Diagnostic Blood Biomarkers in Alzheimer's Disease.

Potential biomarkers for Alzheimer's disease (AD) include amyloid ß1-42 (Aß1-42), t-Tau, p-Tau181, neurofilament light chain (NFL), and neuroimaging biomarkers. Their combined use is useful for diagnosing and monitoring the progress of AD. Therefore, further development of a combination of these biomarkers is essential. We investigated whether plasma NFL/Aß1-42 can serve as a plasma-based primary screening biomarker reflecting brain neurodegeneration and amyloid pathology in AD for monitoring disease progression and early diagnosis. We measured the NFL and Aß1-42 concentrations in the CSF and plasma samples and performed correlation analysis to evaluate the utility of these biomarkers in the early diagnosis and monitoring of AD spectrum disease progression. Pearson's correlation analysis was used to analyse the associations between the fluid biomarkers and neuroimaging data. The study included 136 participants, classified into five groups: 28 cognitively normal individuals, 23 patients with preclinical AD, 22 amyloid-negative patients with amnestic mild cognitive impairment, 32 patients with prodromal AD, and 31 patients with AD dementia. With disease progression, the NFL concentrations increased and Aß1-42 concentrations decreased. The plasma and CSF NFL/Aß1-42 were strongly correlated (r = 0.558). Plasma NFL/Aß1-42 was strongly correlated with hippocampal volume/intracranial volume (r = 0.409). In early AD, plasma NFL/Aß1-42 was associated with higher diagnostic accuracy than the individual biomarkers. Moreover, in preclinical AD, plasma NFL/Aß1-42 changed more rapidly than the CSF t-Tau or p-Tau181 concentrations. Our findings highlight the utility of plasma NFL/Aß1-42 as a non-invasive plasma-based biomarker for early diagnosis and monitoring of AD spectrum disease progression.

Effect of Vibrio-Derived Extracellular Protease vEP-45 on the Blood Complement System.

Vibrio vulnificus is a pathogenic bacterium that can causes wound infections and fetal septicemia. We have reported that V. vulnificus ATCC29307 produces an extracellular zinc-metalloprotease (named vEP-45). Our previous results showed that vEP-45 can convert prothrombin to active thrombin and also activate the plasma kallikrein/kinin system. In this study, the effect of vEP-45 on the activation of the complement system was examined. We found that vEP-45 could proteolytically convert the key complement precursor molecules, including C3, C4, and C5, to their corresponding active forms (e.g., C3a, C3b, C4a, C4b, and C5a) in vitro cleavage assays. C5b production from C5 cleavage mediated by vEP-45 was not observed, whereas the level of C5a was increased in a dose-dependent manner compared to that of the non-treated control. The cleavage of the complement proteins in human plasma by vEP-45 was also confirmed via Western blotting. Furthermore, vEP-45 could convert C3 and C5 to active C3a and C5a as a proinflammatory mediator, while no cleavage of C4 was observed. These results suggest that vEP-45 can activate the complement system involved in innate immunity through an alternative pathway.

Effect of 7-Methylsulfinylheptyl Isothiocyanate on the Inhibition of Melanogenesis in B16-F1 Cells.

Skin aging, characterized by hyperpigmentation, inflammation, wrinkles, and skin cancer, is influenced by intrinsic and extrinsic factors with synergistic effects. Autophagy maintains the homeostatic balance between the degradation, synthesis, and recycling of cellular proteins and organelles, and plays important roles in several cellular and biological processes, including aging. The compound 7-methylsulfinylheptyl isothiocyanate (7-MSI) is a sulfur-containing phytochemical produced by various plants, particularly cruciferous vegetables, with reported anti-inflammatory properties and a role in pathogen defense; however, its effects on skin whitening have not been studied in detail. The purpose of this study was to observe the effects of 7-MSI on skin whitening and autophagy in cultured murine melanoma (B16-F1) cells. Western blotting was used to evaluate the impact of 7-MSI on melanogenesis-, tyrosinase-, and autophagy-associated proteins. The levels of the melanogenesis-associated protein's microphthalmia-associated transcription factor (MITF) and tyrosinase and tyrosinase-related protein-1 were decreased by treatment with 7-MSI under melanogenesis induction. Melanin synthesis also decreased by approximately 63% after treatment with 7-MSI for 73 h, compared with that non-treated controls. In addition, autophagosome formation and the expression levels of the autophagy-related proteins mTOR, p-mTOR, Beclin-1, Atg12, and LC3 were higher in 7-MSI-treated B16-F1 cells than in non-treated cells. These results indicate that 7-MSI can inhibit melanin synthesis in B16-F1 cells by suppressing melanogenesis and autophagy activation and thus can potentially be used as a novel multifunctional cosmetic agent.

Plasma contact factors as novel biomarkers for diagnosing Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia and most of AD patients suffer from vascular abnormalities and neuroinflammation. There is an urgent need to develop novel blood biomarkers capable of diagnosing Alzheimer's disease (AD) at very early stage. This study was performed to find out new accurate plasma diagnostic biomarkers for AD by investigating a direct relationship between plasma contact system and AD. METHODS: A total 101 of human CSF and plasma samples from normal and AD patients were analyzed. The contact factor activities in plasma were measured with the corresponding specific peptide substrates. RESULTS: The activities of contact factors (FXIIa, FXIa, plasma kallikrein) and FXa clearly increased and statistically correlated as AD progresses. We present here, for the first time, the FXIIa cut-off scores to as: > 26.3 U/ml for prodromal AD [area under the curve (AUC) = 0.783, p < 0.001] and > 27.2 U/ml for AD dementia (AUC = 0.906, p < 0.001). We also describe the cut-off scores from the ratios of CSF Aß1-42 versus the contact factors. Of these, the representative ratio cut-off scores of Aß1-42/FXIIa were to be: < 33.8 for prodromal AD (AUC = 0.965, p < 0.001) and < 27.44 for AD dementia (AUC = 1.0, p < 0.001). CONCLUSION: The activation of plasma contact system is closely associated with clinical stage of AD, and FXIIa activity as well as the cut-off scores of CSF Aß1-42/FXIIa can be used as novel accurate diagnostic AD biomarkers.

Alpha-neoendorphin can reduce UVB-induced skin photoaging by activating cellular autophagy.

Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H2DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy.

Cloning, purification and characterization of a recombinant protease with novel thrombolytic activity in human plasma and rat thrombosis models.

BACKGROUND: There is a need to identify and develop novel thrombolytic agents that can directly digest fibrin clots from various biological resources. OBJECTIVE: To clone, express, purify, and characterize a recombinant protease named rvFMP capable of cleaving fibrinogen, fibrin polymer, and cross-linked fibrin in human plasma milieu and rat thrombosis model systems. RESULTS: We cloned a vFMP-encoding gene from the genomic DNA of V. furnissii KCCM41679 using polymerase chain reaction (PCR), expressed in Escherichia coli, and purified rvFMP (stands for recombinant vibrio furnissii metalloprotease). The proteolytic activity of purified rvFMP enzyme could be clearly inhibited by 1,10-phenanthroline and ethylene glycol tetraacetic acid, but not by diisopropyl fluorophosphate, suggesting that it can be a typical metalloprotease. rvFMP showed an effective proteolytic activity in cleaving cross-linked fibrins in human plasma milieu. Remarkably, rvFMP exhibited a clear thrombolytic activity in rat thrombosis models such as ferric chloride-exposed rat carotid artery and carrageenan-treated rat tail. However, rvFMP (1.5 mg/kg) evoked no internal bleeding and also showed no lethal effect in mice. The recombinant enzyme also showed no cytotoxicity and had an inability to induce tumour necrosis factor-α (TNF-α) in Raw264.7 cells. CONCLUSION: rvFMP can be a candidate enzyme capable of being developed as a novel direct-acting thrombolytic agent.

Tranexamic acid inhibits melanogenesis by activating the autophagy system in cultured melanoma cells.

BACKGROUND: As interest in skin beauty increases, the development of new skin whitening agents has attracted substantial attention; however, the action mechanism of the agents developed so far remains largely unknown. Tranexamic acid (TXA) is commonly being used to reduce melanin synthesis in patients with melasma and also used as a raw material for functional whitening cosmetics, although its action mechanism is poorly understood. Autophagy has been well known to be essential for tissue homeostasis, adaptation to starvation, and removal of dysfunctional organelles or pathogens. Recent studies have shown that autophagy regulators might have prominent roles in the initial formation stage of the melanosome, a lysosome-related organelle synthesizing melanin pigments. However, there is still no direct evidence showing a relationship between the activation of the autophagy system and the melanogenesis. OBJECTIVE: To investigate whether TXA can inhibit melanogenesis through the activation of autophagy in a melanoma cell line. METHODS: B16-F1 melanoma cells were treated with TXA and the levels of autophagy- and melanogenesis-related proteins were determined by Western blottings. The direct effect of TXA-mediated autophagy activation on melanin production was further evaluated by transfecting the cells with 60 pmols of small interfering RNAs (siRNAs)-targeting the mechanistic target of rapamycin (mTOR) and the autophagy-related protein 5 (Atg5). RESULTS: The results of Western blottings showed that TXA enhanced the production of autophagy-related proteins such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK)1/2, Beclin-1, Atg12, and light chain 3 (LC3) I-II, whereas it decreased the synthesis of the mTOR complex. Confocal microscopy clearly showed that TXA treatment resulted in the formation of autophagosomes in B16-F1 cells, as revealed by immunostaining with an anti-LC3 antibody. The production of melanogenesis-associated proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 and 2 (TRP1/2), were clearly downregulated by the treatments with TXA. These results suggest that TXA can mediate a decrease in melanin synthesis by alleviating the production of tyrosinase and TRP1/2, along with lowered MITF protein levels. Furthermore, after treatment with TXA, siRNAs- targeting to mTOR and Atg5 increased melanin synthesis by 20% and 40%, respectively, compared to that in non-transfected cells, in a dose-dependent manner. These results further confirmed that TXA can inhibit melanogenesis by activating the autophagy system. CONCLUSION: Collectively, the results demonstrate that TXA can reduce melanin synthesis in melanoma B16-F1 cells by activating the ERK signaling pathway and the autophagy system.