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Protein Lys methylation plays a critical role in numerous cellular processes, but it is challenging to identify Lys methylation in a systematic manner. Here we present an approach combining in silico prediction with targeted mass spectrometry (MS) to identify Lys methylation (Kme) sites at the proteome level. We develop MethylSight, a program that predicts Kme events solely on the physicochemical properties of residues surrounding the putative methylation sites, which then requires validation by targeted MS. Using this approach, we identify 70 new histone Kme marks with a 90% validation rate. H2BK43me2, which undergoes dynamic changes during stem cell differentiation, is found to be a substrate of KDM5b. Furthermore, MethylSight predicts that Lys methylation is a prevalent post-translational modification in the human proteome. Our work provides a useful resource for guiding systematic exploration of the role of Lys methylation in human health and disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1β (HP1β) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1β binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1β in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1β interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although Numb exhibits its tumor-suppressive function in breast cancer in part by binding to and stabilizing p53, it is unknown how the Numb-p53 interaction is regulated in cells. We found that Numb is methylated in its phosphotyrosine-binding (PTB) domain by the lysine methyltransferase Set8. Moreover, methylation uncouples Numb from p53, resulting in increased p53 ubiquitination and degradation. While Numb promotes apoptosis in a p53-dependent manner, the apoptotic function is abolished when Numb is methylated by Set8 or the Lys methylation sites in Numb are mutated. Conversely, the Numb-p53 interaction and Numb-mediated apoptosis are significantly enhanced by depletion of Set8 from cancer cells or by treating the cells with doxorubicin, a chemotherapeutic drug that causes a reduction in the mRNA and protein levels of Set8. Our work identifies the Set8-Numb-p53 signaling axis as an important regulatory pathway for apoptosis and suggests a therapeutic strategy by targeting Numb methylation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

An adult red-legged partridge (Alectoris rufa) presented with concurrent pulmonary carcinoma and severe silicosis. The animal was submitted to the Veterinary Teaching Hospital of the University of Córdoba (Spain) because of respiratory signs, and it died during clinical examination. At postmortem examination, numerous firm, whitish to yellowish nodules involving the lungs, mainly the right lobe, were found. The histopathologic study revealed numerous peribronchiolar large granulomatous lesions composed of macrophages, which showed abundant cytoplasm containing numerous birefringent crystals identified as silicates by transmission electron microscopy. An epithelial neoplasm showing papillary, acinar, and solid patterns occupied large areas of the pulmonary parenchyma. The histopathologic and immunohistochemical features were consistent with a pulmonary carcinoma. Small tumor nests were often located close to the granulomatous lesions. This is the first report of concurrent pneumoconiosis and pulmonary carcinoma in a nonhuman species.

The polycomb repressive complex 2 (PRC2) is the major methyltransferase for H3K27 methylation, a modification critical for maintaining repressed gene expression programs throughout development. It has been previously shown that PRC2 maintains histone methylation patterns during DNA replication in part through its ability to bind to H3K27me3. However, the mechanism by which PRC2 recognizes H3K27me3 is unclear. Here we show that the WD40 domain of EED, a PRC2 component, is a methyllysine histone-binding domain. The crystal structures of apo-EED and EED in complex respectively with five different trimethyllysine histone peptides reveal that EED binds these peptides via the top face of its β-propeller architecture. The ammonium group of the trimethyllysine is accommodated by an aromatic cage formed by three aromatic residues, while its aliphatic chain is flanked by a fourth aromatic residue. Our structural data provide an explanation for the preferential recognition of the Ala-Arg-Lys-Ser motif-containing trimethylated H3K27, H3K9, and H1K26 marks by EED over lower methylation states and other histone methyllysine marks. More importantly, we found that binding of different histone marks by EED differentially regulates the activity and specificity of PRC2. Whereas the H3K27me3 mark stimulates the histone methyltransferase activity of PRC2, the H1K26me3 mark inhibits PRC2 methyltransferase activity on the nucleosome. Moreover, H1K26me3 binding switches the specificity of PRC2 from methylating H3K27 to EED. In addition to determining the molecular basis of EED-methyllysine recognition, our work provides the biochemical characterization of how the activity of a histone methyltransferase is oppositely regulated by two histone marks.

An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of chromatin-binding modules (CBMs) in a cell to control chromatin state, gene expression, and other cellular functions. We have assembled a peptide chip that represents known and putative lysine methylation marks on histones and p53 and probed the chip for binding to a group of CBMs to obtain a comprehensive interaction network mediated by lysine methylation. Interactions revealed by the peptide array screening were validated by in-solution binding assays. This study not only recapitulated known interactions but also uncovered new ones. A novel heterochromatin protein 1 beta (HP1β) chromodomain-binding site on histone H3, H3K23me, was discovered from the peptide array screen and subsequently verified by mass spectrometry. Data from peptide pull-down and colocalization in cells suggest that, besides the H3K9me mark, H3K23me may play a role in facilitating the recruitment of HP1β to the heterochromatin. Extending the peptide array and mass spectrometric approach presented here to more histone marks and CBMs would eventually afford a comprehensive specificity and interaction map to aid epigenetic studies.

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Doñana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Doñana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.

Feline retroviruses are rarely reported in lynx species. Twenty-one Iberian lynx (Lynx pardinus) blood and tissue samples collected from Doñana National Park and Los Villares (Sierra Morena) in southern Spain during 1993-2003 were analyzed by polymerase chain reaction to amplify nucleic acids from feline retroviruses. Six samples were positive for Feline leukemia virus (FeLV), but no samples tested positive for Feline immunodeficiency virus. The BLAST analysis indicated that 5 of the 6 sequences were closely related to FeLV strain Rickard subgroup A, whereas 1 sequence was identical to FeLV. To the authors' knowledge, this is the first report of FeLV in the endangered Iberian lynx.

The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28-44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.

Congenital arteriovenous fistulas of the kidney are very rare. Options for therapy range from embolisation to nephrectomy. It depends on symptoms and extensiveness of the malformations. We report two cases of congenital arteriovenous malformations successfully managed in the first case by embolisation and in the second case by nephrectomy. Renal arteriovenous malformations remain an uncommon clinical problem which causes often diagnostic difficulties. The proper diagnosis can be made on the basis of standard diagnostic procedures such as physical examination, Doppler sonography and spiral computerized tomography.

A wild injured Iberian lynx (Lynx pardinus) was taken from the Sierra Morena population. During the health check small intraerythrocytic piroplasms, morphologically indistinguishable from other feline piroplasms, were observed in Wright-Giemsa-stained blood films. Amplification by polymerase chain reaction of a portion of the 18S nuclear small subunit (NSS) rRNA gene and sequencing revealed similarity of the unknown organism with sequences obtained from Pallas's cat from Mongolia and from a domestic cat in Spain. In a retrospective (1993-2003) study of 50 Iberian lynx tissue samples, no amplifications of the 18S NSS rRNA gene of the organism were obtained. This is the first report of a naturally occurring erythroparasitemia in the Iberian lynx and the first documented case of naturally occurring piroplasm infection in a free-ranging felid from Europe.

Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.

A cutaneous lesion, previously known as "warts", affecting the featherless parts of face and legs has long been recognized in juvenile Spanish Imperial eagles (Aquila adalberti). This paper describes the presentation, microbiological, histopathological, and electron microscopic findings of lesions and diagnosis of poxvirus infection in nine juveniles. Lesions consisted of single or multiple nodules with a crust and surrounded by skin swelling. Seventy-eight percent of the swabs taken from lesions yielded bacterial growth, with Escherichia coli being the most common bacterium isolated. Histopathology revealed typical pox lesions in all cases. Histopathological changes found consisted of proliferative epithelium, with ballooning degeneration of keratinocytes and lymphocyte infiltrates extending into underlying dermis. Avianpox virus was confirmed by the presence of eosinophilic intracytoplasmatic inclusion bodies in the affected cells on light microscopy, and diagnosis confirmation was performed by electron microscopy of biopsies from all nine eagles.

Recent studies clearly show the utility of the carbodiimide protocol to attach the redox-active Fc moiety to the N-terminal side of amino acids and peptides under mild conditions, resulting in stable and often crystalline products that afford themselves to structural analysis by X-ray crystallography. Electrochemical studies of Fc-peptides show that there is a significant influence of the redox potential depending on the amino acid sequence. The Fc moiety is sensitive to structural changes that occur in the peptide to which it is attached. For helical Fc-oligoprolines, the redox potential of the Fc group makes it easier to oxidize as the oligoproline chain increases in length. Nonhelical peptides, having a similar primary but different secondary structure, give rise to very different redox potentials. The ramifications of these findings to biological systems are significant in that they provide further evidence that the redox properties of a metal center are influenced by factors that go beyond the primary ligand sphere and thus for the involvement of long-range interactions. The Fc group is clearly sensitive to the shape of the peptide. These effects are currently under more detailed investigation [45] in order to gain further insight into the electronic structure of these ferrocenoyl peptides. Although we are not yet in a position to distinguish between "sensing" coordination or "sensing" conformation changes, this effect is of interest because it may allow the development of peptidic sensors.

We report the first case of bovine tuberculosis in a free-living Iberian lynx (Lynx pardina), an extremely endangered feline, from Doñana National Park in Spain. The isolate (Mycobacterium bovis) correlates by molecular characterization with other isolates from wild ungulates in the park, strongly suggesting an epidemiologic link. Mycobacterium bovis infects many animal species, with wild and free-ranging domestic ungulates being the main reservoirs in nature (1).

Three anesthetic protocols, each using an alpha-2 agonist sedative in combination with a dissociative anesthetic, were evaluated in 17 captive fallow deer (Cervus dama). The alpha-2 agonist was given first in two of the three protocols: 1) detomidine (0.1-0.2 mg/kg i.m.) followed by tiletamine-zolazepam (3.0-6.3 mg/kg i.m.) and 2) xylazine (0.6-0.9 mg/kg i.m.) followed by tiletamine-zolazepam (4-5 mg/kg i.m.). In the third protocol, xylazine (1.0-6.2 mg/kg i.m.) and ketamine (2.5-5.1 mg/kg i.m.) were given simultaneously. Each of the sedative/anesthetic combinations produced acceptable immobilization in fallow deer and both provide an alternative to narcotic anesthesia.

The evaluation described in this article compared the Therevac SB "mini-enema" with bisacodyl suppositories in the bowel management programs of patients with spinal cord injury (SCI). Of particular interest were (a) determining whether the additional costs of Therevac SB could be justified and (b) identifying the degree of bowel program improvement possible. Fourteen SCI patients were selected from the SCI inpatient unit and the clinic of a Department of Veterans Affairs facility. All patients experienced a delay of at least 45 minutes between insertion of a suppository and the beginning of stool evacuation. Each patient used bisacodyl suppositories for five bowel programs, then Therevac SB for five programs, finally repeating the bisacodyl suppositories for five more programs. Each patient maintained a bowel program log. Ten patients completed the evaluation. Using a MANOVA, the authors found a significant difference between bisacodyl and Therevac SB mean evacuation times for this group. An analysis of direct and indirect costs related to bowel care with the two regimens is also presented.

A back injury prevention program was developed for the Spinal Cord Injury (SCI) nursing staff at the San Diego Veterans Affairs Medical Center (SDVAMC). This preventive program has the following components: 1. All nursing staff are sent to a one hour "back school" in order to learn and practice proper body mechanics. This program is held in the therapy clinic and conducted by a kinesiotherapist. In addition, each nurse must be certified in transfer techniques by our clinical nurse specialist, before performing transfers on patients. 2. All nursing staff participate in 5-10 minutes of stretching, warm-up exercises at the beginning of their shift. These exercises are helpful as injuries are less likely to occur when muscles have been warmed up prior to lifting and moving patients. 3. All nursing staff wear a lumbar sacral back support while on duty. This device will offer physical support to the lower back as well as provide a mental reminder to use proper body mechanics when transferring patients. This back injury prevention program has been in effect since the opening of the SCI unit in March 1989. The rate of low-back injuries on the SCI unit from October 1989 to September 1990 was compared to the rate of low-back injuries on the other hospital units. As a result of this program, there were only four low-back injuries with six lost work days on the SCI unit. This rate compares favorably with the incidence of low-back injuries in other patient care areas.

Four methods of fistulorrhaphy used by us in 96 cases of vesicovaginal fistulas after hysterectomy are described. Obtained results are presented.

Different methods of urinary derivation are described, which are performed in gynecologic patients. Operative technics, their advantages and disadvantages and plans for selection of derivative methods are discussed.

The authors advocate the use of the U-nephrostomy as the routine measure in operative treatment of infected renal stones.

Acid phosphatase (othophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from the human prostate was immobilized by its protein moiety on cyanogen bromide-activated Sepharose, by carbohydrate moiety on Concanavalin-A-Sepharose, and by Schiff base formation with partially oxidized carbohydrate groups on ethylenediamine-Sepharose. The highest retention of enzyme activity, 80%, was found for the noncovalent immobilization on Concanavalin-A-Sepharose. It was demonstrated that the optimal pH changes for the Concanavalin-A-Sepharose and CNBr-Sepharose-enzyme complexes are electrostratic in character. In all cases of immobilization the enzyme has higher thermostability than that for the native enzyme under the same conditions. The effects of the enzyme stabilization were interpreted in terms of the multipoint interaction between the enzyme molecule and the carrier.

The operative repair of retroglandular hypospadias should be individualized. In some cases only resection of hooded foreskin and meatotomy are required. In a small number of properly selected cases a one-stage operation may be performed. In most cases a three-stage repair was carried out by the authors.

Soluble alkaline phosphatase from Thiobacillus thioparus cells was purified about 230-fold. The enzyme had a mol. wt. of 50 000 daltons, optimum pH at 10.5, and was heat-resistant in the presence of diethanolamine. Polyacrylamide-gel electrophoresis demonstrated contamination of the preparation with inactive proteins and the presence of two active bands. The enzyme activity was distinctly stimulated by increasing concentrations of Tris or diethanolamine. In the presence of glycine, 1 mM-Zn2+ enhanced the enzyme activity; in Tris or diethanolamine buffers the activity was stimulated by 1 mM-Mg2+ whereas Zn2+ had a strong inhibitory effect. Glycine at concentrations exceeding 25 mM also inhibited the enzyme. Specificity of the enzyme is fairly broad.