Inositol polyphosphates promote T cell-independent humoral immunity via the regulation of Bruton's tyrosine kinase.

T cell-independent (TI) B cell response is critical for the early protection against pathogen invasion. The regulation and activation of Bruton's tyrosine kinase (Btk) is known as a pivotal step of B cell antigen receptor (BCR) signaling in TI humoral immunity, as observed in patients with X-linked agammaglobulinemia (XLA) experiencing a high incidence of encapsulated bacterial infections. However, key questions remain as to whether a well-established canonical BCR signaling pathway is sufficient to regulate the activity of Btk. Here, we find that inositol hexakisphosphate (InsP6) acts as a physiological regulator of Btk in BCR signaling. Absence of higher order inositol phosphates (InsPs), inositol polyphosphates, leads to an inability to mount immune response against TI antigens. Interestingly, the significance of InsP6-mediated Btk regulation is more prominent in IgM+ plasma cells. Hence, the present study identifies higher order InsPs as principal components of B cell activation upon TI antigen stimulation and presents a mechanism for InsP-mediated regulation of the BCR signaling.

Hydrogen-Bond-Modulated Nucleofugality of SeIII Species to Enable Photoredox-Catalytic Semipinacol Manifolds.

Chemical bond activations mediated by H-bond interactions involving highly electronegative elements such as nitrogen and oxygen are powerful tactics in modern catalysis research. On the contrary, kindred catalytic regimes in which heavier, less electronegative elements such as selenium engage in H-bond interactions to co-activate C-Se σ-bonds under oxidative conditions are elusive. Traditional strategies to enhance the nucleofugality of selenium residues predicate on the oxidative addition of electrophiles onto SeII -centers, which entails the elimination of the resulting SeIV moieties. Catalytic procedures in which SeIV nucleofuges are substituted rather than eliminated are very rare and, so far, not applicable to carbon-carbon bond formations. In this study, we introduce an unprecedented combination of O-H⋅⋅⋅Se H-bond interactions and single electron oxidation to catalytically generate SeIII nucleofuges that allow for the formation of new C-C σ-bonds by means of a type I semipinacol process in high yields and excellent selectivity.

Photolysis of Caged Inositol Pyrophosphate InsP8 Directly Modulates Intracellular Ca2+ Oscillations and Controls C2AB Domain Localization.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living ß-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts ß-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system.

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20-25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.

5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles.

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Pentaindenocorannulene: Properties, Assemblies, and C60 Complex.

Pentaindenocorannulene (C50 H20 , 1), a deep bowl polynuclear aromatic hydrocarbon, accepts 4 electrons, crystallizes in columnar bowl-in-bowl assemblies and forms a nested C60 @12 complex. Spectra, structures and computations are presented.

Extended Corannulenes: Aromatic Bowl/Sheet Hybridization.

Among sheet/sheet polynuclear aromatic hydrocarbon (PAH) hybrids, a buckybowl-graphene hybrid has been used as a model to explore the effects of physical properties of PAHs with distinct planar and bowl regions. Activation of a C(Ar)-F bond was used to synthesize this corannulene/graphenic hybrid. Photophysical and voltammetric studies together with high-level computations revealed curvature and extended π-effects on the properties of these materials.

Serum cholinesterase is an excellent biomarker of liver cirrhosis.

BACKGROUND: Serum cholinesterase (ChE) is an enzyme synthesised by hepatocytes and its serum levels reflect the synthetic function of liver. METHODS: In patients with cirrhosis, liver function tests, PT INR and serum ChE levels were done within a week of enrolment. We studied 178 cirrhosis patients and 154 healthy controls prospectively. Receiver operator characteristics (ROC) curve analysis was employed to compute an optimal cut-off level to distinguish these groups. Correlation between ChE activity and serum bilirubin, albumin, PT INR and MELD score (Model for End-Stage Liver Disease) was analysed. RESULTS: Median serum ChE in cirrhotics was 1590 IU/L (110-8143) compared to controls 7886 IU/L (2022- 21673), p < 0.001. Serum ChE levels below 3506 had a 98.7% sensitivity and 80.3% specificity in predicting cirrhosis. Median serum ChE was higher (p < 0.001) in CC (n = 51) 4246 IU/L (680-8143) compared to DC (n = 127) 1324 IU/L (110-4550). ChE level less than 2385 IU/L had 80.1% sensitivity and 88.2% specificity in predicting DC. Follow-up levels in 25 patients showed good correlation with clinical course. The correlation coefficient between ChE and albumin was -0.67, 0.53 with PT INR and 0.59 with MELD score, (p < 0.001). CONCLUSIONS: Serum ChE is an excellent biomarker of cirrhosis with good sensitivity and specificity. It shows good correlation with serum albumin, PT INR and MELD score. Since it distinguishes DC from CC well, low levels in cirrhosis may serve as a useful prognostic marker of advanced liver disease. Long-term follow-up studies are warranted to define its exact role in clinical practice.

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 µm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

Vitamin B12 deficiency and use of proton pump inhibitors: a systematic review and meta-analysis.

BACKGROUND: Proton pump inhibitors (PPI) may impact the absorption of vitamin B12. We performed a systematic review to ascertain if PPI use increases risk of vitamin B12 deficiency. METHODS: Electronic databases (PubMed, Embase, Scopus) were searched on first of September 2022. We selected studies that compared the frequency of vitamin B12 deficiency in PPI users and non-users. Pooled Odds Ratio (OR) was calculated for the occurrence of vitamin B12 deficiency in PPI users compared to non-users. The risk of bias was assessed using the Newcastle Ottawa scale. RESULTS: Twenty-five studies were included. The pooled OR of vitamin B12 deficiency among PPI users (2852 participants) was higher than non-users (28070 participants) (OR 1.42, 95% CI: 1.16-1.73; I2 = 54%). Overall risk of PPI use among vitamin B12 deficient individuals was higher than those without deficiency (OR 1.49, 1.20-1.85; I2 = 68%). Most studies found no difference between serum vitamin B12 levels among PPI users compared to non-users. CONCLUSION: Although the pooled OR of vitamin B12 deficiency was slightly increased in PPI users, but there was significant heterogeneity, and the pooled OR was too low to imply an association clearly. Better-designed prospective studies in long-term users may clarify the issue. REGISTRATION: This study was not registered on PROSPERO.

Clinically significant findings in patients with focal incidental colorectal abnormalities on positron emission tomography-CT scans.

INTRODUCTION: The aim of this study was to determine the clinical significance of focal incidentally detected colorectal abnormalities on 18 F- Fluoro-2-Deoxy-D-Glucose (FDG) PET-CT scans. METHODS: Retrospective audit of PET-CT scans performed at our institution between 2009 and 2014. Demographic and clinical details were retrieved from electronic patient records. An advanced adenoma was defined as: ≥1 cm in size, tubullo-villous histology, or displaying high grade dysplasia. A high-risk lesion (HRL) was defined as an advanced adenoma or colorectal cancer (CRC). RESULTS: Of the 1911 PET-CT eligible scans, focal incidental colorectal FDG uptake was detected in 99 (5.2%) patients. Colonoscopy was undertaken in 43 (43.4%) patients and 45 FDG-avid sites were evaluated. The commonest site of abnormal FDG uptake was the rectosigmoid region, with 34 (75.6%) of the 45 foci being located in this area. Overall, 23 (53.5%) of these patients had clinically significant pathology. Of the 45 focal PET-CT abnormalities evaluated, 17 (37.8%) were adenomas, of which 11 (24.4%) were advanced adenomas, and six (13.3%) were cancers, with a total of 17 (37.8%) HRLs. Five of the six patients with CRC underwent surgical resection, whilst one had endoscopic resection. The overall survival for the entire cohort was 18 months (range 1-72 months) with those that underwent colonoscopy having higher overall survival compared to those that did not (38 vs. 13.5 months). CONCLUSION: Incidental colorectal abnormalities on PET-CT scans are often clinically significant. However, careful selection for colonoscopy is important due to the poor survival in these patients related to the underlying primary malignancy.

Characterizing Thermodynamics of Protein-Glycosaminoglycan Interactions Using Isothermal Titration Calorimetry.

It has now become increasingly clear that a complete atomic description of how biomacromolecules recognize each other requires knowledge not only of the structures of the complexes but also of how kinetics and thermodynamics drive the binding process. In particular, such knowledge is lacking for protein-glycosaminoglycan (GAG) complexes. Isothermal titration calorimetry (ITC) is the only technique that can provide all of the thermodynamic parameters-enthalpy, entropy, free energy (binding constant), and stoichiometry-from a single experiment. Here we describe different factors that must be taken into consideration in carrying out ITC titrations to obtain meaningful thermodynamic data of protein-GAG interactions.

Conformational transitions of adsorbed proteins on surfaces of varying polarity.

Combining a wide range of protein adsorption experiments (three globular proteins on eight well-defined homogeneous surfaces) with Monte Carlo simulations of lattice proteins at different concentrations and on surfaces of varying "polarity", we explore the extent and rheological behavior of adsorbed proteins as a function of substrate polarity, "on" rate constants (k(a)) and steric parameters (|A(1)|) from the random sequential adsorption model, and demonstrate a folding to unfolding transition upon adsorption. We show that model globular proteins (hen egg lysozyme, ribonuclease A, and insulin dimer) behave similarly with respect to adsorption. Experimentally, above a substrate wettability cos theta > 0.4 (where theta is the sessile contact angle of water on a substrate in air), the adsorbed mass, rigidity, and k(a) of the proteins are diminished, while the steric factor |A(1)| is increased, suggesting a lower packing density. To analyze these results, we have invoked computer simulations. We show that changing surface polarity has two profound effects. First, the amount adsorbed increases as the surfaces become more apolar. Further, the proteins become less stable as their adsorbed amount increased because they gain a large number of interprotein and protein-surface interactions. Finally, apolar surfaces served to reduce the unfolding free energy barriers, further facilitating the reorganizing of proteins on these surfaces. Thus, increasing the nonpolar nature of the surfaces resulted in a more rigid adsorbed layer, in good agreement with the experiments.

Divergent roles of SPINK1 and PRSS2 variants in tropical calcific pancreatitis.

BACKGROUND/AIMS: Tropical calcific pancreatitis (TCP) refers to a type of idiopathic pancreatitis prevalent in Asia. The trypsin inhibitor (SPINK1) N34S variant partially explains the genetic susceptibility to TCP. As anionic trypsinogen (PRSS2) G191R protects against chronic pancreatitis in Europeans, we investigated whether this variant protects from TCP in Indians. METHODS: We enrolled 174 patients and 794 controls from two Indian tertiary care referral hospitals. We analyzed PRSS2 and SPINK1 variants by melting curve analysis, allele-specific discrimination assay, and sequencing. RESULTS: G191R was detected in 1 TCP patient (0.6%) compared to 13 controls (1.6%; OR 0.27, 95% CI 0.03-2.1; p = 0.33). SPINK1 N34S was enriched in the TCP population 67/174 (38.5%) compared to controls 10/234 (4.3%; OR 14, 95% CI 6.9-28.3; p < 0.001). CONCLUSION: G191R PRSS2 is a rare allele in the Indian population and the data suggest a nonsignificant trend towards a protective effect. N34S SPINK1 represents the major genetic risk factor in TCP.

Trehalose Conjugation Enhances Toxicity of Photosensitizers against Mycobacteria.

Trehalose is a natural glucose-derived disaccharide found in the cell wall of mycobacteria. It enters the mycobacterial cell through a highly specific trehalose transporter system. Subsequently, trehalose is equipped with mycolic acid species and is incorporated into the cell wall as trehalose monomycolate or dimycolate. Here, we investigate the phototoxicity of several photosensitizer trehalose conjugates and take advantage of the promiscuity of the extracellular Ag85 complex, which catalyzes the attachment of mycolic acids to trehalose and its analogues. We find that processing by Ag85 enriches and tethers photosensitizer trehalose conjugates directly into the mycomembrane. Irradiation of the conjugates triggers singlet oxygen formation, killing mycobacterial cells more efficiently, as compared to photosensitizers without trehalose conjugation. The conjugates are potent antimycobacterial agents that are, per se, affected neither by permeability issues nor by detoxification mechanisms via drug efflux. They could serve as interesting scaffolds for photodynamic therapy of mycobacterial infections.

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Indian consensus on gastroesophageal reflux disease in adults: A position statement of the Indian Society of Gastroenterology.

The Indian Society of Gastroenterology developed this evidence-based practice guideline for management of gastroesophageal reflux disease (GERD) in adults. A modified Delphi process was used to develop this consensus containing 58 statements, which were generated by electronic voting iteration as well as face-to-face meeting and review of the supporting literature primarily from India. These statements include 10 on epidemiology, 8 on clinical presentation, 10 on investigations, 23 on treatment (including medical, endoscopic, and surgical modalities), and 7 on complications of GERD. When the proportion of those who voted either to accept completely or with minor reservation was 80% or higher, the statement was regarded as accepted. The prevalence of GERD in India ranges from 7.6% to 30%, being < 10% in most population studies, and higher in cohort studies. The dietary factors associated with GERD include use of spices and non-vegetarian food. Helicobacter pylori is thought to have a negative relation with GERD; H. pylori negative patients have higher grade of symptoms of GERD and esophagitis. Less than 10% of GERD patients in India have erosive esophagitis. In patients with occasional or mild symptoms, antacids and histamine H2 receptor blockers (H2RAs) may be used, and proton pump inhibitors (PPI) should be used in patients with frequent or severe symptoms. Prokinetics have limited proven role in management of GERD.

Surface-enhanced nucleation of insulin amyloid fibrillation.

Proteins can interact with biological surfaces such as cell membrane, chaperones, cornea, bone, arteries, veins, and heart cavities of the cardiovascular system and also with non-biological surfaces including dialysis membranes and tubing, catheters, invasive surgical instruments, needles, and artificial implants. Fibrillation of amyloid proteins is implicated in many human diseases, including Alzheimer's, Parkinson's, and type II diabetes. Here, we show that heterogeneous surfaces accelerate the human insulin nucleation process that is the rate-determining step during amyloid fibril formation. The observed shorter lag (nucleation) phase correlates both with surface wettability and surface roughness. Surfaces promote faster nucleation possibly by increasing the local concentration of protein molecules. A composite parameter combining both surface wettability and roughness suggests that the ideal surface for slower nucleation should be hydrophilic and smooth. These findings provide a basis for designing suitable biomaterials and biomedical devices, especially those to resist amyloidosis.

New Synthetic Methods for Phosphate Labeling.

The complexity of phosphorylation pathways and their downstream effects is vast. Synthetic chemistry has been working side by side with biology to develop phosphate labels for biological processes involving phosphorylated compounds. This chapter discusses recently employed methods for the preparation of several phosphate labels. Synthesis of biomolecules and their analogs and other useful or potentially useful phosphate derivatives is discussed.