Search Results (21)

Cytochrome P450 monooxygenases (CYP), crucial detoxification enzymes in insects, are involved in the metabolism of endogenous substances as well as the activation and degradation of exogenous compounds. In this study, T. castaneum was utilized to investigate the roles of TcCYP6K1 and TcCYP9F2 genes influencing in the trehalose metabolism pathway under high-CO₂ stress. By predicting the functional sequences of TcCYP6K1 and TcCYP9F2 genes and analyzing their spatiotemporal expression patterns, it was discovered that both genes belong to the CYP3 group and exhibit high expression levels during the larval stage, decreasing during the pupal stage, while showing high expression in the fatty body, intestine, and malpighian tubules. Furthermore, following the knockdown of TcCYP6K1 and TcCYP9F2 genes in combination with treating larvae with 75% CO₂, it was observed that larval mortality increased, and glycogen content significantly decreased, while trehalose content increased significantly. Additionally, membrane-bound trehalase enzyme activity declined, TPS gene expression was significantly upregulated, GS gene expression was significantly downregulated, and ATP content showed a marked decrease. In conclusion, CYP genes are critical responsive genes of T. castaneum to high CO₂ levels, potentially impacting the insect’s resistance to carbon dioxide through their involvement in the synthesis or breakdown of the carbohydrate metabolism pathway. These findings could serve as a theoretical basis for the utilization of novel pesticides in low-oxygen grain storage techniques and offer new insights for environmentally friendly pest control strategies in grain storage. Full article

Previously, we demonstrated that the ¹⁷⁷Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to ¹⁷⁷Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a ^99mTc-/¹⁷⁷Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel ^99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH₂ (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(−) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and ^99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of ^99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of ^99mTc-HscFv1 and ^99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of ^99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of ^99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging. Full article

Leber hereditary optic neuropathy (LHON) is a rare disorder causing a sudden painless loss of visual acuity in one or both eyes, affecting young males in their second to third decade of life. The molecular background of the LHON is up to 90%, genetically defined by a point mutation in mitochondrial DNA. Recently, an autosomal recessive form of LHON (LHONAR1, arLHON) has been discovered, caused by biallelic variants in the DNAJC30 gene. This study provides the results of the DNAJC30 gene analysis in a large group of 46 Polish patients diagnosed with LHON, together with the clinical characterization of the disease. The c.152A>G (p.Tyr51Cys) substitution in the DNAJC30 gene was detected in all the patients as homozygote or compound heterozygote. Moreover, we identified one novel variant, c.293A>G, p.(Tyr98Cys), as well as two ultra-rare DNAJC30 variants: c.293A>C, p.(Tyr98Ser), identified to date only in one individual affected with LHONAR1, and c.130_131delTC (p.Ser44ValfsTer8), previously described only in two patients with Leigh syndrome. The patients presented here represent the largest group of subjects with DNAJC30 gene mutations described to date. Based on our data, the autosomal recessive form of LHON caused by DNAJC30 gene mutations is more frequent than the mitochondrial form in Polish patients. The results of our study suggest that Sanger sequencing of the single-exon DNAJC30 gene should be a method of choice applied to identify a molecular background of clinically confirmed LHON in Polish patients. This approach will help to reduce the costs of molecular testing. Full article

The purpose of this study was to examine the effects of high temperature on internal egg yolk quality parameters and their possible mechanisms in Huaixiang chickens. This study consisted of two treatments, and each treatment had six replicates with six birds per cage. A total of seventy-two 26-week-old female Huaixiang chickens were randomly divided into a normal-temperature group (NT) and a high-temperature group (HT) for 6 weeks. And these hens were exposed to 25 ± 2 °C and 32 ± 2 °C, respectively. Their relative humidity was maintained at 55–65%. The results showed that the HT group significantly reduced yolk weight, yolk color, and egg weight compared to the NT group (p < 0.05). Heat stress caused vacuolar degeneration of the liver and reduced the absolute liver weight (p < 0.05). Both yolk triglyceride (TG) and liver TG in the HT group were significantly higher than in the NT group (p < 0.05). However, the liver total cholesterol (TC) level in the HT group was remarkably lower than that in the NT group (p < 0.05). Additionally, heat stress remarkably enhanced SREBP-1c, ACACA, and FASN lipid metabolism-related gene mRNA expression levels in Huaixiang chicken liver after 6 weeks of heat exposure (p < 0.05). Furthermore, the HT group had remarkably reduced total amino acid, Cys, and Tyr levels in the yolk when compared with the NT group in our experiment (p < 0.05). In conclusion, heat stress causes egg yolk quality reduction and abnormal lipid metabolism in Huaixiang chickens. These findings provided novel insights into the role of high temperature on egg yolk parameters and the underlying mechanisms in Chinese indigenous laying hens. Full article

Cytochrome P450 monooxygenase (CYP) is one of the three detoxification metabolic enzymes in insects, and is involved in the metabolism and transformation of endogenous substances as well as the activation and degradation of exogenous compounds. This study aims to reveal the molecular mechanism of CYP9E2 in Tribolium castaneum in adapting to high-CO₂ stress. By predicting the sequence function of CYP9E2, analyzing the temporal and spatial expression profile of TcCYP9E2, and using RNAi to silence TcCYP9E2 combined with a high-CO₂ stress treatment, we measured the carbohydrate content, trehalase activity, and gene expression levels in trehalose metabolism of T. castaneum. A bioinformatics analysis showed that the predicted molecular weight of the protein encoded by TcCYP9E2 is 60.15, the theoretical isoelectric point is 8.63, there is no signal peptide, and the protein is hydrophilic. An evolutionary tree analysis showed that TcCYP9E2 belongs to the CYP6 family and belongs to the CYP3 group; and the spatiotemporal expression profile results showed that TcCYP9E2 was highly expressed in the larvae midgut 48 h after injection of dsCYP9E2, with survival rates decreasing with the increase in CO₂ concentration. Under the condition of 75% CO₂, the contents of glycogen, glucose, ATP, and membrane-bound trehalase decreased significantly after the injection of dsCYP9E2. The expression of TRE-1, TRE-2, and GP in trehalose metabolism and energy pathways was significantly downregulated. Full article

Cyclophilins (CyPs) are a family of enzymes involved in protein folding. Trypanosoma cruzi, the causative agent of Chagas disease, has a 19-kDa cyclophilin, TcCyP19, that was found to be secreted in parasite stages of the CL Brener clone and recognized by sera from T. cruzi-infected mice and patients. The levels of specific antibodies against TcCyP19 in T. cruzi-infected mice and subjects before and after drug treatment were measured by an in-house enzyme linked immunosorbent assay (ELISA). Mice in the acute and chronic phase of infection, with successful trypanocidal treatments, showed significantly lower anti-TcCyP19 antibody levels than untreated mice. In children and adults chronically infected with T. cruzi, a significant decrease in the anti-TcCyP19 titers was observed after 12 months of etiological treatment. This decrease was maintained in adult chronic patients followed-up 30–38 months post-treatment. These results encourage further studies on TcCyP19 as an early biomarker of trypanocidal treatment efficiency. Full article

The activation of Wnt/β-catenin signalling is a prerequisite for odontogenesis. APC, a member of the AXIN-CK1-GSK3β-APC β-catenin destruction complex, functions to modulate Wnt/β-catenin signalling to establish regular teeth number and positions. APC loss-of-function mutations are associated with the over-activation of WNT/β-catenin signalling and subsequent familial adenomatous polyposis (FAP; MIM 175100) with or without multiple supernumerary teeth. The ablation of Apc function in mice also results in the constitutive activation of β-catenin in embryonic mouse epithelium and causes supernumerary tooth formation. The objective of this study was to investigate if genetic variants in the APC gene were associated with supernumerary tooth phenotypes. We clinically, radiographically, and molecularly investigated 120 Thai patients with mesiodentes or isolated supernumerary teeth. Whole exome and Sanger sequencing identified three extremely rare heterozygous variants (c.3374T>C, p.Val1125Ala; c.6127A>G, p.Ile2043Val; and c.8383G>A, p.Ala2795Thr) in APC in four patients with mesiodentes or a supernumerary premolar. An additional patient with mesiodens was compound as heterozygous for two APC variants (c.2740T>G, p.Cys914Gly, and c.5722A>T, p.Asn1908Tyr). Rare variants in APC in our patients are likely to contribute to isolated supernumerary dental phenotypes including isolated mesiodens and an isolated supernumerary tooth. Full article

Novel variously substituted thiohydantoin-based dispiro-indolinones were prepared using a regio- and diastereoselective synthetic route from 5-arylidene-2-thiohydantoins, isatines, and sarcosine. The obtained molecules were subsequently evaluated in vitro against the cancer cell lines LNCaP, PC3, HCT^wt, and HCT^(−/−). Several compounds demonstrated a relatively high cytotoxic activity vs. LNCaP cells (IC₅₀ = 1.2–3.5 µM) and a reasonable selectivity index (SI = 3–10). Confocal microscopy revealed that the conjugate of propargyl-substituted dispiro-indolinone with the fluorescent dye Sulfo-Cy5-azide was mainly localized in the cytoplasm of HEK293 cells. P388-inoculated mice and HCT116-xenograft BALB/c nude mice were used to evaluate the anticancer activity of compound 29 in vivo. Particularly, the TGRI value for the P388 model was 93% at the final control timepoint. No mortality was registered among the population up to day 31 of the study. In the HCT116 xenograft model, the compound (170 mg/kg, i.p., o.d., 10 days) provided a T/C ratio close to 60% on day 8 after the treatment was completed. The therapeutic index—estimated as LD₅₀/ED₅₀—for compound 29 in mice was ≥2.5. Molecular docking studies were carried out to predict the possible binding modes of the examined molecules towards MDM2 as the feasible biological target. However, such a mechanism was not confirmed by Western blot data and, apparently, the synthesized compounds have a different mechanism of cytotoxic action. Full article

Chagas disease caused by the protozoan Trypanosoma cruzi is endemic to 21 countries in the Americas, effects approximately 6 million people and on average results in 12,000 deaths annually. Human African Trypanosomiasis (HAT) is caused by the Trypanosoma brucei sub-species, endemic to 36 countries within sub-Saharan Africa. Treatment regimens for these parasitic diseases are complicated and not effective against all disease stages; thus, there is a need to find improved treatments. To identify new molecules for the drug discovery pipelines for these diseases, we have utilised in vitro assays to identify compounds with selective activity against both T. cruzi and T.b. brucei from the Medicines for Malaria Venture (MMV) Pathogen Box compound collection. To prioritise these molecules for further investigation, temporal and wash off assays were utilised to identify the speed of action and cidality of compounds. For translational relevance, compounds were tested against clinically relevant T.b. brucei subspecies. Compounds with activity against T. cruzi cytochrome P450 (TcCYP51) have not previously been successful in clinical trials for chronic Chagas disease; thus, to deprioritise compounds with this activity, they were tested against recombinant TcCYP51. Compounds with biological profiles warranting progression offer important tools for drug and target development against kinetoplastids. Full article

The Sd^a histo-blood group antigen (GalNAcβ1-4(NeuAcα2-3)Galβ-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a−) individuals (2–4% of Europeans) may produce anti-Sd^a, which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a−), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a−) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SID^null candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sd^a synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a−) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg β4GalNAc-T2 variant causes Sd^a-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized. Full article

Amelogenesis imperfecta (AI) is a group of rare genetic diseases affecting the tooth enamel. AI is characterized by an inadequate quantity and/or quality of tooth enamel and can be divided into three major categories: hypoplastic, hypocalcified and hypomaturation types. Even though there are some overlapping phenotypes, hypomaturation AI enamel typically has a yellow to brown discoloration with a dull appearance but a normal thickness indicating a less mineralized enamel matrix. In this study, we recruited four Turkish families with hypomaturation AI and performed mutational analysis using whole exome sequencing. These analyses revealed two novel homozygous mutations in the KLK4 gene: a nonsense mutation in exon 3 (NM_004917.4:c.170C>A, p.(Ser57*)) was found in families 1, 2 and 3 and a missense mutation in exon 6 (c.637T>C, p.(Cys213Arg)) in family 4. Functional analysis showed that the missense mutation transcript could not translate the mutant protein efficiently or generated an unstable protein that lacked functional activity. The two novel inactivating KLK4 mutations we identified caused a hypomaturation AI phenotype similar to those caused by the four previously described KLK4 nonsense and frameshift mutations. This study improves our understanding of the normal and pathologic mechanisms of enamel formation. Full article

This study aimed to investigate the effects of multi-bacteria solid-state fermented diets with different crude fiber (CF) levels on growth performance, nutrient digestibility, and microbial flora of finishing pigs. The multi-bacteria solid-state fermented diets were made up of Lactobacillus amylovorus, Enterococcus faecalis, Bacillus subtilis, and Candida utilis. According to a 2 (factors) × 2 (levels) design, with the two factors being multi-bacteria solid-state fermentation (fed non-fermented diet or multi-bacteria fermentation) or CF levels (fed a basal diet containing 2.52% CF or 7.00% CF), a total of 36 finishing pigs (70.80 ± 5.75 kg) were divided into 4 treatments with 9 barrows per group: (1) pigs fed a diet containing 7.00% CF (HF), (2) pigs fed a multi-bacteria fermentation diet containing 7.00% CF (HFM), (3) pigs fed a diet containing 2.52% CF (LF), and (4) piglets fed a multi-bacteria fermentation diet containing 2.52% CF (LFM). This experiment lasted 28 days. The multi-bacteria solid-state fermented diet increased the backfat thickness (p < 0.05) and apparent total tract nutrient digestibility (ATTD) of CF, neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (CP), 8 amino acids (Trp, Asp, Gly, Cys, Val, Met, Ile, and Leu), total essential amino acids (EAA), total non-essential amino acids (NEEA), and total amino acids (TAA) (p < 0.05). Multi-bacteria solid-state fermented diet increased serum concentrations of HDL-c, ABL, TP, and GLU, the serum enzyme activities of GSH-Px, T-AOC, SOD, and CAT (p < 0.05), the relative abundance of Lactobacillus, Oscillospira, and Coprococcus (p < 0.05), and the abundance of YAMINSYN3-PWY, PWY-7013, GOLPDLCAT-PWY, ARGORNPROST-PWY, and PWY-5022 pathways (p < 0.05). The multi-bacteria solid-state fermented diet reduced the digestion amount of CF, NDF, and ADF (p < 0.05), the serum concentrations of TC, TG, LDL-c, BUN, and MDA (p < 0.05), the relative abundance of Streptococcaceae (p < 0.05), and the abundance of PWY-6470, PWY0-862, HSERMETANA-PWY, LACTOSECAT-PWY, MET-SAM-PWY, PWY-6700, PWY-5347, PWY0-1061, and LACTOSECAT-PWY pathways (p < 0.05). The high-fiber diet increased average daily feed intake (p < 0.05), the serum concentrations of TC, TG, LDL-c, BUN, and MDA (p < 0.05), the relative abundance of Clostridiaceae_Clostridium and Coprococcus (p < 0.05), and the abundance of TCA-GLYOX-BYPASS, GLYCOLYSIS-TCA-GLYOX-BYPASS, and PWY-6906 pathways (p < 0.05). The high-fiber diet reduced chest circumference (p < 0.05) and ATTD of ether extract (EE), CF, NDF, ADF, Ca, CP, 18 amino acids (Trp, Thr, Val, Met, Ile, Leu, Phe, Lys, His, Arg Asp, Ser, Glu, Gly, Ala, Cys, Tyr, and Pro), EAA, NEAA, and TAA (p < 0.05). The high-fiber diet also reduced the serum concentrations of HDL-c, TP, ABL, and GLU, the serum enzyme activities of T-AOC, GSH-Px, SOD, and CAT (p < 0.05), and the relative abundance of Akkermansia and Oscillospira (p < 0.05). There was no significant effect of the interaction between multi-bacteria fermentation and dietary CF levels, except on the digestion amount of CF (p < 0.05). The 7.00% CF had a negative effect on the digestion of nutrients, but multi-bacteria solid-state fermentation diets could relieve this negative effect and increase backfat thickness. High-fiber diets and multi-bacteria solid-state fermentation improved the diversity and abundance of fecal microorganisms in finishing pigs. Full article

Generalized Arterial Calcification of Infancy (GACI) is a rare disease inherited in a recessive manner, with severe and diffuse early onset of calcifications along the internal elastic lamina in large and medium size arteries. The diagnosis results are from clinical manifestations, imaging, histopathologic exams, and genetic tests. GACI is predominantly caused by biallelic pathogenic variant in the ENPP1 gene (GACI1, OMIM#208000) and, to a lesser extent, by pathogenic variants in the ABCC6 gene (GACI2, OMIM#614473). We present a novel variation in the ENPP1 gene identified in a patient clinically diagnosed with GACI and confirmed by genetic investigation and autopsy as GACI type 1. The sequence analysis of the patient’s ENPP1 gene detected two heterozygous variants c.1412A>G (p.Tyr471Cys) and c.1715T>C (p.Leu572Ser). The variant c.1715T>C (p.Leu572Ser) has not been described yet in the literature and in mutation databases. A genetic analysis was also carried out for the parents of the newborn; the heterozygous pathogenic variant c.1412A>G (p.Tyr471Cys) was detected in the mother’s ENPP1 gene, and a sequence analysis of the father’s ENPP1 gene revealed the novel heterozygous variant c.1715T>C (p.Leu572Ser). Our results showed that the variant c.1715T>C (p.Leu572Ser) may have a pathogenic role in the development of GACI type1 (GACI1, OMIM#208000), at least when associated with the pathogenic c.1412A>G (p.Tyr471Cys) variant. The identification of novel mutations potentially enabled genotype/phenotype associations that will ultimately have an impact on clinical management and prognosis for the disease. Full article

We genetically characterized 22 Swiss patients who had been diagnosed with Stargardt disease after clinical examination. We identified in 11 patients (50%) pathogenic bi-allelic ABCA4 variants, c.1760+2T>C and c.4496T>C being novel. The dominantly inherited pathogenic ELOVL4 c.810C>G p.(Tyr270*) and PRPH2-c.422A>G p.(Tyr141Cys) variants were identified in eight (36%) and three patients (14%), respectively. All patients harboring the ELOVL4 c.810C>G p.(Tyr270*) variant originated from the same small Swiss area, identifying a founder mutation. In the ABCA4 and ELOVL4 cohorts, the clinical phenotypes of “flecks”, “atrophy”, and “bull’s eye like” were observed by fundus examination. In the small number of patients harboring the pathogenic PRPH2 variant, we could observe both “flecks” and “atrophy” clinical phenotypes. The onset of disease, progression of visual acuity and clinical symptoms, inheritance patterns, fundus autofluorescence, and optical coherence tomography did not allow discrimination between the genetically heterogeneous Stargardt patients. The genetic heterogeneity observed in the relatively small Swiss population should prompt systematic genetic testing of clinically diagnosed Stargardt patients. The resulting molecular diagnostic is required to prevent potentially harmful vitamin A supplementation, to provide genetic counseling with respect to inheritance, and to schedule appropriate follow-up visits in the presence of increased risk of choroidal neovascularization. Full article

The diagnosis of inherited platelet function disorders (IPFDs) is challenging owing to the unavailability of essential testing methods, including light transmission aggregometry and flow cytometry, in several medical centers in Korea. This study, conducted by the Korean Pediatric Hematology Oncology Group from March 2017 to December 2020, aimed to identify the causative genetic variants of IPFDs in Korean patients using next-generation sequencing (NGS). Targeted exome sequencing, followed by whole-genome sequencing, was performed for diagnosing IPFDs. Of the 11 unrelated patients with suspected IPFDs enrolled in this study, 10 patients and 2 of their family members were diagnosed with Glanzmann thrombasthenia (GT). The variant c.1913+5G>T of ITGB3 was the most common, followed by c.2333A>C (p.Gln778Pro) of ITGB2B. Known variants of GT, including c.917A>C (p.His306Pro) of ITGB3 and c.2975del (p.Glu992Glyfs*), c.257T>C (p.Leu86Pro), and c.1750C>T (p.Arg584*) of ITGA2B, were identified. Four novel variants of GT, c.1451G>T (p.Gly484Val) and c.1595G>T (p.Cys532Phe) of ITGB3 and c.1184G>T (p.Gly395Val) and c.2390del (p.Gly797Valfs*29) of ITGA2B, were revealed. The remaining patient was diagnosed with platelet type bleeding disorder 18 and harbored two novel RASGRP2 variants, c.1479dup (p.Arg494Alafs*54) and c.813+1G>A. We demonstrated the successful application of NGS for the accurate and differential diagnosis of heterogeneous IPFDs. Full article