Genome-wide analysis reveals the patterns of genetic diversity and population structure of 8 Italian local chicken breeds

The aim of this study was to conduct a genome-wide comparative analysis of 8 local Italian chicken breeds (Ermellinata di Rovigo, Millefiori di Lonigo [PML], Polverara Bianca, Polverara Nera, Padovana, Pepoi [PPP], Robusta Lionata, and Robusta Maculata), all under a conservation plan, to understand their genetic diversity and population structure. A total of 152 animals were analyzed using the Affymetrix Axiom 600 K Chicken Genotyping Array. The levels of genetic diversity were highest and lowest in PML and PPP, respectively. The results of genomic inbreeding based on runs of homozygosity (ROH; FROH) showed marked differences among breeds and ranged from 0.161 (PML) to 0.478 (PPP). Furthermore, in all breeds, short ROH (<4 Mb in length) were more frequent than long segments. Patterns of genetic differentiation, model-based clustering, and neighbor networks showed that most breeds formed nonoverlapping clusters and were clearly separate populations. The 2 Polverara breeds shared a similar genetic background and showed the lowest genetic differentiation in comparison with purebred lines; the local populations showed separated groups. PPP and PML were closer to the group of the purebred broiler lines (BRSA, BRSB, BRDA, and BRDB). Six genomic regions are presented as hotspots of autozygosity among the Italian chicken breeds, with candidate genes involved in multiple morphological phenotypes as breast muscle, muscle dry matter content, and body weight. This study is the first exhaustive genome-wide analysis of the diversity of these Italian local chickens from Veneto region. We conclude that breeds have conserved authentic genetic patterns. The results are of significant importance because they will help design and implement conservation strategies. In fact, the conservation of these breeds may also have positive impacts on the local economy, niche traditional markets, and offering a source of high-quality products to consumers. In this context, genomic information may play a crucial role in the management of local breeds

Virtual screening identifies broad-spectrum \u3b2-lactamase inhibitors with activity on clinically relevant serine- and metallo-carbapenemases

Bacteria are known to evade \u3b2-lactam antibiotic action by producing \u3b2-lactamases (BLs), including carbapenemases, which are able to hydrolyze nearly all available \u3b2-lactams. The production of BLs represents one of the best known and most targeted mechanisms of resistance in bacteria. We have performed the parallel screening of commercially available compounds against a panel of clinically relevant BLs: class A CTX-M-15 and KPC-2, subclass B1 NDM-1 and VIM-2 MBLs, and the class C P. aeruginosa AmpC. The results show that all BLs prefer scaffolds having electron pair donors: KPC-2 is preferentially inhibited by sulfonamide and tetrazole-based derivatives, NDM-1 by compounds bearing a thiol, a thiosemicarbazide or thiosemicarbazone moiety, while VIM-2 by triazole-containing molecules. Few broad-spectrum BLs inhibitors were identified; among these, compound 40 potentiates imipenem activity against an NDM-1-producing E. coli clinical strain. The binary complexes of the two most promising compounds binding NDM-1 and VIM-2 were obtained at high resolution, providing strong insights to improve molecular docking simulations, especially regarding the interaction of MBLs with inhibitors

The genetic heritage of Alpine local cattle breeds using genomic SNP data

Background: Assessment of genetic diversity and population structure provides important control metrics to avoid genetic erosion, inbreeding depression and crossbreeding between exotic and locally-adapted cattle breeds since these events can have deleterious consequences and eventually lead to extinction. Historically, the Alpine Arc represents an important pocket of cattle biodiversity with a large number of autochthonous breeds that provide a fundamental source of income for the entire regional economy. By using genotype data from medium-density single nucleotide polymorphism (SNP) arrays, we performed a genome-wide comparative study of 23 cattle populations from the Alpine Arc and three cosmopolitan breeds. Results: After filtering, we obtained a final genotyping dataset consisting of 30,176 SNPs for 711 individuals. The local breeds showed high or intermediate values of genetic diversity compared to the highly selected cosmopolitan breeds. Patterns of genetic differentiation, multidimensional scaling, admixture analysis and the constructed phylogenetic tree showed convergence, which indicates the presence of gene flow among the breeds according to both geographic origin and historical background. Among the most differentiated breeds, we identified the modern Brown cattle. In spite of admixture events, several local breeds have preserved distinctive characteristics, which is probably due to differences in genetic origin and geographic location. Conclusions: This study represents one of the most comprehensive genome-wide analysis of the Alpine cattle breeds to date. Using such a large dataset that includes the majority of the local breeds found in this region, allowed us to expand knowledge on the evaluation and status of Alpine cattle biodiversity. Our results indicate that although many of the analyzed local breeds are listed as endangered, they still harbor a large amount of genetic diversity, even when compared to some cosmopolitan breeds. This finding, together with the reconstruction of the phylogeny and the relationships between these Alpine Arc cattle breeds, provide crucial insights not only into the improvement of genetic stocks but also into the implementation of future conservation strategies

Structural Basis of Enzymatic Activity for the Ferulic Acid Decarboxylase (FADase) from Enterobacter sp. Px6-4

Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an “open-closed” mechanism involving a pocket of 8×8×15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer

CagI Is an Essential Component of the Helicobacter pylori Cag Type IV Secretion System and Forms a Complex with CagL

Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly

A Liposome-Based Mycobacterial Vaccine Induces Potent Adult and Neonatal Multifunctional T Cells through the Exquisite Targeting of Dendritic Cells

BACKGROUND: In the search for more potent and safer tuberculosis vaccines, CAF01 was identified as a remarkable formulation. Based on cationic liposomes and including a synthetic mycobacterial glycolipid as TLR-independent immunomodulator, it induces strong and protective T helper-1 and T helper-17 adult murine responses to Ag85B-ESAT-6, a major mycobacterial fusion protein. Here, we assessed whether these properties extend to early life and how CAF01 mediates its adjuvant properties in vivo. METHODS/FINDINGS: Following adult or neonatal murine immunization, Ag85B-ESAT-6/CAF01 similarly reduced the post-challenge bacterial growth of M. bovis BCG, whereas no protection was observed using Alum as control. This protection was mediated by the induction of similarly strong Th1 and Th17 responses in both age groups. Multifunctional Th1 cells were already elicited after a single vaccine dose and persisted at high levels for at least 6 months even after neonatal priming. Unexpectedly, this potent adjuvanticity was not mediated by a massive targeting/activation of dendritic cells: in contrast, very few DCs in the draining lymph nodes were bearing the labeled antigen/adjuvant. The increased expression of the CD40 and CD86 activation markers was restricted to the minute portion of adjuvant-bearing DCs. However, vaccine-associated activated DCs were recovered several days after immunization. CONCLUSION: The potent adult and neonatal adjuvanticity of CAF01 is associated in vivo with an exquisite but prolonged DC uptake and activation, fulfilling the preclinical requirements for novel tuberculosis vaccines to be used in early life

γδ T cell responses: How many ligands will it take till we know?

γδ T cells constitute a sizeable and non-redundant fraction of the total T cell pool in all jawed vertebrates, but in contrast to conventional αβ T cells they are not restricted by classical MHC molecules. Progress in our understanding of the role of γδ T cells in the immune system has been hampered, and is being hampered, by the considerable lack of knowledge regarding the antigens γδ T cells respond to. The past few years have seen a wealth of data regarding the TCR repertoires of distinct γδ T cell populations and a growing list of confirmed and proposed molecules that are recognised by γδ T cells in different species. Yet, the physiological contexts underlying the often restricted TCR usage and the chemical diversity of γδ T cell ligands remain largely unclear, and only few structural studies have confirmed direct ligand recognition by the TCR. We here review the latest progress in the identification and validation of putative γδ T cell ligands and discuss the implications of such findings for γδ T cell responses in health and disease

Are the heterogeneous nuclear ribonucleoproteins SQUID and Hrb87F involved in the regulation of circadian rhythmicity in Drosophila melanogaster?

The search for new genes involved in circadian rhythmicity in Drosophila melanogaster led to the identification of the RNA binding proteins Hrb87F and SQUID, as partners of Cryptochrome, the photoreceptor responsible for light-synchronization of the circadian clock. In the clock machinery, different post-transcriptional mechanisms have evolved to adjust and consolidate the oscillation of clock genes and proteins in interlocked negative feedback loops. The aim of my PhD project was to study the possible involvement of Hrb87F and SQUID in the post-transcriptional control of the circadian clock in Drosophila. These preliminary data were confirmed by Co-Immunoprecipitation and western-blot experiments using transgenic flies expressing a tagged version of Cry (HACRY) under the control of the driver timGAL4, for SQUID, while for Hrb87F the interaction was validated by yeast two-hybrid assay, in which dCRY was challenged to Hrb87F as prey. To achieve more information about a possible role of the clock in the expression of Squid and Hrb87F in Drosophila head, the analysis of the expression profile of the two hnRNPs (mRNAs and proteins) was performed during the 24 hours in wild type (wt) and clock mutant per0 flies. As for Hrb87F, mRNA levels showed an oscillatory trend in LD (Light-Dark) and DD (Dark-Dark) either in wt and per0 flies; the protein levels oscillate in LD and DD in wt flies, but not in per0, suggesting a potential role for the circadian clock in the translational/posttranslational control of the protein. As for Squid, neither the mRNA nor the protein showed rhythmic expression in LD and DD. Subsequently, the involvement of SQUID and Hrb87F in the generation of the circadian rhythmicity was studied analyzing the locomotor activity pattern of flies’ mutant for each of the two genes. Mutants for both genes showed an impairment of the daily rhythmicity, with a loss of the morning anticipation in LD and low levels of rhythmicity in DD at 29°C, 23°C, 18°C and 15°C, suggesting that Squid and Hrb87F could play a role either in the generation and in the light-synchronization of the circadian behaviour. However, the analysis of locomotor activity in constant light, performed in order to further dissect the possible role of Squid in the light synchronization of the clock, revealed that the light-synchronization of the clock is not impaired in these flies. The expression of period and timeless and the alternative splicing variants in relation to the temperature were also analyzed in wt and Squid mutant brains by using multiplex real time PCR. The results suggest that the expression of period and timeless mRNA is altered in Squid mutant compared to wt both in LD and DD at each temperature. Moreover, the quantities of per unspliced at high temperature and tim unspliced at low temperature are lower both in LD and DD in the mutant. This suggests an increase of the splicing events and thus an involvement of SQUID in the post-transcriptional control of clock genes. In collaboration with Dr Milena Damulewicz (Jagiellonian University of Krakow - Poland), the expression of PERIOD was analyzed in the clock neurons by immunocytochemistry in flies reared at both 18°C and 23°C in LD and DD. At 23°C and 18°C the oscillation of PER in l-LNvs clock neurons is lost, while, in s-LNvs, the accumulation of protein is delayed by 3 hours with a broader peak that reflects a low kinetic of degradation. The analysis of PDF projections from clock neurons shows a profound disorganization of PDF release, suggesting that it can, at least partially, account for the locomotor activity defect observed in the Squid mutant. Taken together, these results highlight the possible involvement of the hnRNPs Hrb87F and SQUID in the generation and maintenance of circadian rhythmicity in Drosophila melanogaster.La ricerca di nuovi geni coinvolti nel controllo della ritmicità circadiana in Drosophila melanogaster ha permesso di identificare una possibile interazione delle due ribonucleoproteine SQUID e HRB87F con CRYPTOCROME. Nella regolazione molecolare dell’orologio circadiano, si sono evoluti diversi eventi di regolazione post trascrizionale che aggiustano e consolidano l’espressione ritmica dei geni orologio e delle corrispettive proteine, secondo un meccanismo a feedback negativo. Per tale motivo lo scopo di questo lavoro è stato studiare e analizzare il possibile coinvolgimento delle hnRNPs nel controllo post-trascrizionale dell’orologio circadiano in Drosophila. In una fase iniziale, per confermare i risultati ottenuti in precedenza, sono stati condotti esperimenti di Co-Immunoprecipitazione e western-blot in mosche transgeniche in grado di esprimere una versione di CRY associata all’epitopo HA (HACRY) sotto il controllo del driver timGal4. L’anticorpo anti-SQUID ha identificato una proteina nel campione co-immunoprecipitato, confermando l’interazione, mentre il legame con HRB87F è stato osservato mediante l’utilizzo del sistema del doppio ibrido di lievito. In seguito è stato valutato il possibile coinvolgimento dell’orologio circadiano nell’espressione delle hnRNPs nell’intera testa del moscerino. I livelli di espressione del mRNA di Hrb87F mostrano un trend oscillatorio in LD (luce-buio) e DD (buio costante), sia nel wt che nel mutante per0, mentre la proteina oscilla in LD e DD nelle mosche wt, ma non nelle per0, suggerendo un possibile ruolo dell’orologio circadiano nel controllo traduzionale o post-traduzionale della proteina. L’analisi dell’espressione del gene Squid ha dimostrato, invece, che né mRNA né proteina vengono espressi in maniera ritmica, né in LD né DD. Successivamente, è stato studiato l’intervento delle due ribonucleoproteine nella generazione della ritmicità circadiana, analizzando l’attività locomotoria di mosche mutanti per entrambi i geni. Tutti i mutanti hanno riportato un’alterazione della ritmicità circadiana, con una perdita dell’anticipazione dell’attività mattutina in LD e bassi livelli di ritmicità in DD a 29°C, 23°C, 18°C e 15°C. I risultati ottenuti suggeriscono che Squid e Hrb87F possano partecipare alla generazione di un fenotipo ritmico nell’attività locomotoria. Questa analisi è stata completata studiando, inoltre, l’attività locomotoria del mutante squid in condizione di luce costante (LL), poiché esibiva un fenotipo associabile a quello in cui è presente un’alterazione del meccanismo di sincronizzazione della luce; si è visto che, le mosche mutanti presentano un risposta in LL paragonabile al wt, sottolineando un corretto funzionamento del meccanismo di sincronizzazione della luce. Abbiamo analizzato i profili di espressione dei geni period e timeless e delle rispettive varianti dovute a splicing alternativo legato alla temperatura. I risultati ottenuti mostrano che l’espressione è alterata nel mutante Squid, rispetto al controllo wt, sia in LD sia in DD ad ogni temperatura considerata, evidenziando che è presente un elevata attività di splicing. In fine, in collaborazione con la Dott. Milena Damulewicz (Università di Jagiellonian di Kracovia - Polonia), è stata condotta un’analisi dell’espressione di PERIOD nei neuroni orologio in mosche mutanti Squid e wt, mediante immunocitochimica a 18°C e 23°C in LD e DD. Si è visto che sia a 23°C sia a 18°C, l’oscillazione di PER nei neuroni orologio l-LNv, viene persa sia in LD sia in DD, mentre nei neuroni s-LNv sia a 23°C sia a 18°C si assiste a un ritardo nell’accumulo della proteina seguito da una cinetica di degradazione più lenta rispetto ai controlli. L’analisi delle proiezioni del neuropeptide PDF ha evidenziato una profonda disorganizzate nel mutante squid. Nel complesso, tutti questi risultati suggeriscono un coinvolgimento di HRB87F e SQUID nella generazione e nel mantenimento della ritmicità circadiana in Drosophila melanogaster

Are the heterogeneous nuclear ribonucleoproteins SQUID and Hrb87F involved in the regulation of circadian rhythmicity in Drosophila melanogaster?

The search for new genes involved in circadian rhythmicity in Drosophila melanogaster led to the identification of the RNA binding proteins Hrb87F and SQUID, as partners of Cryptochrome, the photoreceptor responsible for light-synchronization of the circadian clock. In the clock machinery, different post-transcriptional mechanisms have evolved to adjust and consolidate the oscillation of clock genes and proteins in interlocked negative feedback loops. The aim of my PhD project was to study the possible involvement of Hrb87F and SQUID in the post-transcriptional control of the circadian clock in Drosophila. These preliminary data were confirmed by Co-Immunoprecipitation and western-blot experiments using transgenic flies expressing a tagged version of Cry (HACRY) under the control of the driver timGAL4, for SQUID, while for Hrb87F the interaction was validated by yeast two-hybrid assay, in which dCRY was challenged to Hrb87F as prey. To achieve more information about a possible role of the clock in the expression of Squid and Hrb87F in Drosophila head, the analysis of the expression profile of the two hnRNPs (mRNAs and proteins) was performed during the 24 hours in wild type (wt) and clock mutant per0 flies. As for Hrb87F, mRNA levels showed an oscillatory trend in LD (Light-Dark) and DD (Dark-Dark) either in wt and per0 flies; the protein levels oscillate in LD and DD in wt flies, but not in per0, suggesting a potential role for the circadian clock in the translational/posttranslational control of the protein. As for Squid, neither the mRNA nor the protein showed rhythmic expression in LD and DD. Subsequently, the involvement of SQUID and Hrb87F in the generation of the circadian rhythmicity was studied analyzing the locomotor activity pattern of flies’ mutant for each of the two genes. Mutants for both genes showed an impairment of the daily rhythmicity, with a loss of the morning anticipation in LD and low levels of rhythmicity in DD at 29°C, 23°C, 18°C and 15°C, suggesting that Squid and Hrb87F could play a role either in the generation and in the light-synchronization of the circadian behaviour. However, the analysis of locomotor activity in constant light, performed in order to further dissect the possible role of Squid in the light synchronization of the clock, revealed that the light-synchronization of the clock is not impaired in these flies. The expression of period and timeless and the alternative splicing variants in relation to the temperature were also analyzed in wt and Squid mutant brains by using multiplex real time PCR. The results suggest that the expression of period and timeless mRNA is altered in Squid mutant compared to wt both in LD and DD at each temperature. Moreover, the quantities of per unspliced at high temperature and tim unspliced at low temperature are lower both in LD and DD in the mutant. This suggests an increase of the splicing events and thus an involvement of SQUID in the post-transcriptional control of clock genes. In collaboration with Dr Milena Damulewicz (Jagiellonian University of Krakow - Poland), the expression of PERIOD was analyzed in the clock neurons by immunocytochemistry in flies reared at both 18°C and 23°C in LD and DD. At 23°C and 18°C the oscillation of PER in l-LNvs clock neurons is lost, while, in s-LNvs, the accumulation of protein is delayed by 3 hours with a broader peak that reflects a low kinetic of degradation. The analysis of PDF projections from clock neurons shows a profound disorganization of PDF release, suggesting that it can, at least partially, account for the locomotor activity defect observed in the Squid mutant. Taken together, these results highlight the possible involvement of the hnRNPs Hrb87F and SQUID in the generation and maintenance of circadian rhythmicity in Drosophila melanogaster