Genetic Ablation and Pharmacological Blockade of Bradykinin B1 Receptor Unveiled a Detrimental Role for the Kinin System in Chagas Disease Cardiomyopathy.

Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R-/- mice showed that T. cruzi DNA levels (15 days post infection-dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R-/- hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg9-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15-60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120-160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.

Functionalized Nanogels with Endothelin-1 and Bradykinin Receptor Antagonist Peptides Decrease Inflammatory and Cartilage Degradation Markers of Osteoarthritis in a Horse Organoid Model of Cartilage.

Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this study, we investigated the effects of two functionalized nanogels as DDS. We assessed the effect of chitosan functionalized with a type A endothelin receptor antagonist (BQ-123-CHI) and/or hyaluronic acid functionalized with a type B1 bradykinin receptor antagonist (R-954-HA). The biocompatibility of these nanogels, alone or in combination, was first validated on equine articular chondrocytes cultured under different oxic conditions. Further, in an OA equine organoid model via induction with interleukin-1 beta (IL-1ß), a combination of BQ-123-CHI and R-954-HA (BR5) triggered the greatest decrease in inflammatory and catabolic markers. In basal and OA conditions, BQ-123-CHI alone or in equimolar combinations with R-954-HA had weak pro-anabolic effects on collagens synthesis. These new nanogels, as part of a composite DDS, show promising attributes for treating OA.

The role of kallikrein-kinin and renin-angiotensin systems in COVID-19 infection.

In November 2019 the first cases of a novel acute respiratory syndrome has been reported in Wuhan province, China. Soon after, in January 2020 the World Health Organization declared a pandemic state due to the dissemination of a virus named SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the cause of coronavirus disease 2019 (COVID-19). Being an unknown disease, it is essential to assess not only its main characteristic features and overall clinical symptomatology but also its patient infection mode and propagation to design appropriate clinical interventions and treatments. In this review the pathophysiology of SARS-CoV-2 infection and how the virus enters the cells and activates the immune system are described. The role of three systems involved in the SARS- CoV-2 infection (renin-angiotensin, kinin and coagulation systems) is discussed with the objectives to identify and try to explain several of the events observed during the evolution of the disease and to suggest possible targets for therapeutic interventions.

Primerless Amplification for Circulating Tumor DNA Assays.

A primerless amplification suitable for enrichment of particular genotype cfDNA which is a one-dimensional material has been developed. This primerless amplification coordinated by two thermostable enzymes of endonuclease and proofreading polymerase, functions as a genotype switch in analyzing cfDNA. The endonuclease digests the wild-typed fragments into mega-primer and discriminately destroys the wild-type DNA alleles. The DNA polymerase proofreads the megaprimer and then extends the mega-primer using the mutant DNA as the template. The prototypes of this technology were applied to two hotspot mutations of APC and EGFR with confirmed by DNA sequencing analysis. Genotype switch was then employed to clinical cfDNA assay targeting PIK3CA. Data from the clinical application suggest its potential in early cancer diagnosis.

Leukotrienes: One step in our understanding of asthma.

The steps leading to the discovery of leukotrienes and the researchers that played a major part in this long process are presented. The pharmacology of these exquisitely potent compounds shows that they express bronchoconstrictor activity and numerous cellular effects via very specific receptors. Experimental evidence strongly suggests that these mediators play a significant role in asthma physiopathology. Numerous approaches were taken to block their effects on the lungs and this led to the discovery of selected drugs used for asthma treatment. The complexity of this disease and its treatment is emphasized.

Oxidative stress in the optic nerve and cortical visual area of steptozotocin-induced diabetic Wistar rats: Blockade with a selective bradykinin B1 receptor antagonist.

The role of bradykinin B1 receptors on the oxidative stress as measured by the levels of Na+/K+ ATPase activity, malondialdehyde (MDA) and glutathione (GSH) in male Wistar rat optic nerve and visual cortex area 1 and 4weeks after STZ treatment was studied. Rats were divided into 4 groups (n=6-7): 1. Controls (non-diabetics); 2. Diabetics (65mg/kg streptozotocin, STZ); 3. Diabetics injected with B1 antagonist R-954 (2mg/Kg) during the last 3days of a one week period; 4. Diabetics injected with B1 antagonist R-954 (2mg/Kg) during the last 3days of a 4week period. The results showed that plasma glucose levels increased by up to 4 fold in diabetic rats 1 or 4weeks following the STZ treatment. R-954 treatment did significantly decrease blood glucose levels. Levels of MDA was increased in the plasma of the 1 and 4week diabetic animals whereas the GSH levels were decreased. Both markers returned to normal following R-954 treatment. Na+/K+ ATPase activity significantly decreased in the optic nerve and visual cortex of diabetic rats at 1 and 4weeks but returned to normal following R-954 treatment. MDA levels increased markedly at 1 and 4weeks compared with control levels in the optic nerve but slightly in the visual cortex and returned to control levels in both tissues following R-954 treatment. GSH levels decreased in both tissues at 1 and 4weeks compared with control levels. Following administration of the selective BKB1R antagonist R-954, the levels of GSH returned to normal in both tissues of the 1 and 4week diabetic animals. These results showed that the inducible BKB1 receptors are associated with the oxidative stress in the optic nerve and cortical visual area of diabetic rats and suggested that BKB1-R antagonist R-954 could have a beneficial role in the treatment of diabetic retinopathy.

CRISPR/Cas: A Faster and More Efficient Gene Editing System.

Gene editing technology has been at its mature stage with the successful development of TALENs and CRISPR/Cas enzymes. The genetically modified endonucleases of ZFNs, TALENs, and CRISPR/Cas are widely used in the development of genetically modified cells or organisms. Among the enzymes that possess gene editing ability, CRISPR/Cas is the latest member with high efficiency in gene editing and simplicity in cloning. This review discusses the discovery of CRISPR, the development of the CRISPR/Cas system, and its applications as a new gene editing system.

Preclinical pharmacology, metabolic stability, pharmacokinetics and toxicology of the peptidic kinin B1 receptor antagonist R-954.

We previously showed that R-954 (AcOrn[Oic(2),(αMe)Phe(5),dßNal(7),Ile(8)]desArg(9)-bradykinin) is a potent, selective and stable peptide antagonist of the inducible GPCR kinin B1 receptor. This compound shows potential applications for the treatment of several diseases, including cancer and neurological disturbances of diabetes. To enable clinical translation, more information regarding its pharmacological, pharmacokinetics (PK) and toxicological properties at preclinical stage is warranted. This was the principal objective of the present study. Herein, specificity of R-954 was characterized in binding studies on 133 human molecular targets to reveal minor cross-reactivities against the angiotensin AT2 and the bombesin receptors (110- and 330-fold lower affinity than for B1R, respectively). The pharmacokinetic of R-954 was studied in both normal and streptozotocin-diabetic anaesthetized rats providing half-lives of 1.9-2.7h. R-954 does not appear to be metabolized in the rat circulation and in several rat tissue homogenates, as the kidney, lung and liver. It appears to be excreted as parent drug in the bile (21%) and in urine. A preliminary toxicological profile of R-954 was obtained in rats under various administration routes. R-954 appears to be well tolerated. Overall, these results indicate that R-954 exhibits favorable preclinical pharmacological/PK characteristics and encouraging safety profiles, suitable for early studies in humans.

The role of kinin receptors in cancer and therapeutic opportunities.

Kinins are generated within inflammatory tissue microenvironments, where they exert diverse functions, including cell proliferation, leukocyte activation, cell migration, endothelial cell activation and nociception. These pleiotropic functions depend on signaling through two cross talking receptors, the constitutively expressed kinin receptor 2 (B2R) and the inducible kinin receptor 1 (B1R). We have reviewed evidence, which supports the concept that kinin receptors, especially kinin receptor 1, are promising targets for cancer therapy, since (1) many tumor cells express aberrantly high levels of these receptors; (2) some cancers produce kinins and use them as autocrine factors to stimulate their growth; (3) activation of kinin receptors leads to activation of macrophages, dendritic cells and other cells from the tumor microenvironment; (4) kinins have pro-angiogenic properties; (5) kinin receptors have been implicated in cancer migration, invasion and metastasis; and (6) selective antagonists for either B1R or B2R have shown anti-proliferative, anti-inflammatory, anti-angiogenic and anti-migratory properties. The multiple cross talks between kinin receptors and renin-angiotensin system (RAS) as well as its implications for targeting KKS or RAS for the treatment of malignancies are also discussed. It is expected that B1R antagonists would interfere less with housekeeping functions and therefore would be attractive compounds to treat selected types of cancer. Reliable clinical studies are needed to establish the translatability of these data to human settings and the usefulness of kinin receptor antagonists.

Understanding of and possible strategies to avian influenza outbreak.

Swine flu and avian flu outbreaks have occurred in recent years in addition to seasonal flu. As mortality rate records are not available at the early stage of an outbreak, two parameters may be useful to assess the viral virulence : 1. the time required for the first domestic case in a newly involved region, and 2. the doubling time of new infected cases. Viral virulence is one of the most important factors in guiding short term and immediate responses. Although routine surveillance and repeated vaccination are useful efforts, some novel strategies that may be relevant to prevent and control the spread of influenza among human beings and domestic animals are discussed.

Bradykinin B1 antagonism inhibits oxidative stress and restores Na+K+ ATPase activity in diabetic rat peripheral nervous system.

Diabetic peripheral neuropathy is one the most common complications of diabetes mellitus and frequently results in clinically significant morbidities such as pain, foot ulcers and amputations. The diabetic condition progresses from early functional changes to late, poorly reversible structural changes. The chronic hyperglycemia measured alongside diabetes development is associated with significant damage and failure of various organs. In the present study diabetes was induced in male Wistar rats by a single dose of streptozotocin (STZ) and the association between the BKB1-R and the oxidative stress and Na+-K+ ATPase activity in nervous tissues was analysed. The results showed that the resulting hyperglycemia induced a reduction of the neuronal electrical function integrity and increased oxidative stress in the sciatic nerve homogenates of 30 days diabetic rats. Malondialdehyde (MDA) used as a marker of oxidative stress was elevated whereas Biological Antioxidant Potential (BAP), glutathion (GSH) levels and superoxide dismutase (SOD) activity were decreased. Treatment of the rats 3 days before the end of the 4 week period with the BKB1 antagonist R-954 restored the neuronal activity and significantly attenuated the oxidative stress as shown by the level of the various markers returning close to levels found in control rats. Our results suggest that the BKB1-R subtype is overexpressed in sciatic nerve during the STZ-induced diabetes development as evidenced by inhibitory effects of the BKB1-R antagonist R-954. The beneficial role of BKB1-R antagonist R-954 for the treatment of diabetic neuropathy is also suggested.

Biological and biomedical applications of engineered nucleases.

The development of engineered nucleases is the fruit of a new technological approach developed in the last two decades which has led to significant benefits on genome engineering, particularly on gene therapy. These applications enable efficient and specific genetic modifications via the induction of a double-strand break (DSB) in a specific genomic target sequence, followed by the homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. In addition to the application on gene modification in cells and intact organisms, a number of recent papers have reported that this gene editing technology can be applied effectively to human diseases. With the promising data obtained using engineered endonucleases in gene therapy, it appears reasonable to expect that more diseases could be treated and even be cured in this new era of individualized medicine. This paper first brief introduces the development of engineered nucleases with a special emphasis on zinc-finger nucleases (ZFNs) and transcription activator-like effector (TALE) nucleases (TALENs), and then takes CCR5-based gene therapy as an example to discuss the therapeutic applications of engineered nucleases.

Blockade of early and late retinal biochemical alterations associated with diabetes development by the selective bradykinin B1 receptor antagonist R-954.

The chronic hyperglycemia measured alongside diabetes development is associated with significant long-term damage and failure of various organs. In the present study it was shown that hyperglycemia induced early and long term increases in nitric oxide (NO) levels, kallikrein activity and vascular capillary permeability measured as plasma extravasation, and decreases of Na/K ATPase activity in diabetic rat retina 4 and 12 weeks after streptozotocin (STZ) injection. Treatment of the animals for 5 consecutive days with a novel selective bradykinin B(1) receptor (BKB(1)-R) antagonist R-954 (2mg/kg s.c) at the end of the 4 and 12 week periods highly reduced NO, kallikrein and capillary permeability and increased Na/K ATPase activity in the retina. These results suggest that the BKB(1)-R receptor subtype is over-expressed during the streptozotocin-induced development of diabetes in rat retina as evidenced by the inhibitory effects of the BKB(1)-R antagonist R-954 on NO, kallikrein and vascular permeability increases as well as Na/K ATPase decreases. The beneficial role of the BKB(1)-R antagonist R-954 for the treatment of the diabetic retinopathy is also suggested.

Downregulation of ferroportin 1 expression in hFOB1.19 osteoblasts by hepcidin.

The present study was designed to address the relationship between iron homeostasis and bone metabolism. Cultured hFOB 1.19 osteoblasts were incubated with selected concentrations of hepcidin (50, 100, and 200 nmol/L) for 20 h, harvested for extraction of total RNA and proteins, and the expression of ferroportin 1 was analyzed by RT-PCR and western blotting. The results showed the presence of ferroportin 1 expression in cultured hFOB 1.19 cells. Furthermore, the ferroportin 1 had a similar expression pattern in hFOB cells as in hepatocytes and enterocytes and was downregulated by hepcidin. Our data indicate that osteoblasts are target cells for hepcidin, suggest that hepcidin may have many more targets than previously recognized, and support the role of hepcidin in the development of osteoporosis.

Hepcidin increases intracellular Ca2+ of osteoblast hFOB1.19 through L-type Ca2+ channels.

Hepcidin is a key player in the regulation of iron homeostasis. Several pathological conditions associated with iron overload are attributed to the depressed expression of hepcidin and are often associated with bone diseases including osteoporosis. Hepcidin was suggested to have anti-osteoporosis effects by preventing iron overload. We recently observed that hepcidin could increase intracellular calcium concentration in cultured osteoblast cells. The present study was designed to elucidate the source of the increased intracellular calcium following hepcidin activation. Cultured hFOB1.19 cells were used to test whether there was a dose dependent effect of hepcidin on increasing intracellular calcium. After finding the optimal concentration in increasing intracellular calcium, Cultured hFOB1.19 cells were then divided into three groups: (1) control group, (2) and (3) groups pretreated with either nimodipine (2 × 10(-5)mol/L) or EDTA (2 × 10(-3)mol/L) for 10 min before incubation with hepcidin (100 nmol/L). All cells were stimulated with hepcidin for 60 min and then stained with fluo-3/AM for 40 min before the intracellular calcium was observed using flow cytometry (FCM). As compared with controls, hepcidin treatment significantly increased intracellular calcium concentration. This effect was blocked by nimodipine and EDTA pretreatments which suggested that hepcidin-mediated calcium inflow was mainly through L-type Ca(2+) channels and that the release of intracellular calcium store was not significant. Hepcidin increases of intracellular calcium may be related to its anti-osteoporosis effect but this hypothesis needs further investigation.

The bradykinin B1 receptor antagonist R-954 inhibits Ehrlich tumor growth in rodents.

The present study investigated the effects of a new bradykinin B(1) receptor antagonist, R-954, on the development of Ehrlich ascitic tumor (EAT) induced by the intraperitoneal inoculation of EAT cells in mice and the formation of a solid tumor by the subcutaneous injection of the cells in rat paw. The development of the tumor was associated with an increase in mouse total cell counts in bone marrow (10.8-fold), ascitic fluid (14.6-fold), and blood (12.6-fold). R-954 (2mg/kg, s.c.) significantly reduced the ascitic fluid volume (63.7%) and the mouse weight gain (30.5%) after 10 consecutive days of treatment. The B(1) antagonist as well as the anti-neoplasic drug vincristine also significantly inhibited the increase in total cell count in bone marrow, ascitic fluid, and blood. R-954 reduced significantly the total protein extravasation (57.3%), the production of nitric oxide (56%), PGE(2) production (82%), and TNFα release (85.7%) in mice peritoneal cavity whereas vincristine reduced the release of these inflammatory mediators by 84-94%. The increase in paw edema after intraplantar injection of EAT cells was reduced by approximately 52% by either R-954 or vincristine treatment. In conclusion, this study presents for the first time the antitumoral activity of a new bradykinin B(1) receptor antagonist on ascitic and solid tumors induced by Ehrlich cell inoculation in mice and rats.

A new strategy for next generation sequencing: merging the Sanger's method and the sequencing by synthesis through replacing extension.

The present paper describes a feasible protocol enabling a merge of the classical Sanger sequencing method with the sequencing by DNA synthesis: The cycled proofreading sequencing. This new protocol involves partial chain termination and replacing extension. The first step is done with mixed ddNTP/dNTP targeting one type of nucleotide per subcycle and its relevant replacing extension with unlabeled phosphorothioate-modified dNTP of the same nucleotide type used in partial chain termination. Replacing extension serves as the key step to eliminate fluorescent signals integrated at the previous step, proofread errors, reactivate and synchronize the 3' termini of extended DNA molecules. This microchip-based sequencing method thus inherits advantages from "sequencing-by-termination" and "sequencing-by-extension"; ddNTP is used and reactions are cycled. This microchip-based sequencing method is applicable for reaching the $1000-genome project goal.

Nociceptive tolerance is improved by bradykinin receptor B1 antagonism and joint morphology is protected by both endothelin type A and bradykinin receptor B1 antagonism in a surgical model of osteoarthritis.

INTRODUCTION: Endothelin-1, a vasoconstrictor peptide, influences cartilage metabolism mainly via endothelin receptor type A (ETA). Along with the inflammatory nonapeptide vasodilator bradykinin (BK), which acts via bradykinin receptor B1 (BKB1) in chronic inflammatory conditions, these vasoactive factors potentiate joint pain and inflammation. We describe a preclinical study of the efficacy of treatment of surgically induced osteoarthritis with ETA and/or BKB1 specific peptide antagonists. We hypothesize that antagonism of both receptors will diminish osteoarthritis progress and articular nociception in a synergistic manner. METHODS: Osteoarthritis was surgically induced in male rats by transection of the right anterior cruciate ligament. Animals were subsequently treated with weekly intra-articular injections of specific peptide antagonists of ETA and/or BKB1. Hind limb nociception was measured by static weight bearing biweekly for two months post-operatively. Post-mortem, right knee joints were analyzed radiologically by X-ray and magnetic resonance, and histologically by the OARSI histopathology assessment system. RESULTS: Single local BKB1 antagonist treatment diminished overall hind limb nociception, and accelerated post-operative recovery after disease induction. Both ETA and/or BKB1 antagonist treatments protected joint radiomorphology and histomorphology. Dual ETA/BKB1 antagonism was slightly more protective, as measured by radiology and histology. CONCLUSIONS: BKB1 antagonism improves nociceptive tolerance, and both ETA and/or BKB1 antagonism prevents joint cartilage degradation in a surgical model of osteoarthritis. Therefore, they represent a novel therapeutic strategy: specific receptor antagonism may prove beneficial in disease management.

An excess of G over C nucleotides in mutagenesis of human genetic diseases.

Strand asymmetries in DNA evolution, including indel and single nucleotide substitutions, were reported in prokaryotes. Recently, an excess of G>A over C>T substitutions in hemophilia B patients was recognized in our molecular diagnostic practices. Further analysis demonstrated biased point mutations between sense and antisense strands when unique changes in factor IX were counted. Similar mutation spectra of factor IX and the HGMD prompted us to speculate that the excess of G>A over C>T may be present in genes other than factor IX. Data from nine genes (each has ≥ 100 missense mutations) retrieved from HGMD, international factor IX database, and Dr. Sommer's lab database in the City of Hope National Medical Center, Duarte, CA, USA were analyzed for their point mutation spectra. Similar to factor IX, all genes selected in this study have biased G>A over C>T unique mutations when nonsense mutations were excluded. The biased missense point mutations were recently convincingly documented by the statistic data of categorized missense mutation in HGMD. The consistence of the genetic observation and the genomic data from HGMD strongly indicate that biased point mutations, possibly a phenotypic selection, are more widespread than previously thought. The biased mutations have immediate clinical impact in molecular diagnostics.

In vivo evaluation of cerebral transplantation of resovist-labeled bone marrow stromal cells in Parkinson's disease rats using magnetic resonance imaging.

The effectiveness of Resovist-labeled bone marrow stem cells (BMSCs) was evaluated in vivo following their cerebral transplantation in a model of Parkinson's disease (PD) in rats using MRI, and the MRI findings were further compared with the behavior and histopathological manifestations of these rats. Forty PD rats were randomly assigned into five groups according to the cell doses injected into the rat brain site: control group (normal saline injection) and groups injected with 1 × 10(5), 1.5 × 10(5), 2 × 10(5), and 2.5 × 10(5) BMSCs. Gradient echo T2-weighted images were obtained immediately after cell transplantation and repeatedly taken 1, 4, 8, and 12 week(s) after cell transplantation. The rotational behavior of the animals was observed before and 1, 4, and 8 week(s) after transplantation. The rats were killed after the last MRI scanning, the brain tissues were analyzed by histopathology techniques, and RNAs were extracted for the expression analysis of selected genes using RT-PCR. One week following cell transplantation, all injected sites showed well-defined hypointense areas on MR images, with the most significant effect observed in rats injected with 2 × 10(5) BMSCs. These MR findings in PD rats lasted up to 12 weeks. The effectiveness of BMSC transplantation revealed by MRI was well confirmed by the behavioral and histopathological observations as well as indirectly supported by gene expression analyses. With the use of SPIO labeling, MRI techniques provided a dynamic evaluation of the spatial and temporal changes following cell transplantation and allowed the association analysis among the imaging, functions, and gene expression analysis in rats. These data also suggest the therapeutic potential of transplanted BMSCs. It is reasonable to speculate that the use of MRI in in vivo evaluation of the effect and fate of transplanted cells in various disease models will be beneficial to developing new strategies of cell-based gene therapy.