The Effect of Lactobacillus plantarum Extracellular Vesicles from Korean Women in Their 20s on Skin Aging

Extracellular vesicles, which are highly conserved in most cells, contain biologically active substances. The vesicles and substances interact with cells and impact physiological mechanisms. The skin is the most external organ and is in direct contact with the external environment. Photoaging and skin damage are caused by extrinsic factors. The formation of wrinkles is a major indicator of skin aging and is caused by a decrease in collagen and hyaluronic acid. MMP-1 expression is also increased. Due to accruing damage, skin aging reduces the ability of the skin barrier, thereby lowering the skin’s ability to contain water and increasing the amount of water loss. L. plantarum suppresses various harmful bacteria by secreting an antimicrobial substance. L. plantarum is also found in the skin, and research on the interactions between the bacteria and the skin is in progress. Although several studies have investigated L. plantarum, there are only a limited number of studies on extracellular vesicles (EV) derived from L. plantarum, especially in relation to skin aging. Herein, we isolated EVs that were secreted from L. plantarum of women in their 20s (LpEVs). We then investigated the effect of LpEVs on skin aging in CCD986sk. We showed that LpEVs modulated the mRNA expression of ECM related genes in vitro. Furthermore, LpEVs suppressed wrinkle formation and pigmentation in clinical trials. These results demonstrated that LpEVs have a great effect on skin aging by regulating ECM related genes. In addition, our study offers important evidence on the depigmentation effect of LpEVs.     

Stimulatory Effects of Extracellular Vesicles Derived from Leuconostoc holzapfelii That Exists in Human Scalp on Hair Growth in Human Follicle Dermal Papilla Cells

Human hair follicle dermal papilla cells (HFDPCs) located in hair follicles (HFs) play a pivotal role in hair follicle morphogenesis, hair cycling, and hair growth. Over the past few decades, probiotic bacteria (PB) have been reported to have beneficial effects such as improved skin health, anti-obesity, and immuno-modulation for conditions including atopic dermatitis and inflammatory bowel disease (IBD). PB can secrete 50~150 nm sized extracellular vesicles (EVs) containing microbial DNA, miRNA, proteins, lipids, and cell wall components. These EVs can regulate communication between bacteria or between bacteria and their host. Although numerous biological effects of PB-EVs have been reported, the physiological roles of Leuconostoc holzapfelii (hs-Lh), which is isolated from human scalp tissue, and the extracellular vesicles derived from them (hs-LhEVs) are largely unknown. Herein, we investigated the effects of hs-LhEVs on hair growth in HFDPCs. We show that hs-LhEVs increase cell proliferation, migration, and regulate the cell cycle. Furthermore, hs-LhEVs were found to modulate the mRNA expression of hair-growth-related genes in vitro. These data demonstrate that hs-LhEVs can reduce apoptosis by modulating the cell cycle and promote hair growth by regulation via the Wnt/β-catenin signal transduction pathway.     

重楼中一株产纤维素酶内生真菌的分离及鉴定

从重楼根茎中分离、鉴定具有产纤维素酶活性的内生真菌。采用表面消毒法从重楼块茎中分离内生真菌;用纤维素酶活性CMC平板检测分离菌株的产纤维素酶活性;对高产菌株进行形态学观察和分子生物学测序鉴定;探究影响纤维素酶活力的因素;利用平板法检测该株菌产其他胞外水解酶的活性。从3个来源的重楼中分离出41株内生真菌,通过平板检测发现AS-5、AS-7、AS-9和AS-18菌株能产生纤维素酶,其中AS-9菌株活性最强;通过形态学观察和ITS、LSU序列分析将AS-9菌株鉴定为Setophoma terrestris;该菌在pH值为7.0和温度为28℃时表现出最大纤维素酶活性,紫外线照射对产纤维素酶活性无明显作用;检测发现AS-9菌株同时具有产酪蛋白酶、脂肪酶、天冬酰胺酶、谷氨酰胺酶和脲酶活性。首次在重楼中发现内生真菌Setophoma terrestris,且具有较好的产纤维素酶能力,值得深入研究。     

Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.     

In vitro machine learning-based CAR T immunological synapse quality measurements correlate with patient clinical outcomes

The human immune system consists of a highly intelligent network of billions of independent, self-organized cells that interact with each other. Machine learning (ML) is an artificial intelligence (AI) tool that automatically processes huge amounts of image data. Immunotherapies have revolutionized the treatment of blood cancer. Specifically, one such therapy involves engineering immune cells to express chimeric antigen receptors (CAR), which combine tumor antigen specificity with immune cell activation in a single receptor. To improve their efficacy and expand their applicability to solid tumors, scientists optimize different CARs with different modifications. However, predicting and ranking the efficacy of different "off-the-shelf" immune products (e.g., CAR or Bispecific T-cell Engager [BiTE]) and selection of clinical responders are challenging in clinical practice. Meanwhile, identifying the optimal CAR construct for a researcher to further develop a potential clinical application is limited by the current, time-consuming, costly, and labor-intensive conventional tools used to evaluate efficacy. Particularly, more than 30 years of immunological synapse (IS) research data demonstrate that T cell efficacy is not only controlled by the specificity and avidity of the tumor antigen and T cell interaction, but also it depends on a collective process, involving multiple adhesion and regulatory molecules, as well as tumor microenvironment, spatially and temporally organized at the IS formed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The optimal function of cytotoxic lymphocytes (including CTL and NK) depends on IS quality. Recognizing the inadequacy of conventional tools and the importance of IS in immune cell functions, we investigate a new strategy for assessing CAR-T efficacy by quantifying CAR IS quality using the glass-support planar lipid bilayer system combined with ML-based data analysis. Previous studies in our group show that CAR-T IS quality correlates with antitumor activities in vitro and in vivo. However, current manually quantified IS quality data analysis is time-consuming and labor-intensive with low accuracy, reproducibility, and repeatability. In this study, we develop a novel ML-based method to quantify thousands of CAR cell IS images with enhanced accuracy and speed. Specifically, we used artificial neural networks (ANN) to incorporate object detection into segmentation. The proposed ANN model extracts the most useful information to differentiate different IS datasets. The network output is flexible and produces bounding boxes, instance segmentation, contour outlines (borders), intensities of the borders, and segmentations without borders. Based on requirements, one or a combination of this information is used in statistical analysis. The ML-based automated algorithm quantified CAR-T IS data correlates with the clinical responder and non-responder treated with Kappa-CAR-T cells directly from patients. The results suggest that CAR cell IS quality can be used as a potential composite biomarker and correlates with antitumor activities in patients, which is sufficiently discriminative to further test the CAR IS quality as a clinical biomarker to predict response to CAR immunotherapy in cancer. For translational research, the method developed here can also provide guidelines for designing and optimizing numerous CAR constructs for potential clinical development.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00881920","term_id":"NCT00881920"}}NCT00881920.     

An ecological floating-bed made from dredged lake sludge for purification of eutrophic water

Removal of lake sediments is one effective approach to reduce internal nutrients of eutrophic lakes, the consequence of which is, however, unavoidably exacerbated by the disposal of the dredged sludge. A novel method is reported here to recycle the dredged sludge in an eco-friendly manner by combining solidification/stabilization treatment and floating-bed technology. The objective of the present study was to utilize the dredged lake sludge to produce ecological sludge floating-bed (ESFB) and to evaluate the water purification efficacy of ESFB combined concurrently with Acorus calamus. In doing so, dredged sludge (main material), treated industrial slag (binder) and expanded perlite (lightweight agent) were introduced as the raw materials for the production of ESFB, and then physical strength, water-resistance, toxicity characteristic leaching procedure were measured and field trial was carried out, correspondingly. For the water purification experiment, the mesocosm systems were made to evaluate the nitrogen, phosphorus and Chl-a removal efficacy of planted ESFB. The results show that with the optimum mixing ratio (sludge: 72.5%, BOF slag: 12.5% and expanded perlite: 15%), the solidified product has strong compressive strength of 1.55 MPa and flexural strength of 0.24 MPa. The heavy metal concentrations in the leachate are far lower than the detection limit. The relative softening coefficient and water absorption of solidified product were 90.3% and 11.3%. The results of the consequent water purification experiment suggest that the planted ESFB have promising removal efficiency on nitrogen, phosphorus and Chl-a. The final relative growth rate (RGR) of A. calamus was 0.31. The highest removal rate of total nitrogen (TN), total phosphorus (TP), ammonium nitrogen (NH₄⁺-N), total dissolve phosphorus (TDP) and chlorophyll-a (Chl-a) reached 36.3%, 35.7%, 44.3%, 38.1% and 47.9%, respectively.     

The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action

Five strains of proteolytic rumen bacteria were treated with condensed tannins (CT) purified from Lotus pedunculatus and Lotus corniculatus to investigate their effect on the growth of these bacteria in vitro. Streptococcus bovis NCFB 2476, Eubacterium sp. C124b, Prevotella bryantii B14, Butyrivibrio fibrisolvens H17c, and Clostridium proteoclasticum B316T were tested against 200, 400, and 600 microg CT x mL(-1) extracted from L. pedunculatus and L. corniculatus. In the absence of CT, all bacterial strains showed typical growth and reached maximum optical density (OD) after 6-8 h of incubation in a plant protein medium. Growth of Eubacterium sp., P. bryantii, and B. fibrisolvens was inhibited (P < 0.01-0.001) more by the CT from L. pedunculatus than by the CT from L. corniculatus. All strains continued to grow in the presence of 200 microg x mL(-1) of the CT from L. pedunculatus, but attained significantly (P < 0.05-0.01) lower maximum OD600 values than (minus CT) controls, except for S. bovis. At 400 and 600 microg x mL(-1), the addition of CT from L. pedunculatus inhibited (P < 0.05-0.001) the growth of all bacterial strains tested compared with controls. The growth of Eubacterium sp. and P. bryantii was stimulated for the first 4-6 h of incubation (P < 0.001) by 200 microg x mL(-1) of CT from L. corniculatus, but then declined leading to a significant difference in OD values compared with the controls. At 400 microg x mL(-1), the CT from L. corniculatus reduced (P < 0.05-0.01) the growth of all strains except S. bovis, while 600 microg x mL(-1) inhibited (P < 0.01-0.001) the growth of all strains. To study the mechanism of CT action, the degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; Fraction 1 Leaf protein) was followed after bacterial cells or Rubisco were preincubated with CT extracted from L. corniculatus and L. pedunculatus. Both preincubations decreased LSU degradation, but they differed in their response to polyethylene glycol (PEG) addition. Addition of PEG to CT-Rubisco preincubations negated the effects of CT, while PEG addition to CT-bacteria preincubations did not. This implies that the CT-bacterial interaction is stronger than the CT-Rubisco interaction or the interaction is of a different type. Also, L. pedunculatus CT reduced the degradation of the LSU to a greater extent than the CT from L. corniculatus when preincubated with bacteria.     

Modulation of AMPA receptors by a novel organic nitrate

Positive modulators of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) channels reduce desensitization and alter their gating kinetics. We have discovered a novel compound nitric oxide-mimetic that similarly modulates the AMPA receptor by reducing desensitization. This, designated GT-005, belongs to the organic nitrate family that includes the nitrovasodilator nitroglycerine. In acutely isolated hippocampal neurons, GT-005 enhanced kainate (100 microM)-evoked currents with an EC50 of 1.7+/-0.2 mM and a 176+/-10% maximal increase in the steady-state current response. Similar results were found in cultured hippocampal neurons (EC50 of 1.3+/-0.2 mM and a maximal 83+/-14% increase in the steady-state current response). GT-005 reduced the desensitization of glutamate-evoked currents and slowed the onset of desensitization. This compound also increased the rate of recovery from the desensitized state. With respect to alteration of the excitatory synaptic transmission, GT-005 delayed the decay and increased the frequency of spontaneous miniature excitatory postsynaptic currents (mepsc) recorded in cultured hippocampal neurons.     

Endothelin-1 stimulates leptin production in adipocytes

Leptin is an adipocyte-derived hormone that regulates body fat stores and feeding behavior. In an effort to identify endogenous diffusible modulators of leptin production, we found that endothelin-1 (ET-1) up-regulates leptin expression in adipocytes. ET-1 is as potent and efficacious as insulin in stimulating leptin production in two different adipocyte cell lines. Endothelins stimulate leptin production via the endothelin-A receptor (ET(A)), as judged by a potency rank order of ET-1 ET-3. We detected expression of ET(A) but not ET(B) in both cell lines by Northern blot analysis. In addition, the ET(A)-selective antagonist FR139317 inhibited ET-1-induced leptin expression more potently than did the ET(B)-selective antagonist BQ788. ET-1 and insulin positively interact with each other in increasing leptin production in adipocytes. In primary mouse white fat cells, we detected expression of both ET(A) and ET(B) by Northern blot and in situ hybridization analyses. We conclude that ET-1 stimulates leptin production via the ET(A) receptor in cultured adipocytes.