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Abstract Purpose Determining the primary origin of non-organ-confined neuroendocrine tumors (NETs) for accurate diagnosis and management. Neuroendocrine tumors are rare neoplasms with diverse clinical behaviors. Determining their primary origin remains challenging in cases of non-organ-confined NETs. This study explores the histogenesis of a retroperitoneal, non-functional NET localized between the duodenum and pancreatic head, utilizing advanced molecular diagnostics to elucidate its probable primary source. Methods Initial diagnostic methods, including imaging and histopathology, failed to resolve the tumor’s origin. The tumor was subjected to single-cell RNA sequencing (scRNA-seq) and whole exome sequencing (WES). Publicly available transcriptomic datasets from pancreatic and small intestine NETs were used to develop and validate a molecular gene signature for tissue-of-origin identification. Results The gene signature distinguished pancreatic and small intestine NETs with high accuracy. The tumor cells presented a molecular profile consistent with a pancreatic origin, likely derived from ectopic pancreatic tissue. Conclusions This case demonstrates the value of integrating scRNA-seq and WES for the molecular characterization of complex NETs. Identifying the tumor’s pancreatic origin informed a targeted management approach, avoiding unnecessary systemic treatment and underscoring the potential of single-cell approaches in personalized oncology.

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types, making further research difficult. Here, we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology, doubling rate, ability to form colonies, surface marker analysis, and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology, growth kinetics, and surface marker characterization. In contrast, the serum-containing medium gives better potential for further MSC differentiation into osteogenic, chondrogenic, and adipogenic lineages.

Influenza A viruses (IAVs) pose a serious threat to global health. On the one hand, these viruses cause seasonal flu outbreaks in humans. On the other hand, they are a zoonotic infection that has the potential to cause a pandemic. The most important natural reservoir of IAVs are waterfowl. In this study, we investigated the occurrence of IAV in birds in the Republic of Buryatia (region in Russia). In 2020, a total of 3018 fecal samples were collected from wild migratory birds near Lake Baikal. Of these samples, 11 were found to be positive for the H13N8 subtype and whole-genome sequencing was performed on them. All samples contained the same virus with the designation A/Unknown/Buryatia/Arangatui-1/2020. To our knowledge, virus A/Unknown/Buryatia/Arangatui-1/2020 is the first representative of the H13N8 subtype collected on the territory of Russia, the sequence of which is available in the GenBank database. An analysis of reassortments based on the genome sequences of other known viruses has shown that A/Unknown/Buryatia/Arangatui-1/2020 arose as a result of reassortment. In addition, a reassortment most likely occurred several decades ago between the ancestors of the viruses recently collected in China, the Netherlands, the United States and Chile. The presence of such reassortment emphasizes the ongoing evolution of the H13N8 viruses distributed in Europe, North and East Asia, North and South America and Australia. This study underscores the importance of the continued surveillance and research of less-studied influenza subtypes.

Abstract Background Cystic fibrosis (CF) is one of the most common life-threatening genetic disorders. Around 2000 variants in the CFTR gene have been identified, with some proportion known to be pathogenic and 300 disease-causing mutations have been characterized in detail by CFTR2 database, which complicates its analysis with conventional methods. Methods We conducted next-generation sequencing (NGS) in a cohort of 89 adult patients negative for p.Phe508del homozygosity. Complete clinical and demographic information were available for 84 patients. Results By combining MLPA with NGS, we identified disease-causing alleles in all the CF patients. Importantly, in 10% of cases, standard bioinformatics pipelines were inefficient in identifying causative mutations. Class IV-V mutations were observed in 38 (45%) cases, predominantly ones with pancreatic sufficient CF disease; rest of the patients had Class I-III mutations. Diabetes was seen only in patients homozygous for class I-III mutations. We found that 12% of the patients were heterozygous for more than two pathogenic CFTR mutations. Two patients were observed with p.[Arg1070Gln, Ser466*] complex allele which was associated with milder pulmonary obstructions (FVC 107 and 109% versus 67%, CI 95%: 63-72%; FEV 90 and 111% versus 47%, CI 95%: 37-48%). For the first time p.[Phe508del, Leu467Phe] complex allele was reported, observed in four patients (5%). Conclusion NGS can be a more information-gaining technology compared to standard methods. Combined with its equivalent diagnostic performance, it can therefore be implemented in the clinical practice, although careful validation is still required.

Avian influenza is one of the largest known threats to domestic poultry. Influenza outbreaks on poultry farms typically lead to the complete slaughter of the entire domestic bird population, causing severe economic losses worldwide. Moreover, there are highly pathogenic avian influenza (HPAI) strains that are able to infect the swine or human population in addition to their primary avian host and, as such, have the potential of being a global zoonotic and pandemic threat. Migratory birds, especially waterfowl, are a natural reservoir of the avian influenza virus; they carry and exchange different virus strains along their migration routes, leading to antigenic drift and antigenic shift, which results in the emergence of novel HPAI viruses. This requires monitoring over time and in different locations to allow for the upkeep of relevant knowledge on avian influenza virus evolution and the prevention of novel epizootic and epidemic outbreaks. In this review, we assess the role of migratory birds in the spread and introduction of influenza strains on a global level, based on recent data. Our analysis sheds light on the details of viral dissemination linked to avian migration, the viral exchange between migratory waterfowl and domestic poultry, virus ecology in general, and viral evolution as a process tightly linked to bird migration. We also provide insight into methods used to detect and quantify avian influenza in the wild. This review may be beneficial for the influenza research community and may pave the way to novel strategies of avian influenza and HPAI zoonosis outbreak monitoring and prevention.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene (neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 virus is getting increasingly infective for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild aquatic birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialilation to prevent infection. Tissue specific expression of the sialidases could protect domestic birds from infection.