Epigenetics as a Therapeutic Target in Osteoarthritis.

Osteoarthritis (OA) is a heterogenous, complex disease affecting the integrity of diarthrodial joints that, despite its high prevalence worldwide, lacks effective treatment. In recent years it has been discovered that epigenetics may play an important role in OA. Our objective is to review the current knowledge of the three classical epigenetic mechanisms-DNA methylation, histone post-translational modifications (PTMs), and non-coding RNA (ncRNA) modifications, including microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs)-in relation to the pathogenesis of OA and focusing on articular cartilage. The search for updated literature was carried out in the PubMed database. Evidence shows that dysregulation of numerous essential cartilage molecules is caused by aberrant epigenetic regulatory mechanisms, and it contributes to the development and progression of OA. This offers the opportunity to consider new candidates as therapeutic targets with the potential to attenuate OA or to be used as novel biomarkers of the disease.

Histone Extraction from Human Articular Cartilage for the Study of Epigenetic Regulation in Osteoarthritis.

Osteoarthritis (OA) is a chronic disease that affects articular cartilage, causing its degeneration. Although OA is one of the most prevalent pathologies globally, there are no definitive treatments available. Recently, research has focused on elucidating the complex interplay that takes place between inflammatory processes and epigenetic regulation, showing that histone post-translational modifications (PTMs) can exert a pronounced effect on the expression of OA-related genes. OA chondrocytes enhance the production of interleukin 1ß (IL-1ß) and interleukin 8 (IL-8), which are epigenetically regulated. These cytokines upregulate the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, which promote the extracellular matrix (ECM) destruction. This motivates the study of histone PTMs to investigate the epigenetic regulation of proinflammatory molecules, but the absence of specific protocols to extract histones from human articular cartilage has complicated this task. The lack of effective methods can be explained by the structural complexity and low cellularity of this tissue, which are responsible for the biomechanical properties that allow the movement of the joint but also complicate histone isolation. Here, we provide a histone extraction procedure specifically adapted for cryopreserved human articular cartilage that can be useful to understand epigenetic regulation in OA and accelerate the search for novel strategies.

"Doubly Customizable" Unit for the Generation of Structural Diversity: From Pure Enantiomeric Amines to Peptide Derivatives.

Readily available, low-cost 4R-hydroxy-l-proline (Hyp) is introduced as a "doubly customizable" unit for the generation of libraries of structurally diverse compounds. Hyp can be cleaved at two points, followed by the introduction of new functionalities. In the first cycle, the removal and replacement of the carboxylic group are carried out, followed (second cycle) by the scission of the 4,5-position and manipulation of the resulting chains. In this way, three new chains are generated and can be transformed independently to afford a diversity of products with tailored substituents, such as ß-amino aldehydes, diamines, ß-amino acid derivatives, including N-alkylated ones, or modified peptides. Many of these products are high-profit compounds but, in spite of their commercial value, are still scarce. Moreover, the process takes place with stereochemical control, and either pure R or S isomers can be obtained with small variations of the synthetic route.

Conversion of "Customizable Units" into N-Alkyl Amino Acids and Generation of N-Alkyl Peptides.

An efficient conversion of hydroxyproline "customizable" units into new amino acids with a variety of N-alkyl substituents is described. The process is versatile and can afford valuable N-methyl amino acids and N, O-acetals. In addition, it allows the introduction of N-homoallylic substituents and N-chains with terminal ester, ketone, or cyano groups. These chains could be used for peptide extension or conjugation to other molecules (e.g., by olefin metathesis, peptide ligation, etc.). The transformation is carried out in just two (for R = CH2OAc) or three steps (scission of the pyrrolidine ring, manipulation of the α-chain, and the N-substituent) under mild, metal-free conditions, affording products with high optical purity.

Arritmia cardiaca grave por hipopotasemia. Influencia de las sustancias diuréticas / Severe arrhythmia due to hypokalemia. Influence from diuretic substances

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Kinetics of anti-Fasciola IgG antibodies in serum and milk from dairy cows during lactation, and in serum from calves after feeding colostrum from infected dams.

A study was carried out to compare anti-Fasciola hepatica IgG levels in blood serum and mammary secretions during the entire lactation period in dairy cows experimentally infected with different numbers of F. hepatica metacercariae. The kinetics of specific antibodies passively transferred to the offspring was also studied. The MM3-SERO ELISA, a specific and sensitive method of detecting antibodies against F. hepatica, was used to detect antibodies in milk and serum samples. The progress of infection was monitored by use of the MM3-COPRO ELISA, an immunoassay for detecting Fasciola antigens in faecal samples. The optical density of serum and milk from uninfected control cows remained low throughout the study. In the infected animals, a similar pattern of anti-F. hepatica IgG kinetics was observed in serum and milk throughout the entire observation period. This IgG response was characterized by the early appearance of high levels of specific antibodies in serum (detectable 1-4 weeks pi) and in milk (detectable at the beginning of lactation) and remained invariably high throughout the entire lactation period in cows infected with low-to-moderate infective doses (>or=50 metacercariae). However, in animals administered very low infective doses (or=50 metacercariae, even when samples were diluted al least 1:8. In newborn calves fed colostrum from infected dams, the levels of anti-Fasciola antibodies increased rapidly, then decreased sharply and were no longer detected in calves >12 weeks old. The results indicate that the detection of antibodies in milk samples may provide useful information about the status of F. hepatica infection in dairy herds if repeated analyses are carried out. Analysis of bulk samples may also be an inexpensive way of identifying herds infected with F. hepatica, provided highly sensitive tests are used.

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3).

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.