Molecular Tweezers: Supramolecular Hosts with Broad-Spectrum Biological Applications.

Lysine-selective molecular tweezers (MTs) are supramolecular host molecules displaying a remarkably broad spectrum of biologic activities. MTs act as inhibitors of the self-assembly and toxicity of amyloidogenic proteins using a unique mechanism. They destroy viral membranes and inhibit infection by enveloped viruses, such as HIV-1 and SARS-CoV-2, by mechanisms unrelated to their action on protein self-assembly. They also disrupt biofilm of Gram-positive bacteria. The efficacy and safety of MTs have been demonstrated in vitro, in cell culture, and in vivo, suggesting that these versatile compounds are attractive therapeutic candidates for various diseases, infections, and injuries. A lead compound called CLR01 has been shown to inhibit the aggregation of various amyloidogenic proteins, facilitate their clearance in vivo, prevent infection by multiple viruses, display potent anti-biofilm activity, and have a high safety margin in animal models. The inhibitory effect of CLR01 against amyloidogenic proteins is highly specific to abnormal self-assembly of amyloidogenic proteins with no disruption of normal mammalian biologic processes at the doses needed for inhibition. Therapeutic effects of CLR01 have been demonstrated in animal models of proteinopathies, lysosomal-storage diseases, and spinal-cord injury. Here we review the activity and mechanisms of action of these intriguing compounds and discuss future research directions. SIGNIFICANCE STATEMENT: Molecular tweezers are supramolecular host molecules with broad biological applications, including inhibition of abnormal protein aggregation, facilitation of lysosomal clearance of toxic aggregates, disruption of viral membranes, and interference of biofilm formation by Gram-positive bacteria. This review discusses the molecular and cellular mechanisms of action of the molecular tweezers, including the discovery of distinct mechanisms acting in vitro and in vivo, and the application of these compounds in multiple preclinical disease models.

The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research.

Sarcospan (SSPN) is a 25-kDa transmembrane protein that is broadly expressed at the cell surface of many tissues, including, but not limited to, the myofibers from skeletal and smooth muscles, cardiomyocytes, adipocytes, kidney epithelial cells, and neurons. SSPN is a core component of the dystrophin-glycoprotein complex (DGC) that links the intracellular actin cytoskeleton with the extracellular matrix. It is also associated with integrin α7ß1, the predominant integrin expressed in skeletal muscle. As a tetraspanin-like protein with four transmembrane spanning domains, SSPN functions as a scaffold to facilitate protein-protein interactions at the cell membrane. Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy are caused by the loss of dystrophin at the muscle cell surface and a concomitant loss of the entire DGC, including SSPN. SSPN overexpression ameliorates Duchenne muscular dystrophy in the mdx murine model, which supports SSPN being a viable therapeutic target. Other rescue studies support SSPN as a biomarker for the proper assembly and membrane expression of the DGC. Highly specific and robust antibodies to SSPN are needed for basic research on the molecular mechanisms of SSPN rescue, pre-clinical studies, and biomarker evaluations in human samples. The development of SSPN antibodies is challenged by the presence of its four transmembrane domains and limited antigenic epitopes. To address the significant barrier presented by limited commercially available antibodies, we aimed to generate a panel of robust SSPN-specific antibodies that can serve as a resource for the research community. We created antibodies to three SSPN protein epitopes, including the intracellular N- and C-termini as well as the large extracellular loop (LEL) between transmembrane domains 3 and 4. We developed a panel of rabbit antibodies (poly- and monoclonal) against an N-terminal peptide fragment of SSPN. We used several assays to show that the rabbit antibodies recognize mouse SSPN with a high functional affinity and specificity. We developed mouse monoclonal antibodies against the C-terminal peptide and the large extracellular loop of human SSPN. These antibodies are superior to commercially available antibodies and outperform them in various applications, including immunoblotting, indirect immunofluorescence analysis, immunoprecipitation, and an ELISA. These newly developed antibodies will significantly improve the quality and ease of SSPN detection for basic and translational research.

Examination of SOD1 aggregation modulators and their effect on SOD1 enzymatic activity as a proxy for potential toxicity.

Small-molecule inhibitors of abnormal protein self-assembly are promising candidates for developing therapy against proteinopathies. Such compounds have been examined primarily as inhibitors of amyloid ß-protein (Aß), whereas testing of inhibitors of other amyloidogenic proteins has lagged behind. An important issue with screening compound libraries is that although an inhibitor suitable for therapy must be both effective and nontoxic, typical screening focuses on efficacy, whereas safety typically is tested at a later stage using cells and/or animals. In addition, typical thioflavin T (ThT)-fluorescence-based screens use the final fluorescence value as a readout, potentially missing important kinetic information. Here, we examined potential inhibitors of superoxide dismutase 1 (SOD1) using ThT-fluorescence including the different phases of fluorescence change and added a parallel screen of SOD1 activity as a potential proxy for compound toxicity. Some compounds previously reported to inhibit other amyloidogenic proteins also inhibited SOD1 aggregation at low micromolar concentrations, whereas others were ineffective. Analysis of the lag phase and exponential slope added important information that could help exclude false-positive or false-negative results. SOD1 was highly resistant to inhibition of its activity, and therefore, did not have the necessary sensitivity to serve as a proxy for examining potential toxicity.

Cell surface and extracellular proteins of potentially probiotic Lactobacillus reuteri as an effective mediator to regulate intestinal epithelial barrier function.

The present study aimed to evaluate the potential of cell surface and extracellular proteins in regulation of intestinal epithelial barrier (IEB) function. Eight potentially probiotic L. reuteri strains were evaluated for presence of mapA gene and its expression on co-culturing with the Caco-2 cells. The ability of untreated (Viable), heat-inactivated, 5 M LiCL treated L. reuteri strains as well as their cell-free supernatant (CFS) to modulate expression of IEB function genes (hBD-2, hBD-3, claudin-1 and occludin) was also evaluated. Caco-2 cells were treated with cell surface and extracellular protein extracts and investigated for change in expression of targeted IEB function genes. The results showed that mapA gene is present in all the tested L. reuteri strains and expression of mapA and its receptors (anxA13 and palm) increase significantly on co-culturing of L. reuteri and Caco-2 cells. Also, up-regulated expression of IEB function genes was observed on co-culturing of L. reuteri (viable, heat-inactivated and CFS) and their protein extracts with Caco-2 cells in contrast to down-regulation observed with the pathogenic strain of Salmonella typhi. Therefore, this study concludes that the cell surface and extracellular protein from L. reuteri act as an effective mediator molecules to regulate IEB function.

The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS.

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1's protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.

Hypocholesterolemic effects of Lactobacillus reuteri LR6 in rats fed on high-cholesterol diet.

The bacterium Lactobacillus reuteri LR6, an isolate from breast-fed human infant feces, was tested positive for bile tolerance and bile salt hydrolase activity. It was also evaluated as a potential probiotic with cholesterol-lowering effect in vivo. In this study, 32 male Albino rats were divided into four groups consisting of eight mice per group. For 60 d, group I was fed with normal synthetic diet, group II was fed with cholesterol-enriched diet only, group III was fed with cholesterol-enriched diet supplemented with skimmed milk, and group IV was fed with cholesterol-enriched diet supplemented with L. reuteri LR6-fermented skimmed milk (10(8) cfu/mL). Blood samples were taken to study lipid profile on 0th, 15th, 30th and 60th day. Compared with the control group, the values for total cholesterol (TC), triglyceride (TG), and LDL were reduced significantly in group fed with L. reuteri LR6 but for HDL this difference was not significant. The results indicated that L. reuteri LR6 might be effective as a probiotic with cholesterol-lowering activities.

Antibacterial efficacy of nisin, pediocin 34 and enterocin FH99 against L. monocytogenes, E. faecium and E. faecalis and bacteriocin cross resistance and antibiotic susceptibility of their bacteriocin resistant variants.

The bacteriocin susceptibility of Listeria monocytogenes MTCC 657, Enterococcus faecium DSMZ 20477, E. faecium VRE, and E. faecalis ATCC 29212 and their corresponding bacteriocin resistant variants was assessed. The single and combined effect of nisin and pediocin 34 and enterocin FH99 bacteriocins produced by Pediococcus pentosaceus 34, and E. faecium FH99, respectively, was determined. Pediocin34 proved to be more effective in inhibiting L. monocytogenes MTCC 657. A greater antibacterial effect was observed against E. faecium DSMZ 20477 and E. faecium (VRE) when the a combination of nisin, pediocin 34 and enterocin FH99 were used whereas in case of L. monocytogenes MTCC 657 a combination of pediocin 34 and enterocin FH99 was more effective in reducing the survival of pathogen. Bacteriocin cross-resistance and the antibiotic susceptibility of wild type and their corresponding resistant variants were assessed and results showed that resistance to a bacteriocin may extend to other bacteriocins within the same class and also the acquired resistance to bacteriocins can modify the antibiotic susceptibility/resistance profile of the bacterial species used in the study. According to the hydrophobicity nisin resistant variant of L. monocytogenes was more hydrophobic (p < 0.001), whereas the pediocin 34 and enterocin FH99 resistant variants were less hydrophobic than the wild type strain. Nisin, pediocin 34 and enterocin FH99 resistant variants of E. faecium DSMZ 20477 and E. faecium VRE were less hydrophobic than their wild type counterparts. Nisin resistant E. faecalis ATCC 29212 was less hydrophobic than its wild type counterpart.

Spore germination based assay for monitoring antibiotic residues in milk at dairy farm.

Spore germination based assay involves the transformation of dormant spores of Bacillus stearothermophilus 953 into active vegetative cells. The inhibition of germination process specifically in presence of antibiotic residues was used as a novel approach for monitoring target contaminants in milk. The indicator organism i.e., B. stearothermophilus 953 was initially allowed to sporulate by seeding in sporulation medium and incubating at 55 °C for 18 ± 2 h. The spores exhibited a typical chain behavior as revealed through phase contrast microscopy. The minimal medium inoculated with activated spores was incubated at 64 °C for 2-3 h for germination and outgrowth in presence of specific germinant mixture containing dextrose, whey powder and skimmed milk powder added in specific ratio along with reconstituted milk as negative control and test milk samples. The change in color of the medium from purple to yellow was used as criteria for detection of antibiotic residues in milk. The efficiency of the developed assay was evaluated through a surveillance study on 228 samples of raw, pasteurized and dried milks and results were compared with AOAC approved microbial receptor assay. The presence of antibiotic level was 10.08 % at Codex maximum residual limit having false positive result only in 0.43 % of the samples. The results of the present investigation suggest that developed spore based assay can be a practical solution to dairy industry for its application at farm level, milk processing units, independent testing and R & D centres in order to comply with the legal requirements set by Codex.

Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

Therapeutic Approaches Targeting Protein Aggregation in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease that targets motor neurons (MNs) in the brain and spinal cord. It leads to gradual loss of motor signals to muscles leading to atrophy and weakness. Most patients do not survive for more than 3-5 years after disease onset. Current ALS treatments provide only a small delay of disease progression. Therefore, it is of utmost importance to explore new therapeutic approaches. One of the major hindrances in achieving this goal is poor understanding of causes of the disease. ALS has complex pathophysiological mechanisms in its genetic and sporadic forms. Protein aggregates are a common hallmark of ALS regardless of cause making protein pathways attractive therapeutic targets in ALS. Here, we provide an overview of compounds in different stages of pharmacological development and their protein pathway targets.

In Vivo Implications of Potential Probiotic Lactobacillus reuteri LR6 on the Gut and Immunological Parameters as an Adjuvant Against Protein Energy Malnutrition.

The present study investigated the impact of probiotic Lactobacillus reuteri LR6 on the gut and systemic immunity using protein energy malnourished (PEM) murine model. Thirty male Swiss albino mice were divided into five groups: control (C), malnourished (M), probiotic fermented milk (PFM), skim milk (SM), and bacterial suspension (BS) with six mice per group. Group C was fed with conventional diet throughout the study while the other groups were fed with protein calorie restricted diet until the development of malnutrition. After development of malnutrition, group M was continued with the restricted diet while other groups were fed with re-nourished diet supplemented with PFM, SM, and BS for 1 week, respectively. Thereafter, mice were sacrificed and different histological, microbiological, and immunological parameters were studied. Probiotics feeding in PEM model as fermented product or bacterial suspension improved the intestinal health in terms of intact morphology of colonic crypts, normal goblet cells, and intact lamina propria with no inflammation in large intestine, absence of fibrosis, and no inflammation in spleen. The number of secretory IgA+ cells was significantly higher in group PFM and BS. Also, increase in the phagocytic percentage of the macrophages and bone marrow derived dendritic cells (DCs) were observed in the PFM and BS group in comparison to the group M. In comparison to the group M and SM, lactobacilli, bifidobacteria, and Firmicutes counts were significantly higher in the group PFM and BS. This study concludes that probiotic supplementation to re-nutrition diet could emerge as wonder therapeutics against PEM.

Different Inhibitors of Aß42-Induced Toxicity Have Distinct Metal-Ion Dependency.

Oligomers of amyloid ß-protein (Aß) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of Aß has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn2+ can strongly modulate the aggregation of Aß in vitro and both promote and inhibit the neurotoxicity of Aß, depending on the experimental conditions. Thus, successful inhibitors of Aß self-assembly may have to inhibit the toxicity not only of Aß oligomers themselves but also of Aß-Zn2+ complexes. However, there has been relatively little research investigating the effects of Aß self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of Aß42 C-terminal fragments (CTFs), some of which have been shown to inhibit Aß oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of Aß42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, ß-sheet, and fibril formation by Aß42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with Aß42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to Aß42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both Aß42 oligomerization and Aß42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of Aß42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of Aß42 alone. In agreement with these results, CLR01 inhibited ß-sheet and fibril formation in Aß42-Zn2+ complexes. Our data suggest that, for the development of efficient therapeutic agents, inhibitors of Aß self-assembly and toxicity should be examined in the presence of relevant metal ions and that molecular tweezers may be particularly attractive candidates for therapy development.

Comparison of susceptibility to opsonic killing by in vitro human immune response of Enterococcus strains isolated from dairy products, clinical samples and probiotic preparation.

The genus Enterococcus like other LAB has also been featured in food and probiotic industry for decades due to its specific biochemical traits and beneficial health claims. At the same time, some enterococcal strains present an emerging pool of opportunistic pathogens for humans and are frequently armed with potential virulence factors. Thus, there is a need to assure the safety of enterococci before their use in food and probiotic preparations. Opsonophagocytic assay is an important test for the safety assessment of enterococci. In the present study comparative safety assessment of the different enterococcal strains isolated from dairy products, faeces, clinical samples and a commercial probiotic preparation was carried out by in vitro opsonophagocytic assay. Eleven strains of Enterococcus spp. were tested for their susceptibility to killing by opsonophagocytic assay. Among them, six isolates (Enterococcus faecium strain) were from our previous study (isolated from dairy products and faecal sample), four were from clinical samples and one from a probiotic preparation. Five out of six previous isolates and the isolate from probiotic preparation showed higher susceptibility to killing in contrast to the clinical isolates. The difference in the susceptibility to opsonic killing among the clinical and non-clinical Enterococcus isolates may be attributed to the presence of a capsule in the former, which protect them against the opsonophagocytic killing. Thus, these susceptible E. faecium strains may be designated as safe. However, certain other virulence traits must be evaluated prior to their exploitation in food and probiotic preparations.

Bovicins: The Bacteriocins of Streptococci and Their Potential in Methane Mitigation.

Bovicin is a type AII lantibiotic, possessing two ß-methyllanthionine and a disulfide bridge encoded by bovA gene hitherto unknown a couple of decades ago. Bacteriocins can be useful in directly inhibiting methanogens and/or redirecting H2 to other reductive microorganisms, in particular, propionate producers or reductive acetogens. So far, the role of nisin and bovicin to suppress greenhouse gas (GHG) production under in vitro conditions has been documented. GHG emissions from ruminants are a threat to the environment, because of their role in global warming as well as in climate change. Methane (CH4) produced from livestock farming practices is a potent GHG, comprising 18% of total GHG emissions in the world. Therefore, minimizing enteric CH4 production is quite essential from both the economical livestock production as well as environment perspectives. Strategies for the abatement of CH4 have provided two-way opportunities, viz., improved livestock productivity and reduced GHG emissions. In the past, different strategies have been proposed and tested to mitigate CH4, such as the dietary composition of feeds, ionophores, antibiotics, vaccines, analogues, probiotics, and secondary metabolites of plants and fungi. However, quite a few of these strategies have been adopted at farm level due to their varied effect on animal health and/or residues on animal products. The use of bacteriocins might have potential in inhibiting methanogens in the rumen. A bacteriocin produced by Streptococcus bovis (an isolate from rumen) named bovicin HC5 has been exhibited to decrease CH4 production to an extent of 50%. In this review, authors intend to discuss the sources, structure, biochemical properties, and antimicrobial spectra of bovicins, besides the potential applications with special reference to CH4 mitigation.

Ground water quality in environmentally degraded localities of Panipat city, India.

Asystematic physico-chemical analysis of the groundwater at 41 different locations in Panipat city (Haryana), India has been taken up to evaluate its suitabilityfor domestic purposes. The data revealed considerable variations in the water samples with respect to chemical composition. For the analyzed water samples pH, EC, TDS, TA, TH, Na+, K+, Ca2+, HCO3-, Cl-, SO4(2-) and F(-) varied from 6.6-7.5, 0.09-3.28 mmhoS cm(-1), 700-2100 mg l(-1), 245-1054 mg l(-1) (as CaCO3), 153-520 mg l(-1) (as CaCO3), 57-560 mg l(-1), 5-22 mg l(-1), 36-95 mg l(-1), 298-1285 mg l(-1), 60-311 mgl(-1), 17-786 mg l(-1), 0.24-9.27 mg l(-1) respectively. All samples have high concentration of dissolved salts and all the samples were hard to very hard. Correlation coefficient "r" analysis has been worked out among different water quality parameters. The study shows a positive and significant, correlation of electrical conductivity with total dissolved salts (r = 0.979), total hardness (r = 0.507), sulphate (r = 0.453), total alkalinity (r = 0.725). Total hardness is positively and significantly correlated with magnesium (r = 0.833) and sulphate (r = 0.687). Where as total alkalinity was found to be positively and significantly correlated with bicarbonate (r = 0.992). Fluoride was higher than permissible limits in most of the samples.

Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves.

Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging because of its metastable nature. Antibodies against misfolded SOD1 are useful tools for this purpose, provided their specificity and selectivity are well-characterized. Here, we characterized three recently introduced antimisfolded SOD1 antibodies and compared them with two commercial, antimisfolded SOD1 antibodies raised against the fALS-linked variant G93A-SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti-SOD1 antibodies expected to be insensitive to misfolding. We asked to what extent the antibodies could distinguish between WT and variant SOD1 and between native and misfolded conformations. WT, G93A-SOD1, or E100K-SOD1 were incubated under aggregation-promoting conditions and monitored using thioflavin-T fluorescence, electron microscopy, and dot blots. WT and G93A-SOD1 also were analyzed using native-PAGE/Western blot. The new antimisfolded SOD1 and the commercial antibody B8H10 showed variable reactivity using dot blots but generally showed maximum reactivity at the time misfolded SOD1 oligomers were expected to be most abundant. In contrast, only B8H10 and the control antibodies were reactive in Western blots. Unexpectedly, the polyclonal antibodies showed strong preference for the misfolded form of G93A-SOD1 in dot blots. Surprisingly, antimisfolded SOD1 antibody C4F6 was specific for the apo form of G93A-SOD1 but insensitive to misfolding. Antibody 10C12 showed preference for early misfolded structures, whereas 3H1 bound preferentially to late structures. These new antibodies allow distinction between putative early- and late-forming prefibrillar SOD1 oligomers.

Using Molecular Tweezers to Remodel Abnormal Protein Self-Assembly and Inhibit the Toxicity of Amyloidogenic Proteins.

Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies.

Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens.

Adhesion ability of probiotics is the key factor that decides their colonization in the gastrointestinal tract and potential to inhibit pathogens. Therefore, adhesion ability can be considered as a key determinant for probiotic efficacy. Presents study documents the antagonistic activity of viable/untreated, Lithium chloride (LiCl) treated or heat-killed forms of eight probiotic Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. All strains investigated were able to adhere to Caco-2 cells. L. reuteri strains tested were able to inhibit and displace (P < 0.05) the adhesion of Escherichia coli ATCC25922, Salmonella typhi NCDC113, Listeria monocytogenes ATCC53135, and Enterococcus faecalis NCDC115. The probiotic strain L. reuteri LR6 showed the strongest adhesion and pathogen inhibition ability among the eight L. reuteri strains tested. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting the involvement of various mechanisms. The adhesion and antagonistic potential of the probiotic strains were significantly decreased upon exposure to 5 M LiCl, showing that surface molecules, proteinaceous in nature, are involved. The heat-killed forms of the probiotic L. reuteri strains also inhibited the attachment of selected pathogens to Caco-2 cells. In conclusion, in vitro assays showed that L. reuteri strains, as viable or heat-killed forms, are adherent to Caco-2 cells and are highly antagonistic to pathogens tested in which surface associated proteins play an important role.

Computational On-Chip Imaging of Nanoparticles and Biomolecules using Ultraviolet Light.

Significant progress in characterization of nanoparticles and biomolecules was enabled by the development of advanced imaging equipment with extreme spatial-resolution and sensitivity. To perform some of these analyses outside of well-resourced laboratories, it is necessary to create robust and cost-effective alternatives to existing high-end laboratory-bound imaging and sensing equipment. Towards this aim, we have designed a holographic on-chip microscope operating at an ultraviolet illumination wavelength (UV) of 266 nm. The increased forward scattering from nanoscale objects at this short wavelength has enabled us to detect individual sub-30 nm nanoparticles over a large field-of-view of >16 mm2 using an on-chip imaging platform, where the sample is placed at ≤0.5 mm away from the active area of an opto-electronic sensor-array, without any lenses in between. The strong absorption of this UV wavelength by biomolecules including nucleic acids and proteins has further enabled high-contrast imaging of nanoscopic aggregates of biomolecules, e.g., of enzyme Cu/Zn-superoxide dismutase, abnormal aggregation of which is linked to amyotrophic lateral sclerosis (ALS) - a fatal neurodegenerative disease. This UV-based wide-field computational imaging platform could be valuable for numerous applications in biomedical sciences and environmental monitoring, including disease diagnostics, viral load measurements as well as air- and water-quality assessment.

Stabilization of bovine insulin against agitation-induced aggregation using RNA aptamers.

Stabilization of monomeric insulin is a primary requirement for preserving the efficacy of the final formulation. Degraded and/or aggregated protein as well as the presence of any of the conventional excipients can result in immunogenic or anaphylactic reactions, and reduced bioavailability of the protein drug. The aim of this work was to select novel RNA-based stabilizers of bovine insulin which would recognize and bind to the monomeric protein and help retain its bioactivity. RNA aptamers were selected by an in vitro selection method. They were screened for their ability to inhibit insulin fibrillation using agitation as a stress condition. The in vitro activity of insulin was determined by phosphorylation of downstream proteins in the cell. In vivo bioactivity was determined in a diabetic rat model. RNA aptamers, which bound to insulin with very low dissociation constants and high specificity, were selected. These sequences were aligned and consensus regions were found. The RNA sequences had no effect on the signalling cascade initiated by insulin. The bioactivity of insulin, as measured by its ability to lower plasma glucose level in a diabetic rat model, also remained unchanged. RNA aptamers are a novel class of protein stabilizers which have the ability to disrupt protein-protein interactions and hence inhibit protein aggregation. Their non-toxic and non-immunogenic nature makes such formulations safe for use.