Rechallenge in idiosyncratic drug-induced liver injury: An analysis of cases in two large prospective registries according to existing definitions.

INTRODUCTION: Data on positive rechallenge in idiosyncratic drug-induced liver injury (DILI) are scarce. We aim to analyse the clinical presentation, outcome and drugs associated with positive rechallenge in two DILI registries. METHODS: Cases from the Spanish and Latin American DILI registries were included. Demographics, clinical characteristics and outcome of cases with positive rechallenge according to CIOMS/RUCAM and current definitions were analysed. RESULTS: Of 1418 patients with idiosyncratic DILI, 58 cases had positive rechallenge (4.1%). Patients with positive rechallenge had shorter duration of therapy (p=0.001) and latency (p=0.003). In patients with rechallenge, aspartate transaminase levels were increased (p=0.026) and showed a prolonged time to recovery (p=0.020), albeit no differences were seen in terms of fatal outcomes. The main drug implicated in rechallenge was amoxicillin-clavulanate (17%). The majority of re-exposure events were unintentional (71%). Using both existing definitions of positive rechallenge, there were four cases which exclusively fulfilled the current criteria and five which only meet the historical definition. All cases of positive rechallenge, irrespective of the pattern of damage, fulfilled the criteria of either alanine transaminase (ALT) ≥3 times the upper limit of normal (ULN) and/or alkaline phosphatase (ALP) ≥2 times ULN. CONCLUSIONS: Episodes of rechallenge were characterised by shorter duration of therapy and latency, and longer time to resolution, but did not show an increased incidence of fatal outcome. Based on our findings, ALT ≥3 times ULN and/or ALP ≥2 times ULN, regardless of the pattern of damage, is proposed as a new definition of rechallenge in DILI.

Primary surgical management of anterior pelvic organ prolapse: a systematic review, network meta-analysis and cost-effectiveness analysis.

BACKGROUND: Anterior compartment prolapse is the most common pelvic organ prolapse (POP) with a range of surgical treatment options available. OBJECTIVES: To compare the clinical effectiveness and cost-effectiveness of surgical treatments for the repair of anterior POP. METHODS: We conducted a systematic review of randomised controlled trials comparing surgical treatments for women with POP. Network meta-analysis was possible for anterior POP, same-site recurrence outcome. A Markov model was used to compare the cost-utility of surgical treatments for the primary repair of anterior POP from a UK National Health Service perspective. MAIN RESULTS: We identified 27 eligible trials for the network meta-analysis involving eight surgical treatments tested on 3194 women. Synthetic mesh was the most effective in preventing recurrence at the same site. There was no evidence to suggest a difference between synthetic non-absorbable mesh, synthetic partially absorbable mesh, and biological mesh. The cost-utility analysis, which incorporated effectiveness, complications and cost data, found non-mesh repair to have the highest probability of being cost-effective. The conclusions were robust to model inputs including effectiveness, costs and utility values. CONCLUSIONS: Anterior colporrhaphy augmented with mesh appeared to be cost-ineffective in women requiring primary repair of anterior POP. There is a need for further research on long-term effectiveness and the safety of mesh products to establish their relative cost-effectiveness with a greater certainty. TWEETABLE ABSTRACT: New study finds mesh cost-ineffective in women with anterior pelvic organ prolapse.

Management of mesh complications following surgery for stress urinary incontinence or pelvic organ prolapse: a systematic review.

BACKGROUND: Mesh surgery for stress urinary incontinence or pelvic organ prolapse can result in complications such as mesh exposure, mesh extrusion, voiding dysfunction, dyspareunia, and pain. There is limited knowledge or guidance on the effective management for mesh-related complications. OBJECTIVE: To determine the best management of mesh complications; a systematic review was conducted as part of the national clinical guideline 'Urinary incontinence (update) and pelvic organ prolapse in women: management'. SEARCH STRATEGY: Search strategies were developed for each indication for referral. SELECTION CRITERIA: Relevant interventions included complete or partial mesh removal, mesh division, and non-surgical treatments such as vaginal estrogen. DATA COLLECTION AND ANALYSIS: Characteristics and outcome data were extracted, and as a result of the heterogeneous nature of the data a narrative synthesis was conducted. MAIN RESULTS: Twenty-four studies were included; five provided comparative data and four studies stated the indication for referral. Reported outcomes (including pain, dyspareunia, satisfaction, quality of life, incontinence, mesh exposure, and recurrence) and the reported incidences of these varied widely. CONCLUSIONS: The current evidence base is limited in quantity and quality and does not permit firm recommendations to be made on the most effective management for mesh-related complications. Robust data are needed so that mesh complications can be managed effectively in the future. TWEETABLE ABSTRACT: Systematic review demonstrates that the outcomes following mesh revision surgery are highly variable.

Analysis of FKBP10, SERPINH1, and SERPINF1 genes in patients with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous disorder that causes fragility, deformity, and fractures in bones. A large number of genes that are associated with the disease have been identified in the last decade; this makes the genetic diagnosis of OI more difficult. To improve our knowledge of the genetic mutation profile in OI we used single-stranded conformation polymorphism screening and automated sequencing to investigate the SERPINH1, FKBP10, and SERPINF1 genes, which are related to recessive OI, in 23 unrelated Brazilian patients. Nine rare changes and four common polymorphisms were detected. Most changes were benign genetic variants. In general, changes in the SERPINH1 and SERPINF1 genes were synonymous polymorphisms or missense changes located in non-coding regions. A pathogenic change was found in the FKBP10 gene. The characterization of mutations related to OI in distinct populations can improve our knowledge of the genetic aspects of OI and help us develop molecular strategies for the diagnosis of the disease.

Preoperative somatostatin analogues versus direct transsphenoidal surgery for newly-diagnosed acromegaly patients: a systematic review and meta-analysis using the GRADE system.

UNLABELLED: Whether the preoperative use of somatostatin analogues (SA) improves surgical outcomes in acromegaly is still a matter of debate. OBJECTIVE: We conducted a systematic review of randomized, controlled trials that compared the short-term outcomes of preoperative use of SA (Pre-SA) with direct TSS (No-SA) for the treatment of newly diagnosed acromegaly. METHODS: Embase, Pubmed, Lilacs, and Central Cochrane were used as our data sources. The primary outcomes were no need for any adjuvant treatment 3 months after surgery, based on biochemical results (GH nadir after OGTT <1 µg/L and normal IGF-1 for age and gender), quality of life and mortality. The included trials were analyzed using the Grading of Recommendations Assessment, Development, and Evaluation approach. RESULTS: A total of 2.099 references were identified and two reviewers independently screened the titles and abstracts. From the 14 potentially eligible studies, four were included and ten were excluded due to lack of randomization or different outcomes. A pool of 261 patients was randomly assigned to Pre-SA or No-SA. Meta-analysis of IGF1 normalization showed a significant difference in favor of Pre-SA (RR 2.47; 95% CI 1.66, 3.77). Adding a GH nadir on OGTT ≤1 µg/L, we found a RR of 2.15 (95% CI 1.39, 3.33). Quality of evidence for no need of adjuvant postoperative treatment was moderate, but for improving quality of life was very low and for mortality was absent. CONCLUSION: Pre-SA increases the chance of biochemical control of acromegaly 3 months after TSS in patients harboring GH-secreting pituitary macroadenomas.

Mutational characterization of the P3H1/CRTAP/CypB complex in recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone deformities and fractures. Most cases are caused by autosomal dominant mutations in the type I collagen genes COL1A1 and COL1A2; however, an increasing number of recessive mutations in other genes have been reported. The LEPRE1, CRTAP, and PPIB genes encode proteins that form the P3H1/CRTAP/CypB complex, which is responsible for posttranslational modifications of type I collagen. In general, mutations in these genes lead to severe and lethal phenotypes of recessive OI. Here, we describe sixteen genetic variations detected in LEPRE1, CRTAP, and PPIB from 25 Brazilian patients with OI. Samples were screened for mutations on single-strand conformation polymorphism gels and variants were determined by automated sequencing. Seven variants were detected in patients but were absent in control samples. LEPRE1 contained the highest number of variants, including the previously described West African allele (c.1080+1G>T) found in one patient with severe OI as well as a previously undescribed p.Trp675Leu change that is predicted to be disease causing. In CRTAP, one patient carried the c.558A>G homozygous mutation, predicted as disease causing through alteration of a splice site. Genetic variations detected in the PPIB gene are probably not pathogenic due to their localization or because of their synonymous effect. This study enhances our knowledge about the mutational pattern of the LEPRE1, CRTAP, and PPIB genes. In addition, the results strengthen the proposition that LEPRE1 should be the first gene analyzed in mutation detection studies in patients with recessive OI.

Frequency of multiple endocrine neoplasia type 1 in a group of patients with pituitary adenoma: genetic study and familial screening.

The purpose of this study it was to evaluate the frequency of Multiple Endocrine Neoplasia type 1 (MEN1) in patients with pituitary adenoma and to perform genetic analysis and familial screening of those individuals afflicted with MEN1. 144 patients with pituitary adenoma at Botucatu Medical School, UNESP-Univ Estadual Paulista, were assessed retrospectively for MEN1 during the years of 2005-2011. The patients were evaluated for the presence of primary hyperparathyroidism (PHP) and enteropancreatic tumors. Genetic analysis was performed for the individuals with clinically diagnosed MEN1. Thirteen patients met the diagnostic criteria for MEN1, but three individuals belong to the same family and they were considered as a single MEN1 event, revealing 7.7 % frequency of MEN1 in this patient group. Genetic analysis showed MEN1 mutations in four index cases: IVS4+1 G>A, IVS3-6 C>T, c.1547insC and a new D180A mutation. One patient did not agree to participate in the genetic study and another one was referred for follow up in other hospital. Only polymorphisms were found in the other individuals, one of which was novel. We identified a high frequency of MEN1 in pituitary adenoma patients. Since PHP is one of the most common MEN1 tumor and patients are mostly asymptomatic, we suggest that all pituitary adenoma patients have their calcium profile analyzed.

Insights into Leptocybe invasa resistance in Eucalyptus: phenotyping, genotyping and in silico approaches.

The gall wasp, Leptocybe invasa, poses a significant global threat to Eucalyptus cultivation, by causing substantial economic losses. The objective of this study was to differentiate between resistant and susceptible genotypes by morphological characteristics using image analysis based on the damage caused by the gall wasp. In addition, consensus sequences derived from transposable elements (TEs) and the genome of Eucalyptus spp. Were identified by in silico analysis. Furthermore, another objective was to discriminate Eucalyptus genotypes in response to Leptocybe invasa by conducting molecular analyses involving transposable elements and inter simple sequence markers. For image analysis, the GroundEye ® system was used to collect images of 60 leaves from six genotypes, three of which were resistant and three susceptible. Eucalyptus spp. sequences were obtained from the GenBank database by in silico analysis and pairwise alignments with TE sequences were conducted using BLASTN. Multiple sequence alignment was performed with Clustal Omega, followed by the identification of conserved regions in Jalview. A motif signature was generated using Weblogo. For molecular characterization using ISSR markers and TEs, samples of young leaves were obtained from a total of 80 Eucalyptus seedlings, of which 50 were classified as resistant and 30 as susceptible to L. invasa. It was possible to distinguish gall wasp susceptible and resistant genotypes by image analysis. In silico analysis enabled the identification of conserved regions in the Eucalyptus spp. genome, which were associated with proteins involved in secondary metabolite production, e.g., terpenes, which play a role in the response to L. invasa. The discrimination capacity of TEs and ISSR primers was demonstrated and bands were generated that could be used to identify resistant genotypes. However, increasing the number of markers required to discriminate genotypes in both cases is suggested.

Sex-Specific Effects of Cholesteryl Ester Transfer Protein (CETP) on the Perivascular Adipose Tissue.

Cholesteryl ester transfer protein (CETP) increases the atherosclerosis risk by lowering HDL-cholesterol levels. It also exhibits tissue-specific effects independent of HDL. However, sexual dimorphism of CETP effects remains largely unexplored. Here, we hypothesized that CETP impacts the perivascular adipose tissue (PVAT) phenotype and function in a sex-specific manner. PVAT function, gene and protein expression, and morphology were examined in male and female transgenic mice expressing human or simian CETP and their non-transgenic counterparts (NTg). PVAT exerted its anticontractile effect in aortas from NTg males, NTg females, and CETP females, but not in CETP males. CETP male PVAT had reduced NO levels, decreased eNOS and phospho-eNOS levels, oxidative stress, increased NOX1 and 2, and decreased SOD2 and 3 expressions. In contrast, CETP-expressing female PVAT displayed increased NO and phospho-eNOS levels with unchanged NOX expression. NOX inhibition and the antioxidant tempol restored PVAT anticontractile function in CETP males. Ex vivo estrogen treatment also restored PVAT function in CETP males. Moreover, CETP males, but not female PVAT, show increased inflammatory markers. PVAT lipid content increased in CETP males but decreased in CETP females, while PVAT cholesterol content increased in CETP females. CETP male PVAT exhibited elevated leptin and reduced Prdm16 (brown adipocyte marker) expression. These findings highlight CETP sex-specific impact on PVAT. In males, CETP impaired PVAT anticontractile function, accompanied by oxidative stress, inflammation, and whitening. Conversely, in females, CETP expression increased NO levels, induced an anti-inflammatory phenotype, and preserved the anticontractile function. This study reveals sex-specific vascular dysfunction mediated by CETP.

Does plasma HDL-C concentration interact with whole-body cholesterol metabolism?

This review examines the interactions between plasma high density lipoprotein (HDL) metabolism and whole-body cholesterol economy. More specifically, this review addresses three questions: 1) does plasma HDL-C concentration correlate with the parameters of whole-body cholesterol metabolism? 2) Do variations in cholesterol metabolism interfere with plasma HDL-C concentrations? 3) Are the markers of cholesterol synthesis and intestinal absorption specifically under the control of plasma HDL? The following answers were provided to each question, respectively: 1) plasma HDL influences whole-body cholesterol synthesis rate but the evidence that HDL modifies the total amount of cholesterol absorbed by the intestine is not clearly supported by present investigations; 2) there are suggestions that changes in whole body cholesterol metabolism rates do not interfere with plasma HDL-C concentrations; 3) markers of cholesterol synthesis and absorption may specifically be controlled by plasma HDL-C concentrations regarding the genetic causes of extremely low HDL-C concentrations, although within the general population plasma HDL-C concentration is likely ascribed to insulin resistance or diabetes mellitus.

The origin of the major cystic fibrosis mutation (delta F508) in European populations.

delta F508 is the most frequent cystic fibrosis (CF) mutation and accounts for approximately 70% of CF chromosomes worldwide. Three highly polymorphic microsatellite markers have been used to study the origin and evolution of delta F508 chromosomes in Europe. Haplotype data demonstrate that delta F508 occurred more than 52,000 years ago, in a population genetically distinct from any present European group, and spread throughout Europe in chronologically distinct expansions, which are responsible for the different frequencies of delta F508 in Europe.

Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the baso-lateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Histomorphological evaluation of acellularized bovine pericardium in breast implant coverage.

Bovine pericardium (BP) has been used as a biomaterial for several decades in many medical applications particularly due to its mechanical properties and the high collagen content. In the acellular form it favors faster tissue repair, providing a three-dimensional support for cellular and vascular events observed during tissue repair and due, to a low elastin content, may favor its use as a breast implant cover, resulting in a low possibility of contracture of the biomaterial, preventing the appearance of irregularities during the reconstruction process. Thus, the aim of this study was to evaluate, histomorphologically, the behavior of acellularized bovine pericardium (ABP) as a mammary implant cover in rats. For this purpose, 16 animals were divided into two groups, with eight animals at each biological point: 7 and 15 days after surgery. Of the 16 animals, 32 specimens were obtained: 16 in the experimental group (EG) and 16 in the control group (CG). Throughout this study, none of the studied groups had postoperative complications. Results: The histomorphological results showed, in the two biological points, both in the EG and in the CG, chronic inflammatory infiltrate, leukocyte fibrin exudate, formation of granulation tissue and deposition of collagen fibers, more evident in the EG, regressive along the biological points. At 15 days, the implanted ABP showed initial biointegration with the fibrous capsule and surrounding tissues of the recipient bed. Conclusion: These results indicate that the due to the observed favorable tissue response ABP may be of potential use as a breast implant cover.

Challenges and pitfalls in the characterization of anonymous outlier AFLP markers in non-model species: lessons from an ocellated lizard genome scan.

In the last few years, dozens of studies have documented the detection of loci influenced by selection from genome scans in a wide range of non-model species. Many of those studies used amplified fragment length polymorphism (AFLP) markers, which became popular for being easily applicable to any organism. However, because they are anonymous markers, AFLPs impose many challenges for their isolation and identification. Most recent AFLP genome scans used capillary electrophoresis (CE), which adds even more obstacles to the isolation of bands with a specific size for sequencing. These caveats might explain the extremely low number of studies that moved from the detection of outlier AFLP markers to their actual isolation and characterization. We document our efforts to characterize a set of outlier AFLP markers from a previous genome scan with CE in ocellated lizards (Lacerta lepida). Seven outliers were successfully isolated, cloned and sequenced. Their sequences are noncoding and show internal indels or polymorphic repetitive elements (microsatellites). Three outliers were converted into codominant markers by using specific internal primers to sequence and screen population variability from undigested DNA. Amplification in closely related lizard species was also achieved, revealing remarkable interspecific conservation in outlier loci sequences. We stress the importance of following up AFLP genome scans to validate selection signatures of outlier loci, but also report the main challenges and pitfalls that may be faced during the process.

Variable expressivity of osteogenesis imperfecta in a Brazilian family due to p.G1079S mutation in the COL1A1 gene.

Osteogenesis imperfecta (OI) is a Mendelian disease with genetic heterogeneity characterized by bone fragility, recurrent fractures, blue sclerae, and short stature, caused mostly by mutations in COL1A1 or COL1A2 genes, which encode the pro-α1(I) and pro-α2(I) chains of type I collagen, respectively. A Brazilian family that showed variable expression of autosomal dominant OI was identified and characterized. Scanning for mutations was carried out using SSCP and DNA sequence analysis. The missense mutation c.3235G>A was identified within exon 45 of the COL1A1 gene in a 16-year-old girl diagnosed as having OI type I; it resulted in substitution of a glycine residue (G) by a serine (S) at codon 1079 (p.G1079S). The proband's mother had the disease signs, but without bone fractures, as did five of nine uncles and aunts of the patient. All of them carried the mutation, which was excluded in four healthy brothers of the patient's mother. This is the first description in a Brazilian family with OI showing variable expression; only one among seven carriers for the c.3235G>A mutation developed bone fractures, the most striking clinical feature of this disease. This finding has a significant implication for prenatal diagnosis in OI disease.

Association of Mc1r variants with ecologically relevant phenotypes in the European ocellated lizard, Lacerta lepida.

A comprehensive knowledge on the genetic basis of coloration is crucial to understand how new colour phenotypes arise and how they contribute to the emergence of new species. Variation in melanocortin-1 receptor (Mc1r), a gene that has been reported as a target for repeated evolution in a wide range of vertebrate taxa, was assessed in European ocellated lizards (Lacerta lepida) to search for associations with melanin-based colour phenotypes. Lacerta lepida subspecies' distribution is associated with the three major bio-climatic regions in the Iberian Peninsula. A nonconserved and derived substitution (T162I) was associated with the L. l. nevadensis phenotype (prevalence of brown scales). Another substitution (S172C) was associated with the presence of black scales in both L. l. lepida and L. l. iberica, but no mutations were found to be associated with the higher proportion of black in L. l. iberica. Extensive genotyping of Mc1r along the contact zone between L. l. nevadensis and L. l. lepida revealed low gene flow (only two hybrids detected). The implications of these findings are discussed in the context of previous knowledge about the evolutionary history of ocellated lizards.

Mercury and methyl mercury ratios in caimans (Caiman crocodilus yacare) from the Pantanal area, Brazil.

The Pantanal region is the largest floodplain area in the world and of great biological importance due to its unique flora and fauna. This area is continuously undergoing increasing anthropogenic threats, and has also experienced mercury contamination associated with gold mining and other anthropogenic activities. Pantanal caimans are top-level predators, and, as such, show great potential to accumulate mercury (Hg) by biomagnification. In this study 79 specimens from four locations in the Pantanal were analyzed for total Hg and methyl mercury (MeHg) by cold vapor atomic absorption spectrometry. Total Hg contents ranged from 0.02 to 0.36 µg g(-1) (ww), and most specimens presented MeHg ratios above 70%. One of the sites, impacted by anthropogenic activities, presented significantly higher total Hg in comparison to three less impacted sites, supporting the hypothesis that caimans can, in fact, be considered effective bioindicators of ecosystem health.

Biological behavior of magnesium-substituted hydroxyapatite during bone repair.

The aim of this study was to analyze the biological behavior and osteogenic potential of magnesium (Mg) substituted hydroxyapatite (HA) microspheres, implanted in a critical bone defect, considering that this ion is of great clinical interest, since it is closely associated with homeostasis and bone mineralization. For the purpose of this study, 30 rats were used to compose three experimental groups: GI - bone defect filled with HA microspheres; GII - bone defect filled with HA microspheres replaced with Mg; GIII - empty bone defect; evaluated at biological points of 15 and 45 days. The histological results, at 15 days, showed, in all the groups, a discrete chronic inflammatory infiltrate; biomaterials intact and surrounded by connective tissue; and bone neoformation restricted to the borders. At 45 days, in the GI and GII groups, an inflammatory response of discrete granulomatous chronic type was observed, and in the GIII there was a scarce presence of mononuclear inflammatory cells; in GI and GII, the microspheres were seen to be either intact or fragmented, surrounded by fibrous connective tissue rich in blood vessels; and discrete bone neoformation near the edges and surrounding some microspheres. In GIII, the mineralization was limited to the borders and the remaining area was filled by fibrous connective tissue. It was concluded that the biomaterials were biocompatible and osteoconductive, and the percentage of Mg used as replacement ion in the HA did not favor a greater bone neoformation in relation to the HA without the metal.

Assessment of COMT isolation by HIC using a dual salt system and low temperature.

Sodium citrate (SC) and low temperatures between 7 and 5 degrees C are effective in suppressing aggregation of proteins and may be beneficial to be included during a purification process. In this work, we analyzed the application of dual salt system, ammonium sulfate (AS) and SC on binding and elution conditions of recombinant hSCOMT on typical HIC sorbents. Specifically in butyl and octyl supports, the use of, respectively, 300 mM AS/200 mM SC and 25 mM AS/25 mM SC in the loading buffer resulted in complete binding of COMT. Elution was obtained by decreasing the ionic strength to 0 M of salt. For the delineate goal, it also favorably increased the support chain length while a consequent decrease in the dual ionic strength was observed for hSCOMT retention. In the presence of dual salt systems octyl media exhibited classic HIC behavior, good protein selectivity, an excellent purification factor and reduced denaturation effects of hSCOMT observed with higher salt concentrations. Also the inclusion of temperature control during the elution step appears to be advantageous for greater activity recovery without enzyme aggregation. In fact, these results could allow the prediction of most stabilizing conditions for this termolabile enzyme on the chromatographic stage, regarding salt types and therefore effectiveness to improve HIC selectivity and desirable purity on the target fractions.