Protease inhibitors as a potential agent against visceral Leishmaniasis: A review to inspire future study

Abstract Leishmaniasis is transmitted by sandfly which carries the intracellular protozoa in their midgut. Among visceral, cutaneous and mucocutaneous leishmaniasis, visceral type that is caused by Leishmania donovani is the most lethal one. Findings of leishmanial structure and species took place in 19th century and was initiated by Donovan. Leishmaniasis is still a major concern of health issues in many endemic countries in Asia, Africa, the Americas, and the Mediterranean region. Worldwide1.5-2 million new cases of cutaneous leishmaniasis and 500,000 cases of visceral leishmaniasis are reported each year. Leishmaniasis is endemic in nearly 90 countries worldwide and close to 12 million new cases of leishmaniasis are reported worldwide annually. Studies on antileishmanial drug development is of major concern as leishmaniasis are the second largest parasitic killer in the world and the available drugs are either toxic or costly. The major surface GP63 protease, also known as Zinc- metalloproteases present on the surface of leishmanial promastigotes, can be targeted for drug development. Protease inhibitors targeting such surface proteases show promising results. Different protease inhibitors have been isolated from marine actinobacteria against many infectious diseases. Metabolites produced by these actinobacteria may have greater importance for the discovery and development of new antileishmanial drugs. Hence, this review discusses the background, current situation, treatment, and protease inhibitors from marine actinobacteria for drug development against GP63 molecules.

Effects of ADAM12 abnormal expression on chondrocyte injury and IGFBP-related genes in patients with Kashin-Beck disease / 中华地方病学杂志

Objective:To investigate the role of a disintegrin and metalloprotease 12 (ADAM12) gene in chondrocyte injury in patients with Kashin-Beck disease (KBD) and its impact on genes related to insulin-like growth factor binding protein (IGFBP).Methods:Articular cartilage samples were obtained from 5 patients with KBD and 5 control subjects admitted to Honghui Hospital Affiliated to Xi'an Jiaotong University. Chondrocytes were extracted and cultured in vitro. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect the expression levels of ADAM12 mRNA and protein in chondrocytes of patients with KBD and control subjects, respectively. Subsequently, ADAM12 gene overexpression was performed using lentivirus in chondrocytes of patients with KBD. MTT assay was used to detect changes in cell viability after ADAM12 gene overexpression, and qRT-PCR was used to detect the mRNA expression levels of chondrocyte differentiation related genes SRY-box transcription factor 9 (SOX9) and type Ⅱ collagen (COLⅡ), apoptosis-related gene B-cell lymphoma/leukaemia-2-associated X protein (BAX), and anabolic related genes IGFBP3 and IGFBP5. Results:The expression levels of ADAM12 mRNA and protein in chondrocytes of patients with KBD (0.57 ± 0.05, 0.81 ± 0.07) were significantly lower than those of control subjects (1.00 ± 0.00, 1.00 ± 0.00), and the differences were statistically significant ( t = - 24.50, - 3.61, P < 0.05). The results of MTT assay showed that the cell viability of chondrocytes in ADAM12 overexpression group (1.09 ± 0.05) was higher than that in empty vector control group (1.00 ± 0.08), and the difference was statistically significant ( t = 4.12, P = 0.031). The results of qRT-PCR showed that compared with empty vector control group, the mRNA expression levels of IGFBP3 (2.35 ± 0.79 vs 0.96 ± 0.25), IGFBP5 (2.13 ± 0.30 vs 0.98 ± 0.34), SOX9 (2.92 ± 0.51 vs 0.94 ± 0.36) and COLⅡ (6.45 ± 2.81 vs 0.87 ± 0.19) in ADAM12 overexpression group were significantly increased, and the differences were statistically significant ( t = 3.19, 5.16, 6.27, 4.10, P < 0.05); while the expression level of BAX mRNA (0.31 ± 0.06 vs 1.02 ± 0.22) was significantly decreased, and the difference was statistically significant ( t = - 11.16, P < 0.001). Conclusion:The ADAM12 gene may have a role in inhibiting apoptosis and promoting differentiation in chondrocyte injury in patients with KBD, and its overexpression can increase expression of IGFBP3 and IGFBP5.

Effect of a disintegrin and metalloprotease 17 deletion on production of reactive oxygen species and mitochondrial function in nasopharyngeal carcinoma cells / 中国生物制品学杂志

@#Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17) deletion on the production of reactive oxygen species(ROS) and mitochondrial function in nasopharyngeal carcinoma(NPC) cells.Methods Three groups of ADAM1 7 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1) and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP) by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA) kit and superoxide dismutase(SOD) kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034) and MDA content increased(t=7.415,P=0.038) significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM1 7 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in mitochondrion,reduced MMP,inhibited cell proliferation in vitro,and promoted apoptosis.

Biochemical Properties of Parasite Virulence Factor: Lesson Learned from Leishmania

Leishmania, a parasitic protozoan, a single-celled organism of the genus trypanosomes that are responsible for the disease leishmaniasis. Transmission occured by sandflies of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World. Globally, at least 93 sandfly species are proven or probable vectors. Their primary hosts are vertebrates; Leishmania commonly infects hyraxes, canids, rodents, and humans. Leishmaniasis encompasses diverse clinical syndromes, including cutaneous, mucosal, and potentially life-threatening visceral forms. Three widely known virulence factors belongs to the genus Leishmania include the active compound named proteophosphoglycan (PPG), GP63 metalloprotease and lipophosphoglycan (LPG). these substance established on the surface of the parasite. The aim of this review article is to make an insight of the biochemical characteristics of Leishmania spp virulence factors, the armamentarium that predispose their pathogenesis, its invasion and virulence to the mammalian host.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Expression and role of matrix metalloproteinase in injury related renal transplantation / 器官移植

Matrix metalloproteinase (MMP) is a large class of proteases which can cut or reshape extracellular matrix (ECM) and cell surface proteins. The activity of MMP is regulated by a variety of cytokines, including tissue inhibitor of metalloprotease (TIMP), signal transduction molecules and cell adhesion molecules. The latest research shows that MMP has a role in the pathophysiology process of many acute and chronic kidney diseases. In this article, the classification, expression and distribution in the kidney of MMP and its role in injury related renal transplantation was reviewed.

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential / Pseudomonas spp. e outros micro-organismos psicrotróficos em queijos Minas Frescal inspecionados e não inspecionados: potencial proteolítico, lipolítico e produção de AprX

The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.(AU)

O mais consumido no Brasil, o queijo Minas Frescal (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, pode favorecer a multiplicação de bactérias psicrotróficas deteriorantes. O objetivo deste estudo foi quantificar e comparar Pseudomonas spp. e outras bactérias psicrotróficas em amostras de QMF inspecionadas e não inspecionadas, avaliar o potencial lipolítico, proteolítico e de produção de metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as contagens de bactérias psicrotróficas e Pseudomonas spp., o potencial proteolítico e lipolítico dos isolados e a identificação de potenciais produtores de metaloprotease alcalina (AprX). A média total das contagens de bactérias psicrotróficas foi de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e 4,5 (±5,86) × 108UFC/g nas não inspecionadas, sem diferença significativa (p=0,37). A média de Pseudomonasspp. foi de 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras QMF inspecionadas e não-inspecionadas, respectivamente, sem diferença significativa (p=0,1). Pseudomonas spp. representaram 0,06% e 0,004% de bactérias psicrotróficas encontradas em amostras QMF inspecionadas e não-inspecionadas, respectivamente. Das amostras inspecionadas e não inspecionadas, foram isoladas 694 colônias psicrotróficas e 47 Pseudomonasspp., dos quais 59,9% e 68,1% foram simultaneamente proteolíticos e lipolíticos, respectivamente. Dos 470 isolados de psicrotróficos das amostras de queijo inspecionados e dos 224 isolados das não inspecionadas, 5,74% e 2,23% continham o gene aprX, respectivamente, enquanto 100 e 86,96% das Pseudomonasspp. isoladas em amostras de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX. Esse potencial, no entanto, não determinou a atividade proteolítica em placas desses isolados nas condições avaliadas neste estudo. Do total, 65,63% dos psicrotróficos que continham o gene aprX foram confirmados como Pseudomonasspp., utilizando PCR gênero-específico. A análise filogenética do gene 16S rRNA dos outros psicrotróficos que foram produtores potenciais de AprX os identificou como Serratia spp. (n=7), Raoultella ornithinolytica (n=1) e Acinetobacter schindleri (n=1) nas amostras inspecionadas e Psychrobacter sanguinis (n=1) e Leuconostoc mesenteroides (n=1) nas amostras não inspecionadas. As condições de produção do QMF dessas amostras, atendendo às determinações legais, não são suficientes para controlar a Pseudomonas e outros psicrotróficos relacionados à deterioração. Assim, medidas higiênicas mais rígidas são necessárias durante a produção formal deste tipo de queijo.(AU)

Expressions of Versican and ADAMTS-1 in polycysticovary syndrome patients and their clincal significances / 吉林大学学报(医学版)

Objective: To determine the expressions of Versican and a distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1) in serum in the polycystic ovary syndrome (PCOS) patients, and to clarify their roles in the pathogenesis of PCOS. Methods: A total of 80 patients with PCOS (PCOS group) and 100 healthy women (control group) were selected The heights and body weights of the subjects in two groups were measured; and the body mass index (BMI) was calculated The levels of serum Versican and ADAMTS-1 of the subjects in two groups were measured by enzyme-linked immunosorbent assay (ELISA) method The levels of serum follicle stimulating hormone (FSH), luteinizing hormone (L H), testosterone (T), fasting blood glucose (FBG), fasting insulin of the subjects in two groups were detected; insulin resistance was evaluated according to Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). The correlations between Versican, ADAMTS-1 and the metabolic indexes were analyzed by Pearson linear correlation analysis Results: The serum Versican level of the patients in PCOS group was significantly decreased compared with control group (P = 0 . 004); the serum ADAMTS-1 level was also decreased (P < 0 . 01). The sensitivities, the specificities and the area under receiver operating characteristic (ROC) curve (AUC) of Versican and ADAMTS-1 in prediction of PCOS were 76.74% vs 63. 64% (P = 0 . 018), 52.94% 1)5 40.73% (P = 0 . 009) and 0. 675 (0.550-0.795) vs 0.714 (0 . 6 0 1 - 0. 827) (P = 0 . 032), respectively. The correlation analysis showed that in PCOS group the serum level of Versican of the patients was negatively correlated with FBG (r = - 0 . 7 3 8, P= 0.022), and ADAMTS-1 was negatively correlated with FBG (r = -0.524, P = 0.043). Conclusion: Versican and ADAMTS-1 lowly express in the patients with PCOS. They are negatively correlated with the insulin resistance. They may play inhibitory effects in the occurrence of PCOS.

Comparison of sepsis severity markers in emergency department / 中华急诊医学杂志

Objective This study was to evaluate the values of endothelial cell-specific moleculel (endocan),von Willebrand factor (vWF),and"A disintegrin-like and metalloprotease with thrombospondin type 1 motit"(ADAMTS-13),alone or in combination,in the risk stratification and disease severity assessment of patients with sepsis via comparing the differences of these markers in patients with systemic inflammatory reaction syndrome (SIRS),sepsis,severe sepsis,or septic shock,and healthy volunteers.Methods Clinical data of 301 patients with SIRS or sepsis treated in our Emergency Department from October 2014 to October 2015,were prospectively analyzed.These patients were divided into SIRS,sepsis,severe sepsis,and septic shock groups.40 healthy individuals were selected as control.Endocan,vWF,ADAMTS-13,vWF/ADAMTS-13,and Procalcitonin (PCT) levels were measured,and APACHE Ⅱ score,MEDS score as well as SOFA score were calculated.The all-cause death or survival of each patient was recorded during the 28-day follow-up.Results The endocan,vWF,and vWF/ADAMTS-13 levels significantly increased in patients with SIRS,sepsis,severe sepsis,or septic shock and were positively correlated with disease severity,and were also positively correlated with MEDS score,APACHE Ⅱ score,SOFA score.On the other way,the ADAMTS-13 level gradually declined during disease progression and was negatively correlated with the disease severity,it was also negatively correlated with MEDS score,APACHE Ⅱ score,SOFA score.Conclusions Endocan,vWF,ADAMTS-13,and vWF/ADAMTS-13 ratio are valuable in the risk stratification and disease severity evaluation of sepsis,providing novel sepsis biomarkers in clinic.

Expressions of Versican and ADAMTS-1 in polycysticovary syndrome patients and their clincal significances / 吉林大学学报(医学版)

Objective: To determine the expressions of Versican and a distintegrin and metalloprotease with thrombospondin type l motifs (ADAMTS-1) in serum in the polycystic ovary syndrome (PCOS) patients, and to clarify their roles in the pathogenesis of PCOS. Methods: A total of 80 patients with PCOS ( PCOS group) and 100 healthy women (control group) were selected. The heights and body weights of the subjects in two groups were measured; and the body mass index (BMI) was calculated. The levels of serum Versican and ADAMTS-1 of the subjects in two groups were measured by enzyme-linked immunosorbent assay (ELISA) method. The levels of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), fasting blood glucose (FBG), fasting insulin of the subjects in two groups were detected; insulin resistance was evaluated according to Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). The correlations between Versican, ADAMTS-1 and the metabolic indexes were analyzed by Pearson linear correlation analysis. Results: The serum Versican level of the patients in PCOS group was significantly decreased compared with control group (P=0.004); the serum ADAMTS-1 level was also decreased (P<0.01). The sensitivities, the specificities and the area under receiver operating characteristic (ROC) curve （AUC）of Versican and ADAMTS-1 in prediction of PCOS were 76.74%?vs 63.64% (P=0.018), 52.94% vs 40.73% (P=0.009) and 0675 (0.550-0.795) vs 0.714 (0.601-0.827) (P=0.032), respectively. The correlation analysis showed that in PCOS group the serum level of Versican of the patients was negatively correlated with FBG (r=-0.738, P=0.022), and ADAMTS-1 was negatively correlated with FBG (r=-0.524, P=0.043). Conclusion； Versican and ADAMTS-1 lowly express in the patients with PCOS. They are negatively correlated with the insulin resistance. They may play inhibitory effects in the occurrence of PCOS.

Expresión de TGF-p como indicio temprano de lesión del tejido hepático en la enfermedad de hígado graso no alcohólico / Expression of TGF-p as an early sign of liver tissue injury in nonalcoholic fatty liver disease

Resumen Introducción: la enfermedad de hígado graso no alcohólico (NAFLD) constituye un problema de salud pública asociado con el síndrome metabólico; su patogénesis implica el inicio de una cascada de señalización bioquímica compleja y su estimulación continua podría consolidar un proceso de fibrogénesis en el tejido. El objetivo del estudio fue analizar expresión de genes implicados en daño hepático, en los procesos iniciales de la lesión en pacientes con NAFLD o con factores de riesgo relacionados a esta patología, en búsqueda de biomarcadores moleculares útiles a la práctica clínica tales como TGF-pi, COL1A2 y MMP20. Metodología: estudio analítico de corte transversal. Se estudiaron características epidemiológicas, bioquímicas, y expresión génica de TGF-pU, COL1A2 y MMP20 en tejido hepático, en individuos con factores de riesgo para NAFLD. Resultados: se incluyeron 83 participantes con factores de riesgo asociados a NAFLD, 22 individuos (26.5%) fueron diagnosticados con NAFLD mediante ultrasonografía. Los factores de riesgo hallados fueron hipertensión arterial (50.6%), obesidad (49.4%), diabetes mellitus (34.9%) y dislipidemia (21.7%). La dislipidemia fue significativamente asociada con el riesgo de desarrollar NAFLD (OR=4; p=0.011). Se encontraron diferencias significativas para colesterol total (p<0.05); y una expresión génica de TGF--31 (con NAFLD p<0.0001 y sin NAFLD p<0.0001 frente al control) y COL1A2 (con NAFLD p=0.002 y sin NAFLD p=0.955 frente al control) con un patrón de expresión creciente a mayor grado de lesión hepática. Conclusión: para concluir, sugerimos activación de las vías de señalización que conducen a fibrogénesis en individuos con factores de riesgo para NAFLD, y mucho más acentuada en pacientes con NAFLD.

Abstract Introduction: nonalcoholic fatty liver disease (NAFLD) is a public health problem associated with the metabolic syndrome; its pathogenesis implies the start of a complex biochemical signaling cascade and its continuous stimulation could consolidate a fibrogenesis process in the tissue. The aim of the study was to analyze expression of genes involved in liver damage in the initial processes of the lesion in patients with NAFLD or with risk factors related to this pathology, in search of molecular biomarkers useful to clinical practice such as TGF--31, COL1A2 and MMP20. Methodology: cross-sectional analytical study. Epidemiological, biochemical, and gene expression characteristics of TGF--31, COL1A2 and MMP20 in liver tissue in individuals with risk factors for NAFLD were studied. Results: 83 participants with risk factors associated to NAFLD were included; 22 individuals (26.5%) were diagnosed with NAFLD by ultrasonography. The risk factors found were hypertension (50.6%), obesity (49.4%), diabetes mellitus (34.9%) and dyslipidemia (21.7%). Dyslipidemia was significantly associated with the risk of developing NAFLD (OR = 4; p = 0.011). Significant differences were found for total cholesterol (p <0.05); and a gene expression of TGF-P1 (with NAFLD p <0.0001 and without NAFLD p <0.0001 versus control) and COL1A2 (with NAFLD p = 0.002 and without NAFLD p = 0.955 versus control) with a pattern of increasing expression at higher degree of liver injury. Conclusion: to conclude, we suggest activation of the signaling pathways that lead to fibrogenesis in individuals with risk factors for NAFLD, and much more accentuated in patients with NAFLD.

Proteolytic and lipolytic potential of Pseudomonas spp. from goat and bovine raw milk / Potencial proteolítico e lipolítico de espécies de Pseudomonas do leite cru caprino e bovino

Pseudomonas, the main genus of gram-negative microorganisms isolated from milk, is psychrotrophic, biofilm-forming, and thermo-resistant deteriorating enzyme producers. The aim of this study was to quantify Pseudomonas spp. in goat's and cow's milk produced in the Paraná state, Brazil, to evaluate the deteriorating activity of the isolates at mesophilic and psychrotrophic conditions and to identify, at the species level, the isolates with alkaline metalloprotease (aprX gene) production potential. Microbiological, biochemical and molecular methods were used for isolating, confirming and identifying of isolates. The mean counts were 1.6 (±6.3)x104 and 0.89(±3)x102 CFU/mL for goat and bovine milk samples, respectively, immediately after milking. Of the Pseudomonas colonies isolated from goat milk (n=60), 91.7% showed proteolytic potential when incubated at 35°C/48 h and 80% at 7°C/10 days, and lipolytic potential was observed in 95% of the isolates incubated in mesophilic and 78.3% at refrigeration conditions. From the isolates of bovine milk (n=20), 35% showed proteolytic activity only when incubated at 35°C/48 h, and lipolytic potential was observed in 25% of the isolates incubated at 7°C/10d and 35°C/48h. It was observed that 83.3% and 25% of the isolates genetically confirmed as Pseudomonas spp. of goat and bovine milk showed the potential for alkaline metalloprotease production, with the species P. azotoformans, P. koreensis, P. gessardii, P. monteilii and P. lurida being the most frequent in goat milk and P. aeruginosa the only species identified in cow milk.(AU)

Pseudomonas é o principal gênero de micro-organismos Gram negativos isolados do leite, são psicrotróficos, formadores de biofilmes e produtores de enzimas deteriorantes termodúricas. O objetivo do presente trabalho foi quantificar Pseudomonas spp. no leite de cabras e vacas produzido no estado do Paraná, Brasil, avaliar a atividade deteriorante em temperatura mesofílica e psicrotrófica e identificar, em nível de espécie, os isolados com potencial de produção de metaloprotease alcalina (geneaprX). Foram utilizados métodos microbiológicos, bioquímicos e moleculares para isolamento, confirmação e identificação dos isolados. As contagens médias foram de 1,6 (±6,3) x 104 e 0,9 (±3) x 102 UFC/mL para as amostras de leite caprino e bovino, respectivamente. Dos isolados de Pseudomonas do leite de cabra (n=60), 91,7% demonstraram potencial proteolítico quando incubadas a 35°C/48h e 80% a 7°C/10dias e lipolíticos em 95% dos isolados incubados em mesofilia e em 78,3% dos incubados em temperatura de refrigeração. Dos isolados do leite bovino (n=20), foi verificada atividade proteolítica de 35% apenas quando incubadas a 35°C/48h e lipolítica em 25% dos isolados incubados a 7°C/10d e 35°C/48h. Foi observado que 83,3% e 25% dos isolados confirmados geneticamente como Pseudomonas spp. do leite caprino e bovino, respectivamente, apresentaram o potencial de produção de metaloprotease alcalina, sendo as espécies P. azotoformans, P. koreensis, P. gessardii, P. monteilii e P. lurida as mais frequentes no leite de cabras e P. aeruginosa a única identificada do leite de vacas.(AU)

Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results: Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0-9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion: The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools.(AU)

Serum matrix metalloproteinase-1 level represents disease activity as opposed to fibrosis in patients with histologically proven nonalcoholic steatohepatitis

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) is prevalent in both economically developed and developing countries. Twenty percent of NASH progresses to cirrhosis with/without hepatocellular carcinoma, and there is an urgent need to find biomarkers for early diagnosis and monitoring progression of the disease. Using immunohistochemical and immunoelectron microscopic examination we previously reported that expression of matrix metalloproteinase-1 (MMP-1) increased in monocytes, Kupffer cells and hepatic stellate cells in early stage NASH. The present study investigated whether serum MMP-1 levels reflect disease activity and pharmaceutical effects in NASH patients. METHODS: We measured the serum levels of MMPs, tissue inhibitors of metalloproteinases (TIMPs), and several cytokines/chemokines in patients with histologically proven early and advanced stages of NASH and compared them with those in healthy controls. RESULTS: Serum MMP-1 levels in stage 1 fibrosis, but not in the more advanced fibrosis stages, were significantly higher than in healthy controls (P=0.019). There was no correlation between serum MMP-1 level and fibrosis stage. Serum MMP- 1 levels in NASH patients represented disease activity estimated by serum aminotransferase values during the follow-up period. In contrast, MMP-2, MMP-9 and TIMPs did not change with disease activity. Consistent with the finding that MMP-1 is expressed predominantly in monocytes and Kupffer cells, serum levels of monocyte chemotactic protein-1 and granulocyte-colony stimulating factor were significantly increased in NASH with stage 1 fibrosis. CONCLUSIONS: These results suggest that serum MMP-1 levels represent disease activity and may serve as a potential biomarker for monitoring the progression of NASH.

Expressions of a disintegrin and metalloprotease 17 and epidermal growth factor recepter in esophageal squamous cell carcinoma and their significances / 肿瘤研究与临床

Objective To investigate the expressions of a disintegrin and metalloprotease 17 (ADAM17) and epidermal growth factor recepter (EGFR) in human esophageal squamous cell carcinoma (ESC),and to explore their relationship with clinicopathological characteristics.Methods The paraffin specimens in postoperative pathological tissues of 66 ESC patients in the Third People's Hospital of Hefei from January 2013 to December 2017 were selected.Expressions of ADAM17 and EGFR proteins were examined by using immunohistochemistry in 66 cases of ESC tissues and 33 cases of adjacent tissues of the tumors.The relationship of ADAM17 and EGFR with clinicopathological features was analyzed.Kendall method was used to detect the expression correlation of ADAM17 and EGFR.Results The positive rate of ADAM17 protein in ESC tissues was higher than that in the adjacent tissues of the tumors [68.2 ％ (45/66) vs.33.3 ％ (11/33),x2 =10.874,P =0.001].The positive rate of EGFR protein in ESC tissues was higher than that in the adjacent tissues of the tumors [66.7 ％ (44/66) vs.39.4 ％ (13/33),x2 =6.699,P =0.01].The expressions of ADAM17 and EGFR protein were related with ESC pathological TNM staging,infiltration depth,lymph node metastasis (x2 =4.797,4.890,6.089;8.790,8.766,10.154,respectively,all P ＜ 0.05).ADAM17 expression was positively correlated with EGFR protein (r,=0.368,P ＜ 0.05).Conclusions ADAM17 and EGFR are highly expressed in human ESC.Besides,ADAM17 and EGFR have the interaction in the occurrence and development of esophageal cancer.Joint detection may help to determine the degree of metastasis and evaluate prognosis.

Correlation between extracellular MMP-9 polymorphism and genetic susceptibility of PACG in a Han Chinese population / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To analyze the association of extracellular matrix metalloprotease 9(MMP-9)single nucleotide polymorphism(SNP)with genetic susceptibility of primary angle-closure glaucoma(PACG)in a Han Chinese population. <p>METHODS: Totally 200 PACG patients(PACG group)and 200 healthy people(normal control group)were collected in our hospital from January 2014 to December 2016. Peripheral venous blood was collected and extracted for genomic DNA, the polymerase chain reaction-restriction fragment length polymorphism technique(PCR-RFLP)was applied to detect the alleles and genotypes of rs2250889, rs2274755 and rs2664538 sites in MMP-9 gene. The frequency distribution of alleles and genotypes in the two groups were calculated by chi-square test, and its association with genetic susceptibility of PACG was analyzed. <p>RESULTS: There were no statistical differences in age, gender, body mass index, diastolic blood pressure and systolic blood pressure in the two groups(<i>P</i>>0.05). The genotype frequencies of rs2250889, rs2274755 and rs2664538 sites in MMP-9 gene were in line with Hardy-Weinberg equilibrium. The genotype and allele frequency distribution of rs2250889 and rs2664538 sites were significantly different between the PACG group and the normal control group(<i>P</i><0.05), while the genotype and allele frequency distribution of rs2274755 sites in the two groups had no statistical difference(<i>P</i>>0.05). The subjects whose rs2250889 site carrying the CC genotype was susceptible to PACG. Similarly, the rs2664538 site carrying the GG genotype was susceptible to PACG. <p>CONCLUSION: The rs2250889 and rs2664538 polymorphisms of MMM-9 are correlated with the risk of PACG in a Han Chinese population, while the rs2274755 polymorphism is not related to genetic susceptibility of PACG.

Protective effect of nerve growth factor combined with mecobalamine on optic nerve and influence on MMP-2 and TIMP-2 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the protective effect of nerve growth factor(NGF)combined with mecobalamine on optic nerve and the influence on matrix metalloproteinase-2(MMP-2), tissue inhibitor of matrix metalloprotease-2(TIMP-2). <p>METHODS: Totally 54 patients(73 eyes)with acute angle closure glaucoma undergoing trabeculectomy was included in the study, and the patients were divided into the control group(30 cases, 35 eyes)and combination group(24 cases, 38 eyes)according to the digital table method. The patients in the control group were treated with mecobalamine, and the patients in combination group were treated with NGF combined with mecobalamine. Visual acuity, intraocular pressure, mean light sensitivity(MS), visual field mean defect(MD), retinal nerve fibre layer(RNFL)thickness, the nipple cup/disc ratio, P100 wave incubation period and amplitude of P100 wave, neuron-specific enolase(NSE), nitric oxide(NO), nitric oxide synthase(NOS), matrix metalloproteinase-2(MMP-2), metalloproteinases tissue inhibiting factor-2(TIMP-2)before and after treatment were observed in the two groups. The adverse reactions during medication were supervised. <p>RESULTS: Compared with those before treatment, visual acuity, MS, and P100 wave amplitudes increased, MD and P100 wave latency decreased(<i>P</i><0.05)after treatment, the change ranges in combination group were greater than those in control group(<i>P</i><0.05). There was no significant change in intraocular pressure, RNFL, the nipple cup/disc ratio before and after treatment in the two groups(<i>P</i>>0.05). Compared with those before treatment, serum NO, NOS, MMP-2 levels in the two groups increased, and the levels of NES, TIMP-2, TIMP-2/MMP-2 decreased(<i>P</i><0.05), but the change degrees in combination group were greater than those in control group(<i>P</i><0.05). <p>CONCLUSION: NGF combined with mecobalamine can improve postoperative visual function of glaucoma, which has certain regulatory effects on MMP-2 and TIMP-2.

Uso de doxiciclina como adyuvante en la recuperación postquirúrgica de las lesiones del manguito rotador. Seguimiento a un año / Doxycycline as an adjuvant in postsurgical recovery of rotator cuff injuries. One-year follow-up

Resumen: Antecedentes: La severidad y progresión inevitable de la lesión del manguito rotador ha llevado a experimentar con adyuvantes terapéuticos para disminuir el tiempo de recuperación postquirúrgica, así como mejorar la estructura del tendón en recuperación al inhibir la matriz de metaloproteasas. Objetivo: Evaluar el uso de la doxiciclina como adyuvante en la cicatrización de lesiones hueso-tendón en la reparación quirúrgica del manguito rotador. Material y métodos: Se reclutaron 20 pacientes con lesión del manguito rotador corroborada por imagen con retracción del supraespinoso grado II (Patte) e infiltración grasa de 50% (Goutallier). Fueron divididos en dos grupos: a 10 se les administró doxiciclina, 100 mg cada 24 horas durante un mes, y el resto fueron un grupo control sin doxiciclina. Ambos fueron tratados quirúrgicamente con técnica de doble hilera vía artroscópica, con seguimiento periódico hasta 12 meses mediante escalas de UCLA, Constant y potencia de flexión anterógrada. Resultados: Se encontró recuperación clínica de la lesión en ambos grupos a los 12 meses, mayor potencia de flexión anterógrada en cada uno de los intervalos de medición para el grupo donde se administró la doxiciclina. Durante la evolución del estudio, se mantuvo constante Constant y UCLA; se encontró mejoría considerable con la potencia de flexión anterógrada como valor independiente. Discusión: El uso de doxiciclina podría mejorar de una forma considerable el pronóstico clínico de la reparación artroscópica de mango rotador con el uso de doble hilera, pero aún no sabemos cómo, aunque deberán realizarse estudios adicionales con una muestra mayor.

Abstract: Background: The severity and progression of rotator cuff tears have forced research on new treatment pathways such as metalloprotease inhibition, which has shown a reduction in healing time and improvement in the structure of collagen fibers. Objective: To evaluate the use of doxycycline as a healing enhancer in rotator cuff tears after surgical treatment. Material and methods: 20 patients were included; they were divided into two groups, 10 with the use of doxycycline and 10 without it after arthroscopic repair with one-year follow-up. Doxycycline was given orally, 100 mg once a day for one month. Every subject in the test was diagnosed with rotator cuff tear confirmed by MRI with Patte and Goutallier scores below 2. We used the arthroscopic double row technique. Post-op follow-up was 12 months with clinical scales (UCLA, Constant and forward flexion strength). Results: Both groups reported almost complete healing of rotator cuff tears after surgical treatment during the twelve months of follow-up; forward flexion strength was the only score that reported improvement in the doxycycline group during every check-up. Discussion: Doxycycline use after arthroscopic cuff tear repair could improve the clinical outcome, but we do not know how yet; however larger sample and randomized trials should be developed.

Construction of SH-SY5Y cell line stably expressing human α-secretase adam10 gene promoter luciferase reporter gene / 重庆医学

Objective To construct the luciferase report vector carrying a disintegrin and metalloprotease 10(adam10) gene promoter,to screen its stable expression cell line and to analyze its activity.Methods The genome DNA of human neuroblastoma SH-SY5Y cells was extracted as the template.The adam10 gene promoter was amplified by PCR and was cloned into luciferase reporter vector pGL4.17.The adam10 gene promoter luciferase reporter vector pGL4.17-adam10 was constructed and transfected in to SH-SY5Y cells(pGL4.17 vector without promotoer as the negative control and pGL4.17 vector with CMV promoter as the positive control).Then the stable expression cell line was screened by G418 and its fluorescence activity was detect after treating with 1 tμmol/L retinoic acid(RA) for 4 d.Results About 438 bp adam10 gene promoter was successfully amplified by PCR.The pGL4.17-adam10 vector was correct by pCR and double enzyme digestion identification.The cell line stably expressing adam10 gene promoter was obtained after transfecting SH-SY5Y cells by this vector and screening by G418,which had stronger transcriptional activity by detection;1 μmol/L RA could induce high efficiency expression of adam10 gene promoter.Conclusion Human adam10 gene promoter luciferase vector is successfully constructed.adam10 gene promoter can be stably expressed in SH-SY5Y cells,which provides a basis for deeply studying adam10 gene expression regulation,polymorphism analysis and high-throughput drug screening.

Highlights in the knowledge of brown spider toxins

Abstract Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (440 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.