Point-of-care biomarker assay for rapid multiplexed detection of CRP and IP-10.

Rapid and accurate measurements of immune protein markers are essential for diagnosis and treatment in all clinical settings. The recent pandemic has revealed a stark need for developing new tools and assays that could be rapidly used in diverse settings and provide useful information to clinicians. Here, we describe the development and test application of a novel one-step CRP/IP-10 duplex assay for the LightDeck platform capable of delivering reproducible and accurate measurements in under eight minutes. We used the optimized assay to measure CRP and IP-10 levels in human blood and serum samples from healthy, SARS-CoV-2 (COVID-19) positive, and influenza-like illness (ILI) presenting patients. Our results agreed with previously published analyte levels and enabled us to make statistically significant comparisons relevant to multiple clinical parameters. Our duplex assay is a simple and powerful tool for aiding prognostic decision-making in diverse settings.

Occurrence and trophic magnification profile of triphenyltin compounds in marine mammals and their corresponding food webs.

The occurrence of triphenyltin (TPT) compounds, a highly toxic antifouling biocide, has been documented in marine environments and organisms all over the world. While some studies showed that marine mammals can be used as sentinel organisms to evaluate the pollution status of emerging contaminants in the environment because of their long lifespans and high trophic levels, information regarding the contamination status of TPT in marine mammal species has been limited over the past decade. More importantly, the primary bioaccumulation pathway of TPT in these long-lived apex predators and the corresponding marine food web is still uncertain. Therefore, this study aimed to evaluate the contamination statuses of TPT in two marine mammal species, namely the finless porpoise and the Indo-Pacific humpback dolphin, and assess the trophic magnification potential of TPT along the food webs of these two species, using stable isotope analysis, and chemical analysis with gas chromatography-mass spectrometry. The results showed that TPT is the predominant residue in majority of the analyzed individuals of two marine mammals, with concentrations ranging from 426.2 to 3476.6 ng/g wet weight in their muscle tissues. Our results also demonstrated an exponential increase in the concentration of TPT along the marine food web, indicating that trophic magnification occurs in the respective food webs of the two marine mammals. The range of trophic magnification factors of TPT in the food webs of finless porpoise and Indo-Pacific humpback dolphin was 2.51-3.47 and 2.45-3.39, respectively. These results suggest that high trophic organisms may be more vulnerable to the exposure of TPT-contaminated environments due to the high trophic magnification potential, and thus ecological risk of these compounds ought to be assessed with the consideration of their bioaccumulation potentials in these marine mammals.

Accidental Spill of Palm Stearin Poses Relatively Short-Term Ecological Risks to a Tropical Coastal Marine Ecosystem.

In early August 2017, a serious palm stearin pollution accident occurred in the Pearl River Estuary, South China. While there were already several palm oil related spills around the world, the ecological effects and risks of such accidents to coastal marine environments remain largely unknown. In this study, we found that all seawater and sediment samples collected from six coastal sites were heavily contaminated by palm stearin within 1 week of the accident, and their levels significantly decreased to preaccident levels after four months. Waterborne exposure to palm stearin resulted in growth inhibition to four microalgal species (range of EC50: 9.9-212.6 mg/L) and acute mortality to four invertebrate species (range of LC50: 4.6-409.3 mg/L), while adverse chronic effects of palm stearin on the survival, development, and fecundity of Tigriopus japonicus and on the growth of Oryzias melastigma were observed. On the basis of these results, its interim-predicted no effect concentration was determined as 0.141 mg/L. The hazard quotient of palm stearin greatly exceeded 1 at all sites in August 2017 but returned to <1 at four sites and <2 at the other two sites in November 2017, indicating that its ecological risk was relatively transient and short-term.

Short-term tissue decomposition alters stable isotope values and C:N ratio, but does not change relationships between lipid content, C:N ratio, and Δδ13C in marine animals.

Measures (e.g. δ15N, δ13C, %C, %N and C:N) derived from animal tissues are commonly used to estimate diets and trophic interactions. Since tissue samples are often exposed to air or kept chilled in ice over a short-term during sample preparation, they may degrade. Herein, we hypothesize that tissue decomposition will cause changes in these measures. In this study, we kept marine fish, crustacean and mollusc tissues in air or ice over 120 h (5 days). We found that tissue decomposition in air enriched δ15N (range 0.6‰ to 1.3‰) and δ13C (0.2‰ to 0.4‰), decreased %N (0.47 to 3.43 percentage points from staring values of ~13%) and %C (4.53 to 8.29 percentage points from starting values of ~43%), and subsequently increased C:N ratio (0.14 to 0.75). In air, while such changes to δ13C were relatively minor and therefore likely tolerable, changes in δ15N, %N, %C and C:N ratio should be interpreted with caution. Ice effectively reduced the extent to which decomposition enriched δ15N (≤ 0.4‰) and δ13C (≤ 0.2‰), and eliminated decomposition in C:N ratio, %N and %C. In our second experiment, for fish tissues in either air or ice over 120 h, we observed no effects of decomposition on relationships between lipid content, C:N ratio, and Δδ13C (change in δ13C after lipid removal), which are employed to correct δ13C for samples containing lipid. We also confirmed that lipid in tissues caused large errors when estimating δ13C (mean ± standard error = -1.8‰ ± 0.1‰, range -0.6‰ to -3.8‰), and showed both lipid extraction and mathematical correction performed equally well to correct for lipids when estimating δ13C. We, therefore, recommend that specimens of marine animals should be kept in ice during sample preparation for a short-term, as it is an effective means for minimizing changes of the stable isotope measures in their tissue.

Mechanism of Amyloidogenesis of a Bacterial AAA+ Chaperone.

Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a ß-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation.

The mechanism of membrane disruption by cytotoxic amyloid oligomers formed by prion protein(106-126) is dependent on bilayer composition.

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106-126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106-126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.

Structures of amyloid fibrils formed by the prion protein derived peptides PrP(244-249) and PrP(245-250).

While the formation of amyloid fibrils from diverse peptide and protein sequences is well established, the molecular determinants of structure and assembly are not well understood. In particular, the relationship between amino acid sequence and the type of internal steric zipper packing adopted in amyloid fibrils has not been established. Here we report the structures of two cytotoxic amyloid peptides derived from the mammalian prion protein, PrP(244-249) and PrP(245-250), determined using solid state NMR. While the amino acid composition of these two hexapeptides is very similar (ISFLIF and SFLIFL), the intermolecular interactions that give rise to the intersheet packing within the fibrils differ significantly. PrP(245-250) adopts a class 1 steric zipper, with parallel sheets stacked in an antiparallel face to face arrangement, stabilized by N- to C-terminal salt bridges. PrP(244-249), by contrast, forms two different intersheet interfaces within amyloid fibrils, with parallel opposing sheets in either a face to face (class 3) or face to back (class 2) arrangement. The fibrils formed by this peptide are primarily stabilized by close packing of the hydrophobic side chains, with contributions from side-chain to backbone hydrogen bonding (class 2 only). Thus, the structures presented here provide new insight into the relationship between amino acid sequence and the types of interactions stabilizing amyloid fibrils.

Solid-State NMR characterization of autofluorescent fibrils formed by the elastin-derived peptide GVGVAGVG.

The characterization of the molecular structure and physical properties of self-assembling peptides is an important aspect of optimizing their utility as scaffolds for biomaterials and other applications. Here we report the formation of autofluorescent fibrils by an octapeptide (GVGVAGVG) derived via a single amino acid substitution in one of the hydrophobic repeat elements of human elastin. This is the shortest and most well-defined peptide so far reported to exhibit intrinsic fluorescence in the absence of a discrete fluorophore. Structural characterization by FTIR and solid-state NMR reveals a predominantly ß-sheet conformation for the peptide in the fibrils, which are likely assembled in an amyloid-like cross-ß structure. Investigation of dynamics and the effects of hydration on the peptide are consistent with a rigid, water excluded structure, which has implications for the likely mechanism of intrinsic fibril fluorescence.

Morphology and secondary structure of stable beta-oligomers formed by amyloid peptide PrP(106-126).

The formation of nonfibrillar oligomers has been proposed to be a common element of the aggregation pathway of amyloid peptides. Here we describe the first detailed investigation of the morphology and secondary structure of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). These oligomers have an apparent hydrodynamic radius of approximately 30 nm and are more membrane-active than monomeric or fibrillar PrP(106-126). Circular dichroism and solid state NMR data support formation of an extended beta-strand by the hydrophobic core of PrP(106-126), while negative thioflavin-T binding implies an absence of cross-beta structure in nonfibrillar oligomers.