Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-ß signaling.

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of 'imprinting' by cytokines of the TGF-ß family. We studied mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-ß family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-ß signaling mediated by the cytokine receptor TGFßR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-ß. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-ß signaling in NK cells.

Characterization of transcript enrichment and detection bias in single-nucleus RNA-seq for mapping of distinct human adipocyte lineages.

Single-cell RNA sequencing (scRNA-seq) enables molecular characterization of complex biological tissues at high resolution. The requirement of single-cell extraction, however, makes it challenging for profiling tissues such as adipose tissue, for which collection of intact single adipocytes is complicated by their fragile nature. For such tissues, single-nucleus extraction is often much more efficient and therefore single-nucleus RNA sequencing (snRNA-seq) presents an alternative to scRNA-seq. However, nuclear transcripts represent only a fraction of the transcriptome in a single cell, with snRNA-seq marked with inherent transcript enrichment and detection biases. Therefore, snRNA-seq may be inadequate for mapping important transcriptional signatures in adipose tissue. In this study, we compare the transcriptomic landscape of single nuclei isolated from preadipocytes and mature adipocytes across human white and brown adipocyte lineages, with whole-cell transcriptome. We show that snRNA-seq is capable of identifying the broad cell types present in scRNA-seq at all states of adipogenesis. However, we also explore how and why the nuclear transcriptome is biased and limited, as well as how it can be advantageous. We robustly characterize the enrichment of nuclear-localized transcripts and adipogenic regulatory lncRNAs in snRNA-seq, while also providing a detailed understanding for the preferential detection of long genes upon using this technique. To remove such technical detection biases, we propose a normalization strategy for a more accurate comparison of nuclear and cellular data. Finally, we show successful integration of scRNA-seq and snRNA-seq data sets with existing bioinformatic tools. Overall, our results illustrate the applicability of snRNA-seq for the characterization of cellular diversity in the adipose tissue.

The oxylipin analog CS585 prevents platelet activation and thrombosis through activation of the prostacyclin receptor.

Cardiovascular disease remains the primary cause of morbidity and mortality globally. Platelet activation is critical for maintaining hemostasis and preventing the leakage of blood cells from the vessel. There has been a paucity in the development of new drugs to target platelet reactivity. Recently, the oxylipin 12(S)-hydroxy-eicosatrienoic acid (12-HETrE), which is produced in platelets, was shown to limit platelet reactivity by activating the prostacyclin receptor. Here, we demonstrated the synthesis of a novel analog of 12-HETrE, known as CS585. Human blood and mouse models of hemostasis and thrombosis were assessed for the ability of CS585 to attenuate platelet activation and thrombosis without increasing the risk of bleeding. Human platelet activation was assessed using aggregometry, flow cytometry, western blot analysis, total thrombus formation analysis system, microfluidic perfusion chamber, and thromboelastography. Hemostasis, thrombosis, and bleeding assays were performed in mice. CS585 was shown to potently target the prostacyclin receptor on the human platelet, resulting in a highly selective and effective mechanism for the prevention of platelet activation. Furthermore, CS585 was shown to inhibit platelet function in human whole blood ex vivo, prevent thrombosis in both small and large vessels in mouse models, and exhibit long-lasting prevention of clot formation. Finally, CS585 was not observed to perturb coagulation or increase the risk of bleeding in the mouse model. Hence, CS585 represents a new validated target for the treatment of thrombotic diseases without the risk of bleeding or off-target activation observed with other prostaglandin receptor agonists.

A hexagonal planar transition-metal complex.

Transition-metal complexes are widely used in the physical and biological sciences. They have essential roles in catalysis, synthesis, materials science, photophysics and bioinorganic chemistry. Our understanding of transition-metal complexes originates from Alfred Werner's realization that their three-dimensional shape influences their properties and reactivity1, and the intrinsic link between shape and electronic structure is now firmly underpinned by molecular-orbital theory2-5. Despite more than a century of advances in this field, the geometries of transition-metal complexes remain limited to a few well-understood examples. The archetypal geometries of six-coordinate transition metals are octahedral and trigonal prismatic, and although deviations from ideal bond angles and bond lengths are frequent6, alternative parent geometries are extremely rare7. The hexagonal planar coordination environment is known, but it is restricted to condensed metallic phases8, the hexagonal pores of coordination polymers9, or clusters that contain more than one transition metal in close proximity10,11. Such a geometry had been considered12,13 for [Ni(PtBu)6]; however, an analysis of the molecular orbitals suggested that this complex is best described as a 16-electron species with a trigonal planar geometry14. Here we report the isolation and structural characterization of a simple coordination complex in which six ligands form bonds with a central transition metal in a hexagonal planar arrangement. The structure contains a central palladium atom surrounded by three hydride and three magnesium-based ligands. This finding has the potential to introduce additional design principles for transition-metal complexes, with implications for several scientific fields.

Mast Cell Activation Syndrome: current understanding and research needs.

Mast cell activation syndrome (MCAS) is a term applied to several clinical entities which have gained increased attention from patients and medical providers. While several descriptive publications about MCAS exist, there are many gaps in knowledge resulting in confusion about this clinical syndrome. Whether MCAS is a primary syndrome or exists as a constellation of symptoms in the context of known inflammatory, allergic, or clonal disorders associated with systemic mast cell (MC) activation is not well understood. More importantly, the underlying mechanisms and pathways that lead to MC activation in MCAS patients remain to be elucidated. The purpose of this manuscript is to summarize the known literature, identify gaps in knowledge, and highlight research needs. Several topics are covered: 1) Contextualization of MCAS and MCAS-like endotypes and related diagnostic evaluations; 2) Mechanistic research; 3) Management of typical and refractory symptoms, and 4) MCAS-specific education for patients and healthcare providers.

Vinylic C-H Activation of Styrenes by an Iron-Aluminum Complex.

The oxidative addition of sp2 C-H bonds of alkenes to single-site transition-metal complexes is complicated by the competing π-coordination of the C═C double bond, limiting the examples of this type of reactivity and onward applications. Here, we report the C-H activation of styrenes by a well-defined bimetallic Fe-Al complex. These reactions are highly selective, resulting in the (E)-ß-metalation of the alkene. For this bimetallic system, alkene binding appears to be essential for the reaction to occur. Experimental and computational insights suggest an unusual reaction pathway in which a (2 + 2) cycloaddition intermediate is directly converted into the hydrido vinyl product via an intramolecular sp2 C-H bond activation across the two metals. The key C-H cleavage step proceeds through a highly asynchronous transition state near the boundary between a concerted and a stepwise mechanism influenced by the resonance stabilization ability of the aryl substituent. The metalated alkenes can be further functionalized, which has been demonstrated by the (E)-selective phosphination of the employed styrenes.

Highly Efficient and Scalable p-i-n Perovskite Solar Cells Enabled by Poly-metallocene Interfaces.

Inverted p-i-n perovskite solar cells (PSCs) are easy to process but need improved interface characteristics with reduced energy loss to prevent efficiency drops when increasing the active photovoltaic area. Here, we report a series of poly ferrocenyl molecules that can modulate the perovskite surface enabling the construction of small- and large-area PSCs. We found that the perovskite-ferrocenyl interaction forms a hybrid complex with enhanced surface coordination strength and activated electronic states, leading to lower interfacial nonradiative recombination and charge transport resistance losses. The resulting PSCs achieve an enhanced efficiency of up to 26.08% for small-area devices and 24.51% for large-area devices (1.0208 cm2). Moreover, the large-area PSCs maintain >92% of the initial efficiency after 2000 h of continuous operation at the maximum power point under 1-sun illumination and 65 °C.

Hydrogen Activation with Ru-PN3P Pincer Complexes for the Conversion of C1 Feedstocks.

The hydrogenation of C1 feedstocks (CO and CO2) has been investigated using ruthenium complexes [RuHCl(CO)(PN3P)] as the catalyst. PN3P pincer ligands containing amines in the linker between the central pyridine donor and the phosphorus donors with bulky substituents (tert-butyl (1) or TMPhos (2)) are required to obtain mononuclear single-site catalysts that can be activated by the addition of KOtBu to generate stable five-coordinate complexes [RuH(CO)(PN3P-H)], whereby the pincer ligand has been deprotonated. Activation of hydrogen takes place via heterolytic cleavage to generate [RuH2(CO)(PN3P)], but in the presence of CO, coordination of CO occurs preferentially to give [RuH(CO)2(PN3P-H)]. This complex can be protonated to give the cationic complex [RuH(CO)2(PN3P)]+, but it is unable to activate H2 heterolytically. In the case of the less coordinating CO2, both ruthenium complexes 1 and 2 are highly efficient as CO2 hydrogenation catalysts in the presence of a base (DBU), which in the case of the TMPhos ligand results in a TON of 30,000 for the formation of formate.

Mast Cells in Aspirin-Exacerbated Respiratory Disease.

PURPOSE OF REVIEW: Aspirin-exacerbated respiratory disease (AERD) is a syndrome of high type 2 inflammation and is known to critically involve mast cell activation. The mast cell is an important cell in the baseline inflammatory processes in the upper and lower airway by maintaining and amplifying type 2 inflammation. But it also is prominent in the hypersensitivity reaction to COX-1 inhibition which defines this condition. RECENT FINDINGS: Recent work highlights the mast cell as a focal point in AERD pathogenesis. Using AERD as a specific model of both high type 2 asthma and chronic sinusitis, the role of mast cell activity can be better understood in other aspects of airway inflammation. Further dissecting out the mechanism of COX-1-mediated mast cell activation in AERD will be an important next phase in our understanding of NSAID-induced hypersensitivity as well as AERD pathophysiology.

Identification of HLA-E Binding Mycobacterium tuberculosis-Derived Epitopes through Improved Prediction Models.

Tuberculosis (TB) remains one of the deadliest infectious diseases worldwide, posing great social and economic burden to affected countries. Novel vaccine approaches are needed to increase protective immunity against the causative agent Mycobacterium tuberculosis (Mtb) and to reduce the development of active TB disease in latently infected individuals. Donor-unrestricted T cell responses represent such novel potential vaccine targets. HLA-E-restricted T cell responses have been shown to play an important role in protection against TB and other infections, and recent studies have demonstrated that these cells can be primed in vitro. However, the identification of novel pathogen-derived HLA-E binding peptides presented by infected target cells has been limited by the lack of accurate prediction algorithms for HLA-E binding. In this study, we developed an improved HLA-E binding peptide prediction algorithm and implemented it to identify (to our knowledge) novel Mtb-derived peptides with capacity to induce CD8+ T cell activation and that were recognized by specific HLA-E-restricted T cells in Mycobacterium-exposed humans. Altogether, we present a novel algorithm for the identification of pathogen- or self-derived HLA-E-presented peptides.

Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects.

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than ß-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Lower serum 15-HETE level predicts nasal ILC2 accumulation during COX-1 inhibition in AERD.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is associated with high levels of cysteinyl leukotrienes, prostaglandin D2, and low levels of prostaglandin E2. Further, 15-hydroxyeicosatetraenoic acid (15-HETE) levels may have predictive value in therapeutic outcomes of aspirin desensitization. Accumulation of nasal group 2 innate lymphoid cells (ILC2s) has been demonstrated during COX-1 inhibition in AERD, although the relationships between tissue ILC2 accumulation, reaction symptom severity, and novel lipid biomarkers are unknown. OBJECTIVE: We sought to determine whether novel lipid mediators are predictive of nasal ILC2 accumulation and symptom scores during COX-1 inhibitor challenge in patients with AERD. METHODS: Blood and nasal scraping samples from patients with AERD were collected at baseline and COX-1 inhibitor reaction and then processed for flow cytometry for nasal ILC2s and serum for lipidomic analysis. RESULTS: Eight patients with AERD who were undergoing aspirin desensitization were recruited. Of the 161 eicosanoids tested, 42 serum mediators were detected. Baseline levels of 15-HETE were negatively correlated with the change in numbers of airway ILC2s (r = -0.6667; P = .0428). Docosahexaenoic acid epoxygenase metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) was positively correlated with both changes in airway ILC2s (r = 0.7143; P = .0305) and clinical symptom scores (r = 0.5000; P = .0081). CONCLUSION: Low levels of baseline 15-HETE predicted a greater accumulation of airway ILC2s in patients with AERD who were receiving COX-1 inhibition. Further, increases in the cytochrome P pathway metabolite 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-diHDPA) were associated with increased symptoms and nasal ILC2 accumulation. Future studies to assess how these mediators might control ILC2s may improve the understanding of AERD pathogenesis.

Hereditary hemorrhagic telangiectasia diagnosis: A case report.

This case report presents a 13-year-old patient with a lung nodule identified on a chest radiograph in the emergency department during an evaluation of knee and side pain after a fall. The patient had nosebleeds, family history of hereditary hemorrhagic telangiectasia (HHT) and after chest computed tomography with angiography, the nodule was defined as a single pulmonary arteriovenous malformation (PAVM). Neither parent nor patient had been evaluated for HHT, an autosomal dominant disease, despite the family history. This patient satisfied the clinical criteria for the diagnosis and had a confirmatory genetic test, which led to diagnosis in mother also. The patient's PAVMs were treated, decreasing the risk of life threatening complications. Diagnosing HHT in children is often delayed or missed, even in families with HHT, as in this case report. Without any physical signs or clinical symptoms, families and healthcare providers often dismiss the possibility of the diagnosis. Children with HHT are at the same risk for complications of stroke, anemia, hypoxemia, heart failure and increased morbidity as adults. It is essential to recognize the importance of family history when evaluating children in primary care and urgent settings, as this patient's diagnosis was delayed 13 years. Awareness of HHT signs and symptoms are essential to early referral to an HHT specialist, for diagnosis and management.

Deoxygenative Coupling of CO with a Tetrametallic Magnesium Hydride Complex.

Addition of CO to a tetrametallic magnesium hydride cluster results in both carbon-carbon bond formation and deoxygenation to generate an acetaldehyde enolate [C2OH3]- which remains coordinated to the cluster. To the best of our knowledge, this is the first example of formation of an isolable complex containing an [C2OH3]- fragment from reaction of CO with a metal hydride, and the first example of CO homologation and deoxygenation at a main group metal. DFT studies suggest that key steps in the mechanism involve nucleophilic attack of an oxymethylene on a formyl ligand to generate an unstable [C2O2H3]3- fragment, which undergoes subsequent deoxygenation.

Catalytic, Z-Selective, Semi-Hydrogenation of Alkynes with a Zinc-Anilide Complex.

The reversible activation of dihydrogen with a molecular zinc anilide complex is reported. The mechanism of this reaction has been probed through stoichiometric experiments and density functional theory (DFT) calculations. The combined evidence suggests that H2 activation occurs by addition across the Zn-N bond via a four-membered transition state in which the Zn and N atoms play a dual role of Lewis acid and Lewis base. The zinc hydride complex that results from H2 addition has been shown to be remarkably effective for the hydrozincation of C═C bonds at modest temperatures. The scope of hydrozincation includes alkynes, alkenes, and a 1,3-butadiyne. For alkynes, the hydrozincation step is stereospecific leading exclusively to the syn-isomer. Competition experiments show that the hydrozincation of alkynes is faster than the equivalent alkene substrates. These new discoveries have been used to develop a catalytic system for the semi-hydrogenation of alkynes. The catalytic scope includes both aryl- and alkyl-substituted internal alkynes and proceeds with high alkene: alkane, Z:E ratios, and modest functional group tolerance. This work offers a first example of selective hydrogenation catalysis using zinc complexes.

CD39 and LDHA affects the prognostic role of NLR in metastatic melanoma patients treated with immunotherapy.

BACKGROUND: Identifying response markers is highly needed to guide the treatment strategy in patients with metastatic melanoma. METHODS: A retrospective study was carried out in patients with unresectable/metastatic melanoma (stage IIIb-IV), treated with anti-PD-1 in the first line setting, to better explore the role and the timing of neutrophil/lymphocyte ratio (NLR) as potential biomarker of response. The relationship of NLR with inflammation-immune mediators and the underlying negative effect of raising NLR during immunotherapy, have been investigated with transcriptomic gene analysis. RESULTS: The results confirmed previous findings that a high baseline NLR is associated with a poorer prognosis and with higher serum level of lactate dehydrogenase (LDH), regardless of the presence of brain metastases. The transcriptomic analysis showed that high baseline NLR is associated with a characteristic gene signature CCNA1, LDHA and IL18R1, which correlates with inflammation and tumorigenesis. Conversely, low baseline NLR is associated with the signature CD3, SH2D1A, ZAP70 and CD45RA, linked to the immune-activation. The genes positively associated with NLR (CD39 (ENTPD1), PTEN, MYD88, MMP9 and LDH) are involved in processes of immunosuppression, inflammation and tumor-promoting activity. Increased expression of CD39 correlated with TGFß2, a marker of the N2 neutrophils with immunosuppressive activity. CONCLUSIONS: These results suggest that increasing NLR is associated with an increased neutrophil population, with polarization to the N2 phenotype, and this process may be the basis for the negatively prognostic role of NLR.

Development of Antiplasmodial Peptide-Drug Conjugates Using a Human Protein-Derived Cell-Penetrating Peptide with Selectivity for Infected Cells.

Malaria continues to impose a global health burden. Drug-resistant parasites have emerged to each introduced small-molecule therapy, highlighting the need for novel treatment approaches for the future eradication of malaria. Herein, targeted drug delivery with peptide-drug conjugates (PDCs) was investigated as an alternative antimalarial therapy, inspired by the success of emerging antibody-drug conjugates utilized in cancer treatment. A synthetic peptide derived from an innate human defense molecule was conjugated to the antimalarial drug primaquine (PQ) to produce PDCs with low micromolar potency toward Plasmodium falciparum in vitro. A suite of PDCs with different design features was developed to identify optimal conjugation site and investigate linker length, hydrophilicity, and cleavability. Conjugation within a flexible spacer region of the peptide, with a cleavable linker to liberate the PQ cargo, was important to retain activity of the peptide and drug.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation.

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of ß-globinopathies (sickle cell disease and ß-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

Human tear metabolites associated with nucleoside-signalling pathways in bacterial keratitis.

OBJECTIVE: The study aimed to profile and quantify tear metabolites associated with bacterial keratitis using both untargeted and targeted metabolomic platforms. METHODS: Untargeted metabolomic analysis using liquid-chromatography-Q Exactive-HF mass-spectrometry explored tear metabolites significantly associated with bacterial keratitis (n = 6) compared to healthy participants (n = 6). Differential statistics and principal component analysis determined meaningful metabolite differences between cases and controls. Purines and nucleosides were further quantified and compared between 15 cases and 15 controls in the targeted metabolomic platform using TSQ quantum access triple quadrupole mass spectrometry. Compound quantification was done by plotting the calibration curves and the difference in the compound levels was evaluated using the Wilcoxon rank-sum test. RESULTS: In the untargeted analysis, 49 tear metabolites (27 upregulated and 22 downregulated) were differentially expressed between cases and controls. The untargeted analysis indicated that the purine metabolism pathway was the most affected by bacterial keratitis. Metabolite quantification in the targeted analysis further confirmed the upregulation of xanthine (P = 0.02) and downregulation of adenine (P < 0.0001), adenosine (P < 0.0001) and cytidine (P < 0.0001) in the tears of participants with bacterial keratitis compared to that of healthy participants. CONCLUSIONS: Bacterial keratitis significantly changes the tear metabolite profile, including five major compound classes such as indoles, amino acids, nucleosides, carbohydrates, and steroids. This study also indicates that tear fluids can be used to map the metabolic pathways and uncover metabolic markers associated with bacterial keratitis. Conceivably, the inhibition of nucleoside synthesis may contribute to the pathophysiology of bacterial keratitis because nucleosides are required for maintaining cellular energy homeostasis and immune adaptability.