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•The photoactive and clickable adenosine analog, 8-N3-2ʹ-O-propargyladenosine was synthesized from 8-bromoadenosine in three steps.•Photo-clickable adenosine is a useful new scaffold ...for chemical probes to identify proteins which interact with a variety of adenosine nucleosides and nucleotides.•8-N3-2ʹ-O-propargyladenosine was converted to its NAD derivative, and enzymatically cyclized using Aplasia californica ADP-ribosyl cyclase. The result was a photoactive and clickable analog of the second messenger cyclic ADP-ribose.•Combining 8‑adenosyl and 2ʹ-O-ribosyl substituents into a cyclic ADP-ribose analog was shown to result in a significant decrease in agonist potency when compared to the potency of the two singly substituted analogs.
A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2ʹ-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N3-2ʹ-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5ʹ-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N3-2ʹ-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N3-2ʹ-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N3-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686–26691. 2ʹ-O-propargyl-cADPR was recognized as an agonist which elicited Ca2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N3-2ʹ-O-propargyl cyclic ADP-ribose did not elicit Ca2+ release at low concentration or impact cyclic ADP-ribose mediated Ca2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We synthesized a directed library of compounds to explore the structure–activity relationships of peroxisome proliferator-activated receptor δ (PPARδ) activation relative to mesenchymal stem cell ...(MSC) osteogenesis. Our scaffold used para-substituted cinnamic acids as a polar headgroup, a heteroatom and heterocycle core connecting units, and substituted phenyl groups for the lipophilic tail. Compounds were screened for their ability to increase osteogenesis in MSCs, and the most promising were examined for subunit specificity using a quantitative PPAR transactivation assay. Six compounds were selected for in vivo studies in an ovariectomized mouse model of human postmenopausal osteoporosis. Four compounds improved bone density in vivo, with two (12d and 31a) having activity comparable to that of GW0742, a well-studied PPARδ-selective agonist. 31a (2-methyl-4-N-methyl-N-5-methylene-4-methyl-2-4-(trifluoromethyl)phenylthiazoleaminocinnamic acid) had the highest selectivity for PPARδ compared to other subtypes, its selectivity far exceeding that of GW0742. Our results confirm that PPARδ is a new drug target for possible treatment of osteoporosis via in situ manipulation of MSCs.
A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined ...within the structure of the adenosine nucleoside itself. We synthesized 8-N
-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N
-2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N
-2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N
-cADPR has previously been shown to be an antagonist of cADPR-induced calcium release T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca
release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N
-2'-O-propargyl cyclic ADP-ribose did not elicit Ca
release at low concentration or impact cyclic ADP-ribose mediated Ca
release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate-ribose (cADPR) are the two fraternal twins that act as Ca2+ mobilizing second messengers apart from inositol ...triphosphate (IP3). These Ca2+ mobilizing second messengers regulate various cellular functions like fertilization, insulin secretion, muscle contraction, etc. but the binding proteins for NAADP and cADPR are unknown. There is a high requisite to know about the binding proteins of NAADP and cADPR. To isolate and purify soluble mammalian NAADP binding proteins we propose to use ligand-affinity chromatography. Affinity chromatography is an isolation technique for proteins using `specific-ligand’ approach, by immobilizing an analog of NAADP to a hydrophilic water insoluble resin using a hydrophilic tetraethyleneglycol spacer arm. This affinity resin absorbs the specific proteins on to the resin which can be then eluted by higher ionic strength solvent or using natural ligand NAADP. Affinity chromatography will enable us to obtain the binding proteins of NAADP to identify the proteins by sequencing. To isolate and purify the cADPR binding proteins we propose to synthesize the bifunctional photoaffinity probes consisting of a photoaffinity label and purification site (clickable site). These photoaffinity probes should contain most of structural properties of naturally occurring substrate plus an added photoreactive group (aromatic azide) which has the potential to form an irreversible covalent bond with an amino acid residue located within the active site of the binding protein and an acetylene moiety to conjugate with an azide linked fluorescent or affinity tags for further purification of the protein. This affinity tag will then permit us to rapidly and efficiently enrich for the derivatized target protein. As a bifunctional cADPR analog we synthesized 8-N3-2'-O-propargyl-cADPR by alkylation of 8-bromoadenosine by propargyl bromide, followed by substitution of bromide with hydrazine by treating with anhydrous hydrazine and by diazotization hydrazine was converted to azide. The adenosine derivative was then phosphorylated and coupling to nicotinamide mononucleotide (NMN) to produce 8-N3-2'-O-propargyl-NAD+. The NAD+ analog was then cyclized enzymatically using Aplysia californica ADP ribosyl cyclase to produce the desire bifunctional 8-N3-2'-O-propargyl-cADPR analog.
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•First DBS method being reported for quantification of PF and its metabolites 5-OHPF and N-DPF by utilizing just 10μL of human blood on LC–MS/MS.•Simple sample processing method and ...shorter run time (1.1min) enables this method as high throughput assay.•Method was validated as per regulatory guidelines.•The method is highly sensitive, specific, and no matrix effect was observed and linear from 5.11–1000ng/mL for PF and 5-OHPF & 0.51–100ng/mL for N-DPF.•The method was used in a human PK study and for the first time the reported PK parameters for 3 analytes are matching with product label information.
A highly sensitive, rapid and selective UHPLC–MS/MS method has been developed and validated for quantification of the propafenone (PF), 5-hydroxypropafenone (5-OHPF) and N-depropylpropafenone (N-DPF) on human dried blood spot (DBS). The assay procedure involves a solid–liquid extraction of PF, 5-OHPF and N-DPF and amlodipine (internal standard, I.S.) from dried human DBS cards using water and acetonitrile. The chromatographic resolution was achieved on a BEH C18 column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile with 0.1% formic acid at flow rate of 0.6mL/min. The UHPLC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Total run time of analysis was 1.1min and elution of PF, 5-OHPF, N-DPF and I.S. occurred at 0.69, 0.6, 0.68 and 0.73min, respectively. A detailed method validation was performed as per the regulatory guidelines and the standard curves found to be linear in the range of 5.11–1000ng/mL for PF and 5-OHPF and 0.51–100ng/mL for N-DPF with a correlation coefficient of ≥0.99 for all the drugs. The intra- and inter-day accuracies were in the range of 95.6–107 and 93.5–103; 93.4–106 and 96.3–107 and 87.9–103 and 96.5–102%, for PF, 5-OHPF and N-DPF, respectively. The intra- and inter-day precisions were in the range of 2.50–5.52 and 3.38–5.18; 2.16–6.34 and 3.23–4.94 and 2.63–7.55 and 1.56–10.2%, for PF, 5-OHPF and N-DPF, respectively. The validated assay method was successfully applied to a pharmacokinetic study in humans. The key pharmacokinetic parameters AUC0-∞ and Cmax were 6057±1526, 2002±515 and 525±202 ng*h/mL and 653±183, 295±37.5 and 68.4±13.6ng/mL for PF, 5-OHPF and N-DPF, respectively.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a mechanosensitive bone cell, on the ...migratory behavior of tumor cells. Two cell lines derived from MDA-MB-231 breast cancer cells were transfected with the vinculin tension sensor to quantitatively evaluate the force in focal adhesions of the tumor cell. Tumor cells treated with MLO-A5 osteocyte-conditioned media (CM) decreased the tensile forces in their focal adhesions and decreased their migratory potential. Tumor cells treated with media derived from MLO-A5 cells exposed to fluid flow-driven shear stress (FFCM) increased the tensile forces and increased migratory potential. Focal adhesion tension in tumor cells was also affected by distance from MLO-A5 cells when the two cells were co-cultured, where tumor cells close to MLO-A5 cells exhibited lower tension and decreased cell motility. Overall, this study demonstrates that focal adhesion tension is involved in altered migratory potential of tumor cells, and tumor-osteocyte interactions decrease the tension and motility of tumor cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
This article discusses currently neglected distinctions between control, care, and conviviality in the politics of technology for sustainability. We conceptualize control as the ambition to maintain ...fictitious borders between hierarchically ordered categories such as subjects and objects. This ambition is materialized into a wide range of Modern technological innovations, including genome editing and deep sea mining. Contrasting with control, we conceptualize values of care that constitute socio-technical practices where connections are prioritized over categories and hierarchy is countered with egalitarian commitment. In caring practices, objects are thus treated as subjects, often within political contexts that are dominated by ambitions to control. Building on care, we explore hopes for conviviality as mutualistic autonomy and decolonial self-realization to orient plural socio-technical pathways for moving beyond Modernity. We argue that such pathways are crucial for democratic transformations to sustainability. We illustrate our concepts using two brief case studies of agricultural developments. The first case discusses the politics of control in agricultural biotechnologies in Belgium. The second case reports on care within rural people's coping strategies in a south Indian "green revolution" landscape laden with control. In conclusion, we emphasize the need to situate attempted materializations of control, care, and conviviality in specific historical junctures. Situated understandings of the interplay between control, care, and conviviality can help realize sustainability that does not reproduce the centralizing, control-driven logic of Modern technocratic development.
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CEKLJ, NUK, ODKLJ, UL, UM, UPUK
The quest for neuroprotective factors that can prevent or slow down the progression of retinal degeneration is still ongoing. Acute hypoxic stress has been shown to provide transient protection ...against subsequent damage in the retina. Stanniocalcins – STC1 and STC2 – are secreted glycoproteins that are hypoxia-regulated and were shown to be cytoprotective in various
in vitro
studies. Hence, we investigated the expression of stanniocalcins in the normal, degenerating and hypoxic retina. We show that the expression of
Stc1
and
Stc2
in the retina was detectable as early as postnatal day 10 and persisted during aging. Retinal expression of
Stc2
, but not
Stc1
, was induced in mice in an
in vivo
model of acute hypoxia and a genetic model of chronic hypoxia. Furthermore, we show that HIF1, not HIF2, is responsible for regulating
Stc2
in cells with the molecular response to hypoxia activated due to the absence of von Hippel Lindau protein. Surprisingly,
Stc2
was not normally expressed in photoreceptors but in the inner retina, as shown by laser capture microdissection and immunofluorescence data. The expression of both
Stc1
and
Stc2
remained unchanged in the degenerative retina with an almost complete loss of photoreceptors, confirming their expression in the inner retina. However, the absence of either
Stc1
or
Stc2
had no effect on retinal architecture, as was evident from retinal morphology of the respective knockout mice. Taken together our data provides evidence for the differential regulation of STC1 and STC2 in the retina and the prospect of investigating STC2 as a retinal neuroprotective factor.
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Background:
HFrEF is associated with significant morbidity and mortality. The standard of care is guideline-directed medical therapy (GDMT) consisting of evidence-based pharmacological ...interventions which have demonstrated improved outcomes in HFrEF. However, paltry adherence to GDMT remains an ongoing challenge. We examined in US adults the relationship between social determinants of health (SDOH), demographics, and risk factors on GDMT adherence for HFrEF.
Methods:
The NIH All of Us Research Program started in May 2018 with the goal of enrolling over 1 million US residents. Over 315,000 participants have shared electronic health records, physical measurements, surveys, and lab results. Demographics, risk factors, and SDOH measures were captured for HFrEF in US adults 18 years or older. Patients with HFrEF who were on quadruple therapy (ACE-inhibitor/angiotensin receptor/combination angiotensin receptor-neprilysin inhibitor, beta-blocker, SGLT2 inhibitor, mineralocorticoid antagonist) were compared to those on fewer. Multiple logistic regression analyses were conducted to examine the association between risk factors/SDOH measures and medical therapy optimization.
Results:
In the All of Us data, 6049 patients were identified with HFrEF. Of those patients, 5838 (97%) patients were on less than 4 GDMT, while 210 (3%) patients were on quadruple therapy. Multiple logistics regression showed participants with ASCVD, and diabetes had higher odds (95% CI) of being on quadruple therapy, 2.19 1.28, 3.99 and 9.07 4.88, 18.84, respectively, while hypertension had lower odds (0.64 0.43, 0.95). Race, income, education, and health insurance types did not predict medical therapy optimization.
Conclusion:
Various cardiovascular conditions were associated with higher odds of being on quadruple therapy. However, sociodemographic factors did not show a correlation of predicting the odds of achieving maximal medical therapy.
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