Probe Sets Research Articles

Newborn infant skin gene expression: Remarkable differences versus adults.

At birth, human infants are poised to survive in harsh, hostile conditions. An understanding of the state of newborn skin development and maturation is key to the maintenance of health, optimum response to injury, healing and disease. The observational study collected full-thickness newborn skin samples from 27 infants at surgery and compared them to skin samples from 43 adult sites protected from ultraviolet radiation exposure, as the standard for stable, mature skin. Transcriptomics profiling and gene set enrichment analysis were performed. Statistical analysis established over 25,000 differentially regulated probe sets, representing 10,647 distinct genes, in infant skin compared to adult skin. Gene set enrichment analysis showed a significant increase in 143 biological processes (adjusted p < 0.01) in infant skin, versus adult skin samples, including extracellular matrix (ECM) organization, cell adhesion, collagen fibril organization and fatty acid metabolic process. ECM organization and ECM structure organization were the biological processes in infant skin with the lowest adjusted P-value. Genes involving epidermal development, immune function, cell differentiation, and hair cycle were overexpressed in adults, representing 101 significantly enriched biological processes (adjusted p < 0.01). The processes with the highest significant difference were skin and epidermal development, e.g., keratinocyte differentiation, keratinization and cornification intermediate filament cytoskeleton organization and hair cycle. Enriched Gene Ontology (GO) biological processes also involved immune function, including antigen processing and presentation. When compared to ultraviolet radiation-protected adult skin, our results provide essential insight into infant skin and its ability to support the newborn’s preparedness to survive and flourish, despite the infant’s new environment laden with microbes, high oxygen tension and potential irritants. This fundamental knowledge is expected to guide strategies to protect and preserve the features of unperturbed, young skin.

A novel exome probe set captures phototransduction genes across birds (Aves) enabling efficient analysis of vision evolution.

The diversity of avian visual phenotypes provides a framework for studying mechanisms of trait diversification generally, and the evolution of vertebrate vision, specifically. Previous research has focused on opsins, but to fully understand visual adaptation, we must study the complete phototransduction cascade (PTC). Here, we developed a probe set that captures exonic regions of 46 genes representing the PTC and other light responses. For a subset of species, we directly compared gene capture between our probe set and low-coverage whole genome sequencing (WGS), and we discuss considerations for choosing between these methods. Finally, we developed a unique strategy to avoid chimeric assembly by using "decoy" reference sequences. We successfully captured an average of 64% of our targeted exome in 46 species across 14 orders using the probe set and had similar recovery using the WGS data. Compared to WGS or transcriptomes, our probe set: (1) reduces sequencing requirements by efficiently capturing vision genes, (2) employs a simpler bioinformatic pipeline by limiting required assembly and negating annotation, and (3) eliminates the need for fresh tissues, enabling researchers to leverage existing museum collections. We then utilized our vision exome data to identify positively selected genes in two evolutionary scenarios-evolution of night vision in nocturnal birds and evolution of high-speed vision specific to manakins (Pipridae). We found parallel positive selection of SLC24A1 in both scenarios, implicating the alteration of rod response kinetics, which could improve color discrimination in dim light conditions and/or facilitate higher temporal resolution.

Molecular Cytogenetics in the Era of Chromosomics and Cytogenomic Approaches.

Here the role of molecular cytogenetics in the context of yet available all other cytogenomic approaches is discussed. A short introduction how cytogenetics and molecular cytogenetics were established is followed by technical aspects of fluorescence in situ hybridization (FISH). The latter contains the methodology itself, the types of probe- and target-DNA, as well as probe sets. The main part deals with examples of modern FISH-applications, highlighting unique possibilities of the approach, like the possibility to study individual cells and even individual chromosomes. Different variants of FISH can be used to retrieve information on genomes from (almost) base pair to whole genomic level, as besides only second and third generation sequencing approaches can do. Here especially highlighted variations of FISH are molecular combing, chromosome orientation-FISH (CO-FISH), telomere-FISH, parental origin determination FISH (POD-FISH), FISH to resolve the nuclear architecture, multicolor-FISH (mFISH) approaches, among other applied in chromoanagenesis studies, Comet-FISH, and CRISPR-mediated FISH-applications. Overall, molecular cytogenetics is far from being outdated and actively involved in up-to-date diagnostics and research.

Diagnostic performances of common nucleic acid tests for SARS-CoV-2 in hospitals and clinics: a systematic review and meta-analysis

BackgroundAn optimised standard experimental setup across different hospitals is urgently needed to ensure consistency in nucleic acid test results for SARS-CoV-2 detection. A standard comparison across different nucleic acid tests and their optimal experimental setups is not present. We assessed the performance of three common nucleic acid tests, namely digital PCR (dPCR), quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP), to detect SARS-CoV-2 in clinical settings.MethodsIn this systematic review and meta-analysis we compared sensitivity and specificity of qPCR, dPCR, and LAMP and their performances when different experimental setups (namely specimen type used, use of RNA extraction, primer–probe sets, and RNA extraction methods) are applied. We searched PubMed, BioRxiv, MedRxiv, SciFinder, and ScienceDirect for studies and preprints published between Feb 29 and Dec 15, 2020. Included dPCR, qPCR, and LAMP studies using any type of human specimens should report the number of true-positive, true-negative, false-positive, and false-negative cases with Emergency Use Authorization (EUA)-approved PCR assays as the comparator. Studies with a sample size of less than ten, descriptive studies, case studies, reviews, and duplicated studies were excluded. Pooled sensitivity and specificity were computed from the true and false positive and negative cases using Reitsma's bivariate random-effects and bivariate latent class models. Test performance reported in area under the curve (AUC) of the three nucleic acid tests was further compared by pooling studies with similar experimental setups (eg, tests that used RNA extracted pharyngeal swabs but with either the open reading frame 1ab or the N primer). Heterogeneity was assessed and reported in I2 and τ2.FindingsOur search identified 1277 studies of which we included 66 studies (11 dPCR, 32 qPCR, and 23 LAMP) with 15 017 clinical samples in total in our systematic review and 52 studies in our meta-analysis. dPCR had the highest pooled diagnostic sensitivity (94·1%, 95% CI 88·9–96·6, by Reitsma's model and 95·8%, 54·9–100·0, by latent class model), followed by qPCR (92·7%, 88·3–95·6, and 93·4%, 60·9–99·9) and LAMP (83·3%, 76·9–88·2, and 86·2%, 20·7–99·9), using EUA-approved PCR kits as the reference standard. LAMP was the most specific with a pooled estimate of 96·3% (93·8–97·8) by Reitsma's model and 94·3% (49·1–100·0) by latent class model, followed by qPCR (92·9%, 87·2–96·2, and 93·1%, 47·1–100·0) and dPCR (78·5%, 57·4–90·8, and 73·8%, 0·9–100·0). The overall heterogeneity was I2 0·5% (τ2 2·79) for dPCR studies, 0% (4·60) for qPCR studies, and 0% (3·96) for LAMP studies. AUCs of the three nucleic acid tests were the highest and differed the least between tests (ie, AUC>0·98 for all tests) when performed with RNA extracted pharyngeal swabs using SARS-CoV-2 open reading frame 1ab primer.InterpretationAll three nucleic acid tests consistently perform better with pharyngeal swabs using SARS-CoV-2 open reading frame 1ab primer with RNA extraction. dPCR was shown to be the most sensitive, followed by qPCR and LAMP. However, their accuracy does not differ significantly. Instead, accuracy depends on specific experimental conditions, implying that more efforts should be directed to optimising the experimental setups for the nucleic acid tests. Hence, our results could be a reference for optimising and establishing a standard nucleic acid test protocol that is applicable in laboratories worldwide.FundingUniversity Grants Committee and The Chinese University of Hong Kong.

Adult allergic rhinitis sufferers have unique nasal mucosal and peripheral blood immune gene expression profiles: A case-control study.

BackgroundAllergic rhinitis (AR) is a complex disease involving both mucosal and systemic immune compartments. Greater understanding of the immune networks underpinning AR pathophysiology may assist with further refining disease‐specific biomarkers.ObjectiveTo compare immune gene expression profiles in nasal mucosa and peripheral blood samples between adults with AR and controls without AR.MethodsThis cross‐sectional study included 45 adults with moderate‐severe and persistent AR (37.6 ± 12.8 years; mean ± SD) and 24 adults without AR (36.6 ± 10.2). Gene expression analysis was performed using the NanoString nCounter PanCancer Immune profiling panel (n = 730 immune genes) in combination with the panel plus probe set (n = 30 allergy‐related genes) with purified RNA from peripheral blood and cell lysates prepared from combined nasal lavage and nasal brushing.ResultsOne hundred and thirteen genes were significantly differentially expressed in peripheral blood samples between groups (p < .05). In contrast, 14 genes were differentially expressed in nasal lysate samples between groups (p < .05). Upregulation of allergy‐related genes in nasal mucosa samples in the AR group was observed. Namely, chemokines CCL17 and CCL26 are involved in the chemotaxis of key effector cells and TPSAB1 encodes tryptase, an inflammatory mediator released from activated mast cells and basophils. Six differentially expressed genes (DEGs) were in common between the nasal mucosa and blood samples. In addition, counts of specific DEGs in nasal mucosa samples were positively correlated with eosinophil and dust mite‐specific immunoglobulin E (IgE) counts in blood.Conclusions and Clinical RelevanceDistinct gene expression profiles in blood and nasal mucosa samples were observed between AR sufferers and controls. The results of this study also provide evidence for a close interaction between the local site and systemic immunity. The genes identified in this study contribute to the current knowledge of AR pathophysiology and may serve as biomarkers to evaluate the effectiveness of treatment regimens, or as targets for drug discovery.

Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron Transfer)–based fluorescence quencher

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted “super-substrates” with catalytic constants of up to 15,000,000 M−1s−1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD+ has IC50 values below 200 pM.

Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran.

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig® Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

An isothermal, non-enzymatic, and dual-amplified fluorescent sensor for highly sensitive DNA detection

Abstract Sensitive DNA assays are of importance in life science and biomedical engineering, but they are heavily dependent on thermal cycling programs or enzyme-assisted schemes, which require the utilization of bulky devices and costly reagents. To circumvent such requirements, we developed an isothermal enzyme-free DNA sensing method with dual-stage signal amplification ability based on the coupling use of catalytic hairpin assembly (CHA) and Mg2+-dependent deoxyribozyme (DNAzyme). In this study, the sensing system involves a set of hairpin DNA probes for CHA (ensuring the first stage of signal amplification) as well as ribonucleobase-modified molecular beacons that serve as activatable substrates for DNAzymes (warranting the second stage of signal amplification). An experimentally determined detection limit of about 0.5 pM is achieved with a good linear range from 0.5 to 10 pM. The results from spiked fetal bovine serum samples further confirm the reliability for practical applications. The non-thermal cycling, enzyme-free, and dual-amplified features make it a powerful sensing tool for effective nucleic acid assay in a variety of biomedical applications.

Epidemiology and Molecular Characterization of Seasonal Influenza Viruses in Iraq.

The importance of influenza viruses in respiratory infections in the Middle East, including Iraq, has been historically overlooked. Nowadays, with the pandemic of corona virus disease 2019, the importance of prevention from other respiratory diseases, such as seasonal influenza, can be a critical step in the health management system. Therefore, this study aimed to evaluate the prevalence and seasonal occurrence of influenza viruses in the Iraqi population presented with influenza-like illness (ILI) or severe acute respiratory infection (SARI)within2015-2017. Moreover, this study was conducted to identify the periods with increased influenza transmission for vaccination recommendations in Iraq. In the present study, we presented the cases of infection byinfluenzaA or B viruses. To test influenza virus types A (H1N1 and H3N2) and B, 1,359 throat and nasal swabs were collected from patients with ILI or SARI. Ribonucleic acid was extracted and amplified using a set of primers and probes.The frequency rates of infection were obtained at 1,616 (45%) and 1974 (55%) in females and males, respectively. The mean age of the participants was estimated at 31.71±22.68 with a minimum and maximum ages of 1 month and 96 years, respectively. It was revealed that influenza virus type A was the most predominant with an incidence of 16.2%, followed by type B with 0.33% incidence. It was also found that December was the most prevalent month of being infected by influenza viruses types A and B (30.02% and 0.48%, respectively). Vaccination in September would likely protect the highest number of patients. It was clear that the influenza A virus was predominant over type B. In Iraq, influenza A and B viruses were found in a large percentage of ILI and SARI cases. Additionally, males were reported to be more likely to become infected than females.

A Magnetic Modulation Biosensing-Based Molecular Assay for Rapid and Highly Sensitive Clinical Diagnosis of Coronavirus Disease 2019 (COVID-19)

Rapid and sensitive detection of human pathogens, such as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is an urgent and challenging task for clinical laboratories. Currently, the gold standard for SARS-CoV-2–specific RNA is based on quantitative RT-PCR (RT-qPCR), which relies on target amplification by Taq polymerase and uses a fluorescent resonance energy transfer–based hydrolysis probe. Although this method is accurate and specific, it is also time consuming. Here, a new molecular assay is described that combines a highly sensitive magnetic modulation biosensing (MMB) system, rapid thermal cycling, and a modified double-quenched hydrolysis probe. In vitro transcribed SARS-CoV-2 RNA targets spiked in PCR-grade water, were used to show that the calculated limit of detection of the MMB-based molecular assay was 1.6 copies per reaction. Testing 309 RNA extracts from 170 confirmed RT-qPCR SARS-CoV-2–negative individuals (30 of whom were positive for other respiratory viruses) and 139 RT-qPCR SARS-CoV-2–positive patients (CT ≤ 42) resulted in 97.8% sensitivity, 100% specificity, and 0% cross-reactivity. The total turnaround time of the MMB-based assay is 30 minutes, which is three to four times faster than a standard RT-qPCR. By adjusting the primers and the probe set, the platform can be easily adapted to detect most of the pathogens that are currently being diagnosed by RT-qPCR.

Leveraging telehealth to evaluate infants with prodromal autism spectrum disorder characteristics using the telehealth evaluation of development for infants.

Many families seeking early evaluations for autism spectrum disorder face long waitlists, must often travel to centers with appropriate expertise, and are frequently told by providers to "wait and see." This results in significant stress for families and delayed supports to infants and their caregivers who could benefit. This study evaluated whether telehealth could be used to identify and evaluate infants with early autism spectrum disorder characteristics in the first year of life. In this study, we evaluated 41 infants via telehealth using a standard set of probes and scored behavior related to social communication, play, imitation, and other developmental domains. We found the majority of infants demonstrated elevated likelihood of autism spectrum disorder on both parent-reported questionnaires and examiner-rated behavior. Caregiver ratings of the overall utility of the protocol used in this study were high. Overall, this study demonstrates the feasibility for telehealth-based approaches to evaluate infants' with elevated likelihood of autism spectrum disorder in the first year of life, which could help to improve families' access to care and to expand our capacity to conduct studies evaluating possible intervention supports.

Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.

Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.

Probe design for simultaneous, targeted capture of diverse metagenomic targets

SummaryThe compounding challenges of low signal, high background, and uncertain targets plague many metagenomic sequencing efforts. One solution has been DNA capture, wherein probes are designed to hybridize with target sequences, enriching them in relation to their background. However, balancing probe depth with breadth of capture is challenging for diverse targets. To find this balance, we have developed the HUBDesign pipeline, which makes use of sequence homology to design probes at multiple taxonomic levels. This creates an efficient probe set capable of simultaneously and specifically capturing known and related sequences. We validated HUBDesign by generating probe sets targeting the breadth of coronavirus diversity, as well as a suite of bacterial pathogens often underlying sepsis. In separate experiments demonstrating significant, simultaneous enrichment, we captured SARS-CoV-2 and HCoV-NL63 in a human RNA background and seven bacterial strains in human blood. HUBDesign (https://github.com/zacherydickson/HUBDesign) has broad applicability wherever there are multiple organisms of interest.

A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology.

Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

Application of Genetic Algorithm-Based Support Vector Machine in Identification of Gene Expression Signatures for Psoriasis Classification: A Hybrid Model.

Background Psoriasis is a chronic autoimmune disease impairing significantly the quality of life of the patient. The diagnosis of the disease is done via a visual inspection of the lesional skin by dermatologists. Classification of psoriasis using gene expression is an important issue for the early and effective treatment of the disease. Therefore, gene expression data and selection of suitable gene signatures are effective sources of information. Methods We aimed to develop a hybrid classifier for the diagnosis of psoriasis based on two machine learning models of the genetic algorithm and support vector machine (SVM). The method also conducts gene signature selection. A publically available gene expression dataset was used to test the model. Results A number of 181 probe sets were selected among the original 54,675 probes using the hybrid model with a prediction accuracy of 100% over the test set. A number of 10 hub genes were identified using the protein-protein interaction network. Nine out of 10 identified genes were found in significant modules. Conclusions The results showed that the genetic algorithm improved the SVM classifier performance significantly implying the ability of the proposed model in terms of detecting relevant gene expression signatures as the best features.

Anchoring the CerEla1.0 Genome Assembly to Red Deer (Cervus elaphus) and Cattle (Bos taurus) Chromosomes and Specification of Evolutionary Chromosome Rearrangements in Cervidae.

Simple SummaryThe red deer (Cervus elaphus) de novo genome assembly (CerEla1.0) has provided a great resource for genetic studies in various deer species. In this study, we used gene order comparisons between C. elaphus CerEla1.0 and B. taurus ARS-UCD1.2 genome assemblies and fluorescence in situ hybridization (FISH) with bovine BAC probes to verify the red deer-bovine chromosome relationships and anchor the CerEla1.0 C-scaffolds to karyotypes of both species. We showed the homology between bovine and deer chromosomes and determined the centromere-telomere orientation of the CerEla1.0 C-scaffolds. Using a set of BAC probes, we were able to narrow the positions of evolutionary chromosome breakpoints defining the family Cervidae. In addition, we revealed several errors in the current CerEla1.0 genome assembly. Finally, we expanded our analysis to other Cervidae and confirmed the locations of the cervid evolutionary fissions and orientation of the fused chromosomes in eight cervid species. Our results can serve as a basis for necessary improvements of the red deer genome assembly and provide support to other genetic studies in Cervidae.The family Cervidae groups a range of species with an increasing economic significance. Their karyotypes share 35 evolutionary conserved chromosomal segments with cattle (Bos taurus). Recent publication of the annotated red deer (Cervus elaphus) whole genome assembly (CerEla1.0) has provided a basis for advanced genetic studies. In this study, we compared the red deer CerEla1.0 and bovine ARS-UCD1.2 genome assembly and used fluorescence in situ hybridization with bovine BAC probes to verify the homology between bovine and deer chromosomes, determined the centromere-telomere orientation of the CerEla1.0 C-scaffolds and specified positions of the cervid evolutionary chromosome breakpoints. In addition, we revealed several incongruences between the current deer and bovine genome assemblies that were shown to be caused by errors in the CerEla1.0 assembly. Finally, we verified the centromere-to-centromere orientation of evolutionarily fused chromosomes in seven additional deer species, giving a support to previous studies on their chromosome evolution.

FEMALE GAMETE AND HUMAN EMBRYO SUSCEPTIBILITY TO SARS-COV-2

Objective: To determine the susceptibility of the oocytes, embryos, granulosa and cumulus cells to SARS-CoV-2 infection. Materials and Methods: To address this question, we retrospectively examined the gene expression profiles of SARS-CoV-2-associated receptors and proteases in human granulosa cells (GCs), cumulus cells (CCs), mature oocytes, day 3 embryos, blastocysts and trophectoderm cells obtained from our previously described Affymetrix microarray data from assisted reproduction patients. Human GCs and CCs (n=17), mature oocytes (n=6), and preimplantation embryos (n=20) were analyzed and gene expression levels of receptors and proteases closely related to SARS-CoV-2 infection was reported. For each gene, the number of samples with the probe set 'present', based on the detection call was studied. Each probe set was classified according to the signal intensity value median, as low ( 200). Results: ACE2, BSG, CTSL, CTSA were detectable at high expression level in all mature oocyte samples, while only CTSL was strongly expressed in all day 3 embryos. The most representative dual co-expression of SARS-CoV-2-associated receptor and protease (60% of samples) during the embryonic genome activation stage (EGA) was ACE2-CTSL and BSG-CTSL. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level, and the prevalence of the different dual co-expression of SARS-CoV-2-associated proteases and receptors was optimal (100% of samples). Interestingly, only CTSL was detectable in all trophectoderm samples and a prevalence of 60% was found for the BSG-CTSL co-expression. ACE2, BSG, CTSL and CTSA were present at high expression level in CCs samples. In contrast, ACE2 and BSG expression was very low while CTSL and CTSA showed a high expression level in GCs. A prevalence of 100% was reported for ACE2-CTSL, ACE2-CTSA co-expression for both cell types. In addition, BSG-CTSL and BSG-CTSA co-expression were also present in all CCs against ∼70% in GCs samples. This data suggests a potential risk of SARS-CoV-2 infection either GC or early embryo development. Conclusions: Transcriptomic analyses of SARS-CoV-2-associated receptors and proteases strongly suggest that blastocysts are most permissive to SARS-CoV-2 compared with mature oocytes and day 3 embryos. Impact Statement: Specimens from female genital tract may be considered as potential targets for SARS-CoV-2.

A genome-wide SNP genotyping resource for tropical pine tree species.

We performed gene and genome targeted SNP discovery towards the development of a genome-wide, multispecies genotyping array for tropical pines. Pooled RNA-seq data from shoots of seedlings from five tropical pine species was used to identify transcript-based SNPs resulting in 1.3million candidate Affymetrix SNP probe sets. In addition, we used a custom 40K probe set to perform capture-seq in pooled DNA from 81 provenances representing the natural ranges of six tropical pine species in Mexico and Central America resulting in 563K candidate SNP probe sets. Altogether, 300K RNA-seq (72%) and 120K capture-seq (28%) derived SNP probe sets were tiled on a 420K screening array that was used to genotype 576 trees representing the 81 provenances and commercial breeding material. Based on the screening array results, 50K SNPs were selected for commercial SNP array production including 20K polymorphic SNPs for P. patula, P. tecunumanii, P. oocarpa and P. caribaea, 15K for P. greggii and P. maximinoi, 13K for P. elliottii and 8K for P. pseudostrobus. We included 9.7K ancestry informative SNPs that will be valuable for species and hybrid discrimination. Of the 50K SNP markers, 25% are polymorphic in only one species, while 75% are shared by two or more species. The Pitro50K SNP chip will be useful for population genomics and molecular breeding in this group of pine species that, together with their hybrids, represent the majority of fast-growing tropical and subtropical pine plantations globally.

Novel real-time PCR protocol for the detection of house cricket (Acheta domesticus) in feed

A new real-time PCR protocol was developed for the detection of house cricket (Acheta domesticus) in feeds. A sequence of 97 bp size within the mitochondrial barcode region (cytochrome oxidase c subunit I - COI) was amplified by the chosen specific primer and probe set. The specificity of the PCR system was confirmed with a broad range of plant and animal species. The limit of detection with pure A. domesticus DNA diluted in aquaculture feed DNA was 1 genome copy, corresponding to an absolute amount of 2.14 pg DNA. In addition, the sensitivity was investigated in dependence on different processing treatments of the insect material. The method reliably detected pasteurized (80 °C, 10 min) and oven-dried (60 °C, 3 h) A. domesticus in feed mixtures down to 0.1% (w/w). Practical applicability of the proposed protocol was tested on 33 commercial feeds not declaring the presence of house cricket in the list of ingredients, and on two model feeds spiked in-house with A. domesticus. The method clearly recognized the presence of house cricket in model feeds down to 0.1%, a concentration residing in the range of accidental contamination rather than deliberate addition. It is concluded that the tested system is suitable for practical application, enabling the sensitive and reliable detection of A. domesticus material as a newly allowed component in aquaculture feed.

Generalized spaces of pointwise regularity: toward a general framework for the WLM

In this work we generalize the spaces introduced by Calderón and Zygmund using pointwise conditions emanating from generalized Besov spaces. We give conditions binding the functions belonging to these spaces and their wavelet coefficients. Next, we propose a multifractal formalism based on such spaces which generalizes the so-called wavelet leaders method and show that it is satisfied on a prevalent set.