Elucidating the callus-to-shoot-forming mechanism in Capsicum annuum 'Dempsey' through comparative transcriptome analyses.

BACKGROUND: The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum 'Dempsey', a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum 'Dempsey' shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum 'Dempsey' transcriptome based on comparative transcriptomic analysis across five species. RESULTS: We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum 'Dempsey'. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum 'Dempsey'. CONCLUSION: Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum 'Dempsey' explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

Effects of Heat Stress on the Laying Performance, Egg Quality, and Physiological Response of Laying Hens.

As high temperature and relative humidity (RH) are the main environmental factors causing heat stress, the temperature-humidity index (THI) serves as an indicator of heat stress in livestock animals. This study aimed to determine the effects of heat stress on the laying performance, physiological responses, egg quality, and blood profile of laying hens by subjecting them to environmental conditions with varying THI levels (68-85) for 28 days. The indicators of laying performance, such as feed intake (-30%) and egg production rate (-11%), significantly decreased in the hens exposed to severe heat stress (33 °C, 66% RH) compared to those exposed to thermoneutral conditions (21 °C, 68% RH). Moreover, severe heat stress reduced the egg yolk color, eggshell thickness and strength, and Haugh units of the eggs produced by the laying hens. Furthermore, a significant increase in serum K+ and a decrease in Na+ levels were observed in the hens subjected to severe heat stress compared with those under thermoneutral conditions. Our results indicate that heat stress alters the physiological responses and metabolism of laying hens, resulting in a lower egg quality and production rate.

Imitating Synapse Behavior: Exploiting Off-Current in TPBi-Doped Small Molecule Phototransistors for Broadband Wavelength Recognition.

Phototransistors have gained significant attention in diverse applications such as photodetectors, image sensors, and neuromorphic devices due to their ability to control electrical characteristics through photoresponse. The choice of photoactive materials in phototransistor research significantly impacts its development. In this study, we propose a novel device that emulates artificial synaptic behavior by leveraging the off-current of a phototransistor. We utilize a p-type organic semiconductor, dinaphtho[2,3-b:2',3'- f]thieno[3,2-b]thiophene (DNTT), as the channel material and dope it with the organic semiconductor 2,2',2″-(1,3,5-benzinetriyl)-tris(1-phenyl-1-H-benzimidazole) (TPBi) on the DNTT transistor. Under light illumination, the general DNTT transistor shows no change in off-current, except at 400 nm wavelength, whereas the TPBi-doped DNTT phototransistor exhibits increased off-current across all wavelength bands. Notably, DNTT phototransistors demonstrate broad photoresponse characteristics in the wavelength range of 400-1000 nm. We successfully simulate artificial synaptic behavior by differentiating the level of off-current and achieving a recognition rate of over 70% across all wavelength bands.

Rise in broadly cross-reactive adaptive immunity against human ß-coronaviruses in MERS-recovered patients during the COVID-19 pandemic.

To develop a universal coronavirus (CoV) vaccine, long-term immunity against multiple CoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, Middle East respiratory syndrome (MERS)-CoV, and future CoV strains, is crucial. Following the 2015 Korean MERS outbreak, we conducted a long-term follow-up study and found that although neutralizing antibodies and memory T cells against MERS-CoV declined over 5 years, some recovered patients exhibited increased antibody levels during the COVID-19 pandemic. This likely resulted from cross-reactive immunity induced by SARS-CoV-2 vaccines or infections. A significant correlation in antibody responses across various CoVs indicates shared immunogenic epitopes. Two epitopes-the spike protein's stem helix and intracellular domain-were highly immunogenic after MERS-CoV infection and after SARS-CoV-2 vaccination or infection. In addition, memory T cell responses, especially polyfunctional CD4+ T cells, were enhanced during the pandemic, correlating significantly with MERS-CoV spike-specific antibodies and neutralizing activity. Therefore, incorporating these cross-reactive and immunogenic epitopes into pan-CoV vaccine formulations may facilitate effective vaccine development.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes.

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

Harnessing CRISPR/Cas9 for Enhanced Disease Resistance in Hot Peppers: A Comparative Study on CaMLO2-Gene-Editing Efficiency across Six Cultivars.

The Capsicum annuum Mildew Locus O (CaMLO2) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated CaMLO2 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/CaMLO2single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/CaMLO2sgRNA2-RNP systems by extending their application from the bell pepper 'Dempsey' and the hot pepper 'CM334' to six commercial hot pepper cultivars. Across the six cultivars, CaMLO2sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas CaMLO2sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/CaMLO2sgRNA1-RNP system to achieve efficient gene editing at the CaMLO2 locus in different Capsicum annuum cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with CaMLO2-mediated enhanced disease resistance.

Effect of Fermented Concentrate on Ruminal Fermentation, Ruminal and Fecal Microbiome, and Growth Performance of Beef Cattle.

The impact of fermented concentrate on the growth and rumen health of beef cattle remains an area of emerging research. This study aimed to assess the influence of a fermented concentrate (TRT) compared to a conventional concentrate (CON) on the growth, rumen fermentation characteristics, and microbiota composition in Korean cattle. Using a crossover design, eight cattle were alternately fed TRT and CON diets, with subsequent analysis of feed components, rumen fermentation parameters, and microbial profiles. TRT and CON diets did not differ significantly in their effect on animal growth metrics. However, the TRT diet was associated with reduced digestibility of rapidly degradable carbohydrates and modified rumen fermentation patterns, as evidenced by an elevated pH and increased acetate-to-propionate ratio (p < 0.05). Furthermore, the TRT diet increased the abundance of lactic acid bacteria, Bacillus, and yeast and organic acid levels in the rumen (p < 0.05). Moreover, Lachnospiraceae and Bacteroidales populations in the rumen and fecal Akkermansia abundance increased in the TRT group compared to the CON group. These microbial changes suggest a potential enhancement of the immune system and overall health of the host. Further research on the long-term implications of incorporating fermented concentrate into cattle diets is warranted.

Anti-Diabetic Potential of Sargassum horneri and Ulva australis Extracts In Vitro and In Vivo.

Sargassum horneri (SH) and Ulva australis (UA) are marine waste resources that cause environmental and economic problems when entering or multiplying the coastal waters of Jeju Island. We analyzed their anti-diabetic efficacy to assess their reusability as functional additives. The alpha-glucosidase inhibitory activity of SH and UA extracts was confirmed, and the effect of UA extract was higher than that of SH. After the induction of insulin-resistant HepG2 cells, the effects of the two marine extracts on oxidative stress, intracellular glucose uptake, and glycogen content were compared to the positive control, metformin. Treatment of insulin-resistant HepG2 cells with SH and UA resulted in a concentration-dependent decrease in oxidative stress and increased intracellular glucose uptake and glycogen content. Moreover, SH and UA treatment upregulated the expression of IRS-1, AKT, and GLUT4, which are suppressed in insulin resistance, to a similar degree to metformin, and suppressed the expression of FoxO1, PEPCK involved in gluconeogenesis, and GSK-3ß involved in glycogen metabolism. The oral administration of these extracts to rats with streptozotocin-induced diabetes led to a higher weight gain than that in the diabetic group. Insulin resistance and oral glucose tolerance are alleviated by the regulation of blood glucose. Thus, the SH and UA extracts may be used in the development of therapeutic agents or supplements to improve insulin resistance.

Association between blood pressure and endovascular treatment outcomes differs by baseline perfusion and reperfusion status.

We hypothesized that the association between BP and endovascular treatment (EVT) outcomes would differ by baseline perfusion and recanalization status. We identified 388 ICA or M1 occlusion patients who underwent EVT ≤ 24 h from onset with successful recanalization (TICI ≥ 2b). BP was measured at 5-min intervals from arrival and during the procedure. Systolic BPs (SBP) were summarized as dropmax (the maximal decrease over two consecutive measurements), incmax (the maximal increase), mean, coefficient of variation (cv), and standard deviation. Adequate baseline perfusion was defined as hypoperfusion intensity ratio (HIR) ≤ 0.5; infarct proportion as the volume ratio of final infarcts within the Tmax > 6 s region. In the adequate perfusion group, infarct proportion was closely associated with SBPdropmax (ß ± SE (P-value); 1.22 ± 0.48, (< 0.01)), SBPincmax (1.12 ± 0.33, (< 0.01)), SBPcv (0.61 ± 0.15 (< 0.01)), SBPsd (0.66 ± 0.08 (< 0.01)), and SBPmean (0.71 ± 0.37 (0.053) before recanalization. The associations remained significant only in SBPdropmax, SBPincmax, and SBPmean after recanalization. SBPincmax, SBPcv and SBPsd showed significant associations with modified Rankin Scale score at 3 months in the pre-recanalization period. In the poor perfusion group, none of the SBP indices was associated with any stroke outcomes regardless of recanalization status. BP may show differential associations with stroke outcomes by the recanalization and baseline perfusion status.

Maternal and Fetal Effects of Gestational Vitamin D Concentration.

Most (90%) vitamin D synthesis occurs in the skin using sunlight (ultraviolet rays), and 10% is obtained through food. Vitamin D is an essential nutrient for skeletal growth and maintenance, cell proliferation and differentiation, and immune function. This study investigated whether maternal serum vitamin D concentrations induce maternofetal effects. Hematological analysis, serological changes, and precision fetal ultrasound findings were analyzed by maternal vitamin D concentration in gestational weeks 22-25 to ascertain direct effects on fetal growth. Bone density-vitamin D concentration correlation was analyzed. No hematologic or serological effect of maternal vitamin D concentration was detected; however, the sexually transmitted infection and cross-infection rates were inversely proportional to maternal vitamin D concentration. No significant correlation between vitamin D concentration and vertebral and femoral BMD was detected. For fetal growth, biparietal diameter, head circumference, abdominal circumference, femur length, and humerus length were analyzed. Humerus (p < 0.05) and femur (p < 0.001) lengths were higher in the vitamin D-sufficient group than in the vitamin D-deficient group. Vitamin D concentration did not positively affect hematologic changes and bone density; maternal vitamin D concentration essentially affected fetal bone growth. Vitamin D inhibits sexually transmitted infections in mothers and promotes fetal bone growth. Prevention of vitamin D deficiency, supplementation, or outdoor activities is recommended.

The Immunosuppressive Potential of Cholesterol Sulfate Through T Cell Microvilli Disruption.

Cholesterol (CL) is required for various biomolecular production processes, including those of cell membrane components. Therefore, to meet these needs, CL is converted into various derivatives. Among these derivatives is cholesterol sulfate (CS), a naturally produced CL derivative by the sulfotransferase family 2B1 (SULT2B1), which is widely present in human plasma. CS is involved in cell membrane stabilization, blood clotting, keratinocyte differentiation, and TCR nanocluster deformation. This study shows that treatment of T cells with CS resulted in the decreased surface expression of some surface T-cell proteins and reduced IL-2 release. Furthermore, T cells treated with CS significantly reduced lipid raft contents and membrane CLs. Surprisingly, using the electron microscope, we also observed that CS led to the disruption of T-cell microvilli, releasing small microvilli particles containing TCRs and other microvillar proteins. However, in vivo, T cells with CS showed aberrant migration to high endothelial venules and limited infiltrating splenic T-cell zones compared with the untreated T cells. Additionally, we observed significant alleviation of atopic dermatitis in mice injected with CS in the animal model. Based on these results, we conclude that CS is an immunosuppressive natural lipid that impairs TCR signaling by disrupting microvillar function in T cells, suggesting its usefulness as a therapeutic agent for alleviating T-cell-mediated hypersensitivity and a potential target for treating autoimmune diseases.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Negative association between high temperature-humidity index and milk performance and quality in Korean dairy system: big data analysis.

The aim of this study was to investigate the effects of heat stress on milk traits in South Korea using comprehensive data (dairy production and climate). The dataset for this study comprised 1,498,232 test-day records for milk yield, fat- and protein-corrected milk, fat yield, protein yield, milk urea nitrogen (MUN), and somatic cell score (SCS) from 215,276 Holstein cows (primiparous: n = 122,087; multiparous: n = 93,189) in 2,419 South Korean dairy herds. Data were collected from July 2017 to April 2020 through the Dairy Cattle Improvement Program, and merged with meteorological data from 600 automatic weather stations through the Korea Meteorological Administration. The segmented regression model was used to estimate the effects of the temperature-humidity index (THI) on milk traits and elucidate the break point (BP) of the THI. To acquire the least-squares mean of milk traits, the generalized linear model was applied using fixed effects (region, calving year, calving month, parity, days in milk, and THI). For all parameters, the BP of THI was observed; in particular, milk production parameters dramatically decreased after a specific BP of THI (p < 0.05). In contrast, MUN and SCS drastically increased when THI exceeded BP in all cows (p < 0.05) and primiparous cows (p < 0.05), respectively. Dairy cows in South Korea exhibited negative effects on milk traits (decrease in milk performance, increase in MUN, and SCS) when the THI exceeded 70; therefore, detailed feeding management is required to prevent heat stress in dairy cows.

Overcoming Charge Confinement in Perovskite Nanocrystal Solar Cells.

The small nanoparticle size and long-chain ligands in colloidal metal halide perovskite quantum dots (PeQDs) cause charge confinement, which impedes exciton dissociation and carrier extraction in PeQD solar cells, so they have low short-circuit current density Jsc , which impedes further increases in their power conversion efficiency (PCE). Here, a re-assembling process (RP) is developed for perovskite nanocrystalline (PeNC) films made of colloidal perovskite nanocrystals to increase Jsc in PeNC solar cells. The RP of PeNC films increases their crystallite size and eliminates long-chain ligands, and thereby overcomes the charge confinement in PeNC films. These changes facilitate exciton dissociation and increase carrier extraction in PeNC solar cells. By use of this method, the gradient-bandgap PeNC solar cells achieve a Jsc = 19.30 mA cm-2 without compromising the photovoltage, and yield a high PCE of 16.46% with negligible hysteresis and good stability. This work provides a new strategy to process PeNC films and pave the way for high performance PeNC optoelectronic devices.

Trogocytic molting of T cell microvilli upregulates T cell receptor surface expression and promotes clonal expansion.

Although T cell activation is known to involve the internalization of the T cell antigen receptor (TCR), much less is known regarding the release of TCRs following T cell interaction with cognate antigen-presenting cells. In this study, we examine the physiological mechanisms underlying TCR release following T cell activation. We show that T cell activation results in the shedding of TCRs in T cell microvilli, which involves a combined process of trogocytosis and enzymatic vesiculation, leading to the loss of membrane TCRs and microvilli-associated proteins and lipids. Surprisingly, unlike TCR internalization, this event results in the rapid upregulation of surface TCR expression and metabolic reprogramming of cholesterol and fatty acid synthesis to support cell division and survival. These results demonstrate that TCRs are lost through trogocytic 'molting' following T cell activation and highlight this mechanism as an important regulator of clonal expansion.

Metabolic Discrimination between Adventitious Roots and Standard Medicinal Part of Atractylodes macrocephala Koidz. Using FT-IR Spectroscopy.

This study aims to examine the metabolic discrimination between in vitro grown adventitious roots and the standard medicinal parts of Atractylodes macrocephala. To achieve this goal, firstly, in vitro culture conditions of adventitious roots such as indole-3-butyric acid (IBA) concentrations, types of media, inorganic salt strength of culture medium, and elicitor types and concentrations were optimized. The optimal culture conditions for proliferation of adventitious roots was found to consist of Murashige and Skoog (MS) medium containing 5 mg L-1 IBA. Whole cell extracts from adventitious roots and the standard medicinal parts of A. macrocephala were subjected to Fourier transform infrared spectroscopy (FT-IR). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) from FT-IR spectral data showed that adventitious roots and standard medicinal parts were clearly distinguished in the PCA and PLS-DA score plot. Furthermore, the overall metabolite pattern from adventitious roots was changed depending on the dose-dependent manner of chemicals. These results suggest that FT-IR spectroscopy can be applied as an alternative tool for the screening of higher metabolic root lines and for discriminating metabolic similarity between in vitro grown adventitious roots and the standard medicinal parts. In addition, the adventitious roots proliferation system established in this study can be directly applied as an alternative means for the commercial production of A. macrocephala.

ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

The Effects of Manual Acupuncture on Mitochondrial Fusion and Fission Gene Expression in Rat Spleen.

Background: A significant amount of research has been conducted to establish the validity of acupuncture, and it has been demonstrated through animal disease model studies that acupuncture influences mitochondrial changes. However, to more accurately examine the mechanisms of acupuncture treatment effectiveness in pathological models, it is crucial to investigate changes in disease-free animals. Among various hypotheses regarding the effects of acupuncture on the body, we focused on the result that acupuncture stimulation is related to mitochondria. Objectives: We examined the effects of acupuncture mitochondrial fission and fusionrelated mediators in disease-free Sprague Dawley (SD) rats' spleen meridian acupoints. Methods: SD rats were divided into control, SP1, SP2, SP3, SP5, and SP9 acupuncture groups. Acupuncture was performed at each point for 10 minutes daily for four days. Peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α) and fission protein 1 (Fis1) levels were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), while dynamin-related protein 1 (DRP1), optic atrophy-1 (OPA1), mitofusin-1 (MFN1), and mitofusin-2 (MFN2) levels were assessed via western blotting. Mitochondria protein concentrations and NADH dehydrogenase activity in spleen tissues were measured using enzyme-linked immunosorbent assay (ELISA). Results: PGC-1α expression decreased in the SP1 (p < 0.01), SP5 (p < 0.05), and SP9 (p < 0.05) groups, while Fis1 expression increased in the SP1 (p < 0.01), SP5 (p < 0.01), and SP9 (p < 0.05) groups. DRP1, OPA1, MFN1, and MFN2 levels exhibited no significant changes. Mitochondrial protein concentrations decreased in the SP2 (p < 0.01), SP3 (p < 0.01), SP5 (p < 0.01), and SP9 (p < 0.01) groups, while NADH dehydrogenase activity decreased in the SP2 (p < 0.05) and SP9 (p < 0.05) groups. Conclusion: Acupuncture at the SP9 acupoint influenced the mitochondrial fission pathway by modulating PGC-1α and Fis1 mediators in the rat spleen under non-disease conditions.

T Cell Immunological Synaptosomes: Definition and Isolation.

In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are separated from the T cell body shortly upon TCR stimulation and vesiculated to form T cell microvilli particles (TMPs), a new type of membrane vesicles. TMPs and synaptic ectosomes, which bud from the synaptic cleft, constitute "T cell immunological synaptosomes (TISs)" and act as conveyors of T cell messages or traits to cognate antigen-presenting cells. In practice, it is almost impossible to distinguish between TMPs and synaptic ectosomes. Here, we describe a newly developed protocol to isolate TISs from activated T cells using antibody-immobilized agarose beads and density gradient ultracentrifugation. We further describe the methods for TIS quantification with flow cytometry and to evaluate TIS efficacy on dendritic cells.

T Cell Microvilli: Finger-Shaped External Structures Linked to the Fate of T Cells.

Microvilli are outer membrane organelles that contain cross-linked filamentous actin. Unlike well-characterized epithelial microvilli, T-cell microvilli are dynamic similar to those of filopodia, which grow and shrink intermittently via the alternate actin-assembly and -disassembly. T-cell microvilli are specialized for sensing Ags on the surface of Ag-presenting cells (APCs). Thus, these finger-shaped microprotrusions contain many signaling-related proteins and can serve as a signaling platforms that induce intracellular signals. However, they are not limited to sensing external information but can provide sites for parts of the cell-body to tear away from the cell. Cells are known to produce many types of extracellular vesicles (EVs), such as exosomes, microvesicles, and membrane particles. T cells also produce EVs, but little is known about under what conditions T cells generate EVs and which types of EVs are released. We discovered that T cells produce few exosomes but release large amounsts of microvilli-derived particles during physical interaction with APCs. Although much is unanswered as to why T cells use the same organelles to sense Ags or to produce EVs, these events can significantly affect T cell fate, including clonal expansion and death. Since TCRs are localized at microvilli tips, this membrane event also raises a new question regarding long-standing paradigm in T cell biology; i.e., surface TCR downmodulation following T cell activation. Since T-cell microvilli particles carry T-cell message to their cognate partner, these particles are termed T-cell immunological synaptosomes (TISs). We discuss the potential physiological role of TISs and their application to immunotherapies.