Determination of internal controls for quantitative gene expression of Spodoptera litura under microbial pesticide stress.

Quantitative real-time polymerase chain reaction (qRT-PCR) has become a commonly used method for the quantification of gene expression. However, accurate qRT-PCR analysis requires a valid internal reference for data normalization. To determine the valid reference characterized with low expression variability among Spodoptera litura samples after microbial pesticide treatments, nine housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), arginine kinase, ubiquitin C, actin-5C (ACT5C), actin, ribosomal protein S13 (RPS13), tubulin, acidic ribosomal protein P0 (RPLP0) and ubiquinol-cytochrome c reductase, were evaluated for their suitability using geNorm, Normfinder, BestKeeper, RefFinder and the comparative delta CT methods in this study. S. litura larvae after direct treatment (larvae were immersed in biopesticides), indirect treatment (larvae were fed with biopesticide immersed artificial diets) and comprehensive treatment (larvae were treated with the first two treatments in sequence), respectively with Metarhizium anisopliae, Empedobacter brevis and Bacillus thuringiensis, were investigated. The results indicated that the best sets of internal references were as follows: RPLP0 and ACT5C for direct treatment conditions; RPLP0 and RPS13 for indirect treatment conditions; RPS13 and GAPDH for comprehensive treatment conditions; RPS13 and RPLP0 for all the samples. These results provide valuable bases for further genetic researches in S. litura.

0.5-bit/s/Hz fine-grained adaptive OFDM modulation for bandlimited underwater VLC.

In this paper, we propose and demonstrate a 0.5-bit/s/Hz fine-grained adaptive orthogonal frequency division multiplexing (OFDM) modulation scheme for bandlimited underwater visible light communication (UVLC) systems. Particularly, integer spectral efficiency is obtained by conventional OFDM with quadrature amplitude modulation (QAM) constellations, while fractional spectral efficiency is obtained by two newly proposed dual-frame OFDM designs. More specifically, OFDM with dual-frame binary phase-shift keying (DF-BPSK) is designed to achieve a spectral efficiency of 0.5 bit/s/Hz, while OFDM with dual-frame dual-mode index modulation (DF-DMIM) is designed to realize the spectral efficiencies of 0.5+n bits/s/Hz with n being a positive integer (i.e., n = 1, 2, …). The feasibility and superiority of the proposed 0.5-bit/s/Hz fine-grained adaptive OFDM modulation scheme in bandlimited UVLC systems are successfully verified by simulations and proof-of-concept experiments. Experimental results demonstrate that a significant achievable rate gain of 18.6 Mbps can be achieved by the proposed 0.5-bit/s/Hz fine-grained adaptive OFDM modulation in comparison to the traditional 1-bit/s/Hz granularity adaptive OFDM scheme, which corresponds to a rate improvement of 22.1%.

Dual-targeting CD33/CD123 NANOBODY T-cell engager with potent anti-AML activity and good safety profile.

ABSTRACT: Novel therapies are needed for effective treatment of acute myeloid leukemia (AML). Relapse is common and salvage treatment with cytotoxic chemotherapy is rarely curative. CD123 and CD33, 2 clinically validated targets in AML, are jointly expressed on blasts and leukemic stem cells in >95% of patients with AML. However, their expression is heterogenous between subclones and between patients, which may affect the efficacy of single-targeting agents in certain patient populations. We present here a dual-targeting CD33/CD123 NANOBODY T-cell engager (CD33/CD123-TCE) that was designed to decrease the risk of relapse from possible single antigen-negative clones and to increase coverage within and across patients. CD33/CD123-TCE killed AML tumor cells expressing 1 or both antigens in vitro. Compared with single-targeting control compounds, CD33/CD123-TCE conferred equal or better ex vivo killing of AML blasts in most primary AML samples tested, suggesting a broader effectiveness across patients. In a disseminated cell-line-derived xenograft mouse model of AML, CD33/CD123-TCE cleared cancer cells in long bones and in soft tissues. As cytokine release syndrome is a well-documented adverse effect of TCE, the compound was tested in a cytokine release assay and shown to induce less cytokines compared to a CD123 single-targeting control. In an exploratory single-dose nonhuman primate study, CD33/CD123-TCE revealed a favorable PK profile. Depletion of CD123 and CD33 expressing cells was observed, but there were neither signs of cytokine release syndrome nor clinical signs of toxicity. Taken together, the CD33/CD123 dual-targeting NANOBODY TCE exhibits potent and safe anti-AML activity and promises a broad patient coverage.

Rubus suavissimus S. Lee Extract Alleviates Oxidative Stress and Inflammation in H2O2-Treated Retinal Pigment Epithelial Cells and in High-Fat Diet-Fed Mouse Retinas.

BACKGROUND: Age-related macular degeneration (AMD) is the most common cause of visual disorders in the aged population and is characterized by the formation of retinal pigment epithelium (RPE) deposits and dysfunction/death of the RPE and photoreceptors. It is supposed that both oxidative stress and inflammation play a critical role in the pathogenesis of AMD. The development of therapeutic strategies against oxidative stress and inflammation in AMD is urgently needed. Rubus suavissimus S. Lee (RS), a medicinal plant growing in the southwest region of China, has been used as an herbal tea and medicine for various diseases. METHODS: In this project, we evaluate the therapeutic potential of RS extract for AMD. We prepared RS extracts from dried leaves, which contained the main functional compounds. RESULTS: RS extract significantly increased cell viability, upregulated the expression of antioxidant genes, lowered the generation of malondialdehyde and reactive oxygen species, and suppressed inflammation in H2O2-treated human RPE cells. In the in vivo study, treatment with RS extract attenuated body weight gain, lowered cholesterol and triglyceride levels in the liver and serum, increased antioxidant capacity, and alleviated inflammation in the retina and RPE/choroid of mice fed a high-fat diet. CONCLUSIONS: Our findings suggest that RS extract offers therapeutic potential for treating AMD patients.

Impact of Poloxamer on Crystal Nucleation and Growth of Amorphous Clotrimazole.

Surfactants have been widely used as effective additives to increase the solubility and dissolution rates of amorphous solid dispersions (ASDs). However, they may also generate adverse effects on the physical stability of ASDs. In this study, we systematically investigated the impacts of poloxamer, a frequently used surfactant, on the crystallization of amorphous clotrimazole (CMZ). The added poloxamer significantly decreased the glass transition temperature (Tg) of CMZ and accelerated the growth of Form 1 and Form 2 crystals. It was found that the poloxamer had an accelerating effect on Form 1 and Form 2 but showed a larger accelerating effect on Form 1, which resulted from a combined effect of increased mobility and local phase separation at the crystal-liquid interface. Additionally, the added poloxamer exhibited different effects on nucleation of the CMZ polymorphs, which was more complicated than crystal growth. The nucleation rate of Form 1 was significantly increased by the added poloxamer, and the effect increased with increasing P407 content. However, for Form 2, nucleation was slightly decreased or unchanged. The nucleation of Form 2 may have been influenced by the Form 1 crystallization, and Form 2 converted to Form 1 during nucleation. This study increases our understanding of poloxamer and its impacts on the melt crystallization of drugs.

A Phase Ib/II Study of Ivosidenib with Venetoclax ± Azacitidine in IDH1-Mutated Myeloid Malignancies.

The safety and efficacy of combining the isocitrate dehydrogenase-1 (IDH1) inhibitor ivosidenib (IVO) with the BCL2 inhibitor venetoclax (VEN; IVO + VEN) ± azacitidine (AZA; IVO + VEN + AZA) were evaluated in four cohorts of patients with IDH1-mutated myeloid malignancies (n = 31). Most (91%) adverse events were grade 1 or 2. The maximal tolerated dose was not reached. Composite complete remission with IVO + VEN + AZA versus IVO + VEN was 90% versus 83%. Among measurable residual disease (MRD)-evaluable patients (N = 16), 63% attained MRD--negative remissions; IDH1 mutation clearance occurred in 64% of patients receiving ≥5 treatment cycles (N = 14). Median event-free survival and overall survival were 36 [94% CI, 23-not reached (NR)] and 42 (95% CI, 42-NR) months. Patients with signaling gene mutations appeared to particularly benefit from the triplet regimen. Longitudinal single-cell proteogenomic analyses linked cooccurring mutations, antiapoptotic protein expression, and cell maturation to therapeutic sensitivity of IDH1-mutated clones. No IDH isoform switching or second-site IDH1 mutations were observed, indicating combination therapy may overcome established resistance pathways to single-agent IVO. SIGNIFICANCE: IVO + VEN + AZA is safe and active in patients with IDH1-mutated myeloid malignancies. Combination therapy appears to overcome resistance mechanisms observed with single-agent IDH-inhibitor use, with high MRD-negative remission rates. Single-cell DNA ± protein and time-of-flight mass-cytometry analysis revealed complex resistance mechanisms at relapse, highlighting key pathways for future therapeutic intervention. This article is highlighted in the In This Issue feature, p. 247.

Effects of in vitro simulated digestion and fecal fermentation of polysaccharides from straw mushroom (Volvariella volvacea) on its physicochemical properties and human gut microbiota.

Herein, the fermentation and digestion behavior of Volvariella volvacea polysaccharide (VVP) were examined through the in vitro simulation experiment. The results revealed that succeeding the simulated salivary gastrointestinal digestion, the molecular weight of VVP was reduced by only 8.9 %. In addition, the reducing sugar, uronic acid, monosaccharide composition and Fourier transform infrared spectroscopy characteristics of VVP did not change significantly, which indicate that saliva-gastrointestinal could not digest VVP. However, 48 h of fecal fermentation of VVP dramatically reduced its molecular weight by 40.4 %. Furthermore, the molar ratios of the monosaccharide composition altered considerably due to the degradation of VVP by microorganisms and the metabolysis into different short-chain fatty acids (SCFAs). Meanwhile, the VVP also raised the proportion of Bacteroidetes to Firmicutes and promoted the proliferation of some beneficial bacteria including Bacteroides and Phascolarctobacterium, whereas it inhibited the growth of unfavorable bacteria such as Escherichia-shigella. Therefore, VVP has the potential to have a positive influence on health and hinder diseases by improving the intestinal microbial environment. These findings provide a theoretical foundation to further develop Volvariella volvacea as a healthy functional food.

Discovery of a new polymorph of clotrimazole through melt crystallization: Understanding nucleation and growth kinetics.

Clotrimazole (CMZ) is a classical antifungal drug for studying crystallization. In this study, a new CMZ polymorph (Form 2) was discovered during the process of nucleation and growth rate determination in the melt. High-quality single crystals were grown from melt microdroplets to determine the crystal structure by x-ray diffraction. Form 2 is metastable and exhibits a disordered structure. The crystal nucleation and growth kinetics of the two CMZ polymorphs were systematically measured. Form 2 nucleates and grows faster than the existing form (Form 1). The maximum nucleation rate of Forms 1 and 2 was observed at 50 °C (1.07 Tg). The summary of the maximum nucleation rate temperature of CMZ and the other six organic compounds indicates that nucleation near Tg in the supercooled liquid is a useful approach to discovering new polymorphs. This study is relevant for the discovering new drug polymorphs through an understanding of nucleation and growth kinetics during melt crystallization.

Triple combination targeting methyltransferase, BCL-2, and PD-1 facilitates antileukemia responses in acute myeloid leukemia.

BACKGROUND: A recent breakthrough therapy combining the BCL-2 inhibitor venetoclax with hypomethylating agents (HMAs) targeting DNA methyltransferase has improved outcomes for patients with acute myeloid leukemia (AML), but the responses and long-term survival in older/unfit patients and in patients with relapsed/refractory AML remain suboptimal. Recent studies showed that inhibition of BCL-2 or DNA methyltransferase modulates AML T-cell immunity. METHODS: By using flow cytometry and time-of-flight mass cytometry, the authors examined the effects of the HMA decitabine combined with the BCL-2 inhibitor venetoclax (DAC/VEN therapy) on leukemia cells and T cells in patients with AML who received DAC/VEN therapy in a clinical trial. The authors investigated the response of programmed cell death protein 1 (PD-1) inhibition in the DAC/VEN-treated samples in vitro and investigated the triple combination of PD-1 inhibition with HMA/venetoclax in the trial patients who had AML. RESULTS: DAC/VEN therapy effectively targeted leukemia cells and upregulated the expression of the immune checkpoint-inhibitory receptor PD-1 in T cells while preserving CD4-positive and CD8-positive memory T cells in a subset of patients with AML who were tested. In vitro PD-1 inhibition potentiated the antileukemia response in DAC/VEN-treated AML samples. The combined use of azacitidine, venetoclax, and nivolumab eliminated circulating blasts and leukemia stem cells/progenitor cells and expanded the percentage of CD8-positive memory T cells in an illustrative patient with relapsed AML who responded to the regimen in an ongoing clinical trial. CONCLUSIONS: Immunomodulation by targeting PD-1 enhances the therapeutic effect of combining an HMA and venetoclax in patients with AML.

The Price of Becoming a City: Decentralization and Air Pollution-The Evidence from the Policy of County-to-City Upgrade in China.

It is necessary to reassess the pollution effects of decentralization reforms to improve the future policy design for better economic and social development in the postepidemic era. This study examines the relationship between decentralization and air pollution by exploiting the policy of County-to-City Upgrade in China from 2005 to 2018. Upgrading empowered new cities in fiscal, administrative, and economic matters without changing the political hierarchy. Under the cadre evaluation system, the new county-level city government preferred to increase construction land area and attract more polluting firms to promote economic development, and air pollution became more severe. Heterogeneity tests found that when the new city was located in eastern China or was away from the provincial boundary, decentralization would induce more severe air pollution. Overall, decentralization without a supplementary incentive rule has a negative effect on air pollution.

Bio-nanocomplexes with autonomous O2 generation efficiently inhibit triple negative breast cancer through enhanced chemo-PDT.

As one kind of aggressive cancer, triple-negative breast cancer (TNBC) has become one of the major causes of women mortality worldwide. Recently, combinational chemo-PDT therapy based on nanomaterials has been adopted for the treatment of malignant tumor. However, the efficacy of PDT was partly compromised under tumor hypoxia environment due to the lack of sustainable O2 supply. In this study, CeO2-loaded nanoparticles (CeNPs) with peroxidase activity were synthesized to autonomously generate O2 by decomposing H2O2 within tumor region and reprogramming the hypoxia microenvironment as well. Meanwhile, the compound cinobufagin (CS-1) was loaded for inhibiting TNBC growth and metastasis. Moreover, the hybrid membrane camouflage was adopted to improve the biocompatibility and targeting ability of nanocomplexes. In vitro assay demonstrated that decomposition of H2O2 by CeO2 achieved sustainable O2 supply, which accordingly improved the efficacy of PDT. In turn, the generated O2 improved the cytotoxicity and anti-tumor migration effect of CS-1 by downregulating HIF-1α and MMP-9 levels. In vivo assay demonstrated that the combination of CS-1 and PDT significantly inhibited the growth and distance metastasis of tumor in MDA-MB-231 bearing mice. Thus, this chemo-PDT strategy achieved satisfactory therapeutic effects by smartly utilizing the enzyme activity of nanodrugs and special micro-environment of tumor.

Constellation design of DFT-S-OFDM with dual-mode index modulation in VLC.

In this paper, we for the first time propose a novel partitioning-based constellation design approach for discrete Fourier transform-spread-orthogonal frequency division multiplexing modulation with dual-mode index modulation (DFT-S-OFDM-DM) in visible light communication (VLC) systems. Specifically, two partitioning-based constellation designs, i.e., block-based constellation partitioning and interleaving-based constellation partitioning, are proposed to generate two distinguishable constellation sets for DFT-S-OFDM-DM in VLC, by considering four 8-ary constellations including 8-ary quadrature amplitude modulation (8-QAM), 8-ary phase-shift keying (8-PSK), circular (4,4)-QAM, and circular (7,1)-QAM. The superiority of DFT-S-OFDM-DM using circular (7,1)-QAM constellation with interleaving-based constellation partitioning over other benchmark schemes has been successfully verified by both simulation and experimental results. It is shown by the experimental results that a significant distance extension of 44.6% is obtained by DFT-S-OFDM-DM using circular (7,1)-QAM constellation with interleaving-based constellation partitioning in comparison to DFT-S-OFDM with index modulation achieving the same spectral efficiency of 2.5 bits/s/Hz. It is also demonstrated that the proposed constellation design schemes are also generally applicable to the constellation with an arbitrary shape and an arbitrary size.

The antisolvent coprecipitation method for enhanced bioavailability of poorly water-soluble drugs.

In recent years, poorly water-soluble drug candidates in the drug development pipeline have been a challenging issue for the pharmaceutical industry. Many delivery systems, such as nanocrystals, cocrystals, nanoparticles, and amorphous solid dispersions (ASDs) have been developed to overcome these problems. A large number of methods are utilized to realize the above delivery systems. Among all the preparation methods, the antisolvent coprecipitation method is a relatively simple, cost-effective method, offering many advantages over conventional methods. An overview of recent developments for each solubility enhancement approach using the antisolvent coprecipitation method is presented. This current review details a comprehensive overview of the antisolvent coprecipitation process and its properties, as well as the fundamentals for enhancing the solubility and bioavailability of poorly water-soluble drugs by nanotization, polymorph control with polymers and/or surfactants. Furthermore, this review also presents insights into the factors affecting the antisolvent coprecipitation process.

Non-neglectable therapeutic options for age-related macular degeneration: A promising perspective from traditional Chinese medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Age-related macular degeneration (AMD) is a chronic neurodegenerative disease which causes irreversible central vision loss among the elderly population. Traditional Chinese Medicine (TCM), including formulas, acupuncture and herbs, has been used in the treatment of AMD for thousands of years and is currently used by many AMD patients around the world. AIM OF THE REVIEW: A comprehensive, in-depth literature review examining the use of TCM in the treatment of AMD has yet to be compiled. This review will improve current knowledge relating to the use of TCM and will open new avenues of exploration in developing new drugs for the treatment of AMD. METHODS: A literature search of the PubMed database, Web of Science, Google Scholar and China National Knowledge Infrastructure (CNKI) was performed using relevant terms and keywords related to TCM in the treatment of AMD. Related books, PhD and master's theses were also researched. RESULTS: The TCM-based interpretation of AMD has been used to establish a theoretical foundation for understanding the effect of TCM formulas and acupuncture on AMD. The possible mechanism of action of common Chinese herbs has also been discussed in detail. CONCLUSION: TCM is a promising treatment option of AMD patients. However, lack of rigorous scientific evidence has limited the impact and uptake of TCM therapy. Future research should focus on improving understanding of the mechanism of action and bioactive components of TCM therapies.

Metabolomics in Retinal Diseases: An Update.

Retinal diseases are a leading cause of visual loss and blindness, affecting a significant proportion of the population worldwide and having a detrimental impact on quality of life, with consequent economic burden. The retina is highly metabolically active, and a number of retinal diseases are associated with metabolic dysfunction. To better understand the pathogenesis underlying such retinopathies, new technology has been developed to elucidate the mechanism behind retinal diseases. Metabolomics is a relatively new "omics" technology, which has developed subsequent to genomics, transcriptomics, and proteomics. This new technology can provide qualitative and quantitative information about low-molecular-weight metabolites (M.W. < 1500 Da) in a given biological system, which shed light on the physiological or pathological state of a cell or tissue sample at a particular time point. In this article we provide an extensive review of the application of metabolomics to retinal diseases, with focus on age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), glaucoma, and retinitis pigmentosa (RP).

Gypenosides Alleviate Cone Cell Death in a Zebrafish Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of visual disorders caused by mutations in over 70 genes. RP is characterized by initial degeneration of rod cells and late cone cell death, regardless of genetic abnormality. Rod cells are the main consumers of oxygen in the retina, and after the death of rod cells, the cone cells have to endure high levels of oxygen, which in turn leads to oxidative damage and cone degeneration. Gypenosides (Gyp) are major dammarane-type saponins of Gynostemma pentaphyllum that are known to reduce oxidative stress and inflammation. In this project we assessed the protective effect of Gyp against cone cell death in the rpgrip1 mutant zebrafish, which recapitulate the classical pathological features found in RP patients. Rpgrip1 mutant zebrafish were treated with Gyp (50 µg/g body weight) from two-months post fertilization (mpf) until 6 mpf. Gyp treatment resulted in a significant decrease in cone cell death compared to that of untreated mutant zebrafish. A markedly low level of reactive oxygen species and increased expression of antioxidant genes were detected in Gyp-incubated mutant zebrafish eyes compared to that of untreated mutant zebrafish. Similarly, the activities of catalase and superoxide dismutase and the level of glutathione were significantly increased in Gyp-treated mutant zebrafish eyes compared to that of untreated mutant zebrafish. Gyp treatment also decreased endoplasmic reticulum stress in rpgrip1 mutant eyes. Expression of proinflammatory cytokines was also significantly decreased in Gyp-treated mutant zebrafish eyes compared to that of untreated mutant zebrafish. Network pharmacology analysis demonstrated that the promotion of cone cell survival by Gyp is possibly mediated by multiple hub genes and associated signalling pathways. These data suggest treatment with Gyp will benefit RP patients.

Anti-inflammatory activities of Gardenia jasminoides extracts in retinal pigment epithelial cells and zebrafish embryos.

Age-related macular degeneration (AMD) is the most common cause of visual impairment in developed countries. Inflammation serves a critical role in the pathogenesis of AMD. Gardenia jasminoides is found in several regions of China and is traditionally used as an organic yellow dye but has also been widely used as a therapeutic agent in numerous diseases, including inflammation, depression, hepatic and vascular disorders, which may reflect the variability of functional compounds that are present in Gardenia jasminoides extracts (GJE). To investigate the therapeutic potential of GJE for AMD, ARPE-19 cells were treated with lipopolysaccharide (LPS) or LPS plus GJE. GJE significantly decreased LPS-induced expression of proinflammatory cytokines, including IL-1ß, IL-6 and TNF-α. In the in vivo study, GJE inhibited CuSO4-induced migration of primitive macrophages to the lateral line in zebrafish embryos. GJE also attenuated expression of cytokines (IL-1ß, IL-6 and TNF-α), NFKB activating protein (nkap) and TLR4 in ARPE-19 cells. The results of the present study demonstrated the anti-inflammatory potential of GJE in vitro and in vivo, and suggested GJE as a therapeutic candidate for AMD.

Preclinical Characterization of the Selective JAK1 Inhibitor LW402 for Treatment of Rheumatoid Arthritis.

BACKGROUND: Research on JAK family members as therapeutic targets for autoimmune diseases has brought tofacitinib and baricitinib into clinical for the treatment of rheumatoid arthritis and other autoimmune diseases. Despite the potent efficacy of these first-generation JAK inhibitors, their broad-spectrum JAK inhibition and adverse events warrant development of a JAK1-specific inhibitor to improve their safety profile. METHODS: In this study, we characterized a JAK1-specific inhibitor, LW402, on biochemical and human whole-blood assays. We further evaluated the therapeutic efficacy of LW402 in a rat adjuvant-induced arthritis (rAIA) model and a mouse collagen-induced arthritis (mCIA) model. The safety of LW402 was evaluated in both SpragueDawley rats and cynomolgus monkeys. RESULTS: LW402 exhibited potent nanomolar activity against JAK1 and showed a 45-fold selectivity for inhibition of JAK1- over JAK2-dependent signaling induced by either IL6 or GM-CSF in human whole-blood assays. In the rAIA model, oral dosing of LW402 resulted in a dose-dependent improvement in disease symptoms, including reduction in paw swelling, marked reduction in the inflammatory-cell infiltration to synovial tissue, and protection of articular cartilage and bone from damage. The therapeutic efficacy of LW402 correlated well with the plasma exposure of LW402 and the extent of pSTAT3 inhibition in white blood cells. LW402 also effectively eased disease symptoms in the mCIA model. Toxicity studies in the Sprague Dawley rats and cynomolgus monkeys established a ≥5x therapeutic window for LW402 as drug exposures of toxicity study NOAEL dose and pharmacology study ED50 dose were compared. CONCLUSION: We developed a novel JAK1-specific inhibitor LW402 with potent efficacy in rAIA and mCIA models. We established a good safety profile for LW402 in toxicity studies, and the overall superiority of LW402 should translated well to the clinical setting for the treatment of RA and other autoimmune diseases.

Effect of a TSPO ligand on retinal pigment epithelial cholesterol homeostasis in high-fat fed mice, implication for age-related macular degeneration.

Age-related Macular Degeneration (AMD) is a major cause of sight impairment in the elderly with complex aetiology involving genetics and environment and with limited therapeutic options which have limited efficacy. We have previously shown in a mouse-model of the condition, induced by feeding a high fat diet, that adverse effects of the diet can be reversed by co-administration of the TSPO activator, etifoxine. We extend those observations showing improvements in retinal pigment epithelial (RPE) cells with decreased lipids and enhanced expression of cholesterol metabolism and transport enzymes. Further, etifoxine decreased levels of reactive oxygen species (ROS) in RPE and inflammatory cytokines in RPE and serum. With respect to gut microbiome, we found that organisms abundant in the high fat condition (e.g. in the genus Anaerotruncus and Oscillospira) and implicated in AMD, were much less abundant after etifoxine treatment. The changes in gut flora were associated with the predicted production of metabolites of benefit to the retina including tryptophan and other amino acids and taurine, an essential component of the retina necessary to counteract ROS. These novel observations strengthen earlier conclusions that the mechanisms behind improvements in etifoxine-induced retinal physiology involve an interaction between effects on the host and the gut microbiome.

High-throughput proteomic profiling reveals mechanisms of action of AMG925, a dual FLT3-CDK4/6 kinase inhibitor targeting AML and AML stem/progenitor cells.

FLT3 mutations, which are found in a third of patients with acute myeloid leukemia (AML), are associated with poor prognosis. Responses to currently available FLT3 inhibitors in AML patients are typically transient and followed by disease recurrence. Thus, FLT3 inhibitors with new inhibitory mechanisms are needed to improve therapeutic outcomes. AMG925 is a novel, potent, small-molecule dual inhibitor of FLT3 and CDK4/6. In this study. we determined the antileukemic effects and mechanisms of action of AMG925 in AML cell lines and primary samples, in particular AML stem/progenitor cells. AMG925 inhibited cell growth and promoted apoptosis in AML cells with or without FLT3 mutations. Reverse-phase protein array profiling confirmed its on-target effects on FLT3-CDK4/6-regulated pathways and identified unrevealed signaling network alterations in AML blasts and stem/progenitor cells in response to AMG925. Mass cytometry identified pathways that may confer resistance to AMG925 in phenotypically defined AML stem/progenitor cells and demonstrated that combined blockade of FLT3-CDK4/6 and AKT/mTOR signaling facilitated stem cell death. Our findings provide a rationale for the mechanism-based inhibition of FLT3-CDK4/6 and for combinatorial approaches to improve the efficacy of FLT3 inhibition in both FLT3 wild-type and FLT3-mutated AML.