Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant.

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.

Utilization of Bioinorganic Nanodrugs and Nanomaterials for the Control of Infectious Diseases Using Deep Learning.

As one of the main causes of morbidity and mortality, viral infections have a major impact on the well-being and economics of every nation in the globe. The ability to predictably diagnose viral infections improves the provision of good healthcare as well as the control and prevention of these conditions. Nanomaterials have gained widespread usage in the medical industry recently due to the rapid advancement of nanotechnology and their exceptional chemical and physical qualities, such as their small size and synthesized surface properties. The utilization of nanoparticles for illness detection, surveillance, control, preventive, and therapy, such as the treatment of bacterial infections, is referred to as nanomedicine. Nanomedicine is a comprehensive discipline that is founded on the usage of nanotechnology for clinical objectives. Nanoparticles, which have a nanoscale dimension and exhibit highly controllable optical and physical characteristics as well as the ability to bind to a large variety of chemicals, are among the most popular nanomaterials in nanomedicine. A deep learning framework of autoencoder for categorization study on viral infections is built based on actual hospital patient history of viral infections from August 2015 to August 2020. The information comprises of 10,950 cases, comprising outpatients and inpatients, encompassing the infectious diseases. Of such 10,950 instances, training set made up 70% or 7665 instances, and testing data made up 30% or 3285 instances. The data processing was done using the presented recurrent neural network-artificial bee colony (RNN-ABC) method. Sparse data densifying processes are done through the autoencoder to enhance the system learning outcome. The suggested autoencoder system was also evaluated to other widely used models, including support vector machine, logistic regression, random forest, and Naïve Bayes. In comparison to other approaches, the study's findings demonstrate how well the suggested autoencoder model can predict viral diseases. The methods used for this research can aid in removing reported lags in current monitoring systems, hence reducing society's expenses.

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration.

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

BRCA2-DSS1 interaction is dispensable for RAD51 recruitment at replication-induced and meiotic DNA double strand breaks.

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

A novel mouse model of PMS2 founder mutation that causes mismatch repair defect due to aberrant splicing.

Hereditary non-polyposis colorectal cancer, now known as Lynch syndrome (LS) is one of the most common cancer predisposition syndromes and is caused by germline pathogenic variants (GPVs) in DNA mismatch repair (MMR) genes. A common founder GPV in PMS2 in the Canadian Inuit population, NM_000535.5: c.2002A>G, leads to a benign missense (p.I668V) but also acts as a de novo splice site that creates a 5 bp deletion resulting in a truncated protein (p.I668*). Individuals homozygous for this GPV are predisposed to atypical constitutional MMR deficiency with a delayed onset of first primary malignancy. We have generated mice with an equivalent germline mutation (Pms2c.1993A>G) and demonstrate that it results in a splicing defect similar to those observed in humans. Homozygous mutant mice are viable like the Pms2 null mice. However, unlike the Pms2 null mice, these mutant mice are fertile, like humans homozygous for this variant. Furthermore, these mice exhibit a significant increase in microsatellite instability and intestinal adenomas on an Apc mutant background. Rectification of the splicing defect in human and murine fibroblasts using antisense morpholinos suggests that this novel mouse model can be valuable in evaluating the efficacy aimed at targeting the splicing defect in PMS2 that is highly prevalent among the Canadian Inuits.

Oxytetracycline and Florfenicol Concentrations in Food-Additive Premixes Authorised for Broiler Chickens: Assessing Degree of Agreement with Manufacturers Labelling.

Antimicrobials premixes are the presentation of choice to administer drugs simultaneously to groups of animals in intensive husbandry systems that require treatment for pathologies of bacterial origin. Among the premixes available for use in poultry, florfenicol and oxytetracycline are commonly administered via food or water. However, their actual concentration in premixes must meet on-label statements to ensure plasma concentrations reach effective therapeutic levels. Hence, this work was designed for the purpose of verifying whether the concentration of antimicrobial present in five premixes matched their on-label statement. Three oxytetracycline premixes, and two of florfenicol, were analysed using a Xevo TQ-S micro UPLC-MS/MS, and an ABSciex API4000 HPLC-MS/MS, respectively. Analytical methodologies were implemented and validated, showing an R2 ≥ 0.99 for the calibration curves. Oxytetracycline was detected in these premixes at concentrations exceeding on-label statements by 13.28%, 21.54%, and 29.68%, whereas florfenicol concentrations detected in premixes were 13.06% and 14.75% lower than expected. Consequently, this work shows that the concentration of active ingredients that are present in commercial formulations effectively differ from those stated on premix labels, and it also highlights how unpredictable their range of variability might be. This must be addressed through solid and updated laws that guarantee an effective pharmaceutical product.

Determination of five antimicrobial families in droppings of therapeutically treated broiler chicken by high-performance liquid chromatography-tandem mass spectrometry.

Antimicrobials are currently used in poultry for disease treatment. However, their excretion in bird feces may contaminate the environment. Considering this, the objective of this work was to quantify antimicrobials residues concentrations in therapeutically treated broiler chicken droppings throughout the post-treatment period. For this aim a multiresidue method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was validated. Forty-eight male broiler chickens were distributed and treated with commercial formulations of 5 different antimicrobials. Results showed that oxytetracycline and 4-epi-oxytetracycline, presented the highest concentrations during all sampling period, detecting concentrations of 1471.41 µg kg-1 at the last sampling point (day 22 post-treatment). Florfenicol, tylosin, enrofloxacin, and ciprofloxacin were eliminated and detected in treated chicken droppings until d 18 post-treatment. Sulfachloropyridazine decrease gradually during post-treatment period until day 30. Results demonstrate that studied antimicrobials in treated chicken droppings were eliminated for prolonged periods, therefore becoming a significant route of residues dissemination into the environment.

Detection of Antimicrobial Residues in Poultry Litter: Monitoring a Risk through a Selective and Sensitive HPLC-MS/MS Method.

Tetracyclines, sulphonamides, and quinolones are families of antimicrobials (AMs) widely used in the poultry industry and can excrete up to 90% of AMs administrated, which accumulate in poultry litter. Worryingly, poultry litter is widely used as an agriculture fertilizer, contributing to the spread AMs residues in the environment. The aim of this research was to develop a method that could simultaneously identify and quantify three AMs families in poultry litter by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Samples of AMs free poultry litter were used to validate the method according to 657/2002/EC and VICH GL49. Results indicate that limit of detection (LOD) ranged from 8.95 to 20.86 µg kg-1, while limits of quantitation (LOQ) values were between 26.85 and 62.58 µg kg-1 of tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, enrofloxacin, ciprofloxacin, flumequine, sulfachloropyridazine, and sulfadiazine. Recoveries obtained ranged from 93 to 108%. The analysis of field samples obtained from seven commercial poultry flocks confirmed the adequacy of the method since it detected means concentrations ranging from 20 to 10,364 µg kg-1. This provides us an accurate and reliable tool to monitor AMs residues in poultry litter and control its use as agricultural fertilizer.

Evaluation of Antibiotic Dissemination into the Environment and Untreated Animals, by Analysis of Oxytetracycline in Poultry Droppings and Litter.

Oxytetracycline (OTC) is widely used in broiler chickens. During and after treatment a fraction of OTC is excreted in its original form and as its epimer, 4-epi-OTC in droppings. To address the transfer of OTC into the environment, we evaluated the dissemination of OTC and 4-epi-OTC from treated birds to the environment and sentinels, through the simultaneous analysis of broiler droppings and litter. Male broiler chickens were bred in controlled conditions. One group was treated by orogastric tube with 80 mg kg-1 of OTC and two groups received no treatment (sentinels). OTC+4-epi-OTC were analyzed and detected by a HPLC-MS/MS post the end of treatment. The highest concentrations of OTC+4-epi-OTC were detected in the droppings of treated birds 14-days following the end of treatment (2244.66 µg kg-1), and one day following the end of treatment in the litter (22,741.68 µg kg-1). Traces of OTC+4-epi-OTC were detected in the sentinels' droppings and litter (<12.2 µg kg-1). OTC+4-epi-OTC can be transferred from treated birds to the environment and to other untreated birds. The presence and persistence of OTC+4-epi-OTC in litter could contribute to the selection of resistant bacteria in the environment, increasing the potential hazard to public and animal health.

Efficacy Prediction of Four Pharmaceutical Formulations for Intramammary Administration Containing Aloe vera (L.) Burm. f. Combined With Ceftiofur or Cloxacillin in Lactating Cows as an Alternative Therapy to Treat Mastitis Caused by Staphylococcus aureus.

Synergy or additive effect between Aloe vera (L.) Burm.f. and beta-lactam (ß-lactam) antibiotics has been reported against Staphylococcus aureus, one of the most important etiological agents of cow mastitis. The goal of the present study was to predict the efficacy of intramammary formulations containing the Aloe vera gel extract in the combination with cloxacillin or ceftiofur at low concentrations in lactating cows as an alternative therapy. Each quarter of 20 healthy Holstein Friesian lactating cows were treated with a single dose of one of the following formulations, corresponding to one of these treatment groups: A1, A2, A3, and A4. A1 and A2 contained cloxacillin at 0.25 and 0.5 mg/ml, whereas A3 and A4 contained ceftiofur 0.25 and 0.5 mg/ml, respectively. In addition, all formulations contained 600 mg/ml of an alcoholic extract of Aloe vera. Milk samples were taken at predefined time points. Antibiotics and aloin (active compound of Aloe vera) concentrations were assessed by liquid chromatography mass spectrometry system (LC-MS/MS). Pharmacokinetic profiles were obtained, and the efficacy index, the fraction of dosing interval in which the antimicrobial concentration remains above the minimum inhibitory concentrations (MICs) (T > MIC) for each formulation, was calculated considering MIC values against Staphylococcus aureus ATCC 29213 as obtained for the combination Aloe vera + antibiotic and aloin concentration in the extract. Mammary gland safety assessment was performed for each combination. Values of the main efficacy index for this study, T > MIC (h) for Aloe vera were 23.29, 10.50, 27.50, and 13.89, whereas for cloxacillin or ceftiofur were 19.20, 10.9, 19.74, and 15.63, for A1, A2, A3, and A4, respectively. Only A1 and A3 reached aloin and antibiotic recommended values as predictors of clinical efficacy for cloxacillin, ceftiofur, and aloin (50, 70, and 60%, respectively), assuming a dose interval of 24 h. The efficacy index values obtained suggest that A1 and A3 might be an effective therapy to treat bovine mastitis caused by S. aureus after a single dose. Nevertheless, further trials in S. aureus mastitis clinical cases are mandatory to confirm the efficacy of Aloe vera formulations.

Innate Biomineralization.

In vertebrates, biomineralization is a feature considered unique to mature osteoblasts and odontoblasts by which they synthesize hydroxyapatite (HAP), which is deposited in the collagen matrix to construct endoskeleton. For many decades, the mechanisms that modulate differentiation and maturation of these specialized cells have been sought as a key to understanding bone-remodeling defects. Here, we report that biomineralization is an innate ability of all mammalian cells, irrespective of cell type or maturation stage. This innate biomineralization is triggered by the concomitant exposure of living cells to three indispensable elements: calcium ion, phosphoester salt, and alkaline phosphatase. Any given somatic cell, including undifferentiated mononuclear cells, can undergo a biomineralization process to produce calcium-phosphate agglomerates. The biologically generated minerals under such conditions are composed of genuine HAP crystallites of Ca10(PO4)6(OH)2 and 5-10 nanometer (nm) in size. This discovery will profoundly improve our understanding of bone metabolism and ectopic calcifications.

Assessing the depletion of lincomycin in feathers from treated broiler chickens: a comparison with the concentration of its residues in edible tissues.

Lincomycin is the first antimicrobial agent described for the lincosamide class and it is commonly used for the treatment of infectious enteric and respiratory diseases in poultry. Maximum residue limits (MRLs) in edible tissues have been established for this antimicrobial, however, no regulation has been proposed yet for by-products that are not intended for direct human consumption. Feathers are a by-product from poultry farming that might be used as an ingredient for diets fed to other farm animal species. The presence of antimicrobial residues in them is not monitored in spite of the fact that several studies have proved that they can persist in feathers. Currently though, no evidence has been presented regarding the behaviour of lincomycin in this matrix. Hence, this work intended to assess the depletion of lincomycin residues in feathers of birds treated with therapeutic doses and compare them with those detected in muscle and liver samples. Samples were collected for several days after ceasing treatment from a group of broiler chickens treated with a 25% lincomycin formulation. Methanol and Florisil® columns were used to extract and retain the analyte, and samples were analysed using a triple quadrupole mass spectrometer (API 5500, AB SCIEX™). On day 1 after ceasing treatment, average concentrations of lincomycin detected in feather samples reached up to 8582 µg kg-1 and by day 16, these had only declined by 63%, to an average of 3138 µg kg-1. Lincomycin residues were detected in feathers at every sampling point, even after they were not detectable in edible tissues. Depletion time was 98 days for feathers, considering the LOQ established for the methodology as cut-off value for the calculations. Data showed that lincomycin is highly persistent in feathers, which may result in this matrix becoming a re-entry route for its residues into the food chain.

Optimization of florfenicol dose against Piscirickettsia salmonis in Salmo salar through PK/PD studies.

Salmonid Rickettsial Septicemia (SRS) is the disease of greatest economic importance in the Chilean salmon farming industry, causing high mortality in fish during the final stage of their productive cycle at sea. Since current, commercially available vaccines have not demonstrated the expected efficacy levels, antimicrobials, most commonly florfenicol, are still the main resource for the treatment and control of this pathogen. The aim of this study was to determine the most appropriate single dose of florfenicol, administered through medicated feed, for the treatment of Piscirickettsia salmonis (P. salmonis), using pharmacokinetic/pharmacodynamic (PK/PD) models. Previously, Minimum Inhibitory Concentrations (MICs) of florfenicol were determined for 87 P. salmonis isolates in order to define the epidemiological cut-off point (COWT). The most commonly observed MIC was 0.125 µg mL-1 (83.7%). The COWT value was 0.25 µg mL-1 with a standard deviation of 0.47 log2 µg mL-1 and 0.36 log2 µg mL-1, for Normalized resistance interpretation (NRI) method and ECOFFinder method, respectively. A MIC of 1 µg mL-1 was considered the pharmacodynamic value (PD) to define PK/PD indices. Three doses of florfenicol were evaluated in fish farmed under controlled conditions. For each dose, 150 fish were used and blood plasma samples were collected at different time points (0-48 hours). PK parameters were obtained from curves representing plasma concentrations as a function of time. The results of Monte Carlo simulation indicate that at a dose of 20 mg/Kg l.w. of florfenicol, administered orally as medicated feed, there is 100% probability (PTA) of achieving the desired efficacy (AUC0-24h/MIC>125). According to these results, we suggest that at the indicated dose, the PK/PD cut-off point for florfenicol versus P. salmonis could be 2 µg mL-1 (PTA = 99%). In order to assess the indicated dose in Atlantic salmon, fish were inoculated with P. salmonis LF-89 strain and then treated with the optimized dose of florfenicol, 20 mg/Kg bw for 15 days.

Implementation and Validation of an Analytical Method for Lincomycin Determination in Feathers and Edible Tissues of Broiler Chickens by Liquid Chromatography Tandem Mass Spectrometry.

Recent studies have detected different antimicrobial residues in broiler chicken feathers, where they persisted for longer periods of time and at greater concentrations than in edible tissues. However, until today, lincomycin behaviour in this nonedible tissue has not been assessed yet. Considering this, an analytical methodology to detect and quantify this antibiotic concentration in feathers, muscle, and liver tissues from broiler chickens was implemented and in-house validated. The methodology will allow the determination of the bioaccumulation of this highly persistent antibiotic in feathers of treated birds. For this purpose, 98% lincomycin and 95% lincomycin D3 standards were used. Methanol was selected as the extraction solvent, and Chromabond® Florisil® cartridges were used for the clean-up stage. The separation of analytes was performed through the analytical column SunFire C18 with a running time of 4 minutes, and the instrumental analysis was performed through an LC-MS/MS, with a liquid chromatograph Agilent® 1290 Infinity, coupled to an AB SCIEX® API 5500 mass spectrometer. An internal protocol for an in-house validation was designed based on recommendations from Commission Decision 2002/657/EC and the Guidance document on the estimation of limit of detection and limit of quantification for measurements in the field of contaminants in feed and food. The average retention time for lincomycin was 2.255 min (for quantifier ion, 126.0). The calibration curves showed a coefficient of determination (r 2) greater than 0.99 for all matrices, while recovery levels ranged between 98% and 101%. The limit of detection (LOD) calculated was of 19, 22, and 10 µg·kg-1, and the limit of quantification (LOQ) was of 62, 73, and 34 µg·kg-1 in feathers, muscle, and liver, respectively. This method detects lincomycin in the studied matrices, confidently and accurately, as it is required for designing analytical studies of drug residues in edible and nonedible tissues, such as feathers.

Assessment of Three Antimicrobial Residue Concentrations in Broiler Chicken Droppings as a Potential Risk Factor for Public Health and Environment.

Tetracyclines, sulfonamides and amphenicols are broad spectrum antimicrobial drugs that are widely used in poultry farming. However, a high proportion of these drugs can be excreted at high concentrations in droppings, even after the end of a therapy course. This work intended to assess and compare concentrations of florfenicol (FF), florfenicol amine (FFa), chlortetracycline (CTC), 4-epi-chlortetracycline (4-epi-CTC), and sulfachloropyridazine (SCP) in broiler chicken droppings. To this end, 70 chickens were housed under controlled environmental conditions, and assigned to experimental groups that were treated with therapeutic doses of either 10% FF, 20% CTC, or 10% SCP. Consequently, we implemented and designed an in-house validation for three analytical methodologies, which allowed us to quantify the concentrations of these three antimicrobial drugs using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Our results showed that FF and FFa concentrations were detected in chicken droppings up to day 10 after ceasing treatment, while CTC and 4-epi-CTC were detected up to day 25. As for SCP residues, these were detected up to day 21. Noticeably, CTC showed the longest excretion period, as well as the highest concentrations detected after the end of its administration using therapeutic doses.

Residue Depletion of Florfenicol and Florfenicol Amine in Broiler Chicken Claws and a Comparison of Their Concentrations in Edible Tissues Using LC⁻MS/MS.

Antimicrobial residues might persist in products and by-products destined for human or animal consumption. Studies exploring the depletion behavior of florfenicol residues in broiler chicken claws are scarce, even though claws can enter the food chain directly or indirectly. Hence, this study intended to assess the concentrations of florfenicol (FF) and florfenicol amine (FFA)-its active metabolite-in chicken claws from birds that were treated with a therapeutic dose of florfenicol. Furthermore, concentrations of these analytes in this matrix were compared with their concentrations in edible tissues at each sampling point. A group of 70 broiler chickens were raised under controlled conditions and used to assess residue depletion. Sampling points were on days 5, 10, 20, 25, 30, 35, and 40 after ceasing treatment, thus extending beyond the withdrawal period established for muscle tissue (30 days). Analytes were extracted using HPLC-grade water and acetone, and dichloromethane was used for the clean-up stage. Liquid chromatography coupled to mass spectroscopy detection (LC⁻MS/MS) was used to detect and quantify the analytes. The analytical methodology developed in this study was validated in-house and based on the recommendations described in the Commission Decision 2002/657/EC from the European Union. Analyte concentrations were calculated by linear regression analysis of calibration curves that were fortified using an internal standard of chloramphenicol-d5 (CAF-d5). The depletion time of FF and FFA was set at 74 days in claws, based on a 95% confidence level and using the limit of detection (LOD) as the cut-off point. Our findings show that FF and FFA can be found in chicken claws at higher concentrations than in muscle and liver samples at each sampling point.

Assessment of Consumer Perceptions on the Use of Antimicrobials in Production Animals in Chile.

Successful treatment of diseases that sicken food-producing animals requires the use of antimicrobials. However, these drugs could result in serious consequences for human, animal, and environmental health. The emergence of antimicrobial resistance requires better communication with consumers to inform them about the risks associated with the use of these drugs, as well as the benefits gained from their rational use. This work studied consumer perceptions about the use of antimicrobials in the production of animal origin foods by questioning 72 consumers in nine focus groups. The participants were all inhabitants of the Metropolitan Region, Chile, and were assigned to focus groups of 5 to 12 individuals each, according to their age: age groups I, II, and III spanned age ranges of 25 to 35, 36 to 50, and older than 50 years, respectively. Consumers regarded the use of antimicrobials in farm animals as a relevant and necessary practice, as long as it was done adequately by veterinarians to ensure productivity and to guarantee food quality and safety. Even though most consumers identified antimicrobial use as a potential risk to their health, only a few recognized that antimicrobial resistance could be transferred from animals to humans via food consumption. Also, consumers complained that they did not have access to enough information about foods of animal origin. Consequently, they stated that food industry and regulatory entities should ensure transparency of production processes and also educate the population on this topic. Consumer perceptions about responsible and judicious use of antimicrobials may become an ally in the effort to reduce and improve the use of these drugs in animal production systems.

Determination of sulfachloropyridazine residue levels in feathers from broiler chickens after oral administration using liquid chromatography coupled to tandem mass spectrometry.

Several antimicrobials are routinely used by the poultry farming industry on their daily operations, however, researchers have found for some antimicrobials that their residues persist for longer periods in feathers than they do in edible tissues, and at higher concentrations, as well. But this information is not known for other classes of antimicrobials, such as the sulfonamides. Therefore, this work presents an accurate and reliable analytical method for the detection of sulfachloropyridazine (SCP) in feathers and edible tissues from broiler chickens. This method was also validated in-house and then used to study the depletion of sulfachloropyridazine in those matrices. The experimental group comprised 54 broiler chickens, who were raised under controlled conditions and then treated with a commercial formulation of 10% sulfachloropyridazine for 5 days. Samples were analyzed via LC-MS/MS, using 13C6-sulfamethazine (SMZ-13C6) as an internal standard. Aromatic sulfonic acid solid phase extraction (SPE) cartridges were used to clean up the samples. The Limit of Detection (LOD) for this method was set at 10 µg kg-1 on feathers and liver; and at 5 µg kg-1 on muscle. Within the range of 10-100 µg kg-1, the calibration curves for all matrices presented a determination coefficient greater than 0.96. Our results show, with a 95% confidence level, that sulfachloropyridazine persisted in feathers for up to 55 days after ceasing treatment, and its concentrations were higher than in edible tissues. In consequence, to avoid re-entry of antimicrobial residues into the food-chain, we recommend monitoring and inspecting animal diets that contain feather derivatives, such as feathers meals, because they could be sourced from birds that might have been medicated with sulfachloropyridazine.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens.

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A), tet(B), and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.

BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage.

BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment.