Comparative Hippocampal Proteome and Phosphoproteome in a Niemann-Pick, Type C1 Mouse Model Reveal Insights into Disease Mechanisms.

Niemann-Pick disease, type C (NPC) is a neurodegenerative, lysosomal storage disorder in individuals carrying two mutated copies of either the NPC1 or NPC2 gene. Consequently, impaired cholesterol recycling and an array of downstream events occur. Interestingly, in NPC, the hippocampus displays lysosomal lipid storage but does not succumb to progressive neurodegeneration as significantly as other brain regions. Since defining the neurodegeneration mechanisms in this disease is still an active area of research, we use mass spectrometry to analyze the overall proteome and phosphorylation pattern changes in the hippocampal region of a murine model of NPC. Using 3 week old mice representing an early disease time point, we observed changes in the expression of 47 proteins, many of which are consistent with the previous literature. New to this study, changes in members of the SNARE complex, including STX7, VTI1B, and VAMP7, were identified. Furthermore, we identified that phosphorylation of T286 on CaMKIIα and S1303 on NR2B increased in mutant animals, even at the late stage of the disease. These phosphosites are crucial to learning and memory and can trigger neuronal death by altering protein-protein interactions.

Red blood cell transfusion-induced non-transferrin-bound iron promotes Pseudomonas aeruginosa biofilms in human sera and mortality in catheterized mice.

Transfusion of storage-damaged red blood cells (RBCs) increases non-transferrin-bound iron (NTBI) levels in humans. This can potentially enhance virulence of microorganisms. In this study, Pseudomonas aeruginosa replication and biofilm production in vitro correlated with NTBI levels of transfused subjects (R2 = 0·80; P < 0·0001). Transfusion of stored RBCs into catheterized mice enhanced P. aeruginosa virulence and mortality in vivo, while pre-administration of apotransferrin reduced NTBI levels improving survival (69% vs 27% mortality; P < 0·05). These results suggest that longer RBC storage, by modulating the bioavailability of iron, may increase the risk of P. aeruginosa biofilm-related infections in transfused patients.

Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase.

Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from Escherichia coli DHFR (ecDHFR) as a model, but less studies have used the enzyme from Homo sapiens DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction. The corresponding sites in hsDHFR are F28, 62-PEKN, and 26-PPLR. Each of these sites was studied here through the creation of mutant variants of the enzyme and measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs) on the reaction. F28 is mutated first to M (F28M) and then to the L of the bacterial enzyme (F28L). The KIEs of the F28M variant are larger and more temperature-dependent than wild-type (WT), suggesting a broader and longer average DAD at the TRS. To more fully mimic ecDHFR, we also study a triple mutant of the human enzyme (F32L-PP26N-PEKN62G). Remarkably, the intrinsic KIEs, while larger in magnitude, are temperature-independent like the WT enzymes. We also construct deletion mutations of hsDHFR removing both the 62-PEKN and 26-PPLR sequences. The results mirror those described previously for insertion mutants of ecDHFR. Taken together, these results suggest a balancing act during DHFR evolution between achieving an optimal TRS for hydride transfer and preventing product inhibition arising from the different intercellular pools of NADPH and NADP+ in prokaryotic and eukaryotic cells.

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

KEY POINTS: This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. ABSTRACT: Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Novel in vivo mouse model of shoulder implant infection.

BACKGROUND: Animal models are used to guide management of periprosthetic implant infections. No adequate model exists for periprosthetic shoulder infections, and clinicians thus have no preclinical tools to assess potential therapeutics. We hypothesize that it is possible to establish a mouse model of shoulder implant infection (SII) that allows noninvasive, longitudinal tracking of biofilm and host response through in vivo optical imaging. The model may then be employed to validate a targeting probe (1D9-680) with clinical translation potential for diagnosing infection and image-guided débridement. METHODS: A surgical implant was press-fit into the proximal humerus of c57BL/6J mice and inoculated with 2 µL of 1 × 103 (e3), or 1 × 104 (e4), colony-forming units (CFUs) of bioluminescent Staphylococcus aureus Xen-36. The control group received 2 µL sterile saline. Bacterial activity was monitored in vivo over 42 days, directly (bioluminescence) and indirectly (targeting probe). Weekly radiographs assessed implant loosening. CFU harvests, confocal microscopy, and histology were performed. RESULTS: Both inoculated groups established chronic infections. CFUs on postoperative day (POD) 42 were increased in the infected groups compared with the sterile group (P < .001). By POD 14, osteolysis was visualized in both infected groups. The e4 group developed catastrophic bone destruction by POD 42. The e3 group maintained a congruent shoulder joint. Targeting probes helped to visualize low-grade infections via fluorescence. DISCUSSION: Given bone destruction in the e4 group, a longitudinal, noninvasive mouse model of SII and chronic osteolysis was produced using e3 of S aureus Xen-36, mimicking clinical presentations of chronic SII. CONCLUSION: The development of this model provides a foundation to study new therapeutics, interventions, and host modifications.

Modeling rare diseases with induced pluripotent stem cell technology.

Rare diseases, in totality, affect a significant proportion of the population and represent an unmet medical need facing the scientific community. However, the treatment of individuals affected by rare diseases is hampered by poorly understood mechanisms preventing the development of viable therapeutics. The discovery and application of cellular reprogramming to create novel induced pluripotent stem cell models of rare diseases has revolutionized the rare disease community. Through developmental and functional analysis of differentiated cell types, these stem cell models carrying patient-specific mutations have become an invaluable tool for rare disease research. In this review article, we discuss the reprogramming of samples from individuals affected with rare diseases to induced pluripotent stem cells, current and future applications for this technology, and how integration of genome editing to rare disease research will help to improve our understanding of disease pathogenesis and lead to patient therapies.

Non-invasive three-dimensional imaging of Escherichia coli K1 infection using diffuse light imaging tomography combined with micro-computed tomography.

In contrast to two-dimensional bioluminescence imaging, three dimensional diffuse light imaging tomography with integrated micro-computed tomography (DLIT-µCT) has the potential to realise spatial variations in infection patterns when imaging experimental animals dosed with derivatives of virulent bacteria carrying bioluminescent reporter genes such as the lux operon from the bacterium Photorhabdus luminescens. The method provides an opportunity to precisely localise the bacterial infection sites within the animal and enables the generation of four-dimensional movies of the infection cycle. Here, we describe the use of the PerkinElmer IVIS SpectrumCT in vivo imaging system to investigate progression of lethal systemic infection in neonatal rats following colonisation of the gastrointestinal tract with the neonatal pathogen Escherichia coli K1. We confirm previous observations that these bacteria stably colonize the colon and small intestine following feeding of the infectious dose from a micropipette; invading bacteria migrate across the gut epithelium into the blood circulation and establish foci of infection in major organs, including the brain. DLIT-µCT revealed novel multiple sites of colonisation within the alimentary canal, including the tongue, oesophagus and stomach, with penetration of the non-keratinised oesophageal epithelial surface, providing strong evidence of a further major site for bacterial dissemination. We highlight technical issues associated with imaging of infections in new born rat pups and show that the whole-body and organ bioburden correlates with disease severity.

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100.

The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) has become a model for understanding enzyme catalysis, and yet several details of its mechanism are still unresolved. Specifically, the mechanism of the chemical step, the hydride transfer reaction, is not fully resolved. We found, unexpectedly, the presence of two reactive ternary complexes [enzyme:NADPH:7,8-dihydrofolate (E:NADPH:DHF)] separated by one ionization event. Furthermore, multiple kinetic isotope effect (KIE) studies revealed a stepwise mechanism in which protonation of the DHF precedes the hydride transfer from the nicotinamide cofactor (NADPH) for both reactive ternary complexes of the WT enzyme. This mechanism was supported by the pH- and temperature-independent intrinsic KIEs for the C-H→C hydride transfer between NADPH and the preprotonated DHF. Moreover, we showed that active site residues D27 and Y100 play a synergistic role in facilitating both the proton transfer and subsequent hydride transfer steps. Although D27 appears to have a greater effect on the overall rate of conversion of DHF to tetrahydrofolate, Y100 plays an important electrostatic role in modulating the pKa of the N5 of DHF to enable the preprotonation of DHF by an active site water molecule.

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.

Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

SRC tyrosine kinases regulate neuronal differentiation of mouse embryonic stem cells via modulation of voltage-gated sodium channel activity.

Voltage-gated Na(+) channel activity is vital for the proper function of excitable cells and has been indicated in nervous system development. Meanwhile, the Src family of non-receptor tyrosine kinases (SFKs) has been implicated in the regulation of Na(+) channel activity. The present investigation tests the hypothesis that Src family kinases influence neuronal differentiation via a chronic regulation of Na(+) channel functionality. In cultured mouse embryonic stem (ES) cells undergoing neural induction and terminal neuronal differentiation, SFKs showed distinct stage-specific expression patterns during the differentiation process. ES cell-derived neuronal cells expressed multiple voltage-gated Na(+) channel proteins (Nav) and underwent a gradual increase in Na(+) channel activity. While acute inhibition of SFKs using the Src family inhibitor PP2 suppressed the Na(+) current, chronic inhibition of SFKs during early neuronal differentiation of ES cells did not change Nav expression. However, a long-lasting block of SFK significantly altered electrophysiological properties of the Na(+) channels, shown as a right shift of the current-voltage relationship of the Na(+) channels, and reduced the amplitude of Na(+) currents recorded in drug-free solutions. Immunocytochemical staining of differentiated cells subjected to the chronic exposure of a SFK inhibitor, or the Na(+) channel blocker tetrodotoxin, showed no changes in the number of NeuN-positive cells; however, both treatments significantly hindered neurite outgrowth. These findings suggest that SFKs not only modulate the Na(+) channel activation acutely, but the tonic activity of SFKs is also critical for normal development of functional Na(+) channels and neuronal differentiation or maturation of ES cells.

Preservation of protein dynamics in dihydrofolate reductase evolution.

The hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) is a model for examining how protein dynamics contribute to enzymatic function. The relationship between functional motions and enzyme evolution has attracted significant attention. Recent studies on N23PP Escherichia coli DHFR (ecDHFR) mutant, designed to resemble parts of the human enzyme, indicated a reduced single turnover rate. NMR relaxation dispersion experiments with that enzyme showed rigidification of millisecond Met-20 loop motions (Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J., Benkovic, S. J., and Wright, P. E. (2011) Science 332, 234-238). A more recent study of this mutant, however, indicated that fast motions along the reaction coordinate are actually more dispersed than for wild-type ecDHFR (WT). Furthermore, a double mutant (N23PP/G51PEKN) that better mimics the human enzyme seems to restore both the single turnover rates and narrow distribution of fast dynamics (Liu, C. T., Hanoian, P., French, T. H., Hammes-Schiffer, S., and Benkovic, S. J. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 10159-11064). Here, we measured intrinsic kinetic isotope effects for both N23PP and N23PP/G51PEKN double mutant DHFRs over a temperature range. The findings indicate that although the C-H→C transfer and dynamics along the reaction coordinate are impaired in the altered N23PP mutant, both seem to be restored in the N23PP/G51PEKN double mutant. This indicates that the evolution of G51PEKN, although remote from the Met-20 loop, alleviated the loop rigidification that would have been caused by N23PP, enabling WT-like H-tunneling. The correlation between the calculated dynamics, the nature of C-H→C transfer, and a phylogenetic analysis of DHFR sequences are consistent with evolutionary preservation of the protein dynamics to enable H-tunneling from well reorganized active sites.

Extension and limits of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a "global dynamic network" of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts.

Combination prophylactic therapy with rifampin increases efficacy against an experimental Staphylococcus epidermidis subcutaneous implant-related infection.

The incidence of infections related to cardiac devices (such as permanent pacemakers) has been increasing out of proportion to implantation rates. As management of device infections typically requires explantation of the device, optimal prophylactic strategies are needed. Cefazolin and vancomycin are widely used as single agents for surgical prophylaxis against cardiac device-related infections. However, combination antibiotic prophylaxis may further reduce infectious complications. To model a localized subcutaneous implant-related infection, a bioluminescent strain of Staphylococcus epidermidis was inoculated onto a medical-procedure-grade titanium disc, which was placed into a subcutaneous pocket in the backs of mice. In vivo bioluminescence imaging, quantification of ex vivo CFU from the capsules and implants, variable-pressure scanning electron microscopy (VP-SEM), and neutrophil enhanced green fluorescent protein (EGFP) fluorescence in LysEGFP mice were employed to monitor the infection. This model was used to evaluate the efficacies of low- and high-dose cefazolin (50 and 200 mg/kg of body weight) and vancomycin (10 and 110 mg/kg) intravenous prophylaxis with or without rifampin (25 mg/kg). High-dose cefazolin and high-dose vancomycin treatment resulted in almost complete bacterial clearance, whereas both low-dose cefazolin and low-dose vancomycin reduced the in vivo and ex vivo bacterial burden only moderately. The addition of rifampin to low-dose cefazolin and vancomycin was highly effective in further reducing the CFU harvested from the implants. However, vancomycin-rifampin was more effective than cefazolin-rifampin in further reducing the CFU harvested from the surrounding tissue capsules. Future studies in humans will be required to determine whether the addition of rifampin has improved efficacy in preventing device-related infections in clinical practice.

Microbiota of human breast tissue.

In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly reaching the ducts from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analyzed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria was detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactation. The principal phylum was Proteobacteria. The most abundant taxa in the Canadian samples were Bacillus (11.4%), Acinetobacter (10.0%), Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), Comamonadaceae (5.7%), Gammaproteobacteria (5.0%), and Prevotella (5.0%). In the Irish samples the most abundant taxa were Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%), and Pseudomonas (5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined.

Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice.

BACKGROUND: Although human red blood cell (RBC) units may be refrigerator stored for up to 42 days, transfusion of older RBCs acutely delivers a large bolus of iron to mononuclear phagocytes. Similarly, iron dextran circulates in plasma for hours to days and is progressively cleared by mononuclear phagocytes, which return iron to plasma. Finally, malaria infection continuously delivers iron to macrophages by intra- and extravascular hemolysis. Studies suggest that iron administration increases infectious risk. STUDY DESIGN AND METHODS: To assess the effects of increased iron availability on susceptibility to infection, we infected mice with model Gram-negative intracellular or extracellular pathogens (Salmonella typhimurium or Escherichia coli, respectively), accompanied by RBC transfusion, iron dextran administration, or malarial coinfection. RESULTS: In our mouse models, transfusion of older RBCs exacerbates infection with both Gram-negative pathogens. Although iron dextran exacerbates E. coli infection to a similar extent as transfusion of corresponding amounts of iron, higher iron doses are required to produce comparable effects with S. typhimurium. Coinfection of mice with Plasmodium yoelii and S. typhimurium produces overwhelming Salmonella sepsis. Finally, treating mice with antibiotics abrogates the enhancing effect on E. coli infection of both older RBC transfusion and iron dextran administration. CONCLUSIONS: Transfusion of older RBCs exacerbates Gram-negative infection to a similar extent as malaria coinfection or iron dextran administration. Appropriate antibiotic therapy abrogates the effect of older RBC transfusions on infection with E. coli. Iron delivery to macrophages may be an underappreciated mechanism mediating, at least some, adverse effects of RBC transfusions.

Opening up the optical imaging window using nano-luciferin.

PURPOSE: The objective of this study was to formulate nanoparticles of D-luciferin (Nano-Luc), DiR (Nano-DiR) and dual functional nanoparticles with DiR and luciferin (Nano-LucDiR) for in-vivo imaging as well as tracking of the nanoparticles in tumors. METHODS: Nano-Luc and Nano-LucDiR were prepared using different lipids, and subsequently characterized for loading and entrapment efficiency, physical properties, release profile, toxicity and stability. We utilized Response Surface Methodology (RSM) to optimize the nanoparticles using design of experiment (DOE Vr.8.0). Nano-Luc was evaluated against free luciferin to establish its pharmacokinetic parameters in mice. In-vivo imaging of tumors and tracking of nanoparticles was carried out with an IVIS® Spectrum-CT (Caliper) using xenograft, orthotopic and metastatic tumor models in BALB/c nude mice with different cell lines and different routes of nanoparticle administration (subcutaneous, intraperitoneal and intravenous). RESULTS: Particle size of both Nano-Luc and Nano-LucDiR were found to be <200 nm. Nano-Luc formulation showed a slow and controlled release upto 72 h (90%) in vitro. The optimized Nano-Luc had loading efficiency of 5.0 mg/ml with 99% encapsulation efficiency. Nano-Luc and Nano-LucDiR formulations had good shelf stability. Nano-Luc and Nano-LucDiR enhanced plasma half-life of luciferin compared to free luciferin thus providing longer circulation of luciferin in plasma enabling imaging of tumors for more than 24 h. Nano-LucDiR allowed simultaneous bioluminescent and fluorescent imaging to be conducted, with three-dimensional reconstruct of tumors without losing either signal during the acquisition time. CONCLUSION: Nano-Luc and Nano-LucDiR allowed prolonged reproducible in-vivo imaging of tumors, especially during multimodality 3D imaging.

Glutathione reductase facilitates host defense by sustaining phagocytic oxidative burst and promoting the development of neutrophil extracellular traps.

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.

Theranostic tumor homing nanocarriers for the treatment of lung cancer.

The drugs/strategies to selectively inhibit tumor blood supply have generated interest in recent years for enhancement of cancer therapeutics. The objective of this study was to formulate tumor homing PEGylated CREKA peptide conjugated theranostic nanoparticles of DIM-C-pPhC6H5 (DIM-P) and investigate in vivo antitumor activity as well as evaluate the targeted efficiency to lung tumors using imaging techniques. DIM-P loaded Nanoparticles (NCs-D) were prepared using lipids, and DOGS-NTA-Ni and the surface of NCs-D were modified with PEGylated CREKA peptide (PCNCs-D). PCNCs-D showed 3 fold higher binding to clotted plasma proteins in tumor vasculature compared to NCs-D. PCNCs-D showed 26%±4% and 22%±5% increase in tumor reduction compared to NCs-D in metastatic and orthotopic models respectively. In-vivo imaging studies showed ~40 folds higher migration of PCNCs-Di in tumor vasculature than NCs-Di. Our studies demonstrate the role of PCNCs-D as theranostic tumor homing drug delivery and imaging systems for lung cancer diagnosis and treatment. FROM THE CLINICAL EDITOR: This study demonstrates a very efficient delivery system to address lung cancer growth through blood supply inhibition.

Dishevelled localization and function are differentially regulated by structurally distinct sterols.

The Dishevelled (DVL) family of proteins form supramolecular protein and lipid complexes at the cytoplasmic interface of the plasma membrane to regulate tissue patterning, proliferation, cell polarity, and oncogenic processes through DVL-dependent signaling, such as Wnt/ß-catenin. While DVL binding to cholesterol is required for its membrane association, the specific structural requirements and cellular impacts of DVL-sterol association are unclear. We report that intracellular sterols which accumulate within normal and pathological conditions cause aberrant DVL activity. In silico and molecular analyses suggested orientation of the ß- and α-sterol face within the DVL-PDZ domain regulates DVL-sterol binding. Intracellular accumulation of naturally occurring sterols impaired DVL2 plasma membrane association, inducing DVL2 nuclear localization via Foxk2. Changes to intracellular sterols also selectively impaired DVL2 protein-protein interactions This work identifies sterol specificity as a regulator of DVL signaling, suggests intracellular sterols cause distinct impacts on DVL activity, and supports a role for intracellular sterol homeostasis in cell signaling.