To lyse or not to lyse? Use of intrapleural tissue plasminogen activator and DNase in the management of parapneumonic effusions and empyema

Despite a range of management options, pleural effusions and empyema continue to present therapeutic challenges in the clinical setting. With treatment options ranging from simple use of antibiotics to more complex surgical procedures, several important considerations need be made as to what type of treatment is best for each patient on a case by case basis. One treatment modality of increasing interest is the use of intrapleural fibrinolytics to facilitate drainage of effusions. This presents a viable option especially in patients in whom surgery is not preferred. But, as with many therapeutic approaches, the use of intrapleural fibrinolytics is laden with significant controversies and has been a subject of considerable debate over the last couple of years. With accruing evidence for and against this modality of treatment, the ensuing discussion has been whether or not it should be a routine treatment choice and which group of patients should this consideration be made for. This paper gives a background on the epidemiology and etiology of parapneumonic effusions and empyema and briefly outlines the available options of management. Furthermore, we extensively discuss available evidence on the use of intrapleural fibrinolytics as a management option for parapneumonic effusions and empyema, with particular emphasis on use of tissue plasminogen activator (tPA) and DNase.

A novel DNase like compound that inhibits virus propagation from Asian Green Mussel, Perna viridis (Linn.).

Viral diseases are not only responsible for health related issues but also exert pressure on the State economy. Tropical and subtropical countries have more prevalence of virus associated pathological conditions such as chickenpox, adenovirus related infections, dengue, chickengunya, infectious mononucleosis, etc. Treatment options with effective antiviral drugs are limited and are unfortunately not free from undesirable effects. The Asian Green Mussel, Perna viridis (Linn.) (Mytilidae) are not only important for their evolutionary significance, high caloric index, ecological role in the sequestration of environmental pollutants especially heavy metals, but also are potential source for extraction of therapeutic and bioactive compounds. On the other hand, generally in bivalves, virus mediated mortality is not uncommon. In this study, we made a maiden attempt of exploring DNAse like bioactivity for natural non-protenacious compound(s) extracted from P. viridis. Crude Methanol Extract (CME) of soft tissue of P. viridis and subsequently its partially purified component (PPC) possess exceptional ability to degrade indiscriminately both low and high molecular weight DNAs. In vitro digestions for1, 2 and 3 h with CME and PPC were found to be comparable to commercial (Sigma-Aldrich) enzyme, DNase I. Bioactive assays conducted to evaluate antimicrobial property, have shown that CME and PPC exclusively inhibit viral propagation. Nonetheless, CME & PPC have no effect on the propagation of bacteria (0 mm ZOI). These results indicate the possibility of a source of potential antiviral drug against DNA Group I viruses. Although our study does not provide any data to correlate to any physiological functions of these substances but provides a clue towards an important role in the biology of mussels. Any conclusion at this stage is premature. However, taking into consideration the significantly high virus mediated mortality in bivalves and the antiviral bioactivity of these substances, it appears that mussels have evolved some mechanisms to counteract some viruses.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR

BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

Advances research in chromatin DNaseⅠhypersensitive sites and its transcriptional regulation function / 医学研究生学报

DNaseⅠhypersensitive sites (DHSs) are regions of extreme chromatin which is highly sensitive to DNaseⅠ.Ge-nome-wide mapping DHSs is a powerful method for identifying many different types of regulatory elements within nuclear chromatin .It can help systematically resolve gene and genomic information , and the dynamic molecular mechanism of transcriptional regulation .This paper reviews the distribution of DHSs , the technology of DNase-seq and the research progress in transcriptional regulation functions of DHSs.

Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage.</p><p><b>METHODS</b>Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria.</p><p><b>RESULTS</b>The result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01).</p><p><b>CONCLUSION</b>This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.</p>

Influence of delay in blood-processing on the level of cell-free fetal DNA in maternal plasma sample / 第二军医大学学报

Objective: To investigate the influencing factors of cell-free fetal DNA level in the maternal plasma during blood-processing. Methods: Aliquots of blood samples from pregnant women with male fetus were processed at 6 h and 36 h after sampling. The SRY and β-globin genes and the total DNA level were quantified by real-time quantitative PCR. Death of white blood cells was assayed by flow cytometry after stained with Annexin V/PI. The plasma DNase activity was assayed by radial enzyme-diffusion method and plasma lactate dehydrogenases (LDH) by rate method. Results: A 36 hour delay in blood-processing led to a significant increase in the total DNA and decrease in the fetal DNA (SRY gene) in the maternal plasma. The ratio of fetal DNA decreased from (10.3±5.6) % at 6 h after sampling to (3.0±2.1) % at 36 h after sampling under 4°C (P< 0.05). No dead cells were identified in the blood sample 6 h after sampling; however, apoptosis and necrosis of white blood cells were identified 36 h after sampling. The activity of LDH at 36 h was significantly higher than that at 6 h (P<0.05). Radial enzyme-diffusion result showed that, though greatly decreased at 4°C, the DNase was still able to degrade DNA. Conclusion: Delay in blood-processing can lead to increase of the total free DNA in maternal plasma but decrease of fetal DNA, which might be related to the death of white blood cells and degradation of fetal DNA by plasma DNase, so the extraction of fetal DNA should be done as early as possible after sampling (within 6 h).

The extraction of sperm DNA from mixed stain using DNase-Ⅰ purification combined with alkaline lysis method / 中国法医学杂志

Objective To establish a method of sperm DNA extraction in mixed stain by using DNase-Ⅰ purificationcombined with alkaline lysis method in forensic science.Methods 79 mixed stain samples of criminal cases were collected.Sperm DNA was extracted using the purification of DNase-Ⅰ binding alkaline lysis method.16 STR loci were genotyped with fluorescent multiplex amplification system.The typing results were compared with that of extracted using two-step differential extraction procedure.Results Of all 79 mixed stain samples,64 samples were genotyped successfully by using DNase-Ⅰ purification combined with alkaline lysis method while 57 samples were genotyped successfully with two-step differential extraction procedure.There was significant difference between two methods(P=0.039).The purification of DNase-Ⅰ binding alkaline lysis method had a higher success rate and lower cost than that of two-step differential extraction procedure.Conclusion Purification of DNase-Ⅰ binding alkaline lysis method can increase the typing success rate of the mixed stain samples.The method is simple,rapid and easy to be automated,and suitable for forensic identification test.

Secretion of five extracellular enzymes by strains of chromoblastomycosis agents / Secreção de cinco enzimas extracelulares por amostras de agentes da cromoblastomicose

The gelatinase, urease, lipase, phospholipase and DNase activities of 11 chromoblastomycosis agents constituted by strains of Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana and Exophiala jeanselmei were analyzed and compared. All strains presented urease, gelatinase and lipase activity. Phospholipase activity was detected only on five of six strains of F. pedrosoi. DNase activity was not detected on the strains studied. Our results indicate that only phospholipase production, induced by egg yolk substrate, was useful for the differentiation of the taxonomically related species studied, based on their enzymatic profile.

As atividades gelatinase, urease, lipase, fosfolipase e DNase de 11 agentes da cromoblastomicose constituídos por amostras de Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana e Exophiala jeanselmei foram analisadas e comparadas. Todas as amostras apresentaram atividade urease, gelatinase e lipase. A atividade fosfolipase foi detectada apenas em cinco das seis amostras de F. pedrosoi. A atividade DNase não foi detectada nas amostras estudadas. Os resultados indicam que para a diferenciação entre espécies taxonomicamente relacionadas estudadas, baseado no seu perfil enzimático, apenas a produção de fosfolipase, induzida pelo substrato com gema de ovo, foi útil.

Desenvolvimento e implementação de métodos laboratoriais aplicados ao diagnóstico fenotípico e genotípico do Corynebacterium diptheriae / Development and implementation of laboratory methods applied to phenotypic and genotypic diagnosis of Corynebacterium diphtheriae

A emergência de cepas de Corynebacterium diphtheriae atoxinogênicas como agentes de endocardite e outras infecções sistêmicas aliada ao aumento do número de adultos susceptíveis à difteria enfatizam a necessidade de métodos alternativos para o diagnóstico laboratorial desta doença, especialmente para laboratórios de rotina clínica. Neste estudo avaliou-se a atividade de DNase de 91 amostras de C. diphtheriae (37 toxinogênicas e 54 atoxinogênicas) e de 564 cepas clínicas de bacilo Gram positivo não diftérico. A atividade de DNase foi detectada em todas as amostras de C. diphtheriae examinadas, previamente identificadas por métodos bioquímicos e pelo sistema API Coryne System. Diferentemente, os resultados do teste de DNase foram negativos em 93.9 porcento das cepas clínicas de bacilo Gram positivo não diftérico. Também foi documentado o valor de uma PCR espécie-específica que tem como alvo o gene dtxR como um método para diferenciação entre C. diphtheriae e colônias similares ao gênero Corynebacterium. Os resultados da PCR-dtxR foram positivos para todas as amostras de C. diphtheriae estudadas e foram concordantes com os obtidos através de metodologia bioquímica padrão. Diferentemente, os resultados da PCR-dtxR foram negativos para 100 porcento das 111 amostras de bacilos Gram positivos não diftéricos estudadas. A partir destes resultados, uma PCR multiplex utilizando três pares de oligonucleotídeos iniciadores foi desenvolvida para a detecção do C. diphtheriae e diferenciação em amostras toxinogênicas ou atoxinogênicas. Dois pares de oligonucleotídeos iniciadores têm como alvo as regiões do gene tox relativas aos domínios A e B da toxina diftérica e um terceiro par direcionado para o gene dtxR. Todas as amostras de C. diphtheriae foram identificadas pela reação de PCR multiplex em concordância com os testes bioquímicos padrão e os ensaios de citotoxicidade celular...

The emergence of non-toxigenic Corynebaterium diphtheriae strains as the causative agent of endocarditis and other systemic infections and the significant rise in the percentage of adults susceptible to diphtheria emphasize the need for new laboratory diagnostic procedures. In this study, we examine techniques as alternative procedures for differentiating C. diphtheriae from Corynebacterium-like colonies for the presumptive identification of this pathogen, especially in the diagnosis laboratory. This study evaluated the DNase activity of 91 C. diphtheriae (37 toxigenic and 54 non-toxigenic) and 564 non-diphtherial Gram-positive rod clinical strains. The DNase activity was detected in all C. diphtheriae strains examined, previously identified by both conventional biochemical methods and API Coryne System. Conversely, DNase test results were negative in 93.9 percent of the 564 non-diphtherial Gram-positive rod clinical strains. We also documented the value of a species-specific PCR assay that targets the dtxR gene as a procedure for differentiating C. diphtheriae from Corynebacterium-like colonies. The results of the PCR-dtxR were all positive for 91 C. diphtheriae strains and completely correlated with the standard biochemical methods and commercial identification system for all strains tested. In other hand, the PCR-dtxR results were negative in 100 percent of the 111 non-diphtherial Gram-positive rod strains. Considering these results, a multiplex PCR using three primers pairs was developed for detection of C. diphtheriae infection and differentiation between toxigenic and non-toxigenic strains. Two primer pairs targeted to domains A and B of tox gene and a third primer pair targeted to a region of dtxR gene. All C. diphtheriae strains were diagnosed by the multiplex PCR in agreement with standard biochemical tests and citotoxicity assay in Vero cells. Thus, these tecniques emerged as viable, cost-effective screening methods for C. diphtheriae laboratory...

Effects of DNaseⅠ-treated streptococcal antigens on peripheral blood mononudear cell proliferation in patients with psoriasis vulgaris / 中华皮肤科杂志

Objective To investigate the effects of DNaseⅠ-treated streptococcal antigens on peripheral blood mononuclear cells(PBMC)proliferation in psoriasis patients.Methods PBMC from 15 patients with psoriasis and 10 healthy controls were stimulated with three concentrations(2,10,25?g/mL) of streptococcal antigens(SA), nd DNaseⅠ-treated streptococcal antigens(DNaseⅠ-SA).After 72 h of culture,cellular proliferation was determined by ~3H thymidine incorporation.Results The DNaseⅠ-SA,at all the concentrations tested,was less potent than the corresponding concentrations of SA(P0.05),however,the lower concentrations(2,10?g/mL) of DNaseⅠ-SA were less potent than those of SA(P0.05).Also,the extent of proliferation response to SA and DNaseⅠ-SA was higher in the patients than in the controls(P

DNase, RNase, & RNase Inhibitors as Markers for Hepatocellular Carcinoma / 대한내과학회지

OBJECTIVE: Activities of nucleases (acid DNase and neutral RNase) and RNase inhibitor known to be involved in carcinogenesis and suppression of cancer were determined in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma and were compared with those of the controls. Also studied were nucleases and RNase inhibitor isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients to evaluate the properties and interactions between them. METHOD: Activities of nucleases and RNase inhibitor were measured in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma by ultraviolet spectrophotometry. Nucleases and RNase inhibitor were isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients by DEAE-cellulose column chromatography. As controls, normal tissue of the cancer patients, serum of healthy persons and ascitic fluid of cirrhotic patients were used. RESULT: Activities of DNase, RNase and RNase inhibitor were significantly increased in hepatocellular carcinoma tissue. DNase activity was not detected, RNase activity was increased and RNase inhibitor activity was unchanged in both serum and ascitic fluid of the hepatocellular carcinoma patients. DNase was isolated as a single enzyme and RNase as seven isozymes from the hepatocellular carcinoma tissue. The DNase isolated preferentially cleaved ds DNA over ss DNA and was endonuclease in nature (majority of hydrolytic products of DNA by the DNase were oligodeoxyribonucleotides). Of seven RNase isozymes isolated from the hepatocellular carcinoma tissue, isozyme I exhibited nonsecretory nature of RNase and other six isozymes secretory nature of the enzyme. Activity of RNase isozyme V was greatly increased and the activity of inhibitor complexed with the isozyme V was also increased. RNase in ascitic fluid of the cancer patient was separated into four isozymes, of which isozyme I exhibited mixed form of secretory and nonseretory nature and greatly increased in its activity. RNase isozyme V isolated in the hepatocellular carcinoma tissue was not detected in the ascitic fluid. CONCLUSION: The use of the nucleases and the inhibitor in the cancer tissue as biochemical markers for the hepatocellular carcinoma was suggested. RNase was released into the body fluid from the cancer tissue and could be used as a diagnostic marker for the hepatocellular carcinoma. An important role of the DNase in carcinogenesis of the liver was suggested. RNase isozyme V was limited in the cancer tissue and RNase isozyme I and V and inhibitors associated with these isozymes might be involved in carcinogenesis processes, suppression of cancer and maintenance of hepatocellular carcinoma through their interactions.

Comparison of the Retinal Microvasculature between Normal and Diabetic Rats Using Trypsin & DNase Digestion Technique

Trypsin and DNase digestion technique has become a retinal digestion technique for studying diabetic retinopathy. We tried osmotic digestion method with DNase and compared the quality of preparation of retina and microvascular change with trypsin digestion in normal and diabetic rats. Streptozotocin-induced diabetic rats were sacrificed at 9, 18, 27weeks in 6 rats. Right retinas were digested with 3% trypsin while left wer digested with 0.1% DNase. For control, 18 normal rats were sacrificed at the same time. DNase was superior to trypsin for retinal preparation on the stainability, the degree of separating nonvascular fissue from vascular net, the degree of preservation of vascular net in normal & diabetic rats. In diabetic rats, the number of pericyte decreased significantly with age, which not in normal rats(p=0.0023) We suggest DNase digestion technique as an new alternative for trypsin digestion technique in the study of microvascular change of diabetic retinopathy.

Bacillus thuringiensis crystal protein (δ-endotoxin) gene expression is independent of early sporulation specific functions.

Bacillus thuringiensis produces a parasporal insecticidal crystal protein. The correlation between sporulation and crystal protein production in Bacillus thuringiensis var. israelensis was studied. The strain was made resistant'against streptomycin (StR)- Acrystalliferous (Cry-) cured derivatives and asporogenous acrystalliferous (Spo- Cry-) mutants blocked at an early stage of sporulation were isolated. Plasmid transfer experiments were performed between Sts Spo+ Cry+ (streptomycin sensitive sporogeneous crystalliferous) and StR Spo+ Cry– and also between Sts Spo+ Cry+ and StR Spo– Cry– strains. StR colonies were selected. Insect toxicity was exhibited by the StR isolates in both the cases. The process of crystal formation is, therefore, independent of early sporulative events.

Activation of myosin heavy chain genes during cardiac hypertrophy.

Nick translation of intact rat heart nuclei has shown that the incorporation of [3H]–dATP is greater in hypertrophic heart nuclei than in normal heart nuclei suggesting that hypertrophic heart nuclei have more DNase I sensitive regions than normal heart nuclei. DNase I sensitivity analysis has shown that the rate and extent of digestion of myosin heavy chain genes are greater in hypertrophic than in normal heart nuclei. Dot blot hybridization analysis of myosin heavy chain transcripts from hypertrophic heart nuclei using myosin heavy chain cDNA as probe has shown that the sensitivity of myosin heavy chain genes to DNase I in hypertrophic heart nuclei correlates with myosin heavy chain gene activation and increased number of transcripts.