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Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1–targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy. Human primary and metastatic tumors harbor CD4+ Treg cells that can suppress antitumor immune responses. Bonnal et al. identify an intratumoral type 1 Treg-like CD4+ T cell subset that expresses the transcription factor EOMES, granzyme K and CHI3L2. This EOMES+ T cell subset correlates with disease progression but is responsive to PD-1 checkpoint blockade immunotherapy.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues., The role of epigenetic deregulation in colorectal cancer (CRC) is not fully understood yet. Here the authors use patient-derived organoids, epigenomics and single-cell RNA-seq to reveal that YAP/TAZ are key regulators that bind to active enhancers in CRC and promote tumour survival.

In a scenario of climate change and risk of losing biodiversity, the safeguard of locally adapted breeds and their genetic reservoirs assumes a key role. Furthermore, these breeds are linked to their rearing territory maintenance also through the nutritional, economic or social value of their products. This study focussed on the analysis of milk protein genetic variability of the Piedmontese, the most abundant beef Italian breed, and the Barà-Pustertaler breed, through the testing of individual milk samples by isoelectrofocusing. Allelic and haplotypes frequencies were analysed and the presence of rare variants (CSN1S1*G, 0.7%, and CSN2*C, 0.7%, in Barà and Piedmontese breeds, respectively) was detected. The analysed breeds showed allelic frequencies close to the one found in breeds with good cheesemaking aptitude, like the Italian Brown and Reggiana breeds (e.g. CSN2*B, 20.7% and 16.4%, and CSN3*B, 41,1% and 35%, in Piedmontese and Barà breeds, respectively), and are a good reservoir of alleles that are decreasing (e.g. CSN2*B) in Italian Holstein. Moreover, they have higher frequencies of interesting haplotypes (e.g. BA1B, 8% and 11.2%, and BA2B, 20.8 and 13.1% in Piedmontese and Barà breeds, respectively) that could positively influence composition, nutritional, and technological properties of milk. These breeds could be used to further investigate the effects of variants and haplotypes that could help support the evolution and the changing demands of the dairy sector.Highlights In order to address the problem of climate change effects on animal biodiversity, local breeds should be safeguarded and valorised. The analysed breeds have discrete frequencies of milk protein variants and casein haplotypes associated with good technological properties. Knowing the genetic background of non-dairy commercial breeds is the first step to preserve potentially useful genetic variability.

BackgroundPrevious studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates.MethodsFrom March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome.ResultsTwenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395.ConclusionsThis WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.

The objective of this study was to evaluate the effects of the dietary supplementation of canthaxanthin and cholecalciferol (25OHD3) to broiler breeders and their progenies on their performance. Eighty 25-wk-old Cobb 500 broiler breeders were distributed according to a completely randomized design in 2 experimental treatments, with 10 replicates of 4 birds each. The treatments consisted of the supplementation or not of a corn–soybean meal diet with canthaxanthin + 25OHD3 to broiler breeders fed from 25 to 62 wk of age. Egg production, fertility, hatchability, hatchability of fertile eggs, embryonic mortality, and egg specific gravity were evaluated. Breeders were inseminated at 35, 45, and 62 wk of age, the eggs were incubated, and the performance of the progenies was evaluated. From the progeny of each breeder age, 300 male chicks were distributed according to a completely randomized design in a 2 × 2 factorial arrangement (chicks from breeders supplemented or not with canthaxanthin + 25OHD3 and chicks supplemented or not with canthaxanthin + 25OHD3), totaling 4 treatments with 5 replicates of 15 birds each. Canthaxanthin + 25OHD3 were supplied to the chicks until 21 D of age. The combination of canthaxanthin and 25OHD3, containing 69mg of 25OHD3 and 6g of canthaxanthin, was supplemented at 1kg/t of feed. Breeders supplemented with canthaxanthin + 25OHD3 showed higher egg production, total hatchability, hatchability of fertile eggs, and lower early embryo mortality compared with those fed the control diet. Broilers from breeders fed canthaxanthin + 25OHD3 and supplemented with this additive up to 21 D of age presented a better feed conversion ratio and higher carcass and breast yields than those derived from nonsupplemented breeders.

Local adaptation of animals to the environment can abruptly become a burden when faced with rapid climatic changes such as those foreseen for the Italian peninsula over the next 70 years. Our study investigates the genetic structure of the Italian goat populations and links it with the environment and how genetics might evolve over the next 50 years. We used one of the largest national datasets including 1000 goats from 33 populations across the Italian peninsula collected by the Italian Goat Consortium and genotyped with over 50 k markers. Our results showed that Italian goats can be discriminated in three groups reflective of the Italian geography and its geo-political situation preceding the country unification around two centuries ago. We leveraged the remarkable genetic and geographical diversity of the Italian goat populations and performed landscape genomics analysis to disentangle the relationship between genotype and environment, finding 64 SNPs intercepting genomic regions linked to growth, circadian rhythm, fertility, and inflammatory response. Lastly, we calculated the hypothetical future genotypic frequencies of the most relevant SNPs identified through landscape genomics to evaluate their long-term effect on the genetic structure of the Italian goat populations. Our results provide an insight into the past and the future of the Italian local goat populations, helping the institutions in defining new conservation strategy plans that could preserve their diversity and their link to local realities challenged by climate change.

Introduction Honey is known for exhibiting antibacterial properties, indicating its use as part of traditional medicine since the early ages. With the advent of antibiotic-resistant bacteria, the need for alternative antimicrobials has outpaced the actual development of novel, broad-spectrum antibiotics. Previous research has revolved around the sugar content of honey because its sweetness makes it an attractive food source. However, research assessing the protein and lipid components of honey is lagging behind that of its sugar counterpart. The goal of this investigation was to examine the antimicrobial properties of honey and to identify any distinct proteins or lipids. Methods In order to isolate individual peptides and lipids, the different samples of local and foreign-sourced honeys were dialyzed, and the resulting dialysate proteins were screened via gel electrophoresis (sodium dodecyl-sulfate polyacrylamide gel electrophoresis [SDS-PAGE]) with Coomassie blue and silver stain, while lipids were examined using thin layer chromatography (TLC). To assess antimicrobial potency, a series of Kirby-Bauer disc diffusion assays was performed on Mueller-Hinton agar using different types of raw honey with Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis. The process was then repeated using the peptide extracts from the dialyzed fractions of the honeys. Results The SDS-PAGE trials revealed repetitive promising protein bands across several gels below 75kDa with both Coomassie blue and silver staining. The TLC analysis of varying raw honey samples consistently demonstrated the presence of medium and long-chain fatty acids, likely in the range of C12-C14. In the disc diffusion assays, the greatest amount of inhibition was seen when the honeys were tested as a whole instead of its constituent parts. Conclusion Instead of an individual component acting as the key to honey's action against bacteria, it appears there is a synergistic relationship amongst the sugars, proteins, and lipids that make each honey unique.

The sperm of each avian species and breed have unique characteristics that render them more or less susceptible to the freezing–thawing process, therefore, a suitable cryopreservation protocol that is specific for the sperm of each type of bird is needed. In this context, little information about the common pheasant’s sperm is available. Therefore, the aim of this study was to test different parameters at each step of the process of freezing into pellets and thawing to detect the least deleterious parameter settings. Sixteen different protocols were tested by studying two levels in each of the four steps (dilution, equilibration at 5 °C, final dimethylacetamide concentration, and dimethylacetamide equilibration time) comprising the freezing process. The pheasant sperm exhibited a high susceptibility to the damage caused by freezing into pellets, however, the survival of the sperm reached 29%, and the greatest recovered mobility was 22%. The mobility of the sperm was affected by the dilution and the dimethylacetamide concentration, and the viability of the sperm was affected by the equilibration at 5 °C and the dimethylacetamide equilibration. The protocols that caused the least damage to the pheasant sperm were found to be those with higher dilution rates, 10 min of equilibration at 5 °C, and 6% dimethylacetamide equilibrated for 1 or 5 min. In the present study, we individualise some applicable parameters for certain critical steps of the freezing–thawing process, however, further investigations are needed in order to improve upon and complete a suitable protocol for the cryopreservation and thawing of pheasant sperm.

The intensive use of high-performing strains in poultry production has led to the extinction of several autochthonous chicken breeds and, consequently, loss of genetic variability. Interest in saving biodiversity is growing rapidly and has become a major objective worldwide. The aim of this study was to shed light on the production trends of native Italian poultry breeds and the related market. A questionnaire, which asked about the production cycles, the number of animals and table eggs produced per year and their retail prices was completed by 121 breeders across Italy. The surveyed breeders were divided into two categories: breeders conducting an agrozootechnical farm, referred to as ‘farmers’ (F), and breeders keeping chickens as backyard poultry, referred to as ‘fancy breeders’ (FB). Analysis of the data acquired indicated that animals were mainly slaughtered between 6 and 12 months of age, with F processing more animals per year. The same production trend was observed for table eggs. The recorded retail prices of native chicken products were higher than those for conventional products, but similar to those reported for valuable niche poultry products, such as the Poulet de Bresse in France and organic eggs. Knowledge about these highly valuable markets should be used to encourage the use of local breeds in alternative poultry farming and help protect biodiversity.

The preservation of genetic variability of autochthonous poultry breeds is crucial in global biodiversity. A recent report revealed small breed size and potential risk of extinction of all native Italian poultry breeds; therefore, a correct assessment of their genetic diversity is necessary for a suitable management of their preservation. In this work, we provided an overview of the contribution to poultry biodiversity of some Italian autochthonous breeds reared in conservation centers devoted to local biodiversity preservation. The level of genetic diversity, molecular kinship, inbreeding, contribution to overall genetic diversity, and rate of extinction of each breed were analyzed with a set of 14 microsatellite loci in 17 autochthonous chicken breeds. To evaluate genetic variability, total number (Na), and effective number (Ne) of alleles, observed (Ho) and expected (He) heterozygosity, and F (Wright’s inbreeding coefficient) index were surveyed. The contribution of each analyzed breed to genetic diversity of the whole dataset was assessed using MolKin3.0; global genetic diversity and allelic richness contributions were evaluated. All the investigated loci were polymorphic; 209 alleles were identified (94 of which private alleles). The average number of alleles per locus was 3.62, and the effective number of alleles was 2.27. The Ne resulted lower in all breeds due to the presence of low-frequency alleles that can be easily lost by genetic drift, thus reducing the genetic variability of the breeds, and increasing their risk of extinction. The global molecular kinship was 27%, the average breed molecular kinship was 53%, and the mean inbreeding rate 43%, with a self-coancestry of 78%. Wright’s statistical analysis showed a 41% excess of homozygous due to breed genetic differences (34%) and to inbreeding within the breed (9%). Genetic variability analysis showed that 11 breeds were in endangered status. The contribution to Italian poultry genetic diversity, estimated as global genetic diversity, and ranged from 30.2 to 98.5%. In conclusion, the investigated breeds maintain a unique genetic pattern and play an important role in global Italian poultry biodiversity, providing a remarkable contribution to genetic variability.

Parasites threaten human and animal health globally. It is estimated that more than 60% of people on planet Earth carry at least one parasite, many of them several different species. Unfortunately, parasite studies suffer from duplications and inconsistencies between different investigator groups. Hence, groups need to collaborate in an integrated manner in areas including parasite control, improved therapy strategies, diagnostic and surveillance tools, and public awareness. Parasite studies will be better served if there is coordinated management of field data and samples across multidisciplinary approach plans, among academic and non-academic organizations worldwide. In this paper we report the first 'Living organism-World Molecular Network', with the cooperation of 167 parasitologists from 88 countries on all continents. This integrative approach, the 'Sarcoptes-World Molecular Network', seeks to harmonize Sarcoptes epidemiology, diagnosis, treatment, and molecular studies from all over the world, with the aim of decreasing mite infestations in humans and animals. © 2011 International Society for Infectious Diseases.

Genetic risk for autism spectrum disorder (ASD) has been associated with hundreds of genes spanning a wide range of biological functions. The phenotypic alterations in the human brain resulting from mutations in ASD risk genes remain unclear, and the level at which these alterations converge on shared disease pathology is poorly understood. Here, we leveraged reproducible organoid models of the human cerebral cortex to identify cell type-specific developmental abnormalities associated with haploinsufficiency in three ASD risk genes,SUV420H1(KMT5B),PTEN, andCHD8. We performed comprehensive single-cell RNA-sequencing (scRNA-seq) of over 400,000 cells, and proteomic analysis on individual organoids sampled at different developmental stages to investigate phenotypic convergence among these genes. We find that within a defined period of early cortical development, each of the three mutations demonstrates accelerated development of cortical neurons. Notably, they do so by affecting different neuronal populations: excitatory deep layer (SUV420H1) and callosal (PTEN) neurons, and inhibitory interneurons (CHD8). This work shows that haploinsufficiency in ASD risk genes converge on early developmental defects in the generation of neurons of the cortical microcircuit.

An increasing number of patients with metastatic colorectal cancer (mCRC) are able to receive 3 or more lines of therapy. Treatments in this setting can include regorafenib (an oral multikinase inhibitor), trifluridine/tipiracil hydrochloride (TAS-102), antibodies that target epidermal growth factor receptor for patients with RAS wild-type tumors (if no prior exposure), and, where approved, anti-programmed cell death protein 1 inhibitors for patients with microsatellite instability-high mCRC. Although guidelines describe the available treatment options, few insights are provided to guide selection and sequencing. In this article, we share expert opinion from diverse geographic regions, to offer guidance for best practice when selecting and managing third-line treatment for mCRC. Various factors, including performance status, age, and tumor sidedness, can be used to guide treatment selection. Biomarkers, such as RAS, BRAF, and microsatellite instability, can be useful for treatment stratification. Management of adverse events, to maintain quality of life, is a key consideration and is crucial to best practice in this setting. Common toxicities associated with third-line treatments are hand-foot skin reaction, fatigue, diarrhea, and cytopenias. Patients who receive third-line and later-line treatments should be monitored for these events, especially during the first 2 cycles. Dose modifications can also be used to manage toxicities and to minimize the effect on quality of life, while maximizing treatment benefit. Clinical trials of emerging agents, new treatment combinations, and novel therapies continue the efforts to improve outcomes for patients with mCRC. Sharing expert opinions on best practice for treatment selection and management can ultimately improve outcomes for patients with mCRC.

Bianca di Saluzzo (BS) and Bionda Piemontese (BP) are two Italian chicken breeds, mainly reared for meat production, primarily in antibiotic-free farming. However, technical information on their growth pattern is still missing. At hatching, 150 unsexed chicks of each breed were weighed, labeled, and reared in indoor pens up to 8 w of age. At 8 w of age, the chicks were separated by sex and randomly transferred to growing pens with access to an external paddock (15 birds/pen, 4 pens/sex for each breed). The body weight (BW) was recorded biweekly for each bird, from hatching to 32 w of age. In order to identify an improvement strategy, the objectives of our study were to analyze the growth pattern of these birds using the Gompertz mathematical model and compare results with other chicken breeds. Polymorphism of the PAX7 gene was also analyzed to test its association with growth traits. Both BS and BP are close to unselected native breeds and, among the Italian local poultry, they are confirmed to be slow-growing birds with an intermediate size between heavy and light chicken breeds. Regarding the PAX7 gene, two alleles were found, F and G, and showed an association with the actual BW in the BP females from 14 w of age onwards. The G allele always exhibited a more favorable effect than the F allele. In small size poultry population, a delicate balance between preservation of biodiversity and performance improvement should be considered. Consequently, the most proper way could be an approach based on a mating scheme to keep inbreeding under control, increase growth rate, and improve commercial maturity.

AimsNeurotrophic Tropomyosin Kinase Receptor 1 (NTRK1) gene encodes for the protein Tropomyosin-related kinase A (TRKA). Deregulated activity of TRKA has been shown to have oncogenic potential. We present here the results of an immunohistochemical (IHC) observational cohort study of TRKA expression together with gene copy number (GCN) assessment in various solid tumours.MethodsFormalin-fixed, paraffin-embedded consecutive samples of different tumour types were tested for TRKA expression. Samples showing TRKA IHC staining in at least 10% of cells were analysed by fluorescence in situ hybridisation to assess NTRK1 gene rearrangements and/or individual GCN gain. All patients underwent this molecular assessment within the phase I ALKA-001 clinical trial.Results1043 samples were tested and annotation for histology was available in 1023. Most of the samples were colorectal adenocarcinoma (CRC) (n=550, 52.7%) and lung adenocarcinoma (n=312, 29.9%). 24 samples (2.3%) were biliary tract carcinoma (BTC). Overall, 17 (1.6%) samples were characterised by TRKA IHC expression (four weak, eight moderate, five strong): 9/17 lung adenocarcinoma, 3/17 CRC, 3/17 BTC, 1/17 thyroid cancer and 1/17 cancer of unknown primary. Of these, 1/17 with strong TRKA IHC staining displayed NTRK1 gene rearrangement and 15/17 NTRK1 GCN gain by FISH. No correlation was found between intensity of TRKA IHC staining and number of copies of NTRK1.ConclusionsTRKA expression can be found in 1.6% of solid tumours and can be paralleled by NTRK1 gene rearrangements or mostly GCN gain. The prognostic and translational therapeutic impact of the latter remains to be established.

ObjectiveMutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC).DesignGenome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy.ResultsMethylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival.ConclusionThis five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

The aim of investigation was to evaluate a traceability system to detect industrial chicken meat among indigenous products, considering issues that could affect assignment accuracy. The dataset included 2 Italian indigenous meat breeds, namely Bionda Piemontese (2 ecotypes) and Bianca di Saluzzo, one broiler line, and 3 layer lines. Assignment tests were performed using a standard panel of 28 microsatellite loci. To evaluate effects of inbreeding and substructure on assignment accuracy, a simulated dataset was prepared. Broilers and layers belong to homogeneous populations and never enter the clusters of indigenous breeds. Ambiguity or misallocation are expected between the Bionda ecotypes and between the 2 indigenous breeds, but it is unlikely that niche products provided by Bionda and Bianca will compete with one another. Non-random mating reduces accuracy, but only populations having weak genetic differentiation are involved, namely those that are less interesting to discriminate. The dataset can be used as a reference population to distinguish commercial meat from indigenous meat with great accuracy. Misallocations increase as number of loci decreases, but only within or between the indigenous breeds. A subpanel of the most resolving 14 loci keeps sufficient informative content to provide accuracy and to correctly allocate additional test samples within the reference population. This analytical tool is economically sustainable as a method to detect fraud or mislabeling. Adoption of a monitoring system should increase the value of typical products because the additional burden of molecular analyses would improve commercial grade and perception of quality.

Regenerative medicine aims to restore damaged tissues and mainly takes advantage of human mesenchymal stromal cells (hMSCs), either alone or combined with three-dimensional scaffolds. The scaffold is generally considered a support, and its contribution to hMSC proliferation and differentiation is unknown or poorly investigated. The aim of this study was to evaluate the capability of an innovative three-dimensional gelatin&ndash, chitosan hybrid hydrogel scaffold (HC) to activate the osteogenic differentiation process in hMSCs. We seeded hMSCs from adipose tissue (AT-hMSCs) and bone marrow (BM-hMSCs) in highly performing HC of varying chitosan content in the presence of growing medium (GM) or osteogenic medium (OM) combined with Fetal Bovine Serum (FBS) or human platelet lysate (hPL). We primarily evaluated the viability and the proliferation of AT-hMSCs and BM-hMSCs under different conditions. Then, in order to analyse the activation of osteogenic differentiation, the osteopontin (OPN) transcript was absolutely quantified at day 21 by digital PCR. OPN was expressed under all conditions, in both BM-hMSCs and AT-hMSCs. Cells seeded in HC cultured with OM+hPL presented the highest OPN transcript levels, as expected. Interestingly, both BM-hMSCs and AT-hMSCs cultured with GM+FBS expressed OPN. In particular, BM-hMSCs cultured with GM+FBS expressed more OPN than those cultured with GM+hPL and OM+FBS, AT-hMSCs cultured with GM+FBS presented a lower expression of OPN when compared with those cultured with GM+hPL, but no significant difference was detected when compared with AT-hMSCs cultured with OM+FBS. No OPN expression was detected in negative controls. These results show the capability of HC to primarily and independently activate osteogenic differentiation pathways in hMCSs. Therefore, these scaffolds may be considered no more as a simple support, rather than active players in the differentiative and regenerative process.

The objective of this study was to determine the effect of long-term supplementation of unsaturated oil on lipid metabolism and transcription of genes involved in lipid metabolism in subcutaneous adipose tissue (SAT) of mid-lactating dairy cows. The objective was achieved by supplementing dairy cows with soybean oil (SO, high in linoleic acid) or fish oil (FO, high in EPA and DHA) for 63 days (nine weeks). Cows were fed a control diet with no added lipid, or diets containing SO or FO (n = 5 cows/group). At the onset of the experiment (day 0) and on days 21, 42, and 63 of supplementation, blood and SAT samples were collected from each animal. Oil supplementation increased cholesterol and NEFA in plasma, with a greater effect of SO compared to FO. Concentration of BUN was lower in SO compared to control and FO at the end of the trial. Transcription of few genes was affected by dietary lipids: FABP4 had lowest expression in FO followed by SO and control. ACACA and FASN had higher expression in FO. Transcription of SCAP was higher but expression of INSIG1 was lower in SO. Overall, results revealed that compared to control, SO and FO had lipogenic effect in SAT.

The tropomyosin receptor kinase (TRK) family of receptor tyrosine kinases are encoded by NTRK genes and have a role in the development and normal functioning of the nervous system. Since the discovery of an oncogenic NTRK gene fusion in colorectal cancer in 1986, over 80 different fusion partner genes have been identified in a wide array of adult and paediatric tumours, providing actionable targets for targeted therapy. This review describes the normal function and physiology of TRK receptors and the biology behind NTRK gene fusions and how they act as oncogenic drivers in cancer. Finally, an overview of the incidence and prevalence of NTRK gene fusions in various types of cancers is discussed.

The limited access to functional human brain tissue has led to the development of stem cell-based alternative models. The differentiation of human pluripotent stem cells into cerebral organoids with self-organized architecture has created novel opportunities to study the early stages of the human cerebral formation. Here we applied state-of-the-art label-free shotgun proteomics to compare the proteome of stem cell-derived cerebral organoids to the human fetal brain. We identified 3,073 proteins associated with different developmental stages, from neural progenitors to neurons, astrocytes, or oligodendrocytes. The major protein groups are associated with neurogenesis, axon guidance, synaptogenesis, and cortical brain development. Glial cell proteins related to cell growth and maintenance, energy metabolism, cell communication, and signaling were also described. Our data support the variety of cells and neural network functional pathways observed within cell-derived cerebral organoids, confirming their usefulness as an alternative model. The characterization of brain organoid proteome is key to explore, in a dish, atypical and disrupted processes during brain development or neurodevelopmental, neurodegenerative, and neuropsychiatric diseases. ispartof: FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY vol:7 ispartof: location:Switzerland status: published

Simple Summary A subset of colorectal cancers (CRCs) is characterized by a mismatch repair deficiency that is frequently associated with microsatellite instability (MSI). The compromised DNA repair machinery leads to the accumulation of tumor neoantigens affecting the sensitivity of MSI metastatic CRC to immune checkpoint inhibitors (CPIs), both upfront and in later lines of treatment. However, up to 30% of MSI CRCs exhibit primary resistance to frontline immune based therapy, and an additional subset develops acquired resistance. Here, we first discuss the clinical and molecular features of MSI CRCs and then we review how the loss of antigenicity, immunogenicity, and a hostile tumor microenvironment could influence primary and acquired resistance to CPIs. Finally, we describe strategies to improve the outcome of MSI CRC patients upon CPI treatment. Abstract Immune checkpoint inhibitors (CPIs) represent an effective therapeutic strategy for several different types of solid tumors and are remarkably effective in mismatch repair deficient (MMRd) tumors, including colorectal cancer (CRC). The prevalent view is that the elevated and dynamic neoantigen burden associated with the mutator phenotype of MMRd fosters enhanced immune surveillance of these cancers. In addition, recent findings suggest that MMRd tumors have increased cytosolic DNA, which triggers the cGAS STING pathway, leading to interferon-mediated immune response. Unfortunately, approximately 30% of MMRd CRC exhibit primary resistance to CPIs, while a substantial fraction of tumors acquires resistance after an initial benefit. Profiling of clinical samples and preclinical studies suggests that alterations in the Wnt and the JAK-STAT signaling pathways are associated with refractoriness to CPIs. Intriguingly, mutations in the antigen presentation machinery, such as loss of MHC or Beta-2 microglobulin (B2M), are implicated in initial immune evasion but do not impair response to CPIs. In this review, we outline how understanding the mechanistic basis of immune evasion and CPI resistance in MMRd CRC provides the rationale for innovative strategies to increase the subset of patients benefiting from CPIs.

Simple Summary Mastitis is a worldwide diffused disease usually treated with an excessive use of antibiotics. Therefore, antimicrobial resistance is an important issue to be addressed by scientists. One of the possible solutions to decrease the use of drugs is genetic selection of resistant animals, that is, individuals that can be more resistant to mastitis. In our survey we analyzed Single Nucleotide Polymorphisms (SNPs) in genes known to be involved in both infection resistance and immune system activity. We found a group of SNPs that can be associated to mastitis related phenotypes (namely SCS) and that can be used for selecting resistant animals. An efficient selection is able to improve both animal welfare and quality and safety of animal products Abstract Mastitis is an infectious disease affecting the mammary gland, leading to inflammatory reactions and to heavy economic losses due to milk production decrease. One possible way to tackle the antimicrobial resistance issue stemming from antimicrobial therapy is to select animals with a genetic resistance to this disease. Therefore, aim of this study was to analyze the genetic variability of the SNPs found in candidate genes related to mastitis resistance in Holstein Friesian bulls. Target regions were amplified, sequenced by Next-Generation Sequencing technology on the Illumina® MiSeq, and then analyzed to find correlation with mastitis related phenotypes in 95 Italian Holstein bulls chosen with the aid of a selective genotyping approach. On a total of 557 detected mutations, 61 showed different genotype distribution in the tails of the deregressed EBVs for SCS and 15 were identified as significantly associated with the phenotype using two different approaches. The significant SNPs were identified in intergenic or intronic regions of six genes, known to be key components in the immune system (namely CXCR1, DCK, NOD2, MBL2, MBL1 and M-SAA3.2). These SNPs could be considered as candidates for a future genetic selection for mastitis resistance, although further studies are required to assess their presence in other dairy cattle breeds and their possible negative correlation with other traits.

Objectives The spread of carbapenem-resistant Enterobacteriaceae (CRE) represents one of the most worrisome problems for clinical medicine worldwide. In Italy, the Antibiotic-Resistance-Istituto Superiore di Sanita surveillance network, in collaboration with the Committee for Antimicrobial Agents of the Italian Society of Clinical Microbiologists, promoted a study to investigate the carbapenem-resistance mechanisms, clonal relatedness and capsular typing of a recent collection of carbapenem-resistant Klebsiella pneumoniae (CR-KP). Methods A total of 17 laboratories distributed across Italy collected all consecutive non-replicate CR-KP isolated from invasive infections during two different study periods (2011-12 and 2013). Carbapenemase genes were searched for by filter hybridization and confirmed by PCR and sequencing. KPC-producing K. pneumoniae (KPC-KP) were typed by PFGE and MLST. Capsular types were identified by wzi gene typing. Results Of the collected K. pneumoniae isolates (n = 461), the overall proportion of CR-KP was 36.2% (n = 167). The majority (97%) of the CR-KP were positive for the blaKPC gene. Among the KPC-KP population, nine different STs were detected with the majority of isolates (94%) belonging to the clonal group (CG) 258. A subpopulation that belonged to ST512 and showed an identical PFGE profile represented the majority (57%) of KPC-KP strains, with a countrywide distribution. Capsular characterization showed the predominance of the wzi154, cps-2 capsular type (88.8% of all CG258 strains). ST258 strains were associated with both cps-1 and cps-2 capsular types, while ST512 was associated with cps-2 only. Conclusions Although a trend to a polyclonal evolution of the Italian KPC-KP was noted, this study showed that the KPC-KP population remained largely oligoclonal with the wide diffusion of an ST512 lineage carrying cps-2 capsular type and producing the KPC-3 enzyme.

The study of the nutritional effects of tannins is complex due to the large chemical diversity, consequently, in poultry nutrition the biological responses may vary greatly. The aim of the present study was to evaluate the effect of different levels of dietary Quebracho tannins (QT) on growth and production performance in slow-growing type Muscovy ducks. For this purpose, a 42-d trial was carried out on 126 male ducks (42-d old at start), fed on three levels of dietary QT inclusion in the diet (0% as control diet, vs. 1.5% vs. 2.5% on an as fed basis). Birds were reared under free-range conditions. A linear increase in feed intake as a function of QT inclusion in the diet was observed (p &lt, 0.05). No difference as to final body weight, overall average daily weight gain (ADG) and total feed conversion ratio (FCR) in relation to dietary treatments was observed. Carcass yields were positively improved in QT birds (p &lt, 0.05). No adverse responses were recorded in total blood protein and liver weight. Dietary QT might be safely used up by to 2.5% in 42- to 84-d aged male Muscovy ducks.

In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of 21.93±12.81×106 cells, while at passage 2, it was of 83.05±114.72×106 cells. All final cellular products were free from contamination with aerobic/anaerobic pathogens, mycoplasma, and bacterial endotoxins. The expanded BMSCs expressed CD73, CD90, CD105, and CD146 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Most importantly, nine out of 10 of the cell products formed bone when transplanted in vivo. These validated procedures will serve as the basis for in-house BMSC manufacturing for use in clinical applications in our center.

The feed utilisation of young chicks is characterised by a suboptimal fat digestibility, which can be improved by means of dietary emulsifiers. The objective of this study was to evaluate the effect of dietary Globin on the energy efficiency and digestibility of starter feeds and on the production performance of broilers throughout the whole rearing cycle. A total of 224-day-old ROSS 708 chickens (14 birds/pen, 8 replicates/treatment) were fed ad libitum with either a basal diet (C) or a basal diet with the addition of 0.05% Globin during the starter (d1–10), growing (d10–25) and finisher (d25–35) periods. Nutrient digestibility (aD), protein metabolisability (aMCP), energy efficiency (EE) and net energy for production (NEp) were assessed during the starter period. The average daily feed intake (ADFI), average daily gain (ADG), feed conversion ratio (FCR) and protein efficiency ratio (PER) were measured of each growth period. Globin significantly decreased FCR (p = .020) and increased aDfat (p = .021), EE (p = .028) and NEp (p = .011) during the starter period. aMCP (p = .049) and PER (p = .039) were higher in the Globin group than in the Control group. The increased availability of energy from dietary fat, as a result of Globin supplementation, possibly shifted the use of the absorbed amino acids towards an anabolic metabolism, and this could explain the increased aMCP and PER but similar aDCP. The overall performance was similar between groups, although Globin tended to increase PER (p = .064) overall.Highlights Globin dietary supplementation was studied in broiler chickens. Globin improved digestibility and nutrient efficiency utilization in the first period. The overall performance was similar between groups.

Purpose: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available. Experimental Design: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach. Results: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion. Conclusions: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.

The European Union has implemented breeding programmes to increase scrapie resistance in sheep. A similar approach can be applied also in goats since the K222 allele provides a level of resistance equivalent to that of ARR in sheep. The European Food Safety Authority stated that breeding for resistance could be offered as an option for Member States to control classical scrapie in goats. We assessed the impact of different breeding strategies on PRNP genotype frequencies using a mathematical model that describes in detail the evolution of K222 in two goat breeds, Chamois Coloured and Saanen. Different patterns of age structure and replacement rate were modelled as factors affecting response to selection. Breeding for scrapie resistance can be implemented in goats, even though the initial K222 frequencies in these breeds are not particularly favourable and the rate at which the resistant animals increase, both breeding and slaughtered for meat production, is slow. If the goal is not to achieve the fixation of resistance allele, it is advisable to carry out selection only until a desired frequency of K222-carriers has been attained. Nucleus selection vs. selection on the overall populations is less expensive but takes longer to reach the desired output. The programme performed on the two goat breeds serves as a model of the response the selection could have in other breeds that show different initial frequencies and population structure. In this respect, the model has a general applicability. Electronic supplementary material The online version of this article (10.1186/s13567-018-0518-x) contains supplementary material, which is available to authorized users.

Heart failure is a major cause for premature death. Given the heterogeneity of the heart failure syndrome, identifying genetic determinants of cardiac function and structure may provide greater insights into heart failure. Despite progress in understanding the genetic basis of heart failure through genome wide association studies, the heritability of heart failure is not well understood. Gaining further insights into mechanisms that contribute to heart failure requires systematic approaches that go beyond single trait analysis. We integrated a Bayesian multi-trait approach and a Bayesian networks for the analysis of 10 correlated traits of cardiac structure and function measured across 3387 individuals with whole exome sequence data. While using single-trait based approaches did not find any significant genetic variant, applying the integrative Bayesian multi-trait approach, we identified 3 novel variants located in genes, RGS3, CHD3, and MRPL38 with significant impact on the cardiac traits such as left ventricular volume index, parasternal long axis interventricular septum thickness, and mean left ventricular wall thickness. Among these, the rare variant NC_000009.11:g.116346115C > A (rs144636307) in RGS3 showed pleiotropic effect on left ventricular mass index, left ventricular volume index and maximal left atrial anterior-posterior diameter while RGS3 can inhibit TGF-beta signaling associated with left ventricle dilation and systolic dysfunction.

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Germ cell tumors (GCTs) generally express wild-type p53 protein. Rare p53 mutations may be associated with cisplatin resistance. There is growing interest in the role of cyclins as targets for GCTs. Cyclin B1 is involved in G2/M transition and its overexpression has been reported in tumors carrying nonfunctional p53. Conversely, cyclin B1-specific small interfering RNAs have been shown to dramatically reduce tumor proliferation. We investigated whether a subset of chemotherapy-resistant GCTs overexpressed cyclin B1 as a result of nonfunctional p53, as this would make cyclin B1 a potential therapeutic target. Our data showed that GCTs consistently overexpressed cyclin B1 independently of their responsiveness to chemotherapy or the presence of p53 mutations. Cyclin B1 was overexpressed by GCT cell lines carrying functional p53. Cyclin B1-specific small interfering RNAs only slightly reduced the proliferation of JAR and JEG-3 placental choriocarcinoma cells. Further research into targeting cyclin B1 could provide a novel intervention for GCTs.

Attempts at eradicating metastatic cancers with targeted therapies are limited by the emergence of resistant subclones bearing heterogeneous (epi)genetic changes. We used colorectal cancer (CRC) to test the hypothesis that interfering with an ancestral oncogenic event shared by all the malignant cells (such as WNT pathway alterations) could override heterogeneous mechanisms of acquired drug resistance. Here, we report that in CRC-resistant cell populations, phylogenetic analysis uncovers a complex subclonal architecture, indicating parallel evolution of multiple independent cellular lineages. Functional and pharmacological modulation of WNT signalling induces cell death in CRC preclinical models from patients that relapsed during the treatment, regardless of the drug type or resistance mechanisms. Concomitant blockade of WNT and MAPK signalling restrains the emergence of drug-resistant clones. Reliance upon the WNT–APC pathway is preserved throughout the branched genomic drift associated with emergence of treatment relapse, thus offering the possibility of a common therapeutic strategy to overcome secondary drug resistance., The emergence of sub-clones that are resistant to targeted agents is a major therapeutic obstacle in oncology. Here, using colorectal cancer as a model system, the authors show that interfering with ancestral oncogenic events present in all subclones-like APC-WNT pathway alterations—can restrain the emergence of drug-resistant populations.

Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids. In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids.

Immunoreactive proteins in follicular cells, fibroblasts and endothelial cells were assessed in canine thyroid carcinomas and healthy thyroid glands. No differences were detected in thyrotropin receptor and thyroglobulin staining between cancer and normal tissues, but expression was higher in follicular cells than in fibroblasts. Fibroblast growth factor-2 staining was more intense in healthy follicular cells than in those of carcinomas. Follicular cells in carcinomas presented two- to three-fold greater staining intensity of thyroid transcription factor-1 and proliferating cell nuclear antigen, respectively, than healthy cells, and a similar trend was found for the latter antigen in fibroblasts. Vascular endothelial growth factor staining was more intense in the endothelial cells of tumours than in those of normal tissues. In conclusion, greater expression of factors related to proliferation and angiogenesis was demonstrated in several cell types within thyroid carcinomas compared to healthy tissues, which may represent mechanisms of tumour progression in this disease.

Success in eradicating human cancer with targeted therapies is limited by the emergence of secondary resistance. The prevalent view is that resistance is a fait accompli: when treatment is initiated, tumors already contain drug-resistant mutant cells. However, when cancer cells are challenged with targeted agents, the emergence of drug tolerant ‘persister’ cell population is often observed. Persisters survive exposure to targeted therapies through non-genetic, poorly understood mechanisms, and constitute a reservoir from which genetically divergent, drug-resistant derivatives eventually emerge. Analogously, when bacteria are exposed to stress, such as antibiotic treatment, persister cells can survive and, by switching from high-fidelity to low-fidelity DNA replication process, increase transiently their mutation rate (adaptive mutability), thus improving chances of survival. We used colorectal cancer (CRC) as model system to explore the hypothesis that, in addition to pre-existing drug resistant cells, resistance to targeted therapies could be fostered by a transient increase in genomic instability during treatment, leading to de novo genetic alterations. We found that, when exposed to EGFR and/or BRAF inhibition, mismatch repair proficient (MMRp) CRC cell lines exhibited a down-modulation of MMR and Homologous Recombination (HR) DNA repair genes, and a concomitant up-regulation of error-prone polymerases, resulting in reduced MMR and HR proficiency. Therapy-induced modulation of DNA repair genes was transient and returned to initial levels upon removal of the drugs, or when the cells developed permanent resistance to targeted agents. Notably, MLH1 and MSH2 MMR genes were down-regulated in patients-derived xenografts and tissue samples obtained at clinical response (minimal residual disease) compared to pre-treatment samples, confirming the clinical relevance of our findings. Activation of error-prone polymerases was associated with increased ROS production and accumulation of DNA damage marker γ-H2AX, in a time- a dose-dependent manner upon drug administration. The combination of DNA repair down-modulation and error-prone polymerases upregulation increases mutagenic ability and triggers microsatellite instability under drug-induced stress in CRC cells. Our results demonstrate that cancer cells, like unicellular organisms, evade therapeutic pressures by enhancing mutability. The notion that cancer cells exposed to targeted therapies activate a stress-induced adaptive mutability process may prompt the design of novel therapeutic strategies aimed at interfering with clonal evolution, reducing the generation of new variants during therapeutic treatment. Citation Format: MARIANGELA RUSSO, Giovanni Crisafulli, Alberto Sogari, Nicole Megan Reilly, Sabrina Arena, Simona Lamba, Alice Bartolini, Vito Amodio, Alessandro Magrì, Luca Novara, Ivana Sarotto, Zachary Nagel, Cortt Piett, Alessio Amatu, Andrea Sartore-Bianchi, Salvatore Siena, Andrea Bertotti, Livio Trusolino, Federica Di Nicolantonio, Alberto Bardelli. Adaptive mutability of colorectal cancers in response to targeted therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A120. doi:10.1158/1535-7163.TARG-19-A120

In Italy, since 2005 a breeding plan to increase scrapie resistance has been adopted. The impact of this selection on genetic diversity was assessed on Sambucana, an autochtonous sheep breed reared in southern Piedmont, by analysing the evolution of allele frequencies at different levels: PRNP (prion protein) gene, microsatellite loci on OAR13 (where PRNR maps), and microsatellite loci on other chromosomes, not subjected to selection for scrapie resistance. A total of 147 young rams, 80 born in 2004 and 67 in 2008–2009 were analysed. Evidence of diversity loss was observed for PRNP gene as a consequence of the directional selection. Diversity was affected in the immediate vicinity of PRNP but the effect on more distant loci on the same chromosome was trivial. With regard to neutral markers, lack of heterozygosis with no changeover of allele frequencies was observed suggesting an increase of inbreeding. Mating policies would be sufficient to solve these problems. A selection scheme based on genotyping rams and eliminating carriers of both susceptible and high susceptible alleles is the best way to improve natural resistance to scrapie with low costs and minimal problems in the current conservation programmes targeting rare breeds.

Diabetes mellitus is a global pandemic and continues to increase in numbers and significance. Several pathogenic processes are involved in the development of such disease and these mechanisms could be influenced by genetic, epigenetic and environmental factors. Non-enzymatic glycation reactions of proteins have been strongly related to pathogenesis of chronic diabetic complications. The identification of fructosamine 3-kinase (FN3K), an enzyme involved in protein deglycation, a new form of protein repair, is of great interest. FN3K phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins, suggesting a protective role of this enzyme. Moreover, the variability in FN3K activity has been associated with some polymorphisms in the FN3K gene. Here we argue about genetic studies and evidence of FN3K involvement in diabetes, together with results of our analysis of the FN3K gene on a Caucasian cohort of diabetic patients. Present knowledge suggests that FN3K could act in concert with other molecular mechanisms and may impact on gene expression and activity of other enzymes involved in deglycation process.

Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling.

Dimethyltryptamines are hallucinogenic serotonin-like molecules present in traditional Amerindian medicine (e.g.Ayahuasca)recently associated with cognitive gains, antidepressant effects and changes in brain areas related to attention. Historical and technical restrictions impaired understanding how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids.In silicosystems biology analyses support 5-MeO-DMT’s anti-inflammatory effects and reveal a modulation of proteins associated with long-term potentiation, the formation of dendritic spines, including proteins involved in cellular protrusion formation, microtubule dynamics and cytoskeletal reorganization. These results offer possible mechanistic insights into the neuropsychological changes caused by the ingestion of substances rich in dimethyltryptamines.

Systematic studies of micronutrients during brain formation are hindered by restrictions to animal models and adult post-mortem tissues. Recently, advances in stem cell biology have enabled recapitulation of the early stages of human telencephalon developmentin vitro. In the present work, we analyzed cerebral organoids derived from human pluripotent stem cells by synchrotron radiation X-ray fluorescence in order to measure biologically valuable micronutrients incorporated and distributed into the exogenously developing brain. Our findings indicate that elemental inclusion in organoids is consistent with human brain tissue and involves P, S, K, Ca, Fe and Zn. Occurrence of different concentration gradients also suggests active regulation of elemental transmembrane transport. Finally, the analysis of pairs of elements shows interesting elemental interaction patterns that change from 30 to 45 days of development, suggesting short- or long-term associations, such as storage in similar compartments or relevance for time-dependent biological processes. These findings shed light on which trace elements are important during human brain development and will support studies aimed to unravel the consequences of disrupted metal homeostasis for neurodevelopmental diseases, including those manifested in adulthood.

Immunohistochemical markers (IGF-1, IGF-1R, VEGF, FGF-2, RARα and RXR) were evaluated in healthy canine thyroid glands (n=8) and in follicular-compact (n=8) and compact thyroid carcinomas (n=8). IGF-1, IGF-1R and VEGF expression was higher in fibroblasts and endothelial cells of compact carcinoma than in healthy glands (P < 0.05). Compared to follicular-compact carcinoma, compact carcinoma had higher IGF-1R expression in fibroblasts, and higher FGF-2 expression in endothelial cells (P < 0.05). RARα expression was higher in endothelial cells of compact carcinoma than in those of other groups (P < 0.05). The upregulation of these proliferation- and angiogenesis-related factors in endothelial cells and/or fibroblasts and not in follicular cells of compact carcinoma compared to healthy glands supports the relevance of stromal cells in cancer progression.Keywords: Canine, Histology, Immunohistochemistry, Thyroid carcinoma.

Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression. Cancers deficient in MMR frequently show favourable prognosis and indolent progression. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR-deficient cells was comparable to their proficient counterparts in vitro and on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.

Background Mutations in rat sarcoma (RAS) genes may be a mechanism of secondary resistance in epidermal growth factor receptor inhibitor-treated patients. Tumor-tissue biopsy testing has been the standard for evaluating mutational status; however, plasma testing of cell-free DNA has been shown to be a more sensitive method for detecting clonal evolution. Materials and methods Archival pre- and post-treatment tumor biopsy samples from a phase II study of panitumumab in combination with irinotecan in patients with metastatic colorectal cancer (mCRC) that also collected plasma samples before, during, and after treatment were analyzed for emergence of mutations during/post-treatment by next-generation sequencing and BEAMing. Results The rate of emergence of tumor tissue RAS mutations was 9.5% by next-generation sequencing (n= 21) and 6.3% by BEAMing (n = 16). Plasma testing of cell-free DNA by BEAMing revealed a mutant RAS emergence rate of 36.7% (n = 39). Exploratory outcomes analysis of plasma samples indicated that patients who had emergent RAS mutations at progression had similar median progression-free survival to those patients who remained wild-type at progression. Serial analysis of plasma samples showed that the first detected emergence of RAS mutations preceded progression by a median of 3.6 months (range, -0.3 to 7.5 months) and that there did not appear to be a mutant RAS allele frequency threshold that could predict near-term outcomes. Conclusions This first prospective analysis in mCRC showed that serial plasma biopsies are more inclusive than tissue biopsies for evaluating global tumor heterogeneity; however, the clinical utility of plasma testing in mCRC remains to be further explored. ClinicalTrials.gov Identifier NCT00891930

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders’ statements. The sequence of the two genes, namely Ch1 and Ch2, that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats.

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer mortality worldwide. Survival in the metastatic setting has been gradually improved by the addition to cytotoxic chemotherapy of agents targeting the vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Considerable heterogeneity exists within CRC due to the varied genetic and epigenetic mechanisms involved in differing pathways of carcinogenesis. The knowledge of molecular abnormalities underlying colorectal tumourigenesis and the progression of dysplastic precursors to invasive and ultimately metastatic lesions has advanced in recent years by comprehensive sequencing studies. From these genome-scale analyses, we know that a handful of genes are commonly affected by somatic mutations, whereas recurrent copy-number alterations and chromosomal translocations are rarer in this disease. Even though some of these molecular abnormalities make genes acting as drivers of cancer progression, translation of this recognition for therapeutic purposes is still limited, encompassing only as standard of care the exclusion of RAS-mutated cancers for better selecting patients to candidate to EGFR-targeted therapy with monoclonal antibodies. However, the effort of ameliorating molecular selection should not be considered exhausted by demonstration of RAS and BRAF-induced resistance, as the genomic landscape of response to EGFR blockade has been demonstrated to be wider and dynamically multifaceted. In this chapter we will review main molecular biomarkers of de novo (primary) and acquired (secondary) resistance to EGFR-targeted monoclonal antibodies in metastatic CRC and discuss therapeutic implications.

Increased oxidation of low density lipoprotein (LDL) is characteristic of atherosclerosis. In this frame, high density lipoproteins (HDL) play an important role, being able to remove lipid peroxides (LPOs) and cholesterol from oxidized LDL, so exhibiting a protective role against atherosclerosis. A wide range of reactive compounds lead to the oxidation of methionine (Met) residues with the formation of methionine sulphoxide (MetO) in apolipoprotein A-I (ApoA-I). Consequently, the determination of MetO level can give both an evaluation of oxidative stress and the reduced capability of ApoA-I in LPOs and cholesterol transport. For these reasons, the development of analytical methods able to determine the MetO level is surely of interest, and we report here the results obtained by MALDI mass spectrometry.