Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Heavy metal exposure linked to metabolic syndrome in Korean male firefighters: FRESH cohort cross-sectional analysis.

This study aimed to identify the relationship between blood lead and Cadmium (Cd) concentrations and metabolic syndromes (MetS), including its components (central obesity, hypertriglyceridemia, low high-density lioioritein, hypertension, and hyperglycemia) among Korean firefighters. A total of 965 firefighters of the Enhancement of Safety and Health cohort were analyzed in this study. MetS was defined according to the 2005 revised National Cholesterol Education Program-Adult Treatment Panel III criteria and the Korean Society for the Study of Obesity criteria for waist circumference. The collected data were analyzed using a logistic regression model. Of the 965 participants, 190 (19.7%) had MetS. After adjusting for age, body mass index, smoking, drinking, exercise, shift duty, and main duty position, the Cd level was significantly associated with an increased risk of MetS in the Korean firefighter population (odds ratio [OR] = 1.62, 95% confidence interval [CI] 1.07, 2.46). This association was significant among non-smokers and ex-smokers (OR = 1.58, 95% CI 1.03, 2.43), non-drinkers and ex-drinkers (OR = 1.77, 95% CI 1.06, 2.94), firefighters aged 40 year or older (OR = 1.77, 95% CI 1.10, 2.86), and office administrators (OR = 3.85, 95% CI 1.42, 10.39). This outcome suggests that exposure to Cd is likely to increase risk of MetS among firefighters.

Consideration of the importance of measuring thermal discomfort in biomedical research.

Core temperature stability is the result of a dynamically regulated balance of heat loss and gain, which is not reflected by a simple thermometer reading. One way in which these changes manifest is in perceived thermal comfort, 'feeling too cold' or 'feeling too hot', which can activate stress pathways. Unfortunately, there is surprisingly little preclinical research that tracks changes in perceived thermal comfort in response to either disease progression or various treatments. Without measuring this endpoint, there may be missed opportunities to evaluate disease and therapy outcomes in murine models of human disease. Here, we discuss the possibility that changes in thermal comfort in mice could be a useful and physiologically relevant measure of energy trade-offs required under various physiological or pathological conditions.

The IgH Eµ-MAR regions promote UNG-dependent error-prone repair to optimize somatic hypermutation.

Intoduction: Two scaffold/matrix attachment regions (5'- and 3'-MARsEµ ) flank the intronic core enhancer (cEµ) within the immunoglobulin heavy chain locus (IgH). Besides their conservation in mice and humans, the physiological role of MARsEµ is still unclear and their involvement in somatic hypermutation (SHM) has never been deeply evaluated. Methods: Our study analyzed SHM and its transcriptional control in a mouse model devoid of MARsEµ , further combined to relevant models deficient for base excision repair and mismatch repair. Results: We observed an inverted substitution pattern in of MARsEµ -deficient animals: SHM being decreased upstream from cEµ and increased downstream of it. Strikingly, the SHM defect induced by MARsEµ -deletion was accompanied by an increase of sense transcription of the IgH V region, excluding a direct transcription-coupled effect. Interestingly, by breeding to DNA repair-deficient backgrounds, we showed that the SHM defect, observed upstream from cEµ in this model, was not due to a decrease in AID deamination but rather the consequence of a defect in base excision repair-associated unfaithful repair process. Discussion: Our study pointed out an unexpected "fence" function of MARsEµ regions in limiting the error-prone repair machinery to the variable region of Ig gene loci.

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation.

Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.

Isolation of human and mouse myeloid-derived suppressor cells for metabolic analysis.

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here, we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation, oxidative phosphorylation, and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs, peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol, please refer to Mohammadpour et al. (2021).

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

The effect of polycyclic aromatic hydrocarbons on changes in the brain structure of firefighters: An analysis using data from the Firefighters Research on Enhancement of Safety & Health study.

Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of organic matter, and firefighters are highly exposed to these toxic compounds at fire sites. Exposure to PAHs can cause cognitive decline and neurodegeneration; however, to date, few studies have examined the potential effects of PAH exposure on structural changes in the brain. We aimed to investigate the association between the four types of PAH metabolites and the corresponding changes in neuroimaging markers based on smoking status and hypertension in male firefighters. For this, we utilized the 2-year follow-up data of 301 Korean male firefighters aged over 40 years. The concentrations of four PAH metabolites in urine were measured. Subcortical volume and cortical thickness were estimated using 3 T magnetic resonance imaging of the brain. A generalized linear model was used to investigate the effects of PAHs on changes in the subcortical volume and cortical thickness. We found an association between 1-hydroxyphenathrene (1-OHPHE) and 2-hydroxyfluorene (2-OHF) and changes in several brain regions in all the study participants. Individuals who had never smoked showed significantly thinner frontal (p < 0.001), parietal (p < 0.001), temporal (p < 0.001), and cingulate lobes (p < 0.001) with 1% increase each in the urinary concentration of 1-OHPHE. Hypertension interacted with the concentration of 1-OHPHE to reduce the volume of gray matter and cause cortical thinning in the frontal, parietal, and temporal lobes. Exposure to PAHs may reduce cortical thickness and subcortical volume, which are definitive markers of neurodegeneration. Notably, hypertension can accelerate the degenerative effects of PAHs.

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches.

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which analyze mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

The uncharacterized SANT and BTB domain-containing protein SANBR inhibits class switch recombination.

Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD (KIAA1841, NM_027860), which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein.

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.

Evaluation of Submucosal or Lymphovascular Invasion Detection Rates in Early Gastric Cancer Based on Pathology Section Interval.

PURPOSE: The guidelines for pathological evaluation of early gastric cancer (EGC) recommend wider section intervals for surgical specimens (5-7 mm) than those for endoscopically resected specimens (2-3 mm). Studies in surgically resected EGC specimens showed not negligible lymph node metastasis risks in EGCs meeting the expanded criteria for endoscopic submucosal dissection (ESD). MATERIALS AND METHODS: This retrospective study included 401 EGC lesions with an endoscopic size of ≤ 30 mm detected in 386 patients. Pathological specimens obtained by ESD or surgery were cut into 2-mm section intervals for reference. Submucosal or lymphovascular invasion (LVI) was evaluated arbitrarily in 4- or 6-mm section intervals. McNemar's tests compared the differences between submucosal and LVI. RESULTS: Submucosal invasion was detected in 29.2% (117/401) and LVI in 9.5% (38/401) at 2-mm interval. The submucosal invasion detection rates in 4-mm intervals decreased to 88.0% or 90.6% (both P<0.001), while the LVI detection rates decreased to 86.8% or 57.9% (P=0.025 and P<0.001, respectively). In 6-mm intervals, the submucosal and LVI detection rates decreased further to 72.7-80.3% (P<0.001 for all three sets) and 55.3-63.2% (P<0.001 for all three sets), respectively. Among 150 out-of-indication cases at 2-mm interval, 4-10 (2.7%-6.7%) at 4-mm intervals, and 10-17 (6.7%-11.3%) at 6-mm intervals were misclassified as lesions meeting the curative resection criteria due to the underestimation of submucosal or LVI. CONCLUSIONS: After ESD, the 2-mm wide section interval was suitable for the pathological evaluation of focal submucosal or LVI. Thus, if an EGC lesion meets the expanded criteria for the ESD specimen pathological evaluation, it could be safely followed up.

Cohort Profile: Firefighter Research on the Enhancement of Safety and Health (FRESH), a Prospective Cohort Study on Korean Firefighters.

Firefighters have a high risk of developing cardiovascular and mental disorders due to their physical and chemical environments. However, in Korea, few studies have been conducted on environmental risk of firefighters. The Firefighter Research on the Enhancement of Safety and Health (FRESH) study aimed to discover the risk factors for cardiovascular disease and mental disorders among firefighters. Former and current firefighters were recruited from three university hospitals. A total of 1022 participants completed baseline health examinations from 2016 to 2017. All participants were scheduled for follow-ups every 2 years. Baseline health survey, laboratory testing of blood and urine samples, blood heavy metal concentration, urine polycyclic aromatic hydrocarbons (PAHs) metabolites, stress-related hormone test, natural killer cell activity, as well as physical and mental health examinations that focused on cardiovascular and mental disorders, were conducted. In addition, 3 Tesla (3T) brain magnetic resonance imaging (MRI) and neuropsychological tests were also performed to investigate structural and functional changes in the brains of 352 firefighters aged >40 years or new hires with less than 1 year of service.

AID Phosphorylation Regulates Mismatch Repair-Dependent Class Switch Recombination and Affinity Maturation.

Activation-induced cytidine deaminase (AID) generates U:G mismatches in Ig genes that can be converted into untemplated mutations during somatic hypermutation or DNA double-strand breaks during class switch recombination (CSR). Null mutations in UNG and MSH2 demonstrate the complementary roles of the base excision repair (BER) and mismatch repair pathways, respectively, in CSR. Phosphorylation of AID at serine 38 was previously hypothesized to regulate BER during CSR, as the AID phosphorylation mutant, AID(S38A), cannot interact with APE1, a BER protein. Consistent with these findings, we observe a complete block in CSR in AIDS38A/S38AMSH2-/- mouse B cells that correlates with an impaired mutation frequency at 5'Sµ. Similarly, somatic hypermutation is almost negligible at the JH4 intron in AIDS38A/S38AMSH2-/- mouse B cells, and, consistent with this, NP-specific affinity maturation in AIDS38A/S38AMSH2-/- mice is not significantly elevated in response to NP-CGG immunization. Surprisingly, AIDS38A/S38AUNG-/- mouse B cells also cannot complete CSR or affinity maturation despite accumulating significant mutations in 5'Sµ as well as the JH4 intron. These data identify a novel role for phosphorylation of AID at serine 38 in mismatch repair-dependent CSR and affinity maturation.

Site-specific intestinal DMT1 silencing to mitigate iron absorption using pH-sensitive multi-compartmental nanoparticulate oral delivery system.

Iron is a nutrient metal, but excess iron promotes tissue damage. Since iron chelation therapies exhibit multiple off-target toxicities, there is a substantial demand for more specific approaches to decrease iron burden in iron overload. While the divalent metal transporter 1 (DMT1) plays a well-established role in the absorption of dietary iron, up-regulation of intestinal DMT1 is associated with iron overload in both humans and rodents. Hence, we developed a novel pH-sensitive multi-compartmental particulate (MCP) oral delivery system that encapsulates DMT1 siRNA and validated its efficacy in mice. Using the gelatin NPs coated with Eudragit® L100-55, we demonstrated that DMT1 siRNA-loaded MCPs down-regulated DMT1 mRNA levels in the duodenum, which was consistent with decreased intestinal absorption of orally-administered 59Fe. Together, the Eudragit® L100-55-based oral siRNA delivery system could provide an effective strategy to specifically down-regulate duodenal DMT1 and mitigate iron absorption.

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.

Ginsenosides Rg5 and Rk1 Enriched Cultured Wild Ginseng Root Extract Bioconversion of Pediococcus pentosaceus HLJG0702: Effect on Scopolamine-Induced Memory Dysfunction in Mice.

Wild ginseng is known to contain additional physiologically and pharmacologically active substances than common ginseng. The utilization of this herb can be maximized by altering its composition via tissue culture generating adventitious roots. We enriched the content of specific ginsenosides and investigated their role in ameliorating memory impairment. Cultured wild ginseng root was subjected to extraction, steaming, and fermentation using Pediococcus pentosaceus HLJG0702 to enhance the levels of ginsenosides Rg5 /Rk1. The analysis of product, HLJG0701, confirmed target ginsenosides. We analyzed the inhibitory effect of ginsenoside Rg5/Rk1, HLJG0701 and the raw material on acetylcholinesterase. Further, we performed Morris water maze, Y-maze, and passive avoidance tasks with mice exhibiting memory deficit induced by scopolamine, and we analyzed the concentrations of acetylcholinesterase and acetylcholine in their brains. Studies showed that the levels of ginsenosides Rg5 /Rk1, not found in the raw material, were enhanced in HLJG0701. Ginsenosides and HLJG0701 significantly inhibited acetylcholinesterase unlike the raw material. In all behavioral tasks, HLJG0701 showed memory improvement. It reduced acetylcholinesterase, whereas, it preserved acetylcholine in brain. In conclusion, cultured wild ginseng root extract fermented by P. pentosaceus HLJG0702 contains the distinctive ginsenosides Rg5/Rk1, which may ameliorate memory impairment via inhibition of acetylcholinesterase resulting in increased acetylcholine levels in the brain.

NME proteins regulate class switch recombination.

Class switch recombination (CSR) in B cells involves deletion-recombination at switch (S) region DNA and is important for the diversification of antibody isotypes during an immune response. Here, we identify two NME [NM23/NDPK (nucleoside diphosphate kinase)] isoforms, NME1 and NME2, as novel players in this process. Knockdown of NME2 leads to decreased CSR, while knockdown of the highly homologous NME1 results in increased CSR. Interestingly, these NME proteins also display differential occupancy at S regions during CSR despite their homology; NME1 binds to S regions prior to stimulation, while NME2 binds to S regions only after stimulation. To the best of our knowledge, this represents the first report of a role for these proteins in the regulation of CSR.

Short-term Effect of Ambient Air Pollution on Emergency Department Visits for Diabetic Coma in Seoul, Korea.

OBJECTIVES: A positive association between air pollution and both the incidence and prevalence of diabetes mellitus (DM) has been reported in some epidemiologic and animal studies, but little research has evaluated the relationship between air pollution and diabetic coma. Diabetic coma is an acute complication of DM caused by diabetic ketoacidosis or hyperosmolar hyperglycemic state, which is characterized by extreme hyperglycemia accompanied by coma. We conducted a time-series study with a generalized additive model using a distributed-lag non-linear model to assess the association between ambient air pollution (particulate matter less than 10 µm in aerodynamic diameter, nitrogen dioxide [NO2], sulfur dioxide, carbon monoxide, and ozone) and emergency department (ED) visits for DM with coma in Seoul, Korea from 2005 to 2009. METHODS: The ED data and medical records from the 3 years previous to each diabetic coma event were obtained from the Health Insurance Review and Assessment Service to examine the relationship with air pollutants. RESULTS: Overall, the adjusted relative risks (RRs) for an interquartile range (IQR) increment of NO2 was statistically significant at lag 1 (RR, 1.125; 95% confidence interval [CI], 1.039 to 1.219) in a single-lag model and both lag 0-1 (RR, 1.120; 95% CI, 1.028 to 1.219) and lag 0-3 (RR, 1.092; 95% CI, 1.005 to 1.186) in a cumulative-lag model. In a subgroup analysis, significant positive RRs were found for females for per-IQR increments of NO2 at cumulative lag 0-3 (RR, 1.149; 95% CI, 1.022 to 1.291). CONCLUSIONS: The results of our study suggest that ambient air pollution, specifically NO2, is associated with ED visits for diabetic coma.

Generating and repairing genetically programmed DNA breaks during immunoglobulin class switch recombination.

Adaptive immune responses require the generation of a diverse repertoire of immunoglobulins (Igs) that can recognize and neutralize a seemingly infinite number of antigens. V(D)J recombination creates the primary Ig repertoire, which subsequently is modified by somatic hypermutation (SHM) and class switch recombination (CSR). SHM promotes Ig affinity maturation whereas CSR alters the effector function of the Ig. Both SHM and CSR require activation-induced cytidine deaminase (AID) to produce dU:dG mismatches in the Ig locus that are transformed into untemplated mutations in variable coding segments during SHM or DNA double-strand breaks (DSBs) in switch regions during CSR. Within the Ig locus, DNA repair pathways are diverted from their canonical role in maintaining genomic integrity to permit AID-directed mutation and deletion of gene coding segments. Recently identified proteins, genes, and regulatory networks have provided new insights into the temporally and spatially coordinated molecular interactions that control the formation and repair of DSBs within the Ig locus. Unravelling the genetic program that allows B cells to selectively alter the Ig coding regions while protecting non-Ig genes from DNA damage advances our understanding of the molecular processes that maintain genomic integrity as well as humoral immunity.