Lithocholic Acid Amides as Potent Vitamin D Receptor Agonists

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3, 1] is an active form of vitamin D3 and regulates various biological phenomena, including calcium and phosphate homeostasis, bone metabolism, and immune response via binding to and activation of vitamin D receptor (VDR). Lithocholic acid (LCA, 2) was identified as a second endogenous agonist of VDR, though its potency is very low. However, the lithocholic acid derivative 3 (Dcha-20) is a more potent agonist than 1α,25(OH)2D3, (1), and its carboxyl group has similar interactions to the 1,3-dihydroxyl groups of 1 with amino acid residues in the VDR ligand-binding pocket. Here, we designed and synthesized amide derivatives of 3 in order to clarify the role of the carboxyl group. The synthesized amide derivatives showed HL-60 cell differentiation-inducing activity with potency that depended upon the substituent on the amide nitrogen atom. Among them, the N-cyanoamide 6 is more active than either 1 or 3.

Leukocyte telomere length and response to antiangiogenic therapy in patients with neovascular age-related macular degeneration

Возрастная макулярная дегенерация (ВМД) становится основной причиной потери зрения людьми старше 60 лет. Неоваскулярная форма ВМД характеризуется хориоидальной неоваскуляризацией, основным триггером которой является фактор роста эндотелия сосудов (VEGF), ингибирование которого - современный стандарт лечения. Значительная вариабельность ответа на анти-VEGF-терапию определяет актуальность поиска биологических маркеров - прогностических критериев ответа на лечение. Проведен анализ зависимости ответа 110 пациентов с нВМД на анти-VEGF-терапию от функциональных и анатомических параметров сетчатки (по данным оптической когерентной томографии) и длины теломер лейкоцитов (ДТЛ, оценивали методом количественной ПЦР). В 100 % глаз наблюдали положительную динамику максимально корригированной остроты зрения (МКОЗ). Центральная толщина сетчатки (ЦТС) снизилась после третьей инъекции до 265 [234-306] мкм, к концу периода наблюдения - до 211 [190-262] мкм. Сохранение активности субретинальной неоваскулярной мембраны (СНМ) в конце периода наблюдения коррелировало с более низкими показателями исходной МКОЗ и высокими значениями исходной ЦТС. Выявлена ассоциация ДТЛ с ответом на лечение: у пациентов с большей ДТЛ чаще наблюдали переход активной формы СНМ в неактивную уже после трех инъекций, в то время как при меньшей ДТЛ чаще сохранялась активность СНМ, что определяло потребность в большем числе интравитреальных инъекций. Age-related macular degeneration (AMD) is becoming the leading cause of vision loss in people over 60 years of age. The neovascular form of AMD (nVMD) is characterized by choroidal neovascularization (CNV), the main trigger of which is vascular endothelial growth factor (VEGF), the inhibition of which is the current standard of treatment. Significant variability of response to anti-VEGF therapy determines the relevance of the search for biological markers - prognostic criteria of treatment response. We analyzed the response of 110 nVMD patients to anti-VEGF therapy depending on the functional and anatomical parameters of the retina (according to optical coherence tomography, OCT) and leukocyte telomere length (LTL, was assessed by quantitative PCR). Positive dynamics of best corrected visual acuity (BCVA) was observed in 100 % of eyes. The central retinal thickness (CRT) decreased after the 3rd injection to 265 [234-306] µm, by the end of the observation period - to 211 [190-262] µm. The retention of activity of the subretinal neovascular membrane (SNM) at the end of the observation period correlated with lower values of the initial BCVA and high values of the initial CRT. An association of LTL with response to treatment was revealed: in patients with higher LTL the active form of SNM was more often switched to inactive after three injections, while with lower LTL, the activity of SNM was more often preserved, which determined the need for more intravitreal injections.

Emerging Roles of Vitamin D-Induced Antimicrobial Peptides in Antiviral Innate Immunity

Vitamin D deficiency, characterized by low circulating levels of calcifediol (25-hydroxyvitamin D, 25D) has been linked to increased risk of infections of bacterial and viral origin. Innate immune cells produce hormonal calcitriol (1,25-dihydroxyvitamin D, 1,25D) locally from circulating calcifediol in response to pathogen threat and an immune-specific cytokine network. Calcitriol regulates gene expression through its binding to the vitamin D receptor (VDR), a ligand-regulated transcription factor. The hormone-bound VDR induces the transcription of genes integral to innate immunity including pattern recognition receptors, cytokines, and most importantly antimicrobial peptides (AMPs). Transcription of the human AMP genes β-defensin 2/defensin-β4 (HBD2/DEFB4) and cathelicidin antimicrobial peptide (CAMP) is stimulated by the VDR bound to promoter-proximal vitamin D response elements. HDB2/DEFB4 and the active form of CAMP, the peptide LL-37, which form amphipathic secondary structures, were initially characterized for their antibacterial actively. Notably, calcitriol signaling induces secretion of antibacterial activity in vitro and in vivo, and low circulating levels of calcifediol are associated with diverse indications characterized by impaired antibacterial immunity such as dental caries and urinary tract infections. However, recent work has also provided evidence that the same AMPs are components of 1,25D-induced antiviral responses, including those against the etiological agent of the COVID-19 pandemic, the SARS-CoV2 coronavirus. This review surveys the evidence for 1,25D-induced antimicrobial activity in vitro and in vivo in humans and presents our current understanding of the potential mechanisms by which CAMP and HBD2/DEFB4 contribute to antiviral immunity.

High Dimensional Analyses of Circulating Immune Cells in Psoriatic Arthritis Detects Elevated Phosphorylated STAT3

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.

Insulin Complexation with Cyclodextrins—A Molecular Modeling Approach

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in β-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted β-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5–7 molecules of hydroxypropyl-β-cyclodextrin or sulfobutylether-β-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

Low Temperature Delays Metabolism of Quizalofop in Resistant Winter Wheat and Three Annual Grass Weed Species

Quizalofop-resistant wheat is the core component of the recently commercialized CoAXium™ Wheat Production System. As with other herbicides, quizalofop provides better weed control at early growth stages and under optimum temperature. However, in regions with winter wheat production, quizalofop application may be affected by unpredictable, rapid temperature decreases. Temperature shifts can cause crop injury or impact weed control efficacy. In the following study, we examine the effect of reduced temperature on quizalofop content and metabolism in CoAXium™ winter wheat and three winter weed species: downy brome (Bromus tectorum L.), feral rye (Secale cereale L.), and jointed goatgrass (Aegilops cylindrica Host). Temperature conditions include either 19 or 4.5°C daytime temperatures with tissue sampling over 5 timepoints (1–16 or 18 days after treatment, DAT). Analysis features liquid chromatography coupled to tandem mass spectrometry detection of the active form of quizalofop, quizalofop acid. Quizalofop content trends reveal delayed metabolism under cooler conditions for wheat and weeds. Quizalofop content peaks within 1–2 DAT in the warmer temperatures for all species and decreases thereafter. In contrast, content peaks between 8 and 9 DAT at cooler temperatures except for downy brome. Minimal decreases in content over time generally follow cooler temperature peaks. Further, the absence of differences in maximum quizalofop content in all species suggests absorption and/or de-esterification of quizalofop proherbicide to the active form is not reduced at cooler temperatures. Final dry shoot tissue biomass does not necessarily correspond to differences in metabolism, as biomass of wheat treated with a field rate of quizalofop does not differ between temperatures. Weeds were treated with sublethal doses of quizalofop in order to monitor herbicide metabolism without causing plant death. Under this condition, weed biomass only differs for jointed goatgrass, which has a greater biomass in the cooler temperature.

Effects of Host Genetic Polymorphisms on the Efficacy of the Radical Cure Malaria Drug Primaquine

Malaria is a major cause of death in low-income countries. Malaria relapses are caused by Plasmodium vivax–induced latent liver stage hypnozoites, and relapses contribute significantly to the total disease burden. The goal of malaria elimination is threatened in countries where P. vivax is endemic and relapses remain a key aspect of concern. Targeting of the hypnozoites is crucial for radical cure and this is achieved by primaquine (PQ). In addition to its anti-hypnozoite effects, PQ also possesses gametocidal activity against all malaria causing Plasmodium species and is hence a useful tool to curtail malaria transmission. It is well known that host glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with hemolysis after treatment with PQ. Multiple other host polymorphisms impact on PQ metabolism, potentially affecting drug efficacy. Being a prodrug, PQ requires host factors cytochrome P450 2D6 (CYP2D6), cytochrome P450 NADPH: oxidoreductase (CPR) and monoamine oxidase (MAO) for its metabolism and conversion to active form. The efficacy of PQ in the host is therefore dependent on genetic polymorphisms of these three host genes. The efficacy of PQ is important for clearing reservoirs of P. vivax infection. Here, we have analyzed the known spectrum of genetic polymorphisms for host genes that enable PQ metabolism. It is vital to delineate the polymorphisms that determine the ultimate efficacy of PQ for formulating better malaria elimination strategies in countries with severe malaria burden. Thus population-based studies of these gene variants will provide new insights into the role of host genetics on PQ treatment outcomes.

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

Synthesis of C2-Alkoxy-Substituted 19-Nor Vitamin D3 Derivatives: Stereoselectivity and Biological Activity

The active form of vitamin D3 (D3), 1a,25-dihydroxyvitamn D3 (1,25D3), plays a central role in calcium and bone metabolism. Many structure–activity relationship (SAR) studies of D3 have been conducted, with the aim of separating the biological activities of 1,25D3 or reducing its side effects, such as hypercalcemia, and SAR studies have shown that the hypercalcemic activity of C2-substituted derivatives and 19-nor type derivatives is significantly suppressed. In the present paper, we describe the synthesis of 19-nor type 1,25D3 derivatives with alkoxy groups at C2, by means of the Julia–Kocienski type coupling reaction between a C2 symmetrical A ring ketone and a CD ring synthon. The effect of C2 substituents on the stereoselectivity of the coupling reaction was evaluated. The biological activities of the synthesized derivatives were evaluated in an HL-60 cell-based assay. The a-methoxy-substituted C2α-7a was found to show potent cell-differentiating activity, with an ED50 value of 0.38 nM, being 26-fold more potent than 1,25D3.

Relationship between the Crystal Structure and Tuberculostatic Activity of Some 2-Amidinothiosemicarbazone Derivatives of Pyridine

Tuberculosis remains one of the most common diseases affecting developing countries due to difficult living conditions, the rapidly increasing resistance of M. tuberculosis strains and the small number of effective anti-tuberculosis drugs. This study concerns the relationship between molecular structure observed in a solid-state by X-ray diffraction and the 15N NMR of a group of pyridine derivatives, from which promising activity against M. tuberculosis was reported earlier. It was found that the compounds exist in two tautomeric forms: neutral and zwitterionic. The latter form forced the molecules to adopt a stable, unique, flat frame due to conjugation and the intramolecular hydrogen bond system. As the compounds exist in a zwitterionic form in the crystal state generally showing higher activity against tuberculosis, it may indicate that this geometry of molecules is the “active” form.