The Remarkable and Selective In Vitro Cytotoxicity of Synthesized Bola-Amphiphilic Nanovesicles on Etoposide-Sensitive and -Resistant Neuroblastoma Cells.

Neuroblastoma (NB) is a solid tumor occurring in infancy and childhood. Its high-risk form has currently a survival rate <50%, despite aggressive treatments. This worrying scenario is worsened by drug-induced secondary tumorigenesis and the emergency of drug resistance, calling for the urgent development of new extra-genomic treatments. Triphenyl phosphonium salts (TPPs) are mitochondria-targeting compounds that exert anticancer effects, impair mitochondria functions, and damage DNA at the same time. Despite several biochemical applications, TPP-based bola-amphiphiles self-assembling nanoparticles (NPs) in water have never been tested as antitumor agents. Here, with the aim of developing new antitumor devices to also counteract resistant forms of HR-NB, the anticancer effects of a TPP-based bola-amphiphile molecule have been investigated in vitro for the first time. To this end, we considered the previously synthesized and characterized sterically hindered quaternary phosphonium salt (BPPB). It embodies both the characteristics of mitochondria-targeting compounds and those of bola-amphiphiles. The anticancer effects of BPPB were assessed against HTLA-230 human stage-IV NB cells and their counterpart, which is resistant to etoposide (ETO), doxorubicin (DOX), and many other therapeutics (HTLA-ER). Very low IC50 values of 0.2 µM on HTLA-230 and 1.1 µM on HTLA-ER (538-fold lower than that of ETO) were already determined after 24 h of treatment. The very low cell viability observed after 24 h did not significantly differ from that observed for the longest exposure timing. The putative future inclusion of BPPB in a chemotherapeutic cocktail for HR-NB was assessed by investigating in vitro its cytotoxic effects against mammalian cell lines. These included monkey kidney cells (Cos-7, IC50 = 4.9 µM), human hepatic cells (HepG2, IC50 = 9.6 µM), a lung-derived fibroblast cell line (MRC-5, IC50 = 2.8 µM), and red blood cells (RBCs, IC50 = 14.9 µM). Appreciable to very high selectivity indexes (SIs) have been determined after 24 h treatments (SIs = 2.5-74.6), which provided evidence that both NB cell populations were already fully exterminated. These in vitro results pave the way for future investigations of BPPB on animal models and upon confirmation for the possible development of BPPB as a novel therapeutic to treat MDR HR-NB cells.

Strongly ROS-Correlated, Time-Dependent, and Selective Antiproliferative Effects of Synthesized Nano Vesicles on BRAF Mutant Melanoma Cells and Their Hyaluronic Acid-Based Hydrogel Formulation.

Cutaneous metastatic melanoma (CMM) is the most aggressive form of skin cancer with a poor prognosis. Drug-induced secondary tumorigenesis and the emergency of drug resistance worsen an already worrying scenario, thus rendering urgent the development of new treatments not dealing with mutable cellular processes. Triphenyl phosphonium salts (TPPSs), in addiction to acting as cytoplasmic membrane disruptors, are reported to be mitochondria-targeting compounds, exerting anticancer effects mainly by damaging their membranes and causing depolarization, impairing mitochondria functions and their DNA, triggering oxidative stress (OS), and priming primarily apoptotic cell death. TPP-based bola amphiphiles are capable of self-forming nanoparticles (NPs) with enhanced biological properties, as commonly observed for nanomaterials. Already employed in several other biomedical applications, the per se selective potent antibacterial effects of a TPP bola amphiphile have only recently been demonstrated on 50 multidrug resistant (MDR) clinical superbugs, as well as its exceptional and selective anticancer properties on sensitive and MDR neuroblastoma cells. Here, aiming at finding new molecules possibly developable as new treatments for counteracting CMM, the effects of this TPP-based bola amphiphile (BPPB) have been investigated against two BRAF mutants CMM cell lines (MeOV and MeTRAV) with excellent results (even IC50 = 49 nM on MeOV after 72 h treatment). With these findings and considering the low cytotoxicity of BPPB against different mammalian non-tumoral cell lines and red blood cells (RBCs, selectivity indexes up to 299 on MeOV after 72 h treatment), the possible future development of BPPB as topical treatment for CMM lesions was presumed. With this aim, a biodegradable hyaluronic acid (HA)-based hydrogel formulation (HA-BPPB-HG) was prepared without using any potentially toxic crosslinking agents simply by dispersing suitable amounts of the two ingredients in water and sonicating under gentle heating. HA-BPPB-HA was completely characterized, with promising outcomes such as high swelling capability, high porosity, and viscous elastic rheological behavior.

Microbiological Evaluation of Two Mexican Artisanal Cheeses: Analysis of Foodborne Pathogenic Bacteria in Cotija Cheese and Bola de Ocosingo Cheese by qPCR.

Cotija and Bola de Ocosingo are artisanal ripened cheeses produced in Mexico. Both are made with raw bovine milk from free-grazing cows and with no starter cultures. Unlike culture-based techniques, molecular methods for pathogen detection in food allow a shorter turnaround time, higher detection specificity, and represent a lower microbiological risk for the analyst. In the present investigation, we analyzed 111 cheese samples (95 Cotija and 16 Bola de Ocosingo) by qPCR (TaqMan®) after an enrichment-culture step specific to each foodborne bacterium. The results showed that 100% of the samples were free of DNA from Listeria monocytogenes, Brucella spp., Escherichia coli enterotoxigenic (ETEC), and O157:H7; 9% amplified Salmonella spp. DNA; and 11.7%, Staphylococcus aureus DNA. However, the threshold cycle (Ct) values of the amplified targets ranged between 23 and 30, indicating DNA from non-viable microorganisms. Plate counts supported this assumption. In conclusion, 100% of the cheeses analyzed were safe to consume, and the enrichment step before DNA extraction proved essential to discern between viable and non-viable microorganisms. Hygienic milking, milk handling, cheese manufacturing, and ripening are crucial to achieve an adequate microbiological quality of cheeses made with raw milk.

Novel multidomain peptide self-assembly biomaterials based on bola structure and terminal anchoring: Nanotechnology meets antimicrobial therapy.

To ameliorate the diminished antimicrobial efficiency and physiological stability associated with monomeric antimicrobial peptides (AMPs) molecules, future research will focus on the artificial design of self-assembling peptides to replace monomeric entities, aiming to combat the antibiotic resistance crisis caused by microbial infections. In this study, the "bola" structure was used as the foundational architecture driving molecular self-assembly, with hydrophobic amino acids at the termini to anchor and finely adjust the sequence, thereby organizing a range of novel multidomain peptides (MDPs) templates into an ABA block motif. The results indicate that FW2 (GMSI = 53.94) exhibits the highest selectivity index among all MDPs and can form spherical micelles in an aqueous medium without the addition of any exogenous additives. FW2 exhibited high stability in vitro in the presence of physiological salt ions, serum, and various pH conditions. It exhibited excellent biocompatibility and efficacy both in vivo and in vitro. Furthermore, FW2 strongly interacts with the lipid membrane and employs various synergistic mechanisms, such as reactive oxygen species (ROS) accumulation, collectively driving cellular apoptosis. This study demonstrates a straightforward strategy for designing self-assembling peptides and promotes the advancement of peptide-based biomaterials integration progress with nanotechnology.

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 / Anfotericina B desoxicolato en el neonato y en el lactante pequeño con bolas fúngicas en el riñón: presentación de 2 casos y revisión de la literatura

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.(AU)

Introducción: Las infecciones urinarias fúngicas afectan preferentemente al prematuro gravemente enfermo o al afecto de malformaciones genitourinarias. Las bolas fúngicas son una complicación infrecuente que requiere un diagnóstico y tratamiento precoces para evitar morbimortalidad asociada. La evidencia científica disponible sobre el manejo de las bolas fúngicas por Candida en lactantes pequeños es muy escasa. Métodos: Casos clínicos y revisión de la literatura. Resultados: Se presentan 2 lactantes inmunocompetentes con malformaciones urogenitales que recibieron tratamiento local con anfotericina B desoxicolato junto a terapia sistémica para el tratamiento y la profilaxis del desarrollo de bolas fúngicas por Candida. Identificamos 21 casos similares en la literatura, con muy pocos datos disponibles sobre la preparación y posología, tiempos de permanencia y duración del tratamiento. Se discuten las distintas estrategias de manejo. Conclusiones: La irrigación local con anfotericina B desoxicolato resultó segura y eficaz en el tratamiento y en la profilaxis de las bolas fúngicas por Candida en el lactante pequeño, junto con el tratamiento antifúngico sistémico.(AU)

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.

Polymorphic BolA Ⅰα1α2 binds to constant chains and co-locates in eukaryotic cells / 中国免疫学杂志

Objective:To demonstrate the polymorphism of α chain of bovine major histocompatibility complex(BoLA)classⅠmolecule and domain binding constant chain(Ii).Methods:Total 75 BoLA Iα genes were obtained from three Huaibei cattle and analyzed by molecular biology software;the genes of typical BoLA Iα domains and Ii were cloned,and then inserted into prokaryotic expression plasmid.After induced protein expression;the domains of BoLA Ⅰα chain binding to Ii were detected by pull-down meth-od and Western blot.The recombinant eukaryotic expression plasmids were constructed and the co-localization of BoLA Iα segments with Ii was observed by laser confocal microscopy.Results:Firstly,it was found that there were at least 5 kinds of BoLA Iα in the cloned gene sequence,which were highly polymorphic and they were mainly distributed in the antigen peptide binding region(PBR)of BoLA Ⅰ(α1α2)and cytoplasmic region.Secondly,the prokaryotic recombinant plasmids containing BoLA Ⅰα1α2α3,BoLA Ⅰα1α2 or BoLA Ⅰα 3 were constructed,then they were respectively induced to express and purified,in which,the BoLA Ⅰα1α2α3 and BoLA Ⅰα1α2 had the activity of binding to Ii.Finally,in 293T cells BoLA Ⅰα1α 2α3 or BoLA Ⅰα1α2 was found that could co-localize with Ii,while a single BoLA Ⅰα3 could not.Conclusion:BoLA Ⅰα gene is highly polymorphic.BoLA Ⅰα1α2 is a func-tional fragment that binds to Ii and co-locates intracellular.

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.

Análisis descriptivo y comparativo de los goles conseguidos tras saques de esquina en la liga española 1ª división: temporadas 2015 y 2019 / Descriptive and comparative analysis of the goals achieved after corner kicks in the Spanish league 1st division: 2015 and 2019 seasons / Análise descritiva e comparativa dos gols marcados após escanteios na 1ª divisão do liga espanhola: temporadas de 2015 e 2019

El objetivo del estudio fue describir de manera exhaustiva la manera de obtener goles tras saque de esquina para analizar la existencia de diferencias, entre las temporadas 2014/15 y 2018/19 en la Liga de Primera División española. El estudio pretendió analizar la incidencia real de esta acción del juego e identificar qué variables están asociadas a la eficacia de la acción al analizar todos los saques de esquina que acaban en gol en dichas temporadas. Para ello se construyó una herramienta de registro en la que se incluyeron criterios contextuales y criterios relacionados con el desarrollo de la acción de gol. Como conclusión, los resultados evidencian una estimación de la tendencia hacia un predominio de los saques de esquina directos o de primera acción (2014/15) hacia saques de esquina de segundas acciones (2018/19). Se observó un incremento del número de jugadores que actúan y una pérdida del predominio del remate de cabeza como acción finalizadora. (AU)

The objectives of the study were to exhaustively describe the way of scoring goals after corner kicks in order to analyze the existence of differences, between the 2014/15 (n = 112) and 2018/19 (n = 90) seasons, in the Liga de Spanish First Division. The study aimed to analyze the real incidence of this game action and identify which variables are associated with the effectiveness of the action by analyzing all the SDE actions thatend in a goal in those seasons. As a main conclusion, the results could show a certain evolution of the predominance of direct or first-action corner kicks (2014/15) towards second or third-action corner kicks (2018/19). Increased the number of players involved in the action and a loss of the dominance of the header as a finishing action, probably related to the change in trend from direct corner kicks (2014/15) towardssecond-action corner kicks or rebounds (2018/19). (AU)

Os objetivos do estudo foram descrever exaustivamente a forma de marcar golos após pontapés de canto, de forma a analisar a existência de diferenças, entre as épocas 2014/15 (n=112) e 2018/19 (n=90), na primeira Liga Espanhola. O estudo teve como objetivo analisar a real incidência dessa ação no jogo e identificar quais variáveis que estão associadas à efetividade da ação analisando todas as ações da SDE que terminam em golos nessas temporadas. Como principal conclusão, os resultados puderam evidenciar uma certa evolução da predominância dos pontapés de canto diretos ou de primeira ação (2014/15) para os pontapés de canto de segunda ação (2018/19). Verificou-se ainda um aumento do número de jogadores envolvidos na ação e perdado domínio do cabeceamento como ação de finalização, provavelmente relacionado à mudança na tendência do canto direto (2014/15) para cantos ou ressaltos de segunda ação (2018/19). (AU)

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.

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.

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.

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.