Background Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. Results Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. Conclusion The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

Tripeptides with two consecutive prolines are the shortest and most frequent sequences causing ribosome stalling. The bacterial translation elongation factor P (EF-P) relieves this arrest, allowing protein biosynthesis to continue. A seven amino acids long loop between beta-strands β3/β4 is crucial for EF-P function and modified at its tip by lysylation of lysine or rhamnosylation of arginine. Phylogenetic analyses unveiled an invariant proline in the -2 position of the modification site in EF-Ps that utilize lysine modifications such as Escherichia coli . Bacteria with the arginine modification like Pseudomonas putida on the contrary have selected against it. Focusing on the EF-Ps from these two model organisms we demonstrate the importance of the β3/β4 loop composition for functionalization by chemically distinct modifications. Ultimately, we show that only two amino acid changes in E. coli EF-P are needed for switching the modification strategy from lysylation to rhamnosylation.

The analysis of the function of essential genes in vivo depends on the ability to experimentally modulate levels of their protein products. Current methods to address this are based on transcriptional or post-transcriptional regulation of mRNAs, but approaches based on the exploitation of translation regulation have so far been neglected. Here we describe a toolbox, based on amber suppression in the presence of N-acetyl-l-lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC. The system was used to measure and compare the effects of AcK- and N-(tert-butoxycarbonyl)-l-lysine (BocK) dependent amber suppression in Escherichia coli. We also demonstrate here that, in combination with transcriptional regulation, the system allows protein production to be either totally repressed or gradually adjusted. To identify sequence motifs that provide improved translational regulation, we varied the sequence context of the amber codon and found that insertion of two preceding prolines drastically decreases luminescence. In addition, using LacZ as a reporter, we demonstrated that a strain encoding a variant with a Pro-Pro amber motif can only grow on lactose when AcK is supplied, thus confirming the tight translational regulation of protein output. In parallel, we constructed an E. coli strain that carries an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible version of the AcK-tRNA synthetase (AcKRS) gene on the chromosome, thus preventing mischarging of noncognate substrates. Subsequently, a diaminopimelic acid auxotrophic mutant (ΔdapA) was generated demonstrating the potential of this strain in regulating essential gene products. Furthermore, we assembled a set of vectors based on the broad-host-range pBBR ori that enable the AcK-dependent amber suppression system to control protein output not only in E. coli, but also in Salmonella enterica and Vibrio cholerae.

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Fine PTFE tubes are used in high‐end and high cost devices like flexible Endoscopes where no standard sterilization procedure like autoclaving applies due to material properties. The study of innovative decontamination processes like plasma sterilization request appropriate analysis methods in order to verify the process functionality. In a related research project the IGB‐cycle‐method for contamination and recovery of indicator organisms like Bacillus atrophaeus spores was developed. It was possible to proof the IGB‐cycle‐method to be reproducible and reliable using round robin tests between three laboratories that compared their results in reference to a relevant level of contamination and recovery. Of a relevant contamination more than 7 log10 steps of cfu/specimen could be recovered from the tube surface.

Post-translational modifications (PTM) are the evolutionary solution to challenge and extend the boundaries of genetically predetermined proteomic diversity. As PTMs are highly dynamic, they also hold an enormous regulatory potential. It is therefore not surprising that out of the 20 proteinogenic amino acids, 15 can be post-translationally modified. Even the relatively inert guanidino group of arginine is subject to a multitude of mostly enzyme mediated chemical changes. The resulting alterations can have a major influence on protein function. In this review, we will discuss how bacteria control their cellular processes and develop pathogenicity based on post-translational protein-arginine modifications.

Background There are over 100 known human adenovirus (HAdV) types, which are able to cause a broad variety of different self-limiting but also lethal diseases especially in immunocompromised patients. Only limited information about the pathogenesis and biology of the majority of these virus types is available. In the present study, we performed a systematic screen for coxsackievirus and adenovirus receptor (CAR)-usage of a large spectrum of HAdV types. Methods To study receptor usage we utilized a recombinant HAdV library containing HAdV genomes tagged with a luciferase and GFP encoding transgene. We infected CHO-CAR cells stably expressing the CAR receptor and control cells lacking the CAR receptor with tagged viruses (HAdV3, 14, 16, 50, 10, 24, 27, 37 and 69) and measured luciferase expression levels 26 and for some viruses (AdV10, − 24 and − 27) 52 h post-infection. As positive control, we applied human adenovirus type 5 (HAdV5) known to use the CAR receptor for cell entry. For viruses replication studies on genome level we applied digital PCR. Results Infection of CHO-CAR and CHO-K1 cells at various virus particle numbers per cell (vpc) revealed that HAdV10, 24, and 27 showed similar or decreased luciferase expression levels in the presence of CAR. In contrast, HAdV3, 14, 16, 50, 37 and 69 resulted in increased luciferase expression levels in our initial screening experiments. CAR usage of HAdV3, 14, 50, and 69 was not studied before, and therefore we experimentally confirmed CAR usage for these HAdV as novel viruses utilizing CAR as a receptor. To rule out that replication of HAdV in transduced CHO cells is responsible for increased transduction rates we performed replication assays on virus genome level, which revealed that there is no HAdV replication. Conclusion In the present study, we screened a HAdV library and identified novel human HAdV using the CAR receptor. To our knowledge, this is the first description of CAR usage for HAdV 3, 14, 50, and 69.