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The dry-slaughter process for poultry includes mechanical defeathering followed by a waxing phase, without scalding. The aim of the present work was to study the impact of dry-slaughter procedures on the microbiological quality of poultry carcasses. A total of 161 laying hens were examined. Sponge swab samples were collected from different carcass sites at three successive processing stages (i.e. from the neck after stunning and from the breast after defeathering and after waxing). Each carcass sample was analyzed for counts of total aerobic mesophilic bacteria, coliforms, Escherichia coli, Staphylococcus aureus and the incidence of Salmonella spp. In addition, fecal swab samples collected from each bird were analyzed for the presence of Salmonella spp. The microbiological analysis results showed low levels of carcass contamination for each microorganism, at all stages of the dry-slaughter process. However, an increase in the levels of Staphylococcus aureus was recorded in the post-waxing phase. Salmonella spp. was not found in any of the samples tested. [ABSTRACT FROM AUTHOR]

Rinse water samples were obtained from fifty cattle carcasses processed in a northern Italian slaughterhouse between January and April 2007. The incidence and prevalence of Campylobacter spp. in these samples were determined. Conventional cultural techniques, followed by a rapid latex agglutination test were used for testing. A polymerase chain reaction (PCR) followed by DNA sequencing was used to identify the thermophilic Campylobacter. Campylobacter spp. were detected in four (8%) samples using the cultural methods and in six (12%) samples using molecular techniques. Campylobacter jejuni was the only species detected in these samples. The results indicate a relatively low level of Campylobacter spp. contamination on the examined cattle carcasses. [ABSTRACT FROM AUTHOR]

Canine oral melanoma (COM) is a promising target for immunomodulatory therapies aimed at enhancing the immune system's antitumor response. Given that adipose-derived mesenchymal stem cells (Ad-MSCs) possess immunomodulatory properties through cytokine release, we hypothesized that co-culturing Ad-MSCs and canine peripheral blood mononuclear cells (PBMCs) could stimulate interleukin (IL) production against melanoma cell lines (MCCLs) and help identify therapeutic targets. This study evaluated IL-2, IL-8, and IL-12 expressions in co-culture with MCCL, Ad-MSCs, and PBMCs and assessed the relationship between gene expression, cell viability, and migration. Using four experimental groups in a Transwell insert system to separate cell types, we found that Ad-MSCs can reduce MCCL migration and viability, though the effect may vary depending on each cell line's susceptibility. Furthermore, Ad-MSCs modified IL expression profiles in co-cultured cells. Our findings suggest that Ad-MSCs could have therapeutic potential for COM by inhibiting cell migration and reducing viability. However, deeper insights into Ad-MSC interactions with the tumor microenvironment and melanoma-specific factors will be essential to optimize therapeutic efficacy.

Autism spectrum disorders (ASD) are neurodevelopmental disorders. Genetic factors, along with non-genetic triggers, have been shown to play a causative role. Despite the various causes, a triad of common symptoms defines individuals with ASD; pervasive social impairments, impaired social communication, and repeated sensory-motor behaviors. Therefore, it can be hypothesized that different genetic and environmental factors converge on a single hypothetical neurobiological process that determines these behaviors. However, the cellular and subcellular signature of this process is, so far, not well understood. Here, we performed a comparative study using "omics" approaches to identify altered proteins and, thereby, biological processes affected in ASD. In this study, we mined publicly available repositories for genetic mouse model data sets, identifying six that were suitable, and compared them with in-house derived proteomics data from prenatal zinc (Zn)-deficient mice, a non-genetic mouse model with ASD-like behavior. Findings derived from these comparisons were further validated using in vitro neuronal cell culture models for ASD. We could show that a protein network, centered on VAMP2, STX1A, RAB3A, CPLX2, and AKAP5, is a key convergence point mediating synaptic vesicle release and recycling, a process affected across all analyzed models. Moreover, we demonstrated that Zn availability has predictable functional effects on synaptic vesicle release in line with the alteration of proteins in this network. In addition, drugs that target kinases, reported to regulate key proteins in this network, similarly impacted the proteins' levels and distribution. We conclude that altered synaptic stability and plasticity through abnormal synaptic vesicle dynamics and function may be the common neurobiological denominator of the shared behavioral abnormalities in ASD and, therefore, a prime drug target for developing therapeutic strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Human skin is populated by various microorganisms, the so-called microbiota, such as bacteria, viruses, yeasts, fungi, and archaea. The skin microbiota is in constant contact with the surrounding environment which can alter its eubiotic state. Recently it has been also observed that the application of cosmetic products can alter the balance of the skin microbiota. This effect may be attributed to many factors including the residual activity of the preservatives on the skin. In the present work, we studied the effect of eleven preservatives commonly found in cosmetic products on Propionibacterium acnes, Staphylococcus epidermidis, and Staphylococcus aureus in vitro using 3D skin models and culture-dependent methods. Also, the effect on Histone deacetylase 3 (HDAC3) has been investigated. Among tested combinations, three resulted as the best suitable for restoring a pre-existing dysbiosis since they act moderately inhibiting C. acnes and strongly S. aureus without simultaneously inhibiting the growth of S. epidermidis. The other four combinations resulted as the best suitable for use in topical products for skin and scalp in which it is necessary to preserve the eubiosis of the microbiota. Some of the tested were also able to increase HDAC3 expression. Taking together these data highlight the role of preservatives of skin resident microflora dynamics and could provide a reference for correctly choice preservatives and dosage in cosmetic formulations to preserve or restore homeostasis of skin microbiota.

Black point is a fungal disease of wheat, mainly associated with mycotoxigenic Alternaria species. Affected wheat kernels are characterized by dark brown discolouration of the embryo region and reduction of grain quality. Potential risk is the possible accumulation of Alternaria mycotoxins, alternariol (AOH), alternariol-monomethyl ether (AME), tenuazonic acid (TA), and altenuene (ALT), provided by haemato-toxic, genotoxic, and mutagenic activities. One hundred and twenty durum wheat samples belonging to 30 different genotypes grown in Bologna and Modena areas, in Italy, showing black point symptoms, were analyzed for Alternaria species and their mycotoxin contamination. Alternariol was selected as an indicator of the capability of the Alternaria species to produce mycotoxin in vivo in field conditions. The data showed that Alternaria species occurred in 118 out of 120 wheat kernels samples, with the incidence of infected kernels ranging between 1% and 26%. Moreover, AOH was detected by using a HPLC with a diode array detector (LC-DAD) in 98 out of 120 samples with values ranging between 24 and 262 µg Kg -1 . Ninety-two Alternaria representative strains, previously identified morphologically, were identified at species/section level using gene sequencing, and therefore were analyzed for their mycotoxin profiles. Eighty-four strains, phylogenetically grouped in the Alternaria section, produced AOH, AME, and TA with values up to 8064, 14,341, and 3683 µg g -1 , respectively, analyzed by using a LC-DAD. On the other hand, eight Alternaria strains, included in Infectoriae Section, showed a very low or no capability to produce mycotoxins.

Background: CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins., Methods: We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile., Results: As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions., Conclusions: Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.

Endothelial cells are constantly exposed to blood flow and the resulting frictional force, the wall shear stress, varies in magnitude and direction with time, depending on vasculature geometry. Previous studies have shown that the structure and function of endothelial cells, and ultimately of the vessel wall, are deeply affected by the nature of wall shear stress waveforms. To investigate the in vitro effects of these stimuli, we developed a compact, programmable, real-time operated system based on cone-and-plate geometry, that can be used within a standard cell incubator. To verify the capability to replicate realistic shear stress waveforms, we calculated both analytically and numerically to what extent the system is able to correctly deliver the stimuli defined by the user at plate level. Our results indicate that for radii greater than 25 mm, the shear stress is almost uniform and directly proportional to cone rotation velocity. We further established that using a threshold of 10 Hz of wall shear stress waveform frequency components, oscillating flow conditions can be reproduced on cell monolayer surface. Finally, we verified the capability of the system to perform long-term flow exposure experiments ensuring sterility and cell culture viability on human umbilical vein endothelial cells exposed to unidirectional and oscillating shear stress. In conclusion, the system we developed is a highly dynamic, easy to handle, and able to generate pulsatile and unsteady oscillating wall shear stress waveforms. This system can be used to investigate the effects of realistic stimulations on endothelial cells, similar to those exerted in vivo by blood flow., Competing Interests: All the authors have declared no competing interests.

The arteriovenous fistula has been used for more than 50 years to provide vascular access for patients undergoing hemodialysis. More than 1.5 million patients worldwide have end stage renal disease and this population will continue to grow. The arteriovenous fistula is the preferred vascular access for patients, but its patency rate at 1 year is only 60%. The majority of arteriovenous fistulas fail because of intimal hyperplasia. In recent years, there have been many studies investigating the molecular mechanisms responsible for intimal hyperplasia and subsequent thrombosis. These studies have identified common pathways including inflammation, uremia, hypoxia, sheer stress, and increased thrombogenicity. These cellular mechanisms lead to increased proliferation, migration, and eventually stenosis. These pathways work synergistically through shared molecular messengers. In this review, we will examine the literature concerning the molecular basis of hemodialysis vascular access malfunction., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Intimal hyperplasia (IH) is the first cause of failure of an arteriovenous fistula (AVF). The aim of the present study was to investigate the effects on endothelial cells (ECs) of shear stress waveforms derived from AVF areas prone to develop IH. We used a cone-and-plate device to obtain real-time control of shear stress acting on EC cultures. We exposed human umbilical vein ECs for 48 h to different shear stimulations calculated in a side-to-end AVF model. Pulsatile unidirectional flow, representative of low-risk stenosis areas, induced alignment of ECs and actin fiber orientation with flow. Shear stress patterns of reciprocating flow, derived from high-risk stenosis areas, did not affect EC shape or cytoskeleton organization, which remained similar to static cultures. We also evaluated flow-induced EC expression of genes known to be involved in cytoskeletal remodeling and expression of cell adhesion molecules. Unidirectional flow induced a significant increase in Kruppel-like factor 2 mRNA expression, whereas it significantly reduced phospholipase D1, α4-integrin, and Ras p21 protein activator 1 mRNA expression. Reciprocating flow did not increase Kruppel-like factor 2 mRNA expression compared with static controls but significantly increased mRNA expression of phospholipase D1, α4-integrin, and Ras p21 protein activator 1. Reciprocating flow selectively increased monocyte chemoattractant protein-1 and IL-8 production. Furthermore, culture medium conditioned by ECs exposed to reciprocating flows selectively increased smooth muscle cell proliferation compared with unidirectional flow. Our results indicate that protective vascular effects induced in ECs by unidirectional pulsatile flow are not induced by reciprocating shear forces, suggesting a mechanism by which oscillating flow conditions may induce the development of IH in AVF and vascular access dysfunction., (Copyright © 2016 the American Physiological Society.)

MYCN is an oncogene frequently overexpressed in pediatric solid tumors whereas few evidences suggest his involvement in the pathogenesis of haematologic malignancies. Here we show that MYCN is overexpressed in a relevant proportion (40 to 50%) of adult and pediatric T-cell acute lymphoblastic leukemias (T-ALL). Focusing on pediatric T-ALL, MYCN-expressing samples were found almost exclusively in the TAL1-positive subgroup. Moreover, TAL1 knockdown in T-ALL cell lines resulted in a reduction of MYCN expression, and TAL1 directly binds to MYCN promoter region, suggesting that TAL1 pathway activation could sustain the up-regulation of MYCN. The role of MYCN in T-ALL was investigated by peptide nucleic acid (PNA-MYCN)-mediated transcriptional silencing of MYCN and by siRNAs. MYCN knockdown in T-ALL cell lines resulted in a reduction of cell viability, up to 50%, while no effect was elicited with a mismatch PNA. The inhibitory effect of PNA-MYCN on cell viability was due to a significant increase in apoptosis. PNA-MYCN treatment in pediatric T-ALL samples reduced cell viability of leukemic cells from patients with high MYCN expression, while no effect was obtained in MYCN-negative blast cells. These results showed that MYCN is frequently overexpressed in pediatric T-ALL and suggested his role as a candidate for molecularly-directed therapies.

Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription., Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined., Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity., Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo.

Even if the overall survival of children with cancer is significantly improved over these decades, the cure rate of high-risk pediatric solid tumors such as neuroblastoma, Ewing's sarcoma family tumors or rhabdomiosarcoma remain challenging. Autologous hematopoietic stem cell transplantation (HSCT) allows chemotherapy dose intensification beyond marrow tolerance and has become a fundamental tool in the multimodal therapeutical approach of these patients. Anyway this procedure does not allow to these children an event-free survival approaching more than 50% at 5 years. New concepts of allogeneic HSCT and in particular HLA-mismatched HSCT for high risk solid tumors do not rely on escalation of chemotherapy intensity and tumor load reduction but rather on a graft-versus-tumor effect. We here report an experimental study design of HLA-mismatched HSCT for the treatment of pediatric solid tumors and the inherent preliminary results.

All-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). Nevertheless, most of these patients develop RA resistance and relapse. In an attemp to mimic clinical conditions for the treatment of leukemia, a stably RA-resistant subclone of the human promyelocytic leukemia cell line HL60 (HL60-R) was developed to study the antiproliferative and proapoptotic effect of the retinoid IIF (6-OH-11-O-hydroxyphenantrene) in comparison with RA. Moreover whether the inhibitor of histone deacetylase (HDAC) activity, valproic acid (VPA), could enhance sensitivity to retinoids in HL60-R cells was evaluated. Finally, the effect of IIF on the expression of multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp) was evaluated. It was found that IIF strongly suppressed cell proliferation (as measured by growth curves) and induced apoptosis (as measured by DNA fragmentation and Annexin V detection assays), while RA was practically ineffective. The addition of VPA to IIF accentuated the antiproliferative effect of IIF alone and increased apoptosis; the combination of VPA with RA allowed growth arrest. Moreover IIF caused a reduction of transmembrane transporter expression, particularly of P-gp, as shown by Western blotting. Our results suggest that IIF may be useful in controlling the proliferation of RA-resistant leukemia cells, especially in combination with an HDAC inhibitor, such as VPA.

In cardiac cells the effects of n-3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n-3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n-3 PUFAs is related to a direct modulation of genes in cardiac cells.

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

The MLL-AF9 oncogene - one of the most frequent MLL/HRX/ALL-1 rearrangements found in infantile and therapy-related leukaemias - originates from t(9;11)(p22;q23) and is mainly associated with monocytic acute myeloid leukaemia (AML-M5; FAB-classification). Here, we investigated the MLL-AF9 function by means of an antisense phosphorothioate-oligodeoxyribonucleotide (MLL-AF9-PS-ODNas) using the THP-1 AML-M5 cell line carrying t(9;11). Having confirmed that MLL-AF9-PS-ODNas induces strong inhibition of THP-1 cell growth, but only a moderate increase in apoptosis, we found that MLL-AF9-PS-ODNas did not induce morpho-functional terminal differentiation or restore M-CSF-, G-CSF- or GM-CSF-induced differentiation. Moreover, THP-1 cells showed the same phenotype with/without MLL-AF9-PS-ODNas. In THP-1 cells differentiated to mature macrophage-like cells by PMA/TPA or ATRA, MLL-AF9 expression was downregulated. Thus, in the monocytic lineage, MLL-AF9 may be expressed only in early phases and can induce deregulated amplification in both nonmalignant and malignant cells, maintaining the monocytic phenotype without blocking final maturation. Our findings suggest that: (1) as well as directly promoting cell growth, MLL-AF9 may also indirectly determine phenotype; (2) other leukaemogenic mutations associated with MLL-AF9-related leukaemias should be searched for mainly in processes of resistance to apoptosis (where MLL-AF9 may play only a limited role) and differentiation blockage (where MLL-AF9 may play no role).