Louiza Potamiti

Cep104 is a conserved protein essential for centriole and cilia function, with mutations linked to Joubert Syndrome. We investigated its role in Xenopus embryonic development, revealing that Cep104 is crucial for neural tube closure (NTC) through regulating apical constriction. We show that the role of Cep104 in cilia and hedgehog signalling cannot alone explain the elicited defects. We go on to show that Cep104 localizes to the ends of cytoplasmic microtubules, influencing their stability. Downregulation of CEP104 led to microtubule instability and defects in multiciliated cell intercalation, a process dependent on stable microtubules. Our findings demonstrate that Cep104 functions beyond cilia, playing a significant role in cytoplasmic microtubule dynamics, suggesting that both ciliary and non-ciliary roles are important for neurodevelopment and the pathogenesis of ciliopathies.

Background: In the present study, we aimed to characterize the cytotoxic efficacy of Zebularine either as a single agent or in combination with various isothiocyanates in an in vitro model consisting of human melanoma (A375, Colo-679) as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. Methods: In this model, we have evaluated the anti-melanoma effect of Zebularine (in single and combinatorial protocols) in terms of cell viability, apoptotic induction and alterations in ultrastructural chromatin configuration, protein expression levels of DNA methyltransferases (DNMTs) and associated histone epigenetic marks capable of mediating gene expression. Results: Exposure to Zebularine resulted in dose- and time-dependent cytotoxicity through apoptotic induction in malignant melanoma cells, while neighboring non-tumorigenic keratinocytes remained unaffected. A more profound response was observed in combinational protocols, as evidenced by a further decline in cell viability leading to an even more robust apoptotic induction followed by a differential response (i.e., activation/de-activation) of various apoptotic genes. Furthermore, combined exposure protocols caused a significant decrease of DNMT1, DNMT3A and DNMT3B protein expression levels together with alterations in ultrastructural chromatin configuration and protein expression levels of specific histone modification marks capable of modulating gene expression. Conclusions: Overall, we have developed a novel experimental approach capable of potentiating the cytotoxic efficacy of Zebularine against human malignant melanoma cells while at the same time maintaining a non-cytotoxic profile against neighboring non-tumorigenic keratinocyte (HaCaT) cells.

Antibiotics resistance is expanding amongst pathogenic bacteria. Phage therapy is a revived concept for targeting bacteria with multiple antibiotics resistances. In the present study, we isolated and characterized a novel phage from hospital treatment plant input, using Escherichia coli (E. coli) as host bacterium. Phage lytic activity was detected by using soft agar assay. Whole-genome sequencing of the phage was performed by using Next-Generation Sequencing (NGS). Host range was determined using other species of bacteria and representative genogroups of E. coli. Whole-genome sequencing of the phage revealed that Escherichia phage Ioannina is a novel phage within the Dhillonvirus genus, but significantly diverged from other Dhillonviruses. Its genome is a 45,270 bp linear double-stranded DNA molecule that encodes 61 coding sequences (CDSs). The coding sequence of CDS28, a putative tail fiber protein, presented higher similarity to representatives of other phage families, signifying a possible recombination event. Escherichia phage Ioannina lytic activity was broad amongst the E. coli genogroups of clinical and environmental origin with multiple resistances. This phage may present in the future an important therapeutic tool against bacterial strains with multiple antibiotic resistances.

Surface enhanced Raman spectroscopy (SERS) is becoming an attractive analytical technique for the next generation of breath diagnostics. However, current SERS substrates present challenges related to fabrication cost, complexity, signal uniformity, and reproducibility. Here, a low‐cost, label‐free SERS sensor based on fully solution‐processed decoration of TiO₂ nanowires is demonstrated (NW) with plasmonic Au nanoparticles (NP) followed by the dielectrophoretic self‐assembly into a 3D mesh with high signal to noise ratio. The sensor performance is tested using 4‐aminothiophenol (4‐ATP) as a model analyte in gas phase, at concentrations down to 10 ppb_v, and in solution, with limit of detection ≈2.4 pM. Finally, to explore the sensor capability for breath‐based diagnostics, a proof‐of‐concept experiment is performed with exhaled breath condensates (EBCs). The possibility to discriminate EBCs of individuals with upper respiratory tract infection (URTI) from healthy ones is demonstrated. Multiple SERS spectra (n≈50) from each sample are analyzed using orthogonal partial least squares discriminant analysis (OPLS‐DA), which identifies spectral features representative of URTI in up to 80% of the infection‐related spectra. These results demonstrate the applicability and potential of 1D nanomaterials together with state‐of‐the‐art solution‐processed techniques for the development of low‐cost and compact SERS breath‐based diagnostic platforms for clinical point‐of‐care applications.

The aim of the current study was to (i) extract isolated fractions of watercress flowers enriched in polyphenols, phenethyl isothiocyanate and glucosinolates and (ii) characterize the anticancer mode of action of non-lethal, sub-lethal and lethal concentrations of the most potent extract fraction in primary (A375) and metastatic (COLO-679) melanoma cells as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. Cytotoxicity was assessed via the Alamar Blue assay, whereas ultrastructural alterations in mitochondria and the endoplasmic reticulum were determined via transmission electron microscopy. Mitochondrial membrane depolarization was determined using Mito-MP dye, whereas apoptosis was evaluated through the activation of caspases-3, -8 and -9. Among all extract fractions, the phenethyl isothiocyanate-enriched one (PhEF) possessed significant cytotoxicity against A375 and COLO-679 cells, while HaCaT cells remained relatively resistant at sub-lethal and lethal concentrations. Additionally, ultrastructural subcellular alterations associated with apoptosis were observed by means of increased mitochondrial area and perimeter, decreased cristae density and a shorter distance of the endoplasmic reticulum to the mitochondria, all taking place during “early” time points (2–4 h) of exposure. Moreover, PhEF induced mitochondrial membrane depolarization associated with “late” time points (24 h) of exposure, thereby leading to the activation of intrinsic apoptosis. Finally, the inhibition of cytosolic Ca²⁺ efflux reduced levels of caspases-9 and -3 activity, suggesting the involvement of Ca²⁺ efflux in modulating the activation of intrinsic apoptosis. To conclude, our data demonstrate an association of “early” ultrastructural alterations in mitochondria and the endoplasmic reticulum with the “late” induction of intrinsic apoptosis via the modulation of Ca²⁺ efflux.

Charcot-Marie-Tooth disease type 2A (CMT2A) is the most common hereditary axonal neuropathy caused by mutations in MFN2 encoding Mitofusin-2, a multifunctional protein located in the outer mitochondrial membrane. In order to study the effects of a novel MFN2^K357T mutation associated with early onset, autosomal dominant severe CMT2A, we generated a knock-in mouse model. While Mfn2^K357T/K357T mouse pups were postnatally lethal, Mfn2^+/K357T heterozygous mice were asymptomatic and had no histopathological changes in their sciatic nerves up to 10 months of age. However, immunofluorescence analysis of Mfn2^+/K357T mice revealed aberrant mitochondrial clustering in the sciatic nerves from 6 months of age, in optic nerves from 8 months, and in lumbar spinal cord white matter at 10 months, along with microglia activation. Ultrastructural analyses confirmed dysmorphic mitochondrial aggregates in sciatic and optic nerves. After exposure of 6-month-old mice to lipopolysaccharide, Mfn2^+/K357T mice displayed a higher immune response, a more severe motor impairment, and increased CNS inflammation, microglia activation, and macrophage infiltrates. Overall, ubiquitous Mfn2^K357T expression renders the CNS and peripheral nerves of Mfn2^+/K357T mice more susceptible to mitochondrial clustering, and augments their response to inflammation, modeling some cellular mechanisms that may be relevant for the development of neuropathy in patients with CMT2A.

We aimed to determine genetic diagnosis in the national primary ciliary dyskinesia (PCD) cohort of Cyprus, an island with a high disease prevalence. We used targeted next‐generation sequencing (NGS) of 39 PCD genes in 48 patients of Greek‐Cypriot and other ancestries. We achieved molecular diagnosis in 74% of the unrelated families tested. We identified 24 different mutations in 11 genes, 12 of which are novel. Homozygosity was more common in Greek‐Cypriot than non‐Greek‐Cypriot patients (88% vs 46.2%, p =0.016). Four mutations (DNAH11:c.5095‐2A>G, CFAP300:c.95_103delGCCGGCTCC, TTC25:c.716G>A, RSPH9:c.670+2T>C,) were found in 74% of the diagnosed Greek‐Cypriot families. Patients with RSPH9 mutations demonstrated higher nasal nitric oxide (57nL/min Vs 15 nL/min, p <0.001), higher FEV1 (‐0.89 vs ‐2.37, p=0.018) and FVC (‐1.00 vs ‐2.16, p=0.029) z‐scores than the rest of the cohort. Targeted multigene‐panel NGS is an efficient tool for early diagnosis of PCD, providing insight to disease genetic epidemiology and improved patient stratification.