Synthesized Bis-Triphenyl Phosphonium-Based Nano Vesicles Have Potent and Selective Antibacterial Effects on Several Clinically Relevant Superbugs.

The increasing emergence of multidrug-resistant (MDR) pathogens due to antibiotic misuse translates into obstinate infections with high morbidity and high-cost hospitalizations. To oppose these MDR superbugs, new antimicrobial options are necessary. Although both quaternary ammonium salts (QASs) and phosphonium salts (QPSs) possess antimicrobial effects, QPSs have been studied to a lesser extent. Recently, we successfully reported the bacteriostatic and cytotoxic effects of a triphenyl phosphonium salt against MDR isolates of the Enterococcus and Staphylococcus genera. Here, aiming at finding new antibacterial devices possibly active toward a broader spectrum of clinically relevant bacteria responsible for severe human infections, we synthesized a water-soluble, sterically hindered quaternary phosphonium salt (BPPB). It encompasses two triphenyl phosphonium groups linked by a C12 alkyl chain, thus embodying the characteristics of molecules known as bola-amphiphiles. BPPB was characterized by ATR-FTIR, NMR, and UV spectroscopy, FIA-MS (ESI), elemental analysis, and potentiometric titrations. Optical and DLS analyses evidenced BPPB tendency to self-forming spherical vesicles of 45 nm (DLS) in dilute solution, tending to form larger aggregates in concentrate solution (DLS and optical microscope), having a positive zeta potential (+18 mV). The antibacterial effects of BPPB were, for the first time, assessed against fifty clinical isolates of both Gram-positive and Gram-negative species. Excellent antibacterial effects were observed for all strains tested, involving all the most concerning species included in ESKAPE bacteria. The lowest MICs were 0.250 µg/mL, while the highest ones (32 µg/mL) were observed for MDR Gram-negative metallo-ß-lactamase-producing bacteria and/or species resistant also to colistin, carbapenems, cefiderocol, and therefore intractable with currently available antibiotics. Moreover, when administered to HepG2 human hepatic and Cos-7 monkey kidney cell lines, BPPB showed selectivity indices > 10 for all Gram-positive isolates and for clinically relevant Gram-negative superbugs such as those of E. coli species, thus being very promising for clinical development.

The DRB3 gene of the bovine major histocompatibility complex: discovery, diversity and distribution of alleles in commercial breeds of cattle and applications for development of vaccines.

The bovine Major Histocompatibility Complex (MHC), also known as the Bovine Leucocyte Antigen (BoLA) complex, is the genomic region that encodes the most important molecules for antigen presentation to initiate immune responses. The first evidence of MHC in bovines pointed to a locus containing 2 antigens, one detected by cytotoxic antiserum (MHC class I) and another studied by mixed lymphocyte culture tests (MHC class II). The most studied gene in the BoLA region is the highly polymorphic BoLA-DRB3, which encodes a ß chain with a peptide groove domain involved in antigen presentation for T cells that will develop and co-stimulate cellular and humoral effector responses. BoLA-DRB3 alleles have been associated with outcomes in infectious diseases such as mastitis, trypanosomiasis, and tick loads, and with production traits. To catalog these alleles, 2 nomenclature methods were proposed, and the current use of both systems makes it difficult to list, comprehend and apply these data effectively. In this review we have organized the knowledge available in all of the reports on the frequencies of BoLA-DRB3 alleles. It covers information from studies made in at least 26 countries on more than 30 breeds; studies are lacking in countries that are important producers of cattle livestock. We highlight practical applications of BoLA studies for identification of markers associated with resistance to infectious and parasitic diseases, increased production traits and T cell epitope mapping, in addition to genetic diversity and conservation studies of commercial and creole and locally adapted breeds. Finally, we provide support for the need of studies to discover new BoLA alleles and uncover unknown roles of this locus in production traits.

Nano-lamellar H-ZSM-5 molecular sieves for enhanced indoor air purification: Synthesis and adsorption characterization.

BACKGROUND: The rapid growth of cities has been accompanied by problems with urban air quality, making air pollution challenging to manage. In this situation, people focus on indoor building materials to improve air quality. OBJECTIVE: In this paper, a novel bola-type surfactant was synthesized and used as a template, using ethyl orthosilicate and sodium meta-aluminate as the silicon and aluminum source, in the ratio of n(NaOH): n(NaAIO2): n(SiO2): n(SDA): n(H2O) as 30:2.5:120:5:4800. METHODS: Hydrothermal preparation of ZSM-5 molecular sieves with a nanosheet structure (H-ZSM-5) was accomplished. The manufactured lamellar ZSM-5 molecular sieves were examined using X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and adsorption and desorption techniques. RESULTS: Traditional microporous ZSM-5 had a considerably lower static adsorption of formaldehyde molecules. The findings demonstrated that the nano-lamellar H-ZSM-5 molecular sieve can purify and eliminate larger molecular VOCs inside because it has the ability to adsorb larger molecular diameter VOCs. Additionally, the effectiveness of the adsorption was assessed using toluene vapour molecules with higher molecular diameters. CONCLUSION: The findings demonstrate that the nanosheet H-ZSM-5 molecular sieve can remove bigger molecule VOCs from indoor air and can be utilised to purify indoor spaces. This study offers a fresh approach to indoor environmental cleanup by demonstrating the capability of nano-lamellar H-ZSM-5 molecular sieves for molecular adsorption.

Revision of the sophorolipid biosynthetic pathway in Starmerella bombicola based on new insights in the substrate profile of its lactone esterase.

BACKGROUND: Sophorolipids (SLs) are a class of natural, biodegradable surfactants that found their way as ingredients for environment friendly cleaning products, cosmetics and nanotechnological applications. Large-scale production relies on fermentations using the yeast Starmerella bombicola that naturally produces high titers of SLs from renewable resources. The resulting product is typically an extracellular mixture of acidic and lactonic congeners. Previously, we identified an esterase, termed Starmerella bombicola lactone esterase (SBLE), believed to act as an extracellular reverse lactonase to directly use acidic SLs as substrate. RESULTS: We here show based on newly available pure substrates, HPLC and mass spectrometric analysis, that the actual substrates of SBLE are in fact bola SLs, revealing that SBLE actually catalyzes an intramolecular transesterification reaction. Bola SLs contain a second sophorose attached to the fatty acyl group that acts as a leaving group during lactonization. CONCLUSIONS: The biosynthetic function by which the Starmerella bombicola 'lactone esterase' converts acidic SLs into lactonic SLs should be revised to a 'transesterase' where bola SL are the true intermediate. This insights paves the way for alternative engineering strategies to develop designer surfactants.

The contribution of Alois Alzheimer and Oskar Fischer to the understanding of Pick's complex - (Historical Note) / A contribuição de Alois Alzheimer e Oskar Fischer para a compreensão do complexo de Pick - (Nota Histórica)

Arnold Pick described a series of cases with progressive aphasia, behavioural disorders, and dementia. The post-mortem examination revealed on macroscopy, beside diffuse brain atrophy, also circumscribed (lobar) atrophy of the temporal and/or frontal lobes. The histopathology was not provided. Such kind of cases were soon named after the author, being known for a time as 'Pick's disease', coming to constitute a new nosological group. A time later after the original description, Alois Alzheimer and Oskar Fischer completed microscopic examination of similar cases, where the first author found, on silver impregnation, spheric neuronal inclusions, he named 'argentophilic ball' inclusions, while the second one identified complex cortical changes he named 'spongiform cortical wasting', and additionally a type of swollen cell that was named 'ballooned neuron'. Such microscopic changes became the first histopathological markers of this group of diseases.

Arnold Pick descreveu uma série de casos apresentando, de modo progressivo, afasia, transtornos de comportamento e demência. O exame pós-morte revelou à macroscopia, além de atrofia cerebral difusa, também atrofia circunscrita (lobar) dos lobos temporais e/ou frontais. A histopatologia não foi fornecida. Tal tipo de casos foi logo denominado segundo o autor, sendo conhecido por um período como 'doença de Pick', vindo a constituir um novo grupo nosológico. Algum tempo após a discrição original, Alois Alzheimer e Oskar Fischer perfizeram exame microscópio de casos semelhantes, onde o primeiro autor encontrou inclusões neuronais esféricas à impregnação pela prata, que denominou de 'bola argirofílica', enquanto o segundo identificou alterações corticais complexas às quais denominou 'perda cortical espongiforme', além de um tipo de célula tumefeita que chamou de 'neurônio balonizado'. Tais alterações microscópicas tornaram-se os primeiros marcadores histopatológicos desse grupo de doenças.

First characterization of major histocompatibility complex class II DRB3 diversity in cattle breeds raised in Egypt.

Genes encoding bovine leukocyte antigen (BoLA) enable the immune system to identify pathogens. Therefore, these genes have been used as genetic markers for infectious and autoimmune diseases as well as for immunological traits in cattle. Although BoLA polymorphisms have been reported in various cattle breeds worldwide, they have not been studied in cattle populations in Egypt. In this study, we characterized BoLA-DRB3 in two local Egyptian populations and one foreign population using polymerase chain reaction-sequence-based typing (PCR-SBT) method. Fifty-four previously reported BoLA-DRB3 alleles and eight new alleles (BoLA-DRB3*005:08, *015:07, *016:03, *017:04, *020:02:02, *021:03, *164:01, and *165:01) were identified. Alignment analysis of the eight new alleles revealed 90.7-98.9 %, and 83.1-97.8 % nucleotide and amino acid identities, respectively, with the BoLA-DRB3 cDNA clone NR-1. Interestingly, BoLA-DRB3 in Egyptian cattle showed a high degree of allelic diversity in native (na = 28, hE > 0.95), mixed (na = 61, hE > 0.96), and Holstein (na = 18, hE > 0.88) populations. BoLA-DRB3*002:01 (14.3 %), BoLA-DRB3*001:01 (8.5 %), and BoLA-DRB3*015:01 (20.2 %) were the most frequent alleles in native, mixed, and Holstein populations, respectively, indicating that the genetic profiles differed in each population. Based on the allele frequencies of BoLA-DRB3, genetic variation among Egyptian, Asian, African, and American breeds was examined using Nei's distances and principal component analysis. The results suggested that native and mixed cattle populations were most closely associated with African breeds in terms of their gene pool, whereas Holstein cattle were more distinct from the other breeds and were closely related to Holstein cattle populations from other countries.

ROS-Responsive Bola-Lipid Nanoparticles as a Codelivery System for Gene/Photodynamic Combination Therapy.

The nonviral delivery systems that combine genes with photosensitizers for multimodal tumor gene/photodynamic therapy (PDT) have attracted much attention. In this study, a series of ROS-sensitive cationic bola-lipids were applied for the gene/photosensitizer codelivery. Zn-DPA was introduced as a cationic headgroup to enhance DNA binding, while the hydrophobic linking chains may facilitate the formation of lipid nanoparticles (LNP) and the encapsulation of photosensitizer Ce6. The length of the hydrophobic chain played an important role in the gene transfection process, and 14-TDZn containing the longest chains showed better DNA condensation, gene transfection, and cellular uptake. 14-TDZn LNPs could well load photosensitizer Ce6 to form 14-TDC without a loss of gene delivery efficiency. 14-TDC was used for codelivery of p53 and Ce6 to achieve enhanced therapeutic effects on the tumor cell proliferation inhibition and apoptosis. Results showed that the codelivery system was more effective in the inhibition of tumor cell proliferation than individual p53 or Ce6 monotherapy. Mechanism studies showed that the production of ROS after Ce6 irradiation could increase the accumulation of p53 protein in tumor cells, thereby promoting caspase-3 activation and inducing apoptosis, indicating some synergistic effect. These results demonstrated that 14-TDC may serve as a promising nanocarrier for gene/PDT combination therapy.

Renal fungus balls in neonates and very young infants treated with local amphotericin B deoxycholate: Two case reports and review of the literature.

INTRODUCTION: Fungal urinary tract infections predominantly affect the critically ill premature infant and those with urogenital tract abnormalities. Fungal balls are an uncommon complication which require prompt detection and treatment to prevent morbidity and mortality. The evidence on the management of fungus balls in young infants with Candida urinary tract infections is very scarce. METHODS: Case reports and review of the literature. RESULTS: We report two immunocompetent young infants with urogenital abnormalities that received local amphotericin B deoxycholate, and systemic therapy, for the treatment and prevention of Candida urinary tract infection-associated fungus balls. We identified 21 similar cases in the literature, with very limited data about drug compounding, optimal dosages, dwell times and length of treatment. Different management strategies are discussed. CONCLUSIONS: Amphotericin B deoxycholate local irrigations were safe and effective for the therapeutic management and prophylaxis of Candida fungus balls in young infants, in combination with systemic antifungal therapy.

BolA promotes the generation of multicellular behavior in S. Typhimurium by regulating the c-di-GMP pathway genes yeaJ and yhjH.

The generation of multicellular behavior enhances the stress adaptability, antibiotic resistance, and pathogenic potential of Salmonella enterica serovar Typhimurium (S. Typhimurium), which is challenging for its prevention and control. Therefore, determination of the mechanism of multicellular behavior development is urgently required. Accordingly, this study investigated BolA, a transcription factor that promotes bacterial survival under different stresses. We found that BolA promoted the generation of multicellular behavior. Furthermore, transcriptome analysis revealed that BolA affected the expression of numerous genes, including biofilm formation and motility-related genes. In terms of biofilm formation, compared with the wild-type strain, bolA overexpression (269BolA+) increased the extracellular matrix content (extracellular polysaccharide, extracellular protein, and extracellular DNA (eDNA) by upregulating gene expression, ultimately increasing the biofilm formation ability by 2.56 times. For motility, bolA overexpression inhibited the expression of flagella synthesis genes, resulting in a 91.15 % decrease in motility compared with the wild-type (6 h). Further mechanistic analysis demonstrated that BolA affected the expression of the C-di-GMP pathway genes yeaJ and yhjH, which influenced the generation of multicellular behavior. In terms of biofilms, the extracellular polysaccharide content of 269BolA + ∆Yeaj (bolA overexpression and yeaJ deletion) was reduced by 89.91 % compared with 269BolA+, resulting in a 71.1 % reduction in biofilm forming ability. The motility of the 269∆BolA∆Yhjh (bolA/yhjH double deletion) strain was significantly decreased compared with that of 269∆BolA. Finally, the LacZ gene reporting showed that BolA promoted and inhibited the expression of yeaJ and yhjH, respectively. In conclusion, BolA mainly improves the content of extracellular polysaccharide by promoting the expression of yeaJ, thus enhancing the formation of biofilms. BolA also restricts flagellar synthesis by inhibiting yhjH expression, therefore reducing motility, ultimately promoting multicellular behavior arises. These findings lay a theoretical foundation for the prevention and control of S. Typhimurium.

BoLA-DRB3 Polymorphism Associated with Bovine Leukemia Virus Infection and Proviral Load in Holstein Cattle in Egypt.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most prevalent neoplastic disease of cattle worldwide. The immune response to BLV and disease susceptibility and resistance in cattle are strongly correlated with the bovine leukocyte antigen (BoLA)-DRB3 allelic polymorphism. BLV infection continues to spread in Egypt, in part because the relationships between BLV infection, proviral load in Egypt, and BoLA-DRB3 polymorphism are unknown. Here, we identified 18 previously reported alleles in 121 Holstein cows using a polymerase chain reaction sequence-based typing method. Furthermore, BoLA-DRB3 gene polymorphisms in these animals were investigated for their influence on viral infection. BoLA-DRB3*015:01 and BoLA-DRB3*010:01 were identified as susceptible and resistant alleles, respectively, for BLV infection in the tested Holsteins. In addition, BoLA-DRB3*012:01 was associated with low PVL in previous reports but high PVL in Holstein cattle in Egypt. This study is the first to demonstrate that the BoLA-DRB3 polymorphism confers resistance and susceptibility to PVL and infections of BLV in Holstein cattle in Egypt. Our results can be useful for the disease control and eradication of BLV through genetic selection.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal.

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

DNA supercoiling and regulation of intrinsic ß-lactamase in pathogenic Escherichia coli.

This review addresses the involvement of DNA supercoiling in the development of virulence and antibiotic profiles for uropathogenic Escherichia coli and the emergence of new pathotypes such as strain ST131 (serotype O25:H4). The mechanism suggests a role for topoisomerase enzymes and associated mutations in altering the chromosomal supercoiling state and introducing the required DNA twists for expression of intrinsic ß-lactamase by ampC and certain virulence factors. In Escherichia coli, constitutive hyperexpression of intrinsic ampC is associated with specific mutations in the promoter and attenuator regions. However, many reports have documented the involvement of slow growth interventions in the expression of intrinsic resistance determinants. There is evidence that a stationary phase transcriptional switch protein, "BolA," is involved in the expression of the intrinsic ampC gene under starvation conditions. The process involves changes in the activity of the enzyme "gyrase," which leads to a change in the chromosomal DNA topology. Consequently, the DNA is relaxed, and the expression of the bolA gene is upregulated. The evolution of the extraintestinal pathogenic E. coli strain ST131 has demonstrated successful adaptability to various stress conditions and conferred compensatory mutations that endowed the microbe with resistance to fluoroquinolones and ß-lactams. The results of this study provided new insights into the evidence for the influence of DNA topology in the expression of virulence genes and various determinants of antibiotic resistance (e.g., the intrinsic ampC gene) in Escherichia coli pathotypes.

Low proviral load in the Kumamoto strain of Japanese Brown cattle infected with the bovine leukemia virus.

BACKGROUND: The Kumamoto strain of Japanese Brown (JBRK) cattle is a sub-breed of Wagyu and has a different genetic background than that of Japanese Black (JB) cattle. Bovine leukemia virus (BLV) is the pathogen causing enzootic bovine leukosis (EBL), the predominant type of bovine leukosis (BL). EBL is one of the most common bovine infectious diseases in dairy countries, including Japan. Some host genetic factors, including the bovine leukocyte antigen (BoLA)-DRB3 gene, have been associated with the proviral load (PVL) of BLV and/or onset of EBL. Here, we determined the number of BL cases by analyzing prefectural case records in detail. We measured the PVL of BLV-infected JBRK cattle and compared it with that obtained for other major breeds, JB and Holstein-Friesian (HF) cattle. Finally, the relationship between PVL levels and BoLA-DRB3 haplotypes was investigated in BLV-infected JBRK cattle. RESULTS: We determined the number of BL cases recorded over the past ten years in Kumamoto Prefecture by cattle breed. A limited number of BL cases was observed in JBRK cattle. The proportion of BL cases in the JBRK was lower than that in JB and HF. The PVL was significantly lower in BLV-infected JBRK cattle than that in the JB and HF breeds. Finally, in BLV-infected JBRK cattle, the PVL was not significantly affected by BoLA-DRB3 alleles and haplotypes. BoLA-DRB3 allelic frequency did not differ between BLV-infected JBRK cattle with low PVL and high PVL. CONCLUSIONS: To our knowledge, this is the first report showing that BL occurred less in the JBRK population of Kumamoto Prefecture. After BLV-infection, the PVL was significantly lower in JBRK cattle than that in JB and HF breeds. The genetic factors implicated in maintaining a low PVL have yet to be elucidated, but the BoLA-DRB3 haplotypes are likely not involved.

Synthesis of a photocleavable bola-phosphatidylcholine.

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Regulation of 2,4-diacetylphloroglucinol biosynthesis and biocontrol capacity by the BolA family protein IbaG in Pseudomonas fluorescens 2P24.

The monothiol glutaredoxin GrxD plays an essential role in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and the biocontrol capacity of the soil bacterium Pseudomonas fluorescens 2P24. However, the detailed mechanism underlying GrxD-mediated activation of the production of 2,4-DAPG remains unclear. Here, we found that GrxD directly interacted with IbaG, a BolA protein family member. The mutation of ibaG significantly decreased 2,4-DAPG production. Furthermore, expressing ibaG restored the production of 2,4-DAPG in the grxD ibaG double mutant to wild-type levels in the presence of dithiothreitol, suggesting that IbaG was required for GrxD-mediated regulation of 2,4-DAPG production. Transcriptome sequencing analyses revealed that IbaG plays a global role in gene regulation by affecting the expression of numerous genes throughout the genome. We also demonstrated that IbaG is an important regulator of several cellular processes, including swarming motility, biofilm formation, siderophore production, and acid resistance. Altogether, our data suggest that IbaG has an essential role in 2,4-DAPG production, motility, and biofilm formation. We also propose a regulatory mechanism linking GrxD to 2,4-DAPG production via IbaG. IMPORTANCE The production of 2,4-diacetylphloroglucinol (2,4-DAPG) is positively influenced by the monothiol glutaredoxin GrxD in Pseudomonas fluorescens 2P24. However, the regulatory mechanism underlying GrxD-mediated regulation of 2,4-DAPG biosynthesis is mostly uncharacterized. Here, we show the function of the BolA-like protein IbaG in 2,4-DAPG biosynthesis. We also demonstrate that GrxD directly interacts with IbaG and influences the redox state of IbaG. Altogether, this work provides new insights into the role of the highly conserved IbaG protein in regulating 2,4-DAPG synthesis, biofilm formation, and other biocontrol traits of P. fluorescens.

(1-(4-(5-Phenyl-1,3,4-oxadiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)-methylenyls α,ω-Bisfunctionalized 3- and 4-PEG: Synthesis and Photophysical Studies.

Two new azaheterocycle-based bolas, such as (1-(4-(5-phenyl-1,3,4-oxadiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)-methylenyls α,ω-bisfunctionalized PEGs, were prepared via Cu-catalyzed click reaction between 2-(4-azidophenyl)-5-(aryl)-oxadiazole-1,3,4 and terminal ethynyls derived from PEG-3 and PEG-4. Due to the presence of two heteroaromatic cores and a PEG linker, these bola molecules are considered as promising fluorescent chemosensors for electron-deficient species. As a result of a well-pronounced "turn-off" fluorescence response towards common nitro-explosive components, such as 2,4-dinitrotoluene (DNT) and 2,4,6-trinitrotoluene (TNT), hard-to-detect pentaerythritol tetranitrate (PETN), as well as Hg2+ cation was observed.

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3.

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.

BoLA-DRB3*15:01 allele is associated with susceptibility to early enzootic bovine leukosis onset in Holstein-Friesian and Japanese Black cattle.

Enzootic bovine leukosis (EBL) is typically observed in cattle older than 3 years, but some cases of onset in cattle younger than 3 years have been reported in Japan. BoLA-DRB3 polymorphisms are associated with susceptibility to EBL onset. However, little is known about the relationship between the polymorphisms and EBL onset in young cattle. In the present study, we performed BoLA-DRB3 genotyping in 59 EBL cattle younger than 3 years (25 Holstein-Friesian and 34 Japanese Black) and compared the results with those of 69 EBL cattle older than 3 years (38 Holstein-Friesian and 31 Japanese Black). The BoLA-DRB3*15:01 allele was detected at a frequency of 37.3 % (48.0 % and 29.4 % in Holstein-Friesian and Japanese Black, respectively) and was identified as an early EBL onset susceptibility allele. Nine EBL cattle younger than 3 years (5 Holstein-Friesian and 4 Japanese Black), but only 1 EBL cattle older than 3 years (1 Holstein-Friesian), had a BoLA-DRB3*15:01/*15:01 homozygous genotype. The frequency of the BoLA-DRB3*15:01 allele occurring with a different allele (BoLA-DRB3*015:01/other) in cattle younger than 3 years was 44.1 % (56.0 % Holstein-Friesian and 35.3 % Japanese Black) and significantly higher than that in cattle older than 3 years (28.9 % Holstein-Friesian and 9.7 % Japanese Black) (P = 0.0013). These results suggest that BoLA-DRB3*15:01/*15:01 and BoLA-DRB3*15:01/other genotypes are early EBL onset susceptibility genotypes. The present findings may contribute to cattle breeding selection.

BolA affects the biofilm formation ability, outer membrane permeability and virulence, thus is required for the adaptability of Salmonella enterica serotype Typhimurium to the harsh survival environment.

Salmonella enterica serotype Typhimurium, an important foodborne pathogen with high adaptability to the host's internal and external survival environment, seriously threatens public health. Therefore, to understand the mechanism underlying the high adaptability, this study investigated the transcription factor BolA by constructing BolA deletion strain 269△BolA, complemented strain 269BolAR and overexpression strain 269BolA+ based on WT269. BolA significantly inhibited motility; at 6 h, the BolA overexpression strain (269BolA+) showed 91.2% and 90.7% lower motility than the wild type (WT269) and BolA deletion strain (269△BolA), respectively, by downregulating motility-related flagellar genes. BolA promoted biofilm formation; 269BolA+ showed 3.6-fold and 5.2-fold higher biofilm formation ability than WT269 and 269ΔBolA, respectively, by upregulation biofilm formation-related genes. BolA overexpression downregulated the outer membrane gene OmpF and upregulated OmpC, thereby regulating cell permeability, and reducing the antibacterial effect of vancomycin, which can destruct the outer membrane. BolA improved adaptability; 269△BolA showed higher susceptibility to eight antibiotics and 2.5- and 4-fold lower acid and oxidative stress tolerance, respectively, than WT269. In Caco-2 and HeLa cells, 269△BolA showed 2.8- and 3-fold lower cell adhesion ability, respectively, and 4- and 2-fold lower cell invasion ability, respectively, than WT269, through downregulation of the virulence genes. Thus, BolA expression promotes biofilm formation and balances the membrane permeability, thereby improving the resistance of the strains, and enhances its host cell invasion ability by upregulating bacterial virulence factors. Results of this study suggest that the BolA gene may serve as a potential target of therapeutic or preventative strategies to control Salmonella Typhimurium infections.

Reduction of biofilm formation of Escherichia coli by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter.

BACKGROUND: Escherichia coli is a common cause of biofilm-associated urinary tract infections (UTIs). Biofilm formation in E. coli is responsible for various indwelling medical device-associated infections, including catheter-associated urinary tract infections (CAUTIs). This study aimed to reduce biofilm formation of E. coli ATCC 25922 by knocking out genes involved in quorum sensing (QS) (luxS) and adhesion (fimH and bolA) using the CRISPR/Cas9-HDR approach. METHOD: Single-guide RNAs (sgRNAs) were designed to target luxS, fimH and bolA genes. Donor DNA for homologous recombination was constructed to provide accurate repairs of double-strand breaks (DSBs). A biofilm quantification assay (crystal violet assay) was performed to quantify the biofilm formation of mutant and wild-type strains. Morphological changes in biofilm architecture were confirmed by scanning electron microscopy (SEM). Further application of the biofilm formation of mutant and wild-type strains on urinary catheter was tested. RESULTS: Crystal violet assay showed that the biofilm formation of ΔfimH, ΔluxS, and ΔbolA strains was significantly reduced compared to the wild-type strain (P value<0.001). The percentage of biofilm reduction of mutant strains was as follows: ΔluxS1 77.51 %, ΔfimH1 78.37 %, ΔfimH2 84.17 %, ΔbolA1 78.24 %, and ΔbolA2 75.39 %. Microscopic analysis showed that all mutant strains lack extracellular polymeric substances (EPS) production compared to the wild-type strain, which was embedded in its EPS matrix. The adherence, cell aggregation, and biofilm formation of wild-type strain on urinary catheters were significantly higher compared to ΔfimH, ΔluxS and ΔbolA strains. CONCLUSION: Altogether, our results demonstrated that the knockout of luxS, fimH, and bolA genes reduced EPS matrix production, which is considered the main factor in the development, maturation, and maintenance of the integrity of biofilm. This pathway could be a potential strategy to disrupt E. coli biofilm-associated UTIs. This study suggests that CRISPR/Cas9-HDR system may provide an efficient and site-specific gene editing approach that exhibits a possible antibiofilm strategy through intervention with the QS mechanism and adhesion property to suppress biofilm formation associated with UTI catheter infections.