Characterization of Neowestiellopsis persica A1387 (Hapalosiphonaceae) based on the cpcA, psbA, rpoC1, nifH and nifD gene sequences.

BACKGROUND: Complex descriptions of new strains of cyanobacteria appear very frequently. The main importance of these descriptions concerns potential new substances that they could synthesise, as well as their different properties as a result of their different ecological niches. The main gene used for these descriptions is 16 S with ITS or whole genome sequencing. Neowestiellopsis persica represents a unique example of the influence of ecology on morphological changes, with almost identical 16 S identity. Although our previously described Neowestiellopsis persica strain A1387 was characterized by 16 S analysis, we used different molecular markers to provide a way to separate strains of this genus that are closely related at the genetic level. MATERIALS AND METHODS: In order to conduct an in-depth study, several molecular markers, namely psbA, rpoC1, nifD, nifH and cpcA were sequenced and studied in Neowestiellopsis persica strain A1387. RESULTS: The results of the phylogenetic analysis, based on cpcA, showed that the studied strain A 1387 falls into a separate clade than N. persica, indicating that this signature sequence could be a useful molecular marker for phylogenetic separation of similar strains isolated in the future. CONCLUSIONS: Analysis of strain A1387 based on gene differences confirmed that it is a Neowestiellopsis strain. The morphological changes observed in the previous study could be due to different ecological and cultivation conditions compared to the type species. At the same time, the sequences obtained have increased our understanding of this species and will help in the future to better identify strains belonging to the genus Neowestiellopsis.

Comparative Analysis of Dehydrins from Woody Plant Species.

We conducted analyses on 253 protein sequences (Pfam00257) derived from 25 woody plant species, including trees, shrubs, and vines. Our goal was to gain insights into their architectural types, biochemical characteristics, and potential involvement in mitigating abiotic stresses, such as drought, cold, or salinity. The investigated protein sequences (253) comprised 221 angiosperms (85 trees/shrubs and 36 vines) and 32 gymnosperms. Our sequence analyses revealed the presence of seven architectural types: Kn, KnS, SKn, YnKn, YnSKn, FSKn, and FnKn. The FSKn type predominated in tree and shrub dehydrins of both gymnosperms and angiosperms, while the YnSKn type was more prevalent in vine dehydrins. The YnSKn and YnKn types were absent in gymnosperms. Gymnosperm dehydrins exhibited a shift towards more negative GRAVY scores and Fold Indexes. Additionally, they demonstrated a higher Lys content and lower His content. By analyzing promoter sequences in the angiosperm species, including trees, shrubs, and vines, we found that these dehydrins are induced by the ABA-dependent and light-responsive pathways. The presence of stress- and hormone-related cis-elements suggests a protective effect against dehydration, cold, or salinity. These findings could serve as a foundation for future studies on woody dehydrins, especially in the context of biotechnological applications.

The Influence of Phenol on the Growth, Morphology and Cell Division of Euglena gracilis.

Phenol, a monocyclic aromatic hydrocarbon with various commercial uses, is a major pollutant in industrial wastewater. Euglena gracilis is a unicellular freshwater flagellate possessing secondary chloroplasts of green algal origin. This protist has been widely used for monitoring the biological effect of various inorganic and organic environmental pollutants, including aromatic hydrocarbons. In this study, we evaluate the influence of different phenol concentrations (3.39 mM, 3.81 mM, 4.23 mM, 4.65 mM, 5.07 mM, 5.49 mM and 5.91 mM) on the growth, morphology and cell division of E. gracilis. The cell count continually decreases (p < 0.05-0.001) over time with increasing phenol concentration. While phenol treatment does not induce bleaching (permanent loss of photosynthesis), the morphological changes caused by phenol include the formation of spherical (p < 0.01-0.001), hypertrophied (p < 0.05) and monster cells (p < 0.01) and lipofuscin bodies. Phenol also induces an atypical form of cell division of E. gracilis, simultaneously producing more than 2 (3-12) viable cells from a single cell. Such atypically dividing cells have a symmetric "star"-like shape. The percentage of atypically dividing cells increases (p < 0.05) with increasing phenol concentration. Our findings suggest that E. gracilis can be used as bioindicator of phenol contamination in freshwater habitats and wastewater.

Versatile biotechnological applications of Euglena gracilis.

Euglena gracilis is a freshwater protist possessing secondary chloroplasts of green algal origin. Various physical factors (e.g. UV) and chemical compounds (e.g. antibiotics) cause the bleaching of E. gracilis cells-the loss of plastid genes leading to the permanent inability to photosynthesize. Bleaching can be prevented by antimutagens (i.e. lignin, vitamin C and selenium). Besides screening the mutagenic and antimutagenic activity of chemicals, E. gracilis is also a suitable model for studying the biological effects of many organic pollutants. Due to its capability of heavy metal sequestration, it can be used for bioremediation. E. gracilis has been successfully transformed, offering the possibility of genetic modifications for synthesizing compounds of biotechnological interest. The novel design of the "next generation" transgenic expression cassettes with respect to the specificities of euglenid gene expression is proposed. Moreover, E. gracilis is a natural source of commercially relevant bioproducts such as (pro)vitamins, wax esters, polyunsaturated fatty acids and paramylon (ß-1,3-glucan). One of the highest limitations of large-scale cultivation of E. gracilis is its disability to synthesize essential vitamins B1 and B12. This disadvantage can be overcome by co-cultivation of E. gracilis with other microorganisms, which can synthesize sufficient amounts of these vitamins. Such co-cultures can be used for the effective accumulation and harvesting of Euglena biomass by bioflocculation.

The Vapor Phase of Selected Essential Oils and Their Antifungal Activity In Vitro and In Situ against Penicillium commune, a Common Contaminant of Cheese.

This study aimed to determine the in vitro and in situ antifungal activity of (14) selected essential oils (EOS), namely clove, thyme, red thyme, litsea, eucalyptus, niaouli, fennel, anise, cumin, basil, rosemary, sage, bergamot mint, and marjoram, by vapor contact against the growth of two strains of Penicillium commune (KMi-183 and KMi-402). Furthermore, to exclude the negative effect of EOs on the lactic acid bacteria (LABs) (Streptococcus spp.) on cheeses, their influence was monitored. Next, the sensory evaluation of cheese treated by EOs was evaluated. The results show that litsea and clove EOs were the most effective in the vapor phase against both tested strains. These EOs were characterized by the highest amount of α- (40.00%) and ß-Citral (34.35%) in litsea and eugenol (85.23%) in clove. The antitoxicogenic activity of less effective (in growth inhibition) EOs on cyclopiazonic acid (CPA) production by the tested strains was also observed. The growth of Streptococcus spp. (ranging from 8.11 to 9.69 log CFU/g) was not affected by the EOs in treated cheese. Even though the evaluators recognized some EOs in sensory evaluation by the triangle test, they did not have a negative effect on the taste and smell of the treated cheeses and were evaluated as edible. The antifungal activity of EOs against several types of microscopic fungi and their effect on the sensory properties of treated foods needs to be further tested to achieve the most effective protection of foods from their direct contaminants.

Calpains in cyanobacteria and the origin of calpains.

Calpains are cysteine proteases involved in many cellular processes. They are an ancient and large superfamily of enzymes responsible for the cleavage and irreversible modification of a large variety of substrates. They have been intensively studied in humans and other mammals, but information about calpains in bacteria is scarce. Calpains have not been found among Archaea to date. In this study, we have investigated the presence of calpains in selected cyanobacterial species using in silico analyses. We show that calpains defined by possessing CysPC core domain are present in cyanobacterial genera Anabaena, Aphanizomenon, Calothrix, Chamaesiphon, Fischerella, Microcystis, Scytonema and Trichormus. Based on in silico protein interaction analysis, we have predicted putative interaction partners for identified cyanobacterial calpains. The phylogenetic analysis including cyanobacterial, other bacterial and eukaryotic calpains divided bacterial and eukaryotic calpains into two separate monophyletic clusters. We propose two possible evolutionary scenarios to explain this tree topology: (1) the eukaryotic ancestor or an archaeal ancestor of eukaryotes obtained calpain gene from an unknown bacterial donor, or alternatively (2) calpain gene had been already present in the last common universal ancestor and subsequently lost by the ancestor of Archaea, but retained by the ancestor of Bacteria and by the ancestor of Eukarya. Both scenarios would require multiple independent losses of calpain genes in various bacteria and eukaryotes.

CDH5, a Possible New Candidate Gene for Genetic Testing of Lymphedema.

Background: Expressed by endothelial cells, CDH5 is a cadherin involved in vascular morphogenesis and in the maintenance of vascular integrity and lymphatic function. The main purpose of our study was to identify distinct variants of the CDH5 gene that could be associated with lymphatic malformations and predisposition for lymphedema. Methods and Results: We performed Next Generation Sequencing of the CDH5 gene in 235 Italian patients diagnosed with lymphedema but who tested negative for variants in known lymphedema genes. We detected six different variants in CDH5 five missense and one nonsense. We also tested available family members of the probands. For family members who carried the same variant as the proband, we performed lymphoscintigraphy to detect any lymphatic system abnormalities. Variants were modeled in silico. The results showed that CDH5 variants may contribute to the onset of lymphedema, although further in vitro studies are needed to confirm this hypothesis. Conclusions: Based on our findings, we propose CDH5 as a new gene that could be screened in patients with lymphedema to gather additional evidence.

Recessive multiple epiphyseal dysplasia and Stargardt disease in two sisters.

BACKGROUND: The rapid spread of genome-wide next-generation sequencing in the molecular diagnosis of rare genetic disorders has produced increasing evidence of multilocus genomic variations in cases with a previously well-characterized molecular diagnosis. Here, we describe two patients with a rare combination of skeletal abnormalities and retinal dystrophy caused by variants in the SLC26A2 and ABCA4 genes, respectively, in a family with parental consanguinity. METHODS: Next-generation sequencing and Sanger sequencing were performed to obtain a molecular diagnosis for the retinal and skeletal phenotypes, respectively. RESULTS: Genetic testing revealed that the sisters were homozygous for the p.(Cys653Ser) variant in SLC26A2 and heterozygous for the missense p.(Pro68Leu) and splice donor c.6386+2C>G variants in ABCA4. Segregation analysis confirmed the carrier status of the parents. CONCLUSION: Despite low frequency of occurrence, the detection of multilocus genomic variations in a single disease gene-oriented approach can provide accurate diagnosis even in cases with high phenotypic complexity. A targeted sequencing approach can detect relationships between observed phenotypes and underlying genotypes, useful for clinical management.

NOTCH1: Review of its role in lymphatic development and study of seven families with rare pathogenic variants.

BACKGROUND: We developed a Next-Generation-Sequencing (NGS) protocol to screen the most frequent genetic variants related to lymphedema and a group of candidate genes. The aim of the study was to find the genetic cause of lymphedema in the analyzed patients. METHODS: We sequenced a cohort of 246 Italian patients with lymphatic malformations. In the first step, we analyzed genes known to be linked to lymphedema: 235 out of 246 patients tested negative for the most frequent variants and underwent testing for variants in a group of candidate genes, including the NOTCH1 gene, selected from the database of mouse models. We also performed in silico analysis to observe molecular interactions between the wild-type and the variant amino acids and other protein residues. RESULTS: Seven out of 235 probands, five with sporadic and two with familial lymphedema, were found to carry rare missense variants in the NOTCH1 gene. CONCLUSIONS: Our results propose that NOTCH1 could be a novel candidate for genetic predisposition to lymphedema.

Possible Role of the RORC Gene in Primary and Secondary Lymphedema: Review of the Literature and Genetic Study of Two Rare Causative Variants.

Background: RAR-related Orphan Receptor C (RORC) is a DNA-binding transcription factor and the key transcription factor responsible for differentiation of T helper 17 cells. The RORC gene plays a role in lymphoid organogenesis, thymopoiesis, and lymph node organogenesis. The aim of our study was to determine the possible role of RORC in the development of lymphatic system malformations by combining data from the scientific literature and next-generation sequencing of RORC in lymphedema patients negative for known causative genes. Methods and Results: We sequenced RORC in 235 lymphedema patients negative for known lymphedema-associated genes. We found two probands carrying nonsense RORC variants. Conclusions: We show that RORC is important for normal function of the lymphatic system and that a rare variant with a possible causative effect may imply predisposition for lymphedema.

Segregation Analysis of Rare NRP1 and NRP2 Variants in Families with Lymphedema.

Neuropilins are transmembrane coreceptors expressed by endothelial cells and neurons. NRP1 and NRP2 bind a variety of ligands, by which they trigger cell signaling, and are important in the development of lymphatic valves and lymphatic capillaries, respectively. This study focuses on identifying rare variants in the NRP1 and NRP2 genes that could be linked to the development of lymphatic malformations in patients diagnosed with lymphedema. Two hundred and thirty-five Italian lymphedema patients, who tested negative for variants in known lymphedema genes, were screened for variants in NRP1 and NRP2. Two probands carried variants in NRP1 and four in NRP2. The variants of both genes segregated with lymphedema in familial cases. Although further functional and biochemical studies are needed to clarify their involvement with lymphedema and to associate NRP1 and NRP2 with lymphedema, we suggest that it is worthwhile also screening lymphedema patients for these two new candidate genes.

Mutations in the ARAP3 Gene in Three Families with Primary Lymphedema Negative for Mutations in Known Lymphedema-Associated Genes.

BACKGROUND: ARAP3 is a small GTPase-activating protein regulator, which has important functions in lymphatic vessel organogenesis and modulation of cell adhesion and migration. Mutations in the ARAP3 gene are associated with impaired lymphatic vessel formation. OBJECTIVE: The aim of our study was to determine the genotypes of lymphedema patients in relation to variants in the ARAP3 gene in order to explore its role in the development of lymphedema. METHODS AND RESULTS: We applied next-generation sequencing to DNA samples of a cohort of 246 Italian patients with lymphatic malformations. When we tested probands for known lymphedema genes, 235 out of 246 were negative. Retrospectively, we tested the DNA of these 235 patients for new candidate lymphedema-associated genes, including ARAP3. Three out of 235 probands proved to carry rare missense heterozygous variants in ARAP3. In the case of two families, other family members were also tested and proved negative for the ARAP3 variant, besides being unaffected by lymphedema. According to in silico analysis, alterations due to these variants have a significant impact on the overall structure and stability of the resulting proteins. CONCLUSIONS: Based on our results, we propose that variants in ARAP3 could be included in genetic testing for lymphedema.

TIE1 as a Candidate Gene for Lymphatic Malformations with or without Lymphedema.

TIE1 is a cell surface protein expressed in endothelial cells. Involved in angiogenesis and lymphangiogenesis, including morphogenesis of lymphatic valves, TIE1 is important for lymphatic system functional integrity. The main purpose of this study was to identify different variants in the TIE1 gene that could be associated with lymphatic malformations or dysfunction and predisposition for lymphedema. In a cohort of 235 Italian lymphedema patients, who tested negative for variants in known lymphedema genes, we performed a further test for new candidate genes, including TIE1. Three probands carried different variants in TIE1. Two of these segregated with lymphedema or lymphatic dysfunction in familial cases. Variants in TIE1 could contribute to the onset of lymphedema. On the basis of our findings, we propose TIE1 as a candidate gene for comprehensive genetic testing of lymphedema.

Two rare PROX1 variants in patients with lymphedema.

BACKGROUND: The PROX1 gene is specifically expressed in a subpopulation of endothelial cells that, by budding and sprouting, give rise to the lymphatic system. It also plays a critical role in neurogenesis and during development of many organs, such as the eye lens, liver, and pancreas. METHODS: We used next-generation sequencing (NGS) to sequence the DNA of a cohort of 246 Italian patients with lymphatic malformations. We first investigated 29 known disease-causing genes: 235 of 246 patients tested negative and were then retested for a group of candidate genes, including PROX1, selected from a database of mouse models. The aim of the study was to define these patients' genotypes and explore the role of the candidate gene PROX1 in lymphedema. RESULTS: Two of 235 probands were found to carry rare heterozygous missense variants in PROX1. In silico analysis of these variants-p.(Leu590His) and p.(Gly106Asp)-indicates that the overall protein structure was altered by changes in interactions between nearby residues, leading to functional protein defects. CONCLUSIONS: Our results suggest that PROX1 is a new candidate gene for predisposition to lymphedema.