Amelioration of the brain structural connectivity is accompanied with changes of gut microbiota in a tuberous sclerosis complex mouse model.

Tuberous sclerosis complex (TSC) is a genetic disease that causes benign tumors and dysfunctions in many organs, including the brain. Aside from the brain malformations, many individuals with TSC exhibit neuropsychiatric symptoms. Among these symptoms, autism spectrum disorder (ASD) is one of the most common co-morbidities, affecting up to 60% of the population. Past neuroimaging studies strongly suggested that the impairments in brain connectivity contribute to ASD, whether or not TSC-related. Specifically, the tract-based diffusion tensor imaging (DTI) analysis provides information on the fiber integrity and has been used to study the neuropathological changes in the white matter of TSC patients with ASD symptoms. In our previous study, curcumin, a diet-derived mTOR inhibitor has been shown to effectively mitigate learning and memory deficits and anxiety-like behavior in Tsc2+/- mice via inhibiting astroglial proliferation. Recently, gut microbiota, which is greatly influenced by the diet, has been considered to play an important role in regulating several components of the central nervous system, including glial functions. In this study, we showed that the abnormal social behavior in the Tsc2+/- mice can be ameliorated by the dietary curcumin treatment. Second, using tract-based DTI analysis, we found that the Tsc2+/- mice exhibited altered fractional anisotropy, axial and radial diffusivities of axonal bundles connecting the prefrontal cortex, nucleus accumbens, hypothalamus, and amygdala, indicating a decreased brain network. Third, the dietary curcumin treatment improved the DTI metrics, in accordance with changes in the gut microbiota composition. At the bacterial phylum level, we showed that the abundances of Actinobacteria, Verrucomicrobia, and Tenericutes were significantly correlated with the DTI metrics FA, AD, and RD, respectively. Finally, we revealed that the expression of myelin-associated proteins, myelin bassic protein (MBP) and proteolipid protein (PLP) was increased after the treatment. Overall, we showed a strong correlation between structural connectivity alterations and social behavioral deficits, as well as the diet-dependent changes in gut microbiota composition.

Magnusiomyces Capitatus Lung Nodule in a Patient with Nasopharyngeal and Hepatocellular Carcinoma.

Magnusiomyces capitatus is a dimorphic yeast commonly isolated from the environment and was uncommonly reported as a disease in Asia. It may cause invasive infection in patients with hematological malignancies, especially those with neutropenia, and resulting in high mortality. Herein, we reported a man with nasopharyngeal carcinoma and hepatocellular carcinoma suffered from intermittent fever after pulmonary nodules resection. The histopathology showed yeast-like fungal elements. For further identification, we extracted the tissue DNA from formalin-fixed paraffin-embedded tissue and M. capitatus was confirmed using polymerase chain reaction amplification and sequencing of the ITS region of ribosomal DNA. After a 4-week amphotericin B and flucytosine treatment, his condition recovered well and then was followed by a 3-month oral fluconazole treatment. There was no evidence of recurrence within one year. Our case highlights that nucleic acids obtained from formalin-fixed tissue could be a feasible identification method, especially in those whose culture results are unavailable.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1ß is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.

Pentraxin 3 mediates early inflammatory response and EMT process in human tubule epithelial cells induced by PM2.5.

Pentraxin 3 (PTX3) is a multifunctional molecule that mainly expressed in response to proinflammatory stimuli under physiological and pathological conditions. It is produced in tubule epithelial cells that is involved in the innate immune response and inflammatory reactions in the kidney. However, its role in fine particulate matter (PM2.5)-induced renal injury associated with inflammation remains to be investigated. As a result of PM2.5 exposure, the levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α levels were increased in HK-2 cells. Notably, the mesenchymal phenotypes with migratory abilities of HK-2 cells were found following PM2.5 exposure. The elevated expressions of PTX3 mRNA and protein in response to PM2.5 were tested by RT-PCR and Western blotting respectively. Further determinate the role of PTX3 by siRNA showed lack of PTX3 could increase IL-6 production and promote epithelial-mesenchymal transition (EMT) process, as evidenced by decreased expressions of E-cadherin, and increased expressions of N-cadherin and α-SMA in HK-2 cells following PM2.5 exposure. Our study indicates that PTX3 mediates early inflammatory response and EMT in PM2.5-exposed HK-2 cells, suggesting a counter-regulatory role of PTX3 in the early course of tubule cell injury induced by PM2.5.

Recurrent microangiopathic hemolysis after recovery from complement-mediated hemolytic uremia syndrome during chemotherapy for a CFH-mutated patient with T-lymphoblastic lymphoma.

Complement-mediated hemolytic uremic syndrome (CM-HUS) following chemotherapy for pediatric acute lymphoid neoplasms has rarely been reported. We report the case of an 8-year-old boy with T-lymphoblastic lymphoma (T-LBL) who developed CM-HUS with complement factor H (CFH) mutations (S1191L, V1197A) during induction therapy. Safe administration of chemotherapy after CM-HUS recovery was challenging. By closely monitoring hemolytic and renal parameters during the 2-year treatment period, we observed four episodes of microangiopathic hemolytic anemia (MAHA) with hypocomplementemia and low haptoglobin but no renal dysfunction or thrombocytopenia. Here, we describe the MAHA and CM-HUS episodes in the hopes of elucidating the complex pathophysiology of disorders associated with CFH mutation.

Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits.

TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.

Clinical characteristics and treatment outcomes of SARS-CoV-2 delta variant outbreak, Pingtung, Taiwan, June 2021.

BACKGROUND: An outbreak of SARS-CoV-2 Delta variant infection occurred in Pingtung, Taiwan, in June 2021. In this study, we aimed to elucidate the clinical characteristics of the Delta-variant SARS-CoV-2 infection and the treatment outcome of antiviral agents in patients from Pingtung County in Southern Taiwan. METHODS: A total of 11 patients with Delta-variant COVID-19 were consecutively admitted to a governmental hospital in June 2021. Baseline characteristics and treatment outcome were evaluated. RESULTS: All patients were symptomatic. The most common symptoms were cough (72.7%), followed by fever (54.5%), headache (18.2%) and dysosmia/dysgeusia (18.2%). Two patients developed pneumonia without mechanical ventilation requirement. Compared to patients without pneumonia, those with pneumonia had higher aspartate aminotransferase (AST) (21.0 vs. 126.0 IU/L, P = 0.03) and lactate dehydrogenase (LDH) (143.1 vs. 409.0 IU/mL, P = 0.03), and ferritin (0.2 vs. 2.0 mg/L, P = 0.046) levels. Pneumonia improved after 2-week treatment, and no mortality occurred after 30 days of diagnosis. The median duration of viral shedding duration of viral shedding was 16.5 days (range 11-42 days) (defined by time to repeated negative real-time reverse transcriptase polymerase chain reaction (RT-PCR) or a cycle threshold (CT) value ≥ 30). CONCLUSION: We demonstrated the clinical characteristics of Delta-variant COVID-19 and treatment outcome of antiviral agents. The risk factors attributed to pneumonia were higher serum AST, ferritin, and LDH levels.

Risk factors and predictive markers for early and late-onset neonatal bacteremic sepsis in preterm and term infants.

BACKGROUND: The early detection and prediction of bacteremic sepsis in preterm and term neonates remains a challenging task because of their nonspecific clinical presentations. We aimed to investigate the risk factors associated with bacteremia and find the cutoff values of predictive markers to achieve accurate diagnosis of neonatal bacteremic sepsis. METHODS: Not-doing-well preterm and term neonates with suspected sepsis were retrospectively enrolled between January 2015 and December 2017 in Taipei Veterans General Hospital. Blood culture, hemogram, serum procalcitonin (PCT), and C-reactive protein (CRP) were drawn at the onset of clinical signs and symptoms. All cases were divided to either early-onset or late-onset groups according to postpartum age. Nonparametric statistic, logistic regression, and receiver operating characteristic analysis were performed to evaluate the risk factors and cutoff values for predicting bacteremia. RESULTS: A total of 169 suspected sepsis episodes were analyzed, 68.0% of which had cardiopulmonary dysfunction and 19.5% had perinatal stress. The early-onset group had 123 (72.8%) patients, 4 of which had bacteremia and 119 had nonbacteremia conditions. The late-onset group had 46 (27.2%) patients, 8 of which had bacteremia and 38 had nonbacteremia conditions. Gestational age, birth body weight, Apgar score at 5 minutes, serum PCT, CRP, and platelet (PLT) count in the early-onset group and white blood cell (WBC) count in the late-onset group were substantially different between the patients with bacteremia and nonbacteremia conditions. PCT greater than 27 µg/L (adjusted odd ratio [aOR], 21.6; 95% CI, 1.1-435.1) and thrombocytopenia less than 100 × 109/L (aOR, 38.6; 95% CI, 1.4-1030.3) were predictive markers for bacteremia in the early-onset group. CONCLUSION: Early- and late-onset neonatal sepsis had different risk factors and predictive markers of bacteremia. PCT and PLT count in the early-onset group and WBC count in the late-onset group were accurate diagnostic serum markers for neonatal bacteremic sepsis.

PM2.5 Induces Early Epithelial Mesenchymal Transition in Human Proximal Tubular Epithelial Cells through Activation of IL-6/STAT3 Pathway.

Particulate matter exposure has been known as a potential risk for the global burden of disease, such as respiratory and cardiovascular diseases. Accumulating evidence suggests that PM2.5 (particulate matter with a diameter less than 2.5 µm) is associated with increased risk of kidney disease, but the mechanisms underlying the renal injury caused by PM2.5 remain to be elucidated. This study investigated the effects of PM2.5 on human proximal tubular epithelial (HK-2) cells by monolayer and 3D spheroid cultures and explored the potential mechanisms. The typical morphology of HK-2 cells showed epithelial-mesenchymal transition (EMT), resulting in reduced adhesion and enhanced migration after PM2.5 exposure, and was accompanied by decreased E-cadherin expression and increased vimentin and α-SMA expressions. Exposure to PM2.5 in the HK-2 cells could lead to an increase in interleukin-6 (IL-6) levels and cause the activation of signal transducer and activator of transcription 3 (STAT3), which is involved in EMT features of HK-2 cells. Furthermore, blocking IL-6/STAT3 signaling by an IL-6 neutralizing antibody or STAT3 inhibitor was sufficient to reverse PM2.5-induced EMT characteristics of the HK-2 cells. Our study suggests that PM2.5 could induce early renal tubule cell injury, contributing to EMT change, and the induction of IL-6/STAT3 pathway may play an important role in this process.

The investigation of thermal behaviour and physical properties of several types of contemporary gutta-percha points.

AIM: To analyse the contents and thermal behaviour of several brands of contemporary gutta-percha points due to the variable nature of the components of gutta-percha, and the impact they can have on the physical properties of this unique material during canal filling. METHODOLOGY: Six brands of gutta-percha were investigated: Conform Fit TM Gutta-Percha Points for ProTaper Gold® (PTG) (Dentsply Sirona), ProTaper® Universal Gutta-Percha Points (PTU) (Dentsply Sirona), Autofit TM Feathered Tip Gutta Percha (Kerr), Mtwo® Gutta-Percha (VDW), Gutta Percha Root Canal Points (GC, GC Corporation) and Gutta-Percha Points ISO Color-Coded (ISO; Dentsply Sirona). The organic and inorganic fractions of gutta-percha points were separated by quantitative chemical analysis. Thermal conductivity was detected using a laser flash method. In addition, the thermal behaviour of gutta-percha in response to temperature variations was analysed using differential scanning calorimetry (DSC). Kruskal-Wallis and Dunn tests were applied for comparisons amongst groups for chemical compositions and temperature obtained from DSC. The associations between compositions and thermal conductivity were determined using simple linear regression. A p value <.05 was considered to be statistically significant. RESULTS: There were significant difference in the inorganic fractions of the gutta-percha points in percentage by weight amongst the groups (p < .05). PTG had the lowest thermal conductivity (0.42 W/m K). A positive correlation was observed between the percentage of inorganic fraction and thermal conductivity (r = 0.95). The initial phase changing temperature and peak temperature measured by DSC were significantly different when the ß-form transformed to α-form (p < .05), whereas no significant difference was found during the α-form to the amorphous-phase transition (p > .05). CONCLUSIONS: Chemical compositions and initial phase changing temperature by DSC varied according to the various brands of gutta-percha points. Conform Fit TM gutta-percha had the lowest percentage of inorganic fraction and thermal conductivity amongst these six brands of gutta-percha. Thermal conductivity had the strongest positive correlation with the percentage of inorganic components and zinc, whilst there was a negative correlation to the amount (ratio) of gutta-percha.

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain.

BACKGROUND: Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. METHODS: We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. RESULTS: We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. CONCLUSIONS: Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.

2,3,5,4'-tetrahydroxystilbene-2-O-b-D-glucoside triggers the pluripotent-like possibility of dental pulp stem cells by activating the JAK2/STAT3 axis: Preliminary observations.

Abstract. BACKGROUND/PURPOSE: Although 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG) reportedly has anti-inflammatory properties, its role in inducing the dedifferentiation of human dental pulp stem cells (DPSC) into pluripotent-like stem cells remains to be determined. The purpose of this study is to evaluate the effects of THSG on the pluripotent-like possibility and mechanism of DPSC. MATERIALS AND METHODS: DPSCs were treated with THSG, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTS) assay. Real-time polymerase chain reaction was used to analyze the mRNA expression levels of pluripotency-associated genes and oncogenes and to detect telomerase activity in the cells. Embryoid body formation assay was conducted, and pluripotency-related proteins were identified using Western blotting. Data were analyzed using one-way analysis of variance. RESULTS: Cell viability, telomerase activity, and embryoid body formation were enhanced in THSG-treated DPSCs. The mRNA expression levels of pluripotent-like genes (including Nanog homeobox [NANOG], SRY-box 2 [SOX2], and POU class 5 homeobox 1 [POU5F1/OCT4]) significantly increased after THSG treatment. The expression levels of pluripotency-related genes (Janus kinase-signal transducer 2 [JAK2] and signal transducer and activator of transcription 3 [STAT3]) increased, whereas those of oncogenes (Ras, SRC, HER2, and C-sis) decreased. Furthermore, the expression levels of the phosphorylated JAK2 and STAT3 proteins significantly increased after THSG treatment. CONCLUSION: THSG treatment may enhance the pluripotent-like possibility of DPSC through the JAK2/STAT3 axis. Hence, it may be used as an alternative cell-based therapeutic strategy in regenerative dentistry.

New Era of Immunotherapy in Pediatric Brain Tumors: Chimeric Antigen Receptor T-Cell Therapy.

Immunotherapy, including chimeric antigen receptor (CAR) T-cell therapy, immune checkpoint inhibitors, cancer vaccines, and dendritic cell therapy, has been incorporated as a fifth modality of modern cancer care, along with surgery, radiation, chemotherapy, and target therapy. Among them, CAR T-cell therapy emerges as one of the most promising treatments. In 2017, the first two CAR T-cell drugs, tisagenlecleucel and axicabtagene ciloleucel for B-cell acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL), respectively, were approved by the Food and Drug Administration (FDA). In addition to the successful applications to hematological malignancies, CAR T-cell therapy has been investigated to potentially treat solid tumors, including pediatric brain tumor, which serves as the leading cause of cancer-associated death for children and adolescents. However, the employment of CAR T-cell therapy in pediatric brain tumors still faces multiple challenges, such as CAR T-cell transportation and expansion through the blood-brain barrier, and identification of the specific target antigen on the tumor surface and immunosuppressive tumor microenvironment. Nevertheless, encouraging outcomes in both clinical and preclinical trials are coming to light. In this article, we outline the current propitious progress and discuss the obstacles needed to be overcome in order to unveil a new era of treatment in pediatric brain tumors.

Gene Modified CAR-T Cellular Therapy for Hematologic Malignancies.

With advances in the understanding of characteristics of molecules, specific antigens on the surface of hematological malignant cells were identified and multiple therapies targeting these antigens as neoplasm treatments were developed. Among them, chimeric antigen receptor (CAR) T-cell therapy, which got United States Food and Drug Administration (FDA) approval for relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) as well as for recurrent acute lymphoblastic leukemia (ALL) within the past five years, and for r/r mantle cell lymphoma (MCL) this year, represents one of the most rapidly evolving immunotherapies. Nevertheless, its applicability to other hematological malignancies, as well as its efficacy and persistence are fraught with clinical challenges. Currently, more than one thousand clinical trials in CAR T-cell therapy are ongoing and its development is changing rapidly. This review introduces the current status of CAR T-cell therapy in terms of the basic molecular aspects of CAR T-cell therapy, its application in hematological malignancies, adverse reactions during clinical use, remaining challenges, and future utilization.

TRIM28 Regulates Dlk1 Expression in Adipogenesis.

The tripartite motif-containing protein 28 (TRIM28) is a transcription corepressor, interacting with histone deacetylase and methyltransferase complexes. TRIM28 is a crucial regulator in development and differentiation. We would like to investigate its function and regulation in adipogenesis. Knockdown of Trim28 by transducing lentivirus-carrying shRNAs impairs the differentiation of 3T3-L1 preadipocytes, demonstrated by morphological observation and gene expression analysis. To understand the molecular mechanism of Trim28-mediated adipogenesis, the RNA-seq was performed to find out the possible Trim28-regulated genes. Dlk1 (delta-like homolog 1) was increased in Trim28 knockdown 3T3-L1 cells both untreated and induced to differentiation. Dlk1 is an imprinted gene and known as an inhibitor of adipogenesis. Further knockdown of Dlk1 in Trim28 knockdown 3T3-L1 would rescue cell differentiation. The epigenetic analysis showed that DNA methylation of Dlk1 promoter and differentially methylated regions (DMRs) was not altered significantly in Trim28 knockdown cells. However, compared to control cells, the histone methylation on the Dlk1 promoter was increased at H3K4 and decreased at H3K27 in Trim28 knockdown cells. Finally, we found Trim28 might be recruited by transcription factor E2f1 to regulate Dlk1 expression. The results imply Trim28-Dlk1 axis is critical for adipogenesis.