid capsular assembly of the receptor via N-H···anion interactions. To the best of our knowledge this is the first report on the encapsulation of thiosulfate within dimeric capsular assembly of a neutral receptor. The tight capsular sizes for both anion complexes are quite comparable, whereas the coordination mode of the anions and the hydrogen bonding parameters are significantly varied. The three dimensional solid state structural orientations of the capsular complexes are mainly governed by the Cl···Cl (for thiosulfate complex) and Cl···S (for sulfate complex) halogen bonding interactions. The solution-state binding and encapsulation of oxyanions by N-H···anion hydrogen bonding has also been confirmed by quantitative (1)H NMR titration and 2D NOESY NMR experiments. Both the experiments confirm that in contradiction of 2:1 solid state binding, in solution the studied anions are bound within the pseudocavity of the receptor with 1:1 binding stoichiometry. Moreover, the change in chemical shifts of thiourea -NH protons and the binding constant values suggest the receptor-sulfate interaction is more energetically favorable compared to the receptor thiosulfate interaction.

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis.

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.

Neutral acyclic anion receptor with thiadiazole spacer: halide binding study and halide-directed self-assembly in the solid state.

A halide binding study of a newly synthesized neutral acyclic receptor LH(2) with a thiadiazole spacer has been methodically performed both in solution and in the solid state. Crystal structure analysis of the halide complexes elucidate the fact that fluoride forms an unusual 1:1 hyrogen-bonded complex with monodeprotonated receptor, whereas in the case of other congeners, such as chloride and bromide, the receptor binds two halide anions along with formation of a halide-bridged 1D polymeric chain network by participation of N-H···X(-) and aromatic C-H···X(-) hydrogen-bonding (where X = Cl and Br) interactions. The presence of a rigid thiadiazole spacer presumably opens up enough space for capturing two halide anions by a single receptor molecule, where the coordinated -NH protons are pointed in the same direction with respect to the spacer and eventually favor formation of halide (Cl(-) and Br(-)) induced polymeric architecture, although no obvious chloride- or bromide-directed polymeric assembly is found in solution. A significant red shift of 243 nm in the absorption spectra of LH(2) was solely observed in the presence of excess fluoride anion, which enables LH(2) as an efficient colorimetric sensor for optical detection of fluoride anion (yellow to blue). Furthermore, spectroscopic titration experiments with increasing equivalents of fluoride anion suggest formation of a H-bonded complex with subsequent stepwise deprotonation of two N-H groups, which can be visually monitored by a change in color from yellow to blue via pink.

Oxidative cyclization of thiosemicarbazone: an optical and turn-on fluorescent chemodosimeter for Cu(II).

A weakly fluorescent thiosemicabazone (L(1)H) was found to be a selective optical and "turn-on" fluorescent chemodosimeter for Cu(2+) ion in aqueous medium. A significant fluorescence enhancement along with change in color was only observed for Cu(2+) ion; among the other tested metal ions (viz. Na(+), K(+), Mg(2+), Ca(2+), Cr(3+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), Ag(+), Ni(2+), Co(2+), Fe(3+) and Mn(2+)). The Cu(2+) selectivity resulted from an oxidative cyclization of the weak fluorescent L(1)H into highly fluorescent rigid 4,5-dihydro-5,5-dimethyl-4-(naphthalen-5-yl)-1,2,4-triazole-3-thione (L(2)). The signaling mechanism has been confirmed by independent synthesis with detail characterization of L(2).

Distance measurements within a concatamer of the plasma membrane Cl⁻/HCO3⁻ exchanger, AE1.

AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl⁻/HCO3⁻ exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed at the plasma membrane at levels similar to that of wild-type AE1 and had Cl⁻/HCO3⁻ exchange activity indistinguishable from that of wild-type AE1. Nondenaturing gel electrophoresis revealed that AE1·AE1 does not associate into higher-order oligomers when expressed in HEK293 cells and Xenopus laevis oocytes. The cysteine-less concatamer (AE1·AE1-C⁻) enabled introduction of unique cysteine residues into the whole intramolecular dimer. AE1(Q434C)·AE1(Q434C)-C⁻, with a single cysteine residue in each AE1 subunit, was labeled with the donor Alexa Fluor 488 C(5)-maleimide (AF) and the acceptor tetramethylrhodamine methanethiosulfonate (TMR-MTS). Energy transfer efficiency revealed that the distance between these residues in the AE1 dimer is 49 ± 5 Å. The 72% FRET efficiency observed between AE1(Q434C)·AE1-C⁻ labeled with AF and the lipid bilayer labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate indicates that Q434 is less than 33 Å from the lipid bilayer. We thus provide two distance constraints for the position of Q434, which is located in extracellular loop 1, connecting the first two transmembrane segments of AE1.

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae.

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V(max) and a low K(m) value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl(2) and ZnCl(2) acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 degrees C and pH 8.5 respectively. Enzyme activity was stable at 0-40 degrees C and at alkaline pH.

Antiapoptotic role of S-adenosyl-l-methionine against hydrochloric acid induced cell death in Saccharomyces cerevisiae.

Exposure of stationary phase cells of Saccharomyces cerevisiae to 10 mM HCl (pH approximately 2) resulted in cell death as a function of time (up to 6 h) with most (about 40%-65%) of the cells showing apoptotic features including chromatin condensation along the nuclear envelope, exposure of phosphatidylserine on the outer leaflet of cytoplasmic membrane, and DNA fragmentation. During the first 2 h of acid exposure there was an increase in reactive oxygen species (ROS) level inside cells, with subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents culminating in loss of mitochondrial membrane potential (DeltaPsi(m)). An initial (1 h) event of mitochondrial hyper-polarization with subsequent elevation of ROS level of the acid treated cells was also observed. S-adenosyl-l-methionine (AdoMet; 1 mM) treatment increased the cell survival of the acid stressed cells. It partially scavenged the increased intracellular ROS level by supplementing glutathione through the transsulfuration pathway. It also inhibited acid mediated lipid peroxidation, partially recovered acid evoked loss of DeltaPsi(m) and protected the cells from apoptotic cell death. S-adenosyl di-aldehyde, an indirect inhibitor of the AdoMet metabolic pathway, increased mortality of the acid treated cells. Incubation of acid stressed cells with the antioxidant, N-acetyl-cysteine (1 mM), decreased the cellular mortality, but the same concentration of AdoMet offered more protection by scavenging the free radicals. The ability of AdoMet to scavenge ROS mediated apoptosis may be an important function of this molecule in responding to cellular stress. The study could open a new avenue for detailed investigation on the curative potential of AdoMet against gastric ulcer.