The revealing of a novel lipid transfer protein lineage in green algae.

BACKGROUND: Non-specific lipid transfer proteins (nsLTPs) are a group of small and basic proteins that can bind and transfer various lipid molecules to the apoplastic space. A typical nsLTP carries a conserved architecture termed eight-cysteine motif (8CM), a scaffold of loop-linked helices folding into a hydrophobic cavity for lipids binding. Encoded by a multigene family, nsLTPs are widely distributed in terrestrial plants from bryophytes to angiosperms with dozens of gene members in a single species. Although the nsLTPs in the most primitive plants such as Marchantia already reach 14 members and are divergent enough to form separate groups, so far none have been identified in any species of green algae. RESULTS: By using a refined searching strategy, we identified putative nsLTP genes in more than ten species of green algae as one or two genes per haploid genome but not in red and brown algae. The analyses show that the algal nsLTPs carry unique characteristics, including the extended 8CM spacing, larger molecular mass, lower pI value and multiple introns in a gene, which suggests that they could be a novel nsLTP lineage. Moreover, the results of further investigation on the two Chlamydomonas nsLTPs using transcript and protein assays demonstrated their late zygotic stage expression patterns and the canonical nsLTP properties were also verified, such as the fatty acids binding and proteinase resistance activities. CONCLUSIONS: In conclusion, a novel nsLTP lineage is identified in green algae, which carries some unique sequences and molecular features that are distinguishable from those in land plants. Combined with the results of further examinations of the Chlamydomonas nsLTPs in vitro, possible roles of the algal nsLTPs are also suggested. This study not only reveals the existence of the nsLTPs in green algae but also contributes to facilitating future studies on this enigmatic protein family.

Analysis of Oxidative and Advanced Oxidative Modifications in Hemoglobin of Oral Cancer Patients by Mass Spectrometry.

We are exposed to endogenous reactive oxygen species (ROS) produced during normal aerobic metabolism and by the innate immune systems. Excessive production of ROS is critically important in disease onset and progression. Post-translational oxidative modifications of hemoglobin have been used as a surrogate biomarker for monitoring the oxidative stress in vivo. In this study, the Fenton reaction was used as a model to produce ROS and react with human hemoglobin. After trypsin digestion, the types and sites of modifications were characterized by a nanoflow LC-nanoelectrospray ionization high-resolution mass spectrometer. Besides oxidation at certain sites of Met, His, and Tyr, conversion of histidine to aspartate and hydroxyaspartate was identified at four sites. Furthermore, advanced oxidation of histidine to hydroxyaspartate was identified at two sites. Elevated oxidative stress is tightly associated with oral cancer. The relative extent of modification at the identified sites was quantified relative to the native peptide present in the digest as the reference peptide in hemoglobin from 18 oral cancer patients and 15 healthy control subjects. The results revealed that the extents of oxidation at ß-His-77 and ß-Asp-99 of globin were significantly elevated in oral cancer patients compared to healthy subjects, while the extents of conversion of histidine residues at α-His-20, α-His-50, and ß-His-2 to aspartate were significantly decreased. Furthermore, the advanced oxidation of histidine to hydroxyaspartate at α-His-50 and ß-His-2 is also significantly higher in oral cancer patients than in healthy subjects (p < 0.05). To our knowledge, this advanced oxidation of histidine to hydroxyaspartate is a new post-translational protein modification, and it is found in human hemoglobin isolated from blood. This advanced oxidative modification in hemoglobin might be a potential biomarker to assess oxidative stress in oral cancer patients.

Quantification of tumor necrosis factor-α and matrix metalloproteinases-3 in synovial fluid by a fiber-optic particle plasmon resonance sensor.

The availability of techniques for sensitive detection of early stage osteoarthritis is critical for improving patient health. This study illustrates the feasibility of a fiber-optic particle plasmon resonance (FOPPR) sensor with gold nanoparticles on the unclad region of optical fiber probes for analysis of osteoarthritis biomarkers, tumor necrosis factor-α (TNF-α) and matrix metalloproteinases-3 (MMP-3). Results show that the sensor can achieve a refractive index resolution of 5.18 × 10⁻7 RIU and limits of detection for TNF-α and MMP-3 as low as 8.22 pg ml⁻¹ (0.48 pM) and 34.3 pg ml⁻¹ (1.56 pM), respectively. Additionally, the FOPPR sensor shows a good correlation in determining TNF-α and MMP-3 in synovial fluid with the clinically accepted enzyme-linked immunosorbent assay (ELISA) method. Finally, given the FOPPR sensor's nature of being low-cost, label-free, highly sensitive, real-time, simple-to-operate, the FOPPR sensor could offer potential to monitor biomarkers of various diseases, and provide an ideal technical tool for point-of-care diagnostics.

Fiber Optic Localized Surface Plasmon Resonance Sensor Based on Carboxymethylated Dextran Modified Gold Nanoparticles Surface for High Mobility Group Box 1 (HMGB1) Analysis.

Rapid, sensitive, and reliable detection of high mobility group box 1 (HMGB1) is essential for medical and diagnostic applications due to its important role as a biomarker of chronic inflammation. Here, we report a facile method for the detection of HMGB1 using carboxymethyl dextran (CM-dextran) as a bridge molecule modified on the surface of gold nanoparticles combined with a fiber optic localized surface plasmon resonance (FOLSPR) biosensor. Under optimal conditions, the results showed that the FOLSPR sensor detected HMGB1 with a wide linear range (10-10 to 10-6 g/mL), fast response (less than 10 min), and a low detection limit of 43.4 pg/mL (1.7 pM) and high correlation coefficient values (>0.9928). Furthermore, the accurate quantification and reliable validation of kinetic binding events measured by the currently working biosensors are comparable to surface plasmon resonance sensing systems, providing new insights into direct biomarker detection for clinical applications.

Doubly resonant surface-enhanced Raman scattering on gold nanorod decorated inverse opal photonic crystals.

We present a novel type of surface-enhanced Raman scattering (SERS) substrate constituted of a 3-dimensinal polymeric inverse opal (IO) photonic crystal frame with gold nanorods (Au-NRs) decorating on the top layer. This substrate employs resonant excitation as well as constructive backward scattering of Raman signals to produce large enhancement of SERS output. For the incoming excitation, Au-NRs with appropriate aspect ratio were adopted to align their longitudinal localized surface plasmon band with the excitation laser wavelength. For the outgoing SERS signal, the spectral position of the photonic band gap was tuned to reflect Raman-scattered light constructively. This SERS substrate produces not only strong but also uniform SERS output due to the well control of Au-NRs distribution by the periodic IO structure, readily suitable for sensing applications.

Integrated Graphene Oxide with Noble Metal Nanoparticles to Develop High-Sensitivity Fiber Optic Particle Plasmon Resonance (FOPPR) Biosensor for Biomolecules Determination.

In this research, a direct, simple and ultrasensitive fiber optic particle plasmon resonance (FOPPR) biosensing platform for immunoglobulin G (IgG) detection was developed using a gold nanoparticle/graphene oxide (AuNP/GO) composite as signal amplification element. To obtain the best analytical performance of the sensor, experimental parameters including the surface concentration of GO on the AuNPs, formation time of the GO, the concentration of the anti-IgG and incubation time of anti-IgG were optimized. The calibration plots displayed a good linear relationship between the sensor response (ΔI/I0) and the logarithm of the analyte concentrations over a linear range from 1.0 × 10-10 to 1.0 × 10-6 g/mL of IgG under the optimum conditions. A limit of detection (LOD) of 0.038 ng/mL for IgG was calculated from the standard calibration curve. The plot has a linear relationship (correlation coefficient, R = 0.9990). The analytical performance of present work's biosensor was better than that of our previously reported mixed self-assembled monolayer of 11-mercaptoundecanoic acid/6-mercapto-1-hexanol (MUA/MCH = 1:4) method by about three orders of magnitude. The achieved good sensitivity may be attributed to the synergistic effect between GO and AuNPs in this study. In addition, GO could immobilize more antibodies due to the abundant carboxylic groups on its surface. Furthermore, we also demonstrated that the results from this sensor have good reproducibility, with coefficients of variation (CVs) < 8% for IgG. Therefore, the present strategy provides a novel and convenient method for chemical and biochemical quantification and determination.

Self-referencing fiber optic particle plasmon resonance sensing system for real-time biological monitoring.

We present the design and experimental verification of a self-referencing dual-channel fiber optic particle plasmon resonance (FOPPR) sensing system for compensation of thermal and bulk-composition effects as well as nonspecific adsorption in real-time biosensing of complex samples. A theoretical model is first proposed and then a systematic experimental approach is used to verify the model. The sensing system comprises an analysis fiber sensor and a reference fiber sensor in a single microfluidic chip, where the analysis fiber is functionalized with a recognition molecule. The compensation still works even if the surface coverages of gold nanoparticles on the reference and analysis fibers are not exactly the same. The potential of this approach is illustrated by a model biosensing experiment in which the detection of anti-biotin is compensated for bulk refractive index change, nonspecific adsorption and/or color interference, in various sample media. The percent recovery is 103.2% under both the effects of bulk refractive index change and nonspecific adsorption and is 93.9% under both the effects of color interference and nonspecific adsorption, suggesting that the compensation is effective.