Detection of Bacteroides fragilis Enterotoxin Gene by PCR

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.     

Objective stress monitoring based on wearable sensors in everyday settings

Abstract

Monitoring people’s stress levels has become an essential part of behavioural studies for physical and mental illnesses conducted within the biopsychosocial framework. There have been several stress assessment studies in laboratory-based controlled settings. However, the results of these studies do not always translate effectively to an everyday context. The current state of wearable sensor technology allows us to develop systems measuring the physiological signals reflecting stress 24/7 while capturing the context. In this paper, we present a stress monitoring system that provides objective daily stress measurements in everyday settings based on three physiological signals: electrocardiogram (ECG), photoplethysmogram (PPG), and galvanic skin response (GSR) using Shimmer3 ECG, Shimmer3 GSR+, and Empatica E4 wearable sensors. We perform controlled stress assessment experiments on 17 participants in which we successfully detect stress with a 94.55% accuracy for 10-fold cross-validation and an 85.71% accuracy for subject-wise cross-validation. In everyday settings, the system assesses stress with an 81.82% accuracy. We also examine whether motion artefacts affect stress assessment and filter the low-confidence readings to minimise false alarms.     

Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection

BackgroundDeveloping a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells’ proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection.MethodsUsing 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets.ResultsAn exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature’s negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell–mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature’s negative predictive value was 90.6% and its positive predictive value was 77.8%.ConclusionsOur findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.     

Quantitation of adriamycin in plasma and urine: comparative study of radioimmunoassay and high-performance liquid chromatography methods

The response of tumours to adriamycin, and the cardiotoxicity of the drug, may be related to its pharmacokinetics and plasma levels. Rapid and sensitive methods of adriamycin determination in plasma and urine samples are thus needed. A comparative study shows that high-performance liquid chromatography with fluorimetric detection is a reliable and specific method, but it is relatively slow and sometimes lacks sensitivity. A commercially-available radioimmunoassay kit is convenient, but there is a cross reaction with the major metabolise adriamycinol and unless the assay is combined with an extraction step, it gives erroneously high results.     

Bilateral synchronous breast cancer: mode of detection and comparison of histologic features between the 2 breasts

BACKGROUND: Bilateral synchronous breast cancer is uncommon (accounting for 1.0%-2.6% of all patients with breast cancer), and most physicians do not accumulate a large personal experience of patients with this disease. We reviewed our experience with patients with bilateral synchronous breast cancer, focusing on the mode of detection and histologic features in the 2 breasts. METHODS: The charts of patients who were treated at this institution for bilateral synchronous breast cancer during the 15-year period of 1984 through 1999 were reviewed. Information regarding age, mode of detection, histopathologic features, treatment, and overall survival were analyzed. RESULTS: During the study period, 51 patients (all women) were treated at our institution for bilateral synchronous breast cancer. This comprised 2.1% of all patients (n = 2382 patients) treated for breast cancer during the same period of time. The first cancer was detected by palpation in 81% and by mammography in 14%. The corresponding figures for the contralateral cancer were 24% and 54%, respectively. The histologic type of cancer was identical in the 2 breasts in 29 patients (57%) and was different between the 2 breasts in 22 patients (43%). The overall 10-year survival rate was 63%. CONCLUSIONS: Bilateral synchronous breast cancer is often detected by mammography and is frequently of the same histologic type as the index cancer. A better awareness of the risk for this disease may help detect bilateral breast cancer earlier.     

Introduction of cadmium chloride additive to improve the performance and stability of perovskite solar cells

With the increase in the importance of using green energy sources to meet the world''s energy demands, attempts have been made to push perovskite solar cell technology toward industrialization all around the world. Improving the properties of perovskite materials as the heart of PSCs is one of the methods to fabricate favorable photovoltaic (PV) solar cells based on perovskites. Here, cadmium chloride (CdCl₂) was used as an additive source for the perovskite precursor to improve its PV properties. Results indicated CdCl₂ improves the perovskite growth and tailors its crystalline properties, suggesting boosted charge transport processes in the bulk and interfaces of the perovskite layer with electron–hole transport layers. Overall, by incorporation of 1.0% into the MAPbI₃ layer, a maximum power conversion efficiency of 15.28% was recorded for perovskite-based solar cells, higher than the 12.17% for the control devices. The developed method not only improved the PV performance of devices but also boosted the stability behavior of solar cells due to the passivated domain boundaries and enhanced hydrophobicity in the CdCl₂-based devices.

With the increase in the importance of using green energy sources to meet the world''s energy demands, attempts have been made to push perovskite solar cell technology toward industrialization all around the world.     

Mycobacterial pseudotumor of the skin

Inflammatory pseudotumors have a diverse etiology, mycobacterial pseudotumor (MP) being one of them. MP is a rare entity; it has been reported infrequently in various organs and is extremely rare in the skin. We report a cutaneous MP in an immunosuppressed liver transplant recipient. The lesion consisted mostly of spindle cells, with small numbers of lymphocytes. Conventional acid-fast bacilli (AFB) stain revealed a large number of acid-fast bacilli within spindled histiocytes and the presence of Mycobacterium avium was determined by polymerase chain reaction. Given that the patient had a prior history of cutaneous squamous cell carcinoma resected and reconstructed in the same area, establishing the diagnosis was challenging. Immunohistochemical staining for lysosome-associated membrane protein was strongly positive, suggesting the presence of numerous mature lysosomes within infected spindle cells. Mycobacterial spindle cell pseudotumors can mimic malignant or benign neoplasms and should be considered in differential diagnosis of spindle cell lesions, especially in immunocompromised patients. Further studies are needed to determine mechanisms that permit the survival of mycobacteria within the lesions and that cause this unusual manifestation of infection.