Systematic mapping of TF-mediated cell fate changes by a pooled induction coupled with scRNA-seq and multi-omics approaches.

Transcriptional regulation controls cellular functions through interactions between transcription factors (TFs) and their chromosomal targets. However, understanding the fate conversion potential of multiple TFs in an inducible manner remains limited. Here, we introduce iTF-seq as a method for identifying individual TFs that can alter cell fate toward specific lineages at a single-cell level. iTF-seq enables time course monitoring of transcriptome changes, and with biotinylated individual TFs, it provides a multi-omics approach to understanding the mechanisms behind TF-mediated cell fate changes. Our iTF-seq study in mouse embryonic stem cells identified multiple TFs that trigger rapid transcriptome changes indicative of differentiation within a day of induction. Moreover, cells expressing these potent TFs often show a slower cell cycle and increased cell death. Further analysis using bioChIP-seq revealed that GCM1 and OTX2 act as pioneer factors and activators by increasing gene accessibility and activating the expression of lineage specification genes during cell fate conversion. iTF-seq has utility in both mapping cell fate conversion and understanding cell fate conversion mechanisms.

Metabolic and cell cycle shift induced by the deletion of Dnm1l attenuates the dissolution of pluripotency in mouse embryonic stem cells.

Mitochondria are versatile organelles that continuously change their morphology via fission and fusion. However, the detailed functions of mitochondrial dynamics-related genes in pluripotent stem cells remain largely unclear. Here, we aimed to determine the effects on energy metabolism and differentiation ability of mouse embryonic stem cells (ESCs) following deletion of the mitochondrial fission-related gene Dnml1. Resultant Dnm1l-/- ESCs maintained major pluripotency characteristics. However, Dnm1l-/- ESCs showed several phenotypic changes, including the inhibition of differentiation ability (dissolution of pluripotency). Notably, Dnm1l-/- ESCs maintained the expression of the pluripotency marker Oct4 and undifferentiated colony types upon differentiation induction. RNA sequencing analysis revealed that the most frequently differentially expressed genes were enriched in the glutathione metabolic pathway. Our data suggested that differentiation inhibition of Dnm1l-/- ESCs was primarily due to metabolic shift from glycolysis to OXPHOS, G2/M phase retardation, and high level of Nanog and 2-cell-specific gene expression.

Synthetic recording and in situ readout of lineage information in single cells.

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.

Preischemic transfusion of old packed RBCs exacerbates early-phase warm hepatic ischemia reperfusion injury in rats.

BACKGROUND: Hepatic innate immune cells are considered to play a central role in the early phase of hepatic ischemia reperfusion (IR) injury. Transfusion of old red blood cells (RBCs) is known to prime immune cells, and transfusion before IR may exacerbate liver injury because of the expected hyperresponsiveness of immune cells. MATERIALS AND METHODS: Twenty-four Sprague-Dawley rats were divided into four groups: sham operation (Sham); hepatic IR only (IR Control); and two transfusion groups, preischemic (Pre-T) and postischemic (Post-T), in which allogeneic RBCs stored for 2 weeks were transfused before hepatic IR or after reperfusion, respectively. Partial hepatic ischemia was induced for 90 min, and reperfusion was allowed for 120 min. Serum alanine transaminase levels, area of necrosis, and apoptotic cells were then assessed. Inflammatory (tumor necrosis factor alpha, interleukin 1 beta [IL-1ß], IL-6, IL-10, and cyclooxygenase 2) and oxidative mediators (heme oxygenase 1, superoxide dismutase, and glutathione peroxidase 1) were assessed for elucidating the relevant mechanisms underlying the hepatic injury. RESULTS: Pre-T, but not Post-T, showed increased serum alanine transaminase levels than IR Control (P < 0.05). Area of necrosis was more severe in Pre-T than in IR Control or Post-T (P < 0.01), and apoptotic cells were also more abundant in Pre-T than in IR Control (P < 0.01). tumor necrosis factor alpha and IL-6 levels were higher in Pre-T than in IR Control or Post-T (P < 0.05), with no significant difference in cytoprotective protein levels. CONCLUSIONS: Preischemic transfusion of old RBCs aggravated hepatic injury. Inflammatory cytokines seemed to play a crucial role in liver injury exacerbation. Our results indicate that transfusion before hepatic ischemia may be detrimental.

Antisenescence effect of mouse embryonic stem cell conditioned medium through a PDGF/FGF pathway.

Cellular senescence, an irreversible state of growth arrest, underlies organismal aging and age-related diseases. Recent evidence suggests that aging intervention based on inhibition of cellular senescence might be a promising strategy for treatment of aging and age-related diseases. Embryonic stem cells (ESCs) and ESC conditioned medium (CM) have been suggested as a desirable source for regenerative medicine. However, effects of ESC-CM on cellular senescence remain to be determined. We found that treatment of senescent human dermal fibroblasts with CM from mouse ESCs (mESCs) decreases senescence phenotypes. We found that platelet-derived growth factor BB in mESC-CM plays a critical role in antisenescence effect of mESC-CM through up-regulation of fibroblast growth factor 2. We confirmed that mESC-CM treatment accelerates the wound-healing process by down-regulating senescence-associated p53 expression in in vivo models. Taken together, our results suggest that mESC-CM has the ability to suppress cellular senescence and maintain proliferative capacity. Therefore, this strategy might emerge as a novel therapeutic strategy for aging and age-related diseases.

Clinical Implications for the Comprehensive Interpretation of Radiologic and Immunodiagnostic Tests in Patients Suspected of Parasitic Hepatic Cyst, a Rare Case in Korea.

Cystic echinococcosis (CE) is a representative neglected tropical disease (NTD) with increased morbidity and mortality but is ignored and overlooked in developed countries. Serological and radiographic findings are helpful in distinguishing these parasites; however, conflicting results of these can make it difficult to diagnose if medical knowledge of hepatic parasitic disease, including the etiology, features of imaging, and immunodiagnostic test, is not acquired. We report the case of a male patient with dyspepsia and right epigastric pain who had positive results for cysticercosis antibodies on immunodiagnostic examination. Abdominal ultrasonography revealed two huge communicating cystic lesions measuring 8-11 cm. Further evaluations for cysticercosis of the brain (neurocysticercosis) and eyes (intraocular cysticercosis) were unremarkable throughout the brain imaging test and fundus examination. A laparoscopic right hemi-hepatectomy was performed for diagnosis and treatment. On histopathological examination, diverse stages of Echinococcus granulosus were identified. Albendazole was administered postoperatively, and the patient was also followed up. We should be aware of the etiologies that have been prevalent in parasite infection thought to be the cause of hepatic cysts. Moreover, we make an effort to ascertain the patient's nationality, past travel experiences, and immediate environment, including any animals and pets. We present the case of a patient who was worried about the possibility of liver invasion of cysticercus due to the positivity of the cysticercosis antibody and was ultimately diagnosed with CE.

AI-driven robotic chemist for autonomous synthesis of organic molecules.

The automation of organic compound synthesis is pivotal for expediting the development of such compounds. In addition, enhancing development efficiency can be achieved by incorporating autonomous functions alongside automation. To achieve this, we developed an autonomous synthesis robot that harnesses the power of artificial intelligence (AI) and robotic technology to establish optimal synthetic recipes. Given a target molecule, our AI initially plans synthetic pathways and defines reaction conditions. It then iteratively refines these plans using feedback from the experimental robot, gradually optimizing the recipe. The system performance was validated by successfully determining synthetic recipes for three organic compounds, yielding that conversion rates that outperform existing references. Notably, this autonomous system is designed around batch reactors, making it accessible and valuable to chemists in standard laboratory settings, thereby streamlining research endeavors.

A Simple Method for Generating Cerebral Organoids from Human Pluripotent Stem Cells.

BACKGROUND AND OBJECTIVES: In recent years, brain organoid technologies have been the most innovative advance in neural differentiation research. In line with this, we optimized a method to establish cerebral organoids from feeder-free cultured human pluripotent stem cells. In this study, we focused on the consistent and robust production of cerebral organoids comprising neural progenitor cells and neurons. We propose an optimal protocol for cerebral organoid generation that is applicable to both human embryonic stem cells and human induced pluripotent stem cells. METHODS AND RESULTS: We investigated formation of neuroepithelium, neural tube, and neural folding by observing the morphology of embryoid bodies at each stage during the cerebral organoid differentiation process. Furthermore, we characterized the cerebral organoids via immunocytochemical staining of sectioned organoid samples, which were prepared using a Cryostat and Vibratome. Finally, we established a routine method to generate early cerebral organoids comprising a cortical layer and a neural progenitor zone. CONCLUSIONS: We developed an optimized methodology for the generation of cerebral organoids using hESCs and hiPSCs. Using this protocol, consistent and efficient cerebral organoids could be obtained from hiPSCs as well as hESCs. Further, the morphology of brain organoids could be analyzed through 2D monitoring via immunostaining and tissue sectioning, or through 3D monitoring by whole tissue staining after clarification.

Developmental Potency and Metabolic Traits of Extended Pluripotency Are Faithfully Transferred to Somatic Cells via Cell Fusion-Induced Reprogramming.

As a novel cell type from eight-cell-stage embryos, extended pluripotent stem cells (EPSCs) are known for diverse differentiation potency in both extraembryonic and embryonic lineages, suggesting new possibilities as a developmental research model. Although various features of EPSCs have been defined, their ability to directly transfer extended pluripotency to differentiated somatic cells by cell fusion remains to be elucidated. Here, we derived EPSCs from eight-cell mouse embryos and confirmed their extended pluripotency at the molecular level and extraembryonic differentiation ability. Then, they were fused with OG2+/- ROSA+/- neural stem cells (NSCs) by the polyethylene-glycol (PEG)-mediated method and further analyzed. The resulting fused hybrid cells exhibited pluripotential markers with upregulated EPSC-specific gene expression. Furthermore, the hybrid cells contributed to the extraembryonic and embryonic lineages in vivo and in vitro. RNA sequencing analysis confirmed that the hybrid cells showed distinct global expression patterns resembling EPSCs without parental expression of NSC markers, indicating the complete acquisition of extended pluripotency and the erasure of the somatic memory of NSCs. Furthermore, ultrastructural observation and metabolic analysis confirmed that the hybrid cells rearranged the mitochondrial morphology and bivalent metabolic profile to those of EPSCs. In conclusion, the extended pluripotency of EPSCs could be transferred to somatic cells through fusion-induced reprogramming.

Evaluation of Data Entry Errors and Data Changes to an Electronic Data Capture Clinical Trial Database.

Monitoring of clinical trials includes several disciplines, stakeholders, and skill sets. The aim of the present study was to identify database changes and data entry errors to an electronic data capture (EDC) clinical trial database, and to access the impact of the changes. To accomblish the aim, Target e*CRF was used as the EDC tool for a multinational, dose-finding, multicenter, double-blind, randomized, parallel, placebo-controlled trial to investigate efficacy and safety of a new treatment in men with lower urinary tract symptoms associated with benign prostatic hyperplasia. The main errors observed were simple transcription errors from the paper source documents to the EDC database. This observation was to be expected, since every transaction has an inherant error rate. What and how to monitor must be assessed within the risk-based monitoring section of the comprehensive data monitoring plan. With the advent of direct data entry, and the elimination of the requirement to transcribe from a paper source record to an EDC system, error rates should go down dramatically. In addition, protocol violations and data outside the normal range can be identified at the time of data entry and not days, weeks, and months after the fact.

Extremely delayed solitary cerebral metastasis in patient with T1N0M0 renal cell carcinoma after radical nephrectomy: Case report and literature review.

RATIONALE: Although renal cell carcinoma (RCC) is one of the common origins of brain metastasis, few cases of extremely delayed brain metastasis from RCC, more than 10 years after nephrectomy, have been reported. We present a rare case of extremely delayed brain metastasis from RCC, also performed a literature review to increase knowledge of the characteristics for extremely delayed brain metastasis from RCC. PATIENT CONCERNS: A 72-year-old man presented with right-sided hemiplegia and dysarthria. The patient had a history of radical nephrectomy for RCC with stage T1N0M0 15 years earlier. DIAGNOSIS: Magnetic resonance imaging with contrast revealed a 2-cm sized non-homogenous enhanced mass in the left frontal lobe with peritumoral edema. The pathological examination after surgery reported metastatic clear cell RCC. INTERVENTIONS: A craniotomy for removal of the mass was performed at the time of diagnosis. Stereotactic radiosurgery was performed for the tumor bed 3 weeks after craniotomy, and then, chemotherapy was started 2 months after the SRS. OUTCOMES: Metastasis progressed to multiple organs 6 months after the craniotomy. The patient chose a hospice and no longer visited the hospital. LESSONS: In cases with a history of nephrectomy for RCC, long period follow-up is necessary for monitoring RCC brain metastasis and pathologic diagnosis should be confirmed.

Giant Intradiploic Epidermoid Cyst in the Occipital Bone: A Case Report.

Epidermoid cysts are uncommon intracranial tumors. As one of the extradural types of epidermoid cysts, intradiploic epidermoid cysts are even rarer tumors and occur in any part of the skull. We herein report a rare case of a giant intradiploic epidermoid cyst of the occipital bone. A 57-year-old woman presented with a 1-year history of localized headache in the occipital area. CT and MRI showed an extradural mass measuring 50×70 mm in the occipital bone with bony destruction. The patient underwent surgical resection. The tumor was completely removed with its capsule. There was no extension to the intradural space. The pathological report confirmed that the tumor was an epidermoid cyst. Follow-up MRI 24 months after the operation showed no recurrence. The headache was well controlled without any medications. We report a rare case of intradiploic epidermoid cyst with clinical and radiologic features and surgical treatment. It is important to consider this diagnosis for a patient with persistent regional headache with or without a growing scalp mass.

Current Issues and Technical Advances in Cultured Meat Production: A Review.

As the global population grows, we need a stable protein supply to meet the demands. Although plant-derived protein sources are widely available, animal meat maintains its popularity as a high-quality and savory protein source. Recently, cultured meat, also known as in vitro meat, has been suggested as a meat analog produced through in vitro cell culture technology. Cultured meat has several advantages over conventional meat, such as environmental protection, disease prevention, and animal welfare. However, cultured meat manufacturing is an emerging technology; thus, its further and dynamic development would be pivotal. Commercialization of cultured meat to the public will take a long time but cultured meat undoubtedly will come to our table someday. Here, we discuss the social and economic aspects of cultured meat production as well as the recent technical advances in cultured meat technology.

Inhibition of neural stem cell aging through the transient induction of reprogramming factors.

Adult stem cells age during long-term in vitro culture, and neural stem cells (NSCs), which can self-renew and differentiate into neurons and glial cells, also display reduced differentiation potential after repeated passaging. However, the mechanistic details underlying this process remain unclear. In this study, we found that long-term in vitro culture of NSCs resulted in aging-related upregulation of inflammatory- and endoplasmic reticulum (ER) stress-related genes, including the proinflammatory cytokines interleukin (IL)1ß and IL6, the senescence-associated enzyme matrix metallopeptidase 13 (MMP13), and the ER stress-responsive transcription factor activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, the cyclic and transient induction of four reprogramming factors (POU domain, class 5, transcription factor 1, also known as octamer-binding transcription factor 4; SRY [sex determining region Y]-box 2; Kruppel-like factor 4; and myelocytomatosis oncogene; collectively referred to as OSKM) can inhibit NSC aging, as indicated by the decreased expression of the inflammatory and ER stress-related genes. We used ROSA-4F NSCs, which express OSKM from only one allele, to minimize the potential for full reprogramming or tumor formation during NSC rejuvenation. We expect that this novel rejuvenation method will enhance the potential of NSCs as a clinical approach to the treatment of neurological diseases.

Association of Skeletal Muscle and Adipose Tissue Distribution with Histologic Severity of Non-Alcoholic Fatty Liver.

Adipose tissue and skeletal muscle is associated with non-alcoholic fatty liver disease (NAFLD). This study evaluates the association between body composition and histologic severity in patients with NAFLD. Using the cross-sectional CT images at the level of L3 vertebra and the histologic findings of 178 patients with biopsy-proven NAFLD, we analyzed the correlation of the histologic findings to the skeletal muscle index (SMI), subcutaneous adipose tissue index (SATI), and visceral adipose tissue index (VATI), which is defined as the body composition area (cm2) by height squared (m2). The clinical and laboratory features with body composition were analyzed to determine the risk factors for advanced fibrosis. The VATI significantly increased in severe non-alcoholic steatohepatitis (NASH) or advanced fibrosis. In addition, the VATI was correlated with the NAFLD activity score (NAS) and the fibrosis stage. In multivariate analyses, age (odds ratio (OR), 1.09; 95% confidence interval (CI), 1.02-1.19; p = 0.025), severe NASH (OR, 8.66; 95% CI, 2.13-46.40; p = 0.005), and visceral adiposity (OR, 6.77; 95% CI, 1.81-29.90; p = 0.007) were independently associated with advanced fibrosis in patients with NAFLD. Visceral adiposity is correlated with the histologic severity of NAFLD, which is independently associated with advanced fibrosis.

Calvarial Tuberculosis Presenting with Skin Defect in an Elderly Patient.

BACKGROUND: Tuberculosis is a common disease; however, the prevalence of calvarial tuberculosis is very rare. Most cases of calvarial tuberculosis occur in young patients. We report a rare case of calvarial tuberculosis in an elderly patient. CASE DESCRIPTION: An 89-year-old woman presented with a forehead skin defect. Radiologic imaging showed bony erosion 20 × 10 mm in size with adjacent dural enhancement in the left frontal bone. The patient underwent surgical treatment. Pathology revealed chronic granulomatous inflammation with caseous necrosis, consistent with tuberculosis. Antituberculous medications were prescribed for 6 months. CONCLUSIONS: A careful assessment should be performed to obtain an appropriate diagnosis in cases of osteolytic lesions of the skull.

Genetic and Epigenetic Etiology Underlying Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by difficulties in social interaction, language development delays, repeated body movements, and markedly deteriorated activities and interests. Environmental factors, such as viral infection, parental age, and zinc deficiency, can be plausible contributors to ASD susceptibility. As ASD is highly heritable, genetic risk factors involved in neurodevelopment, neural communication, and social interaction provide important clues in explaining the etiology of ASD. Accumulated evidence also shows an important role of epigenetic factors, such as DNA methylation, histone modification, and noncoding RNA, in ASD etiology. In this review, we compiled the research published to date and described the genetic and epigenetic epidemiology together with environmental risk factors underlying the etiology of the different phenotypes of ASD.

Defect-Passivating Organic/Inorganic Bicomponent Hole-Transport Layer for High Efficiency Metal-Halide Perovskite Device.

In this work, we introduce a bicomponent hole-transport layer, composed of inorganic NiOx and a donor-acceptor-donor (D-A-D)-structured organic small molecule, for p-i-n planar perovskite photovoltaic (PV) cells. The newly designed D-A-D organic hole-transporting material (HTM), (4',4‴-(1,3,4-oxadiazole-2,5-diyl)bis(N,N-bis(4-methoxyphenyl)-[1,1'-biphenyl]-4-amine)), is shown to be an efficient HTM without a dopant, and methoxy functional units, further introduced to the molecules, are confirmed to be beneficial to passivate the defects in the perovskite, which improves the crystallinity of perovskite and suppresses the nonradiative recombination in the devices, consequently enhancing the performances of PV cells (over 20% efficiency from p-i-n architecture). Furthermore, the decreased defect sites along with the UV-blocking property of the HTM in p-i-n architecture are advantageous in improving the stability of the PV devices.

Role of mitochondrial fission-related genes in mitochondrial morphology and energy metabolism in mouse embryonic stem cells.

Mitochondria, the major organelles that produce energy for cell survival and function, dynamically change their morphology via fusion and fission, a process called mitochondrial dynamics. The details of the underlying mechanism of mitochondrial dynamics have not yet been elucidated. Here, we aimed to investigate the function of mitochondrial fission genes in embryonic stem cells (ESCs). To this end, we generated homozygous knockout ESC lines, namely, Fis1-/-, Mff-/-, and Dnm1l-/- ESCs, using the CRISPR-Cas9 system. Interestingly, the Fis1-/-, Mff-/-, and Dnm1l-/- ESCs showed normal morphology, self-renewal, and the ability to differentiate into all three germ layers in vitro. However, transmission electron microscopy showed a significant increase in the cytoplasm to nucleus ratio and mitochondrial elongation in Dnm1l-/- ESCs, which was due to incomplete fission. To assess the change in metabolic energy, we analyzed oxidative phosphorylation (OXPHOS), glycolysis, and the intracellular ATP concentration. The ESC knockout lines showed an increase in OXPHOS, decrease in glycolysis, and an increase in intracellular ATP concentration, which was related to mitochondrial elongation. In particular, the Dnm1l knockout most significantly affected mitochondrial morphology, energy metabolism, and ATP production in ESCs. Furthermore, RNA sequencing and gene ontology analysis showed that the differentially expressed genes in Mff-/- ESCs were distinct from those in Dnm1l-/- or Fis1-/- ESCs. In total, five metabolism-related genes, namely, Aass, Cdo1, Cyp2b23, Nt5e, and Pck2, were expressed in all three knockout ESC lines, and three of them were associated with regulation of ATP generation.

Mitochondrial volume fraction and translation duration impact mitochondrial mRNA localization and protein synthesis.

Mitochondria are dynamic organelles that must precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial surface, the importance of this localization is unclear. As yeast switch to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. Our data point to this change in mitochondrial volume fraction increasing the localization of certain nuclear-encoded mRNAs to the surface of the mitochondria. We show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to mitochondria. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle-specific gene expression through mRNA localization.