Rational design and topochemical synthesis of polymorphs of a polymer

Packing a polymer in different ways can give polymorphs of the polymer having different properties. β-Turn forming peptides such as 2-aminoisobutyric acid (Aib)-rich peptides adopt several conformations by varying the dihedral angles. Aiming at this, a β-turn-forming peptide monomer would give different polymorphs and these polymorphs upon topochemical polymerization would yield polymorphs of the polymer, we designed an Aib-rich monomer N3-(Aib)3-NHCH2-C[triple bond, length as m-dash]CH. This monomer crystallizes as two polymorphs and one hydrate. In all forms, the peptide adopts β-turn conformations and arranges in a head-to-tail manner with their azide and alkyne units proximally placed in a ready-to-react alignment. On heating, both the polymorphs undergo topochemical azide-alkyne cycloaddition polymerization. Polymorph I polymerized in a single-crystal-to-single-crystal (SCSC) fashion and the single-crystal X-ray diffraction analysis of the polymer revealed its screw-sense reversing helical structure. Polymorph II maintains its crystallinity during polymerization but gradually becomes amorphous upon storage. The hydrate III undergoes a dehydrative transition to polymorph II. Nanoindentation studies revealed that different polymorphs of the monomer and the corresponding polymers exhibited different mechanical properties, in accordance with their crystal packing. This work demonstrates the promising future of the marriage of polymorphism and topochemistry for obtaining polymorphs of polymers.Published versionK. M. S. thanks the Department of Science and Technology, Ministry of Science and Technology, Government of India, for a Swarna-Jayanti fellowship (DST/SJF/CSA02/2012-13), and the Science and Engineering Research Board, India, for the research grant (SERB/CRG/000577/2018). All publication charges for this article have been paid for by the Royal Society of Chemistry

Vascular Remodeling and Arterial Calcification Are Directly Mediated by S100A12 (EN-RAGE) in Chronic Kidney Disease

&lt;i&gt;Background:&lt;/i&gt; The proinflammatory cytokine S100A12 (also known as EN-RAGE) is associated with cardiovascular morbidity and mortality in hemodialysis patients. In the cur- rent study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in nonatherosclerosis-prone C57BL/6J mice on normal rodent chow diet, but exposed to the metabolic changes of chronic kidney disease (CKD), would develop vascular disease resembling that observed in patients with CKD. &lt;i&gt;Methods:&lt;/i&gt; CKD was induced in S100A12 transgenic mice and wild-type littermate mice not expressing human S100A12 by surgical ligation of the ureters. The aorta was analyzed after 7 weeks of elevated BUN (blood urea nitrogen), and cultured aortic smooth muscle cells were studied. &lt;i&gt;Results:&lt;/i&gt; We found enhanced vascular medial calcification in S100A12tg mice subjected to CKD. Vascular calcification was mediated, at least in part, by activation of the receptor for S100A12, RAGE (receptor for advanced glycation endproducts), and by enhanced oxidative stress, since inhibition of NADPH-oxidase Nox1 and limited access of S100A12 to RAGE attenuated the calcification and gene expression of osteoblastic genes in cultured vascular smooth muscle cells. &lt;i&gt;Conclusion:&lt;/i&gt; S100A12 augments CKD-triggered osteogenesis in murine vasculature, reminiscent of features associated with enhanced vascular calcification in patients with chronic and end-stage kidney disease.</jats:p

Contrasting Effects of Systemic Monocyte/Macrophage and CD4+ T Cell Depletion in a Reversible Ureteral Obstruction Mouse Model of Chronic Kidney Disease

Using a reversible UUO model (rUUO), we have demonstrated that C57BL/6 mice are susceptible to development of CKD after obstruction-mediated kidney injury while BALB/c mice are resistant. We hypothesized that selective systemic depletion of subpopulations of inflammatory cells during injury or repair might alter the development of CKD. To investigate the impact of modification of Th-lymphocytes or macrophage responses on development of CKD after rUUO, we used an anti-CD4 antibody (GK1.5) or liposomal clodronate to systemically deplete CD4+ T cells or monocyte/macrophages, respectively, prior to and throughout the rUUO protocol. Flow cytometry and immunohistochemistry confirmed depletion of target cell populations. C57BL/6 mice treated with the GK1.5 antibody to deplete CD4+ T cells had higher BUN levels and delayed recovery from rUUO. Treatment of C57BL/6 mice with liposomal clodronate to deplete monocyte/macrophages led to a relative protection from CKD as assessed by BUN values. Our results demonstrate that modulation of the inflammatory response during injury and repair altered the susceptibility of C57BL/6 mice to development of CKD in our rUUO model

Abstract 557: Human S100/calgranulin Accelerates Ectopic Cardiac Calcification and Promotes Cardiac Hypertrophy in Transgenic Mice with Chronic Kidney Disease in a RAGE Dependent Manner

Purpose Chronic kidney disease (CKD) is associated with accelerated cardiovascular disease. Elevated serum S100A12 predicts cardiovascular mortality in patients with end stage renal disease. We test the hypothesis that human S100/calgranulin transgenic mice exposed to the metabolic changes of CKD would develop accelerated cardiovascular disease. Methods A novel humanized mouse with transgenic expression of human S100/calgranulin (hBAC-S100) was generated by expression of a bacterial artificial chromosome of the human S100/calgranulin gene cluster containing genes and regulatory elements for S1008/9 and S100A12 (60kb) in C57BL6/J mice. The hBAC-S100 mice were crossed with the RAGE KO mice (same C57BL6/J strain, gift from Ann Marie Schmidt, NYU) and F4 animals were used for the experiment. CKD was induced in hBAC-S100 and wild type (WT) littermate mice with intact or lacking RAGE signaling by surgical ligation of the ureters. The heart and aorta were analyzed after 10 weeks of elevated blood urea nitrogen. Results hBAC-S100 mice express human S100A12 in circulating myeloid cells and S100A12 was present in the serum of hBAC-S100 mice (25 ng/ml serum), but was not detected in WT mice. Importantly, serum hS100A12 was increased in hBAC-S100 mice with CKD compared to sham operated hBAC-S100 mice (42±17 ng/ml and 25±7 ng/ml, p=0.04). Moreover, hBAC-S100 mice with CKD exhibited ectopic cardiac calcification in fibroblast-rich annulus of the mitral and aortic valves upon Alizarin Red staining. In vivo ECHO showed abnormal mitral valve doppler flow with reduced ratio of early (E) to atrial (A) filling in hBAC-S100 mice with CKD compared to WT-CKD mice (1.17 ±0.1 vs 1.35 ±0.1, p=0.003), indicating diastolic dysfunction. Notably, hBAC-S100 mice with CKD develop cardiac hypertrophy as evidenced by increased heart weight/ body weight ratio and elevated gene expression of hypertrophic and fibrotic markers, such as ANP, β-MHC, TGF-β, CTGF, and Col 1a1. This phenotype was not observed in hBAC-S100 mice lacking RAGE, despite exposure to the same degree of CKD. Conclusion Circulating myeloid derived human S100/calgranulin is associated with the development of ectopic cardiac calcification and cardiac hypertrophy in chronic kidney disease in a RAGE dependent manner. </jats:sec

Asynchronous functional, cellular and transcriptional changes after a bout of eccentric exercise in the rat

Thirty eccentric contractions (ECs) were imposed upon rat dorsiflexors (n = 46) by activating the peroneal nerve and plantarflexing the foot ≈40 deg, corresponding to a sarcomere length change over the range 2.27–2.39 μm for the tibialis anterior and 2.52–2.66 μm for the extensor digitorum longus. Animals were allowed to recover for one of 10 time periods ranging from 0.5 to 240 h, at which time muscle contractile properties, immunohistochemical labelling and gene expression were measured. Peak isometric torque dropped significantly by ≈40 % from an initial level of 0.0530 ± 0.0009 Nm to 0.0298 ± 0.0008 Nm (P < 0.0001) immediately after EC, and then recovered in a linear fashion to control levels 168 h later. Immunohistochemical labelling of cellular proteins revealed a generally asynchronous sequence of events at the cellular level, with the earliest event measured being loss of immunostaining for the intermediate filament protein, desmin. Soon after the first signs of desmin loss, infiltration of inflammatory cells occurred, followed by a transient increase in membrane permeability, manifested as inclusion of plasma fibronectin. The quantitative polymerase chain reaction (QPCR) was used to measure transcript levels of desmin, vimentin, embryonic myosin heavy chain (MHC), myostatin, myoD and myogenin. Compared to control levels, myostatin transcripts were significantly elevated after only 0.5 h, myogenic regulatory factors significantly elevated after 3 h and desmin transcripts were significantly increased 12 h after EC. None of the measured parameters provide a mechanistic explanation for muscle force loss after EC. Future studies are required to investigate whether there is a causal relationship among desmin loss, increased cellular permeability, upregulation of the myoD and desmin genes, and, ultimately, an increase in the desmin content per sarcomere of the muscle

Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background

Chronic kidney disease (CKD) begins with renal injury; the progression thereafter depends upon a number of factors, including genetic background. Unilateral ureteral obstruction (UUO) is a well-described model of renal fibrosis and as such is considered a model of CKD. We used an improved reversible unilateral ureteral obstruction (rUUO) model in mice to study the strain dependence of development of CKD after obstruction-mediated injury. C57BL/6 mice developed CKD after reversal of three or more days of ureteral obstruction as assessed by blood urea nitrogen (BUN) measurements (>40 mg/dl). In contrast, BALB/c mice were resistant to CKD with up to 10 days ureteral obstruction. During rUUO, C57BL/6 mice exhibited pronounced inflammatory and intrinsic proliferative cellular responses, disruption of renal architecture, and ultimately fibrosis. By comparison, BALB/c mice had more controlled and measured extrinsic and intrinsic responses to injury with a return to normal within several weeks after release of ureteral obstruction. Our findings provide a model that allows investigation of the genetic basis of events during recovery from injury that contribute to the development of CKD