Search Results (12)

In this study, the non-destructive determination of pH, total soluble solids (TSS), total acidity (TA), reducing sugars (RS), seed total phenolic content (TPCD), and skin total phenolic content (TPCN) in Ugni Blanc grapes was performed using visible/near-infrared (Vis/NIR) spectroscopy coupled with chemometric quantitative analysis. Diffuse reflectance spectra in the 400–1507 nm range were measured using a handheld Vis–NIR spectrometer, after which the dataset was partitioned using the SPXY algorithm, accounting for joint X-Y distances. Six spectral preprocessing methods and three modeling algorithms, Partial Least Squares (PLS), Support Vector Machine Regression (SVR), and Convolutional Neural Network (CNN), were used to construct quantitative models based on full-wavelength and feature-wavelength data. Feature-based models outperformed full-spectrum models for TA, RS, and TPCN, whereas full-spectrum models performed better for pH, TSS, and TPCD. The optimal models achieved ${\mathit{R}}_{\mathit{p}}^{\mathit{2}}$ values of 0.940, 0.957, 0.913, 0.889, 0.917, and 0.871 and RPD values of 4.074, 4.798, 3.397, 2.998, 2.904, and 2.786, correspondingly. The findings highlight the applicability of Vis/NIR spectroscopy for the accurate and non-destructive prediction of key physicochemical indicators in Ugni Blanc grapes. Full article

The endolysosomal system plays a pivotal role in cellular function. Before reaching lysosomes for degradation, the endocytosed cargoes are sorted at various stages of endosomal trafficking for recycling and/or rerouting. The proper execution of these processes depends on tightly regulated ion fluxes across endolysosomal membranes. Recent studies have demonstrated the importance of two-pore channels (TPCs), including TPC1 and TPC2, in endolysosomal trafficking. These channels are expressed in the membranes of distinct populations of endosomes and lysosomes, where they respond to nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] to conduct Ca²⁺ and Na⁺ release from these acidic organelles. Here, we discuss the potential implications of Ca²⁺ and Na⁺ fluxes mediated by TPCs across endolysosomal membranes in the physiological and pathophysiological functions of these organellar channels. Full article

Type 2 diabetes (T2D) is a chronic metabolic disorder with an estimated prevalence of over 422 million individuals affected globally. Since the advent of genomics, numerous studies have been conducted to elucidate T2D pathogenetic mechanisms and define genetic loci affecting T2D susceptibility. Transcriptomic studies, including bulk and single-cell RNA sequencing, play an important role both in discerning molecular mechanisms of the disease and in identifying potential T2D biomarkers. In this study, we performed bulk RNA-seq of whole blood of nine T2D patients and nine control subjects and performed meta-analysis of these data with seven publicly available blood RNA-seq datasets of T2D patients. Our analysis showed that the changes in the gene expression between different studies show very low concordance; moreover, a substantial number of differentially expressed genes (DEGs) was identified in only three out of eight datasets, with only five DEGs—FBLN2, TPCN1, PC, SHANK1, and PLD4—identified in all three of those datasets. Nevertheless, cross-study meta-analysis identified a broad set of 2065 DEGs, including 713 genes that have not been identified in any of the source studies. These genes showed a significant enrichment of GO terms indicating neutrophil activation and proliferation and included several genes that have not been implicated in type 2 diabetes previously. Taken together, our study highlights challenges associated with biomarker discovery from blood transcriptomics in T2D and suggests novel genes that may be considered as such biomarkers. Full article

Two-pore channel 2 (TPC2) is expressed in endolysosomes throughout the human body, as well as in melanosomes of melanocytes. Melanocytes produce pigment, i.e., melanin, which determines hair and skin color but also protects from UV light. Extensive exposure to UV light is one of the major risk factors for the development of melanoma, which develops from pigment-producing cells, i.e., melanocytes. In recent years, several human TPC2 single nucleotide polymorphisms have been identified to increase the likelihood of carriers presenting with blond hair and hypopigmentation. These variants were all characterized as gain-of-function versions of TPC2. Vice versa, the loss of function of TPC2 increases melanin production and reduces cancer hallmarks such as proliferation, migration, invasion, tumor growth, and metastasis formation. The activity of TPC2 is controlled in a complex manner, with several endogenous ligands as well as a number of interacting proteins being involved. We will discuss here the role of TPC2 in pigmentation and its potential to impact melanoma development and progression and highlight recent findings on Rab7a as an enhancer of TPC2 activity. Full article

Albinism is characterized by a variable degree of hypopigmentation affecting the skin and the hair, and causing ophthalmologic abnormalities. Its oculocutaneous, ocular and syndromic forms follow an autosomal or X-linked recessive mode of inheritance, and 22 disease-causing genes are implicated in their development. Our aim was to clarify the genetic background of a Hungarian albinism cohort. Using a 22-gene albinism panel, the genetic background of 11 of the 17 Hungarian patients was elucidated. In patients with unidentified genetic backgrounds (n = 6), whole exome sequencing was performed. Our investigations revealed a novel, previously unreported rare variant (N687S) of the two-pore channel two gene (TPCN2). The N687S variant of the encoded TPC2 protein is carried by a 15-year-old Hungarian male albinism patient and his clinically unaffected mother. Our segregational analysis and in vitro functional experiments suggest that the detected novel rare TPCN2 variant alone is not a disease-causing variant in albinism. Deep genetic analyses of the family revealed that the patient also carries a phenotype-modifying R305W variant of the OCA2 protein, and he is the only family member harboring this genotype. Our results raise the possibility that this digenic combination might contribute to the observed differences between the patient and the mother, and found the genetic background of the disease in his case. Full article

Maternal parity is an important physiological factor influencing beef cow reproductive performance. However, there are few studies on the influence of different calving periods on early growth and postpartum diseases. Here, we conducted blood transcriptomic analysis on cows of different parities for gene discovery. We used Short Time Series Expression Miner (STEM) analysis to determine gene expression levels in cows of various parities and divided multiple parities into three main periods (nulliparous, primiparous, and multiparous) for subsequent analysis. Furthermore, the top 15,000 genes with the lowest median absolute deviation (MAD) were used to build a co-expression network using weighted correlation network analysis (WGCNA), and six independent modules were identified. Combing with Exon Wide Selection Signature (EWSS) and protein-protein interaction (PPI) analysis revealed that TPCN2, KIF22, MICAL3, RUNX2, PDE4A, TESK2, GPM6A, POLR1A, and KLHL6 involved in early growth and postpartum diseases. The GO and KEGG enrichment showed that the Parathyroid hormone synthesis, secretion, and action pathway and stem cell differentiation function-related pathways were enriched. Collectively, our study revealed candidate genes and gene networks regulating the early growth and postpartum diseases and provided new insights into the potential mechanism of reproduction advantages of different parity selection. Full article

Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca²⁺-mobilizing messenger NAADP and/or the lipid PI(3,5)P₂. TPC currents are regulated by H⁺, Ca²⁺, and Mg²⁺ (luminal and/or cytosolic), as well as protein kinases, and they are impacted by single-nucleotide polymorphisms linked to pigmentation. Bisbenzylisoquinoline alkaloids, flavonoids, and several approved drugs demonstrably block channel activity. Endogenous TPC currents have been recorded from a number of primary cell types and cell lines. Many of the properties of endolysosomal TPCs are recapitulated upon rerouting channels to the cell surface, allowing more facile recording through conventional electrophysiological means. Single-channel analyses have provided high-resolution insight into both monovalent and divalent permeability. The discovery of small-molecule activators of TPC2 that toggle the ion selectivity from a Ca²⁺-permeable (NAADP-like) state to a Na⁺-selective (PI(3,5)P₂-like) state explains discrepancies in the literature relating to the permeability of TPCs. Identification of binding proteins that confer NAADP-sensitive currents confirm that indirect, remote gating likely underpins the inconsistent observations of channel activation by NAADP. Full article

Two pore channels (TPCs) are implicated in vesicle trafficking, virus infection, and autophagy regulation. As Na⁺- or Ca²⁺-permeable channels, TPCs have been reported to be activated by NAADP, PI(3,5)P₂, and/or high voltage. However, a comparative study on the function and regulation of the three mammalian TPC subtypes is currently lacking. Here, we used the electrophysiological recording of enlarged endolysosome vacuoles, inside-out and outside-out membrane patches to examine the three TPCs of rabbit (Oryctolagus cuniculus, or Oc) heterologously expressed in HEK293 cells. While PI(3,5)P₂ evoked Na⁺ currents with a potency order of OcTPC1 > OcTPC3 > OcTPC2, only OcTPC2 displayed a strict dependence on PI(3,5)P₂. Both OcTPC1 and OcTPC3 were activatable by PI3P and OcTPC3 was also activated by additional phosphoinositide species. While OcTPC2 was voltage-independent, OcTPC1 and OcTPC3 showed voltage dependence with OcTPC3 depending on high positive voltages. Finally, while OcTPC2 preferred a luminal pH of 4.6–6.0 in endolysosomes, OcTPC1 was strongly inhibited by extracytosolic pH 5.0 in both voltage-dependent and -independent manners, and OcTPC3 was inhibited by pH 6.0 but potentiated by pH 8.0. Thus, the three OcTPCs form phosphoinositide-activated Na⁺ channels with different ligand selectivity, voltage dependence, and extracytosolic pH sensitivity, which likely are optimally tuned for function in specific endolysosomal populations. Full article

The long search for the environmental trigger of the endemic pemphigus foliaceus (EPF, fogo selvagem) has not yet resulted in any tangible findings. Here, we searched for genetic associations and the differential expression of host genes involved in early viral infections and innate antiviral defense. Genetic variants could alter the structure, expression sites, or levels of the gene products, impacting their functions. By analyzing 3063 variants of 166 candidate genes in 227 EPF patients and 194 controls, we found 12 variants within 11 genes associated with differential susceptibility (p < 0.005) to EPF. The products of genes TRIM5, TPCN2, EIF4E, EIF4E3, NUP37, NUP50, NUP88, TPR, USP15, IRF8, and JAK1 are involved in different mechanisms of viral control, for example, the regulation of viral entry into the host cell or recognition of viral nucleic acids and proteins. Only two of nine variants were also associated in an independent German cohort of sporadic PF (75 patients, 150 controls), aligning with our hypothesis that antiviral host genes play a major role in EPF due to a specific virus–human interaction in the endemic region. Moreover, CCL5, P4HB, and APOBEC3G mRNA levels were increased (p < 0.001) in CD4+ T lymphocytes of EPF patients. Because there is limited or no evidence that these genes are involved in autoimmunity, their crucial role in antiviral responses and the associations that we observed support the hypothesis of a viral trigger for EPF, presumably a still unnoticed flavivirus. This work opens new frontiers in searching for the trigger of EPF, with the potential to advance translational research that aims for disease prevention and treatment. Full article

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible. Full article

Type 2 diabetes (T2D) is a complex disorder that is caused by a combination of genetic, epigenetic, and environmental factors. High-throughput approaches have opened a new avenue toward a better understanding of the molecular bases of T2D. A genome-wide association studies (GWASs) identified a group of the most common susceptibility genes for T2D (i.e., TCF7L2, PPARG, KCNJ1, HNF1A, PTPN1, and CDKAL1) and illuminated novel disease-causing pathways. Next-generation sequencing (NGS)-based techniques have shed light on rare-coding genetic variants that account for an appreciable fraction of T2D heritability (KCNQ1 and ADRA2A) and population risk of T2D (SLC16A11, TPCN2, PAM, and CCND2). Moreover, single-cell sequencing of human pancreatic islets identified gene signatures that are exclusive to α-cells (GCG, IRX2, and IGFBP2) and β-cells (INS, ADCYAP1, INS-IGF2, and MAFA). Ongoing epigenome-wide association studies (EWASs) have progressively defined links between epigenetic markers and the transcriptional activity of T2D target genes. Differentially methylated regions were found in TCF7L2, THADA, KCNQ1, TXNIP, SOCS3, SREBF1, and KLF14 loci that are related to T2D. Additionally, chromatin state maps in pancreatic islets were provided and several non-coding RNAs (ncRNA) that are key to T2D pathogenesis were identified (i.e., miR-375). The present review summarizes major progress that has been made in mapping the (epi)genomic landscape of T2D within the last few years. Full article

Hair color is one of the most visible and heritable traits in humans. Here, we estimated heritability by structural equation modeling (N = 20,142), and performed a genome wide association (GWA) analysis (N = 7091) and a GCTA study (N = 3340) on hair color within a large cohort of twins, their parents and siblings from the Netherlands Twin Register (NTR). Self-reported hair color was analyzed as five binary phenotypes, namely “blond versus non-blond”, “red versus non-red”, “brown versus non-brown”, “black versus non-black”, and “light versus dark”. The broad-sense heritability of hair color was estimated between 73% and 99% and the genetic component included non-additive genetic variance. Assortative mating for hair color was significant, except for red and black hair color. From GCTA analyses, at most 24.6% of the additive genetic variance in hair color was explained by 1000G well-imputed SNPs. Genome-wide association analysis for each hair color showed that SNPs in the MC1R region were significantly associated with red, brown and black hair, and also with light versus dark hair color. Five other known genes (HERC2, TPCN2, SLC24A4, IRF4, and KITLG) gave genome-wide significant hits for blond, brown and light versus dark hair color. We did not find and replicate any new loci for hair color. Full article