High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation.

BACKGROUND: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products. RESULTS: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane. CONCLUSIONS: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Reconfigurable Hanging Drop Microarray Platform for On-Demand Preparation and Analysis of Spheroid Array.

In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.

Understanding the Diversity and Roles of the Ruminal Microbiome.

The importance of ruminal microbiota in ruminants is emphasized, not only as a special symbiotic relationship with ruminants but also as an interactive and dynamic ecosystem established by the metabolites of various rumen microorganisms. Rumen microbial community is essential for life maintenance and production as they help decompose and utilize fiber that is difficult to digest, supplying about 70% of the energy needed by the host and 60-85% of the amino acids that reach the small intestine. Bacteria are the most abundant in the rumen, but protozoa, which are relatively large, account for 40-50% of the total microorganisms. However, the composition of these ruminal microbiota is not conserved or constant throughout life and is greatly influenced by the host. It is known that the initial colonization of calves immediately after birth is mainly influenced by the mother, and later changes depending on various factors such as diet, age, gender and breed. The initial rumen microbial community contains aerobic and facultative anaerobic bacteria due to the presence of oxygen, but as age increases, a hypoxic environment is created inside the rumen, and anaerobic bacteria become dominant in the rumen microbial community. As calves grow, taxonomic diversity increases, especially as they begin to consume solid food. Understanding the factors affecting the rumen microbial community and their effects and changes can lead to the early development and stabilization of the microbial community through the control of rumen microorganisms, and is expected to ultimately help improve host productivity and efficiency.

Antibiotic resistance in livestock, environment and humans: One Health perspective.

Antibiotic resistance (AR) is a complex, multifaceted global health issue that poses a serious threat to livestock, humans, and the surrounding environment. It entails several elements and numerous potential transmission routes and vehicles that contribute to its development and spread, making it a challenging issue to address. AR is regarded as an One Health issue, as it has been found that livestock, human, and environmental components, all three domains are interconnected, opening up channels for transmission of antibiotic resistant bacteria (ARB). AR has turned out to be a critical problem mainly because of the overuse and misuse of antibiotics, with the anticipation of 10 million annual AR-associated deaths by 2050. The fact that infectious diseases induced by ARB are no longer treatable with antibiotics foreshadows an uncertain future in the context of health care. Hence, the One Health approach should be emphasized to reduce the impact of AR on livestock, humans, and the environment, ensuring the longevity of the efficacy of both current and prospective antibiotics.

Complete genome sequence of Enterococcus faecium strain AK_C_05 with potential characteristics applicable in livestock industry.

The Enterococcus faecium (E. faecium) strain AK_C_05 was isolated from cheonggukjang, the Korean traditional food, collected from a local market in South Korea. In this report, we presented the complete genome sequence of E. faecium strain AK_C_05. The genome of E. faecium strain AK_C_05 genome consisted of one circular chromosome (2,691,319 bp) with a guanine + cytosine (GC) content of 38.3% and one circular plasmid (177,732 bp) with a GC content of 35.48%. The Annotation results revealed 2,827 protein-coding sequences (CDSs), 18 rRNAs, and 68 tRNA genes. It possesses genes, which encodes enzymes such as alpha-galactosidase (EC 3.2.1.22), beta-glucosidase (EC 3.2.1.21) and alpha-L-arabinofuranosidase (EC 3.2.1.55) enabling efficient utilization of carbohydrates. Based on Clusters of Orthologous Groups analysis, E. faecium strain AK_C_05 showed specialization in carbohydrate transport and metabolism indicating the ability to generate energy using a variety of carbohydrates.

Evaluation of Fluid Behaviors in a Pushbutton-Activated Microfluidic Device for User-Independent Flow Control.

Although numerous studies have been conducted to realize ideal point-of-care testing (POCT), the development of a user-friendly and user-independent power-free microfluidic platform is still a challenge. Among various methods, the finger-actuation method shows a promising technique that provides a user-friendly and equipment-free way of delivering fluid in a designated manner. However, the design criteria and elaborate evaluation of the fluid behavior of a pushbutton-activated microfluidic device (PAMD) remain a critical bottleneck to be widely adopted in various applications. In this study, we have evaluated the fluid behavior of the PAMD based on various parameters, such as pressing velocity and depth assisted by a press machine. We have further developed a user-friendly and portable pressing block that reduces user variation in fluid behavior based on the evaluation.

Development of the standard mouse model for human bacterial vaginosis induced by Gardnerella vaginalis.

Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a diminished number of protective bacteria in the vaginal flora. Instead, it is accompanied by a significant increase in facultative and strict anaerobes, including Gardnerella vaginalis (G. vaginalis). BV is one of the most common gynecological problems experienced by reproductive age-women. Because an ideal and standard animal model for human BV induced by G. vaginalis is still underdeveloped, the main objective of this study was to develop a mouse model for human BV induced by G. vaginalis to demonstrate the clinical attributes observed in BV patients. A total of 80 female ICR mice were randomly assigned to 4 groups and intravaginally inoculated with different doses of G. vaginalis: NC (uninfected negative control), PC1 (inoculated with 1 × 105 CFU of G. vaginalis), PC2 (inoculated with 1 × 106 CFU of G. vaginalis) and PC3 (inoculated with 1 × 107 CFU of G. vaginalis). The myeloperoxidase (MPO) activity and serum concentrations of cytokines (IL-1ß, IL-10) in mice administered with G. vaginalis were significantly higher than those of the control group. Gross lesion and histopathological analysis of reproductive tract of mice inoculated with G. vaginalis showed inflammation and higher epithelial cell exfoliation compared to the control group. In addition, vaginal swabs from the mice inoculated with G. vaginalis showed the presence of clue cells, which are a characteristic feature of human BV. Altogether, our results suggested that G. vaginalis is sufficient to generate comparable clinical attributes seen in patients with BV.

Cutting-edge knowledge on the roles of phytobiotics and their proposed modes of action in swine.

With the ban on antibiotics in the swine industry, the exploration of alternative options has highlighted phytobiotics as a promising substitute for antibiotic growth promoters, aiming to foster a more sustainable swine industry. Phytobiotics are non-nutritive natural bioactive components derived from plants that offer numerous health benefits. They exhibit antioxidative, antimicrobial, and anti-inflammatory effects. Phytobiotics can be utilized in various forms, including solid, dried, ground, or as extracts, either in crude or concentrated form. They are characterized by low residual levels, a lack of resistance development, and minimal adverse effects. These qualities make phytobiotics an attractive choice for enhancing health and productivity in swine, presenting them as a viable alternative to antibiotics. While there is a general understanding of the effects of phytobiotics, there is still a need for detailed information regarding their effectiveness and mechanisms of action in practical settings. Therefore, the purpose of this mini review was to summarize the current knowledge supporting the roles of phytobiotics and their proposed modes of action, with a specific focus on swine.

Swine gut microbiome associated with non-digestible carbohydrate utilization.

Non-digestible carbohydrates are an unavoidable component in a pig's diet, as all plant-based feeds contain different kinds of non-digestible carbohydrates. The major types of non-digestible carbohydrates include non-starch polysaccharides (such as cellulose, pectin, and hemicellulose), resistant starch, and non-digestible oligosaccharides (such as fructo-oligosaccharide and xylo-oligosaccharide). Non-digestible carbohydrates play a significant role in balancing the gut microbial ecology and overall health of the swine by promoting the production of short chain fatty acids. Although non-digestible carbohydrates are rich in energy, swine cannot extract this energy on their own due to the absence of enzymes required for their degradation. Instead, they rely on gut microbes to utilize these carbohydrates for energy production. Despite the importance of non-digestible carbohydrate degradation, limited studies have been conducted on the swine gut microbes involved in this process. While next-generation high-throughput sequencing has aided in understanding the microbial compositions of the swine gut, specific information regarding the bacteria involved in non-digestible carbohydrate degradation remains limited. Therefore, it is crucial to investigate and comprehend the bacteria responsible for the breakdown of non-digestible carbohydrates in the gut. In this mini review, we have discussed the major bacteria involved in the fermentation of different types of non-digestible carbohydrates in the large intestine of swine, shedding light on their potential roles and contributions to swine nutrition and health.

Correction: Digital Device Exposure and Cognition Levels of Children in Low- and Middle-Income Countries: Cross-sectional Study in Cambodia.

[This corrects the article DOI: .].

Quantifying the narrative flow of imagined versus autobiographical stories.

Lifelong experiences and learned knowledge lead to shared expectations about how common situations tend to unfold. Such knowledge of narrative event flow enables people to weave together a story. However, comparable computational tools to evaluate the flow of events in narratives are limited. We quantify the differences between autobiographical and imagined stories by introducing sequentiality, a measure of narrative flow of events, drawing probabilistic inferences from a cutting-edge large language model (GPT-3). Sequentiality captures the flow of a narrative by comparing the probability of a sentence with and without its preceding story context. We applied our measure to study thousands of diary-like stories, collected from crowdworkers, about either a recent remembered experience or an imagined story on the same topic. The results show that imagined stories have higher sequentiality than autobiographical stories and that the sequentiality of autobiographical stories increases when the memories are retold several months later. In pursuit of deeper understandings of how sequentiality measures the flow of narratives, we explore proportions of major and minor events in story sentences, as annotated by crowdworkers. We find that lower sequentiality is associated with higher proportions of major events. The methods and results highlight opportunities to use cutting-edge computational analyses, such as sequentiality, on large corpora of matched imagined and autobiographical stories to investigate the influences of memory and reasoning on language generation processes.

Correction: Development and Application of a Metaverse-Based Social Skills Training Program for Children With Autism Spectrum Disorder to Improve Social Interaction: Protocol for a Randomized Controlled Trial.

[This corrects the article DOI: 10.2196/35960.].

Intron turnover is essential to the development and pathogenicity of the plant pathogenic fungus Fusarium graminearum.

Intron lariats excised during the splicing process are rapidly degraded by RNA lariat debranching enzyme (Dbr1) and several exonucleases. Rapid turnover of lariat RNA is essential to cellular RNA homeostasis. However, the functions of Dbr1 have not been investigated in filamentous fungi. Here, we characterized the molecular functions of Dbr1 in Fusarium graminearum, a major fungal plant pathogen. Deletion of FgDBR1 resulted in pleiotropic defects in hyphal growth, conidiation, sexual reproduction, and virulence. Through transcriptome analysis, we revealed that the deletion mutant exhibited global accumulation of intron lariats and upregulation of ribosome-related genes. Excessive accumulation of lariat RNA led to reduced overall protein synthesis, causing various phenotypic defects in the absence of FgDBR1. The results of this study demonstrate that a compromised intron turnover process affects development and pathogenesis in this fungus and that Dbr1 function is critical to plant pathogenic fungi.

Digital Device Exposure and Cognition Levels of Children in Low- and Middle-Income Countries: Cross-sectional Study in Cambodia.

BACKGROUND: Policy makers and practitioners in low- and middle-income countries (LMICs) are increasingly focusing on the effectiveness of digital devices in the delivery of medical and educational services to children under resource constraints. It is widely known that digital literacy can be fostered through exposure to and education regarding digital devices, which can improve children's academic performance as well as their search and communication skills in the digital era. However, the correlation between the cognitive function of children and exposure and intensity of the exposure to digital devices has rarely been studied, and the association between digital device exposure and the socioeconomic characteristics and cognitive development of children in LMICs is unknown. OBJECTIVE: This study examines the association among exposure to digital devices, socioeconomic status, and cognitive function in children aged 3 to 9 years in Cambodia. METHODS: We used a survey of 232 children that gathered data on familiarity with digital devices, demographic characteristics, and socioeconomic status, as well as a Cambridge Neuropsychological Test Automated Battery test for cognitive function, to examine the association between possible barriers and factors that may influence the cognitive function of children in 2 Cambodian schools from April 22, 2019, to May 4, 2019. A comparative analysis was performed with and without digital exposure, and an association analysis was performed among the variables from the survey and cognitive function. RESULTS: Significant differences were observed in demographic and socioeconomic characteristics such as school location, family type, and family income according to digital device exposure. The results of the Cambridge Neuropsychological Test Automated Battery tests, except for 1 test related to executive function, indicated no significant differences (P>.05) between group A and group B or among the 4 subgroups. Pretest digital device experience and amount of time spent using digital devices during the test had no significant impacts on the cognitive development of the children. Conversely, the multivariate analyses showed that cognitive function was associated with educational expenses per child, school (location), family type, and family income. CONCLUSIONS: These results provide evidence to policy makers and practitioners on the importance of improving socioeconomic conditions, leading to investment in education by implementing programs for children's cognitive development through digital devices in LMICs.

Development and Application of a Metaverse-Based Social Skills Training Program for Children With Autism Spectrum Disorder to Improve Social Interaction: Protocol for a Randomized Controlled Trial.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by abnormalities in social communication and limited and repetitive behavioral patterns. Children with ASD who lack social communication skills will eventually not interact with others and will lack peer relationships when compared to ordinary people. Thus, it is necessary to develop a program to improve social communication abilities using digital technology in people with ASD. OBJECTIVE: We intend to develop and apply a metaverse-based child social skills training program aimed at improving the social interaction abilities of children with ASD aged 7-12 years. We plan to compare and analyze the biometric information collected through wearable devices when applying the metaverse-based social skills training program to evaluate emotional changes in children with ASD in stressful situations. METHODS: This parallel randomized controlled study will be conducted on children aged 7-12 years diagnosed with ASD. A metaverse-based social skills training program using digital technology will be administered to children who voluntarily wish to participate in the research with consent from their legal guardians. The treatment group will participate in the metaverse-based social skills training program developed by this research team once a week for 60 minutes per session for 4 weeks. The control group will not intervene during the experiment. The treatment group will use wearable devices during the experiment to collect real-time biometric information. RESULTS: The study is expected to recruit and enroll participants in March 2022. After registering the participants, the study will be conducted from March 2022 to May 2022. This research will be jointly conducted by Yonsei University and Dobrain Co Ltd. Children participating in the program will use the internet-based platform. CONCLUSIONS: The metaverse-based Program for the Education and Enrichment of Relational Skills (PEERS) will be effective in improving the social skills of children with ASD, similar to the offline PEERS program. The metaverse-based PEERS program offers excellent accessibility and is inexpensive because it can be administered at home; thus, it is expected to be effective in many children with ASD. If a method can be applied to detect children's emotional changes early using biometric information collected through wearable devices, then emotional changes such as anxiety and anger can be alleviated in advance, thus reducing issues in children with ASD. TRIAL REGISTRATION: Clinical Research Information Service KCT0006859; https://tinyurl.com/4r3k7cmj. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/35960.

Inhibiting Transglutaminase 2 Mediates Kidney Fibrosis via Anti-Apoptosis.

Transglutaminase 2 (TG2) is a calcium-dependent transamidating acyltransferase enzyme of the protein-glutamine γ-glutamyltransferase family implicated in kidney injury. In this study, we identified associations between TG2 and chronic kidney disease (CKD) identified by visualizing TG2 in kidney biopsy samples derived from CKD patients using immunohistochemistry and measuring the plasma TG2 concentrations. Our study revealed a connection between TG2 and the pathological markers of kidney disease. We showed high plasma TG2 levels in samples from patients with advanced CKD. In addition, we observed an increase in TG2 expression in tissues concomitant with advanced CKD in human samples. Moreover, we investigated the effect of TG2 inhibition on kidney injury using cystamine, a well-known competitive inhibitor of TG2. TG2 inhibition reduced apoptosis and accumulation of extracellular molecules (ECM) such as fibronectin and pro-inflammatory cytokine IL-8. Collectively, the increased expression of TG2 that was observed in advanced CKD, hence inhibiting TG2 activity, could protect kidney cells from ECM molecule accumulation, apoptosis, and inflammatory responses, thereby preventing kidney fibrosis.

Improving Printability of Digital-Light-Processing 3D Bioprinting via Photoabsorber Pigment Adjustment.

Digital-light-processing (DLP) three-dimensional (3D) bioprinting, which has a rapid printing speed and high precision, requires optimized biomaterial ink to ensure photocrosslinking for successful printing. However, optimization studies on DLP bioprinting have yet to sufficiently explore the measurement of light exposure energy and biomaterial ink absorbance controls to improve the printability. In this study, we synchronized the light wavelength of the projection base printer with the absorption wavelength of the biomaterial ink. In this paper, we provide a stepwise explanation of the challenges associated with unsynchronized absorption wavelengths and provide appropriate examples. In addition to biomaterial ink wavelength synchronization, we introduce photorheological measurements, which can provide optimized light exposure conditions. The photorheological measurements provide precise numerical data on light exposure time and, therefore, are an effective alternative to the expendable and inaccurate conventional measurement methods for light exposure energy. Using both photorheological measurements and bioink wavelength synchronization, we identified essential printability optimization conditions for DLP bioprinting that can be applied to various fields of biological sciences.

Pilot study of a mobile application-based intervention to induce changes in neural activity in the frontal region and behaviors in children with attention deficit hyperactivity disorder and/or intellectual disability.

Children with neurodevelopmental disorders, such as attention deficit hyperactivity disorder (ADHD) and intellectual disability (ID), need early intervention and continuous treatment. We aimed to investigate the feasibility and acceptability of mobile application-based interventions in children with ADHD and ID in supporting attention and cognitive function. Twenty-six children with ADHD and/or ID with attention and cognition difficulties were recruited. Participants completed a 12-week mobile application-based intervention. To assess whether digital intervention improved attention and cognitive function, we used the Comprehensive Attention Test (CAT), Cambridge Neuropsychological Tests Automated Battery (CANTAB), and electroencephalography (EEG) to examine direct changes in children's behavior and neural activity. Clinicians and parents assessed changes using the Behavior Rating Inventory of Executive Function, Second Edition (BRIEF-2), Korean version of the ADHD Rating Scale (K-ARS), Clinical Global Impression-Improvement Scale, and parental questionnaires. The intervention induced changes in neural activities on EEG and behavior but there were no significant changes in CAT and CANTAB results. Relative theta and alpha power were significantly lower post-intervention in the eyes-open (EO) condition of EEG recording and these changes were mainly observed in the frontal regions of the brain. Parental reports using the BRIEF-2 and K-ARS noted significant improvements in executive function, attention, and hyperactivity-impulsivity. In addition, the clinical impression improved in 60% of participants. These results provide evidence that a mobile application-based intervention has the benefit of supporting children with ADHD and/or ID. Digital intervention could change neural activity and improve children's attention and cognitive function. Given our findings, we suggested that mobile application-based digital therapeutics may have great potential for helping children with neurodevelopmental disorders who need continuous treatment.

Temporally Resolved Electrochemical Interrogation for Stochastic Collision Dynamics of Electrogenerated Single Polybromide Droplets.

In this article, we present electrochemical interrogation for collision dynamics of electrogenerated individual polybromide ionic liquid (PBIL) droplets through chronoamperometry combined with fast scan cyclic voltammetry (CA-FSCV). In the CA mode of CA-FSCV, a Pt ultramicroelectrode (UME) acts as the electrochemical generator for PBIL droplets by holding the oxidation potential for Br- in a given time, while FSCV is repetitively performed at a certain frequency. In the FSCV mode of CA-FSCV, a Pt UME serves as the probe to electrochemically monitor Br3- reduction for an adsorbed PBIL droplet during collision with a high temporal resolution. Based on the newly introduced CA-FSCV, we can estimate the dynamic changes in the following parameters for a short collision time: the contact radius of a PBIL droplet on a Pt UME, the concentration of Br- in the droplet, and the apparent charge transfer rate constant for electro-reduction of Br3- to Br- in the droplet, koapp. Moreover, a computational calculation using molecular dynamics is presented that can explain the change in koapp as a function of time for Br- electrolysis in a PBIL droplet. Based on the quantitative estimation of the above parameters, we suggest a more advanced mechanism for the stochastic electrochemical collision process of a PBIL droplet. These findings are important for understanding QBr2n+1/QBr half redox reactions in aqueous energy storage systems, such as Zn-Br redox flow batteries and Br-related redox enhanced electrochemical capacitors.

A Study of the Possibility of Detecting Pediatric Mild Developmental Delay Through a Serious Game: A Randomized Cluster Trial in Cambodia.

Although early diagnosis of developmental delay is important, there are challenges in identifying cognitive status in developing countries because of limited human and financial resources to perform diagnostic tests. Moreover, diagnosis stability of developmental delay in children using neuropsychological tests (NPTs) can remain unsettled. The aim of this study is (1) to verify the effectiveness of a serious game (DoBrain), (2) to identify existing inconsistencies between NPTs, and (3) to explore the potential of the serious game as a complement to diagnostic tools. Eligible children who had completed results of NPTs were selected (n=119/235; 116/235; case, control). With these children's scores, we performed the Mann- Whitney U test to investigate the effectiveness of the serious game by comparing the improvement of scores in both groups. Among the participants, we additionally selected a case group to identify the potential of the serious game for detecting mild developmental delay. Using the results of the CGI-S as a baseline, we defined the participants whose scores indicated more than mild illness (>=2 points) in at least one area as the suspected group. The score improvement related to memory in case group was greater than that of the control group (p<0.05). Furthermore, four of the NPTs were not inconsistent, and the sensitivity/specificity of DDST-II was the highest score considering CGI-S results as the ground truth (0.43; 0.96). Additionally, games measuring discrimination, velocity, memory, and spatial perception showed statistical significance (p<0.05). This study verifies that the serious game can help specific cognitive areas and suggests that the serious game could be used as a low-cost and unconstrained spatiotemporal alternative to NPTs.