EVALUATION OF SOME HIGH RISK BACTERIAL SPECIES IN THE ORAL MICROBIOME OF HNSCC PATIENTS

In this study we evaluated the composition of seven high risk periodontitis species of microorganisms in the oral microbiome of patients with different forms of head and neck squamous cell carcinoma (HNSCC). The laboratory analyses were performed using the method of quantitative real-time amplification (qPCR) of 26 saliva and mouth swab samples collected from HNSCC patients. Laboratory data regarding the normal or pathological presence of studied species were integrated with patients clinical conditions and some lifestyle factors. The results show that the highest frequency of pathological concentration is in the case of Fusobacterium nucleatum and Tannerella forsythia, while the lowest frequency is observed for microorganisms Aggregatibacter actinomycetemcomitans and Treponema denticola.

Interrogating Biomineralization One Amino Acid at a Time: Amplification of Mutational Effects in Protein-Aided Titania Morphogenesis through Reaction-Diffusion Control

To emulate the control that biomineralizing organisms exert over reactant transport, we construct a countercurrent reaction-diffusion chamber in which an agarose hydrogel regulates the fluxes of inorganic precursor and precipitating...

AC and DC Differential Bridge Structure Suitable for Electrochemical Interfacial Capacitance Biosensing Applications

This paper presents a capacitive differential bridge structure with both AC and DC excitation and balancing capability for low cost electrode-solution interfacial capacitance biosensing applications. The proposed series RC balancing structure offers higher sensitivity, lower susceptibility to common-mode interferences, and drift control. To evaluate the bridge performance in practice, possible effects of initial bridge imbalance due to component mismatches are investigated considering the required resolution of the balancing networks, sensitivity, and linearity. This evaluation is also a guideline to designing the balancing networks, balancing algorithm and the proceeding readout interface circuitry. The proposed series RC bridge structure is implemented along with a custom single frequency real-time amplification/filtering readout board with real-time data acquisition and sine fitting. The main specifications for the implemented structure are 8-bit detection resolution if the total expected fractional capacitance change at the interface is roughly 1%. The characterization and measurement results show the effectiveness of the proposed structure in achieving the design target. The implemented structure successfully achieves distinct detection levels for tiny total capacitance change at the electrode-solution interface, utilizing Microcystin-(Leucine-Arginine) toxin dilutions as a proof of concept.

Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae

Streptococcus agalactiae is a major pathogenic bacterium causing perinatal infections in humans. In the present study, a novel real-time fluorescence loop-mediated isothermal amplification technology was successfully developed and evaluated for the detection of S. agalactiae in a single reaction. Six specific primers were designed to amplify the corresponding six regions of fbs B gene of S. agalactiae, using Bst DNA polymerase with DNA strand displacement activity at a constant temperature for 60 min. The presence of S. agalactiae was indicated by the fluorescence in real-time. Amplification of the targeted gene fragment was optimized with the primer 1 in the current setup. Positive result was only obtained for Sa by Real-LAMP among 10 tested relevant bacterial strains, with the detection sensitivity of 300 pg/µl. Real-LAMP was demonstrated to be a simple and rapid detection tool for S. agalactiae with high specificity and stability, which ensures its wide application and broad prospective utilization in clinical practice for the rapid detection of S. agalactiae.

Simultaneous single-channel multiplex and quantification of carbapenem-resistant genes using multidimensional standard curves

ABSTRACTMultiplexing and absolute quantification of nucleic acids, both have, in their own right, significant and extensive use in biomedical related fields, especially in point-of-care applications. Currently, the ability to detect several nucleic acid targets in a single-reaction scales linearly with the number of targets; an expensive and time-consuming feat. Here, we propose a new methodology based on multidimensional standard curves that extends the use of real-time PCR data obtained by common qPCR instruments. By applying this novel methodology, we achieve simultaneous single-channel multiplexing and enhanced quantification of multiple targets using only real-time amplification data. This is obtained without the need of fluorescent probes, agarose gels, melting curves or sequencing analysis. Given the importance and demand for tackling challenges in antimicrobial resistance, the proposed method is applied to the four most prominent carbapenem-resistant genes: blaOXA-48, blaNDM, blaVIM and blaKPC, which account for 97% of the UK’s reported carbapenemase-producing Enterobacteriaceae.

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing

Background: Confirming the integrity of seed samples in powdered form is important prior to conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. Objective: A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase (Sad1) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). Methods: The assays were performed at a constant temperature of 63°C for 30 min for visual LAMP and 62ºC for 40 min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR® Green or detected as real-time amplification curves. Results: Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. Conclusions: The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. Highlights: LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

Dual enzyme-assisted one-step isothermal real-time amplification assay for ultrasensitive detection of polynucleotide kinase activity

A novel, simple, one-step and one-tube detection method for polynucleotide kinase (PNK) activity based on isothermal real-time amplification assay was proposed.

Evaluation of 18S rRNA PCR in the Diagnosis of Neonatal Fungal Sepsis

Neonatal fungal infections constitute a significant disease burden in India. In the absence of an ideal diagnostic test for the detection of fungal sepsis, the management of sepsis remains enigmatic. At present, blood culture is the method of choice for neonatal fungal sepsis, but it is not 100% sensitive. Together with the requirement for prolonged incubation, blood cultures are of limited utility in investigating neonates for invasive fungal infections, and especially for guiding initial therapy. For quite some time, molecular methods based on polymerase chain reaction (PCR) for detecting microorganisms are gaining prominence in clinical practice, particularly due to their precision, specificity, sensitivity, and shorter reporting time. Amplification of the 18S rRNA subunit gene by PCR from the DNA extracted from patient's blood specifically identifies the causative organism at least at genus level. Here, we evaluate the diagnostic utility of 18S rRNA PCR in neonatal fungal sepsis as part of a prospective cohort study in a tertiary health care setting.

Amplification of local changes along the timescale processing hierarchy

Small changes in word choice can lead to dramatically different interpretations of narratives. How does the brain accumulate and integrate such local changes to construct unique neural representations for different stories? In this study, we created two distinct narratives by changing only a few words in each sentence (e.g., “he” to “she” or “sobbing” to “laughing”) while preserving the grammatical structure across stories. We then measured changes in neural responses between the two stories. We found that differences in neural responses between the two stories gradually increased along the hierarchy of processing timescales. For areas with short integration windows, such as early auditory cortex, the differences in neural responses between the two stories were relatively small. In contrast, in areas with the longest integration windows at the top of the hierarchy, such as the precuneus, temporal parietal junction, and medial frontal cortices, there were large differences in neural responses between stories. Furthermore, this gradual increase in neural differences between the stories was highly correlated with an area’s ability to integrate information over time. Amplification of neural differences did not occur when changes in words did not alter the interpretation of the story (e.g., sobbing to “crying”). Our results demonstrate how subtle differences in words are gradually accumulated and amplified along the cortical hierarchy as the brain constructs a narrative over time.