Development of a nucleic acid-based lateral flow device as a reliable diagnostic tool for respiratory viral infections.

Viral infections continue to pose a significant threat to the public health, leading to high morbidity and mortality rates worldwide. To combat these challenges, early detection and treatment are essential in reducing hospitalizations and preventing severe complications. Simple, inexpensive, and sensitive diagnostic methods are in constant demand in many areas. In this study, we report the development of a nucleic acid-based lateral flow immunoassay device (NALFIA) and demonstrate its successful application in conjunction with a multiplexed reverse-transcription loop-mediated isothermal amplification assay (LAMP) for the detection of SARS-CoV-2 and influenza. In our approach the NALFIA part preparation is independent of the target, and has the potential to ensure widespread use in diagnostics particularly where testing speed is critical such as in respiratory viral infections.•Simple, inexpensive, sensitive and reliable rapid diagnostic tool.•Target independent design.•Effective use for respiratory samples due to practical sample extraction.

Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon.

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

A functional coating to enhance antibacterial and bioactivity properties of titanium implants and its performance in vitro.

Bacterial infections and lack of osseointegration may negatively affect the success of titanium (Ti) implants. In the present study, a functional coating composed of chitosan (CS) microspheres and nano hydroxyapatite (nHA) was prepared to obtain antimicrobial Ti implants with enhanced bioactivity. First, the chitosan microspheres were fixed to Ti surfaces activated by alkali and heat treatment, then nHA coatings were precipitated onto these surfaces. Ciprofloxacin was loaded into the microspheres using two different procedures; encapsulation and diffusion. Scanning electron microscopy micrographs of the modified Ti surfaces showed that the coating was successfully deposited onto the Ti surfaces and stable for 30 days in PBS. The drug was completely released from free microspheres loaded by encapsulation in 21 days whereas only 89% release was observed after immobilization. The burst release also decreased from ca. 55% to ca. 35%. The release was further reduced following the nHA precipitation. The modified Ti surfaces showed antimicrobial activity based on the bacterial time-kill assay using S. aureus, but the efficiency was affected by both nHA precipitation and drug loading strategy. Highest antimicrobial activity was seen in the samples without nHA layer, and when the drug was loaded by diffusion. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed that nHA on the surface enhanced HA growth in simulated body fluid for 3 weeks, showing increased osseointegration potential. Therefore, the proposed coating may be used to prevent Ti implant failure originated from bacterial infection and/or low bioactivity.

Enterobacter Strains Might Promote Colon Cancer.

Many studies have been performed to determine the interaction between bacterial species and cancer. However, there has been no attempts to demonstrate a possible relationship between Enterobacter spp. and colon cancer so far. Therefore, in the present study, it is aimed to investigate the effects of Enterobacter strains on colon cancer. Bacterial proteins were isolated from 11 Enterobacter spp., one Morganella morganii, and one Escherichia coli strains, and applied onto NCM460 (Incell) and CRL1790 (ATCC) cell lines. Cell viability and proliferation were determined in MTS assay. Flow Cytometry was used to detect CD24 level and apoptosis. Real-Time PCR studies were performed to determine NFKB and Bcl2 expression. Graphpad Software was used for statistical analysis. The results showed that proteins, isolated from the Enterobacter spp., have significantly increased cell viability and proliferation, while decreasing the apoptosis of the cell lines tested. The data in the present study indicated that Enterobacter strains might promote colon cancer. Moreover, Enterobacter spp. could be a clinically important factor for colon cancer initiation and progression. Studies can be extended on animal models in order to develop new strategies for treatment.

In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase.

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 mono-cistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR(+) wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase.

Global regulatory systems operating in Bacilysin biosynthesis in Bacillus subtilis.

In Bacillus subtilis, bacilysin is a nonribosomally synthesized dipeptide antibiotic composed of L-alanine and L-anticapsin. The biosynthesis of bacilysin depends on the bacABCDEywfG operon (bac operon)and the adjacent ywfH gene. To elucidate the effects of global regulatory genes on the expression of bac operon, we used the combination of lacZ fusion analysis and the gel mobility shift assays. The cell density-dependent transition state induction of the bac operon was clearly shown. The basal expression level of the bac operon as well as transition state induction of bac is directly ComA dependent. Three Phr peptides, PhrC, PhrF and PhrK, are required for full-level expression of ComA-dependent bac operon expression, but the most important role seemed to be played by PhrC in stimulating bac expression through a RapC-independent manner. Spo0A is another positive regulator which participates in the transition state induction of bac both directly by interacting with the bac promoter and indirectly by repressing abrB expression. AbrB and CodY proteins do not only directly repress the bac promoter, but they also mutually stimulate the transition state induction of bac indirectly, most likely by antagonizing their repressive effects without preventing each other's binding since both proteins can bind to the bac promoter simultaneously.

The novel gene yvfI in Bacillus subtilis is essential for bacilysin biosynthesis.

Using transposon mutagenesis in Bacillus subtilis PY79, three independent mutants defective in production of bacilysin were isolated. To identify the genes in these mutant loci affecting bacilysin biosynthesis, the inserted transposon and its flanking regions were cloned and sequenced from each mutant. Transposon insertions in these three mutants were found to be in the yvfI gene which encodes an unknown protein similar to GntR family transcriptional regulators. For further confirmation, deletion mutants were constructed in which nucleotides 196-314 of the yvfI gene were removed. All resulting yvfI (Delta196-314)::spc deletion mutants exhibited bacilysin-negative phenotypes, as in the case of the yvfI::Tn10::spc insertional mutants. The lacR gene, encoding a transcriptional regulator, resides immediately downstream from the yvfI gene. Therefore, an insertion mutation was created in the lacR gene to demonstrate that the bacilysin negative phenotype is actually due to the mutation in the yvfI gene and not a polar effect of yvfI mutation on the downstream gene. As expected, all resulting lacR mutant derivatives of PY79 still produced bacilysin.