Machine-Learning-Based Clinical Biomarker Using Cell-Free DNA for Hepatocellular Carcinoma (HCC).

(1) Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Although various serum enzymes have been utilized for the diagnosis and prognosis of HCC, the currently available biomarkers lack the sensitivity needed to detect HCC at early stages and accurately predict treatment responses. (2) Methods: We utilized our highly sensitive cell-free DNA (cfDNA) detection system, in combination with a machine learning algorithm, to provide a platform for improved diagnosis and prognosis of HCC. (3) Results: cfDNA, specifically alpha-fetoprotein (AFP) expression in captured cfDNA, demonstrated the highest accuracy for diagnosing malignancies among the serum/plasma biomarkers used in this study, including AFP, aspartate aminotransferase, alanine aminotransferase, albumin, alkaline phosphatase, and bilirubin. The diagnostic/prognostic capability of cfDNA was further improved by establishing a cfDNA score (cfDHCC), which integrated the total plasma cfDNA levels and cfAFP-DNA expression into a single score using machine learning algorithms. (4) Conclusion: The cfDHCC score demonstrated significantly improved accuracy in determining the pathological features of HCC and predicting patients' survival outcomes compared to the other biomarkers. The results presented herein reveal that our cfDNA capture/analysis platform is a promising approach to effectively utilize cfDNA as a biomarker for the diagnosis and prognosis of HCC.

Effect of Fermented Red Ginseng Concentrate Intake on Stool Characteristic, Biochemical Parameters, and Gut Microbiota in Elderly Korean Women.

Fermented red ginseng (FRG) has been used as a general stimulant and herbal medicine for health promotion in Asia for thousands of years. Few studies have investigated the effects of FRG containing prebiotics on the gut microbiota. Here, 29 Korean women aged ≥ 50 years were administered FRG for three weeks to determine its effect on stool characteristics, biochemical parameters, and gut microbiome. Gut microbial DNA was subjected to 16S rRNA V3-V4 region sequencing to assess microbial distribution in different stages. Additionally, the stool consistency, frequency of bowel movements, and biochemical parameters of blood were evaluated. We found that FRG intake improved stool consistency and increased the frequency of bowel movements compared to before intake. Biochemical parameters such as glucose, triglyceride, cholesterol, low-density lipoprotein cholesterol, creatinine, alkaline phosphatase, and lactate dehydrogenase decreased and high-density lipoprotein cholesterol increased with FRG intake. Gut microbiome analysis revealed 20 specific bacteria after three weeks of FRG intake. Additionally, 16 pathways correlated with the 20 specific bacteria were enhanced after red ginseng intake. In conclusion, FRG promoted health in elderly women by lowering blood glucose levels and improving bowel movement frequency. The increase in bacteria observed with FRG ingestion supports these findings.

Different Reactions in Each Enterotype Depending on the Intake of Probiotic Yogurt Powder.

Probiotics can be used as a nutritional strategy to improve gut homeostasis. We aimed to evaluate the intestinal microbiota profile of 18 subjects after ingestion of probiotic yogurt powder (PYP) based on enterotype. The subjects were classified into three enterotypes according to their microbial community: Bacteroides (n = 9, type B), Prevotella (n = 3, type P), and Ruminococcus (n = 6, type R). We performed controlled termination in a transient series that included a control period of three weeks before probiotic intake, PYP intake for three weeks, and a three-week washout period. Fecal microbiota composition was analyzed by sequencing the V3-V4 super variable region of 16S rRNA. Based on the Bristol stool shape scale, abnormal stool shape improved with PYP intake, and bowel movements were activated. The abundance of Faecalibacterium, Eggerthella, and Leuconostoc, which ferment and metabolize glucose, showed a strong correlation with type B Bacteroides, and glucose metabolism improvement was observed in all type B subjects. Alkaline phosphatase was significantly improved only in type B. In addition, the abundance of type B Bacteroides showed a negative correlation with that of Lactobacillus. The abundance of Streptococcus, Agathobacter, and Christensenella, which are involved in lipid metabolism, showed a strong correlation with that of type P Prevotella, and triglyceride metabolism improvement was observed in all type P subjects. The gut microbiota showed only short-term changes after PYP intake and showed resilience by returning to its original state when PYP intake was interrupted. In summary, the different responses to PYP intake may result from the different enterotypes and associated strains; therefore, the probiotic composition should be adjusted based on the individual enterotype.

Individual Identification with Short Tandem Repeat Analysis and Collection of Secondary Information Using Microbiome Analysis.

Forensic investigation is important to analyze evidence and facilitate the search for key individuals, such as suspects and victims in a criminal case. The forensic use of genomic DNA has increased with the development of DNA sequencing technology, thereby enabling additional analysis during criminal investigations when additional legal evidence is required. In this study, we used next-generation sequencing to facilitate the generation of complementary data in order to analyze human evidence obtained through short tandem repeat (STR) analysis. We examined the applicability and potential of analyzing microbial genome communities. Microbiological supplementation information was confirmed for two of four failed STR samples. Additionally, the accuracy of the gargle sample was confirmed to be as high as 100% and was highly likely to be classified as a body fluid sample. Our experimental method confirmed that anthropological and microbiological evidence can be obtained by performing two experiments with one extraction. We discuss the advantages and disadvantages of using these techniques, explore prospects in the forensic field, and highlight suggestions for future research.

Intake of MPRO3 over 4 Weeks Reduces Glucose Levels and Improves Gastrointestinal Health and Metabolism.

Human gut microbiota are involved in different metabolic processes, such as digestion and nutrient synthesis, among others. For the elderly, supplements are a major means of maintaining health and improving intestinal homeostasis. In this study, 51 elderly women were administered MPRO3 (n = 17), a placebo (n = 16), or both (MPRO3: 1 week, placebo: 3 weeks; n = 18) for 4 weeks. The fecal microbiota were analyzed by sequencing the 16S rRNA gene V3-V4 super-variable region. The dietary fiber intake increased, and glucose levels decreased with 4-week MPRO3 intake. Reflux, indigestion, and diarrhea syndromes gradually improved with MPRO3 intake, whereas constipation was maintained. The stool shape also improved. Bifidobacterium animalis, B. pseudolongum, Lactobacillus plantarum, and L. paracasei were relatively more abundant after 4 weeks of MPRO3 intake than in those subjects after a 1-week intake. Bifidobacterium and B. longum abundances increased after 1 week of MPRO3 intake but decreased when the intake was discontinued. Among different modules and pathways, all 10 modules analyzed showed a relatively high association with 4-week MPRO3 intake. The mineral absorption pathway and cortisol biosynthesis and secretion pathways correlated with the B. animalis and B. pseudolongum abundances at 4 weeks. Therefore, 4-week MPRO3 intake decreased the fasting blood glucose level and improved intestinal health and metabolism.

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif.

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapß2) for its transportation into the nucleus. We identify a potential PY-NLS motif ((774)gpgfgssppGaeaapslRyvPY(795)) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X(2-5)PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapß2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapß2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapß2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapß2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapß2 interacts with ULK2 through ULK2's putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.

Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1.

The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, we identified stromal interaction molecule 1 precursor (STIM1) as an auxiliary protein of this epithelial Ca2+channel using confocal microscopy analysis and GST pull-down assay. The STIM1 protein associates specifically with the C-terminal tail of TRPV4 to form a complex. In previous reports, we demonstrated that the serine824 residue of TRPV4 is one of the target phosphorylation sites of serum/glucocorticoid regulated kinase 1 (SGK1). In this report we further identified the role of serine 824 phosphorylation. The TRPV4 mutant S824D (not S824A) exhibited a diminished capacity to bind STIM1. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STIM1 is part of the TRPV4 protein complex. Our observations clearly suggest that the formation of a complex between TRPV4 and STIM1 and its plasma membrane localization are regulated through phosphorylation of serine824 of TRPV4, and that the STIM1-TRPV4 complex plays crucial roles in routing TRPV4 to the plasma membrane from the endoplasmic reticulum and in maintaining its function.

The nuclear localization of glycogen synthase kinase 3ß is required its putative PY-nuclear localization sequences.

Glycogen synthase kinase-3ß(GSK-3ß), which is a member of the serine/threonine kinase family, has been shown to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that GSK-3ß is associated with the karyopherin ß2 (Kap ß2) (102-kDa), which functions as a substrate for transportation into the nucleus. A potential PY-NLS motif ((109)IVRLRYFFY(117)) was observed, which is similar with the consensus PY NLS motif (R/K/H)X(2-5)PY in the GSK-3ß catalytic domain. Using a pull down approach, we observed that GSK-3ß physically interacts with Kap ß2 both in vivo and in vitro. Secondly, GSK-3ß and Kap ß2 were shown to be co-localized by confocal microscopy. The localization of GSK-3ß to the nuclear region was disrupted by putative Kap ß2 binding site mutation. Furthermore, in transient transfection assays, the Kap ß2 binding site mutant induced a substantial reduction in the in vivo serine/threonine phosphorylation of GSK-3ß, where- as the GSK-3ß wild type did not. Thus, our observations indicated that Kap ß2 imports GSK-3ß through its putative PY NLS motif from the cytoplasm to the nucleus and increases its kinase activity.

Endoplasmic Reticulum (ER) Stress Enhances Tip60 (A Histone Acetyltransferase) Binding to the Concanavalin A.

Herein, we report that the concanavalin A binding of Tip60 (a target of the human immunodeficiency virus type 1-encoded transactivator Tat interacting protein 60 KD; a histone acetyltransferase; HAT) is enhanced as the result of endoplasmic reticulum (ER) stress. The cell expression of Tip60 combined with site-directed mutagenesis analysis was used to identify the glutamine 324 residue as the lecithin binding (Concanavalin A; Con A) site. The Tip60 N324A mutant strain, which seems to be the Con A binding-deficient, was attenuated the protein-protein interactions with FE65 and its protein stability, but its ability of G0-G1 cell cycle arrest was not interrupted. Interestingly, both HAT activity and the nuclear localization of Tip60 N324A mutant were enhanced than those of Tip60 WT. Thus, our results indicate that the Con A binding deficient of Tip60 seems to be one of the most pivotal posttranslational modifications (such as N-glycosylation) for its functional regulation signal, which is generated in response to ER stress.

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response.

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1ß and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.

Phosphorylation on the Ser 824 residue of TRPV4 prefers to bind with F-actin than with microtubules to expand the cell surface area.

Previously, we demonstrated that the transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is one of the serum glucocorticoid-induced protein kinase1 (SGK1) authentic substrate proteins, and that the Ser 824 residue of TRPV4 is phosphorylated by SGK1. In this study, we demonstrated that phosphorylation on the Ser 824 residue of TRPV4 is required for its interaction with F-actin, using TRPV4 mutants (S824D; a phospho-mimicking TRPV4 mutant and S824A; a non-phosphorylatable TRPV4 mutant) and its proper subcellular localization. Additionally, we noted that the phosphorylation of the Ser824 residue promotes its single channel activity, Ca(2+) influx, protein stability, and cell surface area (expansion of plasma membrane).

Mutation of a putative S-nitrosylation site of TRPV4 protein facilitates the channel activates.

The transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues. Nitric oxide (NO) as a gaseous signal mediator shows a variety of important biological effects. In many instances, NO has been shown to exhibit its activities via a protein S-nitrosylation mechanism in order to regulate its protein functions. With functional assays via site-directed mutagenesis, we demonstrate herein that NO induces the S-nitrosylation of TRPV4 Ca(2+) channel on the Cys(853) residue, and the S-nitrosylation of Cys(853) reduced its channel sensitivity to 4-α phorbol 12,13-didecanoate and the interaction between TRPV4 and calmodulin. A patch clamp experiment and Ca(2+) image analysis show that the S-nitrosylation of Cys(853) modulates the TRPV4 channel as an inhibitor. Thus, our data suggest a novel regulatory mechanism of TRPV4 via NO-mediated S-nitrosylation on its Cys(853) residue.

Ubiquitylation of Fe65 adaptor protein by neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) via the WW domain interaction with Fe65.

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif ((72)PPLP(75)) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant ((72)PPLP(75) was changed to (72)APLA(75)) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.

The PPLA motif of glycogen synthase kinase 3beta is required for interaction with Fe65.

Glycogen synthase kinase 3beta (GSK 3 beta) is a serine/ threonine kinase that phosphorylates substrates such as beta-catenin and is involved in a variety of biological processes, including embryonic development, metabolism, tumorigenesis, and cell death. Here, we present evidence that human GSK 3beta is associated with Fe65, which has the characteristics of an adaptor protein, possessing a WW domain, and two phosphotyrosine interaction domains, PID1 and PID2. The GSK 3beta catalytic domain also contains a putative WW domain binding motif ((371)PPLA(374)), and we observed, using a pull down approach and co-immuno-precipitation, that it interacts physically with Fe65 via this motif. In addition, we detected co-localization of GSK 3beta and Fe65 by confocal microscopy, and this co-localization was disrupted by mutation of the putative WW domain binding motif of GSK 3beta.Finally, in transient transfection assays interaction of GSK 3 beta (wt) with Fe65 induced substantial cell apoptosis, whereas interaction with the GSK 3beta AALA mutant ((371)AALA(374)) did not, and we noted that phosphorylation of the Tyr 216 residue of the GSK 3beta AALA mutant was significantly reduced compared to that of GSK 3beta wild type. Thus, our observations indicate that GSK 3beta binds to Fe65 through its (371)PPLA(374) motif and that this interaction regulates apoptosis and phosphorylation of Tyr 216 of GSK 3beta.

Regulation Fe65 localization to the nucleus by SGK1 phosphorylation of its Ser566 residue.

Fe65 is characterized as an adaptor precursor (APP) through its PID2 element, as well as with the other members of the APP protein family. With the serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity information, we found that the putative site of phosphorylation in Fe65 by SGK1 is present on its Ser(566) residue in (560)CRVRFLSFLA(569)(X60469). Thus, we demonstrated that Fe65 and the fluorescein-labeled Fe65 peptide FITC-(560)CRVRFLSFLA(569) are phosphorylated in vitro by SGK1. Phosphorylation of the Ser(566) residue was also demonstrated using a Ser566 phospho-specific antibody. The phospho Fe65 was found mainly in the nucleus, while Fe65 S556A mutant was localized primarily to the cytoplasm. Therefore, these data suggest that SGK1 phosphorylates the Ser(566) residue of Fe65 and that this phosphorylation promotes the migration of Fe65 to the nucleus of the cell.

The subcellular localization control of integrin linked kinase 1 through its protein-protein interaction with caveolin-1.

Integrin linked kinase 1 (ILK1), a member of the serine/threonine kinases, has been shown to be crucial for the cell survival, differentiation, and Wnt signaling. Firstly, by using a confocal microscopy and a transfection approach, we obtained the evidence that ILK1 interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. By ILK1 deletion mutant analysis, we characterized the caveolin-1-binding domain in the kinase domain of ILK1. In addition, we found that native ILK1 is associated with endogenous caveolin-1 in COS-1 cells. Secondly, transient transfection assays showed that a reduction in caveolin-1 binding leads to a substantial increase in the serine/threonine phosphorylation of ILK1. Thirdly, caveolin-1 and its scaffolding peptide (amino acids 82-101) functionally suppressed the auto-kinase activity of purified recombinant ILK1 protein. Fourthly, the association of ILK1 with caveolin-1 regulated its cytoplasmic retention; if it was not associated with caveolin-1, it was transported to the nucleus. Fifthly, we also noticed the putative nuclear localization sequences (nls) in ILK1 near the caveolin-1-binding domain. Thus, our data indicate that caveolin-1 regulates ILK1 auto-phosphorylation activity and its subcellular localization via a specific protein-protein interaction through blocking the exposure of its putative nls motif.

The subcellular localization of 3-phosphoinositide-dependent protein kinase is controlled by caveolin-1 binding.

3-Phosphoinositide-dependent protein kinase 1 (PDK1), a member of the serine/threonine kinase family, has been demonstrated to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that PDK1 is associated with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of the caveolae membranes in COS-1. First, we noted the presence of two potential caveolin-1 binding motifs ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306)) in the PDK1 catalytic domain. Using a pull-down approach, we observed that PDK1 interacts physically with caveolin-1 both in vivo and in vitro. Second, we detected the co-localization of PDK1 and caveolin-1 via confocal microscopy. The localization of PDK1 to the plasma membrane was disrupted by caveolin binding. Third, in transient transfection assays, interaction with caveolin-1 induced a substantial reduction in the in vivo serine/threonine phosphorylation of PDK1, whereas the caveolin-1 binding site mutant ((141)FFVKLYFTF(149) and (299)YDFPEKFF(306) change to (141)AFVKLAFTA(149) and (299)ADAPEFLA(306)) did not. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82-101) functionally suppressed the self-phosphorylation and kinase activities of purified recombinant PDK1 protein. Thus, our observations indicated that PDK1 binds to caveolin-1 through its caveolin-binding motifs, and also that the protein-protein interaction between PDK1 and caveolin-1 regulates PDK1 self-phosphorylation, kinase activity, and subcellular localization.