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Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed "committed pre-adipocytes" from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling, and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism., Competing Interests: M.D.W., R.S., H.S., and N.N.M. are employed by AgeX Therapeutics., (© 2024 The Author(s).)

Feline infectious peritonitis (FIP) is a systemic disease in felid species caused by infection with mutated forms of feline coronavirus (FCoV), and outbreaks can devastate exotic felid populations in human care. Feline infectious peritonitis was diagnosed in three of four related juvenile sand cats ( Felis margarita ) from a single institution over a 6-wk period. Case 1 was a 7-mon-old male found deceased with no premonitory signs. Case 2, an 8-mon-old male (littermate to Case 1), and Case 3, a 6-mon-old male (from a different litter with identical parentage), were evaluated for lethargy and anorexia 1 mon after Case 1. Both exhibited transient anisocoria and progressive lethargy, anorexia, and dehydration despite antibiotic and supportive treatment. Approximately 1 wk after initial presentation, Case 2 was humanely euthanized, and Case 3 was found deceased. Necropsy findings included intrathoracic and/or intra-abdominal lymphadenopathy (3/3 cases), bicavitary effusion (2/3), multifocal tan hepatic and intestinal nodules (1/3), and multifocal yellow renal nodules (1/3). Histologically, all cats had severe pyogranulomatous vasculitis in multiple organs, and the presence of FCoV antigen was confirmed using immunohistochemical staining. Next-generation sequencing of the virus from Case 3's affected kidney demonstrated ∼93% homology to the UG-FH8 virus, a serotype 1 feline alphacoronavirus isolated from Denmark. Future research will focus on comparative viral genomic sequencing with the goals of identifying potential sources of FCoV infection and identifying features that may have contributed to the development of FIP in this species.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low-molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage-derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor β solely on activated myeloid cells, we have created a folate-targeted TLR7 agonist (FA-TLR7-54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis-inducing cytokine production. We demonstrate that FA-TLR7-54 reprograms M2-like fibrosis-inducing macrophages into fibrosis-suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7-54 is lethal at fibrosis-suppressing doses, FA-TLR7-54 halts fibrosis without evidence of toxicity. Taken together, FA-TLR7-54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose-limiting systemic toxicities., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Src homology 2 (SH2) domains are composed of weakly conserved sequences of ∼100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra- and intermolecular protein-protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO 4 ) with K d = 14 nM; (iii) binding of cdb3-PO 4 to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO 4 ; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO 4 (but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO 4 to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3-ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties., Competing Interests: The authors declare no conflict of interest.

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

The oxygenation state of erythrocytes is known to impact several cellular processes. As the only known O2-binding protein in red blood cells, haemoglobin has been implicated in the oxygenation-mediated control of cell pathways and properties. Band 3, an integral membrane protein linked to the spectrin/actin cytoskeleton, preferentially binds deoxygenated haemoglobin at its N-terminus, and has been postulated to participate in the mechanism by which oxygenation controls cellular processes. Because the ankyrin-binding site on band 3 is located near the deoxyHb (deoxygenated haemoglobin)-binding site, we hypothesized that deoxyHb might impact the association between band 3 and the underlying erythrocyte cytoskeleton, a link that is primarily established through band 3-ankyrin bridging. In the present paper we show that deoxygenation of human erythrocytes results in displacement of ankyrin from band 3, leading to release of the spectrin/actin cytoskeleton from the membrane. This weakening of membrane-cytoskeletal interactions during brief periods of deoxygenation could prove beneficial to blood flow, but during episodes of prolonged deoxygenation, such as during sickle cell occlusive crises, could promote unwanted membrane vesiculation.

The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal-membrane complex. These pools of ATP are known to directly fuel both the Na(+)/K(+) and Ca(2+) pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na(+)/K(+) or Ca(2+) pump phosphoproteins (E(Na)-P and E(Ca)-P, respectively) from bulk [γ-(32)P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N(3)-ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N(3)-ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool's architecture can be photolabeled. With the aid of an antibody to N(3)-ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.