In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Deciphering the role of charge,hydration, and hydrophobicity for cytotoxic activities and membrane interactions of bile acid based facial amphiphiles

We synthesized four cationic bile acid based facial amphiphiles featuring trimethyl ammonium head groups. We evaluated the role of these amphiphiles for cytotoxic activities against colon cancer cells and their membrane interactions by varying charge, hydration and hydrophobicity. The singly charged cationic Lithocholic acid based amphiphile (LCA-TMA₁) is most cytotoxic, whereas the triply charged cationic Cholic acid based amphiphile (CA-TMA₃) is least cytotoxic. Light microscopy and Annexin-FITC assay revealed that these facial amphiphiles caused late apoptosis. In addition, we studied the interactions of these amphiphiles with model membrane systems by Prodan-based hydration, DPH-based anisotropy, and differential scanning calorimetry. LCA-TMA₁ is most hydrophobic with a hard charge causing efficient dehydration and maximum perturbations of membranes thereby facilitating translocation and high cytotoxicity against colon cancer cells. In contrast, the highly hydrated and multiple charged CA-TMA₃ caused least membrane perturbations leading to low translocation and less cytotoxicity. As expected, Chenodeoxycholic acid and Deoxycholic acid based amphiphiles (CDCA-TMA₂, DCA-TMA₂) featuring two charged head groups showed intermediate behavior. Thus, we deciphered that charge, hydration, and hydrophobicity of these amphiphiles govern membrane interactions, translocation, and resulting cytoxicity against colon cancer cells.     

Rac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migration

14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.     

Integrin and Growth Factor Receptor Alliance in Angiogenesis

A sequence of events in vascular and stromal cells maintained in a highly coordinated manner regulates angiogenesis and tissue remodeling. These processes are mediated by the ability of cells to respond to environmental cues and activate surface integrins. Physiological and pathological processes in vascular biology are dependent on the specificity of important signaling mechanisms that are activated through the association between growth factors, their receptors, integrins, and their specific extracellular matrix ligands. A large body of evidence from in vitro and in vivo models demonstrates the importance of coordination of signals from the extracellular environment that activates specific tyrosine kinase receptors and integrins in order to regulate angiogenic processes in vivo. In addition to complex formation between growth factor receptors and integrins, growth factors and cytokines also directly interact with integrins, depending upon their concentration levels in the environment, and differentially regulate integrin-related processes. Recent studies from a number of laboratories including ours have provided important novel insights into the involvement of many signaling events that improve our existing knowledge on the cross-talk between growth factor receptors and integrins in the regulation of angiogenesis. In this review, our focus will be on updating the recent developments in the field of integrin-growth factor receptor associations and their implications in the vascular processes.     

14-3-3beta-Rac1-p21 activated kinase signaling regulates Akt1-mediated cytoskeletal organization, lamellipodia formation and fibronectin matrix assembly

Akt1 belongs to the three-gene Akt family and functions as a serine-threonine kinase regulating phosphorylation of an array of substrates and mediating cellular processes such as cell migration, proliferation, survival, and cell cycle. Our previous studies have established the importance of Akt1 in angiogenesis and absence of Akt1 resulted in impaired integrin activation, adhesion, migration, and extracellular matrix assembly by endothelial cells and fibroblasts. In this study, we identify the downstream signaling pathways activated by Akt1 in the regulation of these cellular events. We demonstrate here that Akt1 is necessary for the growth factor stimulated activation of 14-3-3beta-Rac1-p21 activated kinase (Pak) pathway in endothelial cells and fibroblasts. While activation of Akt1 resulted in translocation of Rac1 to membrane ruffles, enhanced Rac1 activity, Pak1 phosphorylation, and lamellipodia formation, resulting in enhanced adhesion and assembly of fibronectin, inhibition of Akt1 resulted in inhibition of these processes due to impaired Rac1-Pak signaling. Formation of lamellipodia, adhesion, and fibronectin assembly by myristoylated Akt1 expression in NIH 3T3 fibroblasts was inhibited by co-expression with either dominant negative Rac1 or dominant negative Pak1. In contrast, impaired lamellipodia formation, adhesion, and fibronectin assembly by dominant negative-Akt1 expression was rescued by co-expression with either constitutively active-Rac1 or -Pak1. Moreover, previously reported defects in adhesion and extracellular matrix assembly by Akt1(-/-) fibroblasts could be rescued by expression with either active-Rac1 or -Pak1, implying the importance of Rac1-Pak signaling in growth factor stimulated cytoskeletal assembly, lamellipodia formation and cell migration in endothelial cells and fibroblasts downstream of Akt1 activation.     

Multidrug resistant yeasts in synanthropic wild birds

Background

Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR) assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs).

Methods

Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification).

Results

Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%.

Conclusions

This reliable technique may offer a rapid (<1.5 h) tool that would help clinicians to initiate an appropriate treatment earlier. Further investigations are needed to assess the clinical benefit of this novel strategy as compared to phenotypic methods.     

Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation

Testis- and sperm-specific protein phosphatase, PP1gamma2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1gamma2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1gamma2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1gamma2 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936-1942). The objectives of this study were to determine: 1) stoichiometry of PP1gamma2-sds22 binding and 2) whether PP1gamma2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1gamma2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1gamma2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1gamma2 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP1gamma2 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP1gamma2-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP1gamma2 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1gamma2 (39 kDa), suggesting a 1:1 complex between these two proteins. PP1gamma2 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1gamma2. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1gamma2 during epididymal sperm maturation.