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Aim. This paper aims to bring back into focus the area of the retromolar pad as an important element for the support area and for achieving an effective marginal seal in case of completely edentulous mandible. Material and methods. 24 cases of complete mandibular edentation were analyzed and the characteristics of the retromolar pad were followed. Analyzing the appearance of the retromolar pad, an attempt was made to classify them into the classes proposed by J. Lejoyeux. The clinical implications of various aspects of this area were monitored. Results. In patients with the retromolar pad in the last three classes, difficulties were encountered in adapting the individual tray and during the functional impression; in cases with advanced resorption of the alveolar ridge and unfavorable retromolar pad, a poor marginal seal was obtained. Conclusions. From the analyzed cases, it was reconfirmed that in an old complete mandibular edentation, the retromolar pad can take an oblique or vertical position. Such a situation becomes unfavorable for the retention of the complete denture, for the marginal seal. Knowing the particularities of the completely edentulous prosthetic field and the clinical stages is essential for maintaining denture stability.

Biosurfactants are amphiphilic molecules produced by various microorganisms, with potential applications in different industries or processes. The present study aimed to investigate the potential of food (whey) and industrial (corn extract) by-products to be used as single sources of nutrients for microbial surfactants production by two Pseudomonas strains. The supernatants obtained at the end of the bioprocess were used to form emulsions with heptane, octane, and sunflower oil, in order to determine the emulsification index. Good results were obtained with both Pseudomonas sp., the biggest values of the emulsification index being obtained with Pseudomonas putida ICCF 391 strain, followed by Pseudomonas fluorescens ICCF 392.

Abstract Background Burns are a global health problem affecting the survivors and disrupting many aspects of their lives. It is the second most common injury in rural Nepal accounting 5% of disabilities. Burn Specific Health Scale (BSHS) is a valid and most commonly used tool to measure Health Related Quality of Life (HRQoL) of the patient with Burns. BSHS- B (Brief) has been translated, culturally adapted and validated in multiple languages but not in Nepali. Therefore we aim to translate, culturally adapt and validate the BSHS-B in Nepali language (BSHS-B-Np). Methods Standard guideline was followed to translate the scale into Nepali language. One hundred eleven participants were evaluated to establish the psychometric properties of BSHS-B-Np. Internal consistency, test retest, content validity, discriminant validity and construct validity were assessed using Cronbach’s alpha, Interclass correlation coefficient, Factor analysis, Spearman rank test, and Mann- Whitney U test respectively. Results The Cronbach’s alpha for BSHS-B-Np was 0.93. Test retest inter-class correlation coefficient was between 0.92 and 0.98. The principal component factor analysis with varimax rotation resulted in separation of nine factors explaining 75.19% of total variance. BSHS-B-Np showed good discriminant validity in 35 out of 36 domain correlations confirming the construct of the scale. Furthermore, the scale was able to discriminate between face, upper limb and lower limb injury (p

Introduction: The aim of this study was to obtain and characterize pullulan acetate-based nanoparticles, loaded with an anticancer agent, 5-fluorouracil (5-FU) [...]

Cancer is the second leading cause of death worldwide and one of the most challenging [...]

In the condition of a dynamic environment where the citizens needs and requirements are constantly changing, a particular importance in ensuring a competitive advantage, it assumes strategic approach of the public organization, where marketing becomes a source, a management model necessary for increasing efficiency of the organization. Marketing fulfils four functions in public organization, which reflects a circuit in a continuous movement, aimed to investigate the market, identify community needs, to meet the needs of citizen, therefore lead to increase economic efficiency and permanently connecting the public organization to dynamic environment, this in turn can be achieved by a thorough and efficient market research for the Community that addresses. Therefore the marketing strategy in public organization is a new concept, a new way of thinking, a new orientation which is designed to meet the needs of citizens and to improve the image of public organizations. This paper proposes a comprehensive approach to community development strategies through public marketing. The structure of the paper is represented by introduction, specialized literature review and content. Our research highlights the importance and the need of implementing a marketing strategy in the public organization for local development and beyond.

Migraine is known to be caused by calcitonin gene-related peptide (CGRP), prompting the need for quantitative analysis of CGRP for the clinical treatment of monoclonal antibodies targeting CGRP. Since CGRP is cleaved by proteolytic enzymes post-blood collection, rapid analysis methods are required. In this study, a one-step immunoassay for CGRP was developed using chemically mimicking peptides (mimotopes) with an analysis time of 32 min. Four clones from an Fv-antibody library were screened using two types of monoclonal antibodies against CGRP. Mimotopes for each monoclonal antibody were synthesized into peptides of 15 residues. The binding affinity (K D ) was estimated, and the interaction with monoclonal antibodies was analyzed using docking simulations. Finally, a one-step immunoassay for CGRP was demonstrated using migraine patient samples (n = 57) and healthy volunteer controls (n = 18). The limit of detection (LOD) of one-step immunoassay based on Fremanezumab (mimotope F1) was estimated to be 8.8 pg/mL with the limit of quantification (LOQ) of 125.9 pg/mL. And, the one-step immunoassay based on Galcanezumab (mimotope G7) showed the LOD of 9.4 pg/mL and the LOQ of 84.7 pg/mL. The total analysis time was estimated to be approximately 32 min and the assay results were estimated to be statistically consistent with conventional CGRP assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fv-antibodies targeting the transmembrane protease serine 2 (TMPRSS2) were screened from an Fv-antibody library for inhibiting SARS-CoV-2 infection. Fv-antibodies were derived from the variable region of heavy-chain immunoglobulin G (IgG), which consisted of three complementarity-determining regions (CDRs) and frame regions (FRs). The Fv-antibody library was prepared through site-directed mutagenesis of CDR3 region. The proteolytic cleavage site (S2' site) of TMPRSS2 on the spike protein (SP) of SARS-CoV-2 was used as a screening probe for the library. Two Fv-antibodies were screened and subsequently expressed as soluble recombinant proteins. The binding affinities of the expressed Fv-antibodies were estimated using a surface plasmon resonance (SPR) biosensor. The two expressed Fv-antibodies specifically bound to the active site of TMPRSS2 which interacts with S2' site in the proprotein convertase (PPC) region. The neutralizing activities of the two expressed Fv-antibodies were demonstrated using a cell-based infection assay with pseudo-viruses that expressed the SP of four types of SARS-CoV-2 variants: Wu-1 (D614), Delta (B.1.617.2), Omicron (BA.2), and Omicron (BA.4/5). Additionally, a docking simulation was performed to analyze the interaction between the screened Fv-antibodies and the active sites of TMPRSS2., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Deoxyribonuclease (DNase) I inhibitors have been developed based on proteins, nucleotides and synthetic compounds. In this work, amino acid sequences with the activity of DNase I inhibitor were screened from an Fv-antibody library expressed on the outer membrane of Escherichia coli. The Fv-antibody indicated the heavy chain variable region (V H ) of immunoglobulin G (IgG) and the Fv-antibody library was generated with a randomized complementarity-determining region 3 (CDR3). From the Fv-antibody library, two clones were screened for their binding affinity to DNase I and expressed as soluble recombinant proteins as well as peptides. The binding affinity (K D ) to DNase I was estimated for the expressed Fv-antibodies (73.4 nM for Fv-1 and 89.0 nM for Fv-19) and synthesized peptides (279.2 nM for Peptide-1 and 243.2 nM for Peptide-19) using SPR biosensor. The inhibitory activity (IC 50 ) of the expressed Fv-antibodies (550.0 nM for Fv-1 and 660.2 nM for Fv-19) and synthetic peptides (864.5 nM for Peptide-1 and 974.6 nM for Peptide-19) was measured using agarose-gel assay and TaqMan-like fluorescence assay. These IC 50 values indicated that both expressed Fv-antibodies and synthesized peptides exerted an effective inhibitory activity against DNase I. The interaction between the screened inhibitors and DNase I was analyzed by docking simulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Schistosomiasis is a parasitic disease affecting more than 250 million people worldwide. Schistosomes infect humans by cercariae penetrating the skin in a freshwater environment. Findings obtained more than 100 years prior showed that miracidium develops into cercaria in freshwater snails, though detailed development dynamics have not been elucidated. Although results of histological analyses of development of schistosomes in snails were presented in our previous studies, findings obtained with dynamic imaging have yet to be reported. In the present study, imaging of schistosome infection and dynamics in snails occuring within a short period was performed using fluorescent labeling agents. Labeling of S. mansoni cercariae with carboxyfluorescein succinimidyl ester (CFSE) caused no toxicity, and allowed for monitoring of schistosome dynamics in snails for up to 10 days and release of infective cercariae without fluorescence in 40 days following infection., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, the vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed using ferritin complexes with the immunogenic sequences screened against the SARS-CoV-2 spike protein (SP) from the Fv-antibody library. The Fv-antibody library was prepared on the outer membrane of E. coli by the expression of the V H region of immunoglobulin G (IgG) with a randomized complementarity-determining region 3 (CDR3). Four Fv-antibodies to the receptor-binding domain (RBD) were screened from the Fv-antibody library, which had a comparable binding constant ( K D ) between SARS-CoV-2 SP and the angiotensin-converting enzyme 2 (ACE2) receptor. The binding sites of screened Fv-antibodies on the RBD were analyzed using a docking analysis, and these binding sites were used as immunogenic sequences for the vaccine. The four immunogenic sequences were modified and co-expressed as a part of ferritin which was assembled into a ferritin complex. After the vaccination of ferritin complexes to mice, the anti-sera were analyzed to have a high enough titer. Additionally, the immune responses were found to be activated by vaccination, such as the expression of IgG subclasses and the increased level of cytokines. The neutralizing activity of the anti-sera was estimated using a cell-based infection assay based on pseudo-virus expressing the SP of SARS-CoV-2 variants.

Street fight videos on the internet may provide information about little known aspects of human physical aggression, but their reliability is unclear. Analyses of 100 dyadic fight videos addressing ethological, game theoretic and sex-differentiated questions derived from research on other animals found that prefight verbalizations or gestural signals of nonaggressive or aggressive intent loosely predicted who would strike first and who would win. The head is the preferred strike target. Ordinal severity rankings of different strikes ranged from 1 for spitting to 5 for choking. Half the videos showed briefer, unilateral assaults beginning with one or more high severity strikes, little evidence of escalation and fewer bystander interventions. A quarter of these were sneak attacks. The other videos showed longer fights with reciprocal strikes, some evidence of strike severity escalation and more bystander intervention. Both types were equally injurious. Winner/loser outcomes were reliably identified by postfight behaviors and/or signs of injury. Winners had advantageous prefight resource holding potential (RHP: greater height and/or vigor) significantly more often than losers. Consistent with tendencies for fights to occur between animals of the same sex, there were more male/male and female/female fights and fewer male/female fights than expected from random pairings of men and women in the videos. Female/female fights involved proportionally more hair-pulling, extended bouts of rapidly repeated strikes and longest durations. Bystanders intervened in over half the videos, attempting to separate fighters or help losers more often than they attacked the loser. Carefully selected internet street fight videos can provide important information., (© 2024 The Author(s). Aggressive Behavior published by Wiley Periodicals LLC.)

Background: Hepatocellular carcinoma (HCC) stands as the sixth most prevalent cancer globally, presenting a substantial health challenge, particularly due to late-stage diagnoses that limit treatment effectiveness. Sorafenib, a multi-kinase inhibitor, is the primary chemotherapeutic agent for advanced HCC, but it only extends survival by 2-3 months. However, drug resistance remains a major clinical challenge, necessitating the exploration of new molecular mechanisms, including the role of microRNAs (miRNAs) in sorafenib resistance. In this study, we aimed to identify miRNAs within exosomes derived from sorafenib-resistant HCC cells to elucidate the molecular mechanisms underlying resistance., Methods: Sorafenib-resistant cells were generated by culturing the human HCC cell line Huh7 in a medium containing 20 µM sorafenib for six months. Exosomes were isolated from the conditioned medium 24 h before cell harvest using exosome-depleted serum medium. miRNA sequencing and western blotting were used to analyze the expression profiles of exosomal miRNAs and proteins, respectively. pH measurement was performed to assess pH changes in response to sorafenib treatment and miRNA modulation., Results: A total of 180 exosomal miRNAs were found to be dysregulated between sorafenib-treated control Huh7 (Huh7S) and sorafenib-resistant Huh7 (Huh7RS) cells, as well as between untreated control Huh7 and Huh7RS cells. Among these, miR-6126 was significantly downregulated in Huh7RS cells compared to Huh7S cells. Functional studies using 2-dimensional (D) and 3D cell culture systems revealed that miR-6126 overexpression reduced sorafenib resistance in Huh7RS cells, while its inhibition increased resistance in Huh7 cells. miR-6126 downregulated key proteins involved in cancer stem cell maintenance, such as CD44 and HK2. Furthermore, the pH level was elevated in cells overexpressing miR-6126 following sorafenib treatment, whereas inhibiting miR-6126 resulted in a lower pH., Conclusions: Exosomal miR-6126 plays a pivotal role in sorafenib resistance and tumorigenesis, highlighting its potential as a novel therapeutic target for overcoming drug resistance in HCC., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

For the prevention of SARS-CoV-2 infection, four Fv-antibodies with binding affinity for the ACE2 receptor were screened from an Fv-antibody library. The screened Fv-antibodies were expressed as soluble proteins and estimated to have a high binding affinity, comparable to that between SARS-CoV-2 and the ACE2 receptor. The interaction between the Fv-antibodies and the ACE2 receptor was analyzed using docking simulation, and the significant binding affinity of the screened Fv-antibodies was attributed to the homology in amino acid sequence with the ACE2 receptor. The neutralizing activities of the Fv-antibodies were demonstrated using a cell-based infection assay based on four pseudo-virus types with SARS-CoV-2 variant spike proteins (Wild-type D614, Delta B.1.617.2, and Omicron BA.2, and Omicron BA.4/5)., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants presents challenges in global efforts to transition from the pandemic to an endemic stage. The spike protein of the SARS-CoV-2 virus, which is pivotal for cell entry, exhibits significant mutations in its variants, potentially affecting infectivity and therapeutic efficacy. Recent findings indicate upregulation of the epidermal growth factor receptor (EGFR) pathway, a key target in cancer therapy, by the spike protein of SARS-CoV-2. This study aimed to investigate the activity of the EGFR pathway against SARS-CoV-2 variants and to assess the inhibitory effects of EGFR inhibitors using SARS-CoV variant pseudoviral particles to guide future therapeutic strategies. Omicron variant SARS-CoV pseudoviral particles exhibited heightened infectivity in human angiotensin-converting enzyme 2 (hACE2)-expressing HEK293 and A549 lung cancer cells accompanied by increased EGFR pathway activation in infected cells. Using the EGFR tyrosine kinase inhibitor, osimertinib, we observed a reduction in viral infection rates in hACE2-HEK293 and A549 cells infected with the SARS-CoV-2 variant pseudoviral particles. We conducted further experiments to confirm that the reduction in infection efficacy with osimertinib treatment was not associated to a decrease in cell viability. Furthermore, this inhibitory effect of osimertinib in cell lines was corroborated in a spheroid cell culture model derived from hACE2-A549 cells. These findings suggest the potential application of EGFR-targeted antiviral therapy against highly infectious SARS-CoV-2 variants.IMPORTANCEThe emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is concerning as vaccines designed for one variant need not essentially protect against other novel variants. Therefore, there is an urgent need to identify therapies that can act against multiple novel variants that have heightened virulence compared with the wild type. It has been reported that the spike protein of the SARS-CoV-2 virus elicits an increased expression of the epidermal growth factor receptor (EGFR) pathway. We used this information and examined whether treatment with an EGFR inhibitor, osimertinib, which is already approved for clinical use in cancer therapy, can reduce the infection caused by SARS-CoV-2, wild type, and Omicron and Delta variants, in two cell lines and one spheroid model. The results showed that osimertinib treatment successfully reduced infection efficacy, particularly in variants, and that this effect was not related to a reduction in cell viability, making this a promising strategy for treating SARS-CoV-2 infections., Competing Interests: The authors declare no conflict of interest.

Inhibitors of the epithermal growth factor receptor (EGFR) were screened from an autodisplayed Fv-antibody library using an anti-EGF antibody. The Fv-antibody library was expressed on the outer membrane of Escherichia coli , which corresponds to the heavy chain V H region of immunoglobulin G. The library was constructed by randomizing the CDR3 region of expressed V H regions (11 amino acid residues) by site-directed mutagenesis. Using an anti-EGF antibody as a screening probe, amino acid sequences (CDR3 region) with antibody binding affinity were screened from the Fv-antibody library. These amino acid sequences were considered to have similar chemical properties to EGF, which can bind to EGFR. Two autodisplayed clones with Fv-antibodies against EGFR were screened from the Fv-antibody library, and the screened Fv-antibodies were expressed as soluble proteins. The binding affinity ( K D ) was estimated using an SPR biosensor, and the inhibitory activity of expressed Fv-antibodies was observed for PANC-1 pancreatic tumor cells and T98G glioblastoma cells using Western blot analysis of proteins in the EGFR-mediated signaling pathway. The viability of PANC-1 and T98G cells was observed to decrease via the inhibitory activity of expressed Fv-antibodies. Finally, interactions between Fv-antibodies and EGFR were analyzed by using molecular docking simulations.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive solid tumor. Recently, the uptake of extracellular citrate by the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5 , has been demonstrated to exert profound effects on cancer cell metabolism. However, research on the function of extracellular citrate in PDAC pathogenesis and the relationship between NaCT expression and the tumor metabolic microenvironment is limited. Therefore, we aimed to evaluate the expression of citrate transporters across a spectrum of glucose concentrations in pancreatic cancer and systematically explore the effects of sodium citrate treatment on pancreatic cancer cells at different glucose concentrations. We observed a positive correlation between glucose concentration and NaCT expression in PDAC cell lines. Extracellular sodium citrate significantly reduced cell viability partially due to reduction in intracellular Ca 2+ levels and decreased the migration of human PDAC cells. Furthermore, we observed a decrease in the levels of the stem cell marker prominin I (CD133) following sodium citrate treatment. Notably, the combination treatment of gemcitabine and extracellular sodium citrate exhibited a synergistic anticancer effect in both two-dimensional (2D) and three-dimensional (3D) culture systems. Additionally, we confirmed that pH slightly increased upon administration of sodium citrate, indicating that this could potentially augment the efficacy of gemcitabine. Altogether, these findings suggest that exogenous sodium citrate treatment, particularly in combination with gemcitabine, may represent a novel therapeutic strategy for treating PDAC. This approach holds promise for disrupting PDAC cell metabolism and inhibiting tumor progression., Competing Interests: The authors declare there is no conflict of interests., (© 2024 The Authors.)

Fv-antibodies targeting the proprotein convertase (PPC) region of the SARS-CoV-2 spike protein (SP) were screened from an Fv-antibody library to inhibit SARS-CoV-2 infection. Two selected Fv-antibodies were expressed as soluble recombinant proteins, and their binding affinities were assessed using a surface plasmon resonance biosensor. The binding regions of these Fv-antibodies corresponded to the cleavage sites of furin (S1/S2) and transmembrane serine protease 2 (TMPRSS2, S2'). The neutralizing activities of the two Fv-antibodies were demonstrated using a cell-based infection assay with pseudo-viruses carrying the SP of four different SARS-CoV-2 variants: wild-type (D614), delta (B.1.617.2), omicron (BA.2), and omicron (BA.4/5)., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Outer membrane vesicle-functionalized nanoparticles (OMV-NPs) have attracted significant interest, especially regarding drug delivery applications and vaccines. Here, we report on novel OMV-NPs by applying bioorthogonal click reaction for encapsulating gold nanoparticles (NPs) within outer membrane vesicles (OMVs) by covalent coupling. For this purpose, outer membrane protein A (OmpA), abundant in large numbers (due to 100,000 copies/cell [1]) in OMVs, was modified via the incorporation of the unnatural amino acid p-azidophenylalanine. The azide group was covalently coupled to alkyne-functionalized NPs after incorporation into OmpA. A simplified procedure using low-speed centrifugation (1,000 x g) was developed for preparing OMV-NPs. The OMV-NPs were characterized by zeta potential, Laurdan-based lipid membrane dynamics studies, and the enzymatic activity of functionalized OMVs with surface-displayed nicotinamide adenine dinucleotide oxidase (Nox). In addition, OMVs from attenuated bacteria (ClearColi TM BL21(DE3), E. coli F470) with surface-displayed Nox or antibody fragments were prepared and successfully coupled to AuNPs. Finally, OMV-NPs displaying single-chain variable fragments from a monoclonal antibody directed against epidermal growth factor receptor were applied to demonstrate the feasibility of OMV-NPs for tumor cell targeting., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets., Methods: PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor., Results: Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions., Conclusions: Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT., (© 2024 Wiley Periodicals LLC.)

Background: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explore 11C-acetate PET as an imaging technique for detecting reactive astrogliosis., Methods: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and monocarboxylate transporter 1 (MCT1) expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis., Results: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with a worse prognosis., Conclusions: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose: [ 18 F]fluorodeoxyglucose ([ 18 F]FDG) positron emission tomography/computed tomography (PET/CT) has limitations in prostate cancer (PCa) detection owing to low glycolysis in the primary tumour. Recently, prostate-specific membrane antigen (PSMA) PET/CT has been useful for biochemical failure detection and radioligand therapy (RLT) guidance. However, few studies have evaluated its use in primary prostate tumours using PSMA and [ 18 F]FDG PET/CT. This study aimed to evaluate [ 18 F]PSMA-1007 and [ 18 F]FDG PET/CT for primary tumour detection and understand the association of metabolic heterogeneity with clinicopathological characteristics at staging and postoperatively., Method: This prospective study included 42 index tumours (27 acinar and 15 ductal-dominant) in 42 patients who underwent [ 18 F]PSMA-1007 and [ 18 F]FDG PET/CT and subsequent radical prostatectomy. All patients were followed for a median of 26 mo, and serum prostate-specific antigen levels were measured every 3 mo to evaluate biochemical failure. One-way analysis of variance, Tukey's multiple comparison test, and Fisher's exact test were performed., Results: All 42 index tumours were detected on [ 18 F]PSMA-1007 PET/CT, whereas only 15 were detected on [ 18 F]FDG PET/CT (62.3% vs. 37.7%, p < 0.0001). A high SUV max for [ 18 F]PSMA-1007 was observed in tumours with high Gleason scores (GS 6-7 vs. GS 8-10; 12.1 vs. 20.1, p < 0.05). Tumours with [ 18 F]FDG uptake were mostly ductal dominant (acinar-dominant 4/27; ductal-dominant; 11/15, p < 0.001), with lower [ 18 F]PSMA-1007 uptake than tumours without [ 18 F]FDG uptake (SUVmax 16.58 vs. 11.19, p < 0.001). There were 16.6% (7/42) of patients with pStage IV in whom the primary tumours were [ 18 F]FDG positive. Biochemical failure was observed in 14.8% (4/27) of patients with [ 18 F]FDG negative tumours but in 53.3% (8/15) of patients with [ 18 F]FDG positive tumours (p = 0.013)., Conclusions: [ 18 F]PSMA-1007 PET/CT was superior to [ 18 F]FDG PET/CT in detecting primary PCa. In contrast, tumours with [ 18 F]FDG uptake are associated with larger size, a ductal-dominant type, and likely to undergo metastasis at staging and biochemical failure postoperatively., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The ability to sense and respond to host defenses is essential for pathogen survival. Some mechanisms involve two-component systems (TCS) that respond to host molecules, such as antimicrobial peptides (AMPs) and activate specific gene regulatory pathways to aid in survival. Alongside TCSs, bacteria coordinate cell division proteins, chaperones, cell wall sortases and secretory translocons at discrete locations within the cytoplasmic membrane, referred to as functional membrane microdomains (FMMs). In Group A Streptococcus (GAS), the FMM or "ExPortal" coordinates protein secretion, cell wall synthesis and sensing of AMP-mediated cell envelope stress via the LiaFSR three-component system. Previously we showed GAS exposure to a subset of AMPs (α-defensins) activates the LiaFSR system by disrupting LiaF and LiaS co-localization in the ExPortal, leading to increased LiaR phosphorylation, expression of the transcriptional regulator SpxA2, and altered GAS virulence gene expression. The mechanisms by which LiaFSR integrates cell envelope stress with responses to AMP activity and virulence are not fully elucidated. Here, we show the LiaFSR regulon is comprised of genes encoding SpxA2 and three membrane-associated proteins: a PspC domain-containing protein (PCP), the lipoteichoic acid-modifying protein LafB and the membrane protein insertase YidC2. Our data show phosphorylated LiaR induces transcription of these genes via a conserved operator, whose disruption attenuates GAS virulence and increases susceptibility to AMPs in a manner primarily dependent on differential expression of SpxA2. Our work expands understanding of the LiaFSR regulatory network in GAS and identifies targets for further investigation of mechanisms of cell envelope stress tolerance contributing to GAS pathogenesis.

Monocarboxylate transporter-1 (MCT-1) inhibitors were screened from the Fv-antibody library, which contained complementary determining region 3 with randomized amino acid sequences (11 residues) through site-directed mutagenesis. Fv-antibodies against MCT-1 were screened from the autodisplayed Fv-antibody library. Two clones were screened, and the binding affinity (K D ) against MCT-1 was estimated using flow cytometry. The screened Fv-antibodies were expressed as soluble fusion proteins (Fv-1 and Fv-2) and the K D for MCT-1 was estimated using the SPR biosensor. The inhibitory activity of the expressed Fv-antibodies was observed in HEK293T and Jurkat cell lines by measuring intracellular pH and lactate accumulation. The level of cell viability in HEK293T and Jurkat cell lines was decreased by the inhibitory activity of the expressed Fv-antibodies. The binding properties of the Fv-antibodies to MCT-1 were analyzed using molecular docking simulations. Overall, the results showed that the screened Fv-antibodies against MCT-1 from the Fv-antibody library had high binding affinity and inhibitory activity against MCT-1, which could be used as potential therapeutic drug candidates for the MCT-1 inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acetyl-CoA synthetase 2 (ACSS2)-dependent acetate usage has generally been associated with tumorigenesis and increased malignancy in cancers under nutrient-depleted conditions. However, the nutrient usage and metabolic characteristics of the liver differ from those of other organs; therefore, the mechanism of ACSS2-mediated acetate metabolism may also differ in liver cancer. To elucidate the underlying mechanisms of ACSS2 in liver cancer and acetate metabolism, the relationships between patient acetate uptake and metabolic characteristics and between ACSS2 and tumor malignancies were comprehensively studied in vitro, in vivo and in humans. Clinically, we initially found that ACSS2 expression was decreased in liver cancer patients. Moreover, PET-CT imaging confirmed that lower-grade cancer cells take up more 11 C-acetate but less 18 F-fluorodeoxyglucose ( 18 F-FDG); however, this trend was reversed in higher-grade cancer. Among liver cancer cells, those with high ACSS2 expression avidly absorbed acetate even in a glucose-sufficient environment, whereas those with low ACSS2 expression did not, thereby showing correlations with their respective ACSS2 expression. Metabolomic isotope tracing in vitro and in vivo revealed greater acetate incorporation, greater lipid anabolic metabolism, and less malignancy in high-ACSS2 tumors. Notably, ACSS2 downregulation in liver cancer cells was associated with increased tumor occurrence in vivo. In human patient cohorts, patients in the low-ACSS2 subgroup exhibited reduced anabolism, increased glycolysis/hypoxia, and poorer prognosis. We demonstrated that acetate uptake by ACSS2 in liver cancer is independent of glucose depletion and contributes to lipid anabolic metabolism and reduced malignancy, thereby leading to a better prognosis for liver cancer patients., (© 2024. The Author(s).)

The Fv-antibodies were correponded to V H region of immunoglobulin G, which were composed of three complementarity determining regions (CDRs) for the specific binding of antigens. In this work, the Fv-antibodies against SARS-CoV-2 spike protein (SP) were screened from an autodisplayed Fv-antibody library which was expressed on E. coli outer membrane, and the receptor binding domain (RBD) of SP was used as a screening probe. The screened target clones were analyzed to have quantitative binding properties to the RBD, and the Fv-antibodies from the screened target clones were expressed as soluble proteins. The binding affinity (K D ) of expressed Fv-antibodies to the RBD was estimated to be 70-85 nM using SPR biosensor. The specific binding properties of Fv-antibodies were analyzed for pseudo-virus particles with SARS-CoV-2 SP on the Lenti-virus envelope, such as wild type (Wuhan-1) and variants (Delta, Omicron BA.2, Omicron BA.4/5) using a SPR biosensor. The detection of real SARS-CoV-2 (Wild type, Wuhan-1) based on a SPR biosensor was also presented using the Fv-antibodies with the binding constant (K D ) of cycle threshold value (Ct) = 33.8-32.9 (2.19-4.08 copies/μL) and LOD of 0.67-0.83 copies/μL (Ct = 35.5-35.2). Finally, one-step immunoassay based on switching peptide was demonstrated for the detection of the real SARS-CoV-2 (Wuhan-1) without any washing step. The binding constant (K D ) was estimated to be Ct = 35.2-33.9 (0.83-2.04 copies/μL), and LOD was estimated to be 0.14-0.47 copies/μL (Ct = 37.8-36.0). Considering the LOD of the conventional RT-PCR (Ct = 35), the LOD of the one-step immunoassay based on the switching peptide was determined to be feasible for the medical diagnosis of COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: In several fields, the process of fusing multiple two-dimensional (2D) closed lines is an important step. For instance, this is fundamental in histology and oncology in general. The treatment of a tumor consists of numerous steps and activities. Among them, segmenting the cancer area, that is, the correct identification of its spatial location by the segmentation technique, is one of the most important and at the same time complex and delicate steps. The difficulty in deriving reliable segmentations stems from the lack of a standard for identifying the edges and surrounding tissues of the tumor area. For this reason, the entire process is affected by considerable subjectivity. Given a tumor image, different practitioners can associate different segmentations with it, and the diagnoses produced may differ. Moreover, experimental data show that the analysis of the same area by the same physician at two separate timepoints may result in different lines being produced. Accordingly, it is challenging to establish which contour line is the ground truth. Methods: Starting from multiple segmentations related to the same tumor, statistical metrics and computational procedures could be exploited to combine them for determining the most reliable contour line. In particular, numerous algorithms have been developed over time for this procedure, but none of them is validated yet. Accordingly, in this field, there is no ground truth, and research is still active. Results: In this work, we developed the Two-Dimensional Segmentation Fusion Tool ( TDSFT ), a user-friendly tool distributed as a free-to-use standalone application for MAC , Linux , and Windows , which offers a simple and extensible interface where numerous algorithms are proposed to "compute the mean" (i.e., the process to fuse, combine, and "average") multiple 2D lines. Conclusions: The TDSFT can support medical specialists, but it can also be used in other fields where it is required to combine 2D close lines. In addition, the TDSFT is designed to be easily extended with new algorithms thanks to a dedicated graphical interface for configuring new parameters. The TDSFT can be downloaded from the following link: https://sourceforge.net/p/tdsft., Competing Interests: BK was employed by the Central R&D Center, Medical and Bio Decision (MBD) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Piccinini, Drudi, Pyun, Lee, Kwak, Ku, Carbonaro, Martinelli and Castellani.)

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related mortality worldwide. Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that binds to programmed cell death-1 (PD-1), which is expressed in activated T cells and other immune cells and has been employed in cancer therapy, including HCC. Recently, PD-L1 overexpression has been documented in treatment-resistant cancer cells. Sorafenib is a multikinase inhibitor and the only FDA-approved treatment for advanced HCC. However, several patients exhibit resistance to sorafenib during treatment. This study aimed to assess the effect of glucose deprivation on PD-L1 expression in HCC cells. We used PD-L1-overexpressing HepG2 cells and IFN-γ-treated SK-Hep1 cells to explore the impact of glycolysis on PD-L1 expression. To validate the correlation between PD-L1 expression and glycolysis, we analyzed data from The Cancer Genome Atlas (TCGA) and used immunostaining for HCC tissue analysis. Furthermore, to modulate PD-L1 expression, we treated HepG2, SK-Hep1, and sorafenib-resistant SK-Hep1R cells with rapamycin. Here, we found that glucose deprivation reduced PD-L1 expression in HCC cells. Additionally, TCGA data and immunostaining analyses confirmed a positive correlation between the expression of hexokinase II (HK2), which plays a key role in glucose metabolism, and PD-L1. Notably, rapamycin treatment  decreased the expression of PD-L1 and HK2 in both high PD-L1-expressing HCC cells and sorafenib-resistant cells. Our results suggest that the modulation of PD-L1 expression by glucose deprivation may represent a strategy to overcome PD-L1 upregulation in patients with sorafenib-resistant HCC., (© 2024. The Author(s).)

The lifecycle of schistosomes must be continuously maintained to clarify and understand this parasite in various aspects in laboratory settings. In the previous studies by other researchers, preservation of schistosome larvae or eggs was attempted by freezing with liquid nitrogen or organic chemicals, but frozen schistosomes were substantially impaired. The present study was conducted to determine whether schistosome eggs can be preserved under a non-frozen condition. The results showed that Schistosoma mansoni eggs could be maintained in phosphate-buffered saline at 4 °C, with a high level of infectivity of miracidia to freshwater snails thereafter. Furthermore, the egg hatchability was maintained for up to 12 weeks with weekly exchanges of the medium. The cercariae derived from snails infected with miracidia from preserved eggs were highly infective to mice. This simple schistosome egg preservation method allow researchers to maintain the schistosome lifecycle without freezing or other special procedures., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Serotonin-like mimotopes were screened from the Fv-antibody library to be used as inhibitors against monoamine oxidase A (MAO-A). The Fv-antibody [corresponding to the V H region of immunoglobulin G (IgG)] consists of three complementarity-determining regions and four frame regions. The Fv-antibody library was prepared by site-directed mutagenesis of CDR3, which consists of 11 amino acid residues. Three target clones were screened from the Fv-antibody library, and the binding affinity of the screened clones to the monoclonal anti-serotonin antibody was analyzed using fluorescence-activated cell sorting. The screened Fv-antibodies were expressed as soluble proteins fused with green fluorescence protein. Additionally, the screened CDR3 regions (11 residues) of the selected Fv-antibodies were synthesized as peptides with linking amino acid residues. The binding constants ( K D ) of the three serotonin-like mimotopes (Fv-antibodies and peptides) were estimated using a surface plasmon resonance biosensor. The inhibitory activity (IC 50 ) of the serotonin-like mimotopes (Fv-antibodies and peptides) was estimated separately for MAO-A and MAO-B enzymes and compared with that of conventional inhibitors. Finally, the screened serotonin-like mimotopes were used to treat a cell line (SH-SY5Y, ATCC code: CRL-2266) expressing serotonin receptors. This was done to confirm the following two aspects: (1) the binding of mimotopes to the serotonin receptors on the cell surface and (2) the inhibitory activity of mimotopes against MAO-A enzymes in the cell lysates., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Various extracellular matrix (ECM) in the lungs regulate tissue development and homeostasis, as well as provide support for cell structures. However, few studies regarding the effects of lung cell differentiation using lung-derived ECM (LM) alone have been reported. The present study investigated the capability of lung-derived matrix sheets (LMSs) to induce lung cell differentiation using mouse embryonic stem (ES) cells. Expressions of lung-related cell markers were significantly upregulated in ES-derived embryoid bodies (EBs) cultured on an LMS for two weeks. Moreover, immunohistochemical analysis of EBs grown on LMSs revealed differentiation of various lung-related cells. These results suggest that an LMS can be used to promote differentiation of stem cells into lung cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pancreatic ribonuclease A (RNase A) inhibitors were screened from an autodisplayed Fv-antibody library, which was prepared by randomizing amino acid sequences of the third complementary-determining region (CDR3) within the heavy chain variable region (V H region) of immunoglobulin G (called "Fv-antibody" comprising three CDRs and four frame regions (FRs)) through site-directed mutagenesis. The library was autodisplayed on the outer membrane of Escherichia coli . Target Fv-variants (clones) with specific binding affinity for RNase A were screened using fluorescein-labeled RNase A and flow cytometry. Three Fv variants (clones) were screened, and CDR3 amino acid sequences were analyzed. The screened Fv-antibodies were expressed as soluble proteins, and CDR3 was synthesized into peptides (11 residues). The binding affinity constants ( K D ) of the expressed Fv-antibodies and synthesized peptides to RNase A were estimated using surface plasmon resonance. Fitting analysis based on the adsorption model showed that K D values of the three expressed Fv-antibodies were estimated to be 17.5 ± 4.1, 28.8 ± 9.7, and 33.9 ± 8.9 nM ( n = 3), and those of the three synthesized peptides were 1.3 ± 0.1, 1.3 ± 0.3, and 3.7 ± 1.3 μM ( n = 3). From the RNase activity assay with an RNA probe labeled with fluorophore and quencher, inhibition constants (IC 50 ) of the three expressed Fv-antibodies were estimated to be 90.2, 65.3, and 98.8 nM ( n = 3), and those of the three synthesized peptides were 8.1, 3.6, and 0.4 μM ( n = 3). The activity of RNase inhibitors constituting the expressed Fv-antibodies and synthesized peptides was demonstrated via an RNA cleavage test using the total RNA from HeLa cells., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.

Purpose: Image-guided percutaneous drainage for abscesses is known as a safe and effective treatment. The computed tomography-guided percutaneous drainage kit Drainaway (SB Kawasumi Co., Ltd.), developed on the basis of a modified trocar method, has made it possible to complete the procedure only under computed tomography guidance without radiographic fluoroscopy. This study investigated the feasibility and safety of Drainaway for abscess drainage., Material and Methods: In this retrospective observational study, 28 procedures in 27 patients (18 men and 9 women; age 67.0 ± 12.3 years) who underwent computed tomography-guided drainage using Drainaway between March and December 2021 at seven affiliated hospitals were analyzed. Patients with symptomatic, puncturable on computed tomography and refractory abscesses were included. Technical success (successful drainage with computed tomography alone), primary clinical success (successful drainage with Drainaway alone), secondary clinical success (avoidance of surgery), and complications were evaluated., Results: The sites of the abscesses were the intraperitoneal, retroperitoneal, and thoracic cavities in 19, 5, and 2 patients, respectively, and subcutaneous tissue in 1 patient. The mean size of the abscesses was 7.1 ± 3.4 cm. The technical success rate was 96.4%; the ligament of the puncture route could not be penetrated in one case. The primary clinical success rate was 77.8%, whereas the secondary clinical success rate of catheter upsizing or replacement was 96.3%. Complications included one case of biliary pleurisy that required drainage., Conclusions: Drainaway is a useful device that allows abscess drainage using only computed tomography guidance without radiographic fluoroscopy., Competing Interests: None, (© 2023 Japanese Society of Interventional Radiology.)

Manipulating viral genomes is an essential technique in reverse genetics and recombinant vaccine development. A strategy for manipulating large viral genomes involves introducing their entire genome into bacterial artificial chromosomes and employing Escherichia coli genetic tools. For sequence manipulation on bacterial artificial chromosomes (bacterial artificial chromosomes recombineering), a well-established method that relies on the Escherichia coli strain GS1783, and the template plasmid, pEPKan-S, is often used. This method, known as markerless DNA manipulation, allows for the generation of a recombinant bacterial artificial chromosome that does not retain the selection markers used during recombination. Although this method is highly innovative, there remains room for improvement as the plasmid is currently only available for positive selection. Additionally, differentiating true recombinants from false negatives often proves time-consuming. Consequently, an improved method for bacterial artificial chromosomes recombineering, which utilizes fluorescent proteins, has been developed. This method's core comprises three plasmids containing the I-SceI recognition site, antibiotic resistance genes (ampicillin, kanamycin, and zeocin), and fluorescent genes (YPet, mOrange, and mScarlet). The success or failure of Red recombination can be confirmed via fluorescent signals. To validate this method, the Lassa virus genes were introduced into the bacterial artificial chromosomes, containing the entire genome of the vaccinia virus strain LC16m8. Consequently, the expression of fluorescent protein genes contributed to positive selection, such as blue-white screening and counter-selection during the first and second Red recombination., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

The current study conceptualized body image instability as a maladaptive tendency to vacillate between different self-perceptions of one's overall body image and developed a corresponding measure to assess body image instability. Results from a series of studies of young adult women demonstrated the validity, reliability, and utility of the Vacillating Body Image Scale (VBIS) as a meaningful measure of body image instability. In Study 1, we found that body image instability, as assessed by the VBIS, represents a unidimensional and reliable construct. In Study 2, we found evidence for both the convergent and discriminant validity of the VBIS in relation to other individual differences measures (i.e., self-concept schema, broad personality factors). In Study 3, the concurrent criterion validity of the VBIS was supported for young adult women in relation to a range of adjustment measures. Finally, in Study 4, we found consistent evidence for the incremental validity of the VBIS in predicting subsequent variations in eating disturbances, even after controlling for global self-esteem and self-concept instability. Overall, our findings offer promising support for our contention that body image instability, as measured by the VBIS, represents an important construct for understanding eating-related disturbances and other health outcomes in young adult women.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Misu, Yoshikawa, Sugimoto, Takamatsu, Kurosu, Ouji, Yoshikawa, Shimojima, Ebihara and Saijo.)

Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Schistosomiasis is one of the most prevalent waterborne parasitic diseases affecting humans. In natural conditions, snails are necessary for maintenance of its lifecycle and also required as intermediate hosts to maintain the lifecycle in laboratory settings. In the present study, the location of S. mansoni larvae in Biomphalaria glabrata snails after infection (inoculation of miracidia) was investigated. Larvae were found located in the head-foot (HF) area of B. glabrata snails at 10 days post-infection (DPI), then their location was predominantly changed to the hepatopancreas and ovotestis (HPOT) area by 56 DPI. Next, the effects of extracts from various organs of B. glabrata snails including HF and HPOT for in vitro culturing of S. mansoni larvae were investigated. The HF extract enabled prolonged culturing of S. mansoni larvae. Furthermore, sequential use of that followed by the HPOT extract supported larval development or reproduction of daughter sporocysts. These results may provide important information for identifying essential factors and molecules for culturing Schistosoma larvae in vitro., Competing Interests: Declaration of Competing Interests The authors have no conflicts of interest to declare., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: 11 C-acetate ( 11 C-ACE) uptake on PET/CT was recently discovered to represent reactive astrocytes in the tumor microenvironment. This study aimed at evaluating the role of 11 C-ACE PET/CT as an imaging biomarker of reactive astrogliosis in characterizing different types of gliomas., Methods: In this prospective study, a total of 182 patients underwent 11 C-ACE PET/CT before surgery. The ratio of SUV max of a glioma to the SUV mean of the contralateral choroid plexus ( 11 C-ACE TCR) on PET/CT was calculated. 11 C-ACE TCRs were compared with the World Health Organization grades and isocitrate dehydrogenase 1 ( IDH1 ) mutation status. Grade 2 was considered low-grade tumor, and grades 3 and 4 were considered high-grade tumors., Results: The median 11 C-ACE TCR was significantly higher in IDH1 wild-type (wt) tumors (n = 91) than in IDH1 -mutant (mt) tumors (n = 91) (2.38 vs 1.30, P < 0.001). Of the 91 IDH1 -mt tumors, there were no differences in the median 11 C-ACE TCRs between oligodendrogliomas (ODs) and astrocytic tumors (1.40 vs 1.20, P > 0.05). In grading low- versus high-grade gliomas, the receiver operating characteristic curve analyses showed a higher area under the curve (0.951) in IDH1 -wt tumors than in IDH1 -mt tumors (0.783, P = 0.002). Grade 2 ODs were well differentiated from high-grade gliomas. The 11 C-ACE TCR of grade 3 ODs was significantly lower than that of IDH1 -wt glioblastomas., Conclusions: High 11 C-ACE uptake is associated with high-grade IDH1 -wt tumors, thus facilitating differentiation from high-grade IDH1-mt and low-grade gliomas. In particular, low 11 C-ACE uptake in ODs is advantageous in overcoming the limitation of radiolabeled amino acid tracers., Competing Interests: Conflicts of interest and sources of funding: none declared. This work was supported partially by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2012R1A1A3008042, NRF-2016R1E1A1A01943303, and NRF-2018M3C7A1056898)., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5 /NaCT and SLC16A1 /MCT1 but low SLC2A1 /GLUT1 and HIF1α /HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5 /NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs.

The present study tested a prediction model involving affectivity and dispositional optimism as predictors of suicide risk (i.e., depressive symptoms and suicidal ideation) in young adult Chinese females with and without prior interpersonal violence (IPV) victimization (294 nonvictimized and 94 victimized females). Results of hierarchical regression analyses indicated that negative affectivity was a significant predictor of both depressive symptoms and suicidal ideation for Chinese females, regardless of IPV victimization. Beyond affectivity, dispositional optimism was found to further add to the prediction model of depressive symptoms in both groups, but only for suicidal ideation in the IPV victimized group.

Hair follicle epithelial stem cells (HFSCs), which exist in the bulge region, have important functions for homeostasis of skin as well as hair follicle morphogenesis. Although several methods for isolation of HFSCs using a variety of stem cell markers have been reported, few investigations regarding culture methods or techniques to yield long-term maintenance of HFSCs in vitro have been conducted. In the present study, we screened different types of commercially available culture medium for culturing HFSCs. Among those tested, one type was shown capable of supporting the expression of stem cell markers in cultured HFSCs. However, both the differentiation potential and in vivo hair follicle-inducing ability of HFSCs serially passaged using that optimal medium were found to be impaired, probably because of altered responsiveness to Wnt signaling. The changes noted in HFSCs subjected to a long-term culture suggested that the Wnt signaling-related environment must be finely controlled for maintenance of the cells., (© 2022. The Author(s).)

Inhibitors for monoamine oxidase-B (MAO-B) were screened from an F V library with a randomized complementarity-determining region 3 (CDR3) region using a monoclonal antibody against dopamine. As the first step, the F V library was expressed on the outer membrane of E. coli by site-directed mutagenesis of the randomized CDR3 region. Among the F V library, variants with a binding affinity to monoclonal antibodies against dopamine were screened and cloned. From the comparison of the binding activity of the screened clones to a control clone with a modified F V antibody (only with CDR1 and CDR2), the CDR3 regions of screened clones were determined to directly interact with the monoclonal antibody against dopamine. These CDR3 sequences were then synthesized as mimotopes (mimicking peptides) of dopamine. The inhibitory activity of two mimotopes against MAO-B was analyzed using HeLa cells overexpressing MAO-B, as well as using activated human astrocytes; their inhibitory activity was compared to that of a commercial inhibitor of MAO-B, selegiline. The inhibition efficiency of the two mimotopes (in comparison with selegiline) was estimated to be 67.2% and 69.4% in the HeLa cells and 64.4% and 58.0% in the human astrocytes. The gene expression pattern in astrocytes after treatment with the two mimotopes was also analyzed and compared with that in the human astrocytes treated with selegiline. Finally, the interaction between two mimotopes and MAO-B was analyzed using docking simulation, and the candidate regions of MAO-B for the interaction with each mimotope were explored through the docking simulation.