Lipases: sources, immobilization methods, and industrial applications.

Enzymes are natural catalysts highly specific to the substrate type and operate under mild conditions of temperature, pressure, and pH with high conversion rates, which makes them more efficient than conventional chemical catalysts. The enzymes can be obtained from various sources, animal, vegetable, and microbiological. Lipases are very versatile enzymes, and this has aroused the interest of the industries. However, the great problem of the use of soluble lipases is the high cost of acquisition, low operational stability, and difficulties of recovery, and reuse. Enzymatic immobilization has been suggested as an alternative to reduce the limitations of soluble enzymes, increasing their stability and facilitating recovery, and reuse, significantly reducing the cost of processes involving the use of enzymes. This review presents a discussion on the different immobilization methods for lipase, as well as the challenges of use lipases immobilized on the industrial scale.

Role of rare germline copy number variation in melanoma-prone patients.

AIM: This work evaluates a possible causative role for germline copy number variants (CNVs) in melanoma predisposition. PATIENTS & METHODS: A total of 41 melanoma-prone Brazilian patients were investigated for CNVs using 850K single nucleotide polymorphism arrays. RESULTS: Ten rare CNVs were identified in nine patients, comprising 54 known genes, mostly related to cancer. In silico analyses revealed gene enrichment for cellular development and growth, and proliferation, highlighting five genes directly associated with the melanoma phenotype (ANGPT1, IDH1, PDE5A, HIST1H1B and GCNT2). CONCLUSION: Patients harboring rare CNVs exhibited a decreased age of disease onset, in addition to an overall higher skin cancer predisposition. Our findings suggest that rare CNVs contribute to melanoma susceptibility, and should be taken into account when investigating cancer risk factors.

Lymphovascular invasion and histologic grade are associated with specific genomic profiles in invasive carcinomas of the breast.

Lymphovascular invasion (LVI) and histologic grade are clinical parameters of high prognostic value in breast cancer and indicate the level of tumor aggressiveness. Many studies have focused on the association of breast cancer subtypes with gene expression and chromosomal profiles, but considerably less genomic information is available regarding traditional prognostic factors such as histologic grade and LVI. We studied by array-CGH a group of 57 invasive ductal carcinomas of the breast to outline the DNA copy number aberration (CNA) profile linked to high histologic grades and LVI. Selected CNAs were validated using real-time quantitative PCR (qPCR). Furthermore, gene expression analysis was performed in a subset of 32 of these tumors, and findings were integrated with array-CGH data. Our findings indicated an accumulation of genomic alterations in high-grade breast tumors compared to low-grade samples. Grade III tumors showed higher number of CNAs and larger aberrations than low-grade tumors and displayed a wide range of chromosomal aberrations, which were mainly 5p, 8q, 10p, 17q12, and 19 gains, and 3p, 4, 5q proximal, 9p, 11p, 18q, and 21 losses. The presence of LVI, a well-established prognostic marker, was not significantly associated with increased genomic instability in comparison to breast tumors negative for LVI, considering the total number of chromosomal alterations. However, a slightly increase in the frequency of specific alterations could be detected in LVI-positive group, such as gains at 5p, 16p, 17q12, and 19, and losses at 8p, 11q, 18q, and 21. Three newly reported small-scale rearrangements were detected in high-risk tumors (LVI-positive grade III) harboring putative breast cancer genes (amplicons at 4q13.3 and 11p11.2, and a deletion at 12p12.3). Furthermore, gene expression analysis uncovered networks highlighting S100A8, MMP1, and MED1 as promising candidate genes involved in high-grade and LVI-positive tumors. In summary, a group of genomic regions could be associated with high-risk tumors, and expression analysis pinpointed candidate genes deserving further investigation. The data has shed some light on the molecular players involved in two highly relevant prognostic factors and may further add to the understanding of the mechanisms of breast cancer aggressiveness.

Large germline copy number variations as predisposing factor in childhood neoplasms.

AIMS: Constitutive genetic factors are believed to predispose to cancer in children. This study investigated the role of rare germline copy number variations (CNVs) in pediatric cancer predisposition. PATIENTS & METHODS: A total of 54 patients who developed cancer in infancy were screened by array-CGH for germline CNVs. RESULTS: In total, 12 rare CNVs were detected, including a Xq27.2 triplication, and two >1.8 Mb deletions: one of them at 13q31, containing only RNA genes, and another at 3q26.33-q27.1, in a patient with congenital malformations. Detected rare CNVs are significantly larger than those identified in controls, and encompass genes never implicated in cancer predisposition. CONCLUSION: Our results suggest that constitutive CNVs contribute to the etiology of pediatric neoplasms, revealing new candidate genes for tumorigenesis.

Li-Fraumeni-like syndrome associated with a large BRCA1 intragenic deletion.

BACKGROUND: Li-Fraumeni (LFS) and Li-Fraumeni-like (LFL) syndromes are associated to germline TP53 mutations, and are characterized by the development of central nervous system tumors, sarcomas, adrenocortical carcinomas, and other early-onset tumors. Due to the high frequency of breast cancer in LFS/LFL families, these syndromes clinically overlap with hereditary breast cancer (HBC). Germline point mutations in BRCA1, BRCA2, and TP53 genes are associated with high risk of breast cancer. Large rearrangements involving these genes are also implicated in the HBC phenotype. METHODS: We have screened DNA copy number changes by MLPA on BRCA1, BRCA2, and TP53 genes in 23 breast cancer patients with a clinical diagnosis consistent with LFS/LFL; most of these families also met the clinical criteria for other HBC syndromes. RESULTS: We found no DNA copy number alterations in the BRCA2 and TP53 genes, but we detected in one patient a 36.4 Kb BRCA1 microdeletion, confirmed and further mapped by array-CGH, encompassing exons 9-19. Breakpoints sequencing analysis suggests that this rearrangement was mediated by flanking Alu sequences. CONCLUSION: This is the first description of a germline intragenic BRCA1 deletion in a breast cancer patient with a family history consistent with both LFL and HBC syndromes. Our results show that large rearrangements in these known cancer predisposition genes occur, but are not a frequent cause of cancer susceptibility.

Synthesis and characterization of a material derived from 4-mercaptobenzoic acid: A novel platform for oligonucleotide immobilization.

This paper reports the electrochemical modification of pencil carbon graphite electrodes with a polymeric material derived from 4-mercaptobenzoic acid. Acidic solutions (pH 0 and 5.02) yielded an insulating polymeric film with anionic permselective properties. Scanning Electron Microscopy (SEM) analysis showed a complete coverage of the carbon graphite electrodes with a laminar-like polymeric structure. Different characterization studies indicate that the carboxyl group remained unchanged since the absorbance peak and oxidation potential did not change with the increase in pH at the pKa accounting for the carboxyl/carboxylate redox transition. The functionalized matrix was activated using carbodiimide, succinimide and an amine-modified oligonucleotide. The immobilization and hybridization processes were successfully verified using the redox electroactive indicator methylene blue, where better electrochemical signals were obtained when compared with the traditional self-assembled monolayer system. The selectivity of the system was verified using a noncomplementary target where no significant difference in electric current was observed when compared to the system containing only the probe. The method showed a good linear correlation coefficient (r2=0.9915), low limit of detection (1.17nmolL-1), and an acceptable precision (RSD=2.75%). The proposed method is suitable for further studies using different sequences of oligonucleotides.

An inherited small microdeletion at 15q13.3 in a patient with early-onset obsessive-compulsive disorder.

Copy number variations (CNVs) have been previously associated with several different neurodevelopmental psychiatric disorders, such as autism, schizophrenia, and attention deficit hyperactivity disorder (ADHD). The present study consisted of a pilot genome-wide screen for CNVs in a cohort of 16 patients with early-onset obsessive-compulsive disorder (OCD) and 12 mentally healthy individuals, using array-based comparative genomic hybridization (aCGH) on 44K arrays. A small rare paternal inherited microdeletion (∼64 kb) was identified in chromosome 15q13.3 of one male patient with very early onset OCD. The father did not have OCD. The deletion encompassed part of the FMN1 gene, which is involved with the glutamatergic system. This finding supports the hypothesis of a complex network of several genes expressed in the brain contributing for the genetic risk of OCD, and also supports the glutamatergic involvement in OCD, which has been previously reported in the literature.

Array-CGH as an adjuvant tool in cytogenetic diagnosis of pediatric MDS and JMML.

Myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) are rare clonal hematopoietic diseases presented in the childhood. Both diseases exhibit abnormal karyotype and/or monosomy of chromosome 7 in a subgroup of patients. We screened for copy number variations (CNVs) by array-comparative genomic hybridization (aCHG) the DNA from bone marrow of six MDS and four JMML pediatric patients. Array-CGH analysis identified five cases (50%) with monosomy 7, disclosing the chromosome 7 monosomy in two patients whose samples could not be evaluated by other methods. We identified CNVs in six patients, one of which displayed loss of LMO2, an oncogene that plays a central role in hematopoietic development. Our results suggest that array-CGH is a reliable and accurate technique to identify genomic alterations in MDS and JMML.

Mining gene expression signature for the detection of pre-malignant melanocytes and early melanomas with risk for metastasis.

BACKGROUND: Metastatic melanoma is a highly aggressive skin cancer and currently resistant to systemic therapy. Melanomas may involve genetic, epigenetic and metabolic abnormalities. Evidence is emerging that epigenetic changes might play a significant role in tumor cell plasticity and metastatic phenotype of melanoma cells. PRINCIPAL FINDINGS: In this study, we developed a systematic approach to identify genes implicated in melanoma progression. To do this, we used the Affymetrix GeneChip Arrays to screen 34,000 mouse transcripts in melan-a melanocytes, 4C pre-malignant melanocytes, 4C11- non-metastatic and 4C11+ metastatic melanoma cell lines. The genome-wide association studies revealed pathways commonly over-represented in the transition from immortalized to pre-malignant stage, and under-represented in the transition from non-metastatic to metastatic stage. Additionally, the treatment of cells with 10 µM 5-aza-2'-deoxycytidine (5AzaCdR) for 48 hours allowed us to identify genes differentially re-expressed at specific stages of melan-a malignant transformation. Treatment of human primary melanocytes with the demethylating agent 5AzaCdR in combination to the histone deacetylase inhibitor Trichostatin A (TSA) revealed changes on melanocyte morphology and gene expression which could be an indicator of epigenetic flexibility in normal melanocytes. Moreover, changes on gene expression recognized by affecting the melanocyte biology (NDRG2 and VDR), phenotype of metastatic melanoma cells (HSPB1 and SERPINE1) and response to cancer therapy (CTCF, NSD1 and SRC) were found when Mel-2 and/or Mel-3-derived patient metastases were exposed to 5AzaCdR plus TSA treatment. Hierarchical clustering and network analyses in a panel of five patient-derived metastatic melanoma cells showed gene interactions that have never been described in melanomas. SIGNIFICANCE: Despite the heterogeneity observed in melanomas, this study demonstrates the utility of our murine melanoma progression model to identify molecular markers commonly perturbed in metastasis. Additionally, the novel gene expression signature identified here may be useful in the future into a model more closely related to translational research.

Avaliação parasitológica e bacteriológica do gelo comercializado na cidade de Juazeiro do Norte – CE / Parasitological and bacteriological evaluation of the ice sold in the city of Juazeiro do Norte - CE

A água é a matéria-prima utilizada na produção de gelo e a adoção de práticas rigorosas de higiene em sua fabricação, manuseio, embalagem, conservação e distribuição torna-se indispensável. O método de congelamento não é capaz de destruir os patógenos contaminantes, pois os mesmos permanecem em estado de latência até que o produto entre em processo de fusão. O estudo teve como objetivo avaliar as condições parasitológicas e bacteriológicas do gelo comercializado para consumo no município de Juazeiro do Norte, CE. Trata-se de um estudo analítico e transversal, realizado a partir de análises microbiológicas, por meio da técnica dos tubos múltiplos, e parasitológicas, pelo método de Hoffmann Modificado, técnica de Faust e pesquisa de Cryptosporidium spp. pela técnica de Ziehl-Neelsen Modificado, conhecido como coloração de Kinyoun. Em quinze amostras adquiridas em diferentes bairros deste município, 66,7% apresentaram coliformes totais e 33,3% coliformes termotolerantes. Na avaliação parasitológica, 33,3% estavam contaminadas por Giardia lamblia e 6,6% por Cryptosporidium spp. As irregularidades higienicossanitárias, que abordam desde o processo de fabricação do produto até a sua distribuição nos estabelecimentos, são as possíveis causas de contaminações parasitárias e microbiológicas. Reforça-se, portanto, a necessidade de estabelecer estratégias a fim de detectar os possíveis pontos de contaminação do gelo, buscando solucionar o problema e, assim, prevenir os consumidores de possíveis infecções e parasitoses, além de garantir a segurança deste alimento.

Since this is the raw material used in the production of ice, it is essential to adopt strict hygiene practices in their manufacturing, handling, packaging, storage and distribution. Since the freezing method is not able to destroy contaminating pathogens because the microorganisms remain in the sleep state until the product goes into melting process. The objective of this study was to evaluate the parasitological and bacteriological ice conditions marketed for consumption in Juazeiro do Norte, Ceará. It's about an analytic and transversal study, conducted from microbiological, using the technique of multiple tubes, and parasitological through the modified method of Hoffmann, Faust technic and detection of Cryptosporidium spp. by Ziehl-Neelsen Modified, known as Kinyoun staining. In fifteen samples, acquired in different neighborhoods of the city, 66,7% had total coliforms and 33,3% thermotolerant coliforms. In parasitological evaluation, 33.3% were contaminated with Giardia lamblia and 6,6% with Cryptosporidium spp. The hygienic and sanitary irregularities, which range from the manufacturing process of the product to distribution establishments, are possible causes of parasitic and microbiological contamination. Therefore, it reinforces the need to provide strategies to detect the possible points of contamination of ice, seeking to solve the problem and then prevent consumers from possible infections, in addition to ensure the safety of food.

Telomere-centromere-driven genomic instability contributes to karyotype evolution in a mouse model of melanoma.

Aneuploidy and chromosomal instability (CIN) are hallmarks of most solid tumors. These alterations may result from inaccurate chromosomal segregation during mitosis, which can occur through several mechanisms including defective telomere metabolism, centrosome amplification, dysfunctional centromeres, and/or defective spindle checkpoint control. In this work, we used an in vitro murine melanoma model that uses a cellular adhesion blockade as a transforming factor to characterize telomeric and centromeric alterations that accompany melanocyte transformation. To study the timing of the occurrence of telomere shortening in this transformation model, we analyzed the profile of telomere length by quantitative fluorescent in situ hybridization and found that telomere length significantly decreased as additional rounds of cell adhesion blockages were performed. Together with it, an increase in telomere-free ends and complex karyotypic aberrations were also found, which include Robertsonian fusions in 100% of metaphases of the metastatic melanoma cells. These findings are in agreement with the idea that telomere length abnormalities seem to be one of the earliest genetic alterations acquired in the multistep process of malignant transformation and that telomere abnormalities result in telomere aggregation, breakage-bridge-fusion cycles, and CIN. Another remarkable feature of this model is the abundance of centromeric instability manifested as centromere fragments and centromeric fusions. Taken together, our results illustrate for this melanoma model CIN with a structural signature of centromere breakage and telomeric loss.

Avaliação parasitológica e bacteriológica do gelo comercializado na cidade de Juazeiro do Norte - CE

A água é a matéria-prima utilizada na produção de gelo e a adoção de práticas rigorosas de higiene em sua fabricação, manuseio, embalagem, conservação e distribuição toma-se indispensável. O método de congelamento não é capaz de destruir os patógenos contaminantes, pois os mesmos permanecem em estado de latência até que o produto entre em processo de fusão. O estudo teve como objetivo avaliar as condições parasitológicas e bacteriológicas do gelo comercializado para consumo no município de Juazeiro do Norte, CE. Trata-se de um estudo analítico e transversal, realizado a partir de análises microbiológicas, por meio da técnica dos tubos múltiplos, e parasitológicas, pelo método de Hoffmann Modificado, técnica de Faust e pesquisa de Cryptosporidium spp. pela técnica de Ziehl-Neelsen Modificado, conhecido como coloração de Kinyoun. Em quinze amostras adquiridas em diferentes bairros deste município, 66,7% apresentaram coliformes totais e 33,3% coliformes termotolerantes. Na avaliação parasitológica, 33,3% estavam contaminadas por Giardia lamblia e 6,6% por Cryptosporidium spp. As irregularidades higienicossanitárias, que abordam desde o processo de fabricação do produto até a sua distribuição nos estabelecimentos, são as possíveis causas de contaminações parasitárias e microbiológicas. Reforça-se, portanto, a necessidade de estabelecer estratégias a fim de detectar os possíveis pontos de contaminação do gelo, buscando solucionar o problema e, assim, prevenir os consumidores de possíveis infecções e parasitoses, além de garantir a segurança deste alimento.

Since this is the raw material used in the production of ice, it is essential to adopt strict hygiene practices in their manufacturing, handling, packaging, storage and distribution. Since the freezing method is not able to destroy contaminating pathogens because the microorganisms remain in the sleep state until the product goes into melting process. The objective of this study was to evaluate the parasitological and bacteriological ice conditions marketed for consumption in Juazeiro do Norte, Ceará. It's about an analytic and transversal study, conducted from microbiological, using the technique of multiple tubes, and parasitological through the modified method of Hoffmann, Faust technic and detection of Cryptosporidium spp. by Ziehl-Neelsen Modified, known as Kinyoun staining. In fifteen samples, acquired in different neighborhoods of the city, 66,7% had total coliforms and 33,3% thermotolerant coliforms. /n parasitological evaluation, 33.3% were contaminated with Giardia lamblia and 6,6% with Cryptosporidium spp. The hygienic and sanitary irregularities, while range from the manufacturing process of the product to distribution establishments, are possible causes of parasite and microbiological contamination. Therefore, it reinforces the need to provide strategies to detect the possible points of contamination of ice, seeking to solve the problem and then prevent consumers from possible infections, in addition to ensure the safety of food.