Cytotoxic Complexes of Sodium Oleate with β-Lactoglobulin

pre-printA complex of α-lactalbumin and oleic acid has previously been shown to induce apoptosis in cancer cells in a number of in vitro and in vivo trials. This complex is called HAMLET or BAMLET, depending on the origin of α-la (human/bovine alpha-lactalbumin made lethal to tumour cells). In the current study, it was shown that bovine β-lactoglobulin (β-lg), upon binding sodium oleate (NaOle), the salt of oleic acid, also acquires cytotoxicity towards tumour cells (human monocytic cells U937), analogously to HAMLET/BAMLET complexes. The properties of the complex were characterized using FIR spectroscopy, HPLC and SDS-PAGE. It was shown that the level of covalent oligomerization (dimers and trimers) of β-lg increased with increasing the molar ratio of sodium oleate NaOle:β-lg in the preparation procedure. At the same time, increasing the molar ratio of NaOle:β-lg increased the cytotoxicity of the complex. The increase in cytotoxicity appeared to be dependent on the amount of bound NaOle in the complex, but not on the content of multimeric forms of β-lg. The NaOle/β-lg complex also showed similarity with BAMLET in penetrating the cell membrane and co-localizing with the cell nucleus. Furthermore, DNA fragmentation studies suggested that tumour cells (U937) treated with the complex died by apoptosis, as in the case of BAMLET, and healthy cells appeared to be less affected by treatment, as shown with model rat adrenal pheochromocytoma cells PC12. In conclusion, β-lg and NaOle can form complexes with apoptosis-inducing qualities comparable to those of BAMLET.The work was funded by the Irish Dairy Research Trust and The Department of Agriculture (Food Institutional Research Measure – FIRM project 08RDTMFRC650) under the National Development Plan 2007-2013. K. Lišková was funded under the TeagascWalsh Fellowship Scheme

Photoactivatable organometallic pyridyl ruthenium(II) arene complexes

The synthesis and characterization of a family of piano-stool RuII arene complexes of the type [(η6-arene)Ru(N,N′)(L)][PF6]2, where arene is p-cymene (p-cym), hexamethylbenzene (hmb), or indane (ind), N,N′ is 2,2′-bipyrimidine (bpm), 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (phendio), or 4,7-diphenyl-1,10-phenanthroline (bathophen), and L is pyridine (Py), 4-methylpyridine (4-MePy), 4-methoxypyridine (4-MeOPy), 4,4′-bipyridine (4,4′-bpy), 4-phenylpyridine (4-PhPy), 4-benzylpyridine (4-BzPy), 1,2,4-triazole (trz), 3-acetylpyridine (3-AcPy), nicotinamide (NA), or methyl nicotinate (MN), are reported, including the X-ray crystal structures of [(η6-p-cym)Ru(bpm)(4-MePy)]2+ (2), [(η6-p-cym)Ru(bpm)(4-BzPy)]2+ (6), [(η6-p-cym)Ru(bpm)(trz)]2+ (7), [(η6-p-cym)Ru(phen)(Py)]2+ (10), and [(η6-ind)Ru(bpy)(Py)]2+ (13). These complexes can selectively photodissociate the monodentate ligand (L) when excited with UVA or white light, allowing strict control of the formation of the reactive aqua species [(η6-arene)Ru(N,N′)(OH2)]2+ that otherwise would not form in the dark. The photoproducts were characterized by UV–vis absorption and 1H NMR spectroscopy. DFT and TD-DFT calculations were employed to characterize the excited states and to obtain information on the photochemistry of the complexes. All the RuII pyridine complexes follow a relatively similar photochemical L-ligand dissociation mechanism, likely to occur from a series of 3MC triplet states with dissociative character. The photochemical process proved to be much more efficient when UVA-range irradiation was used. More strikingly, light activation was used to phototrigger binding of these potential anticancer agents with discriminating preference toward 9-ethylguanine (9-EtG) over 9-ethyladenine (9-EtA). Calf thymus (CT)-DNA binding studies showed that the irradiated complexes bind to CT-DNA, whereas the nonirradiated forms bind negligibly. Studies of CT-DNA interactions in cell-free media suggest combined weak monofunctional coordinative and intercalative binding modes. The RuII arene complexes [(η6-p-cym)Ru(bpm)(Py)]2+ (1), [(η6-p-cym)Ru(bpm)(4-MeOPy)]2+ (3), [(η6-p-cym)Ru(4,4′-bpy)]2+ (4), [(η6-hmb)Ru(bpm)(Py)]2+ (8), [(η6-ind)Ru(bpm)(Py)]2+ (9), [(η6-p-cym)Ru(phen)(Py)]2+ (10), [(η6-p-cym)Ru(bathophen)(Py)]2+ (12), [(η6-p-cym)Ru(bpm)(NA)]2+ (15), and [(η6-p-cym)Ru(bpm)(MN)]2+ (16) were cytotoxic toward A2780 human ovarian cancer cell line in the absence of photoirradiation (IC50 values in the range of 9.0–60 μM)

Bipyrimidine ruthenium(II) arene complexes : structure, reactivity and cytotoxicity

The synthesis and characterization of complexes [(η6-arene)Ru(N,N′)X][PF6], where arene is para-cymene (p-cym), biphenyl (bip), ethyl benzoate (etb), hexamethylbenzene (hmb), indane (ind) or 1,2,3,4-tetrahydronaphthalene (thn), N,N′ is 2,2′-bipyrimidine (bpm) and X is Cl, Br or I, are reported, including the X-ray crystal structures of [(η6-p-cym)Ru(bpm)I][PF6], [(η6-bip)Ru(bpm)Cl][PF6], [(η6-bip)Ru(bpm)I][PF6] and [(η6-etb)Ru(bpm)Cl][PF6]. Complexes in which N,N′ is 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione or 4,7-diphenyl-1,10-phenanthroline (bathophen) were studied for comparison. The RuII arene complexes undergo ligand-exchange reactions in aqueous solution at 310 K; their half-lives for hydrolysis range from 14 to 715 min. Density functional theory calculations on [(η6-p-cym)Ru(bpm)Cl][PF6], [(η6-p-cym)Ru(bpm)Br][PF6], [(η6-p-cym)Ru(bpm)I][PF6], [(η6-bip)Ru(bpm)Cl][PF6], [(η6-bip)Ru(bpm)Br][PF6] and [(η6-bip)Ru(bpm)I][PF6] suggest that aquation occurs via an associative pathway and that the reaction is thermodynamically favourable when the leaving ligand is I > Br ≈ Cl. pK a* values for the aqua adducts of the complexes range from 6.9 to 7.32. A binding preference for 9-ethylguanine (9-EtG) compared with 9-ethyladenine (9-EtA) was observed for [(η6-p-cym)Ru(bpm)Cl][PF6], [(η6-hmb)Ru(bpm)Cl]+, [(η6-ind)Ru(bpm)Cl]+, [(η6-thn)Ru(bpm)Cl]+, [(η6-p-cym)Ru(phen)Cl]+ and [(η6-p-cym)Ru(bathophen)Cl]+ in aqueous solution at 310 K. The X-ray crystal structure of the guanine complex [(η6-p-cym)Ru(bpm)(9-EtG-N7)][PF6]2 shows multiple hydrogen bonding. Density functional theory calculations show that the 9-EtG adducts of all complexes are thermodynamically preferred compared with those of 9-EtA. However, the bmp complexes are inactive towards A2780 human ovarian cancer cells. Calf thymus DNA interactions for [(η6-p-cym)Ru(bpm)Cl][PF6] and [(η6-p-cym)Ru(phen)Cl][PF6] consist of weak coordinative, intercalative and monofunctional coordination. Binding to biomolecules such as glutathione may play a role in deactivating the bpm complexes

ALG3-CDG: a patient with novel variants and review of the genetic and ophthalmic findings

BACKGROUND: ALG3-CDG is a rare autosomal recessive disease. It is characterized by deficiency of alpha-1,3-mannosyltransferase caused by pathogenic variants in the ALG3 gene. Patients manifest with severe neurologic, cardiac, musculoskeletal and ophthalmic phenotype in combination with dysmorphic features, and almost half of them die before or during the neonatal period. CASE PRESENTATION: A 23 months-old girl presented with severe developmental delay, epilepsy, cortical atrophy, cerebellar vermis hypoplasia and ocular impairment. Facial dysmorphism, clubfeet and multiple joint contractures were observed already at birth. Transferrin isoelectric focusing revealed a type 1 pattern. Funduscopy showed hypopigmentation and optic disc pallor. Profound retinal ganglion cell loss and inner retinal layer thinning was documented on spectral-domain optical coherence tomography imaging. The presence of optic nerve hypoplasia was also supported by magnetic resonance imaging. A gene panel based next-generation sequencing and subsequent Sanger sequencing identified compound heterozygosity for two novel variants c.116del p.(Pro39Argfs*40) and c.1060 C > T p.(Arg354Cys) in ALG3. CONCLUSIONS: Our study expands the spectrum of pathogenic variants identified in ALG3. Thirty-three variants in 43 subjects with ALG3-CDG have been reported. Literature review shows that visual impairment in ALG3-CDG is most commonly linked to optic nerve hypoplasia

Phenotype Variability in Czech Patients Carrying PAX6 Disease-Causing Variants

The aim of this study was to report PAX6 disease-causing variants in six Czech families, to describe the associated phenotypes, and to perform functional assessment of the splice site variants. Detailed ophthalmic examination was performed. The PAX6 coding region was directly sequenced in three probands. Two probands were analysed by exome sequencing and one by genome sequencing. The effect of two variants on pre-mRNA splicing was evaluated using an exon trapping assay. Six different heterozygous PAX6 variants were identified, with c.111_120del and c.1183+1G˃T being novel. Both c.1183+1G˃T and c.1032+1G>A were proved to cause aberrant splicing with exon skipping and subsequent frameshift. The phenotypic features were variable between and within families. One individual, aged 31 years, presented with mild unilateral ptosis accompanied by aniridia in the right eye, partial aniridia in the left eye, and bilateral congenital cataracts, without marked foveal hypoplasia. Bilateral microcornea, partial aniridia, congenital cataracts, and a large posterior segment coloboma were found in another proband, aged 32 years. One child, aged 8 years, had bilateral high myopia, optic nerve colobomas, anterior polar cataracts, but no iris defects. Another individual, aged 46 years, had bilateral congenital ptosis, iris hypoplasia, keratopathy with marked fibrovascular pannus, anterior polar cataract, and foveal hypoplasia combined with impaired glucose tolerance. However, his daughter, aged 11 years, showed classical features of aniridia. Our study extends the genetic spectrum of PAX6 disease-causing variants and confirms that the associated phenotypic features may be very broad and different to the 'classical' aniridia

Should patients with kearns-sayre syndrome and corneal endothelial failure be genotyped for a TCF4 trinucleotide repeat, commonly associated with fuchs endothelial corneal dystrophy?

The aim of this study was to describe the ocular phenotype in a case with Kearns-Sayre syndrome (KSS) spectrum and to determine if corneal endothelial cell dysfunction could be attributed to other known distinct genetic causes. Herein, genomic DNA was extracted from blood and exome sequencing was performed. Non-coding gene regions implicated in corneal endothelial dystrophies were screened by Sanger sequencing. In addition, a repeat expansion situated within an intron of TCF4 (termed CTG18.1) was genotyped using the short tandem repeat assay. The diagnosis of KSS spectrum was based on the presence of ptosis, chronic progressive external ophthalmoplegia, pigmentary retinopathy, hearing loss, and muscle weakness, which were further supported by the detection of ~6.5 kb mtDNA deletion. At the age of 33 years, the proband’s best corrected visual acuity was reduced to 0.04 in the right eye and 0.2 in the left eye. Rare ocular findings included marked corneal oedema with central corneal thickness of 824 and 844 µm in the right and left eye, respectively. No pathogenic variants in the genes, which are associated with corneal endothelial dystrophies, were identified. Furthermore, the CTG18.1 genotype was 12/33, which exceeds a previously determined critical threshold for toxic RNA foci appearance in corneal endothelial cells

Non-Penetrance for Ocular Phenotype in Two Individuals Carrying Heterozygous Loss-of-Function ZEB1 Alleles

ZEB1 loss-of-function (LoF) alleles are known to cause a rare autosomal dominant disorder—posterior polymorphous corneal dystrophy type 3 (PPCD3). To date, 50 pathogenic LoF variants have been identified as disease-causing and familial studies have indicated that the PPCD3 phenotype is penetrant in approximately 95% of carriers. In this study, we interrogated in-house exomes (n = 3616) and genomes (n = 88) for the presence of putative heterozygous LoF variants in ZEB1. Next, we performed detailed phenotyping in a father and his son who carried a novel LoF c.1279C>T; p.(Glu427*) variant in ZEB1 (NM_030751.6) absent from the gnomAD v.2.1.1 dataset. Ocular examination of the two subjects did not show any abnormalities characteristic of PPCD3. GnomAD (n = 141,456 subjects) was also interrogated for LoF ZEB1 variants, notably 8 distinct heterozygous changes presumed to lead to ZEB1 haploinsufficiency, not reported to be associated with PPCD3, have been identified. The NM_030751.6 transcript has a pLI score ≥ 0.99, indicating extreme intolerance to haploinsufficiency. In conclusion, ZEB1 LoF variants are present in a general population at an extremely low frequency. As PPCD3 can be asymptomatic, the true penetrance of ZEB1 LoF variants remains currently unknown but is likely to be lower than estimated by the familial led approaches adopted to date

Novel disease-causing variants and phenotypic features of X-linked megalocornea

Purpose: The aim of the study was to describe the phenotype and molecular genetic causes of X-linked megalocornea (MGC1). We recruited four British, one New Zealand, one Vietnamese and four Czech families. // Methods: All probands and three female carriers underwent ocular examination and Sanger sequencing of the CHRDL1 gene. Two of the probands also had magnetic resonance imaging (MRI) of the brain. // Results: We identified nine pathogenic or likely pathogenic and one variant of uncertain significance in CHRDL1, of which eight are novel. Three probands had ocular findings that have not previously been associated with MGC1, namely pigmentary glaucoma, unilateral posterior corneal vesicles, unilateral keratoconus and unilateral Fuchs heterochromic iridocyclitis. The corneal diameters of the three heterozygous carriers were normal, but two had abnormally thin corneas, and one of these was also diagnosed with unilateral keratoconus. Brain MRI identified arachnoid cysts in both probands, one also had a neuroepithelial cyst, while the second had a midsagittal neurodevelopmental abnormality (cavum septum pellucidum et vergae). // Conclusion: The study expands the spectrum of pathogenic variants and the ocular and brain abnormalities that have been identified in individuals with MGC1. Reduced corneal thickness may represent a mild phenotypic feature in some heterozygous female carriers of CHRDL1 pathogenic variants

Phenotypic features of CRB1-associated early-onset severe retinal dystrophy and the different molecular approaches to identifying the disease-causing variants

PURPOSE: The aim of this study was to determine the molecular genetic basis of an early-onset severe retinal dystrophy in three unrelated consecutive patients of Czech origin and to describe their ocular phenotype. METHODS: DNA samples from two probands were analyzed using a genotyping microarray (Asper) followed by either target analysis of 43 genes implicated in retinal disorders by next generation sequencing or whole-exome sequencing, respectively. The third proband underwent conventional Sanger sequencing of CRB1 based on her ocular findings. RESULTS: All three probands harboured a known disease-causing mutation c.2843G>A; p.(Cys948Tyr) in the CRB1 gene. One individual was homozygous for this mutation, while in the other two probands c.2308G>A; p.(Gly770Ser) and c.3121A>G; p.(Met1041Val) were also identified in the heterozygous state, respectively. Both variants were novel and evaluated by in silico analysis as pathogenic. A false-negative result was observed in one of the two samples examined by the genotyping microarray. Disease onset in all patients was before the age of 7 years. Hypermetropic refractive error, bilateral nummular retinal pigmentation, retinal thickening and cystoid spaces in the macula were observed in two probands, aged 6 and 7 years. The third proband, aged 28 years, had bone spicule-like pigmentary changes associated with increased retinal nerve fiber layer. CONCLUSIONS: The first study reporting on the molecular genetic cause of non-syndromic early-onset severe retinal dystrophy in Czech patients identified one homozygous and two compound heterozygote probands with CRB1 mutations. Retina nerve fibre layer measurements should be considered an integral part of the clinical evaluation of retinal dystrophies. Detailed clinical examination and imaging can both direct molecular screening and help to confirm or refute disease causation of identified variants