Single-cell analysis reveals cellular and molecular factors counteracting HPV-positive oropharyngeal cancer immunotherapy outcomes.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV-positive) is associated with better clinical outcomes than HPV-negative OPSCC. However, the clinical benefits of immunotherapy in patients with HPV-positive OPSCC remain unclear. METHODS: To identify the cellular and molecular factors that limited the benefits associated with HPV in OPSCC immunotherapy, we performed single-cell RNA (n=20) and T-cell receptor sequencing (n=10) analyses of tonsil or base of tongue tumor biopsies prior to immunotherapy. Primary findings from our single-cell analysis were confirmed through immunofluorescence experiments, and secondary validation analysis were performed via publicly available transcriptomics data sets. RESULTS: We found significantly higher transcriptional diversity of malignant cells among non-responders to immunotherapy, regardless of HPV infection status. We also observed a significantly larger proportion of CD4+ follicular helper T cells (Tfh) in HPV-positive tumors, potentially due to enhanced Tfh differentiation. Most importantly, CD8+ resident memory T cells (Trm) with elevated KLRB1 (encoding CD161) expression showed an association with dampened antitumor activity in patients with HPV-positive OPSCC, which may explain their heterogeneous clinical outcomes. Notably, all HPV-positive patients, whose Trm presented elevated KLRB1 levels, showed low expression of CLEC2D (encoding the CD161 ligand) in B cells, which may reduce tertiary lymphoid structure activity. Immunofluorescence of HPV-positive tumors treated with immune checkpoint blockade showed an inverse correlation between the density of CD161+ Trm and changes in tumor size. CONCLUSIONS: We found that CD161+ Trm counteracts clinical benefits associated with HPV in OPSCC immunotherapy. This suggests that targeted inhibition of CD161 in Trm could enhance the efficacy of immunotherapy in HPV-positive oropharyngeal cancers. TRIAL REGISTRATION NUMBER: NCT03737968.

Self-powered semitransparent WS2/LaVO3vertical-heterostructure photodetectors by employing interfacial hexagonal boron nitride.

Two-dimensional (2D) semiconductor and LaVO3materials with high absorption coefficients in the visible light region are attractive structures for high-performance photodetector (PD) applications. Insulating 2D hexagonal boron nitride (h-BN) with a large band gap and excellent transmittance is a very attractive material as an interface between 2D/semiconductor heterostructures. We first introduce WS2/h-BN/LaVO3semitransparent PD. The photo-current/dark current ratio of the device exhibits a delta-function characteristic of 4 × 105at 0 V, meaning 'self-powered'. The WS2/h-BN/LaVO3PD shows up to 0.27 A W-1responsivity (R) and 4.6 × 1010cm Hz1/2W-1detectivity (D*) at 730 nm. Especially, it was confirmed that theD* performance improved by about 5 times compared to the WS2/LaVO3device at zero bias. Additionally, it is suggested that the PD maintains 87% of its initialRfor 2000 h under the atmosphere with a temperature of 25 °C and humidity of 30%. Based on the above results, we suggest that the WS2/h-BN/LaVO3heterojunction is promising as a self-powered optoelectronic device.

Two weeks dose range-finding and four weeks repeated dose oral toxicity study of a novel reversible monoamine oxidase B inhibitor KDS2010 in cynomolgus monkeys.

A novel reversible monoamine oxidase B inhibitor, KDS2010, has been developed as a therapeutic candidate for neurodegenerative diseases. This study investigated its potential toxicity in non-human primates before human clinical trials. Daily KDS2010 doses (25, 50, or 100 mg/kg) were orally administered to cynomolgus monkeys (1 animal/sex/group, 4 males and 4 females) for 2 weeks to determine the dose range. One male was moribund, and one female was found dead in the 100 mg/kg/day group. One male was also found dead in the 50 mg/kg/day group. The death was considered an adverse effect in both sexes since distal tubules/collecting duct dilation and hypertrophy in the epithelium of the papillary duct were observed in their kidneys. Based on dose range finding results, KDS2010 (10, 20, or 40 mg/kg/day) was administered orally for 4 weeks, and animals were given 2 weeks for recovery. No significant changes were observed during daily clinical observations and macro-and microscopic examinations, including body weight, food consumption, hematology, clinical chemistry, and organ weight. And, the kidney was seen as the primary target organ of KDS2010 in the 2 weeks study, but no adverse effect was observed in the 4 weeks study. Therefore, 40 mg/kg/day is considered the no-observed-adverse-effect level in both sexes of cynomolgus monkeys. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00182-4.

A phased array ultrasound system with a robotic arm for neuromodulation.

BACKGROUND: Ultrasonic neuromodulation (UNMOD) provides a non-invasive brain stimulation. However, the high-resolution region-specificity of UNMOD with a single element transducer combined with a mechanical positioning system could have limits due to the intrinsic positioning error from mechanical systems. OBJECTIVE/HYPOTHESIS: A phased array system could lead to highly selective neuromodulation with electronic control. METHODS: A specialized phased-array system with a robotic arm is implemented for a rhesus monkey model. Various primary motor cortex areas related to tail, hand, and mouth were stimulated with a 200 µm step size. The ultrasonic parameters were ISPTA of 840 mW/cm2, pulse repetition frequency of 100 Hz, and a 5% duty factor at 600 kHz. The induced movement were recorded and analyzed. RESULTS: Separate digits, mouth, and tongue motions were successfully induced by electronically controlling the focus. The identical body part movement could be induced when the focus was moved back to the identical primary motor cortex with electronic control. Accordingly, the reproducibility of UNMOD could be partially validated with rhesus monkey model. CONCLUSION: A phased-array system appears to have a potential for the non-invasive and region-selective neuromodulation method.

Fermented Aronia melanocarpa Inhibits Melanogenesis through Dual Mechanisms of the PI3K/AKT/GSK-3ß and PKA/CREB Pathways.

UV light causes excessive oxidative stress and abnormal melanin synthesis, which results in skin hyperpigmentation disorders such as freckles, sunspots, and age spots. Much research has been carried out to discover natural plants for ameliorating these disorders. Aronia melanocarpa contains various polyphenolic compounds with antioxidative activities, but its effects on melanogenesis have not been fully elucidated. In this study, we investigated the inhibitory effect of fermented Aronia melanocarpa (FA) fermented with Monascus purpureus on melanogenesis and its underlying mechanism in the B16F10 melanoma cell line. Our results indicate that FA inhibited tyrosinase activity and melanogenesis in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. FA significantly downregulated the PKA/CREB pathway, resulting in decreased protein levels of tyrosinase, TRP-1, and MITF. FA also inhibited the transcription of MITF by increasing the phosphorylation levels of both GSK3ß and AKT. Interestingly, we demonstrated that these results were owing to the significant increase in gallic acid, a phenolic compound of Aronia melanocarpa produced after the fermentation of Monascus purpureus. Taken together, our research suggests that Aronia melanocarpa fermented with Monascus purpureus acts as a melanin inhibitor and can be used as a potential cosmetic or therapeutic for improving hyperpigmentation disorders.

Prognostic role of surgical margins in patients undergoing transoral robotic surgery after neo-adjuvant chemotherapy.

PURPOSE: To define if positive and close surgical margins are associated to worse prognosis in patients who underwent transoral robotic surgery (TORS) after neoadjuvant chemotherapy (NCT). METHODS: A retrospective cohort study was carried out at a tertiary referral center. The primary outcome was local-regional control (LRC), and the results were summarized with hazard ratios (HR) and 95% confidence intervals (CIs). RESULTS: A total of 308 patients (median age: 62.0, IQR: 55.0-68.2) were included. Univariable analysis showed a significant reduced LRC for patients with positive margins (HR = 1.82, 95% CI: 1.02-3.24). However, they were not associated with worse LRC after adjusting for adverse tumor variables (HR = 0.81, 95% CI: 0.40-1.65). ROC analysis was performed on 123 patients with negative margins (AUC: 0.54) measuring an optimal threshold of 1.25 mm (sensitivity = 60.0%; specificity = 50.5%). Univariable analysis showed non-significant differences between close and wide negative margins (HR = 1.44, 95% CI: 0.59-3.54). CONCLUSIONS: A positive surgical margin is not an independent predictor of tumor control and survival. A threshold of 1.25 mm was identified as the most appropriate to define close margins, but no difference was measured after distinguishing negative margins in close and wide margins.

Immersive Virtual Tasks with Motor and Cognitive Components: A Feasibility Study of Adults and Older Adult Fallers and Nonfallers.

The objective of the present study was to compare the feasibility, safety, and satisfaction of an immersive virtual reality system developed specifically for cognitive-sensory-motor training among older adult fallers and nonfallers and adult individuals. This was a cross-sectional observational study, and 20 adults, 20 nonfaller older adults, and 20 faller older adults were assessed. The primary outcome was feasibility assessed with safety and satisfaction measures. Safety outcomes were associated with adverse events occurred during the experience with the immersive virtual reality system (IVRS), assessed through the Simulator Sickness Questionnaire and by registering the falls, pain, or any discomfort reported by the participants. Satisfaction was assessed with a structured questionnaire, answered after 10 minutes of experiencing the IVRS. The dates were assessed with one-way analysis of variance or the Kruskal-Wallis test and Bonferroni post hoc test. The results showed that the IVRS was safe and the participants related good satisfaction with the system. Most of participants related no symptoms (93.6 percent) or light cybersickness symptoms (6.0 percent). There were no occurrences of falls or pain associated with the IVRS. The IVRS was feasible for adults and nonfaller and faller older adults.

Whole-genome sequence of Macaca fascicularis: liver tissue.

OBJECTIVES: Thrombocytopenia is a condition that causes a low amount of blood platelets. Platelets are blood cells that play an essential role in blood coagulation. Therefore, thrombocytopenia can put the patient at risk for mild to severe bleeding. Thrombocytopenia is caused by a decrease in platelet production in the bone marrow or by a drug or immune system problem when production is normal. In particular, in some ASO-induced thrombocytopenia, the mechanism is not clear. Therefore, whole genome sequencing (WGS) was performed to discover genetic differences that affect thrombocytopenia and individual susceptibility to drugs between normal and reduced platelet monkeys despite administering the same ASO. DATA DESCRIPTION: Three antisense oligonucleotide (ASO) substances were injected into the subcutaneous tissue of monkeys for 12 weeks in two experiments. The monkeys were classified into three groups: monkeys with thrombocytopenia, monkeys without thrombocytopenia, and control monkeys not treated with ASO substances. Whole genome sequencing data was generated using liver tissues of monkeys. These data will be useful for identifying genetic differences that affect thrombocytopenia and drug sensitivity.

The Anti-Inflammatory Activities of Fermented Curcuma That Contains Butyrate Mitigate DSS-Induced Colitis in Mice.

Inflammatory bowel disease is characterized by a radical imbalance of inflammatory signaling pathways in the gastrointestinal tract, and it is categorized into two diseases, such as Crohn's disease and ulcerative colitis. In this study, we investigated anti-inflammatory activities using fermented Curcuma that contains butyrate (FB). Nitric oxide production in RAW 264.7 cells and the expression of inducible nitric oxide synthase in the intestinal mucosa appears to be enhanced in active ulcerative colitis. Here, the cytotoxicity, physiological activity, and anti-inflammatory efficacy of FB in colitis animals were investigated. To verify the anti-inflammatory effect, this study was conducted using the dextran sulfate sodium (DSS)-induced colitis mice model. As a result, non-toxicity was confirmed, and anti-inflammatory effects were revealed by inducing a reduction of LPS-induced NO production. In the DSS-induced colitis, reduced weight was recovered and a decrease in inflammatory factors Ig-E and TNF-α in the mesenteric lymph node (MLN) and spleen was induced, and it was confirmed to help with the morphological remodeling of the intestine. In conclusion, this paper suggests that FB can help to alleviate intestinal inflammation and to improve the intestinal environment, with the help of morphological remodeling.

Sodium butyrate ameliorates neurotoxicity and exerts anti-inflammatory effects in high fat diet-fed mice.

The prevalence of high-fat diet consumption-related disorders is increasing, and it is often associated with oxidative stress, inflammation, and dysregulation in the brain may lead to neurodegenerative diseases (NDDs). Our study aims to evaluate the neuroprotective effects of sodium butyrate (NaB) on HFD-fed mice. In this study, four-week-old male C57Bl/6NTac mice were divided into three groups; the control group, the HFD group, and the HFD + NaB group where mice received 11 mg/kg body weight of NaB with HFD. Western blotting, reverse transcription-PCR, and ELISA were used for biochemical analysis of brain specimens. We found that NaB restored bodyweight and attenuated P-53, Bcl-2-associated X protein (BAX), and caspase cascades in the brains of HFD-fed mice. In addition. NaB reduced the expressions of proinflammatory cytokines and positively modulated antioxidant biomarkers. NaB treatment upregulated the expression of the growth factor-related factors PPARγ, CREB, and BDNF in the brain tissues of HFD-fed mice. Furthermore, we found that NaB significantly ameliorated glucocorticoid receptor and NLRP3 inflammasome expression. Based on our findings, NaB suppressed apoptotic and inflammatory cytokines and enhanced the expression of endogenous antioxidants in brain tissues of HFD-fed mice. Our data strongly suggests that NaB could be utilized as an effective therapeutic agent for NDDs.

Multidisciplinary aerodigestive program at a children's hospital: A protocol for a prospective observational study.

BACKGROUND: Children with complex chronic multisystemic diseases frequently require care from multiple pediatric subspecialists. The aerodigestive program is a multidisciplinary program that diagnoses and treats pediatric patients with complex multi-systematic problems affecting airway, breathing, feeding, swallowing, or growth. The aim of this study is to present the protocol of the aerodigestive program of a children's hospital. METHODS AND DESIGN: This study is a prospective study to evaluate and compare the overall improvement of patients' objective and subjective conditions before and after the AeroDigestive Team (ADT) program. Among children from 1 month to 18 years of age, patients with complex problems of the airway, breathing, feeding, swallowing, or growth meeting at least two parameters of the inclusion criteria were enrolled. The overall process included referral based on the inclusion criteria, enrollment of ADT program with informed consents, interview and questionnaire for assessing patients' medical condition, prescheduling appointment, multi-specialists' evaluation, monthly team meetings, wrap-up discussion with the patients and family, therapeutic intervention, and follow-up at 6 months with the assessment of outcome measures. The outcome was evaluated objectively and subjectively. The objective outcome measure was divided into surgical or medical intervention, assessment of changes in medical condition, and follow-up study. Both caregiver interviews and questionnaires using a scoring system were used as subjective outcome measures before and after the ADT program. Children were scheduled to be followed-up at 6 months after the interventions or ADT meeting. DISCUSSION: The aerodigestive program is expected to provide comprehensive and multidisciplinary management of children with complex airway and digestive tract disorders.

Development of a Multidisciplinary Aerodigestive Program: An Institutional Experience.

We share our experience on the implementation of a multidisciplinary aerodigestive program comprising an aerodigestive team (ADT) so as to evaluate its feasibility. We performed a retrospective chart review of the patients discussed at the monthly ADT meetings and analyzed the data. A total of 98 children were referred to the ADT during the study period. The number of cases increased steadily from 3.5 cases per month in 2019 to 8.5 cases per month in 2020. The median age of patients was 34.5 months, and 55% were male. Among the chronic comorbidities, neurologic disease was the most common (85%), followed by respiratory (36%) and cardiac (13%) disorders. The common reasons for consultation were suspected aspiration (56%), respiratory difficulty (44%), drooling/stertor (30%), regurgitation/vomiting (18%), and feeding/swallowing difficulty (17%). Following discussions, 58 patients received active interventions, including fundoplication, gastrostomy, laryngomicrosurgery, tracheostomy, and primary dilatation of the airway. According to the questionnaire of the caregiver, the majority agreed that the main symptoms and quality of life of patients had improved (88%), reducing the burden on caregivers (77%). Aerodigestive programs may provide comprehensive and multidisciplinary management for children with complex airway and digestive tract disorders.

A strategic approach to apply bacterial substances for increasing metabolite productions of Euglena gracilis in the bioreactor.

Bacterial extracellular polymeric substances (EPS) are promising materials that have a role in enhancing growth, metabolite production, and harvesting efficiency. However, the validity of the EPS effectiveness in scale-up cultivation of microalgae is still unknown. Therefore, in order to verify whether the bacterial metabolites work in the scale-up fermentation of microalgae, we conducted a bioreactor fermentation following the addition of bacterial EPS derived from the marine bacterium, Pseudoalteromonas sp., to Euglena gracilis. Various culture strategies (i.e., batch, glucose fed-batch, and glucose and EPS fed-batch) were conducted to maximize metabolite production of E. gracilis in scale-up cultivation. Consequently, biomass and paramylon concentrations in the continuous glucose and EPS-treated culture were enhanced by 3.0-fold and 4.2-fold (36.1 ± 1.4 g L-1 and 25.6 ± 0.1 g L-1), respectively, compared to the non-treated control (12.0 ± 0.3 g L-1 and 6.1 ± 0.1 g L-1). Also, the supplementation led to the enhanced concentrations of α-tocopherols and total fatty acids by 3.7-fold and 2.8-fold, respectively. The harvesting efficiency was enhanced in EPS-supplemented cultivation due to the flocculation of E. gracilis. To the best of our knowledge, this is the first study that verifies the effect of bacterial EPS in scale-up cultivation of microalgae. Also, our results showed the highest paramylon productivity than any other previous reports. The results obtained in this study showed that the scale-up cultivation of E. gracilis using bacterial EPS has the potential to be used as a platform to guide further increases in scale and in the industrial environment. KEY POINTS: Effect of EPS on Euglena gracilis fermentation was tested in bioreactor scale. EPS supplement was effective for the paramylon, α-tocopherol, and lipid production. EPS supplement induced the flocculation of E. gracilis.

A comparative study on intraocular pressure under various anesthetics in cynomolgus monkeys (Macaca fascicularis).

BACKGROUND: Nonhuman primates (NHPs) are superior model for ocular research due to its morphological and physiological similarities with humans. Thus, the effect of four different anesthetic combinations [ketamine (10 mg/kg), ketamine + xylazine (7 + 0.6 mg/kg), zoletil (4 mg/kg), and zoletil + xylazine (4 + 0.2 mg/kg)] on intraocular pressure (IOP) was determined in cynomolgus monkeys. RESULTS: The administration of ketamine + xylazine or zoletil + xylazine resulted in lower IOP compared to ketamine or zoletil alone. Moreover, the IOP in male monkeys was higher than in females. The difference between the right and left eye was not found. CONCLUSIONS: Anesthetics affected the IOP, and gender differences should be considered when measuring the IOP of nonhuman primates (NHPs).

Effect of human acellular dermal matrix (Megaderm™) on infra-auricular depressed deformities, Frey's syndrome, and first bite syndrome following parotidectomy: a multi-center prospective study.

BACKGROUND: Parotidectomy is the primary treatment for parotid gland tumors. However, complications may include a prominent facial scar or infra-auricular depressed deformity, Frey's syndrome, first bite syndrome, or other facial pain, numbness, and paralysis. Acellular dermal matrix (ADM) has been widely used to prevent these complications in parotid surgery, but there have been no prospective, multi-center trials documenting its efficacy. This study evaluates the effectiveness of ADM implantation in preventing infra-auricular depressed deformity, Frey's syndrome and first bite syndrome after parotidectomy. METHODS: We analyzed 51 cases of standard parotidectomy and 58 cases of parotidectomy with implantation of Megaderm™ ADM through prospective multicenter trial. Acute complications including infection, seroma, hematoma, skin necrosis, and acute parotid area pain were evaluated 1 week postoperatively. Clinician grading of Frey's syndrome and blinded clinician evaluation of infra-auricular depressed deformities were conducted at 3, 6, and 12 months. Patients evaluated subjective satisfaction with neck appearance, Frey's syndrome quality, and acute parotid area pain at 3, 6, and 12 months. RESULTS: There was a higher incidence of seroma in the Megaderm™ group than in the control group at week 1. The incidence and total clinician-evaluated Frey's syndrome scores were significantly lower in the Megaderm™ group than in the control group at 3, 6, and 12 months. Both the objective and subjective evaluations of the facial contour showed a better outcome in the Megaderm™ group compared to the control group at 3, 6, and 12 months. There were no significant differences between the groups in the patient-reported Frey's syndrome quality scores at 3, 6, and 12 months, but the Megaderm™ group reported significantly less acute pain than the control group. CONCLUSIONS: ADM implantation can effectively reduce the occurrence of Frey's syndrome, infra-auricular depressed deformity, and first bite syndrome after parotidectomy. ADM may be especially advantageous in complex parotidectomy cases when significant complications are expected.

Overexpression of poliovirus receptor is associated with poor prognosis in head and neck squamous cell carcinoma patients.

PURPOSE: We aimed to investigate the prognostic value of multiple immune cell markers including programmed death-ligand 1 (PD-L1) and poliovirus receptor (PVR) in head and neck squamous cell carcinoma (HNSCC) using archival tumor tissues METHODS: Patients diagnosed with HNSCC who have undergone surgical resection in 2005-2012 were included. Correlations between PVR and PD-L1 expression and patient characteristics were analyzed by analysis of variance. The Kaplan-Meier method and log-rank test were used to estimate survival. P values < 0.05 were considered statistically significant. RESULTS: In total, 375 primary tumor tissues were analyzed using immunohistochemistry. High PVR expression was associated with a poor prognosis in terms of overall survival (OS) and recurrence-free survival (RFS), and tumors with high PVR expression were associated with a short OS. PD-L1 tumor expression did not have a prognostic impact on survival. Univariate analysis revealed that OS and RFS were affected by age and p16 and PVR expression; multivariate analysis revealed that age and p16 and PVR expression were the most important determinants of RFS. CONCLUSION: PVR overexpression is a poor prognostic factor in patients with HNSCC and co-targeting PVR and PD-L1 may be a promising therapeutic option that needs further investigation.

Application of electrical treatment on Euglena gracilis for increasing paramylon production.

Paramylon also called ß-1,3-glucan is a value-added product produced from Euglena gracilis. Recently, researchers have developed various strategies for the enhanced paramylon production, among which electrical treatment for microbial stimulation can be an alternative owing to the applicability to large-scale cultivation. In this study, we applied the electrical treatment for enhanced paramylon production and found the proper treatment conditions. Under the treatment with platinum electrodes at 10 mA, the paramylon production of treated cells was significantly increased about 2.5-fold, compared to those of the untreated cells, although the density of cells was maintained due to considerable stress. The size of treated cells became larger, possibly due to the increased level of paramylon production within the cells. Accordingly, the contents of glucose uptake, glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), and uridine diphosphoglucose (UDPG) were shifted to appropriate states for the process of paramylon synthesis under the treatment. The increased level of transcripts encoding glucan synthase-like 2 (EgGSL2) was also confirmed via droplet digital PCR (ddPCR) under the treatment. Overall, this study makes a major contribution to research on electrical stimulation and provides new insights into E. gracilis metabolism like paramylon synthesis. KEY POINTS: • Electrical treatment induced the paramylon production and morphological change of Euglena gracilis. • The glucose uptake of E. gracilis was increased during the electrical treatment, fueling the paramylon synthesis.

Comprehensive Analysis of Clinicopathologic Factors Predictive of an Unfavorable Prognosis in Patients With Acinic Cell Carcinoma of the Parotid Gland.

OBJECTIVES: In subset of patients, acinic cell carcinoma (AcCC) exhibits aggressive features such as recurrence, distant metastasis, and mortality. This study aimed to investigate clinicopathologic factors influencing patients' prognosis and to identify adverse features predictive of an unfavorable prognosis. METHODS: Between January 2000 and December 2016, 59 patients with AcCC were enrolled in this study. RESULTS: The patients' 5-year overall survival rate was 93.3%, and their 5-year recurrence-free survival rate was 80.5%. During the study period, recurrence occurred in 10 patients. The mean time to recurrence after surgery was 26 months (range, 5-60 months). During the study period, three patients died from the disease. Univariate analysis showed that sex, surgical extent, extranodal extension, T classification, and TNM stage were significantly associated with disease recurrence. Multivariate analysis showed that, among the clinicopathologic factors included in the analysis, only TNM stage displayed a statistically significant correlation with disease recurrence. CONCLUSION: Surgical treatment alone yielded good results for AcCC, and additional treatment did not affect the recurrence-free survival rate or the overall survival rate, even when the resection margin was less than 1 mm. Other pathologic factors did not show prognostic significance for disease recurrence or death.

In Vivo Monitoring of Intracellular Metabolite in a Microalgal Cell Using an Aptamer/Graphene Oxide Nanosheet Complex.

Real-time sensing and imaging of intracellular metabolites in living cells are crucial tools for the characterization of complex biological processes, including the dynamic fluctuation of metabolites. Therefore, additional efforts are required to develop in vivo detection strategies for the visualization and quantification of specific target metabolites, particularly in microalgae. In this study, we developed a strategy to monitor a specific microalgal metabolite in living cells using an aptamer/graphene oxide nanosheet (GOnS) complex. As a proof-of-concept, ß-carotene, an antioxidant pigment that accumulates in most microalgal species, was chosen as a target metabolite. To achieve this, a ß-carotene-specific aptamer was selected through graphene oxide-assisted systematic evolution of ligands by exponential enrichment (GO-SELEX) and characterized thereafter. The aptamer could sensitively sense the changes in the concentration of ß-carotene (i.e., the target metabolite) and more specifically bind to ß-carotene than to nontargets. The selected aptamer was labeled with a fluorophore (fluorescein; FAM) and allowed to form an aptamer/GOnS complex that protected the aptamer from nucleic cleavages. The aptamer/GOnS complex was delivered into the cells via electroporation, thus enabling the sensitive monitoring of ß-carotene in the cell by quantifying the aptamer fluorescence intensity. The results suggest that our biocompatible strategy could be employed to visualize and semiquantify intracellular microalgae metabolites in vivo, which holds a great potential in diverse fields such as metabolite analysis and mutant screening.