HLA-DM captures partially empty HLA-DR molecules for catalyzed removal of peptide.

The mechanisms of HLA-DM-catalyzed peptide exchange remain uncertain. Here we found that all stages of the interaction of HLA-DM with HLA-DR were dependent on the occupancy state of the peptide-binding groove. High-affinity peptides were protected from removal by HLA-DM through two mechanisms: peptide binding induced the dissociation of a long-lived complex of empty HLA-DR and HLA-DM, and high-affinity HLA-DR-peptide complexes bound HLA-DM only very slowly. Nonbinding covalent HLA-DR-peptide complexes were converted into efficient HLA-DM binders after truncation of an N-terminal peptide segment that emptied the P1 pocket and disrupted conserved hydrogen bonds to HLA-DR. HLA-DM thus binds only to HLA-DR conformers in which a critical part of the binding site is already vacant because of spontaneous peptide motion.

The Geroprotective Drug Candidate CMS121 Alleviates Diabetes, Liver Inflammation, and Renal Damage in db/db Leptin Receptor Deficient Mice.

db/db mice, which lack leptin receptors and exhibit hyperphagia, show disturbances in energy metabolism and are a model of obesity and type 2 diabetes. The geroneuroprotector drug candidate CMS121 has been shown to be effective in animal models of Alzheimer's disease and aging through the modulation of metabolism. Thus, the hypothesis was that CMS121 could protect db/db mice from metabolic defects and thereby reduce liver inflammation and kidney damage. The mice were treated with CMS121 in their diet for 6 months. No changes were observed in food and oxygen consumption, body mass, or locomotor activity compared to control db/db mice, but a 5% reduction in body weight was noted. Improved glucose tolerance and reduced HbA1c and insulin levels were also seen. Blood and liver triglycerides and free fatty acids decreased. Improved metabolism was supported by lower levels of fatty acid metabolites in the urine. Markers of liver inflammation, including NF-κB, IL-18, caspase 3, and C reactive protein, were lowered by the CMS121 treatment. Urine markers of kidney damage were improved, as evidenced by lower urinary levels of NGAL, clusterin, and albumin. Urine metabolomics studies provided further evidence for kidney protection. Mitochondrial protein markers were elevated in db/db mice, but CMS121 restored the renal levels of NDUFB8, UQCRC2, and VDAC. Overall, long-term CMS121 treatment alleviated metabolic imbalances, liver inflammation, and reduced markers of kidney damage. Thus, this study provides promising evidence for the potential therapeutic use of CMS121 in treating metabolic disorders.

Zinc Borate Hydrolysis.

The crystalline zinc borate phase ZnB3O4(OH)3, known in commerce as 2ZnO·3B2O3·3.5H2O, is an important industrial material used as a fire-retardant synergist in polymers, a source of micronutrients in agriculture, and a preservative in building materials. It lends durability to wood composite building materials by inhibiting attack by wood destroying organisms. The hydrolysis chemistry of this zinc borate is relevant to its industrial use. ZnB3O4(OH)3 exhibits incongruent solubility, reversibly hydrolyzing at neutral pH to insoluble Zn(OH)2 and soluble B(OH)3. It is sparingly soluble with a room temperature solubility of 0.270 wt% in terms of its equivalent oxide components in solution, comprising 0.0267 wt% B2O3 and 0.003 wt% ZnO. Aspects of the hydrolysis chemistry of zinc borate under neutral pH conditions are discussed.

High-dimensional single-cell proteomics analysis identifies immune checkpoint signatures and therapeutic targets in ulcerative colitis.

Immune checkpoints are regulators of immune cells and play key roles in the modulation of immune responses. The role of checkpoints in autoimmune disease is poorly understood but likely to be central since checkpoint inhibition during cancer treatment can cause autoimmunity. We generated a high-dimensional single-cell proteomics data set from PBMCs of healthy individuals and patients with ulcerative colitis (UC) by mass cytometry, enabling systems-wide analyses of immune cell frequencies and cell type-specific expression patterns of 12 immune checkpoints. Subtle but significant changes in immune cell frequencies and checkpoint expression were observed between UC patients on different treatment regimens and between patients and healthy controls. Most strikingly, UC patients showed a reduced number of peripheral NK-cells and those cells showed an altered phenotype including increased TIGIT expression. Based on these results, we modulated NK-cell function ex vivo through targeting of TIGIT pathway members. In summary, we describe a pattern of changes in immune cell abundance and checkpoint expression as a basis for UC patient stratification and we show modulation of a corresponding immune cell subset through checkpoint targeting. Our approach can be used for the identification of pathogenic immune cell subsets and guide target selection in autoimmunity and chronic inflammation.

Several targets involved in Alzheimer's disease amyloidogenesis are affected by morin and isoquercitrin.

Long-term consumption of phytochemicals has been associated with a decreased risk of dementia. The modes of action of two flavonols (morin and isoquercitrin) and two flavanones (hesperidin and neohesperidin) were characterized as single-compound drugs in several Alzheimer's disease (AD)-related assays. First, these phytochemicals were assayed in an amyloid toxicity model (MC65 cells). Second, we examined the activity of the flavonoids in cell-free assays against ß- and γ-secretases and acetylcholinesterase activities, as well as agents able to modify the fibrillogenesis of the amyloid ß-peptide. Additionally, they were assayed against glutamate-induced oxytosis, as scavengers of reactive oxygen species (ROS), as inhibitors of caspase-3, -8 and -9 activation and as modulators of the chymotrypsin-like activity of the ubiquitin-proteasome system. Morin and isoquercitrin, unlike flavanones, exhibited significant activities as ß- and γ-secretase inhibitors, as well as capacity to inhibit Aß aggregation and favor its disaggregation. Flavonols and flavanones showed ROS scavenger activity (P < 0.05), attenuation of caspase-3 and -9 activation (P < 0.05) and restoration of the reduced chymotrypsin-like activity of proteasome 20S (P < 0.05) upon H2O2 exposure of APPswe cells. Flavanones failed to protect against glutamate-induced oxytosis. These findings provide new insight into the anti-amyloidogenic effects of morin and isoquercitrin.

A Brief History of Adherons: The Discovery of Brain Exosomes.

Although exosomes were first described in reticulocytes in 1983, many people do not realize that similar vesicles had been studied in the context of muscle and nerve, beginning in 1980. At the time of their discovery, these vesicles were named adherons, and they were found to play an important role in both cell-substrate and cell-cell adhesion. My laboratory described several molecules that are present in adherons, including heparan sulfate proteoglycans (HSPGs) and purpurin. HSPGs have since been shown to play a variety of key roles in brain physiology. Purpurin has a number of important functions in the retina, including a role in nerve cell differentiation and regeneration. In this review, I discuss the discovery of adherons and how that led to continuing studies on their role in the brain with a particular focus on HSPGs.

Hydrated Zinc Borates and Their Industrial Use.

Zinc borates are important chemical products having industrial applications as functional additives in polymers, bio-composites, paints and ceramics. Of the thirteen well documented hydrated binary zinc borates, Zn[B3O4(OH)3] (2ZnO∙3B2O3∙3H2O) is manufactured in the largest quantity and is known as an article of commerce as 2ZnO∙3B2O3∙3.5H2O. Other hydrated zinc borates in commercial use include 4ZnO∙B2O3∙H2O, 3ZnO∙3B2O3∙5H2O and 2ZnO∙3B2O3∙7H2O. The history, chemistry, and applications of these and other hydrated zinc borate phases are briefly reviewed, and outstanding problems in the field are highlighted.

Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

Age is, by far, the greatest risk factor for Alzheimer's disease (AD), yet few AD drug candidates have been generated that target pathways specifically associated with the aging process itself. Two ubiquitous features of the aging brain are the intracellular accumulation of aggregated proteins and inflammation. As intraneuronal amyloid protein is detected before markers of inflammation, we argue that old, age-associated, aggregated proteins in neurons can induce inflammation, resulting in multiple forms of brain toxicities. The consequence is the increased risk of old, age-associated, neurodegenerative diseases. As most of these diseases are associated with the accumulation of aggregated proteins, it is possible that any therapeutic that reduces intracellular protein aggregation will benefit all.-Currais, A., Fischer, W., Maher, P., Schubert, D. Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

Structure and Properties of Sodium Enneaborate, Na2[B8O11(OH)4]·B(OH)3·2H2O.

Millions of tons of sodium borates are used annually by global industries in diverse applications important to modern society. The Na2O-B2O3-H2O phase diagram in the 0-100 °C temperature range contains 13 unique hydrated crystalline sodium borates, including five important industrial products. Structures were previously reported for each of these except for that having the highest boron content, known as sodium enneaborate, Na4B18O29·11H2O or 2Na2O·9B2O3·11H2O (1). Here we report the single-crystal structure of 1, revealing the structural formula Na2[B8O11(OH)4]·B(OH)3·2H2O, and describe some of its properties and relationships to other sodium borates. The structure of 1 features linear polyborate chains composed of the repeating [B8O11(OH)4]2- fundamental building blocks with interstitial water and boric acid molecules integrated by extensive H bonding. Interstitial sodium cations occur in groups of four with interatomic distances of 3.7830(6) and 3.7932(8) Å. Upon heating, 1 initially becomes amorphous and then crystallizes as α-Na2B8O13 along with amorphous B2O3. Notably, α-Na2B8O13 contains octaborate fundamental building blocks that are topologically equivalent to those in 1. Compound 1 crystallizes in the monoclinic space group P21/n with a = 10.2130(8) Å, b = 12.940(1) Å, c = 12.457(1) Å, ß = 93.070(2)°, V = 1644.0(2) Å3, and Z = 2.

Solid-State Synthesis and Structure of the Enigmatic Ammonium Octaborate: (NH4)2[B7O9(OH)5]·3/4B(OH)3·5/4H2O.

The compound known since the 19th century as ammonium octaborate was structurally characterized revealing the ammonium salt of the ribbon isomer of the heptaborate anion, [B7O9(OH)5](2-), with boric acid and water molecules. Of composition (NH4)2B7.75O12.63·4.88H2O, it approximates the classical ammonium octaborate composition (NH4)2B8O13·6H2O and has the structural formula {(NH4)2[B7O9(OH)5]}4·3B(OH)3·5H2O. It spontaneously forms at room temperature in solid-state mixtures of ammonium tetraborate and ammonium pentaborate. It crystallizes in the monoclinic space group P21/c with a = 11.4137(2) Å, b = 11.8877(2) Å, c = 23.4459(3) Å, ß = 90.092(1)°, V = 3181.19(8) Å(3), and Z = 2 and contains well-ordered ammonium cations and [B7O9(OH)5](2-) anions and disordered B(OH)3 and H2O molecules linked by extensive H bonding. Expeditious solid-state formation of the heptaborate anion under ambient conditions has important implications for development of practical syntheses of industrially useful borates.

Selecting for neurogenic potential as an alternative for Alzheimer's disease drug discovery.

INTRODUCTION: Neurons die in Alzheimer's disease (AD) and are not effectively replaced. An alternative approach to maintain nerve cell number is to identify compounds that stimulate the proliferation of endogenous neural stem cells in old individuals to replace lost neurons. However, unless a neurogenic drug is also neuroprotective, the replacement of lost neurons will not be sufficient to stop disease progression. METHODS: The neuroprotective AD drug candidate J147 is shown to enhance memory, improve dendritic structure, and stimulate cell division in germinal regions of the brains of very old mice. Based on the potential neurogenic potential of J147, a neuronal stem cell screening assay was developed to optimize derivatives of J147 for human neurogenesis. RESULTS: The best derivative of J147, CAD-031, maintains the neuroprotective and memory enhancing properties of J147, yet is more active in the human neural stem cell assays. DISCUSSION: The combined properties of neuroprotection, neurogenesis, and memory enhancement in a single drug are more likely to be effective for the treatment of age-associated neurodegenerative disorders than any individual activity alone.

Modulation of 5-lipoxygenase in proteotoxicity and Alzheimer's disease.

The accumulation of intracellular ß amyloid (Aß) may be one of the factors leading to neuronal cell death in Alzheimer's disease (AD). Using a pyrazole called CNB-001, which was selected for its ability to reduce intracellular Aß, we show that the activation of the eIF2α/ATF4 arm of the unfolded protein response is sufficient to degrade aggregated intracellular Aß. CNB-001 is a potent inhibitor of 5-lipoxygenase (5-LOX), decreases 5-LOX expression, and increases proteasome activity. 5-LOX inhibition induces eIF2α and PERK (protein kinase R-like extracellular signal-regulated kinase) phosphorylation, and HSP90 and ATF4 levels. When fed to AD transgenic mice, CNB-001 also increases eIF2α phosphorylation and HSP90 and ATF4 levels, and limits the accumulation of soluble Aß and ubiquitinated aggregated proteins. Finally, CNB-001 maintains the expression of synapse-associated proteins and improves memory. Therefore, 5-LOX metabolism is a key element in the promotion of endoplasmic reticulum dysfunction, and its inhibition under conditions of stress is sufficient to reduce proteotoxicity both in vivo and in vitro.

CMS121: a novel approach to mitigate aging-related obesity and metabolic dysfunction.

BACKGROUND: Modulated by differences in genetic and environmental factors, laboratory mice often show progressive weight gain, eventually leading to obesity and metabolic dyshomeostasis. Since the geroneuroprotector CMS121 has a positive effect on energy metabolism in a mouse model of type 2 diabetes, we investigated the potential of CMS121 to counteract the metabolic changes observed during the ageing process of wild type mice. METHODS: Control or CMS121-containing diets were supplied ad libitum for 6 months, and mice were sacrificed at the age of 7 months. Blood, adipose tissue, and liver were analyzed for glucose, lipids, and protein markers of energy metabolism. RESULTS: The CMS121 diet induced a 40% decrease in body weight gain and improved both glucose and lipid indexes. Lower levels of hepatic caspase 1, caspase 3, and NOX4 were observed with CMS121 indicating a lower liver inflammatory status. Adipose tissue from CMS121-treated mice showed increased levels of the transcription factors Nrf1 and TFAM, as well as markers of mitochondrial electron transport complexes, levels of GLUT4 and a higher resting metabolic rate. Metabolomic analysis revealed elevated plasma concentrations of short chain acylcarnitines and butyrate metabolites in mice treated with CMS121. CONCLUSIONS: The diminished de novo lipogenesis, which is associated with increased acetyl-CoA, acylcarnitine, and butyrate metabolite levels, could contribute to safeguarding not only the peripheral system but also the aging brain. By mimicking the effects of ketogenic diets, CMS121 holds promise for metabolic diseases such as obesity and diabetes, since these diets are hard to follow over the long term.

Metabolism of a potent neuroprotective hydrazide.

Using a drug discovery scheme for Alzheimer's disease (AD) that is based upon multiple pathologies of old age, we identified a potent compound with efficacy in rodent memory and AD animal models. Since this compound, J147, is a phenyl hydrazide, there was concern that it can be metabolized to aromatic amines/hydrazines that are potentially carcinogenic. To explore this possibility, we examined the metabolites of J147 in human and mouse microsomes and mouse plasma. It is shown that J147 is not metabolized to aromatic amines or hydrazines, that the scaffold is exceptionally stable, and that the oxidative metabolites are also neuroprotective. It is concluded that the major metabolites of J147 may contribute to its biological activity in animals.

Attenuation of Age-Related Hearing Impairment in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice Treated with Fatty Acid Synthase Inhibitor CMS121.

In the senescence-accelerated mouse prone 8 (SAMP8) mouse model, oxidative stress leads to premature senescence and age-related hearing impairment (ARHI). CMS121 inhibits oxytosis/ferroptosis by targeting fatty acid synthase. The aim of our study was to determine whether CMS121 is protective against ARHI in SAMP8 mice. Auditory brainstem responses (ABRs) were used to assess baseline hearing in sixteen 4-week-old female SAMP8 mice, which were divided into two cohorts. The control group was fed a vehicle diet, while the experimental group was fed a diet containing CMS121. ABRs were measured until 13 weeks of age. Cochlear immunohistochemistry was performed to analyze the number of paired ribbon-receptor synapses per inner hair cell (IHC). Descriptive statistics are provided with mean ± SEM. Two-sample t-tests were performed to compare hearing thresholds and paired synapse count across the two groups, with alpha = 0.05. Baseline hearing thresholds in the control group were statistically similar to those of the CMS121 group. At 13 weeks of age, the control group had significantly worse hearing thresholds at 12 kHz (56.5 vs. 39.8, p = 0.044) and 16 kHz (64.8 vs. 43.8, p = 0.040) compared to the CMS121 group. Immunohistochemistry showed a significantly lower synapse count per IHC in the control group (15.7) compared to the CMS121 group (18.4), p = 0.014. Our study shows a significant reduction in ABR threshold shifts and increased preservation of IHC ribbon synapses in the mid-range frequencies among mice treated with CMS121 compared to untreated mice.

Transcriptional profiling upon T cell stimulation reveals down-regulation of inflammatory pathways in T and B cells in SLE versus Sjögren's syndrome.

Systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) share clinical as well as pathogenic similarities. Although previous studies suggest various abnormalities in different immune cell compartments, dedicated cell-type specific transcriptomic signatures are often masked by patient heterogeneity. Here, we performed transcriptional profiling of isolated CD4, CD8, CD16 and CD19 lymphocytes from pSS and SLE patients upon T cell stimulation, in addition to a steady-state condition directly after blood drawing, in total comprising 581 sequencing samples. T cell stimulation, which induced a pronounced inflammatory response in all four cell types, gave rise to substantial re-modulation of lymphocyte subsets in the two autoimmune diseases compared to healthy controls, far exceeding the transcriptomic differences detected at steady-state. In particular, we detected cell-type and disease-specific down-regulation of a range of pro-inflammatory cytokine and chemokine pathways. Such differences between SLE and pSS patients are instrumental for selective immune targeting by future therapies.

Human Th17- and IgG3-associated autoimmunity induced by a translocating gut pathobiont.

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases. One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

Suppressive effects of a pyrazole derivative of curcumin on airway inflammation and remodeling.

To advance the control of airway epithelial cell function and asthma, we investigated the effects of a new curcumin derivative, CNB001, which possesses improved pharmacological properties. Normal human bronchial epithelial (NHBE) cells were stimulated with synthetic double-stranded RNA, Poly(I:C). CNB001 significantly suppressed IL-6, TNF-α, and GM-CSF production by NHBE cells, and did so more effectively than did curcumin or dexamethasone (DEX). CNB001 significantly inhibited the decrease of E-cadherin mRNA expression and increase of vimentin mRNA expression observed in NHBE cells induced by a combination of TGF-ß1 and TNF-α, which are markers of airway remodeling. In NHBE cells stimulated by TGF-ß1, CNB001 significantly downregulated the level of active serine peptidase inhibitor clade E member (SERPINE) 1, which is also reported to be related to airway remodeling. Whereas DEX alone significantly increased the active SERPINE1 level, the combination of DEX and CNB001 significantly suppressed active SERPINE1. In addition, CNB001 significantly suppressed neutrophil infiltration, IL-6, TNF-α, IL-13 and active SERPINE1 production in bronchoalveolar lavage fluid of the murine asthma model, which was not observed in the case of DEX. In conclusion, the curcumin derivative, CNB001, is a promising candidate to treat asthma associated with neutrophilic airway inflammation and remodeling.

Cannabinol inhibits oxytosis/ferroptosis by directly targeting mitochondria independently of cannabinoid receptors.

The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics.