Ketolysis drives CD8+ T cell effector function through effects on histone acetylation.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.

LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.

Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

Rare case of epithelioid hemangioendothelioma in the right innominate vein-An innovative diagnostic approach.

BACKGROUND AND AIMS: Epithelioid hemangioendothelioma is a rare malignant vascular tumor with limited literature. AIMS: We reported an innovative endovascular biopsy of the right innominate vein tumor. MATERIALS AND METHODS: Endovascular suction thrombectomy was performed with multipurpose catheter and constant negative pressure under fluoroscopic guidance. RESULTS: Epithelioid hemangioendothelioma was diagnosed preoperatively and a complete margin-free tumor resection with patch repair of the right innominate vein was achieved via sternotomy. DISCUSSION: Preoperatively diagnosis is usually not available due to lesions' location. Identifying malignant vascular tumors becomes valuable to guide the surgical treatment. CONCLUSIONS: In this case report, this innovative endovascular approach led to a rare preoperative diagnosis of EHE and subsequent margin-free resection.

Integrin-Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor-Specific Delivery Achieve MYCN Silencing with Improved Survival.

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor-targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor-mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non-targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide-dependent indicating integrin-mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN-amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor-targeting, with minimal clearance by the liver and so enable delivery of tumor-targeted siRNA therapeutics.

Serologic Responses in Healthy Adult with SARS-CoV-2 Reinfection, Hong Kong, August 2020.

In March 2020, mild signs and symptoms of coronavirus disease developed in a healthy 33-year-old man in Hong Kong. His first infection did not produce virus neutralizing antibodies. In August, he had asymptomatic reinfection, suggesting that persons without a robust neutralizing antibody response might be at risk for reinfection.

Hepatitis B infection acquired after haematopioetic stem cell transplant through horizontal mode.

A 22 month old child with thalassaemia major received unrelated umbilical cord blood transplantation. She was born to mother of HBsAg carrier and received hepatitis B immunoglobulin at birth and hepatitis B vaccination. She was HBsAg negative and anti-HBs positive before transplantation. After transplant, she was taken care by her mother and found to be HBsAg positive at 2 year post-transplant. Genotyping of the mother's and child's HBV status confirmed to be of same genotype and demonstrated horizontal transmission in post-transplant setting. Passive immunization of HBV may be considered in early post-transplant phase to prevent horizontal transmission of HBV, and antiviral treatment of the carer should be offered to prevent transmission of infection to immunocompromised child.

Homeobox and Polycomb target gene methylation in human solid tumors.

DNA methylation is an epigenetic mark that plays an important role in defining cancer phenotypes, with global hypomethylation and focal hypermethylation at CpG islands observed in tumors. These methylation marks can also be used to define tumor types and provide an avenue for biomarker identification. The homeobox gene class is one that has potential for this use, as well as other genes that are Polycomb Repressive Complex 2 targets. To begin to unravel this relationship, we performed a pan-cancer DNA methylation analysis using sixteen Illumina HM450k array datasets from TCGA, delving into cancer-specific qualities and commonalities between tumor types with a focus on homeobox genes. Our comparisons of tumor to normal samples suggest that homeobox genes commonly harbor significant hypermethylated differentially methylated regions. We identified two homeobox genes, HOXA3 and HOXD10, that are hypermethylated in all 16 cancer types. Furthermore, we identified several potential homeobox gene biomarkers from our analysis that are uniquely methylated in only one tumor type and that could be used as screening tools in the future. Overall, our study demonstrates unique patterns of DNA methylation in multiple tumor types and expands on the interplay between the homeobox gene class and oncogenesis.

Thymus antibody-secreting cells possess an interferon gene signature and are preferentially expanded in young female mice.

Antibody-secreting cells (ASCs) are key contributors to humoral immunity through immunoglobulin production and the potential to be long-lived. ASC persistence has been recognized in the autoimmune thymus (THY); however, only recently has this population been appreciated in healthy THY tissue. We showed that the young female THY was skewed toward higher production of ASCs relative to males. However, these differences disappeared with age. In both sexes, THY ASCs included Ki-67+ plasmablasts which required CD154(CD40L) signals for their propagation. Single cell RNA-sequencing revealed that THY ASCs were enriched for an interferon responsive transcriptional signature relative to those from bone marrow and spleen. Flow cytometry confirmed that THY ASCs had increased levels of Toll-like receptor 7 as well as CD69 and major histocompatibility complex class II. Overall, we identified fundamental aspects of THY ASC biology which may be leveraged for future in depth studies of this population in both health and disease.

PPARγ and C/EBPα response to acute cold stress in brown adipose tissue.

Brown adipose tissue (BAT) has the ability to burn calories as heat. Utilizing BAT thermogenesis is thus an attractive way to combat obesity. However, the transcriptional network resulting in the lipid synthesis to oxidation shift during thermogenesis is not completely understood. Here, we report the regulation of two master regulators of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), during acute cold stress in BAT. We found PPARγ dissociates from DNA in a fifth of its binding sites and these include Cebpa enhancers, leading to decreased C/EBPα expression. This dissociation requires PPARγ binding to activating ligands and is thus modulated by diet. Meanwhile, PPARα also detaches from DNA, and co-activator PGC1α associates with ERRα as part of a transcriptional network regulating lipid metabolism. Subsequent global replacement of C/EBPα by C/EBPß and its associated transcriptional machinery is required for upregulation of structural lipid synthesis despite general upregulation of fatty acid oxidation.

Single-embryo RNA sequencing for continuous and sex-specific gene expression analysis on Drosophila.

Exploring early embryonic gene expression is challenging due to the rate of development and the limited material available. Here, we present a protocol for ordering Drosophila embryos along a developmental pseudo-time trajectory and determining the sex of the embryos using RNA-seq data. We describe steps for sample collection, RNA isolation, RNA-seq, and RNA-seq data processing. We then detail the establishment of a continuous transcriptome dataset for assessing gene expression throughout early development and in a sex-specific manner. For complete details on the use and execution of this protocol, please refer to Pérez-Mojica et al.1.

Optimal HSF1 activation in response to acute cold stress in BAT requires nuclear TXNIP.

While TXNIP (thioredoxin interacting protein) in the plasma membrane and vesicular location is known to negatively regulate cellular glucose uptake by facilitating glucose transporter endocytosis, the function of TXNIP in the nucleus is far less understood. Herein, we sought to determine the function of nuclear TXNIP in vivo, using a new HA-tagged TXNIP knock-in mouse model. We observed that TXNIP can be found in the nucleus of a variety of cells from different tissues including hepatocytes (liver), enterocytes (small intestine), exocrine cells (pancreas), and brown adipocytes (BAT). Further investigations into the role of nuclear TXNIP in BAT revealed that cold stress rapidly and transiently activated HSF1 (heat shock factor 1). HSF1 interaction with TXNIP during its activation is required for optimal HSF1 directed cold shock response in BAT.

Differential effects of sugar and fat on adipose tissue inflammation.

Obese individuals experience low grade inflammation initiated within their adipose tissue. However, the early events that lead to the release of these inflammatory factors from adipose tissue are poorly characterized. To separate glucose effects from lipid effects on adipose tissue, we used an adipose-specific TXNIP knockout model where excess basal glucose influx into adipocytes led to modest increase in adiposity without using high fat diet. We found an uncoupling of two events that are generally presumed to be coregulated: (1) an increase of adipose tissue macrophage (ATM) number; and (2) pro-inflammatory activation of ATMs. These two events are associated with different triggering signals: elevated free fatty acids output and extracellular matrix remodeling with increased ATM number, whereas decreased adiponectin level with activated ATM. This separation reflects non-overlapping pathways regulated by glucose and lipids in adipocytes, and neither group alone is sufficient to elicit the full inflammatory response in adipose tissue.

Continuous transcriptome analysis reveals novel patterns of early gene expression in Drosophila embryos.

The transformative events during early organismal development lay the foundation for body formation and long-term phenotype. The rapid progression of events and the limited material available present major barriers to studying these earliest stages of development. Herein, we report an operationally simple RNA sequencing approach for high-resolution, time-sensitive transcriptome analysis in early (≤3 h) Drosophila embryos. This method does not require embryo staging but relies on single-embryo RNA sequencing and transcriptome ordering along a developmental trajectory (pseudo-time). The resulting high-resolution, time-sensitive mRNA expression profiles reveal the exact onset of transcription and degradation for thousands of transcripts. Further, using sex-specific transcription signatures, embryos can be sexed directly, eliminating the need for Y chromosome genotyping and revealing patterns of sex-biased transcription from the beginning of zygotic transcription. Our data provide an unparalleled resolution of gene expression during early development and enhance the current understanding of early transcriptional processes.

Establishment of paternal methylation imprint at the H19/Igf2 imprinting control region.

The insulator model explains the workings of the H19 and Igf2 imprinted domain in the soma, where insulation of the Igf2 promoter from its enhancers occurs by CTCF in the maternally inherited unmethylated chromosome but not the paternally inherited methylated allele. The molecular mechanism that targets paternal methylation imprint establishment to the imprinting control region (ICR) in the male germline is unknown. We tested the function of prospermatogonia-specific broad low-level transcription in this process using mouse genetics. Paternal imprint establishment was abnormal when transcription was stopped at the entry point to the ICR. The germline epimutation persisted into the paternal allele of the soma, resulting in reduced Igf2 in fetal organs and reduced fetal growth, consistent with the insulator model and insulin-like growth factor 2 (IGF2)'s role as fetal growth factor. These results collectively support the role of broad low-level transcription through the H19/Igf2 ICR in the establishment of its paternal methylation imprint in the male germ line, with implications for Silver-Russell syndrome.

Cysteine-rich intestinal protein 1 is a novel surface marker for human myometrial stem/progenitor cells.

Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better markers. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers. CRIP1 expression was found highly upregulated by both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

Deficiency of the Polycomb Protein RYBP and TET Methylcytosine Oxidases Promotes Extensive CpG Island Hypermethylation and Malignant Transformation.

Hypermethylation of CpG islands (CGI) is a common feature of cancer cells and predominantly affects Polycomb-associated genomic regions. Elucidating the underlying mechanisms leading to DNA hypermethylation in human cancer could help identify chemoprevention strategies. Here, we evaluated the role of Polycomb complexes and 5-methylcytosine (5mC) oxidases in protecting CGIs from DNA methylation and observed that four genes coding for components of Polycomb repressive complex 1 (PRC1) are downregulated in tumors. Inactivation of RYBP, a key activator of variant PRC1 complexes, in combination with all three 5mC oxidases (TET proteins) in nontumorigenic bronchial epithelial cells led to widespread hypermethylation of Polycomb-marked CGIs affecting almost 4,000 target genes, which closely resembled the DNA hypermethylation landscape observed in human squamous cell lung tumors. The RYBP- and TET-deficient cells showed methylation-associated aberrant regulation of cancer-relevant pathways, including defects in the Hippo tumor suppressor network. Notably, the quadruple knockout cells acquired a transformed phenotype, including anchorage-independent growth and formation of squamous cell carcinomas in mice. This work provides a mechanism promoting hypermethylation of CGIs and shows that such hypermethylation can lead to cell transformation. The breakdown of a two-pronged protection mechanism can be a route towards genome-wide hypermethylation of CGIs in tumors. SIGNIFICANCE: Dysfunction of the Polycomb component RYBP in combination with loss of 5-methylcytosine oxidases promotes widespread hypermethylation of CpG islands in bronchial cells and induces tumorigenesis, resembling changes seen in human lung tumors.

Cysteine-Rich Intestinal Protein 1 is a Novel Surface Marker for Myometrial Stem/Progenitor Cells.

Myometrial stem/progenitor cells (MyoSPCs) have been proposed as the cells of origin for uterine fibroids, which are benign tumors that develop in the myometrium of most reproductive age women, but the identity of the MyoSPC has not been well established. We previously identified SUSD2 as a possible MyoSPC marker, but the relatively poor enrichment in stem cell characteristics of SUSD2+ over SUSD2- cells compelled us to find better discerning markers for more rigorous downstream analyses. We combined bulk RNA-seq of SUSD2+/- cells with single cell RNA-seq to identify markers capable of further enriching for MyoSPCs. We observed seven distinct cell clusters within the myometrium, with the vascular myocyte cluster most highly enriched for MyoSPC characteristics and markers, including SUSD2. CRIP1 expression was found highly upregulated in both techniques and was used as a marker to sort CRIP1+/PECAM1- cells that were both enriched for colony forming potential and able to differentiate into mesenchymal lineages, suggesting that CRIP1+/PECAM1- cells could be used to better study the etiology of uterine fibroids.

Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory.

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

[Metastatic lung tumor from colon cancer with cavitation: report of a case].

A 55-year-old man underwent rectal amputation for rectal cancer in August 2005. A tiny thin-walled cavity lesion in his left S1+2 was found on computed tomography (CT) of the chest in November 2008. The cavity lesion in the left S1+2 gradually increased in size over 3 months and positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) showed FDG accumulation at the lesion. Videoassisted thoracoscopic (VATS) wedge resection was performed to make a definite diagnosis in March 2009. The pathological findings revealed a metastatic lung tumor from the rectal cancer. It is necessary to consider the possibility of metastatic lung tumors in a case with the cavity lesions especially in patients with a history of colon cancer.

Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome.

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.