Efficacy of High-Altitude Biofilm-Forming Novel Bacillus subtilis Species as Plant Growth-Promoting Rhizobacteria on Zea mays L.

With the global population explosion, the need for increasing crop productivity is reaching its peak. The significance of organic means of cultivation including biofertilizers and biopesticides is undeniable in this context. Over the last few decades, the use of rhizobacteria to induce crop productivity has gained particular interest of researchers. Of these, several Bacillus spp. have been known for their potential plant growth-promoting and phyto-pathogenic actions. Keeping this background in mind, this study was formulated with an aim to unravel the PGPR and phyto-pathogenic potency of Bacillus sp. isolated from extreme environmental conditions, viz. high-altitude waters of Ganges at Gangotri (Basin Extent Longitude Latitude-73° 2' to 89° 5' E 21° 6' to 31° 21' N). Based on recent studies showing the impact of biofilm on bacterial PGPR potency, three novel strains of Bacillus subtilis were isolated on basis of their extremely high biofilm-producing abilities (BRAM_G1: Accession Number MW006633; BRAM_G2: Accession Numbers MT998278-MT998280; BRAM_G3: Accession Number MT998617), and were tested for their PGPR properties like nutrient sequestration, growth hormone production (IAA, GA3), stress-responsive enzyme production (ACC deaminase) and lignocellulolytic and agriculturally important enzyme productions. The strains were further tested for the plethora of metabolites (liquid and VOCs) exuded by them. Finally, the strains both in individually and in an association, i.e. consortium was tested on a test crop, viz. Zea mays L., and the data were collected at regular intervals and the results were statistically analysed. In the present study, the role of high-altitude novel Bacillus subtilis strains as potent PGPR has been analysed statistically.

M281, an anti-FcRn antibody, inhibits IgG transfer in a human ex vivo placental perfusion model.

BACKGROUND: The transfer of pathogenic immunoglobulin G antibodies from mother to fetus is a critical step in the pathophysiology of alloimmune and autoimmune diseases of the fetus and neonate. Immunoglobulin G transfer across the human placenta to the fetus is mediated by the neonatal Fc receptor, and blockade of the neonatal Fc receptor may provide a therapeutic strategy to prevent or minimize pathological events associated with immune-mediated diseases of pregnancy. M281 is a fully human, aglycosylated monoclonal immunoglobulin G1 antineonatal Fc receptor antibody that has been shown to block the neonatal Fc receptor with high affinity in nonclinical studies and in a phase 1 study in healthy volunteers. OBJECTIVE: The objective of the study was to determine the transplacental transfer of M281 and its potential to inhibit transfer of immunoglobulin G from maternal to fetal circulation. STUDY DESIGN: To determine the concentration of M281 required for rapid cellular uptake and complete saturation of the neonatal Fc receptor in placental trophoblasts, primary human villous trophoblasts were incubated with various concentrations of M281 in a receptor occupancy assay. The placental transfer of M281, immunoglobulin G, and immunoglobulin G in the presence of M281 was studied using the dually perfused human placental lobule model. Immunoglobulin G transfer was established using a representative immunoglobulin G molecule, adalimumab, a human immunoglobulin G1 monoclonal antibody, at a concentration of 270 µg/mL. Inhibition of immunoglobulin G transfer by M281 was determined by cotransfusing 270 µg/mL of adalimumab with 10 µg/mL or 300 µg/mL of M281. Concentrations of adalimumab and M281 in sample aliquots from maternal and fetal circuits were analyzed using a sandwich enzyme-linked immunosorbent assay and Meso Scale Discovery assay, respectively. RESULTS: In primary human villous trophoblasts, the saturation of the neonatal Fc receptor by M281 was observed within 30-60 minutes at 0.15-5.0 µg/mL, suggesting rapid blockade of neonatal Fc receptor in placental cells. The transfer rate of adalimumab (0.23% ± 0.21%) across dually perfused human placental lobule was significantly decreased by 10 µg/mL and 300 µg/mL of M281 to 0.07 ± 0.01% and 0.06 ± 0.01%, respectively. Furthermore, the transfer rate of M281 was 0.002% ± 0.02%, approximately 100-fold lower than that of adalimumab. CONCLUSION: The significant inhibition of immunoglobulin G transfer across the human placental lobule by M281 and the minimal transfer of M281 supports the development of M281 as a novel agent for the treatment of fetal and neonatal diseases caused by transplacental transfer of alloimmune and autoimmune pathogenic immunoglobulin G antibodies.

A Quantitative Microtiter Assay for Sialylated Glycoform Analyses Using Lectin Complexes.

Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin-enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This "customizable" ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis.

Synthesis and biological evaluation of a unique heparin mimetic hexasaccharide for structure-activity relationship studies.

To date, the structure-activity relationship studies of heparin/heparan sulfate with their diverse binding partners such as growth factors, cytokines, chemokines, and extracellular matrix proteins have been limited yet provide early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the test assays.

Characterization of heparin-protein interaction by saturation transfer difference (STD) NMR.

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin-protein complex. We believe this approach opens up additional application avenues to further investigate heparin-protein interactions.

Identification of a novel structure in heparin generated by sequential oxidative-reductive treatment.

Unfractionated heparin is isolated from animal organs, predominantly porcine intestinal mucosa, and goes through an extensive process of purification before it can be used for pharmaceutical purposes. While the structural microheterogeneity of heparin is predominantly biosynthetically imprinted in the Golgi, subsequent steps involved in the purification and manufacture of commercial heparin can lead to the introduction of additional modifications. Postheparin crisis of 2008, it has become increasingly important to identify what additional structural diversity is introduced as a function of the purification process and thus can be determined as being heparin-related, as opposed to being an adulterant or contaminant, e.g., oversulfated chondroitin sulfate. Our study focuses on the identification of a previously unreported structure in heparin that arises due to specific steps used in the manufacturing process. This structure was initially observed as a disaccharide peak in a complete enzymatic digest of heparin, but its presence was later identified in the NMR spectra of intact heparin as well. Structural elucidation experiments involved isolation of this structure and analysis based on multidimensional NMR and liquid chromatography coupled with mass spectrometry (LC-MS). Heparin was also subjected to specific chemical reactions to determine which steps in the manufacturing process are responsible for this novel structure. Our results allowed for the definitive assignment of the structure of this novel process-related modification and enabled an identification of the putative steps in the process that give rise to the structure.

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Bioactivity screening of partially desulfated low-molecular-weight heparins: a structure/activity relationship study.

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.

Identification of a novel structure in heparin generated by potassium permanganate oxidation.

The worldwide heparin contamination crisis in 2008 led health authorities to take fundamental steps to better control heparin manufacture, including implementing appropriate analytical and bio-analytical methods to ensure production and release of high quality heparin sodium product. Consequently, there is an increased interest in the identification and structural elucidation of unusually modified structures that may be present in heparin. Our study focuses on the structural elucidation of species that give rise to a signal observed at 2.10 ppm in the N-acetyl region of the 1H NMR spectrum of some pharmaceutical grade heparin preparations. Structural elucidation experiments were carried out using homonuclear (COSY, TOSCY and NOESY) and heteronuclear (HSQC, HSQC-DEPT, HMQC-COSY, HSQC-TOCSY, and HMBC) 2D NMR spectroscopy on both heparin as well as heparin-like model compounds. Our results identify a novel type of oxidative modification of the heparin chain that results from a specific step in the manufacturing process used to prepare heparin.

Disruption of the pollen-expressed FERONIA homologs ANXUR1 and ANXUR2 triggers pollen tube discharge.

The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Novel, potent THC/anandamide (hybrid) analogs.

The structure-activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1-3 were designed. In these hybrids the aromatic ring of THC-DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K(i)=8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.

Novel compounds that interact with both leukotriene B4 receptors and vanilloid TRPV1 receptors.

The aim of this study was to investigate the interaction of a series of novel compounds with leukotriene B(4) receptors (BLT) and vanilloid receptor (TRPV1). First, we characterized leukotriene B(4) (LTB(4)) ethanolamide. In guinea pig isolated lung parenchyma, LTB(4) ethanolamide antagonized the contractile action of LTB(4) with an apparent K(B) value of 7.28 nM. Using a Boyden chamber assay, we demonstrated that this compound stimulated human neutrophil migration in a similar manner to LTB(4) but with lower efficacy. In rat TRPV1 (rTRPV1)-expressing Chinese hamster ovary (CHO) cells and dorsal root ganglion (DRG) neurons, LTB(4) and LTB(4) ethanolamide acted as low-efficacy agonists, increasing intracellular calcium concentration ([Ca(2+)](i)) in a capsazepine-sensitive manner. These results prompted us to hypothesize that a molecule may possess pharmacophores such that it is capable of dual antagonism of BLT and TRPV1 receptors. Two novel compounds, N-[2-fluoro-4-[3-(11 hydroxyheptadec-8-enyl)-thioureiomethyl]-phenyl]-methanesulfonamide (O-3367) and N-[4-[3-(11 hydroxyheptadec-8-enyl)-thioureio-methyl]-phenyl]-methanesulfonamide (O-3383), were synthesized. In human neutrophils, both compounds acted as antagonists, significantly attenuating the BLT receptor-mediated ability of LTB(4) to induce migration, with pIC(50) values of 7.22 +/- 0.17 and 5.95 +/- 0.16, respectively. In rTRPV1-expressing CHO cells, they caused a significant rightward shift in the log concentration-response curve for the TRPV1 receptor agonist capsaicin (3-methoxy-4-hydroxy)benzyl-8-methyl-6-nonenamide). In DRG neurons O-3367 significantly attenuated the capsaicin-induced increases in [Ca(2+)](i) with a pIC(50) value of 5.94 +/- 0.004. O-3367 and O-3383 represent novel structural templates for generating compounds possessing dual antagonism at BLT and TRPV1 receptors. In view of the crucial role of both TRPV1 and BLT receptors in the pathophysiology of inflammatory conditions, such compounds may betoken a novel class of highly effective therapeutics.

Biomimetic synthesis of a water soluble conducting molecular complex of polyaniline and lignosulfonate.

A new biomimetic route for the synthesis of a conducting molecular complex of polyaniline (Pani) and a natural polyelectrolyte, lignosulfonate (LGS) is presented. A poly(ethylene glycol) modified hematin (PEG-hematin) was used to catalyze the polymerization of aniline in the presence of LGS to form a Pani/LGS complex. UV-vis, FTIR, conductivity and TGA studies for the LGS-polyaniline complex indicate the presence of a thermally stable and electrically conductive form of polyaniline. Also the presence of LGS in this complex, an inexpensive byproduct from pulp processing, provides a unique combination of properties such as electronic conductivity, processability and biodegradability. The use of this conductive complex for corrosion protection is also proposed.