Influence of Dead Cells Killed by Industrial Biocides (BAC and DBNPA) on Biofilm Formation.

Industrial biocides aim to keep water systems microbiologically controlled and to minimize biofouling. However, the resulting dead cells are usually not removed from the water streams and can influence the growth of the remaining live cells in planktonic and sessile states. This study aims to understand the effect of dead Pseudomonas fluorescens cells killed by industrial biocides-benzalkonium chloride (BAC) and 2,2-dibromo-3-nitrilopropionamide (DBNPA)-on biofilm formation. Additionally, the effect of different dead/live cell ratios (50.00% and 99.99%) was studied. The inoculum was recirculated in a Parallel Plate Flow Cell (PPFC). The overall results indicate that dead cells greatly affect biofilm properties. Inoculum with DBNPA-dead cells led to more active (higher ATP content and metabolic activity) and thicker biofilm layers in comparison to BAC-dead cells, which seems to be linked to the mechanism of action by which the cells were killed. Furthermore, higher dead cell ratios (99.99%) in the inoculum led to more active (higher culturability, metabolic activity and ATP content) and cohesive/compact and uniformly distributed biofilms in comparison with the 50.00% dead cell ratio. The design of future disinfection strategies must consider the contribution of dead cells to the biofilm build-up, as they might negatively affect water system operations.

Pseudomonas fluorescens Cells' Recovery after Exposure to BAC and DBNPA Biocides.

A proper assessment of the effects of biocides on bacterial cells is key to the prevention of antimicrobial resistance and the implementation of suitable biocidal programmes. It is particularly relevant regarding the ability of dead-labelled cells to recover their functional processes once the biocide is removed. In the present work, we studied how Pseudomonas fluorescens cells previously exposed to different concentrations of BAC (benzalkonium chloride) and DBNPA (2,2-Dibromo-3-nitrilopropionamide) behave upon the restoration of optimum growth conditions. The following indicators were evaluated: culturability, membrane integrity, metabolic activity (resazurin), cellular energy (ATP), and cell structure and morphology (transmission electron microscopy (TEM)). The results demonstrated that cells previously labelled as 'dead' recovered to a greater extent in all indicators. Only cells previously exposed to BAC at 160 mg/L (concentration above the MBC) showed significant reductions on all the evaluated indicators. However, the obtained values were much higher than the 'death' thresholds found for the autoclaved cells. This suggests that cells exposed to this concentration take more time to rebuild their functional processes. The recovery of DBNPA-treated cells did not seem to be related to the biocide concentration. Finally, a reflection on what kind of cells were able to recover (remaining cells below the detection limit and/or dormant cells) is also presented.

A Multi-Purpose Approach to the Mechanisms of Action of Two Biocides (Benzalkonium Chloride and Dibromonitrilopropionamide): Discussion of Pseudomonas fluorescens' Viability and Death.

Biocides are widely used in water treatment for microbiological control. The rise of antimicrobial resistance and the need to assure properly managed water systems require a better understanding of the mechanisms of action of biocides and of their impact on cell's viability as a function of dosage concentrations. The present work addresses these two aspects regarding the biocides benzalkonium chloride (BAC) and dibromonitrilopropionamide (DBNPA)-two biocides commonly found in the water treatment industry. For that, the following parameters were studied: culturability, membrane integrity, metabolic activity, cellular energy, and the structure and morphology of cells. Also, to assess cell's death, a reliable positive control, consisting of cells killed by autoclave (dead cells), was introduced. The results confirmed that BAC is a lytic biocide and DBNPA a moderate electrophilic one. Furthermore, the comparison between cells exposed to the biocides' minimum bactericidal concentrations (MBCs) and autoclaved cells revealed that other viability parameters should be taken into consideration as "death indicators." The present work also shows that only for the concentrations above the MBC the viability indicators reached values statistically similar to the ones observed for the autoclaved cells (considered to be definitively dead). Finally, the importance of considering the biocide mechanism of action in the definition of the viability parameter to use in the viable but non-culturable (VBNC) determination is discussed.

New Functionalized Macroparticles for Environmentally Sustainable Biofilm Control in Water Systems.

Reverse osmosis (RO) depends on biocidal agents to control the operating costs associated to biofouling, although this implies the discharge of undesired chemicals into the aquatic environment. Therefore, a system providing pre-treated water free of biocides arises as an interesting solution to minimize the discharge of chemicals while enhancing RO filtration performance by inactivating bacteria that could form biofilms on the membrane system. This work proposes a pretreatment approach based on the immobilization of an industrially used antimicrobial agent (benzalkonium chloride-BAC) into millimetric aluminum oxide particles with prior surface activation with DA-dopamine. The antimicrobial efficacy of the functionalized particles was assessed against Escherichia coli planktonic cells through culturability and cell membrane integrity analysis. The results showed total inactivation of bacterial cells within five min for the highest particle concentration and 100% of cell membrane damage after 15 min for all concentrations. When reusing the same particles, a higher contact time was needed to reach the total inactivation, possibly due to partial blocking of immobilized biocide by dead bacteria adhering to the particles and to the residual leaching of biocide. The overall results support the use of Al2O3-DA-BAC particles as antimicrobial agents for sustainable biocidal applications in continuous water treatment systems.

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.

Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, but devastating genetic disease characterized by segmental premature aging, with cardiovascular disease being the main cause of death. Cells from HGPS patients accumulate progerin, a permanently farnesylated, toxic form of Lamin A, disrupting the nuclear shape and chromatin organization, leading to DNA-damage accumulation and senescence. Therapeutic approaches targeting farnesylation or aiming to reduce progerin levels have provided only partial health improvements. Recently, we identified Remodelin, a small-molecule agent that leads to amelioration of HGPS cellular defects through inhibition of the enzyme N-acetyltransferase 10 (NAT10). Here, we show the preclinical data demonstrating that targeting NAT10 in vivo, either via chemical inhibition or genetic depletion, significantly enhances the healthspan in a Lmna G609G HGPS mouse model. Collectively, the data provided here highlights NAT10 as a potential therapeutic target for HGPS.

Synthetic lethality between PAXX and XLF in mammalian development.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.

Resolving the chemical heterogeneity of natural organic matter: new insights from comprehensive two-dimensional liquid chromatography.

For the purpose of resolving the chemical heterogeneity of natural organic matter (NOM), comprehensive two-dimensional liquid chromatography (LC×LC) was employed for the first time to map the hydrophobicity versus molecular weight (MW) distribution of two well-known complex organic mixtures: Suwannee River Fulvic Acids (SR-FA) and Pony Lake Fulvic Acids (PL-FA). Two methods have been developed using either a conventional reversed-phase (RP) silica column or a mixed-mode hydrophilic interaction column operating under aqueous RP mode in the first dimension, and a size-exclusion column in the second dimension. The LC×LC fractions were screened on-line by UV at 254 nm, molecular fluorescence at excitation/emission wavelengths (λ(Exc)/λ(Em)) of 240/450 nm, and by evaporative light scattering. The MW distributions of these two NOM samples were further characterized by number (Mn) and weight (Mw) average MW, and by polydispersity (Mw/Mn). Findings suggest that the combination of two independent separation mechanisms is promising in extend the range of NOM separation. For the cases where NOM separation was accomplished, smaller Mw group fractions seem to be related to a more hydrophobic nature. Regardless of the detection method, the complete range of MW distribution provided by both comprehensive LC×LC methods was found to be lower than those reported in the literature.

Ultrasonographic and radiographic determination of egg development of jurarás (Kinosternon scorpioides) in captivity / Determinação ultrassonográfica e radiográfica do desenvolvimento de ovos de jurarás em cativeiro

Kinosternon scorpioides (Linnaeus, 1766), with its common name of jurará, is a quite variable species of turtles, and many different names have been applied to populations throughout its range. Currently, however, four subspecies are considered valid as K. scorpioides arises from southern Panama over most of northern South America and is found in Ecuador, northern Peru, southern Bolivia, northern Argentina, eastern Guyana and Brazil. Thus, an ultrasonographic and radiographic study was performed in order to describe the morphology and development of eggs of 20 female jurará mud turtles K. scorpioides, from September 2005 to August 2006. In the first month, the ovarian cycle was characterized by absence of vitellogenic follicles, atresic follicles or oviduct eggs. From October 2005 to March 2006 on, ultrasonographic scanning allowed to establish the growing vitellogenic follicles. Vitellogenic follicles were observed with spherical to ovoid shapes, with a surrounding echogenic yolk, a nonechogenic albumin layer, and a high echogenic shell. The oviduct eggs were identified by radiography just 180 days after beginning the experiment, when the shell became enough mineralized to impress the radiographic film. This experiment allowed to obtain by means of the 7.5 MHz linear probe images with adequate resolution and penetration for visualization of follicles. Successive ultrasonographic examinations of 20 K. scorpioides females allowed to access initial stages of vitellogenic follicles and oviduct eggs, and radiographic examination revealed to be an easy technique to assess oviduct eggs and to allow evaluation of egg development in jurarás, from 6 months on.

A tartaruga Kinosternon scorpioides (Linnaeus, 1766), com nome popular de jurará, é uma espécie bastante variável, e muitos nomes diferentes são atribuídos às suas populações, de acordo com sua localização geográfica. Atualmente, quatro subespécies foram validadas como K. scorpioides surgindo desde o extremo Sul do Panamá e norte da América do Sul, podendo ser encontradas ainda no Equador, no norte do Peru, sul da Bolívia, norte da Argentina, leste da Guiana e no Brasil. Desde modo, foi realizado um estudo ultrassonográfico e radiológico, a fim de descrever a morfologia e o desenvolvimento dos ovos de K. scorpioides em cativeiro, utilizando-se 20 fêmeas, no período de setembro de 2005 a agosto de 2006. No primeiro mês, o ciclo ovariano foi caracterizado pela ausência de folículos vitelogênicos, atrésicos ou presença de ovos em ovidutos. De outubro de 2005 a março de 2006 a varredura ultrassonográfica permitiu determinar o crescimento vitelogênico dos folículos, os quais foram observados com um formato que variou de ovóide a esférico, envolvidos por um conteúdo ecogênico, uma camada não-ecogência de albumina e uma casca de elevada ecogenicidade. Ovos presentes em ovidutos foram identificados por radiografias apenas 180 dias após início do experimento, quando a casca tornou-se suficientemente mineralizada para impressionar o filme radiográfico. Um transdutor linear de 7,5 MHz promoveu imagens com resolução e penetração adequada para visibilização de folículos. Exames ultrassonográficos sucessivos em fêmeas K. scorpioides permitiram determinar os estágios iniciais de desenvolvimento de folículos vitelogênicos e o exame radiográfico mostrou-se como técnica simples para avaliar a presença de ovos em ovidutos, permitindo fazer inferências sobre desenvolvimento de ovos em jurarás, a partir dos 6 meses de pós-observação por ultrassonografia.

Avaliação ultra-sonográfica do sistema urinário, figado e útero do macaco-prego, Cebus apella / Ultrasonographic evaluation of the urinary system, liver and uterus of Cebus apella monkey

O macaco-prego, Cebus apella, é muito difundido no norte e sul da Amazônia Legal Brasileira e no Cerrado. Estes animais encontram-se rotineiramente submetidos à caça predatória, aumentando assim a necessidade de preservação desta espécie silvestre. Realizou-se um estudo ultra-sonográfico de 10 macacos-prego como forma de descrever a anatomia ultra-sonográfica normal de sua cavidade abdominal. A vesícula urinária apresentou parede com espessura média 0,2cm e em posição anatômica cuja topografia permitiu contato com as paredes do corpo do útero e cólon descendente. À varredura abdominal caudal foi visualizada a aorta, veia cava caudal e veia ilíaca direita. O fígado foi visto em varredura sagital e transversal, possibilitando a observação da vesícula biliar e vasos hepáticos. A varredura renal demonstrou com precisão a pelve, seio renal e relação cortico-medular. O comprimento médio de ambos os rins foi de 6,24±0,31cm, não existindo diferença estatística entre o rim direito e esquerdo (Teste t de Student e ANOVA). O volume renal foi 2,37±0,18cm³. Os coeficientes de Correlação de Pearson entre os comprimentos renais direito e esquerdo e entre volumes renais direito e esquerdo foram dispostos como r = 0,74 e 0,51. As espessuras médias para a região cortical e medular foram 0,75±0,11cm e 0,39±0,06cm, respectivamente. O coeficiente de correlação para a relação cortico-medular entre os rins direito e esquerdo foi de r = 0,19. O exame ultrasonográfico mostrou-se como uma técnica eficiente, nãoinvasiva, rápida e reprodutível, que provê dados importantes aos profissionais da área de clínica e cirurgia de animais silvestres.

The Brown Capuchin, Cebus apella, has a wide distribution in the northern and southern Brazilian Amazon region and in the Cerrado (savanna). These monkeys are usually submitted to predatory chase, increasing the need for preservation of this wild animal species. An ultrasonographic examination of 10 Brown Capuchins was made in order to describe the normal ultrasonographic anatomy of their abdominal cavity. The urinary bladder revealed its wall thickness with an average of 0.2cm, the topographic situation of which allowed close relation with the wall of uterus and descendent colon. Using caudal abdominal scan, images of aorta, caudal vena cava and right iliac vein were obtained. Liver was accessible for examination by sagittal and cross-section ultrasound, allowing visualization of gallbladder and hepatic vessels. Renal scan allowed accuracy to evidence the echogenicity differences between pelvis, renal sinus, as well as the cortical-medullary relationship. The mean length of the kidneys was 6.24±0.31cm, and no significant differences were observed between left and right kidney length (Student's t-test and ANOVA). The renal volume obtained was 2.37±0.18cm³. Correlation Coefficients of Pearson between right and left renal length and between right and left renal volume were r = 0.74 and 0.51. Mean thickness for cortical and medullar regions was 0.75±0.11 and 0.39±0.06cm, respectively. Correlation Coefficient of corticomedullar relation between right and left renal was r = 0.19. Examination by ultrasound was considered an efficient, noninvasive, fast and repeatable technique which provides useful data for clinicians and surgeons engaged in wild animal medicine.

Positive reaction for cysticercosis and multicentric anaplastic oligoastrocytoma.

INTRODUCTION: An unusual case of positive immunological testing for cysticercosis in the cystic fluid obtained from an anaplastic oligoastrocytoma is presented. CASE REPORT: A 15-year-old boy was admitted with multiple brain lesions. The biggest was a cyst with a mural node and neurocysticercosis was suspected. In order to relieve intracranial pressure, the cyst was punctured and the immunological testing for cysticercosis was positive, reinforcing the clinical suspicion and leading to a clinical trial with albendazole and steroids. As the patient deteriorated the cystic lesion was removed and the diagnosis of anaplastic oligoastrocytoma was established. A second lesion was eventually approached and the histopathological diagnosis of both specimens concurred. DISCUSSION: Although some authors believe that chronic inflammatory changes following neurocysticercosis could induce the formation of brain tumors, this association may be a mere coincidence. In our case no clinical evidence of a prior infestation by Cysticercus was found. In fact, an exhaustive examination of the specimens did not reveal any areas of inflammatory reaction. We believe that the similarity of the glioma and cysticercosis antigens may be the cause of the positive reactions in the cystic fluid.