Microneedle patch-based enzyme-linked immunosorbent assay to quantify protein biomarkers of tuberculosis.

There is a clinical need for differential diagnosis of the latent versus active stages of tuberculosis (TB) disease by a simple-to-administer test. Alpha-crystallin (Acr) and early secretory antigenic target-6 (ESAT-6) are protein biomarkers associated with the latent and active stages of TB, respectively, and could be used for differential diagnosis. We therefore developed a microneedle patch (MNP) designed for application to the skin to quantify Acr and ESAT-6 in dermal interstitial fluid by enzyme-linked immunosorbent assay (ELISA). We fabricated mechanically strong microneedles made of polystyrene and coated them with capture antibodies against Acr and ESAT-6. We then optimized assay sensitivity to achieve a limit of detection of 750 pg/ml and 3,020 pg/ml for Acr and ESAT-6, respectively. This study demonstrates the feasibility of an MNP-based ELISA for differential diagnosis of latent TB disease.

Crumbs, Galla and Xpd are required for Kinesin-5 regulation in mitosis and organ growth in Drosophila.

Xeroderma Pigmentosum D (XPD, also known as ERCC2) is a multi-functional protein involved in transcription, DNA repair and chromosome segregation. In Drosophila, Xpd interacts with Crumbs (Crb) and Galla to regulate mitosis during embryogenesis. It is unknown how these proteins are linked to mitosis. Here, we show that Crb, Galla-2 and Xpd regulate nuclear division in the syncytial embryo by interacting with Klp61F, the Drosophila mitotic Kinesin-5 associated with bipolar spindles. Crb, Galla-2 and Xpd physically interact with Klp61F and colocalize to mitotic spindles. Knockdown of any of these proteins results in similar mitotic defects. These phenotypes are restored by overexpression of Klp61F, suggesting that Klp61F is a major effector. Mitotic defects of galla-2 RNAi are suppressed by Xpd overexpression but not vice versa. Depletion of Crb, Galla-2 or Xpd results in a reduction of Klp61F levels. Reducing proteasome function restores Klp61F levels and suppresses mitotic defects caused by knockdown of Crb, Galla-2 or Xpd. Furthermore, eye growth is regulated by Xpd and Klp61F. Hence, we propose that Crb, Galla-2 and Xpd interact to maintain the level of Klp61F during mitosis and organ growth.

8-Epi-xanthatin induces the apoptosis of DU145 prostate carcinoma cells through signal transducer and activator of transcription 3 inhibition and reactive oxygen species generation.

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in many human cancers. We tried to find STAT3 inhibitors from natural sources and found that Xanthium fruit extracts decreased phosphorylation of STAT3-Y705. 8-Epi-xanthatin (EXT) was isolated from the extracts. When DU145 cancer cells were treated with EXT, p-STAT3-Y705 was decreased with an IC50 of 3.2 µM. EXT decreased the expression of STAT3 target genes, such as cyclin A, cyclin D1, and BCL-2, and induced PARP cleavage, indicating apoptotic cell death. Downregulation of EXT-induced p-STAT3-Y705 was rescued by pretreating DU145 cells with antioxidants, such as N-acetyl-L-cysteine (NAC), indicating that reactive oxygen species (ROS) were involved in the EXT-induced inhibition of STAT3 activation. Furthermore, we proved the association of EXT with STAT3 protein by using a drug affinity responsive target stability (DARTS) assay and a cellular thermal shift assay (CETSA). EXT inhibited proliferation of DU145 cells with a GI50 of 6 µM and reduced tumor growth in mice xenografted with DU145 cells. Immunoblotting showed that phosphorylation of STAT3-Y705 was lower in EXT-treated tumor tissue than in control tissues. Collectively, we found that EXT binds to, and inhibits, STAT3 activation and could be a lead compound for anticancer therapy.

PLEKHA8P1 Promotes Tumor Progression and Indicates Poor Prognosis of Liver Cancer.

Hepatocellular carcinoma (HCC) records the second-lowest 5-year survival rate despite the avalanche of research into diagnosis and therapy. One of the major obstacles in treatment is chemoresistance to drugs such as 5-fluorouracil (5-FU), making identification and elucidation of chemoresistance regulators highly valuable. As the regulatory landscape grows to encompass non-coding genes such as long non-coding RNAs (lncRNAs), a relatively new class of lncRNA has emerged in the form of pseudogene-derived lncRNAs. Through bioinformatics analyses of the TCGA LIHC dataset, we have systematically identified pseudogenes of prognostic value. Initial experimental validation of selected pseudogene-derived lncRNA (PLEKHA8P1) and its parental gene (PLEKHA8), a well-studied transport protein in Golgi complex recently implicated as an oncogene in both colorectal and liver cancer, indicates that the pseudogene/parental gene pair promotes tumor progression and that their dysregulated expression levels affect 5-FU-induced chemoresistance in human HCC cell line FT3-7. Our study has thus confirmed cancer-related functions of PLEKHA8, and laid the groundwork for identification and validation of oncogenic pseudogene-derived lncRNA that shows potential as a novel therapeutic target in circumventing chemoresistance induced by 5-FU.

Preparation of Low-Diacylglycerol Cocoa Butter Equivalents by Hexane Fractionation of Palm Stearin and Shea Butter.

Herein, we prepared 1,3-dipalmitoyl-2-oleoyl glycerol (POP)-rich fats with reduced levels of diacylglycerols (DAGs), adversely affecting the tempering of chocolate, via two-step hexane fractionation of palm stearin. DAG content in the as-prepared fats was lower than that in POP-rich fats obtained by previously reported conventional two-step acetone fractionation. Cocoa butter equivalents (CBEs) were fabricated by blending the as-prepared fats with 1,3-distearoyl-2-oleoyl glycerol (SOS)-rich fats obtained by hexane fractionation of degummed shea butter. POP-rich fats achieved under the best conditions for the fractionation of palm stearin had a significantly lower DAG content (1.6 w/w%) than that in the counterpart (4.6 w/w%) prepared by the previously reported method. The CBEs fabricated by blending the POP- and SOS-rich fats in a weight ratio of 40:60 contained 63.7 w/w% total symmetric monounsaturated triacylglycerols, including 22.0 w/w% POP, 8.6 w/w% palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol, 33.1 w/w% SOS, and 1.3 w/w% DAGs, which was not substantially different from the DAG content in cocoa butter (1.1 w/w%). Based on the solid-fat content results, it was concluded that, when these CBEs were used for chocolate manufacture, they blended with cocoa butter at levels up to 40 w/w%, without distinctively altering the hardness and melting behavior of cocoa butter.

Structural and biophysical properties of RIG-I bound to dsRNA with G-U wobble base pairs.

Retinoic acid-inducible gene I (RIG-I) is responsible for innate immunity via the recognition of short double-stranded RNAs in the cytosol. With the clue that G-U wobble base pairs in the influenza A virus's RNA promoter region are responsible for RIG-I activation, we determined the complex structure of RIG-I ΔCARD and a short hairpin RNA with G-U wobble base pairs by X-ray crystallography. Interestingly, the overall helical backbone trace was not affected by the presence of the wobble base pairs; however, the base pair inclination and helical axis angle changed upon RIG-I binding. NMR spectroscopy revealed that RIG-I binding renders the flexible base pair of the influenza A virus's RNA promoter region between the two G-U wobble base pairs even more flexible. Binding to RNA with wobble base pairs resulted in a more flexible RIG-I complex. This flexible complex formation correlates with the entropy-favoured binding of RIG-I and RNA, which results in tighter binding affinity and RIG-I activation. This study suggests that the structure and dynamics of RIG-I are tailored to the binding of specific RNA sequences with different flexibility.

Systematic editing of synthetic RIG-I ligands to produce effective antiviral and anti-tumor RNA immunotherapies.

Retinoic acid-inducible gene I (RIG-I) recognizes double-stranded viral RNAs (dsRNAs) containing two or three 5' phosphates. A few reports of 5'-PPP-independent RIG-I agonists have emerged, but little is known about the molecular principles underlying their recognition. We recently found that the bent duplex RNA from the influenza A panhandle promoter activates RIG-I even in the absence of a 5'-triphosphate moiety. Here, we report that non-canonical synthetic RNA oligonucleotides containing G-U wobble base pairs that form a bent helix can exert RIG-I-mediated antiviral and anti-tumor effects in a sequence- and site-dependent manner. We present synthetic RNAs that have been systematically modified to enhance their efficacy and we outline the basic principles for engineering RIG-I agonists applicable to immunotherapy.

Design, synthesis, and biological evaluation of C7-functionalized DMXAA derivatives as potential human-STING agonists.

STING, a central protein in the innate immune response to cytosolic DNA, has emerged as a hot target for the development of vaccine-adjuvants and anticancer drugs. The discovery of potent human-STING (hSTING) agonist is expected to revolutionize the current cancer immunotherapy. Inspired by the X-ray crystal structure of DMXAA (5,6-dimethylxanthenone-4-acetic acid) and hSTINGG230I complex, we designed various DMXAA derivatives that contain a hydrogen bonding donor/acceptor or a halide at the C7 position. While 7-bromo- and 7-hydroxyl-DMXAA showed notable binding to mouse-STING (mSTING), our newly synthesized C7-functionalized DMXAA derivatives did not bind to hSTING. Nevertheless, our newly developed synthetic protocol for the C7-functionalization of DMXAA would be applicable to access other C7-substituted DMXAA analogues as potential hSTING agonists.

Anti-Obesity Effect of Standardized Extract of Microalga Phaeodactylum tricornutum Containing Fucoxanthin.

Fucoxanthin (FX), a marine carotenoid found in macroalgae and microalgae, exhibits several beneficial effects to health. The anti-obesity activity of FX is well documented, but FX has not been mass-produced or applied extensively or commercially because of limited availability of raw materials and complex extraction techniques. In this study, we investigated the anti-obesity effect of standardized FX powder (Phaeodactylum extract (PE)) developed from microalga Phaeodactylum tricornutum as a commercial functional food. The effects of PE on adipogenesis inhibition in 3T3-L1 adipocytes and anti-obesity in high-fat diet (HFD)-fed C57BL/6J mice were evaluated. PE and FX dose-dependently decreased intracellular lipid contents in adipocytes without cytotoxicity. In HFD-fed obese mice, PE supplementation for six weeks decreased body weight, organ weight, and adipocyte size. In the serum parameter analysis, the PE-treated groups showed attenuation of lipid metabolism dysfunction and liver damage induced by HFD. In the liver, uncoupling protein-1 (UCP1) upregulation and peroxisome proliferator activated receptor γ (PPARγ) downregulation were detected in the PE-treated groups. Additionally, micro computed tomography revealed lower fat accumulation in PE-treated groups compared to that in the HFD group. These results indicate that PE exerts anti-obesity effects by inhibiting adipocytic lipogenesis, inducing fat mass reduction and decreasing intracellular lipid content, adipocyte size, and adipose weight.

Posttraumatic Symptoms and Change of Complicated Grief among Bereaved Families of the Sewol Ferry Disaster: One Year Follow-up Study.

BACKGROUND: The objective of this study was to determine whether severity and severity change of coexisting psychiatric symptoms might affect change of complicated grief (CG) regarding the Sewol ferry disaster. METHODS: Data from a cross-sectional survey were obtained 18 months (Time 1) and 30 months (Time 2) after the disaster. We ascertained sociodemographic variables and variables obtained from self-reporting questionnaires (i.e., CG, depression, anxiety, post-traumatic stress disorder [PTSD], insomnia, embitterment, and suicidal risk) among 56 bereaved family members. RESULTS: Severity of other psychiatric symptoms at Time 1 had no effect on change of CG at Time 2. However, changes in severity of PTSD over a year affected change of CG. CONCLUSION: It is important to evaluate changes in severity of PTSD and its treatment during management of CG, especially when it involves bereaved families experiencing a traumatic accident.

High sensitivity temperature measurement via mask-free hybrid polymer long period fiber grating.

Long-period fiber gratings (LPFGs) are useful for environmental sensing under conditions of high corrosiveness and electromagnetic interference. Most LPFGs are fabricated by coherent or high-power UV illumination of an optical fiber under an amplitude mask, resulting in narrow and environmentally-dependent band rejection. We present a hybrid LPFG waveguide fabricated without an amplitude mask through polymer self-assembly under low-power incoherent UV illumination, which demonstrates high-temperature sensitivity in its transmission spectrum compared to LPFG sensors based purely on silica waveguides. A sensitivity of 1.5 nm °C -1 is obtained experimentally for attenuation near 1180 nm, and a sensitivity of 4.5 nm °C -1 with a low random error was obtained with a composite of attenuation bands. Finite element method simulations and coupling mode theory reveal this to be due to a thermo-optic coefficient one order of magnitude greater than that of fused silica. The device has potential for a simple and inexpensive transmission intensity based temperature sensor consisting of an infrared light source, the LPFG, a bandpass filter, and a photodiode.

Highly birefringent V-groove liquid core fiber.

We report a new efficient light guidance along a liquid core using an open V-groove. Guiding properties were analyzed using finite element method in terms of the single mode guidance condition, and the corresponding modal birefringence. We experimentally demonstrated a silica V-groove fiber with an opening angle of 40°, which was spliced to single mode fibers at both ends. A liquid with the refractive index of 1.455 was filled to serve as a core along a maximum length of 47cm. We confirmed the single mode guidance and birefringence consistent to theory, which will enable polarimetric liquid sensing.

Crude Ecklonia cava Flake Extracts Attenuate Inflammation through the Regulation of TLR4 Signaling Pathway in LPS-Induced RAW264.7 Cells.

We investigated the beneficial effects of the crude Ecklonia cava flake (CEF), which is a residual product after polyphenol extraction from Ecklonia cava, on inflammation in LPS-stimulated RAW264.7 cells. A group of five different CEF extracts was obtained by a preparation process using water, hydrochloric acid or temperature. We observed that large-size (>19 kDa) CEF extract, which was extracted with water at 95 °C (CEF-W, 95 °C), suppressed the production of inflammatory cytokines by inhibiting its mRNA expression in LPS-induced RAW264.7 cells. TLR4 signaling involvements were negatively regulated by CEF-W, 95 °C. CEF-W, 95 °C repressed the translocation of NF-κB from cytoplasm into nucleus in LPS-induced RAW264.7 cells. CEF-W, 95 °C attenuated the phosphorylation of TBK1 and IRF3 by inhibiting the phosphorylation of ERK. Taken together, we demonstrated that large-size CEF-W, 95 °C may act as a negative regulator of inflammation through the suppression of TLR4 signaling constituents in LPS-induced RAW264.7 cells.

Seapolynol Extracted from Ecklonia cava Inhibits Adipocyte Differentiation in Vitro and Decreases Fat Accumulation in Vivo.

Seapolynol (SN) is a polyphenol mixture derived from Ecklonia cava. We evaluated the effects of SN on lipid accumulation in adipocytes, zebrafish, and mice. SN effectively inhibited lipid accumulation in three experimental models by suppressing adipogenic factors. Triglyceride synthetic enzymes such as diacylglycerol acyltransferase 1 (DGAT1) and GPAT3 were also downregulated by SN. This SN-induced inhibition of adipogenic factors was shown to be due to the regulatory effect of SN on early adipogenic factors; SN downregulated the expression of Krueppel-like factor 4 (KLF4), KLF5, CCAAT-enhancer-binding protein ß (C/EBPß), C/EBPδ, and Protein C-ets-2 (ETS2), while KLF2, an anti-early adipogenic factor, was upregulated by SN. SN-mediated inhibition in early adipogenesis was closely correlated with the inhibition of mitotic clonal expansion via cell cycle arrest. SN inhibited cell cycle progression by suppressing cell cycle regulators, such as cyclin A, cyclinD, and pRb but increased p27, a cell cycle inhibitor. In a mouse study, SN effectively reduced body weight and plasma lipid increases induced by a high-fat diet; triglycerides, total cholesterol, and low-density lipoprotein (LDL) levels were markedly reduced by SN. Moreover, SN remarkably improved high-fat-diet-induced hepatic lipid accumulation. Furthermore, SN activated AMP-activated protein kinase-α (AMPKα), an energy sensor, to suppress acetyl-coA carboxylase (ACC), inhibiting lipid synthesis. Our study suggests that SN may be an edible agent that can play a positive role in prevention of metabolic disorders.

Microphysiological Blood-Brain Barrier Systems for Disease Modeling and Drug Development.

The blood-brain barrier (BBB) is a highly controlled microenvironment that regulates the interactions between cerebral blood and brain tissue. Due to its selectivity, many therapeutics targeting various neurological disorders are not able to penetrate into brain tissue. Pre-clinical studies using animals and other in vitro platforms have not shown the ability to fully replicate the human BBB leading to the failure of a majority of therapeutics in clinical trials. However, recent innovations in vitro and ex vivo modeling called organs-on-chips have shown the potential to create more accurate disease models for improved drug development. These microfluidic platforms induce physiological stressors on cultured cells and are able to generate more physiologically accurate BBBs compared to previous in vitro models. In this review, different approaches to create BBBs-on-chips are explored alongside their application in modeling various neurological disorders and potential therapeutic efficacy. Additionally, organs-on-chips use in BBB drug delivery studies is discussed, and advances in linking brain organs-on-chips onto multiorgan platforms to mimic organ crosstalk are reviewed.

Eye-movement reveals word order effects on comparative sentences in older adults using a verb-final language.

Objectives: This study aimed to examine age-related differences in the comprehension of Korean comparative sentences with varying word orders by employing both offline and online measures, and to investigate how variations in word order affect sentence processing across different age groups. Methods: A total of 52 monolingual native Korean speakers, 26 young adults, and 26 older adults, completed a sentence-picture-matching task under two word order conditions: comparative-first and nominative-first. Offline measures included accuracy and response time, while an online method involved eye-tracking within the Visual World Paradigm. Data analyses were performed using linear and generalized linear mixed-effects models. Results: Older adults demonstrated lower accuracy and longer response times compared to younger individuals. Distinctive fixation patterns were observed, particularly in the sentential-final phrase, across different age groups. Specifically, nominative-first sentences elicited greater target advantage scores among younger adults, whereas older adults showed higher scores in comparative-first sentences. Conclusion: The study highlights the potential of comparative sentences in elucidating age-related changes in sentence comprehension. These differences were evident not only in offline tasks but also in real-time processing, as evidenced by eye-tracking data. The findings suggest distinct processing strategies employed by young and older adults and underscore the importance of considering both syntactic and semantic cues in sentence comprehension.

The Relative Contribution of Facial and Body Information to the Perception of Cuteness.

Faces and bodies both provide cues to age and cuteness, but little work has explored their interaction in cuteness perception. This study examines the interplay of facial and bodily cues in the perception of cuteness, particularly when these cues convey conflicting age information. Participants rated the cuteness of face-body composites that combined either a child or adult face with an age-congruent or incongruent body alongside manipulations of the head-to-body height ratio (HBR). The findings from two experiments indicated that child-like facial features enhanced the perceived cuteness of adult bodies, while child-like bodily features generally had negative impacts. Furthermore, the results showed that an increased head size significantly boosted the perceived cuteness for child faces more than for adult faces. Lastly, the influence of the HBR was more pronounced when the outline of a body's silhouette was the only available information compared to when detailed facial and bodily features were presented. This study suggests that body proportion information, derived from the body's outline, and facial and bodily features, derived from the interior surface, are integrated to form a unitary representation of a whole person in cuteness perception. Our findings highlight the dominance of facial features over bodily information in cuteness perception, with facial attributes serving as key references for evaluating face-body relationships and body proportions. This research offers significant insights into social cognition and character design, particularly in how people perceive entities with mixed features of different social categories, underlining the importance of congruency in perceptual elements.

Preliminary investigation of a combined herbal extract of Aruncus dioicus, Cirsium nipponicum, and Ocimum basilicum for halitosis.

BACKGROUND: Halitosis, the unpleasant odor in the oral cavity, has garnered increased attention and concern due to the growing significance of social interaction. SGE-107, a blend of 3 botanical drugs-Korean goat's beard, Cirsium tanakae, and Basil-with caffeic acid as its indicator component. This study aims to investigate the efficacy of SGE-107 in treating halitosis in patients with mild gastrointestinal symptoms. METHODS: We enrolled 25 participants with oral malodor and dyspeptic symptoms. We assessed the severity of halitosis using the visual analog scale. Throughout a 4-week period of administering SGE-107, we evaluated both objective and subjective parameters, including the halitosis-associated life-quality test, the Korean gastrointestinal symptom rating scale, levels of volatile sulfur compounds, salivary flow rate, oral moisture, tongue index, Winkel tongue coating index, and tongue temperature. RESULTS: After the intervention period, both the visual analog scale (5.88 ±â€…1.03 vs 2.38 ±â€…0.93, P < .001) and the scores of the halitosis-associated life-quality test (31.21 ±â€…11.78 vs 13.83 ±â€…6.38, P < .001) showed significant reductions. The proportion of participants with abnormal levels of methyl mercaptan (a volatile sulfur compound) also significantly decreased (17, 70.8% vs 9, 37.5%, P = .039). Furthermore, there were significant improvements in reflux, constipation, diarrhea, and the total scores on the Korean gastrointestinal symptom rating scale. Throughout the study period, only 2 participants experienced mild adverse events. CONCLUSION: SGE-107 appears to be a safe and effective treatment for halitosis-associated with gastrointestinal symptoms; nevertheless, the limited sample size necessitates further large-scale randomized, controlled studies to confirm our findings.

Immobilized Candida antarctica lipase B as an sn-1,3 regiospecific biocatalyst for the interesterification of triacylglycerols with fatty acid ethyl esters.

Candida antarctica lipase B (CALB) is regarded as non-regiospecific. This study aimed to investigate the regiospecificity of CALB in the solvent-free interesterification of high-oleic sunflower oil with stearic acid ethyl ester for 1,3-distearoyl-2-oleoylglycerol (SOS)-rich fat preparation using a packed bed reactor. The content ratio of 1,2-distearoyl-3-oleoylglycerol (SSO) to SOS (denoted by SSO/SOS content) obtained using Lipozyme 435 (a commercially immobilized CALB; 0-4.1%), at residence times (1-32 min) was similar to that obtained using Lipozyme RM IM (0-3.0%), but lower than that obtained using Lipozyme TL IM (6.0-39.4%). When immobilized on Lewatit VP OC 1600, Lipozyme CALB had an SSO/SOS content of 0-10.4%, which was greater than that of Palatase 20,000 L (0-1.1%) but was lower than that of Lipozyme TL 100 L (8.8-97.7%). Our findings suggest that immobilized CALB shows distinct sn-1,3 regiospecificity in the interesterification of triacylglycerol with fatty acid ethyl esters.