Immunohistochemistry (IHC) and immunofluorescence (IF) are fundamental molecular biology techniques to assess protein expression. However, the melanin present normally in the eye in the uveal tract (choroid, iris, and ciliary body) and the retinal pigment epithelium (RPE) poses a significant challenge for IHC and IF. This is because melanin interferes with both chromogenic and fluorescent detection methods. Additionally, formalin fixation, which is commonly used for IHC, can result in shrinkage and loss of cellular detail in the eye. This protocol provides an optimized approach using Davidson’s fixative with a hydrogen peroxide bleaching step to eliminate melanin interference in the mouse eye, improving the quality and interpretability of IHC analyses of the uveal tract and RPE. It is particularly useful for the analysis of uveal melanoma.
Laurenz De Cock, Erika D’haenens, Lies Vantomme
et al.
Abstract RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification.
Catarina Leal, Ales Eichmeier, Kateřina Štůsková
et al.
With a reduction in available chemical treatments, there is an increased interest in biological control of grapevine trunk diseases. Few studies have investigated the impact of introducing beneficial microorganisms in the rhizosphere on the existing indigenous soil microbiome. In this study, we explored the effect of two biocontrol agents (BCAs), Trichoderma atroviride SC1 (Ta SC1) (Vintec; Certis Belchim) and Bacillus subtilis PTA-271 (Bs PTA-271), on the grapevine rhizosphere bacterial and fungal microbiome as well as plant defense expression using high-throughput amplicon sequencing and quantitative real-time polymerase chain reaction (PCR), respectively. Additionally, we quantified both Ta SC1 and Bs PTA-271 in the rhizosphere over time using droplet digital PCR. The fungal microbiome was more affected by factors such as soil type, BCA treatment, and sampling time compared with the bacterial microbiome. Specifically, Ta SC1 application produced negative impacts on fungal diversity, whereas application of BCAs did not affect bacterial diversity. Interestingly, the survival and establishment of both BCAs showed opposite trends depending on the soil type, indicating that the physicochemical properties of soils have a role in BCA establishment. Fungal co-occurrence networks were less complex than bacterial networks but highly impacted by Ta SC1 application. Soils treated with Ta SC1 presented more complex and stable co-occurrence networks, with a higher number of positive correlations. Induced grapevine defenses also differed according to the soil, being more affected by BCA inoculation on sandy soil. The findings of this research emphasize the complex relationships among microorganisms in the rhizosphere and highlight the significance of taking into account various factors, such as soil type, sampling time, and BCA treatment, and their influence on the structure and dynamics of microbial communities.
Magdalena Sevilla-González, Maria Fernanda Garibay-Gutiérrez, Arsenio Vargas-Vázquez
et al.
Background: A risk haplotype in SLC16A11 characterized by alterations in fatty acid metabolism emerged as a genetic risk factor associated with increased susceptibility to type 2 diabetes (T2D) in Mexican population. Its role on treatment responses is not well understood. Objectives: We aimed to determine the impact of the risk haplotype on the metabolomic profile during a lifestyle intervention (LSI). Methods: We recruited Mexican-mestizo individuals with ≥1 prediabetes criteria according to the American Diabetes Association with a body mass index between 25 and 45 kg/m2. We conducted a 24-wk quasiexperimental LSI study for diabetes prevention. Here, we compared longitudinal plasma liquid chromatography/mass spectrometry metabolomic changes between carriers and noncarriers. We analyzed the association of risk haplotype with metabolites leveraging repeated assessments using multivariable-adjusted linear mixed models. Results: Before the intervention, carriers (N = 21) showed higher concentrations of hippurate, C16 carnitine, glycine, and cinnamoylglycine. After 24 wk of LSI, carriers exhibited a deleterious metabolomic profile. This profile was characterized by increased concentrations of hippurate, cinnamoglycine, xanthosine, N-acetylputrescine, L-acetylcarnitine, ceramide (d18:1/24:1), and decreased concentrations of citrulline and phosphatidylethanolamine. These metabolites were associated with higher concentrations of total cholesterol, triglycerides, and low density lipoprotein cholesterol. The effect of LSI on the risk haplotype was notably more pronounced in its impact on 2 metabolites: methylmalonylcarnitine (β: −0.56; P-interaction = 0.014) and betaine (β: −0.64; P-interaction = 0.017). Interestingly, lower consumption across visits of polyunsaturated (β: −0.038; P = 0.017) fatty acids were associated with higher concentrations of methylmalonylcarnitine. Covariates for adjustment across models included age, sex, genetic ancestry principal components, and body mass index. Conclusions: Our study highlights the persistence of deleterious metabolomic patterns associated with the risk haplotype before and during a 24-wk LSI. We also emphasize the potential regulatory role of polyunsaturated fatty acids on methylmalonylcarnitine concentrations suggesting a route for improving interventions for individuals with high-genetic risk.
Nutrition. Foods and food supply, Nutritional diseases. Deficiency diseases
Climate change is now evident and severe water shortage due to unpredictable raining season along with extended summers is expected to hamper crop production across the globe. Application of nanoparticle based formulations is one of the most sought after approach that is being explored currently to alleviate drought stress impact on plants. The present study was aimed to evaluate the potential of biosynthesized silica nanoparticles (silica NPs) in improving the drought tolerance of wheat. Four different concentrations of silica NPs (30, 60, 90, and 120 ppm) were used to treat wheat plants grown under two irrigation regimes- 50% soil moisture content (drought) and 100% soil moisture content (well-watered). The induced drought caused a prominent reduction in both - the crop yield and the morphological parameters of the crop. Foliar application of silica NPs at all concentrations, increased the plant's tolerance towards water stress but 60 ppm concentration was found to be most effective amongst all. After treatment with silica NPs at 60 ppm concentration, the plant height increased by 8.28%, spikes per plant by 98%, seeds per spike by 12.4%, and thousand seed weight by 37.5% as compared to the control. Besides this, expression levels of four drought-stress responsive genes-ABC1, Wdhn13, CHP, and EXP2 was also studied. We observed an enhanced expression of all the stress genes after treatment with silica nanoparticles in wheat plants grown under water deficient conditions, clearly supporting the influence of NP treatment at gene/molecular level. In nutshell, we conclude that silica nanoparticles have the potential to significantly ameliorate the negative impact of drought stress by reviving plant growth and modulating gene expression.
The aim of this trial was to investigate the effects of dietary protein and lipid levels on growth performance and flesh quality of largemouth bass (Micropterus salmoides). A 3 × 3 two-factor design was used, including three crude protein levels of 46%, 50%, 54% (P46, P50, P54) and three crude lipid levels of 8%, 12%, 16% (L8, L12, L16). Thus, a total of nine diets (P46/L8, P46/L12, P46/L16, P50/L8, P50/L12, P50/L16, P54/L8, P54/L12 and P54/L16) were prepared and fed to large-size largemouth bass with initial body weight of 109.7 g for 84 days. At the same dietary protein level, weight gain rate (WGR), protein efficiency rate (PER), viscerosomatic index and polyunsaturated fatty acids (PUFAs) percent in flesh showed an increasing trend with increasing the dietary lipid level, while feed conversion ratio (FCR) showed a decreasing trend. At P46 and P50, whole fish crude protein, muscle crude protein, total collagen content, hardness, springiness, chewiness and shear force increased, and steaming loss and freezing loss decreased with increasing the dietary lipid level, while the above indicators showed an opposite trend at P54. At P50, the protein retention (PR) of 16% crude lipid group was significantly higher than that in the 8% and 12% crude lipid groups (P < 0.05). At the same dietary lipid level, the 50% and 54% crude protein groups presented higher WGR and lower FCR than the 46% crude protein group. The P54/L16 group had the highest WGR (184.4%) and lowest FCR (0.92) among all the groups, but the P50/L16 group presented similar WGR and FCR to the P54/L16 group (P > 0.05). The P50/L16 group showed the highest PER, PR, whole fish protein, muscle protein, total collagen, PUFAs, muscle hardness, springiness, chewiness, shear force and the lowest steaming loss. In conclusion, based on growth performance and flesh quality, the appropriate dietary protein and lipid requirements for large-size largemouth bass were 50% and 16%, respectively.
Rice blast, caused by Magnaporthe oryzae (M. oryzae), is one of the most destructive diseases threatening rice production worldwide. Development of resistant cultivars using broad-spectrum resistance (R) genes with high breeding value is the most effective and economical approach to control this disease. In this study, the breeding potential of Pigm gene in geng/japonica rice breeding practice in Jiangsu province was comprehensively evaluated. Through backcross and marker-assisted selection (MAS), Pigm was introduced into two geng rice cultivars (Wuyungeng 32/WYG32 and Huageng 8/HG8). In each genetic background, five advanced backcross lines with Pigm (ABLs) and the same genotypes as the respective recurrent parent in the other 13 known R gene loci were developed. Compared with the corresponding recurrent parent, all these ABLs exhibited stronger resistance in seedling inoculation assay using 184 isolates collected from rice growing regions of the lower region of the Yangtze River. With respect to panicle blast resistance, all ABLs reached a high resistance level to blast disease in tests conducted in three consecutive years with the inoculation of seven mixed conidial suspensions collected from different regions of Jiangsu province. In natural field nursery assays, the ABLs showed significantly higher resistance than the recurrent parents. No common change on importantly morphological traits and yield-associated components was found among the ABLs, demonstrating the introduction of Pigm had no tightly linked undesirable effect on rice economically important traits and its associated grain weight reduction effect could be probably offset by others grain weight genes or at least in the background of the aforementioned two varieties. Notably, one rice line with Pigm, designated as Yangnonggeng 3091, had been authorized as a new variety in Jiangsu province in 2021, showing excellent performance on both grain yield and quality, as well as the blast resistance. Together, these results suggest that the Pigm gene has a high breeding value in developing rice varieties with durable and broad-spectrum resistance to blast disease.
Marta Alonso-Hearn, Gerard Badia-Bringué, Maria Canive
Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease or paratuberculosis (PTB), with important animal health and economic implications. There are no therapeutic strategies to control this disease, and vaccination with inactivated vaccines is limited in many countries because it can interfere with the intradermal test used for bovine tuberculosis detection. Thus, infected animals either get culled after a positive ELISA or fecal PCR result or die due to clinical disease. In this study, we review recent studies aimed to discover genetic markers which could help to identify and select cattle less susceptible and more resilient to PTB. In recent years, the genotyping and subsequent imputation to whole-genome sequence (WGS) has allowed the identification of single-nucleotide polymorphisms (SNPs), quantitative trait loci (QTL), and candidate genes in the Bos taurus genome associated with susceptibility to MAP infection. In most of these genome-wide association studies (GWAS), phenotypes were based on ante-mortem test results including serum ELISA, milk ELISA, and detection of MAP by fecal PCR and bacteriological culture. Cattle infected with MAP display lesions with distinct severity but the associations between host genetics and PTB-associated pathology had not been explored until very recently. On the contrary, the understanding of the mechanisms and genetic loci influencing pathogen resistance, and disease tolerance in asymptomatic individuals is currently very limited. The identification of long-time asymptomatic cattle that is able to resist the infection and/or tolerate the disease without having their health and milk production compromised is important for disease control and breeding purposes.
Mohammed Alyahya, Taghrid Aloraini, Youseef Al-Harbi
et al.
Background: Physicians and geneticists face challenges in making accurate diagnoses during clinical evaluations; affecting patients and clinicians. The aim of this study was to estimate the hit rate of the non-consanguineous population. Moreover, prevalence of the genetic disorder in both the consanguineous and non- consanguineous population of Saudi Arabia at King Abdulaziz Medical City in Riyadh data.
Methods: We reviewed 681 families and 1563 individuals with 2,565,335 variants in the King Abdullah International Medical Research Center (KAIMRC) Genomic Database (KGD), Riyadh, Saudi Arabia. All the ES requests were obtained from the physician and clinical geneticist of KAIMRC, and the test was performed either in-house or in a College of American Pathologists accredited laboratory center for clinical purposes.
Results: A total of 151 non-consanguineous individuals with exome sequencing requests in the population genomic database of King Abdullah International Medical Research Center was considered for the study. In total, 27 had disease-causing variants, and the hit rate was 27/151 (18%). Among the 28 different variants in the 27 individuals, 50% were de novo variants and 50% inherited. The hit rate of the variants causing autosomal recessive disorders was 12/28 (42.8%), autosomal dominant disorders 13/28 (46.4%), and X-linked disorders 3/28 (10.7%).
Conclusion: Non-consanguineous marriages have a lower risk of genetic disorders, and reducing consanguinity reduces the risk of genetic disorders by two to three times.
[JBCGenetics 2022; 5(2.000): 37-42]
Sofia Antunes-Duarte, Maria Mendonça-Sanches , Rita Pimenta
et al.
Hailey-Hailey disease (HHD) is a rare autosomal dominant acantholytic dermatosis. It is characterized by a recurrent eruption of vesicles, erosions, and scaly erythematous plaques involving intertriginous areas and first occurring after puberty, mostly in the third or fourth decade. In 2000, mutations in the ATP2C1 gene on band 3q22.1, encoding the secretory pathway Ca2+/Mn2+-ATPase protein 1(hSPCA1), have been identified as the cause of HHD. We report the identification of two novel mutations of ATP2C1 gene in two Portuguese patients, which expands the spectrum of ATP2C1 mutations underlying HHD and provides useful information for genetic counseling.
Chinmay Saha, Sayantan Laha, Raghunath Chatterjee
et al.
Altered expression of protein coding gene (PCG) and long non-coding RNA (lncRNA) have been identified in SARS-CoV-2 infected cells and tissues from COVID-19 patients. The functional role and mechanism (s) of transcriptional regulation of deregulated genes in COVID-19 remain largely unknown. In the present communication, reanalyzing publicly available gene expression data, we observed that 66 lncRNA and 5491 PCG were deregulated in more than one experimental condition. Combining our earlier published results and using different publicly available resources, it was observed that 72 deregulated lncRNA interacted with 3228 genes/proteins. Many targets of deregulated lncRNA could also interact with SARS-CoV-2 coded proteins, modulated by IFN treatment and identified in CRISPR screening to modulate SARS-CoV-2 infection. The majority of the deregulated lncRNA and PCG were targets of at least one of the transcription factors (TFs), interferon responsive factors (IRFs), signal transducer, and activator of transcription (STATs), NFκB, MYC, and RELA/p65. Deregulated 1069 PCG was joint targets of lncRNA and TF. These joint targets are significantly enriched with pathways relevant for SARS-CoV-2 infection indicating that joint regulation of PCG could be one of the mechanisms for deregulation. Over all this manuscript showed possible involvement of lncRNA and mechanisms of deregulation of PCG in the pathogenesis of COVID-19.
Mourad A. M. Aboul-Soud, Alhusain J. Alzahrani, Amer Mahmoud
The discovery of induced pluripotent stem cells (iPSCs) has made an invaluable contribution to the field of regenerative medicine, paving way for identifying the true potential of human embryonic stem cells (ESCs). Since the controversy around ethicality of ESCs continue to be debated, iPSCs have been used to circumvent the process around destruction of the human embryo. The use of iPSCs have transformed biological research, wherein increasing number of studies are documenting nuclear reprogramming strategies to make them beneficial models for drug screening as well as disease modelling. The flexibility around the use of iPSCs include compatibility to non-invasive harvesting, and ability to source from patients with rare diseases. iPSCs have been widely used in cardiac disease modelling, studying inherited arrhythmias, neural disorders including Alzheimer’s disease, liver disease, and spinal cord injury. Extensive research around identifying factors that are involved in maintaining the identity of ESCs during induction of pluripotency in somatic cells is undertaken. The focus of the current review is to detail all the clinical translation research around iPSCs and the strength of its ever-growing potential in the clinical space.
Marjo J. den Broeder, Jarle Ballangby, Leonie M. Kamminga
et al.
Abstract Background Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility may in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. The epigenetic modifier enhancer of zeste 2 (Ezh2) is a histone H3K27 methyltransferase implicated to play a role in lipid metabolism and adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate. We used the environmental chemical tributyltin (TBT) as a positive control, as this chemical is known to act on lipid metabolism via EZH-mediated pathways in mammals. Results Zebrafish embryos (0–5 days post-fertilization, dpf) exposed to non-toxic concentrations of PF-06726304 acetate (5 μM) and TBT (1 nM) exhibited increased lipid accumulation. Changes in chromatin were analyzed by the assay for transposase-accessible chromatin sequencing (ATAC-seq) at 50% epiboly (5.5 hpf). We observed 349 altered chromatin regions, predominantly located at H3K27me3 loci and mostly more open chromatin in the exposed samples. Genes associated to these loci were linked to metabolic pathways. In addition, a selection of genes involved in lipid homeostasis, adipogenesis and genes specifically targeted by PF-06726304 acetate via altered chromatin accessibility were differentially expressed after TBT and PF-06726304 acetate exposure at 5 dpf, but not at 50% epiboly stage. One gene, cebpa, did not show a change in chromatin, but did show a change in gene expression at 5 dpf. Interestingly, underlying H3K27me3 marks were significantly decreased at this locus at 50% epiboly. Conclusions Here, we show for the first time the applicability of ATAC-seq as a tool to investigate toxicological responses in zebrafish. Our analysis indicates that Ezh2 inhibition leads to a partial primed state of chromatin linked to metabolic pathways which results in gene expression changes later in development, leading to enhanced lipid accumulation. Although ATAC-seq seems promising, our in-depth assessment of the cebpa locus indicates that we need to consider underlying epigenetic marks as well.
Santiago Radío, Santiago Radío, Rafael Sebastián Fort
et al.
Most signals involved in post-transcriptional regulatory networks are located in the untranslated regions (UTRs) of the mRNAs. Therefore, to deepen our understanding of gene expression regulation, delimitation of these regions with high accuracy is needed. The trypanosomatid lineage includes a variety of parasitic protozoans causing a significant worldwide burden on human health. Given their peculiar mechanisms of gene expression, these organisms depend on post-transcriptional regulation as the main level of gene expression control. In this context, the definition of the UTR regions becomes of key importance. We have developed UTR-mini-exon (UTRme), a graphical user interface (GUI) stand-alone application to identify and annotate 5′ and 3′ UTR regions in a highly accurate way. UTRme implements a multiple scoring system tailored to address the issue of false positive UTR assignment that frequently arise because of the characteristics of the intergenic regions. Even though it was developed for trypanosomatids, the tool can be used to predict 3′ sites in any eukaryote and 5′ UTRs in any organism where trans-splicing occurs (such as the model organism C. elegans). UTRme offers a way for non-bioinformaticians to precisely determine UTRs from transcriptomic data. The tool is freely available via the conda and github repositories.
Jean-Étienne R. L. Morlighem, Chen Huang, Qiwen Liao
et al.
Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.
Glutathione S-transferase (GST) genes play a protective role against oxidative stress and may influence disease risk and drug pharmacokinetics. In this study, massive multiscalar trait profiling across a large population of mice derived from a cross between C57BL/6J (B6) and DBA2/J (D2)--the BXD family--was combined with linkage and bioinformatic analyses to characterize mechanisms controlling GST expression and to identify downstream consequences of this variation. Similar to humans, mice show a wide range in expression of GST family members. Variation in the expression of Gsta4, Gstt2, Gstz1, Gsto1, and Mgst3 is modulated by local expression QTLs (eQTLs) in several tissues. Higher expression of Gsto1 in brain and liver of BXD strains is strongly associated (P < 0.01) with inheritance of the B6 parental allele whereas higher expression of Gsta4 and Mgst3 in brain and liver, and Gstt2 and Gstz1 in brain is strongly associated with inheritance of the D2 parental allele. Allele-specific assays confirmed that expression of Gsto1, Gsta4, and Mgst3 are modulated by sequence variants within or near each gene locus. We exploited this endogenous variation to identify coexpression networks and downstream targets in mouse and human. Through a combined systems genetics approach, we provide new insight into the biological role of naturally occurring variants in GST genes.