Ectopic expression of a grapevine alkaline α-galactosidase seed imbibition protein VvSIP enhanced salinity tolerance in transgenic tobacco plants.

Alpha-galactosidase seed imbibition protein (VvSIP) isolated from Vitis vinifera is up-regulated upon salt stress and mediates osmotic stress responses in a tolerant grapevine cultivar. So far, little is known about the putative role of this stress-responsive gene. In the present study, VvSIP function was investigated in model tobacco plants via Agrobacterium-mediated genetic transformation. Our results showed that overexpression of VvSIP exhibited increased tolerance to salinity at germination and late vegetative stage in transgenic Nicotiana benthamiana compared to the nontransgenic plants based on the measurement of the germination rate and biomass production. High salt concentrations of 200 and 400 mM NaCl in greenhouse-grown pot assay resulted in better relative water content, higher leaf osmotic potential, and leaf water potential in transgenic lines when compared to the wild-type (WT) plants. These physiological changes attributed to efficient osmotic adjustment improved plant performance and tolerance to salinity compared to the WT. Moreover, the VvSIP-expressing lines SIP1 and SIP2 showed elevated amounts of chlorophyll with lower malondialdehyde content indicating a reduced lipid peroxidation required to maintain membrane stability. When subjected to high salinity conditions, the transgenic tobacco VvSIP exhibited higher soluble sugar content, which may suggest an enhancement of the carbohydrate metabolism. Our findings indicate that the VvSIP is involved in plant salt tolerance by functioning as a positive regulator of osmotic adjustment and sugar metabolism, both of which are responsible for stress mitigation. Such a candidate gene is highly suitable to alleviate environmental stresses and thus could be a promising candidate for crop improvement.

Late-onset Fabry disease due to a new (p.Pro380Leu) pathogenic variant of GLA Gene.

Fabry disease is a rare X-linked lysosomal storage disorder due to pathogenic variants of the galactosidase alpha (GLA) gene, leading to a deficiency of alpha-galactosidase A. The inadequate enzymatic activity leads to progressive glycosphingolipids accumulation within tissues and subsequent multi-systemic dysfunction, with predominant involvement of heart, kidney, and nervous system. Two subtypes are recognized: the classic type and the late-onset type. We here describe the clinical characteristics of a patient with late-onset Fabry disease carrying a not previously identified GLA gene variant. This 50-year-old man came to hospital because of an acute ischemic stroke. He also complained of acroparesthesia and had angiokeratomas in the nape and the back. Blood alpha-galactosidase A activity was low, plasmatic lyso-Gb3 level was borderline, cardiac MRI showed cardiac fibrosis, brain MRI documented cerebrovascular disease, and skin biopsy revealed small fiber neuropathy without globotriaosylceramide-3 skin deposits. Genetic study by means of targeted next-generation sequencing analysis disclosed a missense substitution c.1139C>T (p.Pro380Leu) in the GLA gene. We suggest that this novel variant should be considered as pathogenic and associated with a late-onset variant of Fabry disease with a predominant neurological phenotype.

Generation of the induced pluripotent stem cell line UKWNLi005-A derived from a patient with the GLA mutation c.376A > G of unknown pathogenicity in Fabry disease.

Human dermal fibroblasts (HDF) were obtained by skin punch biopsy from a 51-year old man with suspected Fabry disease (FD), carrying the hemizygous c.376A > G variant in the α-galactosidase A gene (GLA). Cultured HDF were reprogrammed to induced pluripotent stem cells (iPSC) using a non-modified RNA-based transfection protocol. GLA-S126G-iPSC exhibit typical embryonic stem cell-like morphology, normal karyotype, expression of all tested pluripotency markers, and three germ layer differentiation potential. We provide a novel patient-specific cell line that can be used to investigate a genetic variation of yet unknown significance.

Circular RNA-based biomarkers in blood of patients with Fabry disease and related phenotypes.

BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disease caused by mutations in the galactosidase α gene. Deficient activity of α-galactosidase A leads to glycosphingolipid accumulations in multiple organs. Circular RNAs represent strong regulators of gene expression. Their circular structure ensures high stability in blood. We hypothesised that blood-based circular RNA profiles improve phenotypic assignment and therapeutic monitoring of Fabry disease. METHODS: A genome-wide circular RNA expression analysis was performed in blood of genetically diagnosed patients with Fabry disease (n=58), age-matched and sex-matched healthy volunteers (n=14) and disease control patients with acute kidney injury (n=109). Most highly dysregulated circular RNAs were validated by quantitative real-time PCR. Circular RNA biomarker sensitivity, specificity, predictive values and area under the curve (AUC) were determined. Linear regression analyses were conducted for validated circular RNA biomarkers and clinical patient characteristics. RESULTS: A distinct circular RNA transcriptome signature identified patients with Fabry disease. Level of circular RNAs hsa_circ_0006853 (AUC=0.73), hsa_circ_0083766 (AUC=0.8) and hsa_circ_0002397 (AUC=0.8) distinguished patients with Fabry disease from both healthy controls and patients with acute kidney injury. Hsa_circ_0002397 was, furthermore, female-specifically expressed. Circular RNA level were significantly related to galactosidase α gene mutations, early symptoms, phenotypes, disease severities, specific therapies and long-term complications of Fabry disease. CONCLUSION: The discovery of circular RNA-based and Fabry disease-specific biomarkers may advance future diagnosis of Fabry disease and help to distinguish related phenotypes.

A late-onset male Fabry disease patient with somatic mosaicism of a classical GLA mutation: a case report.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA). Male patients of FD develop early sign and symptoms in childhood or adolescence. However, "de novo somatic mosaicism" is rare and might be developed a relatively mild phenotype despite carrying a classic type. A 34-year-old male patient visited with foamy urine. Renal biopsy findings were consistent with FD. Leukocyte α-galactosidase activity was markedly reduced at 5.3 nmol/hr/mg (normal range, 25-126). Sequence analysis of the patient's GLA gene identified mosaicism for the mutation GLA[NM_000169.2] c.820=/G>C. This mutation results in a substitution of the amino acid in position 274 from glycine to arginine. However, no family members showed FD-related symptoms, and the daughter of the patient was also tested for paternity and was identified as a real biological daughter, but DNA sequence analysis for FD showed no mutations. Based on these results, we diagnosed the patients as de novo mutation with somatic mosaicism. Next generation sequencing turned out that 58% of the readings had the mutated allele in buccal cells, 84% in blood, and 85% in urine, when 100% should be expected in a hemizygous affected male confirming the presence of somatic mosaicisms. The patient has been on treatment for enzyme replacement therapy (agalsidase-ß, 1.0 mg/kg biweekly) for past 9 years and has maintained normal renal function (serum creatinine 1.0 mg/dL) with mild albuminuria (123 mg/g Cr). Therefore, this case suggests somatic mosaicism is one of important phenotype modifiers.

A Pathogenic Galactosidase A Mutation Coexisting With an MYBPC3 Mutation in a Female Patient With Hypertrophic Cardiomyopathy.

The coexistence of GLA (Pro259Ser, c.775C>T) and MYBPC3 (c.1351+2T>C) mutations was found in a female patient with hypertrophic cardiomyopathy. Histology documented abundant vacuolisation with osmiophilic lamellar bodies and positive Gb3 immunohistochemistry. In the presence of a hypertrophic cardiomyopathy phenotype, the systematic search for unusual findings is mandatory to rule out a phenocopy.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2'-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2'-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2'-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2'-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Fabry Nephropathy: An Evidence-Based Narrative Review.

Fabry disease (FD) is a rare, X-linked disorder caused by mutations in the GLA gene encoding the enzyme α-galactosidase A. Complete or partial deficiency in this enzyme leads to intracellular accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in many cell types throughout the body, including the kidney. Progressive accumulation of Gb3 in podocytes, endothelial cells, epithelial cells, and tubular cells contribute to the renal symptoms of FD, which manifest as proteinuria and reduced glomerular filtration rate leading to renal insufficiency. A correct diagnosis of FD, although challenging, has considerable implications regarding treatment, management, and counseling. The diagnosis may be confirmed by demonstrating the enzyme deficiency in males and by identifying the specific GLA gene mutation in male and female patients. Treatment with enzyme replacement therapy, as part of the therapeutic strategy to prevent complications of the disease, may be beneficial in stabilizing renal function or slowing its decline, particularly in the early stages of the disease. Emergent treatments for FD include the recently approved chaperone molecule migalastat for patients with amenable mutations. The objective of this report is to provide an updated overview on Fabry nephropathy, with a focus on the most relevant aspects of its epidemiology, diagnosis, pathophysiology, and treatment options.

Targeting of products of genes to tumor sites using adoptively transferred A-NK and T-LAK cells.

Despite successes in animals, cytokine gene expression selectively in human tumors is difficult to achieve owing to lack of efficient delivery methods. Since interleukin (IL)-2-activated natural killer (A-NK) and phytohemagglutinin and IL-2 activated killer T (T-LAK) cells, as previously demonstrated, localize and accumulate in murine lung tumor metastases following adoptive transfer, we transduced them to test their ability to deliver products of genes selectively to tumors. Assessments of transduction efficiency in vitro demonstrated that adenoviral transduction consistently resulted in high (>60%) transduction rates and substantial expression of transgenes such as GFP, Red2, luciferase, beta-galactosidase and mIL-12 for at least 4 days. In vivo experiments illustrated that Ad-GFP transduced A-NK and Ad-Red2 (RFP) transduced T-LAK or mIL-12 transduced A-NK cells localized 10-50-fold more or survived significantly better than mock transduced cells, respectively, within lung metastases than in the surrounding normal lung tissue. Most importantly, mIL-12 transduced A-NK cells provided a significantly greater antitumor response than non-transduced A-NK cells. Thus, adoptive transfer of A-NK and T-LAK cells represents an efficient method for targeting products of genes to tumor sites.

Mixed hematopoietic molecular chimerism results in permanent transgene expression from retrovirally transduced hepatocytes in mice.

BACKGROUND: Cytotoxic immune elimination of transduced hepatocytes may limit gene therapy for inherited liver diseases. Using beta-galactosidase as a marker gene, we studied whether creation of mixed beta-galactosidase molecular hematopoietic chimerism could induce tolerance to beta-galactosidase-transduced hepatocytes. METHODS: Molecular hematopoietic chimerism was established in irradiated recipient mice by transplantation of either a mixture of wild-type and beta-galactosidase-transgenic bone marrow or autologous bone marrow stem cells that were transduced with beta-galactosidase lentiviral vectors. After transplantation, mice were hepatectomized and injected with beta-galactosidase recombinant retroviruses to transduce regenerating hepatocytes. We monitored the presence of beta-galactosidase-expressing hepatocytes as well as the appearance of anti-beta-galactosidase antibodies during the time. RESULTS: In control animals, anti-beta-galactosidase antibodies and cytotoxic T-lymphocyte (CTL) response developed as early as 3 weeks after gene transfer. Transduced hepatocytes disappeared concomitantly. In bone marrow transplanted mice, tolerance could be observed in a significant proportion of animals. Tolerance resulted in permanent liver transgene expression and was absent unless a chimerism above 1% was achieved, demonstrating a threshold effect. CONCLUSIONS: Creation of a molecular hematopoietic chimerism can result in transgene tolerance and evade immune rejection of retrovirally transduced hepatocytes. This strategy may be useful for hepatic inherited diseases in which the transgene product behaves as a non-self protein.

Spacer-arm modulated gene delivery efficacy of novel cationic glycolipids: design, synthesis, and in vitro transfection biology.

Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids 1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described. The results of the present structure-activity investigation convincingly demonstrate that the in vitro gene delivery efficacies of galactosylated cationic glycolipids are strikingly dependent on the absence of a spacer-arm between the open-form galactose and the positively charged nitrogen atom in their headgroup region. While the cationic glycolipids 1-3 with no headgroup spacer unit between the positively charged nitrogen and galactose showed high in vitro gene transfer efficacies in all four cells (lipids 1 and 2 with myristyl and palmityl tails, respectively, being the most efficacious), lipids 4-6 with five-carbon spacer units between the quaternized nitrogen and galactose heads were essentially transfection incompetent. The transfection inhibiting role of the five-carbon spacer unit in the headgroup region of the present novel class of cationic lipids was demonstrated by both beta-galactosidase reporter gene expression and histochemical X-gal staining assays. Results of MTT assay-based cell viability measurements in representative MCF7 cells show that cell viabilities of lipoplexes (lipid:DNA complexes) prepared from all the lipids 1-6 are remarkably high. Thus, possibilities of differential cellular cytotoxicities playing any key role behind the strikingly contrasting transfection properties of lipids 1-3 with no spacer and lipids 4-6 with a spacer unit in the headgroup regions was ruled out. Electrophoresis gel patterns in DNase I sensitivity assays are consistent with more free DNA (accessible to DNase I) being present in lipoplexes of lipids 4-6 than in lipoplexes of lipids 1-3. Thus, the results of our DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 may play an important role in abolishing their in vitro gene transfer efficacies.

Induction of pulmonary angiogenesis by adenoviral-mediated gene transfer of vascular endothelial growth factor.

BACKGROUND: We hypothesized that gene transfer of vascular endothelial growth factor (VEGF) mediated by an adenovirus vector might induce pulmonary artery angiogenesis in a lamb model of pulmonary artery hypoplasia. METHODS: Thirteen fetal lambs had left pulmonary artery banding at 106 days of gestation. Following birth, 3 groups were divided: VEGF group (n = 5) and beta-GAL group (n = 4) received an adenoviral vector encoding respectively for human VEGF165 and for galactosidase A. A control group (n = 4) had neither gene nor virus. Viral suspensions were selectively instilled in the left bronchus 6.5 days after birth. Five nonoperated lambs constituted the normal group. Euthanasia was performed at 30 days of age. Gene transfer was confirmed by blue coloration of left lung obtained with Xgal solution in an additional experiment. Histomorphometric evaluation was performed. All groups were compared with ANOVA test and paired test was used to compare right and left lung in each animal. RESULTS: Left lung was similarly hypoplastic in all operated lambs. Left pulmonary artery hypoplasia present in all operated groups was significantly less pronounced in VEGF group. The number of pleural arteries was similarly increased in left lung of all operated lambs. Left lung arterial density was higher in VEGF group than in all other groups. The percentage of parenchyma of left lung was lower in beta-GAL group than in all others, partially returned to normal in VEGF group. CONCLUSIONS: In this model, transbronchial VEGF gene transfer induces pulmonary angiogenesis, proximal pulmonary artery growth and contributes to lung parenchyma recovery.

Widespread defects in the primary olfactory pathway caused by loss of Mash1 function.

MASH1, a basic helix-loop-helix transcription factor, is widely expressed by neuronal progenitors in the CNS and PNS, suggesting that it plays a role in the development of many neural regions. However, in mice lacking a functional Mash1 gene, major alterations have been reported in only a few neuronal populations; among these is a generalized loss of olfactory receptor neurons of the olfactory epithelium. Here, we use a transgenic reporter mouse line, in which the cell bodies and growing axons of subsets of central and peripheral neurons are marked by expression of a tau-lacZ reporter gene (the Tattler-4 allele), to look both more broadly and deeply at defects in the nervous system of Mash1-/- mice. In addition to the expected lack of olfactory receptor neurons in the main olfactory epithelium, developing Mash1-/-;Tattler-4+/- mice exhibited reductions in neuronal cell number in the vomeronasal organ and in the olfactory bulb; the morphology of the rostral migratory stream, which gives rise to olfactory bulb interneurons, was also abnormal. Further examination of cell proliferation, cell death, and cell type-specific markers in Mash1-/- animals uncovered parallels between the main olfactory epithelium and the vomeronasal organ in the regulation of sensory neuron development. Interestingly, this analysis also revealed that, in the olfactory epithelium of Mash1-/- animals, there is an overproduction of proliferating cells that co-express markers of both neuronal progenitors and supporting cells. This finding suggests that olfactory receptor neurons and olfactory epithelium supporting cells may share a common progenitor, and that expression of Mash1 may be an important factor in determining whether these progenitors ultimately generate neurons or glia.

Neurological presentation of Fabry's disease in a 52 year old man.

Fabry's disease is an X linked inborn error of metabolism due to deficient activity of the lysosomal enzyme alpha galactosidase A. Previously unrecognised Fabry's disease presenting in a 52 year old man being investigated for progressive dysarthria and ataxia is discussed. Brain magnetic resonance imaging suggested the presence of small vessel disease but skin biopsy (done to exclude cerebral autosomal dominant arteriopathy with subcortical infarcts and leucencephalopathy) showed typical changes of Fabry's disease. This diagnosis was confirmed by subsequent enzyme assays. The authors contend that Fabry's disease should be excluded, at least on clinical grounds, in patients with otherwise unexplained cerebrovascular disease.

Embryo transfer experiments and ovarian transplantation identify the ovary as the only site in which nuclear receptor interacting protein 1/RIP140 action is crucial for female fertility.

Spatial and temporal regulation of gene expression by a number of different nuclear receptors is critical in female reproduction. In this study we investigated whether the nuclear receptor corepressor nuclear receptor interacting protein 1 (Nrip1)/RIP140, which is essential for ovulation, is also required for postovulatory events, leading to pregnancy and parturition. Expression analysis indicated that Nrip1 is present in the uterus in stromal and glandular epithelial cells, primary decidual cells, and subsequently in differentiating decidual cells at the anti-mesometrial side of the implantation site. It also indicated a temporal regulation of Nrip1 in the corpora lutea at different stages of pregnancy, with increased levels at midgestation at approximately d 9.5 postcoitum (pc). By performing both embryo and ovarian transfer experiments we demonstrate that, provided the block to ovulation is by-passed, Nrip1(-/-) mice are capable of establishing and maintaining pregnancies. However, although the majority of offspring derived from ovarian transplantation survived, approximately 50% of embryos were resorbed by d 13.5 pc after embryo transfer, and the majority of pups were stillborn or died soon thereafter. Thus, although Nrip1 is differentially expressed in the reproductive tract, we conclude that the ovary is the only site in which its action is essential for fertility, with a crucial role in ovulation and a secondary role in the maintenance of pregnancy.

Adenoviral gene transfer restores lysyl hydroxylase activity in type VI Ehlers-Danlos syndrome.

Type VI Ehlers-Danlos syndrome is a disease characterized by disturbed lysine hydroxylation of collagen. The disease is caused by mutations in lysyl hydroxylase 1 gene and it affects several organs including the cardiovascular system, the joint and musculoskeletal system, and the skin. The skin of type VI Ehlers-Danlos syndrome patients is hyperelastic, scars easily, and heals slowly and poorly. We hypothesized that providing functional lysyl hydroxylase 1 gene to the fibroblasts in and around wounds in these patients would improve healing. In this study we tested the feasibility of transfer of the lysyl hydroxylase 1 gene into fibroblasts derived from rats and a type VI Ehlers-Danlos syndrome patient (in vitro) and into rat skin (in vivo). We first cloned human lysyl hydroxylase 1 cDNA into a recombinant adenoviral vector (Ad5RSV-LH). Transfection of human type VI Ehlers-Danlos syndrome fibroblasts (about 20% of normal lysyl hydroxylase 1 activity) with the vector increased lysyl hydroxylase 1 activity in these cells to near or greater levels than that of wild type, unaffected fibroblasts. The adenoviral vector successfully transfected rat fibroblasts producing both beta-galactosidase and lysyl hydroxylase 1 gene activity. We next expanded our studies to a rodent model. Intradermal injections of the vector to the abdominal skin of rats produced lysyl hydroxylase 1 mRNA and elevated lysyl hydroxylase 1 activity, in vivo. These data suggest the feasibility of gene replacement therapy to modify skin wound healing in type VI Ehlers-Danlos syndrome patients.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines.

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription-PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.