A Novel Method to Assess Antimicrobial Susceptibility in Commensal Oropharyngeal Neisseria—A Pilot Study

Commensal Neisseria provide a reservoir of resistance genes that can be transferred to the pathogens Neisseria gonorrhoeae and N. meningitidis in the human oropharynx. Surveillance programs are thus needed to monitor resistance in oropharyngeal commensal Neisseria, but currently the isolation and antimicrobial susceptibility testing of these commensals is laborious, complex and expensive. In addition, the posterior oropharyngeal/tonsillar swab, which is commonly used to sample oropharyngeal Neisseria, is poorly tolerated by many individuals. We evaluated an alternative non-invasive method to isolate oropharyngeal commensal Neisseria and to detect decreased susceptibility to azithromycin using selective media (LBVT.SNR) with and without azithromycin (2 µg/mL). In this pilot study, we compared paired posterior oropharyngeal/tonsillar swabs and oral rinse-and-gargle samples from 10 participants and demonstrated that a similar Neisseria species diversity and number of colonies were isolated from both sample types. Moreover, the proportion of Neisseria colonies that had a decreased susceptibility to azithromycin was similar in the rinse samples compared to the swabs. This pilot study has produced encouraging data that a simple protocol of oral rinse-and-gargle and culture on plates selective for commensal Neisseria with and without a target antimicrobial can be used as a surveillance tool to monitor antimicrobial susceptibility in commensal oropharyngeal Neisseria. Larger studies are required to validate these findings.

Improvement of field-deployable metagenomic virus detection by a simple pretreatment method.

As both the current COVID-19 pandemic and earlier pandemics have shown, animals are the source for some of the deadliest viral pathogens, which can spread to humans. Therefore, early detection at the point of incidence is crucial to both prevent and understand the threats posed to human health from pathogens in animal reservoirs. Often, the exact genetic nature of these zoonotic pathogens is unknown and advanced laboratory facilities do not exist in most field settings and therefore the development of methods for unbiased metagenomic and point of incidence detection is crucial in order to identify novel viral pathogens in animals with zoonotic and pandemic potential. Here we addressed some of these issues by developing a metagenomic Nanopore next-generation sequencing (mNGS) method for nucleic acids extracted from clinical samples from patients with SARS-CoV-2. To reduce the non-RNA viral genetic components in the samples, we used DNase pretreatment in a syringe followed by filtration and found that these pretreatments increased the number of SARS-CoV2 reads by > 500-fold compared with no pretreatment. The simple protocol, described here, allows for fast (within 6 hours) metagenomic detection of RNA viruses in biological samples exemplified by SARS-CoV-2 detection in clinical throat swabs. This method could also be applied in field settings for point of incidence detection of virus pathogens, thus eliminating the need for transport of infectious samples, cold storage and a specialized laboratory.

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

HaloTag labeling technology has introduced unrivaled potential in protein chemistry, molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, tetramethylrhodamine and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We show that highly efficient labelling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from a wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates a need for expensive and laborious protein engineering.

III–V nanowires for ammonia detection

Ammonia is an inorganic agent found both in nature and in the human body, which is of great interest for modern sensory applications. Here we use GaP, GaN and GaAs epitaxial nanowires as sensitive elements of the ammonia sensors fabricated via a simple protocol on the platform with golden interdigital contacts. Impedancemetry is used to study change of the device properties with addition of ammonia to the reference medium (water). GaP and GaN-based devices exhibit sufficient response to the ammonia presence with the detection limit lower than 10ppm. This work is aimed at fabrication and study of precise, technological and relatively cheap ammonia sensors compatible with a liquid medium, and motivated by the possibility of using this type of adsorption sensors in medical, environmental equipment and biological purposes.

Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics

The CRISPR diagnostic (CRISPR-Dx) technology that employs the trans-cleavage activities has shown great potential in diagnostic sensitivity, specificity, convenience, and portability, and has been recognized as the next-generation diagnostic methods. However, due to the lack of standardized definition of Cas trans-cleavage enzymatic units, it is difficult to standardize the present CRISPR-Dx systems, which have undoubtedly impeded the development of the CRISPR-Dx industry. To solve the problem, we here first systematically optimized the reaction systems for Cas12a, and then defined its trans-cleavage units (transU), which we believe will be of great importance and interest to researchers in both molecular diagnostic industry and basic research. Moreover, a simple protocol was provided to facilitate a step-by-step measurement of the Cas12a transU, which can also act as a reference for the definition of the transU for other Cas proteins.

Demonstration of Modbus Protocol for Robot Communication Using C#

Purpose: The Modbus is the trusted name in the industrial automation communication domain. It is a pretty simple protocol to implement and so very popular to the industrial communication personnel. Nowadays, Some industrial robots are also capable of communicating through Modbus. So our robot researchers frequently face the challenge of communicating with Modbus-enabled devices or robots. They need to know the protocol in detail before integrating it into their project. Its learning curves are a bit higher because of the lack of document which is practical oriented. The protocol selection, packet structure, CRC, or LRC calculation need to maintain precisely as standards; otherwise, the Modbus exception may happen. We experience those scenarios. Through our practical experience, we learned what is required for a new researcher who wants to implement Modbus in their project. In this paper, we demonstrate the Modbus packet structure and implement it with several practical examples. Finally, to test the written code, we provide simple tools which are easy to use and customizable. The researcher can easily integrate into their research project. The complete project source code is available in Github. Design/Methodology/Approach: The Modbus is the standard protocol to communicate between or among the devices. We need a better understanding of it and interface software to test around all aspects. Here we described some practical examples. The GUI is created using C# language inside the Microsoft Visual Studio. The application has several capabilities. In the TCP/IP mode, It can be a server or client. In RTU mode, it can play as a Master or slave device. We can also run two instances in a single system. To communicate between two running apps in RTU mode, we need virtual loopback software, two physical comm port, or two USB to Serial modules. For Modbus TCP mode, we can test within the system using the localhost address (127.0.0.1) or need an IP address for a different. Findings/results: The robot researcher can find helpful information about communicating the robot through the Modbus protocol. The practical example can help them to create packet purser. The functional CRC algorithm code can be used for better understanding and implementation into their project. Originality/Value: This work has some different features than other available utilities. We added features based on our research needs. Our created application is a little bit different from a professional approach. Various display formats are available in our GUI. That makes a difference in the originality of this work. Our GUI can be master, slave, server, or client, which is rarely available. Paper Type: Experimental-based Research.

Tropane and Related Alkaloid Skeletons via a Radical [3+3]-Annulation Process

A mild and simple protocol for the synthesis of 8-azabicyclo[3.2.1]octane and 9-azabicyclo[3.3.1]nonane derivatives is described. It provides these valuable bicyclic alkaloid skeletons in good yields and high levels of diastereoselectivity from simple and readily available starting materials using visible-light photoredox catalysis. This unprecedented annulation process takes advantage of the unique reactivity of ethyl 2-(acetoxymethyl)acrylate as a 1,3-bis radical acceptor and of cyclic N,N-dialkylanilines as radical 1,3-bis radical donors. The success of this process relies on efficient electron transfer processes and highly selective deprotonation of aminium radical cations leading to the key α-amino radical intermediates.

Tropane and Related Alkaloid Skeletons via a Radical [3+3]-Annulation Process

A mild and simple protocol for the synthesis of 8-azabicyclo[3.2.1]octane and 9-azabicyclo[3.3.1]nonane derivatives is described. It provides these valuable bicyclic alkaloid skeletons in good yields and high levels of diastereoselectivity from simple and readily available starting materials using visible-light photoredox catalysis. This unprecedented annulation process takes advantage of the unique reactivity of ethyl 2-(acetoxymethyl)acrylate as a 1,3-bis radical acceptor and of cyclic N,N-dialkylanilines as radical 1,3-bis radical donors. The success of this process relies on efficient electron transfer processes and highly selective deprotonation of aminium radical cations leading to the key α-amino radical intermediates.