Factor-inhibiting HIF (FIH) promotes lung cancer progression.

Factor-inhibiting HIF (FIH) is an asparagine hydroxylase that acts on hypoxia-inducible factors (HIFs) to control cellular adaptation to hypoxia. FIH is expressed in several tumor types, but its impact in tumor progression remains largely unexplored. We observed that FIH was expressed on human lung cancer tissue. Deletion of FIH in mouse and human lung cancer cells resulted in an increased glycolytic metabolism, consistent with increased HIF activity. FIH-deficient lung cancer cells exhibited decreased proliferation. Analysis of RNA-Seq data confirmed changes in the cell cycle and survival and revealed molecular pathways that were dysregulated in the absence of FIH, including the upregulation of angiomotin (Amot), a key component of the Hippo tumor suppressor pathway. We show that FIH-deficient tumors were characterized by higher immune infiltration of NK and T cells compared with FIH competent tumor cells. In vivo studies demonstrate that FIH deletion resulted in reduced tumor growth and metastatic capacity. Moreover, high FIH expression correlated with poor overall survival in non-small cell lung cancer (NSCLC). Our data unravel FIH as a therapeutic target for the treatment of lung cancer.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

The spike of SARS-CoV-2 promotes metabolic rewiring in hepatocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a multi-organ damage that includes hepatic dysfunction, which has been observed in over 50% of COVID-19 patients. Liver injury in COVID-19 could be attributed to the cytopathic effects, exacerbated immune responses or treatment-associated drug toxicity. Herein we demonstrate that hepatocytes are susceptible to infection in different models: primary hepatocytes derived from humanized angiotensin-converting enzyme-2 mice (hACE2) and primary human hepatocytes. Pseudotyped viral particles expressing the full-length spike of SARS-CoV-2 and recombinant receptor binding domain (RBD) bind to ACE2 expressed by hepatocytes, promoting metabolic reprogramming towards glycolysis but also impaired mitochondrial activity. Human and hACE2 primary hepatocytes, where steatosis and inflammation were induced by methionine and choline deprivation, are more vulnerable to infection. Inhibition of the renin-angiotensin system increases the susceptibility of primary hepatocytes to infection with pseudotyped viral particles. Metformin, a common therapeutic option for hyperglycemia in type 2 diabetes patients known to partially attenuate fatty liver, reduces the infection of human and hACE2 hepatocytes. In summary, we provide evidence that hepatocytes are amenable to infection with SARS-CoV-2 pseudovirus, and we propose that metformin could be a therapeutic option to attenuate infection by SARS-CoV-2 in patients with fatty liver.

Methionine adenosyltransferase 1a antisense oligonucleotides activate the liver-brown adipose tissue axis preventing obesity and associated hepatosteatosis.

Altered methionine metabolism is associated with weight gain in obesity. The methionine adenosyltransferase (MAT), catalyzing the first reaction of the methionine cycle, plays an important role regulating lipid metabolism. However, its role in obesity, when a plethora of metabolic diseases occurs, is still unknown. By using antisense oligonucleotides (ASO) and genetic depletion of Mat1a, here, we demonstrate that Mat1a deficiency in diet-induce obese or genetically obese mice prevented and reversed obesity and obesity-associated insulin resistance and hepatosteatosis by increasing energy expenditure in a hepatocyte FGF21 dependent fashion. The increased NRF2-mediated FGF21 secretion induced by targeting Mat1a, mobilized plasma lipids towards the BAT to be catabolized, induced thermogenesis and reduced body weight, inhibiting hepatic de novo lipogenesis. The beneficial effects of Mat1a ASO were abolished following FGF21 depletion in hepatocytes. Thus, targeting Mat1a activates the liver-BAT axis by increasing NRF2-mediated FGF21 secretion, which prevents obesity, insulin resistance and hepatosteatosis.

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate.

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals.

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.

Study of 17 X-STRs in Native American and Mestizo populations of Central America for forensic and population purposes.

In the present work, an extensive analysis of the X-chromosomal pool of Native American and Mestizo groups of Central America (Guatemala, El Salvador, Nicaragua, and Panama) has been carried out. Allele and haplotype frequency databases, as well as other forensic parameters for these populations, are presented. The admixture analysis supports the tri-hybrid composition in terms of ancestry in the Mestizo populations, with a predominant Native American contribution (54-69%), followed by European (19-28%) and African contributions (12-19%). Pairwise FST genetic distances highlight the genetic proximity between the northernmost Central American populations, especially among admixed populations. The unique and complex nature of this area, where populations from different origins intercrossed, as well as the informativity of X-STR data, highpoint the great interest of this genetic study. Furthermore, the X-chromosome databases for Central American populations here provided will be not only useful for forensic and population purposes not only in the target countries but also in the host countries.

Hypoxia Promotes Syndecan-3 Expression in the Tumor Microenvironment.

The syndecan (Sdc) family is comprised of four members of cell surface molecules (Sdc-1 to 4) with different biological functions. Syndecan-3 (Sdc-3) is known to be mainly expressed in the brain and nervous tissue and plays a key role in development, cell adhesion, and migration. Recent studies point to important roles for Sdc-3 in inflammatory disease, but the patterns of expression and significance of Sdc-3 in cancer remains unexplored. Here we show that Sdc-3 expression is upregulated on several cancer types, especially in solid tumors that are known to be hypoxic. The Cancer Genome Atlas program (TCGA) data demonstrated that Sdc-3 expression in the tumor microenvironment positively correlates with a hypoxia gene signature. To confirm a potential cause-effect, we performed experiments with tumor cell lines showing increased expression upon in vitro exposure to 1% oxygen or dimethyloxalylglycine, an inhibitor of prolyl hydroxylases, indicating that Sdc-3 expression is promoted by hypoxia inducible factors (HIFs). HIF-1α was responsible for this upregulation as confirmed by CRISPR-engineered tumor cells. Using single-cell RNA sequencing data of melanoma patients, we show that Sdc-3 is expressed on tumor associated macrophages, cancer cells, and endothelial cells. Syndecan-3 expression positively correlated with a macrophage gene signature across several TCGA cancer types. In vitro experiments demonstrated that hypoxia (1% oxygen) or treatment with IFN-γ stimulate Sdc-3 expression on RAW-264.7 derived macrophages, linking Sdc-3 expression to a proinflammatory response. Syndecan-3 expression correlates with a better patient overall survival in hypoxic melanoma tumors.

Variability in Cerebrospinal Fluid MicroRNAs Through Life.

The development of the human brain starts in the first weeks of embryo differentiation. However, there are many relevant neurodevelopmental processes that take place after birth and during lifespan. Such a fine and changing scenario requires the coordinated expression of thousands of genes to achieve the proper specialization and inter-connectivity. In this context, microRNAs (miRNAs), which can modulate mRNA stability and translation, are gaining recognition for their involvement in both brain development and neurodevelopmental disorders. Therefore, cerebrospinal fluid (CSF) miRNAs should be perfectly differentiated in relevant age periods. In this study, we aimed to highlight the biological variability of miRNA expression in the CSF throughout life, which is also crucial for biomarker discovery in CNS pathologies, especially in children, where they are desperately needed. We analyzed the CSF microRNAome of 14 healthy children (aged 0-7.4 years) by smallRNA-Seq and compared it with previously published data in adults (N = 7) and elders (N = 11). miR-423-5p and miR-22-3p were overexpressed in the < 1 and > 3 years groups, respectively. Additionally, we detected 18 miRNAs that reached their highest peak of expression at different time-points during the lifespan and sets of miRNAs that were exclusively expressed in a specific age group. On the contrary, miR-191-5p showed stable expression in CSF from the first year of life. Our results remark the complex differential miRNA expression profile that can be observed through life, which underlines the need for including appropriate age-matched controls when the expression of CSF miRNAs is analyzed in different pathological contexts. Graphical abstract.

A Comprehensive Study of Vesicular and Non-Vesicular miRNAs from a Volume of Cerebrospinal Fluid Compatible with Clinical Practice.

Cerebrospinal fluid (CSF) microRNAs (miRNAs) have emerged as potential biomarkers for minimally invasive diagnosis of central nervous system malignancies. However, despite significant advances in recent years, this field still suffers from poor data reproducibility. This is especially true in cases of infants, considered a new subject group. Implementing efficient methods to study miRNAs from clinically realistic CSF volumes is necessary for the identification of new biomarkers. Methods: We compared six protocols for characterizing miRNAs, using 200-µL CSF from infants (aged 0-7). Four of the methods employed extracellular vesicle (EV) enrichment step and the other two obtained the miRNAs directly from cleared CSF. The efficiency of each method was assessed using real-time PCR and small RNA sequencing. We also determined the distribution of miRNAs among different CSF shuttles, using size-exclusion chromatography. Results: We identified 281 CSF miRNAs from infants. We demonstrated that the miRNAs could be efficiently detected using only 200 µL of biofluid in case of at least two of the six methods. In the exosomal fraction, we found 12 miRNAs that might be involved in neurodevelopment. Conclusion: The Norgen and Invitrogen protocols appear suitable for the analysis of a large number of miRNAs using small CSF samples.

Cell-free DNA as a biomarker in diffuse large B-cell lymphoma: A systematic review.

Cell-free DNA (cfDNA), which is DNA released from cells into the circulation, is one of the most promising non-invasive biomarkers in cancer. This approach could be of interest for the management of Diffuse Large B-Cell Lymphoma (DLBCL) patients, which is the most common non-Hodgkin lymphoma. Then, the aim of this systematic review was to define the utility of cfDNA in this disease. Selected articles were classified in four groups, depending on the aspects of cfDNA studied, i.e. concentration, methylation, IgH gene rearrangements, and somatic mutations. While concentration and methylation of cfDNA need to be further analyzed, IgH gene rearrangements and somatic mutations seem to be the most promising biomarkers to date. Their detection has been shown to allow disease monitoring and early prediction of relapse. Although more efforts and standardization of techniques are needed, studying cfDNA in liquid biopsy may help improve the outcome of DLBCL patients.

Genetic variation of 17 X-chromosome STR loci in Tunisian population of Nabeul.

In the present study, the genetic variations of 17 X-STR markers (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS6801, DXS7423, DXS6809, DXS6799, DXS7132, DXS9902, DXS6800, DXS6789, DXS10075, DXS10079, DXS6807, and DXS6803) were analyzed in 139 unrelated individuals in Nabeul, aiming to perform an X-STR database for anthropological and forensic purposes. Our results indicate that DXS6809 was the most polymorphic locus, whereas DXS6807 was the least informative marker. In addition, the obtained values for the statistical parameters of forensic interest, i.e., the power of discrimination in males (PDM) and females (PDF), as well as the mean exclusion chance in duos (MECD) and trios (MECT) have demonstrated that this panel of 17 X-STRs is highly informative and useful for forensic application and anthropological research. Additionally, pairwise genetic distances based on FST were calculated between Nabeul population and other populations extracted from the literature. Genetic distances were represented in a non-metric MDS plot and clustering of populations according to their geographic locations and their historical relationship was detected.

Differentially methylated CpG regions analyzed by PCR-high resolution melting for monozygotic twin pair discrimination.

Discrimination between monozygotic (MZ) twins is a forensic limitation when using conventional DNA profiling techniques for human identification. Recent works based on epigenetics seem to open a new way to solve this issue due to methylation status of MZ twins change during their lifetime. Methylation analysis through BeadChip platforms allows the study up to 850 K CpG sites revealing that numerous differential methylation regions exist between MZ twins. However, this methodology is difficult to implement in forensic laboratories. On the contrary, PCR-HRM (High Resolution Melting) technology is one of the easiest methods for analyzing DNA methylation and it has been capable to discriminate between MZ twins. The purpose of this study is to contribute with new differential methylation regions in MZ twins to those that have been previously studied through PCR-HRM. Here, we have selected 6 CpG regions located at the ITGA2B, ASPA, PDE4C, ZIC5, USP11 and NOP14 loci that have shown methylation status variation during lifetime. The study has been carried out from saliva-derived DNA of 18 MZ twin pairs. The most discriminating regions were those located at ITGA2B, ASPA and ZIC5 loci showing significant within-pair differences in 44.4% of the cases. Non evidences of relation between age and significant differences between MZ twins were found, although the 50% of MZ twin pairs were discrimnated in the oldest age range (59-66 years old). These results support the use of these regions to increase the number of epigenetics age-related markers available to discriminate between MZ twins in a pair by PCR-HRM in forensic laboratories.

A genetic overview of Atlantic coastal populations from Europe and North-West Africa based on a 17 X-STR panel.

The forensic use of X-STRs requires the creation of allele and haplotype frequency databases in the populations where they are going to be used. Recently, an updated Spanish allele and haplotype frequency database for the new 17 X-STR panel has been created, being the only database available up to now for this new multiplex. In order to broaden the forensic applicability of the 17 X-STR panel, 513 individuals from four different populations located on the Atlantic Coast of Europe and North-West Africa have been studied, i.e. Brittany (France), Ireland, northern Portugal, and Casablanca (Morocco). Allele and haplotype frequency databases, as well as parameters of forensic interest for these populations are presented. The obtained results showed that the 17 X-STR panel constitutes a highly discriminative tool for forensic identification and kinship testing in the studied populations. Furthermore, we aimed to study if these populations located on the Atlantic coast actually share alike allele and haplotype frequency distributions since they have experienced genetic exchanges throughout history. This would allow creating larger forensic databases that include several genetically similar populations for its use in forensic casework. For this purpose, pairwise FST genetic distances between the analyzed populations and others from the Atlantic Coast previously studied with the 17 X-STR panel or the ten coincident markers included in the decaplex of the GHEP-ISFG were estimated. Our results suggest that certain nearby populations located on the European Atlantic coast could have underwent episodes of genetic interchange as they have not shown statistically significant differentiation between them. However, the population of Casablanca showed significant differentiation with the majority of the European populations. Likewise, the autochthonous Basque Country and Brittany populations have shown distinctive allele frequency distributions between them. Therefore, these findings seem to support that the use of independent allele and haplotype frequency databases for each population instead of a global database would be more appropriate for forensic purposes.

Forensic Spanish allele and haplotype database for a 17 X-STR panel.

The currently developed 17 X-STR panel (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS6801, DXS7423, DXS6809, DXS6799, DXS7132, DXS9902, DXS6800, DXS6789, DXS10075, DXS10079, DXS6807, and DXS6803) offers a highly discriminative tool for forensic identification and kinship testing. With the aim of providing a global Spanish population X-STR database, we present haplotype and allele frequencies and parameters of forensic interest for the 17 X-STR panel obtained from 593 unrelated individuals from Alicante, Aragon, the Basque Country, Andalusia, Galicia, Madrid, and Barcelona that represent the most populated regions of the Spanish Peninsular territory. The seven populations were compared to test possible population genetic substructures. The lack of significant differences among the studied Spanish populations supports the use of the allele and haplotype frequency database presented herein as a global Spanish population sample useful for statistical evaluation in forensic casework. After conducting the LD plots derived from HapMap and pairwise linkage disequilibrium tests, DXS7132, DXS10075, and DXS10079 markers were included in a cluster and haplotype frequencies were calculated. The improvement in the forensic parameters for the Spanish population using 17 X-STRs in comparison to the previous 10 X-STR allele frequencies database is also shown.

Development of a new highly efficient 17 X-STR multiplex for forensic purposes.

Currently, two of the most widely used X-chromosome STR (X-STR) multiplexes are composed by ten (GHEP-ISFG decaplex) and 12 markers (Investigator Argus X-12 Kit). The number of markers included is a drawback for complex relative testing cases, likewise the large size of some amplicons difficult their application to degraded samples. Here, we present a new multiplex of 17 X-STRs with the aim of increasing both the resolution power and forensic applicability. This newly proposed set includes the X-STRs of the GHEP-ISFG decaplex, four X-STRs from the Investigator Argus X-12 Kit, three of them also included in the decaplex, and six additional more. In order to ensure the allele designation, an allelic ladder was developed. The validation of the present multiplex was carried out according to the revised guidelines by the SWGDAM (Scientific Working Group on DNA Analysis Methods). A total of 488 unrelated individuals from four different continents were analyzed. The forensic efficiency evaluation showed high values of combined power of discrimination in males (≥0.999999996) and females (≥0.999999999999995) as well as combined paternity exclusion probabilities in trios (≥0.99999998) and duos (≥0.999996). The results presented herein have demonstrated that the new 17 X-STR set constitutes a high-resolution alternative to the current X-STR multiplexes.