PURION® processed human amnion chorion membrane allografts retain material and biological properties supportive of soft tissue repair.

The reparative properties of amniotic membrane allografts are well-suited for a broad spectrum of specialties. Further enhancement of their utility can be achieved by designing to the needs of each application through the development of novel processing techniques and tissue configurations. As such, this study evaluated the material characteristics and biological properties of two PURION® processed amniotic membrane products, a lyophilized human amnion, intermediate layer, and chorion membrane (LHACM) and a dehydrated human amnion, chorion membrane (DHACM). LHACM is thicker; therefore, its handling properties are ideal for deep, soft tissue deficits; whereas DHACM is more similar to a film-like overlay and may be used for shallow defects or surgical on-lays. Characterization of the similarities and differences between LHACM and DHACM was conducted through a series of in vitro and in vivo studies relevant to the healing cascade. Compositional analysis was performed through histological staining along with assessment of barrier membrane properties through equilibrium dialysis. In vitro cellular response was assessed in fibroblasts and endothelial cells using cell proliferation, migration, and metabolic assays. The in vivo cellular response was assessed in an athymic nude mouse subcutaneous implantation model. The results indicated the PURION® process preserved the native membrane structure, nonviable cells and collagen distributed in the individual layers of both products. Although, LHACM is thicker than DHACM, a similar composition of growth factors, cytokines, chemokines and proteases is retained and consequently elicit comparable in vitro and in vivo cellular responses. In culture, both treatments behaved as potent mitogens, chemoattractants and stimulants, which translated to the promotion of cellular infiltration, neocollagen deposition and angiogenesis in a murine model. PURION® processed LHACM and DHACM differ in physical properties but possess similar in vitro and in vivo activities highlighting the impact of processing method on the versatility of clinical use of amniotic membrane allografts.

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells.

Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.

Robust synthesis of 2'-azido modified RNA from 2'-amino precursors by diazotransfer reaction.

Azides are versatile bioorthogonal reporter moieties that are commonly used for site-specific labeling and functionalization of RNA to probe its biology. The preparation of azido modified nucleic acids by solid-phase synthesis is problematic due to the inherent reactivity of P(III) species with azides according to the Staudinger reaction. Various strategies have been developed to bypass this limitation and are often time-consuming, low-yielding and labor-intensive. In particular, the synthesis of RNA with internal 2'-azido modifications is restricted to a single approach that employs P(V) chemistry instead of the widely used P(III) phosphoramidite chemistry. To fill this methodological gap, we present a novel convenient path toward 2'-azido RNA from readily accessible 2'-amino RNA through treatment with the diazotizing reagent fluorosulfuryl azide (FSO2N3). A diazotransfer reaction was established for oligoribonucleotides of different lengths and secondary structures. The robustness of the approach was further demonstrated for RNAs containing multiple 2'-azido moieties and for RNAs containing other sensitive modifications such as thiouridine or methylated nucleobases with a positive charge. The synthetic ease of generating 2'-azido RNA will pave the way for biotechnological applications, in particular for siRNA technologies and for referencing the growing number of RNA metabolic labeling approaches that rely on 2'-azido nucleosides.

Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling.

Metabolic labeling has emerged as a powerful tool to endow RNA with reactive handles allowing for subsequent chemical derivatization and processing. Recently, thiolated nucleosides, such as 4-thiouridine (4sU), have attracted great interest in metabolic labeling-based RNA sequencing approaches (TUC-seq, SLAM-seq, TimeLapse-seq) to study cellular RNA expression and decay dynamics. For these and other applications (e.g. PAR-CLIP), thus far only the naked nucleoside 4sU has been applied. Here we examined the concept of derivatizing 4sU into a 5'-monophosphate prodrug that would allow for cell permeation and potentially improve labeling efficiency by bypassing the rate-limiting first step of 5' phosphorylation of the nucleoside into the ultimately bioactive 4sU triphosphate (4sUTP). To this end, we developed robust synthetic routes towards diverse 4sU monophosphate prodrugs. Using metabolic labeling assays, we found that most of the newly introduced 4sU prodrugs were well tolerated by the cells. One derivative, the bis(4-acetyloxybenzyl) 5'-monophosphate of 4sU, was also efficiently incorporated into nascent RNA.

Synthesis of N 4-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis.

The growing interest in 3-methylcytidine (m3C) originates from the recent discoveries of m3C modified tRNAs in humans as well as its intensively debated occurrence in mRNA. Moreover, m3C formation can be catalyzed by RNA without the assistance of proteins as has been demonstrated for a naturally occurring riboswitch fold using the methylated form of its cognate ligand as cofactor. Additionally, new RNA sequencing methods have been developed to detect this modification in transcriptome-wide manner. For all these reasons, an increasing demand for synthetic m3C containing oligoribonucleotides is emerging. Their chemical synthesis relies on RNA solid-phase synthesis using phosphoramidite building blocks. Here, we describe a facile synthetic path towards N4-acetylated 2'-O-TBDMS- and 2'-O-TOM m3C phosphoramidites to provide an optimal toolbox for solid-phase synthesis of m3C containing RNA. Supplementary Information: The online version contains supplementary material available at 10.1007/s00706-022-02896-x.

Dehydrated human amniotic membrane regulates tenocyte expression and angiogenesis in vitro: Implications for a therapeutic treatment of tendinopathy.

Tendon injuries are among the most common ailments of the musculoskeletal system. Prolonged inflammation and persistent vasculature are common complications associated with poor healing. Damaged tendon, replaced with scar tissue, never completely regains the native structural or biomechanical properties. This study evaluated the effects of micronized dehydrated human amnion/chorion membrane (µdHACM) on the inflammatory environment and hypervascularity associated with tendinopathy. Stimulation of human tenocytes with interleukin-1 beta (IL1ß) induced the expression of inflammatory and catabolic markers, resulting in secretion of active MMPs and type 3 collagen that is associated with a degenerative phenotype. Treatment with µdHACM diminished the effects of IL1ß, reducing the expression of inflammatory genes, proteases, and extracellular matrix components, and decreasing the presence of active MMP and type 3 collagen. Additionally, a co-culture model was developed to evaluate the effects of µdHACM on angiogenesis associated with tendinopathy. Micronized dHACM differentially regulated angiogenesis depending upon the cellular environment in which it was placed. This phenomenon can be explained in part through the detection of both angiogenic protagonists and antagonists in µdHACM. Observations from this study identify a mechanism by which µdHACM regulates inflammatory processes and angiogenesis in vitro, two key pathways implicated in tendinopathic injuries.

Dehydrated Human Amniotic Membrane Inhibits Myofibroblast Contraction through the Regulation of the TGFß‒SMAD Pathway In Vitro.

Excessive fibrosis affects more than 100 million patients yearly, leading to the accumulation of extracellular matrix that compromises tissue architecture and impedes its function. Intrinsic properties of the amniotic membrane have alluded to its potential to inhibit excessive fibrosis; therefore, this study aimed to investigate the effects of dehydrated human amnion/chorion membrane (dHACM) on dermal fibroblasts and their role in fibrotic pathways. Human dermal fibroblasts were stimulated with TGFß1, triggering myofibroblast-like characteristics in vitro. Subsequent addition of dHACM in the continued presence of TGFß1 inhibited downstream signaling, leading to a reduction in the expression of known fibrotic and extracellular matrix genes. In addition, dHACM decreased alpha-smooth muscle actin, a stress filament responsible for contractile activity in scarring. The functional outcome of these effects was observed in an ex vivo model for cellular contraction. Hyperactivation of TGFß signaling increased the contractile capacity of myofibroblasts embedded within a collagen substrate. Simultaneous addition of dHACM treatment prevented the marked contraction, which is likely a direct result of the inhibition of TGFß signaling mentioned earlier. These observations may support the use of dHACM in the regulation of fibroblast activity as it relates to tissue fibrosis.

Synthesis of O 6-alkylated preQ1 derivatives.

A naturally occurring riboswitch can utilize 7-aminomethyl-O 6-methyl-7-deazaguanine (m6preQ1) as cofactor for methyl group transfer resulting in cytosine methylation. This recently discovered riboswitch-ribozyme activity opens new avenues for the development of RNA labeling tools based on tailored O 6-alkylated preQ1 derivatives. Here, we report a robust synthesis for this class of pyrrolo[2,3-d]pyrimidines starting from readily accessible N 2-pivaloyl-protected 6-chloro-7-cyano-7-deazaguanine. Substitution of the 6-chloro atom with the alcoholate of interest proceeds straightforward. The transformation of the 7-cyano substituent into the required aminomethyl group turned out to be challenging and was solved by a hydration reaction sequence on a well-soluble dimethoxytritylated precursor via in situ oxime formation. The synthetic path now provides a solid foundation to access O 6-alkylated 7-aminomethyl-7-deazaguanines for the development of RNA labeling tools based on the preQ1 class-I riboswitch scaffold.

Dehydrated human amniotic membrane modulates canonical Wnt signaling in multiple cell types in vitro.

Canonical Wnt signaling is a major pathway known to regulate diverse physiological processes in multicellular organisms. Signaling is tightly regulated by feedback mechanisms; however, persistent dysregulation of this pathway is implicated in the progression of multiple disease states. In this study, proteomic analysis identified endogenous Wnt antagonists in micronized dehydrated human amnion/chorion membrane (µdHACM); thereby, prompting a study to further characterize the intrinsic properties of µdHACM as it relates to Wnt activity, in vitro. A TCF/LEF reporter cell line demonstrated the general ability of µdHACM to inhibit ß-catenin induced transcription activity. Furthermore, in vitro systems, modeling elevated Wnt signaling, were developed in relevant cell types including tenocytes, synoviocytes, and human dermal fibroblasts (HDFs). Stimulation of these cells with Wnt3A resulted in translocation of ß-catenin to the nucleus and increased expression of Wnt related genes. The subsequent addition of µdHACM, in the continued presence of Wnt-stimulus, mitigated the downstream effects of Wnt3A in tenocytes, synoviocytes, and HDFs. Nuclear localization of ß-catenin was abated with corresponding reduction of Wnt related gene expression. These data demonstrate the in vitro regulation of canonical Wnt signaling as an inherent property of µdHACM and a novel mechanism of action.

A natural riboswitch scaffold with self-methylation activity.

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.

Correction of cilia structure and function alleviates multi-organ pathology in Bardet-Biedl syndrome mice.

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive ciliopathy affecting multiple organs. The development of potential disease-modifying therapy for BBS will require concurrent targeting of multi-systemic manifestations. Here, we show for the first time that monosialodihexosylganglioside accumulates in Bbs2-/- cilia, indicating impairment of glycosphingolipid (GSL) metabolism in BBS. Consequently, we tested whether BBS pathology in Bbs2-/- mice can be reversed by targeting the underlying ciliary defect via reduction of GSL metabolism. Inhibition of GSL synthesis with the glucosylceramide synthase inhibitor Genz-667161 decreases the obesity, liver disease, retinal degeneration and olfaction defect in Bbs2-/- mice. These effects are secondary to preservation of ciliary structure and signaling, and stimulation of cellular differentiation. In conclusion, reduction of GSL metabolism resolves the multi-organ pathology of Bbs2-/- mice by directly preserving ciliary structure and function towards a normal phenotype. Since this approach does not rely on the correction of the underlying genetic mutation, it might translate successfully as a treatment for other ciliopathies.

Epithelial propionyl- and butyrylcholine as novel regulators of colonic ion transport.

BACKGROUND AND PURPOSE: The colonic surface epithelium produces acetylcholine, released after the binding of propionate to GPCRs for this short-chain fatty acid (SCFA). This epithelial acetylcholine then induces anion secretion via stimulation of acetylcholine receptors. The key enzyme responsible for acetylcholine synthesis, choline acetyltransferase, is known to be unselective as regards the fatty acid used for esterification of choline. As the colonic epithelium is permanently exposed to high concentrations of different SCFAs produced by bacterial fermentation, we investigated whether choline esters other than acetylcholine, propionylcholine and butyrylcholine, are produced by the colonic epithelium, too, and whether these 'atypical' esters are able to stimulate the acetylcholine receptors involved in the regulation of colonic ion transport. EXPERIMENTAL APPROACH: Desorption electrospray ionization mass spectroscopy (DESI-MS), Ussing chamber and Ca(2+) -imaging experiments were performed on rat distal colon. KEY RESULTS: DESI-MS analyses revealed the production of acetylcholine, propionylcholine and butyrylcholine in the surface epithelium. Relative expression rates were 2-3% in comparison with acetylcholine. In Ussing chamber experiments, both atypical choline esters caused a concentration-dependent increase in short-circuit current, that is, stimulated anion secretion. Inhibitor experiments in the absence and presence of the submucosal plexus revealed the involvement of neuronal and epithelial acetylcholine receptors. While butyrylcholine obviously stimulated both nicotinic and muscarinic receptors, propionylcholine predominantly acted on muscarinic receptors. CONCLUSIONS AND IMPLICATIONS: These results suggest a novel pathway for communication between intestinal microbes producing SCFA and the host via modification of epithelial production of choline esters involved in the paracrine regulation of the colonic epithelium.

Differences in the timing and magnitude of Pkd1 gene deletion determine the severity of polycystic kidney disease in an orthologous mouse model of ADPKD.

Development of a disease-modifying therapy to treat autosomal dominant polycystic kidney disease (ADPKD) requires well-characterized preclinical models that accurately reflect the pathology and biochemical changes associated with the disease. Using a Pkd1 conditional knockout mouse, we demonstrate that subtly altering the timing and extent of Pkd1 deletion can have a significant impact on the origin and severity of kidney cyst formation. Pkd1 deletion on postnatal day 1 or 2 results in cysts arising from both the cortical and medullary regions, whereas deletion on postnatal days 3-8 results in primarily medullary cyst formation. Altering the extent of Pkd1 deletion by modulating the tamoxifen dose produces dose-dependent changes in the severity, but not origin, of cystogenesis. Limited Pkd1 deletion produces progressive kidney cystogenesis, accompanied by interstitial fibrosis and loss of kidney function. Cyst growth occurs in two phases: an early, rapid growth phase, followed by a later, slow growth period. Analysis of biochemical pathway changes in cystic kidneys reveals dysregulation of the cell cycle, increased proliferation and apoptosis, activation of Mek-Erk, Akt-mTOR, and Wnt-ß-catenin signaling pathways, and altered glycosphingolipid metabolism that resemble the biochemical changes occurring in human ADPKD kidneys. These pathways are normally active in neonatal mouse kidneys until repressed around 3 weeks of age; however, they remain active following Pkd1 deletion. Together, this work describes the key parameters to accurately model the pathological and biochemical changes associated with ADPKD in a conditional mouse model.

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis.

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.

Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis.

Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.

Genetic and functional abnormalities of the melatonin biosynthesis pathway in patients with bipolar disorder.

Patients affected by bipolar disorder (BD) frequently report abnormalities in sleep/wake cycles. In addition, they showed abnormal oscillating melatonin secretion, a key regulator of circadian rhythms and sleep patterns. The acetylserotonin O-methyltransferase (ASMT) is a key enzyme of the melatonin biosynthesis and has recently been associated with psychiatric disorders such as autism spectrum disorders and depression. In this paper, we analysed rare and common variants of ASMT in patients with BD and unaffected control subjects and performed functional analysis of these variants by assaying the ASMT activity in their B-lymphoblastoid cell lines. We sequenced the coding and the regulatory regions of the gene in a discovery sample of 345 patients with BD and 220 controls. We performed an association study on this discovery sample using common variants located in the promoter region and showed that rs4446909 was significantly associated with BD (P= 0.01) and associated with a lower mRNA level (P< 10(-4)) and a lower enzymatic activity (P< 0.05) of ASMT. A replication study and a meta-analysis using 480 independent patients with BD and 672 controls confirmed the significant association between rs4446909 and BD (P= 0.002). These results correlate with the general lower ASMT enzymatic activity observed in patients with BD (P= 0.001) compared with controls. Finally, several deleterious ASMT mutations identified in patients were associated with low ASMT activity (P= 0.01). In this study, we determined how rare and common variations in ASMT might play a role in BD vulnerability and suggest a general role of melatonin as susceptibility factor for BD.