Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated. METHODS: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. RESULTS: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. CONCLUSIONS: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

Background: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating the regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is a key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has not yet been elucidated. Methods: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 hours post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. Results: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. Conclusions: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Bacterial Decolonization for Prevention of Radiation Dermatitis: A Randomized Clinical Trial.

Importance: Evidence-based approaches for the prevention of acute radiation dermatitis (ARD) are limited, and additional strategies are necessary to optimize care. Objective: To determine the efficacy of bacterial decolonization (BD) to reduce ARD severity compared with standard of care. Design, Setting, and Participants: This phase 2/3 randomized clinical trial was conducted from June 2019 to August 2021 with investigator blinding at an urban academic cancer center and enrolled patients with breast cancer or head and neck cancer receiving radiation therapy (RT) with curative intent. Analysis was performed on January 7, 2022. Interventions: Intranasal mupirocin ointment twice daily and chlorhexidine body cleanser once daily for 5 days prior to RT and repeated for 5 days every 2 weeks through RT. Main Outcomes and Measures: The primary outcome as planned prior to data collection was the development of grade 2 or higher ARD. Based on wide clinical variability of grade 2 ARD, this was refined to grade 2 ARD with moist desquamation (grade 2-MD). Results: Of 123 patients assessed for eligibility via convenience sampling, 3 were excluded, and 40 refused to participate, with 80 patients in our final volunteer sample. Of 77 patients with cancer (75 patients with breast cancer [97.4%] and 2 patients with head and neck cancer [2.6%]) who completed RT, 39 were randomly assigned BC, and 38 were randomly assigned standard of care; the mean (SD) age of the patients was 59.9 (11.9) years, and 75 (97.4%) were female. Most patients were Black (33.7% [n = 26]) or Hispanic (32.5% [n = 25]). Among patients with breast cancer and patients with head and neck cancer (N = 77), none of the 39 patients treated with BD and 9 of the 38 patients (23.7%) treated with standard of care developed ARD grade 2-MD or higher (P = .001). Similar results were observed among the 75 patients with breast cancer (ie, none treated with BD and 8 [21.6%] receiving standard of care developed ARD grade ≥2-MD; P = .002). The mean (SD) ARD grade was significantly lower for patients treated with BD (1.2 [0.7]) compared with patients receiving standard of care (1.6 [0.8]) (P = .02). Of the 39 patients randomly assigned to BD, 27 (69.2%) reported regimen adherence, and only 1 patient (2.5%) experienced an adverse event related to BD (ie, itch). Conclusions and Relevance: The results of this randomized clinical trial suggest that BD is effective for ARD prophylaxis, specifically for patients with breast cancer. Trial Registration: ClinicalTrials.gov Identifier: NCT03883828.

Association of Staphylococcus aureus Colonization With Severity of Acute Radiation Dermatitis in Patients With Breast or Head and Neck Cancer.

Importance: Pathogenesis of acute radiation dermatitis (ARD) is not completely understood. Pro-inflammatory cutaneous bacteria may contribute to cutaneous inflammation after radiation therapy. Objective: To evaluate whether nasal colonization with Staphylococcus aureus (SA) before radiation therapy is associated with ARD severity in patients with breast or head and neck cancer. Design, Setting, and Participants: This prospective cohort study with observers blinded to colonization status was conducted from July 2017 to May 2018 at an urban academic cancer center. Patients aged 18 years or older with breast or head and neck cancer and plans for fractionated radiation therapy (≥15 fractions) with curative intent were enrolled via convenience sampling. Data were analyzed from September to October 2018. Exposures: Staphylococcus aureus colonization status before radiation therapy (baseline). Main Outcomes and Measures: The primary outcome was ARD grade using the Common Terminology Criteria for Adverse Event Reporting, version 4.03. Results: Among 76 patients analyzed, mean (SD) age was 58.5 (12.6) years and 56 (73.7%) were female. All 76 patients developed ARD: 47 (61.8%) with grade 1, 22 (28.9%) with grade 2, and 7 (9.2%) with grade 3. The prevalence of baseline nasal SA colonization was higher among patients who developed grade 2 or higher ARD compared with those who developed grade 1 ARD (10 of 29 [34.5%] vs 6 of 47 [12.8%]; P = .02, by χ2 test). Conclusions and Relevance: In this cohort study, baseline nasal SA colonization was associated with development of grade 2 or higher ARD in patients with breast or head and neck cancer. The findings suggest that SA colonization may play a role in the pathogenesis of ARD.

The Role of B Lymphocyte Subsets in Adipose Tissue Development, Metabolism, and Aging.

Adipose tissue contains resident B lymphocytes (B cells) with varying immune functions and mechanisms, depending on the adipose depot type and location. The heterogeneity of B cells and their functions affect the immunometabolism of the adipose tissue in aging and age-associated metabolic disorders. B cells exist in categorizations of subsets that have developmental or phenotypic differences with varying functionalities. Subsets can be categorized as either protective or pathogenic depending on their secretion profile or involvement in metabolic maintenance. In this article, we summarized recent finding on the B cell heterogeneity and discuss how we can utilize our current knowledge of adipose resident B lymphocytes for potential treatment for age-associated metabolic disorders. © 2022 American Physiological Society. Compr Physiol 12: 1-13, 2022.

The Role of Fat Reducing Agents on Adipocyte Death and Adipose Tissue Inflammation.

Deoxycholic Acid (DCA), which is an FDA-approved compound for the reduction of submental fat, has evolved through an unanticipated and surprising sequence of events. Initially, it was used as a solvent for Phosphatidylcholine (PDC), which was thought to promote lipolysis, but it was later proven to be the bioactive component of the formula and is currently widely used as Kybella. It has also been used off-label to treat other types of fat deposits like lipomas, HIV lipodystrophy, and excess orbital fat. Despite widespread clinical use, there has been no consensus clarifying the mechanisms of DCA and PDC alone or in combination. Furthermore, despite PDC's removal from the FDA-approved formula, some studies do suggest it plays an important role in fat reduction. To provide some clarity, we conducted a PubMed search and reviewed 41 articles using a comprehensive list of terms in three main categories, using the AND operator: 1) Phosphatidylcholines 2) Deoxycholic Acid, and 3) Lipoma. We isolated articles that studied PDC, DCA, and a PDC/DCA compound using cell biology, molecular and genetic techniques. We divided relevant articles into those that studied these components using histologic techniques and those that utilized specific cell death and lipolysis measurement techniques. Most morphologic studies indicated that PDC/DCA, DCA, and PDC, all induce some type of cell death with accompanying inflammation and fibrosis. Most morphologic studies also suggest that PDC/DCA and DCA alone are non-selective for adipocytes. Biochemical studies describing PDC and DCA alone indicate that DCA acts as a detergent and rapidly induces necrosis while PDC induces TNF-α release, apoptosis, and subsequent enzymatic lipolysis after at least 24 hours. Additional papers have suggested a synergistic effect between the two compounds. Our review integrates the findings of this growing body of literature into a proposed mechanism of fat reduction and provides direction for further studies.

Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases.

Beige Adipocyte Maintenance Is Regulated by Autophagy-Induced Mitochondrial Clearance.

Beige adipocytes gained much attention as an alternative cellular target in anti-obesity therapy. While recent studies have identified a number of regulatory circuits that promote beige adipocyte differentiation, the molecular basis of beige adipocyte maintenance remains unknown. Here, we demonstrate that beige adipocytes progressively lose their morphological and molecular characteristics after withdrawing external stimuli and directly acquire white-like characteristics bypassing an intermediate precursor stage. The beige-to-white adipocyte transition is tightly coupled to a decrease in mitochondria, increase in autophagy, and activation of MiT/TFE transcription factor-mediated lysosome biogenesis. The autophagy pathway is crucial for mitochondrial clearance during the transition; inhibiting autophagy by uncoupled protein 1 (UCP1(+))-adipocyte-specific deletion of Atg5 or Atg12 prevents beige adipocyte loss after withdrawing external stimuli, maintaining high thermogenic capacity and protecting against diet-induced obesity and insulin resistance. The present study uncovers a fundamental mechanism by which autophagy-mediated mitochondrial clearance controls beige adipocyte maintenance, thereby providing new opportunities to counteract obesity.

A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function.

Activation of brown and beige fat can reduce obesity and improve glucose homeostasis through nonshivering thermogenesis. Whether brown or beige fat also secretes paracrine or endocrine factors to promote and amplify adaptive thermogenesis is not fully explored. Here we identify Slit2, a 180 kDa member of the Slit extracellular protein family, as a PRDM16-regulated secreted factor from beige fat cells. In isolated cells and in mice, full-length Slit2 is cleaved to generate several smaller fragments, and we identify an active thermogenic moiety as the C-terminal fragment. This Slit2-C fragment of 50 kDa promotes adipose thermogenesis, augments energy expenditure, and improves glucose homeostasis in vivo. Mechanistically, Slit2 induces a robust activation of PKA signaling, which is required for its prothermogenic activity. Our findings establish a previously unknown peripheral role for Slit2 as a beige fat secreted factor that has therapeutic potential for the treatment of obesity and related metabolic disorders.

Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure.

Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under ß3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the ß3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

A creatine-driven substrate cycle enhances energy expenditure and thermogenesis in beige fat.

Thermogenic brown and beige adipose tissues dissipate chemical energy as heat, and their thermogenic activities can combat obesity and diabetes. Herein the functional adaptations to cold of brown and beige adipose depots are examined using quantitative mitochondrial proteomics. We identify arginine/creatine metabolism as a beige adipose signature and demonstrate that creatine enhances respiration in beige-fat mitochondria when ADP is limiting. In murine beige fat, cold exposure stimulates mitochondrial creatine kinase activity and induces coordinated expression of genes associated with creatine metabolism. Pharmacological reduction of creatine levels decreases whole-body energy expenditure after administration of a ß3-agonist and reduces beige and brown adipose metabolic rate. Genes of creatine metabolism are compensatorily induced when UCP1-dependent thermogenesis is ablated, and creatine reduction in Ucp1-deficient mice reduces core body temperature. These findings link a futile cycle of creatine metabolism to adipose tissue energy expenditure and thermal homeostasis. PAPERCLIP.

Genetic and functional characterization of clonally derived adult human brown adipocytes.

Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and its activation to a state of increased energy expenditure is believed to protect against the development of obesity. Even though the existence of BAT in adult humans has been widely appreciated, its cellular origin and molecular identity remain elusive largely because of high cellular heterogeneity within various adipose tissue depots. To understand the nature of adult human brown adipocytes at single cell resolution, we isolated clonally derived adipocytes from stromal vascular fractions of adult human BAT from two individuals and globally analyzed their molecular signatures. We used RNA sequencing followed by unbiased genome-wide expression analyses and found that a population of uncoupling protein 1 (UCP1)-positive human adipocytes possessed molecular signatures resembling those of a recruitable form of thermogenic adipocytes (that is, beige adipocytes). In addition, we identified molecular markers that were highly enriched in UCP1-positive human adipocytes, a set that included potassium channel K3 (KCNK3) and mitochondrial tumor suppressor 1 (MTUS1). Further, we functionally characterized these two markers using a loss-of-function approach and found that KCNK3 and MTUS1 were required for beige adipocyte differentiation and thermogenic function. The results of this study present new opportunities for human BAT research, such as facilitating cell-based disease modeling and unbiased screens for thermogenic regulators.

A combination of exercise and capsinoid supplementation additively suppresses diet-induced obesity by increasing energy expenditure in mice.

Exercise effectively prevents the development of obesity and obesity-related diseases such as type 2 diabetes. Capsinoids (CSNs) are capsaicin analogs found in a nonpungent pepper that increase whole body energy expenditure. Although both exercise and CSNs have antiobesity functions, the effectiveness of exercise with CSN supplementation has not yet been investigated. Here, we examined whether the beneficial effects of exercise could be further enhanced by CSN supplementation in mice. Mice were randomly assigned to four groups: 1) high-fat diet (HFD, Control), 2) HFD containing 0.3% CSNs, 3) HFD with voluntary running wheel exercise (Exercise), and 4) HFD containing 0.3% CSNs with voluntary running wheel exercise (Exercise + CSN). After 8 wk of ingestion, blood and tissues were collected and analyzed. Although CSNs significantly suppressed body weight gain under the HFD, CSN supplementation with exercise additively decreased body weight gain and fat accumulation and increased whole body energy expenditure compared with exercise alone. Exercise together with CSN supplementation robustly improved metabolic profiles, including the plasma cholesterol level. Furthermore, this combination significantly prevented diet-induced liver steatosis and decreased the size of adipocyte cells in white adipose tissue. Exercise and CSNs significantly increased cAMP levels and PKA activity in brown adipose tissue (BAT), indicating an increase of lipolysis. Moreover, they significantly activated both the oxidative phosphorylation gene program and fatty acid oxidation in skeletal muscle. These results indicate that CSNs efficiently promote the antiobesity effect of exercise, in part by increasing energy expenditure via the activation of fat oxidation in skeletal muscle and lipolysis in BAT.

Aligning LC peaks by converting gradient retention times to retention index of peptides in proteomic experiments.

MOTIVATION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. RESULTS: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k(0) (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (t(g)) to log k(0). Using the optimized function, t(g) values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale.

Informatics for peptide retention properties in proteomic LC-MS.

Retention times in HPLC yield valuable information for the identification of various analytes and the prediction of peptide retention is useful for the identification of peptides/proteins in LC-MS-based proteomics. Informatics methods such as artificial neural networks and support vector machines capable of solving nonlinear problems made possible the accurate modeling of quantitative structure-retention relationships of peptides (including large polymers) up to 5 kDa to which classical linear models cannot be applied, as well as the proteome-wide prediction of peptide retention. Proteome-wide retention prediction and accurate mass-information facilitate the identification of peptides in complex proteomic samples. In this review, we address recent developments in solid informatics methods and their application to peptide-retention properties in 'bottom-up' shotgun proteomics. We also describe future prospects for the standardization and application of retention times.