Detection Of DNA Fragmentation Research Articles

PBDEs disrupt homeostasis maintenance and regeneration of planarians due to DNA damage, proliferation and apoptosis anomaly

Polybrominated diphenyl ethers (PBDEs) are widely used as brominated flame retardants in the manufacturing industry, belonging to persistent organic pollutants in the environment. Planarians are the freshwater worms, with strong regenerative ability and extreme sensitivity to environmental toxicants. This study aimed to evaluate the potential acute comprehensive effects of PBDE-47/-209 on freshwater planarians. Methods to detect the effects include: detection of oxidative stress, observation of morphology and histology, detection of DNA fragmentation, and detection of cell proliferation and apoptosis. In the PBDE-47 treatment group, planarians showed increased oxidative stress intensity, severe tissue damage, increased DNA fragmentation level, and increased cell proliferation and apoptosis. In the PBDE-209 treatment group, planarians showed decreased oxidative stress intensity, slight tissue damage, almost unchanged DNA fragmentation level and apoptosis, proliferation increased only on the first day after treatment. In conclusion, both PBDE-47 and PBDE-209 are dangerous environmental hazardous material that can disrupt planarians homeostasis, while the toxicity of PBDE-47 is sever than PBDE-209 that PBDE-47 can lead to the death of planarians.

Detecting DNA damage in stored blood samples

Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Isolation and Purification of the Anti-Bacterial Compound of Cinnamon Oil Extracted from South India

Cinnamon oil is derived from the leaves of the cinnamon tree, the essential oil of the plant is extracted by steam distillation, while its bark is used to make cinnamon spice. The present study was conducted to isolate the most important bioactive compounds from cinnamon oil extracted from South India. The thin-layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) were used to determine and estimate the isolates obtained. It found that the organic phase's highest retention time was 5.433, and the peak area percentage was 88.8 %. The organic phase sample had a high peak area percentage, indicating that there was a lot of bioactive chemical in it. The broad absorption band at 3270 cm-1 is due to the (-OH) group, 1670 and 1623 cm-1 to the C=C group, 1449 and 1381 cm-1 to the (-C-H) group, and the band at 1120 cm-1 to the (-C-O) group. A pseudo molecular ion peak at m/z 275 for a [M+H] +. This study resulted in finding a peak in the positive mode mass spectra, indicating a molecular weight of 274 and the molecular formula of C15H14O5.1H 13CNMR investigations verified the presence of phenol hydroxide groups, two aromatic ring systems, methylene groups, and a phenoxy carbon in the chemical composition of the compound. Moreover, the composition is quite similar to catechins, and this chemical is separated from the organic phase of cinnamon oil. The potent inhibitory activity of the newly synthesized catechin antagonist against S.aureus and E.coli was found to assess cell death by detection of DNA fragments using agarose gel electrophoresis and SEM analysis.

A fully functional palmtop microchip electrophoresis analyzer with laser-induced fluorescence detection

Microchip electrophoresis (MCE) is an ideal tool for rapid on-site analysis of multiple components, therefore the demand for portable MCE devices is increasing. In this work, a fully functional palmtop MCE analyzer was built, integrating units of sample injection, laser-induced fluorescence (LIF) detection, high-voltage (HV), imaging and data acquisition. Frames were fabricated with 3D printing to encapsulate miniaturized components selected with balanced consideration on size, cost and performance. The LIF unit contains a laser diode as the excitation light source, a collimating lens used in the commercial laser diode as the focusing lens and an avalanche photodiode (APD) for the laser distance meter as the photoelectric converter. The sample injection was achieved by negative pressure induced pinching. The total cost of the device is approximately $600 with a size of 89 × 98 × 98 mm3 and a weight of 350 g. It can be powered by a laptop or a mobile power bank. The limit of detection (LOD) measured under a typical MCE condition for sodium fluorescein could be 110 pM. The applicability of the device was demonstrated with the detection of rhodamines and DNA fragments.

Investigation of Microbial Translocation, TLR and VDR Gene Polymorphisms, and Recurrence Risk in Stage III Colorectal Cancer Patients.

Simple SummaryGut microbial dysbiosis and microbial passage into the peripheral blood leads to colorectal cancer (CRC) and disease progression. Toll-like (TLR) and vitamin D (VDR) receptors play important role in the immune modulation and polymorphisms that may increase CRC risk. The aim of the current study was to demonstrate the prognostic value of microbial DNA fragments in the blood of stage III CRC patients and correlate such microbial detection to TLR/VDR polymorphisms. TLR/VDR polymorphisms and presence of microbial DNA in CRC patients highlight their role in cancer development and progression.Gut microbial dysbiosis and microbial passage into the peripheral blood leads to colorectal cancer (CRC) and disease progression. Toll-like (TLR) and vitamin D (VDR) receptors play important role in the immune modulation and polymorphisms that may increase CRC risk and death rates. The aim of the current study was to demonstrate the prognostic value of microbial DNA fragments in the blood of stage III CRC patients and correlate such microbial detection to TLR/VDR polymorphisms. Peripheral blood was collected from 132 patients for the detection of microbial DNA fragments, and TLR/VDR gene polymorphisms. In the detection of various microbial DNA fragments, TLR and VDR polymorphisms was significantly higher compared to healthy group. Homozygous individuals of either TLR or VDR polymorphisms had significantly higher detection rates of microbial DNA fragments. Mutational and MSI status were significantly correlated with TLR9 and VDR polymorphisms. Significantly shorter disease-free survival was associated with patients with BRAF mutated tumors and ApaI polymorphisms, whereas shorter overall survival was associated with the detection of C. albicans. The detection of B. fragilis, as demonstrated by the multivariate analysis, is an independent poor prognostic factor for shorter disease-free survival. TLR/VDR genetic variants were significantly correlated with the detection of microbial fragments in the blood, and this in turn is significantly associated with tumorigenesis and disease progression.

Loop-mediated isothermal amplification of PBAN gene for molecular diagnosis of Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae)

• LAMP assay for PBAN of the whitefly was developed. • The PBAN-based LAMP primer set showed specific amplification of the target region. • The PBAN-based LAMP assay can be applicable for monitoring B. tabaci biotype Q. The tobacco whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is a serious pest that transmits tomato yellow leaf curl virus (TYLCV) in tomato. The tobacco whitefly exhibits morphological similarity with the greenhouse whitefly, Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) which also damages various plants in the greenhouse. Therefore, it is very important to quickly and accurately diagnose whiteflies for applying pest management strategies. In this study, we used a loop-mediated isothermal amplification (LAMP) assay to quickly and effectively detect B. tabaci . Primer sets were investigated for the specificity of B. tabaci by visible detection of the target DNA fragment. Two primer sets for pheromone biosynthesis activating neuropeptide (PBAN) gene were obtained from transcriptomic analysis of B. tabaci biotype Q. The PBAN-based LAMP primer set showed specific amplification of the target region. The optimal conditions for B. tabaci detection were 60 °C for 60 min with four LAMP primers of PBAN. The minimum amount of genomic DNA required for visible detection was 100 ng. These results suggest that the PBAN-based LAMP assay can be applicable for field monitoring of B. tabaci .

Qualitative and Quantitative Detection of Mealworm DNA in Raw and Commercial Food Products Using Real-Time PCR

Considering food safety and an increasing public awareness of the ingredients, production process and origin of foods, the application of insects as food requires the development of tests for the reliable identification of their presence. The aim of the study was (1) the determination of appropriate modifications of the selected method for isolating the DNA of two life stages of mealworm, i.e., larva and adult, from commercial food products; (2) the determination of the method parameters for the qualitative and quantitative analysis of mealworm contents based on the detection of a species-specific mitochondrial DNA fragment, using real-time PCR; (3) the application of a method to test the commercial food products of mealworm. A total of nine species of adult insect were investigated (field cricket, Dubia cockroach, Madagascar cockroach, banded cricket, migratory locust, yellow mealworm, superworm, house fly and lacewing), theirlarvaes (yellow mealworms and superworms) and thirteen commercial food products (dried whole insects, powder and granules) representing various insect species and origins which were purchased from the European market. The obtained results showed that the efficiency of the modification of the DNA extraction method is dependent on the life stage of the mealworm. We proved the high sensitivity of the test, with the range of the method being 0.1–100%; we also proved the biological specificity in this range, and the linearity. The linearity of the test was also statistically verified using the Fisher–Snedecor test. One-way variance analysis showed statistically significant differences between the cT values of the two mealworm life stages studied, and similarly, between the threshold cycle (cT) values of adult forms. In contrast, for the inside group of mealworm larvae, there was no significant difference observed between the results of the cT values. The test is effective for processed food products and may be used to monitor food. The research proved the suitability of the applied method for the analysis of samples that are commercially available as food for exotic animals. The hereby-developed method is based on widely used laboratory techniques, and does not require any additional investment in equipment. The availabilityof such a methodallows for the verification of the accuracy of the declared species component of the food products.

Controversies in the Interpretation of Liquid Biopsy Data in Lymphoma.

The rapid evolution of genomic technologies over the last years has led to the development of different methods for the detection, measurement and analysis of cell-free DNA fragments (cfDNA) which are shed into the bloodstream by apoptotic cells and circulate at a low concentration in plasma. In cancer patients, the proportion of tumor-derived cfDNA is defined as circulating tumor DNA. This analysis, commonly known as liquid biopsy, allows to access tumor DNA through a simple blood sampling and therefore without the need of an invasive tissue biopsy. For this reason, this tool may have several clinical applications in terms of diagnosis, prognosis, and monitoring of minimal residual disease. However, there are still several critical issues that need to be resolved. In this review, we will discuss some of the controversies around this method and its potential clinical applications.

CTREP-finder: A web service for quick identification and visualization of clean transgenic and genome-edited plants

To feed 10 billion people in 30 years from now will heavily depend on new higher yield, yet safe, biotech crop varieties, which are developed using biotechnologies, particularly genetic transformation and genome editing. A clean transgenic plant (CTP) contains only the defined transgene, whilst a clean edited plant (CEP) is transgene-free. Currently, it is difficult and time-consuming to identify and locate transgenes within plant genomes due to the complex nature of transfer DNA (T-DNA) or plasmid backbone integration, which has led to a major obstacle in mass scale production of clean transgenic/edited plants (CTREPs). Here we have built a web-based portal, CTREP-finder, for fast detection, characterization and visualization of foreign DNA fragments in plants, based on next-generation whole genome sequencing data. We invented a novel bioinformatics strategy to handle three scenarios of transgene integration and successfully identified CTREPs by testing 132 samples. Furthermore, we compared the CTREP-finder with three public programs, FED, transgeneR and TDNAscan, and it showed that the CTREP-finder outperforms those programs particularly on the localization of transgene integration. The use of CTREP-finder needs little requirement for bioinformatics expertise. Therefore, it is a user-friendly web-based portal that could be used by plant breeders and regulatory agencies for the rapid selection/detection of CTREPs for crop breeding and production.

Detection and Validation of Circular DNA Fragments Using Nanopore Sequencing.

Occurrence of extra-chromosomal circular DNA is a phenomenon frequently observed in tumor cells, and the presence of such DNA has been recognized as a marker of adverse outcome across cancer types. We here describe a computational workflow for identification of DNA circles from long-read sequencing data. The workflow is implemented based on the Snakemake workflow management system. Its key step uses a graph-theoretic approach to identify putative circular fragments validated on simulated reads. We then demonstrate robustness of our approach using nanopore sequencing of selectively enriched circular DNA by highly sensitive and specific recovery of plasmids and the mitochondrial genome, which is the only circular DNA in normal human cells. Finally, we show that the workflow facilitates detection of larger circular DNA fragments containing extrachromosomal copies of the MYCN oncogene and the respective breakpoints, which is a potentially useful application in disease monitoring of several cancer types.

Mechanisms of the host immune response and helminth-induced pathology during Trichobilharzia regenti (Schistosomatidae) neuroinvasion in mice.

Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools.

Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system.

The fabrication of nanopores with a matched pore size, and the existence of multiple interferents make the reproducible detection of small-sized molecules by means of solid-state nanopores still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructures related to the existence of small-sized targets. In particular, a DNA tetrahedron with a well-defined 3D nanostructure is the ideal candidate for use as a signal transducer. Here, we demonstrate the detection of an L1-encoding gene of HPV18 as a test DNA target sequence in a reaction buffer solution, where long single-stranded DNA linking DNA tetrahedra onto the surface of the magnetic beads is cleaved by a target DNA-activated CRISPR-cas12 system. The DNA tetrahedra are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) one signal transducer can be used to detect different targets; (2) a glassy nanopore with a pore size much larger than the target DNA fragment can boost the tolerance of the contaminants and interferents which often degrade the performance of a nanopore sensor.

Label- and enzyme-free plasmon-enhanced single molecule fluorescence detection of HIV DNA fragments based on a catalytic hairpin assembly.

We developed a label- and enzyme-free single molecule fluorescence counting strategy for HIV DNA fragments detection. The nucleic acid biosensor consists of a 5' terminal connected with a triangular gold nanoplate, 3' terminal rich in guanine hairpin probe (HP1) and a hairpin probe HP2 complementary to the partial sequence of HP1. Without the existence of the target DNA, the DNA fragment rich in the guanine region is locked in a hairpin structure and cannot form a G-quadruplex, hence NMM exhibits a low fluorescence signal. When the target DNA exists, the hairpin assembly will trigger a strand displacement amplification reaction that produces a great number of G-quadruplexes, and the fluorescence brightness of NMM will be enhanced. The plasmon resonance effect of the triangular gold nanoplates will further amplify the fluorescence signal. This method can analyze the target DNA with high sensitivity and selectivity, and the detection limit is 0.83 fM. The analysis of the HIV DNA fragments in diluted human serum samples was successfully achieved, and the recovery rate was 92%-104%. Because of its easy operation and low cost, it has broad development potential in biochemical analysis and clinical applications.

Полиморфизм ДНК собак. VI. Индивидуализирующие ампликоны

To detect DNA polymorphism in dogs and their wild relatives, a fairly simple method was used in the form of virtual amplification of certain random areas of their genomes using multiplex RAPD analysis with primers with arbitrary nucleotide sequences, which does not require prior knowledge of the sequences of nitrogenous bases of the detected DNA fragments. At the same time, information about the complete genomes of the studied species predicts the expected results, which makes it possible to reject unsuitable primers before conducting "wet" experiments. The multiplex RAPD analysis conducted in silico with 12 decameric primers, the sequences of which exclude the formation of homo- and heterodimers in PCR, showed both genomic similarities and differences between different canid species. If amplicons common to all the studied dog samples are removed from the final results of such virtual amplification, leaving only significant ones that actually act as individualizing, then it is potentially possible to DNA-cataloging each canine specimen with the probability of a random coincidence of their genomic barcodes on average one event per quadrillion.

A novel self-enhancement NCNDs-BPEI-Ru nanocomposite with highly efficient electrochemiluminescence as signal probe for ultrasensitive detection of MTB

Herein, a novel self-enhanced Ru(Ⅱ)-based nanocomposite (NCNDs-BPEI-Ru), synthesized by covalently connecting nitrogen-doped carbon nanodots (NCNDs) and tris (4,4′-dicarboxylicacid-2,2′-bipyridyl) ruthenium (II) dichloride (Ru(dcbpy)3Cl2) through branched polyethyleneimine (BPEI), could be used as an electrochemiluminescence (ECL) signal probe for ultrasensitive detection of mycobacterium tuberculosis (MTB) DNA fragment. Without any extraneous coreactants, NCNDs-BPEI-Ru could generate an ultra-strong initial ECL signal on the glassy carbon electrode (GCE) (signal-on) due to the coreaction from BPEI and NCNDs to Ru(dcbpy)3Cl2. Then, self-assembled DNA nanotubes embedding methylene blue molecules (MB-DNANTs) were modified on the nanocomposite to effectively quench the ECL intensity of NCNDs-BPEI-Ru owing to the quenching effect of MB for Ru(dcbpy)3Cl2 (signal-off). Finally, the 3D DNA nanomachine, formed by a DNA-functionalized magnetic beads (DNA-MBs) could transform trace target MTB into massive output DNA, and the MB-DNANTs could be disintegrated to get ECL signal recovery (signal-on) due to hybridization of output DNA and the S2 in DNANTs. Therefore, the proposed biosensor exhibited predominant performance for the detection of MTB DNA fragment with a low detection limit of 1.4 aM, which showed great application value in the detection of biomarkers in clinical diagnosis.

Response of MCF-7 Breast Cancer Cells Overexpressed with P-Glycoprotein to Apoptotic Induction after Photodynamic Therapy.

Multidrug resistance (MDR) has posed a significant threat to cancer treatment and has led to the emergence of a new therapeutic regime of photodynamic therapy (PDT) to curb the menace. The PDT modality employs a photosensitiser (PS), excited at a specific wavelength of light to kill cancer cells. In the present study, we used a zinc phthalocyanine tetrasulfonic acid PS to mediate the photodynamic killing of MCF-7 cells overexpressed with P-glycoprotein (P-gp) and investigate the response to cell death induction. After photodynamic treatment, MCF-7 cells undergo cell death, and indicators like Annexin V/PI staining, DNA fragmentation, and measurement of apoptotic protein expression were investigated. Results showed increased externalisation of phosphatidylserine protein, measured as a percentage in flow cytometry indicative of apoptotic induction. This expression was significant (p < 0.006) for the untreated control cells, and there was no detection of DNA fragments after a laser fluence of 20 J/cm2. In addition, a statistically significant difference (p < 0.05) was seen in caspase 8 activity and Bax protein expression. These findings were indicative of apoptotic induction and thus seem to represent the extrinsic apoptotic pathway. This study shows the role of PDT in the treatment of a resistant phenotype breast cancer.

In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii

Toxoplasma gondiiis an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline tonew derivatives improved the anti-T. gondiiactivity 3.4 fold. The most active derivative presented ED50in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.

Novel methods to detect ROS in viable spermatozoa of native semen samples

A crucial issue in male infertility work-up is to have reliable methods to detect oxidative stress in native semen samples. Here, we explored flow cytometric detection of Reactive Oxygen Species (ROS) in viable spermatozoa using native semen samples. To this aim, we challenged three fluorescent probes: CM-H2DCFDA, CellROX Green and MitoSOX Red. After excluding all non-sperm cells, each probe was coupled to a suitable stain to eliminate also semen apoptotic bodies and non-viable spermatozoa: Merocyanine 540 (M540) for CM-H2DCFDA and CellROX Green, and LIVE/DEAD Fixable Green Dead Cell Stain (LD-G) for MitoSOX Red. We found that CM-H2DCFDA was confined in the sperm midpiece, whereas CellROX Green and MitoSOX Red were localized in the head of spermatozoa. Treatment with H2O2 highly increased MitoSOX Red fluorescence (36.20 ± 5.24 vs 18.02 ± 2.25, %, p < 0.01), but not, or only slightly, the labelling with CMH2DCFDA (2.57 ± 1.70 vs 2.77 ± 1.43, p > 0.05) and CellROX Green (5.34 ± 3.18 vs 3.76 ± 2.04, p < 0.05), respectively. Menadione treatment highly increased CellROX Green (10.13 ± 5.85 vs 3.82 ± 2.70, p < 0.01) and MitoSOX Red (69.20 ± 27.14 vs 21.18 ± 7.96, %, p < 0.05), but not CM-H2DCFDA fluorescence (8.30 ± 11.56 vs 7.30 ± 9.19, p > 0.05). Further, only MitoSOX Red was able to detect spontaneous ROS generation during in vitro sperm incubation. We also detected DNA fragmentation by Comet and SCD Assay after sorting MitoSOX Red positive and negative sperm viable fractions. Results indicated that MitoSOX labelling in viable spermatozoa was strictly associated to sperm DNA fragmentation. In conclusion, MitoSOX Red/LD-G appears to be a promising method to detect oxidative stress in human semen for male infertility work-up.

The application of shotgun metagenomics to the diagnosis of granulomatous amoebic encephalitis due to Balamuthia mandrillaris: a case report

BackgroundGranulomatous amoebic encephalitis (GAE) is an infrequent and fatal infectious disease worldwide. Antemortem diagnosis in this condition is very difficult because clinical manifestations and neuroimaging are nonspecific.Case presentationA 60-year-old Japanese woman was admitted with a chief complaint of left homonymous hemianopsia. Brain-MRI showed extensive necrotizing lesions enhanced by gadolinium, in the right frontal lobe, right occipital lobe, and left parietal lobe. Epithelioid granulomas of unknown etiology were found in the biopsied brain specimens. Shotgun metagenomic sequencing using a next-generation sequencer detected DNA fragments of Balamuthia mandrillaris in the tissue specimens. The diagnosis of granulomatous amoebic encephalitis was confirmed using an amoeba-specific polymerase chain reaction and immunostaining on the biopsied tissues.ConclusionsShotgun metagenomics is useful for the diagnosis of central nervous system infections such as GAE wherein the pathogens are difficult to identify.