A Review on Natural Sources Derived Protein Nanoparticles as Anticancer Agents.

Cancer notably carcinoma represents a prominent health challenge worldwide. A variety of chemotherapeutic agents are being used to deal with a variety of carcinomas. However, these delivering agents not only enter the targeted site but also affect normal tissues yielding poor therapeutic outcomes. Chemotherapeutic-associated problems are being attributed to drug non-specificity resulting from poor drug delivery systems. These problems are now being solved using nanomedicine, which entails using nanoparticles as drug delivery systems or nanocarriers. This nanoparticle-based drug delivery system enhances clinical outcomes by enabling targeted delivery, improving drug internalization, enhanced permeability, easy biodistribution, prolonged circulation and enhanced permeability rate, thereby improving the therapeutic effectiveness of several anticancer agents. Natural Protein-based Nanoparticles (PNPs) such as ferritin, lipoprotein, and lectins from natural sources have gained extensive importance at a scientific community level as nanovehicle for effective drug delivery and photo acoustic labeling replacing several synthetic nanocarriers that have shown limited therapeutic outcomes. The bioavailability of PNP, the chance of genetic engineering techniques to modify their biological properties made them one of the important raw material sources for drug delivery research. This current review highlighted different chemotherapeutic agents used in the treatment of some carcinomas. It also focused on the wide variety of natural protein sources derived nanoparticles (NPs) as anticancer delivery of agents for cancer therapy.

XFEL and NMR Structures of Francisella Lipoprotein Reveal Conformational Space of Drug Target against Tularemia.

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia.

Lipopolysaccharide derived alginate coated Hepatitis B antigen loaded chitosan nanoparticles for oral mucosal immunization.

Mucosal administration of vaccine can produce a strong immune response. Antigens adhere to "M-cells", present at the intestinal mucosa and the M-cells produce immunity after actively transporting luminal antigens to the underlying immune cells. The objective of the present study was to prepare and characterize alginate coated chitosan nanoparticles (ACNPs) loaded with HBsAg as an antigen to produce immunity; additionally anchored with lipopolysaccharide (LPS) as an adjuvant. Ionic gelation method was used to prepare chitosan nanoparticles (CNPs) which were loaded with HBsAg and stabilized by alginate coating to protect from gastric environment. Results showed that the prepared LPS-HB-ACNPs were small and spherical with mean particle size 605.23 nm, polydispersity index 0.234 and Zeta potential -26.2 mV and could effectively protect antigen at GIT in acidic medium. HB-ANCPs were stable during storage at 4 ± 1 and 27 ± 2 °C. Anchoring with LPS showed increased immunity as compared to other formulations. Additionally, NPs elicited significant sIgA at mucosal secretions and IgG antibodies in systemic circulation. Thus, the prepared LPS anchored alginate coated chitosan NPs may be a promising approach as a vaccine delivery system for oral mucosal immunization.

Targeting Lipoprotein Biogenesis: Considerations towards Antimicrobials.

Decades have passed without approval of a new antibiotic class. Several companies have recently halted related discovery efforts because of multiple obstacles. One promising route under research is to target the lipoprotein maturation pathway in light of major recent findings and the virulence roles of lipoproteins. To support the future design of selective drugs, considerations and priority-setting are established for the main lipoprotein processing enzymes (Lgt, LspA, and Lnt) based on microbiology, biochemistry, structural biology, chemical design, and pharmacology. Although not all bacterial species will be similarly impacted by drug candidates, several advantages make LspA a top target to pursue in the development of novel antibiotics effective against bacteria that are resistant to existing drugs.

Bread enriched with phytosterols with or without curcumin modulates lipoprotein profiles in hypercholesterolaemic individuals. A randomised controlled trial.

We previously demonstrated that the combination of phytosterols (PS) and curcumin administered as dietary supplements significantly lowers LDL-cholesterol (LDL-C) more than either treatment alone. The aim of this study was to investigate the effects of this combination in a novel food (bread) on plasma lipid profiles in hypercholesterolaemic individuals. In a double-blinded, placebo-controlled, 2 × 2 factorial trial, participants were randomised to receive bread fortified with placebo (PL), 2.3 g PS (PS), 228 mg curcumin (CC) or a combination of 2.3 g PS and 228 mg CC (PS-CC) daily for four weeks. Primary outcomes were fasting plasma lipids [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG)] and secondary outcomes were plasma LDL-particle (LDL-P) profile: LDL-P number and LDL-P size. Cardiovascular disease (CVD) risk (Framingham Risk Algorithm) was also explored. There was no significant difference between PL and CC or PS and PS-CC on blood lipids or CVD risk; therefore, groups were pooled for final analysis: the PL and CC group (PL-C, n = 36) and the PS and PS-CC group (PS-C, n = 39). PS-C significantly lowered TC (-0.52 mmol L-1, p < 0.0001), LDL-C (-0.49 mmol L-1, p < 0.0001) and CVD risk (-1.1 absolute %, p = 0.0005) compared to the PL-C group. Reductions from baseline in the PS-C group compared to that in the PL-C group were 7.6% and 10.6% for TC and LDL-C, respectively, and statistically significant (p < 0.0001). CVD-risk in the PS-C group reduced significantly (-12.7%) compared to that in the PL-C group (p = 0.0005). HDL-C and TG remained unchanged. The LDL-P number significantly decreased in the PS-C group by 124.33 nmol L-1 compared to that in the PL-C group (p = 0.005) and both groups showed a significant decrease in LDL-P size (p < 0.01); however, the absolute nm change in LDL-P size did not differ between groups and the percent change in LDL-P size in the PS-C group was borderline significant (-0.89%, p = 0.05) compared to that in the PL-C group. Regular consumption of PS-enriched bread with or without curcumin lowers blood cholesterol; however, curcumin alone did not influence blood lipids. Bread may be a convenient means of delivering PS with greater compliance for reducing the blood cholesterol concentration.

Superoxide-producing lipoprotein fraction from Stevia leaves: definition of specific activity.

BACKGROUND: Stevia rebaudiana Bertoni has various pharmacological actions, which includes antidiabetic, antioxidant, anti-inflammatory activities. The superoxide and consequently NADPH oxidase (Nox) are relevant targets involved in biological effects of Stevia. The presence of NADPH-containing superoxide-producing lipoprotein (suprol) in Stevia leaves has not yet been tested. The mechanism of producing superoxide radicals (O2-) by suprol was determined in vitro, which is associated with the electron transfer from NADPH in the composition of suprol by traces of transition metal ions (Fe3+ or Cu2+) to molecular oxygen, turning it into O2-. It is expected that the therapeutic efficacy of Stevia leaves is caused by specific activity of superoxide-producing lipoprotein fraction. METHODS: For the first time, from the dry leaves of Stevia the NADPH-containing superoxide-producing lipoprotein was isolated and purified. The specific content of suprol (milligrams in 1 g of Stevia leaves- mg/g) was determined after desalination of suprol and lyophilization. RESULTS: According to the method provided, the specific content of the isolated suprol from Stevia's leaves was up to 4.5 ± 0.2 mg / g (yields up to 68.5 ± 4.7%, p < 0.05, n = 6). Nox forms a stable complex with suprol. The optical absorption spectrum of the Nox-suprol complex represents the overlapping suprol and Nox spectra, with a certain background increase and characteristic features of optical absorption for Nox. Due to O2- producing capacity suprol-Nox complex discolors KMnO4 solutions, Coomassie brilliant blue, restores nitrotetrazolium blue to formazan and oxidizes epinephrine to adrenochrome. The oxidation activity of adrenaline is 50.3 ± 5.1 U / mg / ml (p < 0.05, n = 6). CONCLUSION: Superoxide-producing lipoprotein fraction-Nox complex from Stevia leaves (membranes) can modulate redox regulated signaling pathways and may play a positive role in type-2 diabetes by means of adrenaline oxidation mechanism.

Site-Specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction.

Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplAW37V) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif-a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DAinv). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

Novel cremochylomicrons for improved oral bioavailability of the antineoplastic phytomedicine berberine chloride: Optimization and pharmacokinetics.

Berberine chloride (BER) is an antineoplastic phytomedicine that combat non-Hodgkin lymphoma. BER suffers from low oral bioavailability due to p-glycoprotein efflux and first-pass metabolism. Lymphatic drug targeting recently gained a profound attention due to circumventing hepatic first-pass metabolism and targeting lymph diseases. Therefore, novel BER-loaded cremochylomicrons were elaborated to mitigate BER drawbacks and enhance its lymphatic targeting and bioavailability. Optimized cremochylomicron was prepared with 2.5%w/v Cremophor El and 12.5% w/w berberine content. Promising in vitro characteristics (particle size = 175.6 nm and entrapment efficiency = 95.5%) were obtained. Lyophilized system showed high colloidal stability over 6 months. In addition in vivo pharmacokinetics study demonstrated significant enhancement (>2fold) in the rate and extent of absorption in cremochylomicron over free BER. Moreover, cremochylomicrons demonstrated in significant increase in mean residence time and volume of distribution with decreased intestinal drug clearance as a result of efflux inhibition. In another avenue, a significant reduction in BER absorption (43%) in presence of cycloheximide inhibitor was obtained confirming the lymphatic targeting ability of cremochylomicrons. In conclusion, berberine-loaded cremochylomicron could be considered as a promising nanoplatform for targeting lymphatic system and improving BER oral bioavailability with lower dose and side effects.

Nonpyrogenic Molecular Adjuvants Based on norAbu-Muramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo.

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).

Lichenysin-geminal amino acid-based surfactants: Synergistic action of an unconventional antimicrobial mixture.

Recently it has been demonstrated that catanionic mixtures of oppositely charged surfactants have improved physicochemical-biological properties compared to the individual components. Isotherms of mixtures of an anionic biosurfactant (lichenysin) and a cationic aminoacid surfactant (C3(LA)2) indicate a strong interaction suggesting the formation of a new "pseudo-surfactant". The antimicrobial properties of the mixture lichenysin and C3(LA)2 M80:20, indicate a synergistic effect of the components. The mechanism of action on the bacterial envelope was assessed by flow cytometry and Transmission Electron Microscopy.

Lutein and atherosclerosis: Belfast versus Toulouse revisited.

In 1995 we reported that mean plasma lutein concentrations in salaried men and women from Toulouse in Southern France were double those in subjects recruited from general practitioner lists in Belfast, Northern Ireland. At the time incidence of coronary heart disease (CHD) in Southern France was among the lowest in Europe and was much higher in Northern Ireland. Plasma lutein is a biomarker of vegetable and fruit intake and evidence suggests that high concentrations are generally associated with better cardiometabolic health. At the time we speculated like others that role of the carotenoids may well have been to prevent oxidation of lipid in the lipoproteins and so reduce the uptake of oxidised lipid by macrophages and its deposition within the intimal layers of the major arteries as plaque. It is now widely accepted that CHD is an inflammatory disease and that macrophages within plaque together with tissue damage contribute to this inflammation. Stimulated macrophages release cytokines to activate the immune system both locally and systemically. Precursor complement proteins in the blood are activated to assist immune cells in phagocytosis and cell repair. Individuals with a history of arteriosclerosis display significantly higher concentrations of complement factors C3 and C3a than subjects without such a history. Metabolism of C3 via the alternate complement pathway can give rise to the membrane attack complex (MAC) which creates a hole or pore in pathogens or host cells, killing the cell. Recent studies in patients with early age related macular disease (AMD) who also exhibit similar elevated concentrations of complement proteins in their blood, showed supplementation with lutein progressively decreased the amount of the MAC and other complement factors in the blood. Lutein was used in the supplementation experiments because it is an important constituent of macular pigment. Thus the healthier cardiometabolic features displayed by the people in Toulouse may have been due to the effects of concurrent high concentrations of plasma lutein on the immune system and complement in particular. Other carotenoids may exert similar antioxidant effects but we and others found no differences in antioxidant nutrients between subjects in Toulouse and Belfast or between subjects with asymptomatic markers of atherosclerosis and controls.

Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging.

Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm(-1)) and lipid (~2845 cm(-1)) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

Lipoprotein binding to anionic biopolyelectrolytes and the effect of glucose on nanoplaque formation in arteriosclerosis and Alzheimer's disease.

Arteriosclerosis with its clinical sequelae (cardiac infarction, stroke, peripheral arterial occlusive disease) and vascular/Alzheimer dementia not only result in far more than half of all deaths but also represent dramatic economic problems. The reason is, among others, that diabetes mellitus is an independent risk factor for both disorders, and the number of diabetics strongly increases worldwide. More than one-half of infants in the first 6months of life have already small collections of macrophages and macrophages filled with lipid droplets in susceptible segments of the coronary arteries. On the other hand, the authors of the Bogalusa Heart Study found a strong increase in the prevalence of obesity in childhood that is paralleled by an increase in blood pressure, blood lipid concentration, and type 2 diabetes mellitus. Thus, there is a clear linkage between arteriosclerosis/Alzheimer's disease on the one hand and diabetes mellitus on the other hand. Furthermore, it has been demonstrated that distinct apoE isoforms on the blood lipids further both arteriosclerotic and Alzheimer nanoplaque formation and therefore impair flow-mediated vascular reactivity as well. Nanoplaque build-up seems to be the starting point for arteriosclerosis and Alzheimer's disease in their later full clinical manifestation. In earlier work, we could portray the anionic biopolyelectrolytes syndecan/perlecan as blood flow sensors and lipoprotein receptors in cell membrane and vascular matrix. We described extensively molecular composition, conformation, form and function of the macromolecule heparan sulfate proteoglycan (HS-PG). In two supplementary experimental settings (ellipsometry, myography), we utilized isolated HS-PG for in vitro nanoplaque investigations and isolated human coronary artery segments for in vivo tension measurements. With the ellipsometry-based approach, we were successful in establishing a direct connection on a molecular level between diabetes mellitus on the one side and arteriosclerosis/Alzheimer's disease on the other side. Application of glucose at a concentration representative for diabetics and leading to glycation of proteins and lipids, entailed a significant increase in arteriosclerotic and Alzheimer nanoplaque formation. IDLapoE4/E4 was by far superior to IDLapoE3/E3 in plaque build-up, both in diabetic and non-diabetic patients. Recording vascular tension of flow-dependent reactivity in blood substitute solution and under application of different IDLapoE isoforms showed an impaired vasorelaxation for pooled IDL and IDLapoE4/E4, thus confirming the ellipsometric investigations. Incubation in IDLapoE0/E0 (apoE "knockout man"), however, resulted in a massive flow-mediated contraction, also complemented by strongly aggregated nanoplaques. In contrast, HDL was shown to present a powerful protection against nanoplaque formation on principle, both in the in vitro model and the in vivo scenario on the endothelial cell membrane. The competitive interplay with LDL is highlighted through the flow experiment, where flow-mediated, HDL-induced vasodilatation remains untouched by additional incubation with LDL. This is due to the four times higher affinity for the proteoglycan receptor of HDL as compared to LDL. Taken together, the studies demonstrate that while simplistic, the ellipsometry approach and the endothelial-mimicking proteoglycan-modified surfaces provide information on the initial steps of lipoprotein-related plaque formation, which correlates with findings on endothelial cells and blood vessels, and afford insight into the role of lipoprotein deposition and exchange phenomena at the onset of these pathophysiologies.

Consumption of high-dose vitamin C (1250 mg per day) enhances functional and structural properties of serum lipoprotein to improve anti-oxidant, anti-atherosclerotic, and anti-aging effects via regulation of anti-inflammatory microRNA.

Background Although the health effects of vitamin C are well known, its physiological effect on serum lipoproteins and microRNA still remain to be investigated, especially daily consumption of a high dosage. Objectives To investigate the physiological effect of vitamin C on serum lipoprotein metabolism in terms of its anti-oxidant and anti-glycation activities, and gene expression via microRNA regulation. Methods We analyzed blood parameters and lipoprotein parameters in young subjects (n = 46, 22 ± 2 years old) including smokers who consumed a high dose of vitamin C (1250 mg) daily for 8 weeks. Results Antioxidant activity of serum was enhanced with the elevation of Vit C content in plasma during 8 weeks consumption. In the LDL fraction, the apo-B48 band disappeared at 8 weeks post-consumption in all subjects. In the HDL fraction, apoA-I expression was enhanced by 20% at 8 weeks, especially in male smokers. In the lipoprotein fraction, all subjects showed significantly reduced contents of advanced glycated end products and reactive oxygen species (ROS). Triglyceride (TG) contents in each LDL and HDL fraction were significantly reduced in all groups following the Vit C consumption, suggesting that the lipoprotein was changed to be more anti-inflammatory and atherogenic properties. Phagocytosis of LDL, which was purified from each individual, into macrophages was significantly reduced at 8-weeks post-consumption of vitamin C. Anti-inflammatory and anti-senescence effects of HDL from all subjects were enhanced after the 8-weeks consumption. The expression level of microRNA 155 in HDL3 was reduced by 49% and 75% in non-smokers and smokers, respectively. Conclusion The daily consumption of a high dose of vitamin C for 8 weeks resulted in enhanced anti-senescence and anti-atherosclerotic effects via an improvement of lipoprotein parameters and microRNA expression through anti-oxidation and anti-glycation, especially in smokers.

Colorimetric polymer assay for the diagnosis of plasma lipids atherogenic quality in hypercholesterolemic patients.

OBJECTIVE: Hypercholesterolemia (increased blood cholesterol level) is considered a major risk factor for developing atherosclerotic diseases. As such, alerting individuals on hypercholesterolemic conditions is a crucial component in averting onset of atherosclerosis and its outcome-cardiovascular diseases. While common diagnostic tools such as cholesterol and lipoproteins determination are widely employed for hypercholesterolemia screening, their effectiveness has been questioned since they do not shed light on critical physiological factors like lipid oxidation and inflammation levels, which constitute prominent determinants for development of atherosclerotic diseases. The objective of this study is to develop a simple assay for identifying hypercholesterolemia, and assessing the impact of therapeutic treatments. METHODS: We developed a diagnostic assay based upon color transformations of polydiacetylene, a unique conjugated polymer, upon interactions with blood plasma obtained from healthy individuals, hypercholesterolemic patients, hypercholesterolemic patients treated with statin, and hypercholesterolemic patients treated with statin together with pomegranate extracts. The color transformations of the polymer were monitored through desktop color scanning combined with colorimetric image analysis. RESULTS: We show that the colorimetric assay was able to distinguish among plasma. Bio-analytical characterization reveals that the distinct colorimetric responses likely arise from interactions with plasma lipoproteins. Importantly, the colorimetric changes are not simply correlated with the relative abundance of cholesterol (or other lipids) in the plasma of hypercholesterolemic or healthy patients, but also reflect the presence of oxidized and inflamed species. CONCLUSIONS: This paper introduces a simple color assay for detection of hypercholesterolemia and monitoring the effect of therapies directed at mitigating this physiological condition. The colorimetric system might constitute a novel platform for assessing patient vulnerability towards the development of atherosclerosis.

Therapeutic epitopes of Leptospira LipL32 protein and their characteristics.

Two LipL32-specific mouse monoclonal antibodies (mAbLPF1 and mAbLPF2) which neutralized Leptospira-mediated hemolysis in vitro and rescued hamsters from lethal Leptospira infection were produced. In this communication, locations and characteristics of the protective epitopes of the mAbs were studied by using a truncated LipL32 recombinant protein based-immunoassay and phage consensus mimotope identification and multiple alignments. The mAbLPF1 epitope consisted of P243, L244, I245, H246, L252 and Q253 on the LipL32 protein; it is mapped on the surface-exposed region of non-continuous ß13-turn and C-terminal amphipathic α6 helix with hydrophobic patch, contributing to phospholipid/host cell adhesion and membrane insertion on one side, and hydrophilic, acidic and basic amino acid residues on another side. The epitope peptide of the mAbLPF2 is linear 122PEEKSMPHW130 and located on surface-exposed α1 and α2 between ß7 and ß8 that bound to several host constituents. Both epitopes are highly conserved among the pathogenic and intermediately pathogenic Leptospira spp. and are absent from the LipL32 superfamily proteins of other microorganisms. This study not only enlightens the molecular mechanisms of the therapeutic mAbLPF1 and mAbLPF2, but also elaborates the potential of the two LipL32 regions as diagnostic and vaccine targets for leptospirosis.

Varied metal-binding properties of lipoprotein PsaA in Streptococcus pneumoniae.

Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn(2+) and Zn(2+) transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn(2+) and Zn(2+) binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA-Mn(2+) has a lower thermal stability than PsaA-Zn(2+), possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA-Mn(2+) binding is a fast first-order reaction, whereas PsaA-Zn(2+) binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn(2+) to Zn(2+). The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae.

In vivo magnetic enrichment, photoacoustic diagnosis, and photothermal purging of infected blood using multifunctional gold and magnetic nanoparticles.

Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic.

Preformulation characterization of an aluminum salt-adjuvanted trivalent recombinant protein-based vaccine candidate against Streptococcus pneumoniae.

The preformulation of a trivalent recombinant protein-based vaccine candidate for protection against Streptococcus pneumoniae is described both in the presence and in the absence of aluminum salt adjuvants. The biophysical properties of the three protein-based antigens, fragments of pneumococcal surface adhesion A (PsaA), serine-threonine protein kinase (StkP), and protein required for cell wall separation of group B streptococcus (PcsB), were studied using several spectroscopic and light scattering techniques. An empirical phase diagram was constructed to assess the overall conformational stability of the three antigens as a function of pH and temperatures. A variety of excipients were screened on the basis of their ability to stabilize each antigen using intrinsic fluorescence spectroscopy and circular dichroism spectroscopy. Sorbitol, sucrose, and trehalose stabilized the three proteins in solution. The addition of manganese also showed a drastic increase in the thermal stability of SP1650 in solution. The adsorption and desorption processes of each of the antigens to aluminum salt adjuvants were evaluated, and the stability of the adsorbed proteins was then assessed using intrinsic fluorescence spectroscopy and Fourier transform infrared spectroscopy. All the three proteins showed good adsorption to Alhydrogel. PsaA was destabilized when adsorbed onto Alhydrogel® and adding sodium phosphate showed a stabilizing effect. PcsB was found to be stabilized when adsorbed to Alhydrogel®, and no destabilizing or stabilizing effects were seen in the case of StkP.

Differential effects of the trans-18:1 isomer profile of partially hydrogenated vegetable oils on cholesterol and lipoprotein metabolism in male F1B hamsters.

Trans-fatty acid consumption from partially hydrogenated vegetable oil (PHVO) has been positively associated with multiple cardiovascular disease risk factors and events. This study was designed to examine the effects of trans-fatty acid isomer profile of PHVO on plasma lipids and lipoproteins and hepatic expression of key genes involved in cholesterol and fatty acid metabolism. Thirty-three male F(1)B strain Syrian Golden Hamsters were allocated to 1 of 3 hypercholesterolemic diets containing (5% by weight): 1) tristearin [control fat (CON)]; 2) partially hydrogenated high-oleic acid sunflower oil (PH-SUN); or 3) partially hydrogenated high-linoleic acid safflower oil (PH-SAF). PH-SUN contained more trans-4 to trans-10 18:1 compared with PH-SAF, which contained more trans-11 to trans-16 18:1. The addition of both PHVO to the diet increased plasma total cholesterol concentrations relative to CON, but only PH-SUN increased the plasma ratio of non-HDL:HDL cholesterol compared with CON. PH-SUN increased VLDL (total, large, and medium) and IDL particle concentrations while decreasing total, medium, and small HDL particle concentrations relative to CON. Both PHVO diets increased the hepatic cholesterol ester concentration, whereas the hepatic TG concentration was lower in PH-SUN compared with PH-SAF and CON. Levels of hepatic LDL receptor, HMG-CoA reductase, and sterol response element binding protein 1 mRNA were specifically reduced in the PH-SUN group compared to the CON group. Expression of SREBP1c was upregulated in both PHVO groups compared to CON, whereas only the PH-SAF group had higher levels of the lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 compared to CON. These results indicate that differences in the trans-fatty acid profile of PHVO can differentially affect lipid and lipoprotein metabolism.