The pediatric gut bacteriome and virome in response to SARS-CoV-2 infection

IntroductionSince the beginning of the SARS-CoV-2 pandemic in early 2020, it has been apparent that children were partially protected from both infection and the more severe forms of the disease. Many different mechanisms have been proposed to explain this phenomenon, including children’s frequent exposure to other upper respiratory infections and vaccines, and which inflammatory cytokines they are more likely to produce in response to infection. Furthermore, given the presence of SARS-CoV-2 in the intestine and its ability to infect enterocytes, combined with the well described immunomodulatory capabilities of the microbiome, another potential contributing factor may be the presence of certain protective microbial members of the gut microbiota (GM).MethodsWe performed shotgun metagenomic sequencing and profiled both the bacteriome and virome of the GM of pediatric SARS-CoV-2 patients compared to healthy, age-matched subjects.ResultsWe found that, while pediatric patients do share some pro-inflammatory microbial signatures with adult patients, they also possess a distinct microbial signature of protective bacteria previously found to be negatively correlated with SARS-CoV-2 infectivity and COVID-19 severity. COVID-19 was also associated with higher fecal Cytomegalovirus load, and with shifts in the relative abundances of bacteriophages in the GM. Furthermore, we address how the preventative treatment of COVID-19 patients with antibiotics, a common practice especially in the early days of the pandemic, affected the bacteriome and virome, as well as the abundances of antimicrobial resistance and virulence genes in these patients. DiscussionTo our knowledge, this is the first study to address the bacteriome, virome, and resistome of pediatric patients in response to COVID-19 and to preventative antibiotics use

Drosophila melanogaster as a model for mitochondrial biology, mitochondrial disease and neurological disorders.

Drosophila melanogaster has a long history of being used as an animal model for a wide variety of human diseases, including genetic diseases, neurodegeneration and alcoholism. Despite the fact that Drosophila was first used as an animal model over 100 years ago, it still remains an extremely relevant model today, thanks to its short life cycle, its low cost ease to rear in laboratory conditions and the sophistication of the molecular tools available for genetic manipulation in Drosophila melanogaster. This model also has far less genetic redundancy with respect to mammals, making the study of the role of certain genes far more straightforward, and yet despite this, still possesses an ortholog for 75% of human disease-causing genes. All of these properties contribute to the relevance of this model and were taken advantage of during this project. In the first part of this project, Drosophila melanogaster was used as a model for mitochondrial deoxynucleotide transport. The Drosophila homolog CG18317 of the yeast gene RIM2, which was previously reported to be a pyrimidine dNTP transporter, was characterized. Knock-out (K.O.) flies for gene CG18317, here referred to as drim2, were characterized for mitochondrial function and mtDNA integrity. The two human homologs for this gene, PNC-1 and SLC25A36 were also expressed in this mutant background, in order to investigate the functional homology of these genes and confirm the validity of this model for human mitochondrial dNTP transport. This project also focuses on further characterizing a K.O. fly line for dTTC19, a gene whose human homolog has already been tied to mitochondrial encephalopathy and psychosis in humans. This characterization was also accompanied by the generation of three K.O. lines which express the dTTC19 gene in a mutant background, in order to finally confirm that the entirety of the mutant phenotype is due to the absence of transcription of the dTTC19 gene. Finally, this project attempts to propose a new protocol which will enable researchers to use Drosophila melanogaster as a model for neurological disorders which present with antisocial symptoms. A protocol was developed to investigate social behaviour in Drosophila melanogaster and to demonstrate that subtle changes in either dopamine levels or previous social contact can have dramatic effects on their social interactions. We therefore propose that Drosophila can also be a useful model for the investigation of the genetic factors involved in diseases which present with antisocial behaviour such as autism, obsessive compulsive disorder, depression and so forth. In conclusion, this project takes full advantage of Drosophila melanogaster as an animal model for mitochondrial biology and disease. Furthermore, it proposes yet another way in which Drosophila can be used as a model which has not yet been done.Drosophila melanogaster ha una lunga storia come animale modello per tante malattie umane, incluse le malattie genetiche, la neurodegenerazione e l’alcolismo. Anche se Drosophila fu inizialmente utilizzata come animale modello più di 100 anni fa, rimane comunque un modello rilevante oggi grazie al suo ciclo vitale breve, il suo basso costo e la sofisticazione degli attrezzi molecolari disponibili per la sua manipolazione genetica. Questo modello ha anche meno ridondanza genetica rispetto ai mammiferi, rendendo lo studio della funzione di questi geni molto più diretto, ma malgrado questo possiede un ortologo per 75% dei geni legati a malattie umane. Tutte queste proprietà contribuiscono alla sua rilevanza come modello e sono state sfruttate durante questo progetto. In primis, Drosophila melanogaster è stata usata come modello per il trasporto mitocondriale di deossinucleotidi. Il gene RIM2 in lievito, che è stato precedentemente caratterizzato come trasportatore mitocondriale di deossinucleotidi pirimidinici, ha un omologo in Drosophila: CG18317, qui chiamato drim2, che è stato caratterizzato in questo progetto. Questo gene è stato rimosso in vivo e la funzione mitocondriale e l’integrità del mtDNA sono state caratterizzate. I due omologhi umani per questo gene, PNC-1 e SLC25A36, sono stati espressi nel mutante, per determinare l’omologia funzionale di questi geni e per confermare la validità di questo mutante come modello per il trasporto mitocondriale umano di deossinucleotidi. Questo progetto si è anche focalizzato su una caratterizzazione più approfondita di una linea mutante per dTTC19, un omologo di un gene umano che è già stato collegato alla encefalopatia mitocondriale e la psicosi. Questa caratterizzazione è stata accompagnata dalla generazione di tre linee mutanti che esprimono dTTC19, per confermare che il fenotipo mutante osservato sia dovuto alla mancanta trascrizione di dTTC19. In fine, questo progetto propone un nuovo protocollo che, nella nostra opinione, permetterà di utilizzare Drosophila melanogaster come modello per disturbi neurologici che presentano con sintomi asociali. Un protocollo è stato sviluppato per studiare il comportamento sociale in Drosophila melanogaster e per dimostrare che piccole differenze nei livelli di dopamina o nel contatto sociale dopo l’eclosione possono avere effetti drammatici sulle interazioni sociali in Drosophila. Proponiamo che Drosophila può essere un modello utile per lo studio dei fattori genici coinvolti nelle malattie che presentano con comportamento asociale come l’autismo, il disturbo ossessivo compulsivo, la depressione eccetera. In conclusione, questo progetto sfrutta interamente Drosophila melanogaster come animale modello per la biologia e le malattie mitocondriali. In più, propone un nuovo modo per utilizzare Drosophila come modello che non è stato finora sfruttato

Drosophila melanogaster as a model for mitochondrial biology, mitochondrial disease and neurological disorders.

Drosophila melanogaster has a long history of being used as an animal model for a wide variety of human diseases, including genetic diseases, neurodegeneration and alcoholism. Despite the fact that Drosophila was first used as an animal model over 100 years ago, it still remains an extremely relevant model today, thanks to its short life cycle, its low cost ease to rear in laboratory conditions and the sophistication of the molecular tools available for genetic manipulation in Drosophila melanogaster. This model also has far less genetic redundancy with respect to mammals, making the study of the role of certain genes far more straightforward, and yet despite this, still possesses an ortholog for 75% of human disease-causing genes. All of these properties contribute to the relevance of this model and were taken advantage of during this project. In the first part of this project, Drosophila melanogaster was used as a model for mitochondrial deoxynucleotide transport. The Drosophila homolog CG18317 of the yeast gene RIM2, which was previously reported to be a pyrimidine dNTP transporter, was characterized. Knock-out (K.O.) flies for gene CG18317, here referred to as drim2, were characterized for mitochondrial function and mtDNA integrity. The two human homologs for this gene, PNC-1 and SLC25A36 were also expressed in this mutant background, in order to investigate the functional homology of these genes and confirm the validity of this model for human mitochondrial dNTP transport. This project also focuses on further characterizing a K.O. fly line for dTTC19, a gene whose human homolog has already been tied to mitochondrial encephalopathy and psychosis in humans. This characterization was also accompanied by the generation of three K.O. lines which express the dTTC19 gene in a mutant background, in order to finally confirm that the entirety of the mutant phenotype is due to the absence of transcription of the dTTC19 gene. Finally, this project attempts to propose a new protocol which will enable researchers to use Drosophila melanogaster as a model for neurological disorders which present with antisocial symptoms. A protocol was developed to investigate social behaviour in Drosophila melanogaster and to demonstrate that subtle changes in either dopamine levels or previous social contact can have dramatic effects on their social interactions. We therefore propose that Drosophila can also be a useful model for the investigation of the genetic factors involved in diseases which present with antisocial behaviour such as autism, obsessive compulsive disorder, depression and so forth. In conclusion, this project takes full advantage of Drosophila melanogaster as an animal model for mitochondrial biology and disease. Furthermore, it proposes yet another way in which Drosophila can be used as a model which has not yet been done.Drosophila melanogaster ha una lunga storia come animale modello per tante malattie umane, incluse le malattie genetiche, la neurodegenerazione e l’alcolismo. Anche se Drosophila fu inizialmente utilizzata come animale modello più di 100 anni fa, rimane comunque un modello rilevante oggi grazie al suo ciclo vitale breve, il suo basso costo e la sofisticazione degli attrezzi molecolari disponibili per la sua manipolazione genetica. Questo modello ha anche meno ridondanza genetica rispetto ai mammiferi, rendendo lo studio della funzione di questi geni molto più diretto, ma malgrado questo possiede un ortologo per 75% dei geni legati a malattie umane. Tutte queste proprietà contribuiscono alla sua rilevanza come modello e sono state sfruttate durante questo progetto. In primis, Drosophila melanogaster è stata usata come modello per il trasporto mitocondriale di deossinucleotidi. Il gene RIM2 in lievito, che è stato precedentemente caratterizzato come trasportatore mitocondriale di deossinucleotidi pirimidinici, ha un omologo in Drosophila: CG18317, qui chiamato drim2, che è stato caratterizzato in questo progetto. Questo gene è stato rimosso in vivo e la funzione mitocondriale e l’integrità del mtDNA sono state caratterizzate. I due omologhi umani per questo gene, PNC-1 e SLC25A36, sono stati espressi nel mutante, per determinare l’omologia funzionale di questi geni e per confermare la validità di questo mutante come modello per il trasporto mitocondriale umano di deossinucleotidi. Questo progetto si è anche focalizzato su una caratterizzazione più approfondita di una linea mutante per dTTC19, un omologo di un gene umano che è già stato collegato alla encefalopatia mitocondriale e la psicosi. Questa caratterizzazione è stata accompagnata dalla generazione di tre linee mutanti che esprimono dTTC19, per confermare che il fenotipo mutante osservato sia dovuto alla mancanta trascrizione di dTTC19. In fine, questo progetto propone un nuovo protocollo che, nella nostra opinione, permetterà di utilizzare Drosophila melanogaster come modello per disturbi neurologici che presentano con sintomi asociali. Un protocollo è stato sviluppato per studiare il comportamento sociale in Drosophila melanogaster e per dimostrare che piccole differenze nei livelli di dopamina o nel contatto sociale dopo l’eclosione possono avere effetti drammatici sulle interazioni sociali in Drosophila. Proponiamo che Drosophila può essere un modello utile per lo studio dei fattori genici coinvolti nelle malattie che presentano con comportamento asociale come l’autismo, il disturbo ossessivo compulsivo, la depressione eccetera. In conclusione, questo progetto sfrutta interamente Drosophila melanogaster come animale modello per la biologia e le malattie mitocondriali. In più, propone un nuovo modo per utilizzare Drosophila come modello che non è stato finora sfruttato

Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs

Chromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms

A disease-associated Aifm1 variant induces severe myopathy in knockin mice

Objective: Mutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo. Methods/results: Here we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling. Conclusion: Our findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients. Keywords: Akt/mTOR signaling, Apoptosis-inducing factor (AIF), 1C metabolism, Mitochondria, Mitochondrial diseases, Oxidative phosphorylatio

Case Report: The impact of severe cryptosporidiosis on the gut microbiota of a pediatric patient with CD40L immunodeficiency

Cryptosporidium parvum is a protozoan parasite and one of the leading causes of gastroenteritis in the world, primarily affecting very young children and immunocompromised patients. While infection is usually self-limiting, it can become chronic and even lethal in these vulnerable populations, in whom Cryptosporidium treatments are generally ineffective, due to their acting in concert with a functioning immune system. Here, we describe a case of chronic cryptosporidiosis in a European child with severe CD40L immunodeficiency infected with Cryptosporidium parvum of the IIa20G1 subgenotype, a lineage which has thus far only ever been described in the Middle East. After years of on-off treatment with conventional and non-conventional anti-parasitic drugs failed to clear parasitosis, we performed targeted metagenomics to observe the bacterial composition of the patient's gut microbiota (GM), and to evaluate fecal microbiota transplantation (FMT) as a potential treatment option. We found that C. parvum infection led to significant shifts in GM bacterial composition in our patient, with consequent shifts in predicted intestinal functional signatures consistent with a state of persistent inflammation. This, combined with the patient's poor prognosis and increasing parasitic burden despite many rounds of anti-parasitic drug treatments, made the patient a potential candidate for an experimental FMT procedure. Unfortunately, given the many comorbidities that were precipitated by the patient's immunodeficiency and chronic C. parvum infection, FMT was postponed in favor of more urgently necessary liver and bone marrow transplants. Tragically, after the first liver transplant failed, the patient lost his life before undergoing FMT and a second liver transplant. With this case report, we present the first description of how cryptosporidiosis can shape the gut microbiota of a pediatric patient with severe immunodeficiency. Finally, we discuss how both our results and the current scientific literature suggest that GM modulations, either by probiotics or FMT, can become novel treatment options for chronic Cryptosporidium infection and its consequent complications, especially in those patients who do not respond to the currently available anti-parasitic therapies

Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.</p