Targeting Endogenous Mechanisms of Brain Resilience for the Treatment and Prevention of Alzheimer's Disease.

Alzheimer's disease is a neurodegenerative disorder which contributes to millions of cases of dementia worldwide. The dominant theoretical models of Alzheimer's disease propose that the brain passively succumbs to disruptions in proteostasis, neuronal dysfunction, inflammatory and other processes, ultimately leading to neurodegeneration and dementia. However, an emerging body of evidence suggests that the adult brain is endowed with endogenous mechanisms of resilience which may enable individuals to remain cognitively intact for years despite underlying pathology. In this brief review, we discuss evidence from basic neuroscience and clinical research which demonstrates the existence of endogenous molecular signaling pathways that can promote resilience to neurodegeneration. The p75 neurotrophin receptor provides one such pathway of resilience due to its role as a fundamental signaling switch which determines neuronal survival or degeneration. We highlight a series of preclinical studies targeting the p75 neurotrophin receptor in mouse models which demonstrate resilience to amyloid. We briefly discuss the design and goals of a recent clinical trial of p75 neurotrophin receptor modulation in patients with mild to moderate Alzheimer's disease. Unique challenges for developing therapeutics and biomarkers which are optimized for targeting and detecting endogenous mechanisms of resilience are also discussed. Altogether, this review motivates further trial work of therapeutics modulating the p75 neurotrophin receptor and other deep biology targets.

La telerehabilitación durante la pandemia COVID-19: experiencia de las familias de niños con condiciones crónicas complejas / Telerehabilitation during the COVID-19 pandemic: experience of families of children with complex chronic conditions

La telerehabilitación ha sido una solución rápida y efectiva para la atención de pacientes durante la pandemia COVID-19. Nuestro objetivo ha sido describir la experiencia de las familias de niños con condiciones crónicas complejas (CCC) asistidos con la modalidad de telerehabilitación durante la pandemia. Materiales y métodos: Se ha realizado un estudio observacional, descriptivo y transversal de tipo encuesta online. Se encuestaron familias de niños (1 mes a 5 años de edad) con al menos una CCC que recibieron seguimiento interdisciplinario a distancia durante la pandemia de 2 o más áreas de rehabilitación (kinesiología, fonoaudiología y terapia ocupacional) pertenecientes al Servicio de Kinesiología del Hospital de Pediatría Juan P Garrahan, durante marzo a octubre de 2020. Resultados: El 88,3% de los participantes (n=43) se expresó satisfecho con la modalidad de telerehabilitación. Entre los facilitadores seleccionados por los participantes se mencionan la reducción de tiempos, mayor frecuencia de consultas, disminución de gastos por traslados y mayor comodidad, mientras que entre las barreras se destacaron: el no vínculo con profesionales, alargue en tiempos de tratamiento, problemas de conectividad. El análisis estadístico no indicó diferencias significativas entre quienes se reportaron como más satisfechos según su estrato social (alto o bajo), la disponibilidad de wifi propio, o respecto a la edad del niño. Se reportaron diferencias significativas en cuanto a la distancia al hospital (p=0.034). Conclusión: Esta forma de intervención ofreció nuevas posibilidades de atención que podrían considerarse a futuro en el seguimiento de nuestros pacientes. (AU)

Telerehabilitation has been a fast and effective solution in patient care during the COVID-19 pandemic. Our aim was to describe the experience of families of children with complex chronic conditions (CCC) treated through telerehabilitation during the pandemic. Materials and methods: An observational, descriptive, cross-sectional, online survey study was conducted. Families of children (1 month to 5 years of age) with at least one CCC who received interdisciplinary remote follow-up during the pandemic from two or more rehabilitation areas (physical therapy, speech therapy, and occupational therapy) belonging to the Department of Physical Therapy of Hospital de Pediatría Juan P Garrahan, from March to October 2020, were surveyed. Results: 88.3% of the participants (n=43) expressed satisfaction with the telerehabilitation modality. Among the facilitators selected by the participants, the following were mentioned: time saving, greater frequency of consultations, reduction of travel expenses and greater comfort, while among the barriers, the following stood out: no connection with the professionals, longer treatment times, connectivity problems. Statistical analysis did not show significant differences between those who reported being more satisfied according to socioeconomic level (high or low), availability of their own wifi connection, or age of the child. A significant difference was found for distance to the hospital (p=0.034). Conclusion: This type of intervention provided new possibilities of care that could be considered in the future follow-up of our patients (AU)

Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.

Disfagia pediátrica: rol del fonoaudiólogo como parte de un equipo interdisciplinario en el Hospital Juan P. Garrahan / Dysphagia in children: The role of the speech therapist in the interdisciplinary team of Hospital Juan P. Garrahan

Introducción: El 80% de los niños con condiciones médicas crónicas complejas presentan alteración de la conducta alimentaria. Dada la heterogeneidad de los diagnósticos y la complejidad en el manejo de la disfagia pediátrica, es muy importante la intervención de equipos especializados. Objetivo: Evaluación de la evolución de los niños con trastornos de la deglución y/o conducta alimentaria atendidos durante el año 2017 por el equipo interdisciplinario de deglución y conducta alimentaria, del Hospital de Pediatría Juan P. Garrahan. Material y métodos: Estudio analítico, prospectivo y longitudinal con la intervención de un equipo interdisciplinario. Se incluyeron todos los pacientes evaluados durante el año 2017. Se realizó evaluación clínica de la deglución que permitió detectar dificultades durante el momento de la alimentación. Se dieron pautas de estimulación y modificación de consistencias y se derivó a tratamiento oportuno. Se midió porcentaje de destete de soporte nutricional (SN) y/o aumento del aporte por vía oral. Resultados: evaluamos 153 pacientes, 39% mujeres, 90% menores de 3 años de edad y el 75% en apoyo nutricional. El 72% presentó trastorno de la deglución exclusivamente o asociado a trastorno de la conducta alimentaria. El 68% fue seguido en más de una oportunidad. El 36% de los pacientes que ingresaron con requerimiento de SN lograron el destete (sin diferencia significativa entre los que tenían o no trastorno deglutorio p=0.85 y los que tenían o no traqueostomía p=0,88) y 40% aumentó el aporte por vía oral dentro del grupo que no logró el destete. Se observó una diferencia estadísticamente significativa en el destete de los pacientes que concurrieron al espacio de la clínica con respecto a los que no(p=0,016) y mayor tiempo de intervención entre quienes lograron el destete y quienes no, 5,2 ± 3,1 vs 3,45 ± 3,1 meses (p=0,0099). Conclusión: Es esencial el trabajo interdisciplinario y especializado en niños con trastornos de la deglución. La intervención del fonoaudiólogo como parte del equipo es fundamental para una detección precoz y correcto abordaje de la disfagia (AU)

Of all children with chronic complex medical conditions, 80% have eating behavior disorders. Given the heterogeneity of the diagnoses and the complexity of the management of dysphagia in children, intervention of a specialized medical team is essential. Objective: Evaluation of the outcome in children with swallowing and/or eating behavior disorders seen during 2017 by the interdisciplinary group of swallowing and eating behavior disorders at Hospital de Pediatría Juan P. Garrahan. Material and methods: An analytical, prospective and longitudinal study with intervention by an interdisciplinary team. All patients evaluated during 2017 were included. Swallowing was clinically assessed to identify eating disorders. Indications were given for stimulation and food consistency and patients were referred for adequate treatment. The rates of weaning from nutritional support (NS) and/or increase of oral food intake were measured. Results: 153 patients were evaluated, 39% were female, 90% younger than 3 years of age, and 75% AN. Overall, 72% had swallowing difficulties only or associated with an eating behavior disorder; 68% was followed on more than one occasion. Thirty-six percent of the patients who were admitted with NS requirement could be weaned (without a significant difference between those who had a swallowing disorder and those who did not p=0.85 and those that did and did not have a tracheostomy p=0.88) and oral food intake increased in 40% of the patients in the group that could not be weaned. A statistically significant difference was found in the weaning of patients who attended the clinic and those who did not (p=0.016) and longer intervention time between those who could be weaned and those who could not, 5.2 ± 3.1 vs 3.45 ± 3.1 months (p=0.0099). Conclusion: Interdisciplinary and specialized care is necessary for children with swallowing disorders. Intervention of a speech therapist as part of the team is fundamental for the early detection and adequate management of dysphagia (AU)

7,12-Dimethylbenz(a)anthracene-induced genotoxicity on bone marrow cells from mice phenotypically selected for low acute inflammatory response

Exposure to polycyclic aromatic hydrocarbon (PAH) environmental contaminants has been associated with the development of mutations and cancer. 7,12-Dimethylbenz(a)anthracene ( DMBA), a genotoxic agent, reacts with DNA directly, inducing p53-dependent cytotoxicity resulting in cell death by apoptosis or giving rise to cancer. DMBA metabolism largely depends on activation of the aryl hydrocarbon receptor (AhR). Mice phenotypically selected for high (AIRmax) or low (AIRmin) acute inflammatory response present a complete segregation of Ahr alleles endowed with low (Ahr(d)) or high (Ahr(b1)) affinity to PAHs, respectively. To evaluate the role of AhR genetic polymorphism on the bone marrow susceptibility to DMBA, AIRmax and AIRmin mice were treated with a single intraperitoneal injection of DMBA (50 mg/kg b.w.) in olive oil. Bone marrow cells (BMCs) were phenotyped by both flow cytometry and cytoslide preparations. Despite a significant decrease in total cell count in BM from AIRmin mice, there was an increase of blast cells and immature neutrophils at 1 and 50 days after DMBA treatment, probably due to a cell-cycle blockade at the G1/S transition leading to immature stage cell production. A panel of proteins related to cell cycle regulation was evaluated in immature BM cells (Lin(-)) by Western Blot, and DNA damage and repair were measured using an alkaline version of the Comet assay. In Lin(-) cells isolated from AIRmin mice, high levels were found in both p53 and p21 protein contents in contrast with the low levels of CDK4 and Ciclin D1. Evaluation of DNA repair in DMBA-treated BMCs, indicated long-lasting genotoxicity and cytotoxicity in BMC from AIRmin mice and a blockade of cell cycle progression. On the other hand, AIRmax mice have a high capacity of DNA damage repair and protection. These mechanisms can be associated with the differential susceptibility to the toxic and carcinogenic effects of DMBA observed in these mice. (C) 2015 Elsevier B.V. All rights reserved.

Occurrence of Vibrio vulnificus in mussel farms from the Varano lagoon environment.

AIMS: Monitoring the occurrence of the human pathogen Vibrio vulnificus in a mussel farm located in the lagoon of Varano (Italy). METHODS AND RESULTS: A total of 72 samples of mussel, water and sediment, collected from two locations of Varano lagoon in the Gargano peninsula, during a 7-month survey, were analysed. Isolation and PCR characterization of six V. vulnificus environmental genotype strains revealed that this pathogen was isolated when with T was above 22 °C and salinity ranged between 22.7 and 26.4‰. No significant correlation of the occurrence of V. vulnificus with water pH or salinity was observed. Moreover, 8% of mussel samples were found to be contaminated by V. vulnificus. All of that positive mussel samples originated from the same sampling station. CONCLUSION: It is suggested that warmer season are risky to eat raw or undercooked bivalve molluscs in the local area. SIGNIFICANCE AND IMPACT OF THE STUDY: To increase knowledge about environmental conditions that may affect the occurrence of waterborne pathogen Vibrio vulnificus in seafood.

[The role of endoscopy in gastroenteropancreatic neuroendocrine tumors]. / Il ruolo dell'endoscopia nei tumori neuroendocrini gastroenteropancreatici.

Versione italiana Riassunto: Il ruolo dell'endoscopia nei tumori neuroendocrini gastroenteropancreatici. L. Magno, L. Sivero, V. Napolitano, S. Ruggiero, G. Fontanarosa, S. Massa I tumori neuroendocrini (NET) gastro-entero-pancreatici (GEP) sono neoplasie rare che originano dalle cellule neuroendocrine del tubo digerente e del pancreas. L'endoscopia digestiva e l'ecoendoscopia rivestono un ruolo importante nella diagnosi, stadiazione e sorveglianza dei pazienti con NET. Inoltre, in casi selezionati, le tecniche endoscopiche operative consentono il trattamento di queste neoplasie in fase precoce. English version Summary: The role of endoscopy in gastroenteropancreatic neuroendocrine tumors. L. Magno, L. Sivero, V. Napolitano, S. Ruggiero, G. Fontanarosa, S. Massa Gastroenteropancreatic (GEP) neuroendocrine tumors (NET) are rare neoplasia arisen from neuroendocrine cells present in the gut mucosa and pancreas. Digestive endoscopy and endoscopic ultrasonography play a relevant role in NET diagnosis, stadiation and surveillance. Moreover, in selected patients, surgical endoscopy allows the tratment of these cancers at an early stage.

Immunomodulatory activity of a plant extract containing human papillomavirus 16-E7 protein in human monocyte-derived dendritic cells.

This study reports the immunomodulatory activity on human monocyte derived dendritic cells (MDDCs) of a vaccine preparation shown to be effective against an HPV16-related tumour in an animal model. The vaccine is composed of extract from Nicotiana benthamiana leaves containing HPV16 E7 protein expressed by a potato virus X-derived vector (NbPVX-E7). The effect of the extract was evaluated on MDDC differentiation and maturation by monitoring the phenotypic expression of specific markers. The results show that NbPVX-E7 does not induce monocyte differentiation to dendritic cells, but does induce MDDC maturation. Plant extract does not influence MDDC-uptake of E7-FITC while it significantly improves the Ovalbumin-FITC uptake, considered as a model antigen. Importantly, NbPVX-E7-pulsed MDDCs/PBMCs are able to prime human blood-derived lymphocytes from healthy individuals to induce HPV16 E7-specific cytotoxic activity. This is a propaedeutic study for a possible use of E7-containing plant extract in human immunotherapy of HPV-related lesions.

Occurrence of Giardia and Cryptosporidium in Italian water supplies.

A total of 21 samples: raw water (RW) samples; water samples after coagulation with aluminium sulfate (clarified water: CW); and water after chlorination (treated water: TW) from a water purification plant that treats river surface water from the neighbourhood of Foggia (Italy), were analysed for the presence of Giardia cysts and Cryptosporidium oocysts. Bacteriological indicator of faecal contamination (total and faecal coliforms, faecal streptococci,), total bacterial count at 22 and 36 degrees C and physicochemical parameters (turbidity, temperature, pH) were evaluated. Cryptosporidium oocysts were not found in any samples examined, while Giardia cysts were found only in RW samples, with the maximal concentration of 8 cysts/100 l. A positive correlation was found between the Giardia densities and quality parameters such as TC, FC and TBC at 22 degrees C. Giardia levels in raw water samples correlated (p < 0.05) with TC, FC and with temperature. No other water quality parameters was consistently correlated with cysts level.

In Vivo PCR-DGGE analysis of Lactobacillus plantarum and Oenococcus oeni populations in red wine.

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety "Primitivo di Puglia"), a polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30-60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.

Genetic structure at range edge: low diversity and high inbreeding in Southeast Asian mangrove (Avicennia marina) populations.

Understanding the genetic composition and mating systems of edge populations provides important insights into the environmental and demographic factors shaping species' distribution ranges. We analysed samples of the mangrove Avicennia marina from Vietnam, northern Philippines and Australia, with microsatellite markers. We compared genetic diversity and structure in edge (Southeast Asia, and Southern Australia) and core (North and Eastern Australia) populations, and also compared our results with previously published data from core and southern edge populations. Comparisons highlighted significantly reduced gene diversity and higher genetic structure in both margins compared to core populations, which can be attributed to very low effective population size, pollinator scarcity and high environmental pressure at distribution margins. The estimated level of inbreeding was significantly higher in northeastern populations compared to core and southern populations. This suggests that despite the high genetic load usually associated with inbreeding, inbreeding or even selfing may be advantageous in margin habitats due to the possible advantages of reproductive assurance, or local adaptation. The very high level of genetic structure and inbreeding show that populations of A. marina are functioning as independent evolutionary units more than as components of a metapopulation system connected by gene flow. The combinations of those characteristics make these peripheral populations likely to develop local adaptations and therefore to be of particular interest for conservation strategies as well as for adaptation to possible future environmental changes.

Environmental stress response in wine lactic acid bacteria: beyond Bacillus subtilis.

Lactic acid bacteria (LAB) constitute a heterogeneous group of bacteria that are traditionally used to produce fermented foods. The industrialization of food transformations has increased the economical importance of LAB, as they play a crucial role in the development of the organoleptic and hygienic quality of fermented products. However, the strains selected for industrial purposes, should tolerate adverse conditions encountered in industrial processes, either during starter handling and storage (freeze-drying, freezing, or spray-drying) or during food processing in which abiotic stresses such as heat, cold, acidity, and high concentration of NaCl or ethanol are common. Wine LAB have to deal with several stresses including an acidic pH, a high alcoholic content, non optimal growth temperatures, and growth-inhibitory compounds such as fatty acids and tannins, originated from yeast and bacteria metabolism. Wine LAB have developed several mechanisms to escape or to tolerate wine conditions. They carry out a malolactic fermentation in this stressful environment. In addition to the regulation of the expression of specific genes, bacteria have evolved adaptive networks to face the challenges of a changing environment and to survive under conditions of stress. The so called Global Regulatory Systems control the simultaneous expression of a large number of genes in response to a variety of environmental stress factors. CIRCE sequences able to bind the HrcA repressor, sigma(B) dependent promoters and CtsR regulatory elements have been observed in several genes identified from wine LAB. Improved knowledge of regulators and a better understanding of LAB stress responses could constitute a basis of comparison with the well known model microorganisms, Escherichia coli and Bacillus subtilis. Moreover, it can provide an important insight into improving current industrial starter strains.

Exploiting the plant secretory pathway to improve the anticancer activity of a plant-derived HPV16 E7 vaccine.

The human papillomavirus 16 (HPV16) E7 oncoprotein can be considered a "tumor-specific antigen", and therefore it represents a promising target for a therapeutic vaccine against HPV-associated tumors. Efficient production of E7 protein with a plant-based transient expression system has been already described and it was demonstrated that E7-containing crude plant extracts confer partial protection against tumor challenge in a mouse model system. Before adopting the plant-based system as a cost-effective method for the production of an E7-based anti-cancer vaccine, some aspects, such as the oncoprotein yield, need further investigation. In the present study, we report the transient expression, mediated by a potato virus X (PVX)-derived vector, of the E7 protein targeted to the secretory system of Nicotiana benthamiana plants by using a plant-derived signal sequence. Targeting the antigen to the secretory pathway enhanced the E7 protein expression levels about five-fold. Mice immunized by s.c. administration with crude foliar extracts containing E7 showed strong stimulation of cell-mediated immune response after five boosters, as detected by ELISPOT. After challenging with the E7-expressing C3 tumor cells, tumor growth was completely inhibited in 80% of the vaccinated animals and a drastic reduction of tumor burden was observed in the remaining tumor-affected mice. These data demonstrate that, by enhancing E7 yield, it is possible to improve the anti-cancer activity of the plant-based experimental vaccine and open the way for a large-scale production of the E7 protein which could be purified or used as in planta formulation, also suitable for oral therapeutic vaccination.

A beta-glucosidase gene isolated from wine Lactobacillus plantarum is regulated by abiotic stresses.

AIMS: Little genetic information exists on the ability of wine lactic acid bacteria (LAB) to hydrolyse glycoconjugates during malolactic fermentation. We tried to fill this important gap by characterizing a gene codifying for a putative beta-glucosidase enzyme from wine Lactobacillus plantarum and from a commercial strain of Oenococcus oeni. METHODS AND RESULTS: The coding region of the putative beta-glucosidase gene is 1400 nucleotides long and started with an ATG codon. The gene is widespread among LAB and the highest identity was observed between the nucleotide of L. plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum and O. oenibeta-glucosidase gene. The protein sequence deduced from the isolated genes has a calculated molecular mass of 61.19 kDa. Furthermore, the expression of the beta-glucosidase gene in L. plantarum strain was analysed, under several stress, by reverse transcriptase (RT)-PCR and Northern-blot analysis. The gene was apparently regulated by abiotic stresses such as temperature, ethanol and pH. CONCLUSIONS: The beta-glucosidase gene is widespread among LAB and its expression is probably regulated by a wide range of abiotic stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of temperature and ethanol on the L. plantarumbeta-glucosidase gene may be useful to explain the differences found in beta-glucosidase activity reported in wines by several authors.

Cloning and characterization of the hsp 18.55 gene, a new member of the small heat shock gene family isolated from wine Lactobacillus plantarum.

Using a molecular approach based on PCR, RT-PCR and northern blot analysis, a new member of the small heat shock family of wine, Lactobacillus plantarum, was cloned and characterized. The protein sequence deduced from the isolated gene had a calculated molecular mass of 18.548 kDa and was therefore named HSP 18.55. The gene codes for a protein homologous to the previously characterized HSP 19.3 and HSP 18.5 and is co-transcribed with an upstream gene of unknown function. Analysis of the 5' flanking region of the hsp 18.55 gene revealed the presence of putative cis elements able to bind alternative sigma factor sigma(B). Based on its structure, the gene was classified as belonging to class II of the heat shock genes according to Bacillus subtilis nomenclature for shock-responsive genes. Expression of the newly identified small heat shock gene, analyzed by RT-PCR and northern blot analysis, was induced by a wide range of abiotic stresses including heat, cold and ethanol, suggesting that the small family of heat shock genes is probably involved in the general stress response in wine L. plantarum. Moreover, the expression of hsp 18.5, hsp 18.55 and hsp 19.3 genes, analyzed over a complete culture cycle, revealed that early growing cells contained substantial amounts of hsp 18.5, hsp 18.55 and hsp 19.3 mRNAs, which rapidly declined upon entry into stationary phase.

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater.

AIMS: Developing and evaluating a rapid real-time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre-enrichment step before DNA extraction. METHODS AND RESULTS: Wastewater samples were collected from a dairy farm producing mozzarella cheese and located in Puglia (south of Italy). Plate count and other microbial assays were performed 1 h after sampling. Wastewater samples were artificially inoculated with 10(4), 10(7) and 10(8) cells ml(-1) of E. coli O157:H7, strain EDL933. PCR protocols for stx1, stx2 and eae genes were first tested on pure DNA extracted from type strains, in order to optimize the amplification conditions and reagent concentration before real-time PCR experiments. Three specific fragments of ca 106, 150 and 200 bp corresponding to genes eae, stx1 and stx2, respectively, were obtained. Real-time PCR experiments were performed with DNA extracted from dairy and manure wastewater samples inoculated with 10(4), 10(7) and 10(8) colony-forming units (CFU) ml(-1) of E. coli O157:H7 strain EDL 933. The sensitivity limit of the assay was 10(-1) pg microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-1) for stx1 gene respectively. CONCLUSIONS: A real-time PCR protocol has been developed and used in order to identify potential pathogens in dairy wastewater, in which previous methods (including standard PCR) failed to work. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle and dairy wastewater samples produced from mozzarella cheese factories may harbour verocytotoxin-producing E. coli. The availability of rapid and sensitive molecular methods may be useful to monitor the persistence of verocytotoxin-producing E. coli in general and to assess the effectiveness of wastewater treatments.

Synthesis and biological evaluation of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides as novel synthetic HDAC inhibitors.

A new series of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides 1-3 have been prepared, and their anti-HDAC (against maize HD2, HD1-B, and HD1-A enzymes) activities have been assessed. Cinnamyl-hydroxyamides bearing acylamino substituents at the C2 position of the benzene ring (compounds 1a-g) showed very low HDAC inhibiting activities, with IC(50) values in the high micromolar range. By shifting the same acylamino groups from C2 to C3 (compounds 2a-g) as well as C4 (compounds 3a-f) position of the benzene ring, a number of highly potent HDAC inhibitors have been obtained. In the anti-HD2 assay 3c (IC(50) = 11 nM) was the most potent compound, being >11600-, 4.5-, and 10-fold more potent than sodium valproate, SAHA, and HC-toxin, respectively, and showing the same activity as trapoxin. HD1-B and HD1-A assays have been performed to screen the inhibitory action of 1-3 against mammalian class I (HD1-B) and class II (HD1-A) HDAC homologous enzymes. From the corresponding IC(50) data, a selectivity ratio has been calculated. In general, compounds 1-3 showed no or little selectivity towards the class II homologue HD1-A, the most selective being 2a with class II selectivity ratio = 4.3. About the inhibitory potency, the 4-(2-naphthoylamino)cinnamyl-N-hydroxyamide 3f showed the highest inhibiting effect against the two enzymes (IC(50-HD1-B) = 36 nM; IC(50-HD1-A) = 42 nM). Selected 2 and 3 compounds will be evaluated to determine their antiproliferative and cyto differentiating activities on HL-60 cells.