Antibody-Mediated Activation of FGFR1 Induces FGF23 Production and Hypophosphatemia

The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs) can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb) in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.     

Assembly and replication of HIV-1 in T cells with low levels of phosphatidylinositol-(4,5)-bisphosphate

HIV-1 Gag assembles into virus particles predominantly at the plasma membrane (PM). Previously, we observed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] is essential for Gag binding to the plasma membrane and virus release in HeLa cells. In the current study, we found that PI(4,5)P(2) also facilitates Gag binding to the PM and efficient virus release in T cells. Notably, serial passage of HIV-1 in an A3.01 clone that expresses polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), yielded an adapted mutant with a Leu-to-Arg change at matrix residue 74 (74LR). Virus replication in T cells expressing 5ptaseIV was accelerated by the 74LR mutation relative to replication of wild type HIV-1 (WT). This accelerated replication of the 74LR mutant was not due to improved virus release. In control T cells, the 74LR mutant releases virus less efficiently than does the WT, whereas in cells expressing 5ptaseIV, the WT and the 74LR mutant are similarly inefficient in virus release. Unexpectedly, we found that the 74LR mutation increased virus infectivity and compensated for the inefficient virus release. Altogether, these results indicate that PI(4,5)P(2) is essential for Gag-membrane binding, targeting of Gag to the PM, and efficient virus release in T cells, which in turn likely promotes efficient virus spread in T cell cultures. In T cells with low PI(4,5)P(2) levels, however, the reduced virus particle production can be compensated for by a mutation that enhances virus infectivity.     

Enhanced bioefficacy of Bacillus thuringiensis var. kurstaki against Spodoptera litura (Lepidoptera: Noctuidae) through particle size reduction and formulation as a suspension concentrate

Technical powder of DOR Bt-127, a local isolate of Bacillus thuringiensis (Bt) var. kurstaki, containing 105?µm particles was subjected to milling in a planetary ball mill. Dynamic light scattering of samples drawn at 30?min intervals revealed reduced particle sizes ranging 1171–210?nm. Alkali-soluble protein was higher at 153–175?mg/g in milled Bt powders over 140?mg/g in unmilled Bt powder. All milled powders gave higher mortality of Spodoptera litura (Fabricius) larvae in comparison to the unmilled Bt powder. Bt powder from 60?min milling containing 559?nm particles with a low polydispersity index of 0.338 gave a lower LC₅₀ value of 1.35?mg/ml against third instar S. litura larvae in comparison to 2.04?mg/ml for unmilled Bt powder. LC₅₀ value of the suspension concentrate (SC) formulation developed with 559?nm Bt particles was 2.84?µl/ml containing only 0.95?mg Bt. Field evaluation of the SC formulation against S. litura on castor revealed highest per cent larval reduction of 92.4 and 96.2 at concentrations of 2.5 and 3.0?ml/l, respectively.     

Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient gag membrane binding

Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P₂ depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P₂ depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P₂-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P₂. To examine a putative Gag interaction with PI(4,5)P₂, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P₂. Using this assay, we observed that PI(4,5)P₂ significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P₂ for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P₂ binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P₂-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P₂ on the membrane and that the MA basic domain mediates this interaction.     

Suspension concentrate formulation of Bacillus thuringiensis var. kurstaki for effective management of Helicoverpa armigera on sunflower (Helianthus annuus)

A local strain DOR Bt-1 belonging to Bacillus thuringiensis var. kurstaki (Bt.k) was multiplied through solid state fermentation and the resulting technical powder was milled and sieved to obtain particles of various sizes. Efficacy of Bt.k. against larvae of Helicoverpa armigera was found to increase with the decrease in particle size. Boric acid was found to be synergistic to DOR Bt-1 technical powder. LC₅₀ of the Bt and boric acid mixture (75:25) was lower at 89.63 mg/100 mL in comparison to Bt alone at 116.75 mg/100 mL. A suspension concentrate formulation with DOR Bt-1 technical was developed using boric acid as an adjuvant. The formulation was found to be highly effective against H. armigera in laboratory bioassays with an LC₅₀ of 185 µL (containing 53.36 mg Bt). The formulation gave effective control of H. armigera on sunflower within 3 days after spray even at the lowest dose of 1.0 mL/L under field conditions.     

Polybacterial Periodontal Pathogens Alter Vascular and Gut BH4/nNOS/NRF2-Phase II Enzyme Expression

Periodontal disease is a highly prevalent chronic inflammatory disease and is associated with complex microbial infection in the subgingival cavity. Recently, American Heart Association supported a century old association between periodontal disease and atherosclerotic vascular disease. We have recently shown that polybacterial periodontal infection led to aortic atherosclerosis and modulation of lipid profiles; however the underlying mechanism(s) has not been yet demonstrated. Altered nitric oxide (NO) synthesis and tetrahydrobiopterin (BH₄), a cofactor for nitric oxide synthases (NOS) has long been shown to be associated with vascular dysfunction and gastrointestinal motility disorders. We sought to examine the mechanism of periodontal infection leading to altered vascular and gastrointestinal smooth muscle relaxation, focusing on the BH₄/nNOS pathways. In addition, we also have investigated how the antioxidant system (NRF2-Phase II enzyme expression) in vascular and GI specimens is altered by oral infection. Eight week old male ApoE^null mice were either sham-infected or infected orally for 16 weeks with a mixture of major periodontal bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia to induce experimental periodontitis. Serum, vascular (mesenteric), stomach, and colon specimens were collected at the end of periodontal pathogen infection. Bacterial infection induced significant (p<0.05) reductions in the levels of BH₄,in ratio of BH₄:BH₂+B and also in nitric oxide levels compared to sham-infected controls. In addition, we identified a significant (p<0.05) reduction in eNOS dimerization, nNOS dimerization and protein expression of BH₄ biosynthesis enzymes; GCH-1, DHFR and NRF2 & Phase II enzymes in infected mice versus controls in both mesenteric artery and colon tissues. However, we found no differences in nNOS/BH₄ protein expression in stomach tissues of infected and sham-infected mice. This suggests that a polybacterial infection can cause significant changes in the vascular and colonic BH₄/nNOS/NRF2 pathways which might lead to impaired vascular relaxation and colonic motility.     

Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag

The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P(2) interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P(2) dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P(2) is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P(2). Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P(2). Furthermore, membrane binding of HTLV-1 Gag in vitro was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P(2) and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.     

Role of the EphB2 receptor in autophagy,apoptosis and invasion in human breast cancer cells

The Eph and Ephrin proteins, which constitute the largest family of receptor tyrosine kinases, are involved in normal tissue development and cancer progression. Here, we examined the expression and role of the B-type Eph receptor EphB2 in breast cancers. By immunohistochemistry using a progression tissue microarray of human clinical samples, we found EphB2 to be expressed in benign tissues, but strongly increased in cancers particularly in invasive and metastatic carcinomas. Subsequently, we found evidence that EphB2, whose expression varies in established cell breast lines, possesses multiple functions. First, the use of a DOX-inducible system to restore EphB2 function to low expressers resulted in decreased tumor growth in vitro and in vivo, while its siRNA-mediated silencing in high expressers increased growth. This function involves the onset of apoptotic death paralleled by caspases 3 and 9 activation. Second, EphB2 was also found to induce autophagy, as assessed by immunofluorescence and/or immunoblotting examination of the LC3, ATG5 and ATG12 markers. Third, EphB2 also has a pro-invasive function in breast cancer cells that involves the regulation of MMP2 and MMP9 metalloproteases and can be blocked by treatment with respective neutralizing antibodies. Furthermore, EphB2-induced invasion is kinase-dependent and is impeded in cells expressing a kinase-dead mutant EphB2. In summary, we identified a mechanism involving a triple role for EphB2 in breast cancer progression, whereby it regulates apoptosis, autophagy, and invasion.     

Monitoring Powder Blend Homogeneity Using Light-Induced Fluorescence

Light-induced fluorescence (LIF) was evaluated as a process analytical technology to monitor blend homogeneity and establish a relationship with high-performance liquid chromatography (HPLC). Secondary aims for this study included a determination of blend steady-state, acceptable mixing time interval, and mixing end point. Also, identification of potential “dead spots” in the 124 L intermediate bulk container mixing tote was explored. Individual samples from 13 sample locations were collected at 0.25, 0.5, 0.75, 1, 2, 5, 10, and 20 min and analyzed using LIF and HPLC. LIF and HPLC methods showed similar mixing profiles. A coefficient of determination (R ²) of 0.86 (p value < 0.0001) was obtained for a second-degree polynomial bivariate fit of LIF counts by HPLC percent label claim (%LC). A significant linear relationship was determined between LIF percent relative standard (%RSD) and HPLC %RSD (R ² = 0.97, p < 0.0001). The LIF steady-state, acceptable mixing time interval, and mixing end point were determined to be 1–20, 2–20, and 2 min, respectively. The steady-state, acceptable mixing time interval, and mixing end point determined by HPLC were 1–20, 5–10, and 5 min, respectively. The Tukey–Kramer honestly significant difference analysis of HPLC %LC by sample location at 5 and 10 min mixing times showed that there was a statistical difference between the HPLC %LC group means at two blender locations.