Diagnostic utility of DNA integrity number as an indicator of sufficient DNA quality in next-generation sequencing-based genomic profiling.

OBJECTIVES: DNA integrity number (DIN) is a metric for assessing DNA degradation, calculated based on electrophoresis using the Agilent TapeStation System. The utility of DIN as a diagnostic indicator of sufficient DNA quality in clinical next-generation sequencing (NGS) has not been well described. METHODS: We evaluated the DINs of 166 tumor formalin-fixed, paraffin-embedded (FFPE) tissue samples submitted for 124-gene panel sequencing. We also investigated a new metric on the electropherogram that could improve the predictive accuracy of the DIN. RESULTS: A DIN cutoff of 2.5 discriminated samples with successful analysis (n = 143) from samples with failed analysis (n = 23), with a sensitivity of 0.84 and a specificity of 0.78 (area under the curve [AUC] = 0.88). The DIN was positively correlated with the mean coverage (r = 0.72, P < .0001) but could not discriminate success from failure when the DIN was below 2.5 (negative predictive value, 0.44). We introduced a new metric, the peak/base ratio, that distinguished success from failure with higher accuracy than the DIN (cutoff = 1.6; sensitivity = 0.98, specificity = 0.83, and AUC =0.96). CONCLUSIONS: To predict successful NGS, the DNA quality of FFPE tissue can be easily and reliably assessed using the DIN and peak/base ratio.

Novel species identification and deep functional annotation of electrogenic biofilms, selectively enriched in a microbial fuel cell array.

In this study, electrogenic microbial communities originating from a single source were multiplied using our custom-made, 96-well-plate-based microbial fuel cell (MFC) array. Developed communities operated under different pH conditions and produced currents up to 19.4 A/m3 (0.6 A/m2) within 2 days of inoculation. Microscopic observations [combined scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS)] revealed that some species present in the anodic biofilm adsorbed copper on their surface because of the bioleaching of the printed circuit board (PCB), yielding Cu2 + ions up to 600 mg/L. Beta- diversity indicates taxonomic divergence among all communities, but functional clustering is based on reactor pH. Annotated metagenomes showed the high presence of multicopper oxidases and Cu-resistance genes, as well as genes encoding aliphatic and aromatic hydrocarbon-degrading enzymes, corresponding to PCB bioleaching. Metagenome analysis revealed a high abundance of Dietzia spp., previously characterized in MFCs, which did not grow at pH 4. Binning metagenomes allowed us to identify novel species, one belonging to Actinotalea, not yet associated with electrogenicity and enriched only in the pH 7 anode. Furthermore, we identified 854 unique protein-coding genes in Actinotalea that lacked sequence homology with other metagenomes. The function of some genes was predicted with high accuracy through deep functional residue identification (DeepFRI), with several of these genes potentially related to electrogenic capacity. Our results demonstrate the feasibility of using MFC arrays for the enrichment of functional electrogenic microbial consortia and data mining for the comparative analysis of either consortia or their members.

COVID-19 serological survey using micro blood sampling.

During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

Metabolic engineering of a novel strain of electrogenic bacterium Arcobacter butzleri to create a platform for single analyte detection using a microbial fuel cell.

Electrogenic bacteria metabolize organic substrates by transferring electrons to the external electrode, with subsequent electricity generation. In this proof-of-concept study, we present a novel strain of a known, electrogenic Arcobacter butzleri that can grow primarily on acetate and lactate and its electric current density is positively correlated (R2 = 0.95) to the COD concentrations up to 200 ppm. Using CRISPR-Cas9 and Cpf1, we engineered knockout Arcobacter butzleri mutants in either the acetate or lactate metabolic pathway, limiting their energy metabolism to a single carbon source. After genome editing, the expression of either acetate kinase, ackA, or lactate permease, lctP, was inhibited, as indicated by qPCR results. All mutants retain electrogenic activity when inoculated into a microbial fuel cell, yielding average current densities of 81-82 mA/m2, with wild type controls reaching 85-87 mA2. In the case of mutants, however, current is only generated in the presence of the substrate for the remaining pathway. Thus, we demonstrate that it is possible to obtain electric signal corresponding to the specific organic compound via genome editing. The outcome of this study also indicates that the application of electrogenic bacteria can be expanded by genome engineering.

A case of non-alcoholic steatohepatitis complicated with severe acute pancreatitis induced by decreased lipoprotein lipase and hepatic triglyceride lipase activity levels in a young Japanese woman.

We report a case of non-alcoholic steatohepatitis complicated with acute pancreatitis induced by hypertriglyceridemia in a young Japanese woman. A precise examination of the lipid profile showed decreased lipoprotein lipase (LPL) and hepatic triglyceride lipase activity levels, while the LPL mass was at the minimum level of the normal range.

Interplay between bladder microbiota and urinary antimicrobial peptides: mechanisms for human urinary tract infection risk and symptom severity.

Resident bacterial communities (microbiota) and host antimicrobial peptides (AMPs) are both essential components of normal host innate immune responses that limit infection and pathogen induced inflammation. However, their interdependence has not been investigated in the context of urinary tract infection (UTI) susceptibility. Here, we explored the interrelationship between the urinary microbiota and host AMP responses as mechanisms for UTI risk. Using prospectively collected day of surgery (DOS) urine specimens from female pelvic floor surgery participants, we report that the relative abundance and/or frequency of specific urinary microbiota distinguished between participants who did or did not develop a post-operative UTI. Furthermore, UTI risk significantly correlated with both specific urinary microbiota and ß-defensin AMP levels. Finally, urinary AMP hydrophobicity and protease activity were greater in participants who developed UTI, and correlated positively with both UTI risk and pelvic floor symptoms. These data demonstrate an interdependency between the urinary microbiota, AMP responses and symptoms, and identify a potential mechanism for UTI risk. Assessment of bacterial microbiota and host innate immune AMP responses in parallel may identify increased risk of UTI in certain populations.

Urinary bacteria in adult women with urgency urinary incontinence.

INTRODUCTION AND HYPOTHESIS: This study's aims were to detect and quantify bacterial DNA in the urine of randomized trial participants about to undergo treatment for urinary urgency incontinence (UUI) without clinical evidence of urinary tract infection (UTI) and to determine if the presence of bacterial DNA in baseline urine relates to either baseline urinary symptoms or UTI risk after urinary tract instrumentation. METHODS: Women without clinical evidence of baseline UTI were randomized to cystoscopic onabotulinum toxin A injection and oral placebo medication versus cystoscopic placebo injection and active oral medication. Bacterial DNA in participants' catheterized urine was measured by quantitative polymerase chain reaction (qPCR). RESULTS: Bacterial DNA was detected in the urine of 38.7 % of participants (60 out of 155). In these 60 qPCR-positive participants, baseline daily UUI episodes were greater than in the 95 qPCR-negative participants (5.71 [±2.60] vs 4.72 [±2.86], p = 0.004). Neither symptom severity by questionnaire nor treatment outcome was associated with qPCR status or with qPCR level in qPCR-positive subjects. In contrast, the presence of urinary bacterial DNA was associated with UTI risk: only 10 % of the qPCR-positive women developed a UTI post-treatment, while 24 % of the qPCR-negative women did so. The median qPCR level for qPCR-positive samples did not differ significantly by UTI status (UTI 2.58 × 10(5) vs no UTI 1.35 × 10(5) copies/mL, p = 0.6). CONCLUSIONS: These results may indicate a urinary bacterial contribution to both baseline UUI and the risk of post-treatment UTI.

[Case of acute necrotizing esophagitis associated with AA amyloidosis secondary to bronchiectasis].

An 82-year-old woman with a history of bronchiectasis for 20 years was admitted to our hospital with anorexia and diarrhea. Sigmoidoscopy showed multiple mucosal erythematous areas and erosions. Histologic examination with Congo red stain revealed massive amyloid deposition around the submucosal vessels as well as in the parenchyma of the mucosa and submucosa. With immunohistochemistry, the diagnosis of secondary/reactive AA amyloidosis was confirmed. Esophagogastroduodenoscopy demonstrated diffuse dark brown mucosa, establishing the diagnosis of acute necrotizing esophagitis. Ischemia associated with amyloid deposition of the vessels in the esophagus was considered to be a possible etiology of acute necrotizing esophagitis. Additionally, gastric outlet obstruction and gastroesophageal reflux associated with gastroduodenal erosions caused by amyloid deposition were supposed to be another factor. Amyloid deposition in the esophageal mucosa may cause a reduction in mucosal defense that is responsible for the pathogenesis. We report the first case of acute necrotizing esophagitis associated with amyloidosis.

[Molecular diagnosis of lung cancer in association with treatment of choice].

Recent advances in lung cancer research have enabled significant progress in our understanding of the molecular pathogenesis and treatment for lung cancer. For example, EGFR tyrosine kinase inhibitors have been developed, and a subset of patients show marked therapeutic responses to this treatment. Subsequently, this super-response was revealed to be associated with an EGFR mutation that was identified in 2005. Currently, EGFR tyrosine kinase inhibitors are available for first-line treatments of patients with advanced non-small-cell lung cancer when the tumor is positive for an EGFR mutation. More recently, similar marked responses to an ALK inhibitor in patients with ALK fusion lung cancers were demonstrated. In this article, we review such recent advances in lung cancer, focusing on EGFR mutation and ALK fusion..

Evidence of uncultivated bacteria in the adult female bladder.

Clinical urine specimens are usually considered to be sterile when they do not yield uropathogens using standard clinical cultivation procedures. Our aim was to test if the adult female bladder might contain bacteria that are not identified by these routine procedures. An additional aim was to identify and recommend the appropriate urine collection method for the study of bacterial communities in the female bladder. Consenting participants who were free of known urinary tract infection provided urine samples by voided, transurethral, and/or suprapubic collection methods. The presence of bacteria in these samples was assessed by bacterial culture, light microscopy, and 16S rRNA gene sequencing. Bacteria that are not or cannot be routinely cultivated (hereinafter called uncultivated bacteria) were common in voided urine, urine collected by transurethral catheter (TUC), and urine collected by suprapubic aspirate (SPA), regardless of whether the subjects had urinary symptoms. Voided urine samples contained mixtures of urinary and genital tract bacteria. Communities identified in parallel urine samples collected by TUC and SPA were similar. Uncultivated bacteria are clearly present in the bladders of some women. It remains unclear if these bacteria are viable and/or if their presence is relevant to idiopathic urinary tract conditions.

Cycleave polymerase chain reaction method is practically applicable for V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)/V-raf murine sarcoma viral oncogene homolog B1 (BRAF) genotyping in colorectal cancer.

Activating V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and V-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene mutations are important predictive markers for antiepidermal growth factor receptor chemotherapy in colorectal cancer (CRC). However, a rapid and accurate assay for KRAS/BRAF mutation detection from routine pathological specimens is lacking in clinical practice. We applied the cycleave polymerase chain reaction (PCR) method to routine KRAS/BRAF genotyping of CRC patients at our institution from 2001 to 2009. The accuracy of cycleave PCR genotyping was shown by the high concordance with reverse transcriptase-PCR-coupled direct sequencing. KRAS gene mutations were analyzed successfully from small biopsy or cytology specimens. Although some surgical specimens could not be evaluated by cycleave PCR, corresponding biopsy specimens could be used instead. This PCR failure observed for some biopsy specimens may have been a result of the use of formalin fixation, as overfixation of surgical specimens by formalin impaired PCR amplification. In conclusion, cycleave PCR is practically applicable to KRAS/BRAF genotyping using small amounts of biopsied tumor cells. Care must be taken in the selection of pathological specimens for KRAS/BRAF testing.

[Fundamental evaluation of HCV core antigen method comparison with Cobas Amplicor HCV monitor v2.0 (high range method)].

Quantitative measurement of hepatitis C virus (HCV) has been performed by PCR method. However, PCR method has problems such as a special instrument, a complicated manual skill and a high cost. Recently, simple and highly sensitive HCV core antigen (Ag) method has been developed. We performed fundamental evaluation of HCV core Ag method, and compared HCV core Ag method with HCV PCR high-range method. The intra-assay and inter-assay variation coefficients for HCV core Ag were calculated to be within the ranges of 1.0-11.3% and 0.8-9.3%, respectively. The test of dilution linearity revealed the unstableness in the vicinity of a cut-off level of 50 fmol/L. Based on the result of the high-range method; sensitivity, specificity, positive predictive value, negative predictive value, and agreement rate were 97.0%, 100%, 100%, 82.0%, and 96.5%, respectively. The correlation between the HCV core Ag method and the high-range method was r = 0.87. Cost per sample and time from sample preparation to final report for HCV core Ag were cheaper and shorter than those of HCV PCR method, respectively. We consider that the HCV core Ag method seems to be useful as the quantitative measurement of HCV with respect to rapidness, easiness and low cost.

[Associations between glycated hemoglobin levels and atherosclerotic risk factors in urban communities].

PURPOSE: To prevent coronary heart disease and type 2 diabetes mellitus, we need to focus on "the deadly quartet" (coexistence of upper-body obesity, glucose intolerance, hypertriglyceridemia, and hypertension), and the multiple risk factor syndrome related to insulin resistance. As few urban community-based population studies have evaluated the correlation between glycated hemoglobin A1c (HbA1c) levels and risks of life-style related disease, we investigated this parameter and its correlation with atherosclerotic risk factors in participants of health check ups in two communities in Kanagawa prefecture. We also examined whether these correlations were affected by difference between the two groups. METHODS: The study populations comprised male and female residents aged 40-79 in two communities (A and B cities) in 1998. Age, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides (TG), GOT, GPT, Uric acid (UA), and gamma GTP were considered as atherosclerotic risk factors. Firstly we calculated correlation coefficients with HbA1c level. Secondly, logistic regression analyses were performed with HbA1c as the dependent variable, and risk factors correlated with HbA1c significantly and variable for each community as independent variables. To assess whether community differences affect associations between HbA1c levels and risk factors, we added interaction terms as independent variables in the logistic regression analysis. RESULTS: 1. There were no significant interaction terms while significant positive associations were observed between HbA1c and age, BMI, and levels of TC and gamma GTP for men in both communities. A significant negative association was observed between HbA1c and UA.. 2. Age, BMI, and the levels of SBP, TG, GPT, and gamma GTP were positively associated, and GOT was negatively associated with the HbA1c levels in women in both communities. Each community had its own association between TC and HbA1c. CONCLUSIONS: The findings that TC is associated with HbA1c in men, and that BMI and the level of TC are linked with HbA1c in women are consistent with previous results for Japanese. The association between HbA1c and TG in women was newly observed for Japanese. Furthermore, the levels of gamma GTP in both sexes and also GPT in women are associated with HbA1c. From these results, investigating linkage between HbA1c level and atherosclerotic risk factors was thought to be appropriate for estimating accumulation of multiple risk factors in the community.