Novel Probiotic Candidates in Artisanal Feta-Type Kefalonian Cheese: Unveiling a Still-Undisclosed Biodiversity.

Autochthonous dairy lactic acid bacteria (LAB) isolates encompass a natural source of starter, adjunct, or probiotic candidates. In this context, traditionally manufactured, using exclusively animal rennet, Feta-type cheeses were collected from five farms located in different regions of Kefalonia island (Greece). The primary objective of this study was to isolate and characterize novel LAB, thereby exploring the unmapped microbial communities of Kefalonian Feta-type cheese and identifying new potential probiotics. The initial screening, included a preliminary gastrointestinal (GI) tolerance assessment (acidic conditions and bile salts), followed by their safety evaluation (hemolytic activity and antibiotic susceptibility). Based on the preliminary screening, selected strains underwent molecular identification and were further investigated for their probiotic attributes (lysozyme and phenol resistance, antimicrobial traits, antidiabetic aspects, cholesterol reduction and adhesion, adhesion to Caco-2 cells, and milk acidification potential). The results showed that 49, out of the 93 retrieved isolates, exhibited resistance to GI conditions, whereas 18 met the safety criteria. The molecular identification revealed strains belonging to the species Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and Lacticaseibacillus paracasei. The selected rod-shaped 14 isolates displayed a potential probiotic character. The best-performing isolates concerning cholesterol assimilation and adhesion, α-glucosidase inhibition, and epithelial adherence were Lpb. plantarum F89, F162, and F254 and Lcb. paracasei F214 and F216, whereas Lcb. paracasei F70 showed potential as a defined strain starter. The present study explores for the first time the biodiversity of traditionally fermented microbial communities in Kefalonian Feta-type cheese, revealing novel potential probiotic strains that can contribute to the development of innovative functional food products.

Novel Wild-Type Pediococcus and Lactiplantibacillus Strains as Probiotic Candidates to Manage Obesity-Associated Insulin Resistance.

As the food and pharmaceutical industry is continuously seeking new probiotic strains with unique health properties, the aim of the present study was to determine the impact of short-term dietary intervention with novel wild-type strains, isolated from various sources, on high-fat diet (HFD)-induced insulin resistance. Initially, the strains were evaluated in vitro for their ability to survive in simulated gastrointestinal (GI) conditions, for adhesion to Caco-2 cells, for bile salt hydrolase secretion, for cholesterol-lowering and cellular cholesterol-binding ability, and for growth inhibition of food-borne pathogens. In addition, safety criteria were assessed, including hemolytic activity and susceptibility to antibiotics. The in vivo test on insulin resistance showed that mice receiving the HFD supplemented with Pediococcus acidilactici SK (isolated from human feces) or P. acidilactici OLS3-1 strain (isolated from olive fruit) exhibited significantly improved insulin resistance compared to HFD-fed mice or to the normal diet (ND)-fed group.

A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels.

As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.

Characterization of the Abracl-Expressing Cell Populations in the Embryonic Mammalian Telencephalon.

Abracl (ABRA C-terminal-like protein) is a small, non-typical winged-helix protein that shares similarity with the C-terminal domain of the protein ABRA (Actin-Binding Rho-Activating protein). The role of Abracl in the cell remains elusive, although in cancer cells, it has been implicated in proliferation, migration and actin dynamics. Our previous study showed that Abracl mRNA was expressed in the dividing cells of the subpallial subventricular zone (SVZ), in the developing cortical plate (CP), and in the diencephalic SVZ; however, the molecular identities of the Abracl-expressing cell populations were not defined in that work. In this study, we use double immunofluorescence to characterize the expression of Abracl on sections of embryonic murine (E11.5-E18.5) and feline (E30/31-E33/34) telencephalon; to this end, we use a battery of well-known molecular markers of cycling (Ki67, Ascl1, Dlx2) or post-mitotic (Tubb3, Gad65/67, Lhx6 and Tbr1) cells. Our experiments show that Abracl protein has, compared to the mRNA, a broader expression domain, including, apart from proliferating cells of the subpallial and diencephalic SVZ, post-mitotic cells occupying the subpallial and pallial mantle (including the CP), as well as subpallial-derived migrating interneurons. Interestingly, in late embryonic developmental stages, Abracl was also transiently detected in major telencephalic fiber tracts.

Assessment of the Anti-Amyloidogenic Properties of Essential Oils and Their Constituents in Cells Using a Whole-Cell Recombinant Biosensor.

Essential oils exhibit numerous medicinal properties, including antimicrobial, anti-inflammatory and antioxidant effects. Recent studies also indicate that certain essential oils demonstrate anti-amyloidogenic activity against ß-amyloid, the protein implicated in Alzheimer's disease. To investigate whether the anti-aggregating properties of essential oils extend to α-synuclein, the protein involved in Parkinson's disease, we constructed and employed a whole-cell biosensor based on the split-luciferase complementation assay. We validated our biosensor by using baicalein, a known inhibitor of α-synuclein aggregation, and subsequently we tested eight essential oils commonly used in food and the hygienic industry. Two of them, citron and sage, along with their primary components, pure linalool (the main constituent in citron essential oil) and pure eucalyptol (1,8-cineole, the main constituent in sage essential oil), were able to reduce α-syn aggregation. These findings suggest that both essential oils and their main constituents could be regarded as potential components in functional foods or incorporated into complementary Parkinson's disease therapies.

Molecular Genetics of Microcephaly Primary Hereditary: An Overview.

MicroCephaly Primary Hereditary (MCPH) is a rare congenital neurodevelopmental disorder characterized by a significant reduction of the occipitofrontal head circumference and mild to moderate mental disability. Patients have small brains, though with overall normal architecture; therefore, studying MCPH can reveal not only the pathological mechanisms leading to this condition, but also the mechanisms operating during normal development. MCPH is genetically heterogeneous, with 27 genes listed so far in the Online Mendelian Inheritance in Man (OMIM) database. In this review, we discuss the role of MCPH proteins and delineate the molecular mechanisms and common pathways in which they participate.

HSPC280, a winged helix protein expressed in the subventricular zone of the developing ganglionic eminences, inhibits neuronal differentiation.

Winged helix proteins have critical roles in a variety of developmental processes. During a screening for genes expressed in the developing forebrain, we identified HSPC280, a non-typical winged helix protein, which shares similarity with a protein-protein interaction domain found in the proteins of the actin-binding Rho-activating protein family. In this work, we analyzed HSPC280 expression during mouse development as well as during neuronal differentiation of mouse Neuro2a cells. HSPC280 expression is tightly regulated; during mouse development, it was detected predominantly in the ganglionic eminences of the ventral telencephalon, from their appearance at E11.5 to P0, with the highest levels between E13.5 and E15.5, a period that correlates with the peak of neurogenesis in these structures. Comparative expression analysis of HSPC280 with Dlx2, cyclinD2 and Lhx6 revealed that, within the ganglionic eminences, HSPC280 was restricted in the proliferating cell population of the subventricular zone, in a pattern similar to that of cyclinD2. Finally, we showed that HSPC280 is a nuclear protein which, when overexpressed in Neuro2a cells, it inhibited neuronal differentiation in vitro, suggesting its involvement in the mechanisms controlling neural progenitor cells proliferation.

Zinc-based fixation for high-sensitivity in situ hybridization: a nonradioactive colorimetric method for the detection of rare transcripts on tissue sections.

Nonradioactive colorimetric in situ hybridization (NoRISH) has been widely applied to analyze gene expression at the single-cell level. Zinc fixation is time efficient and provides excellent tissue morphology. Furthermore, it improves the preservation of the RNA, facilitating the detection of rare transcripts or the identification of expressing cells scattered within a tissue. Here we present a rapid, highly sensitive NoRISH method that uses a zinc-salt-based fixative and is especially suitable for the study of genes expressed at low levels and/or in a small number of cells within a structure.

A rapid and highly sensitive method of non radioactive colorimetric in situ hybridization for the detection of mRNA on tissue sections.

BACKGROUND: Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels. METHODOLOGY/PRINCIPAL FINDINGS: A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50-60% shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFA-fixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method. CONCLUSIONS/SIGNIFICANCE: Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highly sensitive, and especially suitable to implement in the study of genes expressed at low levels and/or in sparse cells within a structure.