A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels.

Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge.

In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry.

Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors.

Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

Background: Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B.

Combining CRISPR-Cas12a with terminal deoxynucleotidyl transferase dependent reporter elongation for pathogen detection using lateral flow test strips.

CRISPR-Cas (CC)-based detection technologies have some exceptional features, which hold the promise of developing into the next-generation diagnostic platforms. One of these features is the ability to trigger non-specific single-stranded DNA/RNA cleavage activity after specific target recognition and Cas enzyme activation. This cleavage activity can be visualized either by single-stranded DNA/RNA fluorescence resonance energy transfer quenching reporters or via lateral flow strips, which separate and detect the cleaved reporters.

Phylogenetic analysis of the bacterial Pro-Pro-endopeptidase domain reveals a diverse family including secreted and membrane anchored proteins.

Pro-Pro-endopeptidases (PPEP, EC 3.4.24.

Immobilized Regenerable Active Chlorine within a Zirconium-Based MOF Textile Composite to Eliminate Biological and Chemical Threats.

The most recent global health crisis caused by the SARS-CoV-2 outbreak and the alarming use of chemical warfare agents highlight the necessity to produce efficient protective clothing and masks against biohazard and chemical threats. However, the development of a multifunctional protective textile is still behind to supply adequate protection for the public. To tackle this challenge, we designed multifunctional and regenerable N-chlorine based biocidal and detoxifying textiles using a robust zirconium metal-organic framework (MOF), UiO-66-NH, as a chlorine carrier which can be easily coated on textile fibers.

Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles.

Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specific functions of two enzymes, CRISPR-Cas12a and terminal deoxynucleotidyl transferase (TdT), we developed a DNA detection assay that generates copper nanoparticles (CuNPs) that are easily visible to the naked eye under UV-light; we named this detection assay Cas12a Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP).

Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks.

To improve the preparedness against exposure to highly pathogenic bacteria and to anticipate the wide variety of bacteria that can cause bloodstream infections (BSIs), a safe, unbiased and highly accurate identification method was developed. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method can identify highly pathogenic bacteria, their near-neighbors and bacteria that are common causes of BSIs directly from positive blood culture flasks. The developed Peptide-Based Microbe Detection Engine (http://proteome2pathogen.

Data evidencing slow anaerobic digestion in emergency treatment and disposal of infectious animal carcasses.

Burial of infectious and potentially infectious livestock and poultry animals is the most common response to an emergency situation. The data set summarizes 22-week-long experiment that simulates the environment found within conventional burial trenches for emergency disposal of animal carcasses, worldwide, sometimes with a topical application of quicklime as it is required in the Republic of Korea. This data set shows the rarely presented evidence of the extremely slow decay of animal carcasses.

Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacter xylosoxidans.

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans.

Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic insights into the differences in substrate specificity within the PPEP family.

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen (PPEP-1) cleaves two cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family.

Lab-scale evaluation of aerated burial concept for treatment and emergency disposal of infectious animal carcasses.

Nearly 55,000 outbreaks of animal disease were reported to the World Animal Health Information Database between 2005 and 2016. To suppress the spread of disease, large numbers of animal mortalities often must be disposed of quickly and are frequently buried on the farm where they were raised. While this method of emergency disposal is fast and relatively inexpensive, it also can have undesirable and lasting impacts (slow decay, concerns about groundwater contamination, pathogens re-emergence, and odor).

Covalent attachment and Pro-Pro endopeptidase (PPEP-1)-mediated release of Clostridium difficile cell surface proteins involved in adhesion.

In the past decade, Clostridium difficile has emerged as an important gut pathogen. This anaerobic, Gram-positive bacterium is the main cause of infectious nosocomial diarrhea. Whereas much is known about the mechanism through which the C.

Method for sampling and analysis of volatile biomarkers in process gas from aerobic digestion of poultry carcasses using time-weighted average SPME and GC-MS.

A passive sampling method, using retracted solid-phase microextraction (SPME) - gas chromatography-mass spectrometry and time-weighted averaging, was developed and validated for tracking marker volatile organic compounds (VOCs) emitted during aerobic digestion of biohazardous animal tissue. The retracted SPME configuration protects the fragile fiber from buffeting by the process gas stream, and it requires less equipment and is potentially more biosecure than conventional active sampling methods. VOC concentrations predicted via a model based on Fick's first law of diffusion were within 6.

Efficacy of NH3 as a secondary barrier treatment for inactivation of Salmonella Typhimurium and methicillin-resistant Staphylococcus aureus in digestate of animal carcasses: Proof-of-concept.

Managing the disposal of infectious animal carcasses from routine and catastrophic disease outbreaks is a global concern. Recent research suggests that burial in lined and aerated trenches provides the rapid pathogen containment provided by burial, while reducing air and water pollution potential and the length of time that land is taken out of agricultural production. Survival of pathogens in the digestate remains a concern, however.

Typing Pseudomonas aeruginosa Isolates with Ultrahigh Resolution MALDI-FTICR Mass Spectrometry.

The introduction of standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms in the medical microbiological practice has revolutionized the way microbial species identification is performed on a daily basis. To a large extent, this is due to the ease of operation. Acquired spectra are compared to profiles obtained from cultured colonies present in a reference spectra database.

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix.

Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831.

The Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro-Pro endopeptidase (PPEP-1). PPEP-1 cleaves C. difficile cell surface proteins (e.

Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation.

Covalent attachment of surface proteins to the cell wall of Gram-positive bacteria requires a sortase-mediated transpeptidation reaction. In almost all Gram-positive bacteria, the housekeeping sortase, sortase A, recognizes the canonical recognition sequence LPXTG (X=any amino acid). The human pathogen Clostridium difficile carries a single putative sortase gene (cd2718) but neither transpeptidation activity nor specificity of CD2718 has been investigated.

Capillary-electrophoresis mass spectrometry for the detection of carbapenemases in (multi-)drug-resistant Gram-negative bacteria.

In a time in which the spread of multidrug resistant microorganisms is ever increasing, there is a need for fast and unequivocal identification of suspect organisms to supplement existing techniques in the clinical laboratory, especially in single bacterial colonies. Mass-spectrometry coupled with efficient peptide separation techniques offer great potential for identification of resistant-related proteins in complex microbiological samples in an unbiased manner. Here, we developed a capillary electrophoresis-electrospray ionization-tandem mass spectrometry CE-ESI-MS/MS bottom-up proteomics workflow for sensitive and specific peptide analysis with the emphasis on the identification of β-lactamases (carbapenemases OXA-48 and KPC in particular) in bacterial species.

Hydroxyl radical formation in batch and continuous flow ultrasonic systems.

The creation of free radicals by ultrasonic cavitation is the main mechanism that leads to chemical degradation of target pollutants and the process is considered an alternative advanced oxidation technology. The goal of this study was to compare the effects of batch and continuous flow ultrasonic systems on the formation of hydroxyl radicals. Ultrasonic batch experiments were conducted in two reactors (small and large) using a standard 20kHz catenoidal titanium horn at varying amplitudes and sonication times.

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars.

Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile.

Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging.

A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.

Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease.