Genotype-phenotype of autosomal dominant polycystic kidney disease in Malta.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple renal cysts causing kidney enlargement and end-stage renal disease (ESRD) in half the patients by 60 years of age. The aim of the study was to determine the genetic aetiology in Maltese patients clinically diagnosed with ADPKD and correlate the clinical features. METHODS: A total of 60 patients over 18 years of age clinically diagnosed with ADPKD were studied using a customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results. RESULTS: Genetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population. The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1. CONCLUSIONS: This study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.

PRKCSH serves as a potential immunological and prognostic biomarker in pan-cancer.

Protein kinase C substrate 80K-H (PRKCSH) plays a crucial role in the protein N-terminal glycosylation process, with emerging evidence implicating its involvement in tumorigenesis. To comprehensively assess PRKCSH's significance across cancers, we conducted a pan-cancer analysis using data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Cancer Cell Line Encyclopedia (CCLE). We assessed aberrant PRKCSH mRNA and protein expression, examined its prognostic implications, and identified correlations with clinical features, tumor mutational burden (TMB), microsatellite instability (MSI), and tumor immunity across cancer types. We explored PRKCSH gene alterations, DNA methylation, and their impact on patient prognosis. Gene Set Enrichment Analysis (GSEA) and single-cell analysis revealed potential biological roles. Additionally, we investigated drug susceptibility and conducted Connectivity Map (Cmap) analysis. Key findings revealed that PRKCSH exhibited overexpression in most tumors, with a significant association with poor overall survival (OS) in six cancer types. Notably, PRKCSH expression demonstrated variations across disease stages, primarily increasing in advanced stages among eleven tumor types. Moreover, PRKCSH exhibited significant correlations with TMB in five cancer categories, MSI in eight, and displayed associations with immune cell populations in pan-cancer analysis. Genetic variations in PRKCSH were identified across 26 tumor types, suggesting favorable disease-free survival. Furthermore, PRKCSH methylation displayed a significant negative correlation with its expression in 27 tumor types, with a marked decrease compared to normal tissues in ten tumors. Cmap predicted 24 potential therapeutic small molecules in over four cancer types. This study highlights that PRKCSH, as a potential oncogene, may be a promising prognostic marker and therapeutic target of immunotherapy for a range of malignancies.

PRKCSH contributes to TNFSF resistance by extending IGF1R half-life and activation in lung cancer.

Tumor necrosis factor superfamily (TNFSF) resistance contributes to the development and progression of tumors and resistance to various cancer therapies. Tumor-intrinsic alterations involved in the adaptation to the TNFSF response remain largely unknown. Here, we demonstrate that protein kinase C substrate 80K-H (PRKCSH) abundance in lung cancers boosts oncogenic IGF1R activation, leading to TNFSF resistance. PRKCSH abundance is correlated with IGF1R upregulation in lung cancer tissues. Specifically, PRKCSH interacts with IGF1R and extends its half-life. The PRKCSH-IGF1R axis in tumor cells impairs caspase-8 activation, increases Mcl-1 expression, and inhibits caspase-9, leading to an imbalance between cell death and survival. PRKCSH deficiency augmented the antitumor effects of natural killer (NK) cells, representative TNFSF effector cells, in a tumor xenograft IL-2Rg-deficient NOD/SCID (NIG) mouse model. Our data suggest that PRKCSH plays a critical role in TNFSF resistance and may be a potential target to improve the efficacy of NK cell-based cancer therapy.

Biochemical, Toxicological, and in Silico Aspects of Trillium govanianum Wall. ex D.Don (Trilliaceae): A Rich Source of Natural Bioactive Compounds.

Trillium govanianum is a high-value medicinal herb, having multifunctional traditional and culinary uses. The present investigation was carried out to evaluate the phytochemical, biological and toxicological parameters of the T. govanianum Wall. ex D. Don (Family: Trilliaceae) roots collected from Azad Kashmir, Pakistan. Phytochemical profiling was achieved by determining total bioactive contents (total phenolic and flavonoid contents) and UHPLC-MS analysis. For biological evaluation, antioxidant activities (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, and metal chelation assays) and enzyme inhibition activities (against AChE, BChE, glucosidase, amylase, and tyrosinase) were performed. Moreover, cytotoxicity was assessed against three human carcinoma cell lines (MDA-MB-231, CaSki, and DU-145). The tested extract was found to contain higher total phenolics (7.56 mg GAE/g dry extract) as compared to flavonoid contents (0.45 mg RE/g dry extract). Likewise, for the antioxidant activity, higher CUPRAC activity was noted with 39.84 mg TE/g dry extract values. In the case of enzyme assays, higher activity was pointed out against the cholinesterase, glucosidase and tyrosinase enzymes. The plant extract displayed significant cytotoxicity against the cell lines examined. Moreover, the in-silico studies highlighted the interaction between the important phytochemicals and tested enzymes. To conclude, the assessed biological activity and the existence of bioactive phytochemicals in the studied plant extract may pave the way for the development of novel pharmaceuticals.

Sex, Genotype, and Liver Volume Progression as Risk of Hospitalization Determinants in Autosomal Dominant Polycystic Liver Disease.

BACKGROUND & AIMS: Autosomal dominant polycystic liver disease is a rare condition with a female preponderance, based mainly on pathogenic variants in 2 genes, PRKCSH and SEC63. Clinically, autosomal dominant polycystic liver disease is characterized by vast heterogeneity, ranging from asymptomatic to highly symptomatic hepatomegaly. To date, little is known about the prediction of disease progression at early stages, hindering clinical management, genetic counseling, and the design of randomized controlled trials. To improve disease prognostication, we built a consortium of European and US centers to recruit the largest cohort of patients with PRKCSH and SEC63 liver disease. METHODS: We analyzed an international multicenter cohort of 265 patients with autosomal dominant polycystic liver disease harboring pathogenic variants in PRKCSH or SEC63 for genotype-phenotype correlations, including normalized age-adjusted total liver volumes and polycystic liver disease-related hospitalization (liver event) as primary clinical end points. RESULTS: Classifying individual total liver volumes into predefined progression groups yielded predictive risk discrimination for future liver events independent of sex and underlying genetic defects. In addition, disease severity, defined by age at first liver event, was considerably more pronounced in female patients and patients with PRKCSH variants than in those with SEC63 variants. A newly developed sex-gene score was effective in distinguishing mild, moderate, and severe disease, in addition to imaging-based prognostication. CONCLUSIONS: Both imaging and clinical genetic scoring have the potential to inform patients about the risk of developing symptomatic disease throughout their lives. The combination of female sex, germline PRKCSH alteration, and rapid total liver volume progression is associated with the greatest odds of polycystic liver disease-related hospitalization.

ERVW-1 Activates ATF6-Mediated Unfolded Protein Response by Decreasing GANAB in Recent-Onset Schizophrenia.

Schizophrenia, a mental disorder, afflicts 1% of the worldwide population. The dysregulation of homeostasis in the endoplasmic reticulum (ER) has been implicated in schizophrenia. Moreover, recent studies indicate that ER stress and the unfolded protein response (UPR) are linked to this mental disorder. Our previous research has verified that endogenous retrovirus group W member 1 envelope (ERVW-1), a risk factor for schizophrenia, is elevated in individuals with schizophrenia. Nevertheless, no literature is available regarding the underlying relationship between ER stress and ERVW-1 in schizophrenia. The aim of our research was to investigate the molecular mechanism connecting ER stress and ERVW-1 in schizophrenia. Here, we employed Gene Differential Expression Analysis to predict differentially expressed genes (DEGs) in the human prefrontal cortex of schizophrenic patients and identified aberrant expression of UPR-related genes. Subsequent research indicated that the UPR gene called XBP1 had a positive correlation with ATF6, BCL-2, and ERVW-1 in individuals with schizophrenia using Spearman correlation analysis. Furthermore, results from the enzyme-linked immunosorbent assay (ELISA) suggested increased serum protein levels of ATF6 and XBP1 in schizophrenic patients compared with healthy controls, exhibiting a strong correlation with ERVW-1 using median analysis and Mann-Whitney U analysis. However, serum GANAB levels were decreased in schizophrenic patients compared with controls and showed a significant negative correlation with ERVW-1, ATF6, and XBP1 in schizophrenic patients. Interestingly, in vitro experiments verified that ERVW-1 indeed increased ATF6 and XBP1 expression while decreasing GANAB expression. Additionally, the confocal microscope experiment suggested that ERVW-1 could impact the shape of the ER, leading to ER stress. GANAB was found to participate in ER stress regulated by ERVW-1. In conclusion, ERVW-1 induced ER stress by suppressing GANAB expression, thereby upregulating the expression of ATF6 and XBP1 and ultimately contributing to the development of schizophrenia.

Selectivity Tuning by Natural Deep Eutectic Solvents (NADESs) for Extraction of Bioactive Compounds from Cytinus hypocistis-Studies of Antioxidative, Enzyme-Inhibitive Properties and LC-MS Profiles.

In the present study, the extracts of Cytinus hypocistis (L.) L using both traditional solvents (hexane, ethyl acetate, dichloromethane, ethanol, ethanol/water, and water) and natural deep eutectic solvents (NADESs) were investigated in terms of their total polyphenolic contents and antioxidant and enzyme-inhibitive properties. The extracts were found to possess total phenolic and total flavonoid contents in the ranges of 26.47-186.13 mg GAE/g and 0.68-12.55 mg RE/g, respectively. Higher total phenolic contents were obtained for NADES extracts. Compositional differences were reported in relation to antioxidant potential studied by several assays (DPPH: 70.19-939.35 mg TE/g, ABTS: 172.56-4026.50 mg TE/g; CUPRAC: 97.41-1730.38 mg TE/g, FRAP: 84.11-1534.85 mg TE/g). Application of NADESs (choline chloride-urea 1:2, a so-called Reline) allowed one to obtain the highest number of extracts having antioxidant potential in the radical scavenging and reducing assays. NADES-B (protonated by HCl L-proline-xylitol 5:1) was the only extractant from the studied solvents that isolated a specific fraction without chelating activity. Reline extract exhibited the highest acetylcholinesterase inhibition compared to NADES-B and NADES-C (protonated by H2SO4 L-proline-xylitol 5:1) extracts, which showed no inhibition. The NADES extracts were observed to have higher tyrosinase inhibitory properties compared to extracts obtained by traditional organic solvents. Furthermore, the NADES extracts were relatively better inhibitors of the diabetic enzymes. These findings provided an interesting comparison in terms of total polyphenolic content yields, antioxidant and enzyme inhibitory properties (cholinesterase, amylase, glucosidase, and tyrosinase) between traditional solvent extracts and NADES extracts, used as an alternative. While the organic solvents showed better antioxidant activity, the NADES extracts were found to have some other improved properties, such as higher total phenolic content and enzyme-inhibiting properties, suggesting functional prospects for their use in phytonutrient extraction and fractionation. The obtained results could also be used to give a broad overview of the different biological potentials of C. hypocistis.

Glycoprotein α-Subunit of Glucosidase II (GIIα) is a novel prognostic biomarker correlated with unfavorable outcome of urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is among the most prevalent malignancies. The muscle-invasive bladder cancer (MIBC) shows an invasive feature and has poor prognosis, while the non-muscle invasive bladder cancer (NMIBC) shows a better prognosis as compared with the MIBC. However, a significant proportion (10%-30%) of NMIBC cases progress to MIBC. Identification of efficient biomarkers for the prediction of the course of UC remains challenging nowadays. Recently, there is an emerging study showed that post-translational modifications (PTMs) by glycosylation is an important process correlated with tumor angiogenesis, invasion and metastasis. Herein, we reported a data-driven discovery and experimental validation of GANAB, a key regulator of glycosylation, as a novel prognostic marker in UC. METHODS: In the present study, we conducted immunohistochemistry (IHC) assay to evaluate the correlation between the expression levels of GANAB protein and the prognosis of UC in our cohort of 107 samples using whole slide image (WSI) analysis. In vitro experiments using RNAi were also conducted to investigate the biological functions of GANAB in UC cell lines. RESULTS: We observed that positive GANAB protein expression was significantly correlated with poor prognosis of UC in our cohort, with p-value of 0.0017 in Log-rank test. Notably, tumor cells at the invasive front of the tumor margin showed stronger GANAB expression than the tumor cells inside the tumor body in UCs. We further validated that the elevated expression levels of GANAB were significantly correlated with high grade tumors (p-values of 1.72 × 10-10), advanced stages (6.47 × 10-6), and elevated in luminal molecular subtypes. Moreover, knocking-down GANAB using RNAi in UM-UC-3 and T24 cells inhibited cell proliferation and migration in vitro. Knockdown of GANAB resulted in cell cycle arrest at G1 phase. We demonstrated that GANAB mediated HIF1A and ATF6 transcriptional activation in the ER stress signaling, and regulated the gene expression of cell cycle-related transcriptional factors E2F7 and FOXM1. CONCLUSIONS: The elevated expression of GANAB is a novel indicator of poorer prognosis of UC. Our data suggests that GANAB is not only a new and promising prognostic biomarker for UC, but also may provide important cues for the development of PTM-based therapeutics for UC treatment.

Potential role of PRKCSH in lung cancer: bioinformatics analysis and a case study of Nano ZnO.

PRKCSH, also known as glucosidase II beta, functions as a contributor to lung tumorigenesis by regulating the cell cycle in a p53-dependent manner under severe environmental stress. However, the prognostic value and molecular mechanisms by which the level of PRKCSH is significantly increased in cancer cells are not clearly understood. Here, we first generated a biological profile of PRKCSH expression changes in cancers by analysing bioinformatic data from cancer databases. We found that higher PRKCSH expression was correlated with a poorer prognosis and greater infiltration of most immune cell types in patients with lung cancer. In particular, PRKCSH expression showed significant negative correlations with the level of STAT6 (r = -0.31, p < 0.001) in lung cancer tissues. We further found that PRKCSH deficiency promoted G2/M arrest in response to zinc oxide nanoparticle (Nano ZnO) treatment in A549 cells. With regard to the mechanism, PRKCSH deficiency may induce STAT6 translocation to the nucleus to activate p53 expression through binding to the p53 promoter region from -365 bp to +126 bp. Eventually, activated p53 contributed to Nano-ZnO-induced G2/M arrest in lung cancer cells. Taken together, our data provide new insights into immunotherapy target choices and the prognostic value of PRKCSH. Since the G2/M cell cycle checkpoint is crucial for lung cancer prognosis, targeting PRKCSH expression to suppress the activation of the STAT6/p53 pathway is a potential therapeutic strategy for managing lung cancer.

Hydro-alcoholic extract of Morus nigra reduces fasting blood glucose and HbA1c% in diabetic patients, probably via competitive and allosteric interaction with alpha-glucosidase enzyme; a clinical trial and in silico analysis.

OBJECTIVES: 1-Deoxynojirimycin (1-DNJ), the main active component found in Morus nigra (black mulberry) is reported to be effective in controlling diabetes. We have evaluated the effect of hydro-alcoholic extract of M. nigra leaves on the fasting blood glucose (FBS) and hemoglobin A1c% (HbA1c%) in diabetic patients. Furthermore, we compared the interaction of 1-DNJ and glucose molecules with the alpha-glucosidase enzyme, which has a critical role in the lysis of glucose-based polymers in human cells. METHODS: 4% hydro-alcoholic extract was prepared from black mulberry leaves. Patients in treatment (n=50) and control (n=50) groups received 3 mL extract or placebo in water, respectively, and three times a day. Fasting blood glucose and HbA1c% were evaluated before and after three months of evaluation. Potential binding sites of 1-DNJ or glucose on the enzyme glucosidase found by docking study. Docking scores were obtained using an energy minimization method by Molegro Virtual Docker software. The Mean ± SD of each variable was compared between groups at the 95% significant level. RESULTS: Age mean ± SD was equal to 54.79 ± 9.203 (38-69) years. There was no significant difference between intervention and placebo groups considering FBS (p=0.633) but was for HbA1c% (p=0.0011), before treatment. After three months, both FBS and HbA1c% were significantly reduced in patients under mulberry leaves extract-treatment. FBS changed was from 182.23 ± 38.65 to 161.23 ± 22.14 mg/dL in treatment group (p<0.001) and from 178.45 ± 39.46 to 166.23 ± 29.64 mg/dL in control group (p<0.001). HbA1c was changed from 7.23 ± 0.25 to 6.13 ± 0.61% in treatment group (p<0.001) and from 7.65 ± 0.85 to 7.12 ± 0.33% in control group (p=0.854). Docking results showed that 1-DNJ binds more efficiently, and with a significant score than glucose, to human alpha-glucosidase. CONCLUSIONS: This clinical trial and virtual analysis showed that a hydro-alcoholic extract of black mulberry (M. nigra) leaf may be efficient in reducing the blood glucose and HbA1c% in diabetic patients. Furthermore, docking studies propose a competitive and allosteric regulation for herbal ingredients. Drug-development could be based on the presented idea in this report.

Regulation and role of glycophagy in skeletal muscle energy metabolism.

Glycophagy is the autophagic degradation of glycogen via the lysosomal enzyme GAA/alpha-acid glucosidase. Glycophagy is considered a housekeeping process to degrade poorly branched glycogen particles, but the regulation and role of glycophagy in skeletal muscle metabolism remains enigmatic. Herein, prior muscle contraction promoted glycogen supercompensation 24 and 48 h post contraction, an effect associated with reduced glycophagy. Moreover, NOTCH or cAMP signaling promoted glycophagy, whereas acute glycophagy deficiency rewired cell metabolism by reducing glycolysis and enhancing AMPK and PPAR signaling and fatty acid and glutamine metabolism. These metabolic adaptations were associated with reduced inflammation and triglyceride content but enhanced phosphoinositide 3-kinase (PI3K)-AKT/protein kinase B signaling and insulin action, the latter of which was abolished by exogenous oxidative stress. Collectively, these data suggest glycophagy is dynamically regulated, while the function of glycophagy can be extended beyond a housekeeping process to having an additional role in regulating energy metabolism and insulin action.Abbreviations: AMPK, AMP-activated protein kinase; ASM, acid soluble metabolites; cAMP, cyclic adenosine monophosphate; EPS, electrical pulse stimulation; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GAA, glucosidase, alpha, acid; mTOR, mechanistic target of rapamycin kinase; NAD, nicotinamide adenine dinucleotide; PARP, poly (ADP-ribose) polymerase family; PI3K, phosphoinositide 3-kinase; PPAR, peroxisome proliferator activated receptor ; PYGM, muscle glycogen phosphorylase; STBD1, starch binding domain 1; TFEB, transcription factor EB.

CircPrkcsh, a circular RNA, contributes to the polarization of microglia towards the M1 phenotype induced by spinal cord injury and acts via the JNK/p38 MAPK pathway.

Spinal cord injury (SCI) is a complex pathological change that includes primary SCI and gradually evolves into secondary SCI. Accumulating evidence demonstrates that circular RNAs (circRNAs) are involved in the pathology of a variety of neurological diseases and injuries. However, the characteristics and function of circRNAs in SCI have yet to be elucidated. Although previous research demonstrated that circPrkcsh induces astrocytes to produce inflammatory factors and chemokines, the precise function and mechanism of circPrkcsh in microglia after SCI remains unknown. In this study, we constructed a mouse model of SCI by applying a SCI impactor. Quantitative Real-time PCR and Fluorescence in situ hybridization analysis revealed that circPrkcsh was upregulated in the microglia of SCI mice when compared to sham-operated mice. Gain- or loss-of-function experiments and in vivo assays further indicated that circPrkcsh promotes microglia M1 polarization both in vivo and in vitro. Furthermore, bioinformatics analysis, dual-luciferase assays, and RNA immunoprecipitation assays, confirmed that circPrkcsh serves as a competing endogenous RNA (ceRNA) to promote the expression of MEKK1 mRNA by sponging miR-488. Double knockout rescue experiments further showed that circPrkcsh regulates the MEKK1/JNK/p38 MAPK pathway via miR-488. Our research provides a better understanding of the mechanism of circPrkcsh in SCI and demonstrates that the circPrkcsh/miR-488/Mekk1 axis is a promising regulatory method for the treatment of SCI.

Dose-response evaluation of urinary cadmium and kidney injury biomarkers in Chinese residents and dietary limit standards.

BACKGROUND: Cadmium (Cd) is a common heavy metal that mainly causes renal damage. There is a lack of research on the large-scale and systematic evaluation of the association between urinary Cd (U-Cd) and various effect biomarkers among Chinese residents. METHODS: Based on the establishment process of dietary Cd limit standards by European Food Safety Authority (EFSA), the dose-response relationships between U-Cd and four biomarkers, ß2-microglobulin (ß2-MG), N-acetyl-ß-glucosidase (NAG), microalbumin (mALB), and retinol binding Protein (RBP) were explored, respectively. Toxicokinetic model was used to derive the dietary Cd exposure limit for Chinese residents after critical U-Cd concentration was calculated. RESULTS: As the sensitive biomarkers of renal injury, ß2-MG and NAG were selected to estimate the 95% confidence interval lower limit of the U-Cd benchmark dose (BMDL5) to be 3.07 and 2.98 µg/g Cr, respectively. Dietary Cd exposure limit was calculated to be 0.28 µg/kg bw/day (16.8 µg/day, based on the body weight of 60 kg), which was lower than the average Chinese Cd exposure (30.6 µg/day) by the China National Nutrient and Health Survey. CONCLUSION: This study established an overall association between U-Cd and renal injury biomarkers, and explored the Chinese dietary Cd exposure limits, which helps improve Chinese Cd exposure risk assessment and provides a reference basis for formulating reasonable exposure standards.

Clinical genetic diagnostics in Danish autosomal dominant polycystic kidney disease patients reveal possible founder variants.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common heritable kidney disease. ADPKD leads to cysts, kidney enlargement and end-stage renal disease. ADPKD is mainly caused by variants in PKD1 and PKD2, with truncating PKD1 variants causing the most severe phenotype. This study aimed to characterize variants in Danish patients referred for screening of genes related to cystic kidney disease. METHODS: 147 families were analysed for variants in PKD1, PKD2 and GANAB using next generation sequencing and multiplex ligation-dependent probe amplification. If a variant was identified, relatives were analysed for the specific variant using Sanger sequencing. RESULTS: A pathogenic or possibly pathogenic variant was identified in 87% (103/118) of patients suspected to suffer from ADPKD, according to the requisition form. In total, 112 pathogenic or possibly pathogenic variants were observed, of which 94 were unique; 74 (79%) in PKD1 and 20 (21%) in PKD2, while 41 variants were novel. No variants in GANAB were observed. Ten recurrent variants were observed in 26 (26%) families. These were either PKD2 variants (N = 6) or non-truncating PKD1 variants (N = 4). Five of these were likely founder variants. CONCLUSIONS: The distribution of pathogenic or possibly pathogenic variants in the Danish ADPKD population is similar to that in other populations, except that recurrent truncating PKD1 variants appear to be rare, i.e. founder variants tend to be variant types associated with a mild phenotype. Patients with a mild phenotype may remain undiagnosed, consequently the frequency of founder variants and prevalence of ADPKD may be underestimated.

Biological activity, phytochemical parameters, and potential bioaccessibility of wheat bread enriched with powder and microcapsules made from Saskatoon berry.

The aim of the present study was to determine the impact of Saskatoon powder addition on phytochemical parameters, biological activity, and nutritional value of wheat bread. Supplementation increased phenolics content up to 72% in the bread with 6% powder content. This increase was reflected in the improved antioxidative properties of breads, especially after their supplementation with the microencapsulated additives (an increase by 93% in the bread enriched with 6% of powder covered with maltodextrin). The in vitro digestion released the antioxidative compounds, leading to higher bioaccessibility of the breads enriched with the microencapsulated powders. The highest inhibition of activities of cyclooxygenase 1 and -2, as well as amylase and glucosidase was recorded for the breads enriched with the additive microencapsulated with maltodextrin and inulin. Thus, Saskatoon berry powders, especially the microencapsulated ones, may be used as functional components in designing innovative bakery products.

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

Novel GANAB variants associated with polycystic liver disease.

BACKGROUND: Polycystic liver disease (PLD) is an inherited disorder characterized by numerous cysts in the liver. Autosomal dominant polycystic kidney and liver disease (ADPKD and ADPLD, respectively) have been linked to pathogenic GANAB variants. GANAB encodes the α-subunit of glucosidase II (GIIα). Here, we report the identification of novel GANAB variants in an international cohort of patients with the primary phenotype of PLD using molecular inversion probe analysis. RESULTS: Five novel GANAB variants were identified in a cohort of 625 patients with ADPKD or ADPLD. In silico analysis revealed that these variants are likely to affect functionally important domains of glucosidase II α-subunit. Missense variant c.1835G>C p.(Arg612Pro) was predicted to disrupt the structure of the active site of the protein, likely reducing its activity. Frameshift variant c.687delT p.(Asp229Glufs*60) introduces a premature termination codon predicted to have no activity. Two nonsense variants (c.2509C>T; p.(Arg837*), and c.2656C>T; p.(Arg886*)) and splice variant c.2002+1G>C, which causes aberrant pre-mRNA splicing and affecting RNA processing, result in truncated proteins and are predicted to cause abnormal binding of α- and ß-subunits of glucosidase II, thus affecting its enzymatic activity. Analysis of glucosidase II subunits in cell lines shows expression of a truncated GIIα protein in cells with c.687delT, c.2509C>T, c.2656C>T, and c.2002+1G>C variants. Incomplete colocalization of the subunits was present in cells with c.687delT or c.2002+1G>C variants. Other variants showed normal distribution of GIIα protein. CONCLUSIONS: We identified five novel GANAB variants associated with PLD in both ADPKD and ADPLD patients supporting a common pathway in cystogenesis. These variants may lead to decreased or complete loss of enzymatic activity of glucosidase II which makes GANAB a candidate gene to be screened in patients with an unknown genetic background.

Health-promoting activities of edible seaweed extracts from Chilean coasts: assessment of antioxidant, anti-diabetic, anti-inflammatory and antimicrobial potential / Actividades promotoras de la salud de extractos de algas comestibles de las costas chilenas: evaluación del potencial antioxidante, antidiabético, antiinflamatorio y antimicrobiano

ABSTRACT The aim of this study was to evaluate the antioxidant, antidiabetic, anti-inflammatory and antimicrobial activities of three edible seaweed extracts from Chilean coasts: Pyropia orbicularis, Ulva spp, and Durvillaea antarctica. Seaweed extracts in methanol and 70% acetone were performed to evaluate antioxidant and antidiabetic activities, whereas 60% methanol was used to measure anti-inflammatory and antimicrobial activities. Acetone extracts from D. antarctica had the highest total phenolic content and consequently exhibited the strongest antioxidant activity, while methanol extract of this seaweed presented the highest α-glucosidase inhibition (IC50= 0.004 mg mL-1). In the tests against E. coli and Penicillium sp., the extracts obtained from Ulva spp. were the most effective and exhibited the maximum anti-inflammatory effect against phorbol 12-myristate 13-acetate irritant agent (61.8% inhibition) in mice. Results indicated that all evaluated Chilean seaweed extracts are promising candidates for application in functional foods and in the pharmaceutical industry.

RESUMEN El objetivo de este estudio fue evaluar las actividades antioxidantes, antidiabéticas, antiinflamatorias y antimicrobianas de los extractos de tres algas marinas comestibles de las costas Chilenas (Pyropia orbicularis, Ulva spp. y Durvillaea antarctica). Se realizaron extractos de algas marinas en metanol y acetona al 70% para evaluar las actividades antioxidantes y antidiabéticas, mientras que el metanol al 60% se usó para actividades antiinflamatorias y antimicrobianas. Los extractos de acetona de D. antarctica tuvieron el mayor contenido de fenoles totales (TPC) y, en consecuencia, exhibieron la mayor actividad antioxidante, mientras que el extracto metanólico de estas algas presentó la mayor inhibición de la α-glucosidasa (IC50= 0,004 mg mL-1). En las pruebas contra E. coli y Penicillium sp., los extractos obtenidos de Ulva spp., actuaron como los más efectivos y exhibieron el máximo efecto antiinflamatorio contra el agente irritante de forbol 12-miristato 13-acetato (TPA) (inhibición del 61,8%) en ratones. Por lo tanto, los resultados indican que todos los extractos de algas chilenas evaluados pueden ser candidatos prometedores para su aplicación en alimentos funcionales y en las industrias farmacéuticas.

Ganab Haploinsufficiency Does Not Cause Polycystic Kidney Disease or Polycystic Liver Disease in Mice.

BACKGROUND: Heterozygous GANAB mutations that can cause autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD) have been described previously, but their roles in ADPKD and PLD are largely unknown. With the increase in polycystic kidney disease caused by GANAB gene mutations in recent years, a suitable animal model is still needed to further explore the pathogenic role of this gene. METHODS: To construct a mouse model of Ganab gene deletion, we analyzed the Ganab gene structure and designed two CRISPR-/Cas9-based targeting strategies. The Cas9/sgRNA we constructed was microinjected into fertilized mouse eggs to obtain chimeric F0 mice. Mice with stable genotypes were selected from offspring born after mating F0 mice with wild-type mice. RESULTS: We found that homozygous mutation of the Ganab gene in C57BL/6 mice resulted in early embryonic lethality, and there were no cysts in the kidneys or livers of Ganab +/- mice. Additionally, Ganab protein expression was reduced by at least 50%, while the expression of ADPKD proteins (PC1 and PC2) and acetylated tubulin was not affected in the Ganab +/- kidney. However, the Ganab +/- mice did not show any abnormal clinical phenotypes after birth and failed to reveal renal tubule dilatation or any abnormalities of the glomeruli in the Ganab +/- kidney. CONCLUSIONS: Homozygous Ganab mutations are lethal in the fetal stage, and Ganab haploinsufficiency does not cause kidney or liver cysts in mice, suggesting that it may not be the causative gene in polycystic kidney disease.

Identification and Characterization of a-Glucosidase Inhibition Flavonol Glycosides from Jack Bean (Canavalia ensiformis (L.) DC.

Although the intake of jack bean (Canavalia ensiformis (L.) DC.), an underutilized tropical legume, can potentially decrease the risk of several chronic diseases, not much effort has been directed at profiling the polyphenolics contained therein. Hence, this work aimed to identify and quantify the dominant jack bean polyphenolics, which are believed to have antioxidant and other bioactivities. Four major compounds were detected and identified as kaempferol glycosides with three or four glycoside units. Their structures were established based on UV-visible, 1d, 2D NMR, and HR-ESI-MS analyses. Specifically, kaempferol 3-O-a-l-rhamnopyranosyl (1®6)- b-d-glucopyranosyl (1®2)-b-d-galactopyranosyl-7-O-[3-O-o-anisoyl]-a-l-rhamnopyranoside was detected for the first time, while the other three compounds have already been described in plants other than jack bean. This new compound was found to have a higher a-glucosidase inhibition activity compared to acarbose.