A Review of the CACNA Gene Family: Its Role in Neurological Disorders.

Calcium channels are specialized ion channels exhibiting selective permeability to calcium ions. Calcium channels, comprising voltage-dependent and ligand-gated types, are pivotal in neuronal function, with their dysregulation is implicated in various neurological disorders. This review delves into the significance of the CACNA genes, including CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1G, and CACNA1H, in the pathogenesis of conditions such as migraine, epilepsy, cerebellar ataxia, dystonia, and cerebellar atrophy. Specifically, variants in CACNA1A have been linked to familial hemiplegic migraine and epileptic seizures, underscoring its importance in neurological disease etiology. Furthermore, different genetic variants of CACNA1B have been associated with migraine susceptibility, further highlighting the role of CACNA genes in migraine pathology. The complex relationship between CACNA gene variants and neurological phenotypes, including focal seizures and ataxia, presents a variety of clinical manifestations of impaired calcium channel function. The aim of this article was to explore the role of CACNA genes in various neurological disorders, elucidating their significance in conditions such as migraine, epilepsy, and cerebellar ataxias. Further exploration of CACNA gene variants and their interactions with molecular factors, such as microRNAs, holds promise for advancing our understanding of genetic neurological disorders.

Correction: Szymanowicz et al. Headache and NOTCH3 Gene Variants in Patients with CADASIL. Neurol. Int. 2023, 15, 1238-1252.

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Epigenetics and the neurodegenerative process.

Neurological diseases are multifactorial, genetic and environmental. Environmental factors such as diet, physical activity and emotional state are epigenetic factors. Environmental markers are responsible for epigenetic modifications. The effect of epigenetic changes is increased inflammation of the nervous system and neuronal damage. In recent years, it has been shown that epigenetic changes may cause an increased risk of neurological disorders but, currently, the relationship between epigenetic modifications and neurodegeneration remains unclear. This review summarizes current knowledge about neurological disorders caused by epigenetic changes in diseases such as Alzheimer's disease, Parkinson's disease, stroke and epilepsy. Advances in epigenetic techniques may be key to understanding the epigenetics of central changes in neurological diseases.

Headache and NOTCH3 Gene Variants in Patients with CADASIL.

Autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular disease characterized by recurrent strokes, cognitive impairment, psychiatric symptoms, apathy, and migraine. Approximately 40% of patients with CADASIL experience migraine with aura (MA). In addition to MA, CADASIL patients are described in the literature as having migraine without aura (MO) and other types of headaches. Mutations in the NOTCH3 gene cause CADASIL. This study investigated NOTCH3 genetic variants in CADASIL patients and their potential association with headache types. Genetic tests were performed on 30 patients with CADASIL (20 women aged 43.6 ± 11.5 and 10 men aged 39.6 ± 15.8). PCR-HRM and sequencing methods were used in the genetic study. We described three variants as pathogenic/likely pathogenic (p.Tyr189Cys, p.Arg153Cys, p.Cys144Arg) and two benign variants (p.Ala202=, p.Thr101=) in the NOTCH3 gene and also presented the NOTCH3 gene variant (chr19:15192258 G>T), which has not been previously described in the literature. Patients with pathogenic/likely pathogenic variants had similar headache courses. People with benign variants showed a more diverse clinical picture. It seems that different NOTCH3 variants may contribute to the differential presentation of a CADASIL headache, highlighting the diagnostic and prognostic value of headache characteristics in this disease.

Disorders of Endogenous and Exogenous Antioxidants in Neurological Diseases.

In diseases of the central nervous system, such as Alzheimer's disease (AD), Parkinson's disease (PD), stroke, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and even epilepsy and migraine, oxidative stress load commonly surpasses endogenous antioxidative capacity. While oxidative processes have been robustly implicated in the pathogenesis of these diseases, the significance of particular antioxidants, both endogenous and especially exogenous, in maintaining redox homeostasis requires further research. Among endogenous antioxidants, enzymes such as catalase, superoxide dismutase, and glutathione peroxidase are central to disabling free radicals, thereby preventing oxidative damage to cellular lipids, proteins, and nucleic acids. Whether supplementation with endogenously occurring antioxidant compounds such as melatonin and glutathione carries any benefit, however, remains equivocal. Similarly, while the health benefits of certain exogenous antioxidants, including ascorbic acid (vitamin C), carotenoids, polyphenols, sulforaphanes, and anthocyanins are commonly touted, their clinical efficacy and effectiveness in particular neurological disease contexts need to be more robustly defined. Here, we review the current literature on the cellular mechanisms mitigating oxidative stress and comment on the possible benefit of the most common exogenous antioxidants in diseases such as AD, PD, ALS, HD, stroke, epilepsy, and migraine. We selected common neurological diseases of a basically neurodegenerative nature.

Modern therapies of nonsmall cell lung cancer.

Lung cancer (LC), particularly nonsmall cell lung cancer (NSCLC), is one of the most prevalent types of neoplasia worldwide, regardless of gender, with the highest mortality rates in oncology. Over the years, treatment for NSCLC has evolved from conventional surgery, chemotherapy, and radiotherapy to more tailored and minimally invasive approaches. The use of personalised therapies has increased the expected efficacy of treatment while simultaneously reducing the frequency of severe adverse effects (AEs). In this review, we discuss established modern approaches, including immunotherapy and targeted therapy, as well as experimental molecular methods like clustered regularly interspaced short palindromic repeat (CRISPR) and nanoparticles. These emerging methods offer promising outcomes and shorten the recovery time for various patients. Recent advances in the diagnostic field, including imaging and genetic profiling, have enabled the implementation of these methods. The versatility of these modern therapies allows for multiple treatment options, such as single-agent use, combination with existing conventional treatments, or incorporation into new regimens. As a result, patients can survive even in the advanced stages of NSCLC, leading to increased survival indicators such as overall survival (OS) and progression-free survival (PFS).

The cannabinoid receptors system in horses: Tissue distribution and cellular identification in skin.

BACKGROUND: The endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid-based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become disordered in pathological conditions. Cannabinoid receptors may be a possible therapeutic target in skin diseases. OBJECTIVES: To characterize the distribution and cellular expression of CBRs in the skin of horses under normal conditions. ANIMALS: Fifteen healthy horses. METHODS: Using full-thickness skin punch biopsy samples, skin-derived primary epidermal keratinocytes and dermal-derived cells, we performed analysis of Cnr1 and Cnr2 genes using real-time PCR and CBR1 and CBR2 protein expression by confocal microscopy and Western blotting. RESULTS: Normal equine skin, including equine epidermal keratinocytes and dermal fibroblast-like cells, all exhibited constant gene and protein expression of CBRs. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results represent a starting point for developing and translating new veterinary medicine-based pharmacotherapies using ECS as a possible target.

The smoking estrogens - a potential synergy between estradiol and benzo(a)pyrene.

According to recent statistics, Lung Cancer (LC) is one of the most frequently diagnosed tumor types, representing nearly 12% of all global cancer cases. Moreover, in recent years, an increased mortality rate and incidence of this cancer were observed, especially among nonsmokers. Lung cancer patients are often characterized by poor prognosis and low survival rates, which encourages the scientific community to investigate the biochemical and molecular processes leading to the development of this malignancy. Furthermore, the mechanisms of LC formation and progression are not yet fully elucidated due to their high complexity, as well as a multitude of environmental, genetic, and molecular factors involved. Even though LC's association with exposure to cigarette smoke is indisputable, current research provides evidence that the development of this cancer can also be affected by the presence of estrogens and their interaction with several tobacco smoke components. Hence, the main goal of this brief review was to investigate reports of a possible synergy between 17ß estradiol (E2), the most biologically active estrogen, and benzo(a)pyrene (BaP), a strongly carcinogenic compound produced as a result of incomplete tobacco combustion. The literature sources demonstrate a possible carcinogenic synergy between estrogens, especially E2, and BaP, a toxic tobacco smoke component. Therefeore, the combined effect of disturbed estrogen production in cancer cells, as well as the molecular influence exerted by BaP, could explain the increased aggressiveness and rate of LC development. Summarizing, the synergistic effect of these risk factors is an interesting area of further research.

PARP1 as a Marker of an Aggressive Clinical Phenotype in Cutaneous Melanoma-A Clinical and an In Vitro Study.

(1) Background: Poly(ADP-ribose) polymerase 1) (PARP1) is a pleiotropic enzyme involved in several cellular processes, e.g., DNA damage repair, regulation of mitosis, and immune response. Little is known about the role of PARP1 in melanoma development and progression. We aimed to investigate the prognostic significance of PARP1 expression in cutaneous melanoma through evaluation of mRNA and protein levels of PARP1 in normal melanocytes and melanoma cell lines, as well as in patients' tissue material from surgical resections. (2) Methods: An in vitro model was based on two types of normal human melanocytes (HEMn-DP and HEMn-LP) and four melanoma cell lines (A375, WM1341D, Hs294T, and WM9). PARP1 mRNA gene expression was estimated using real-time polymerase chain reaction (RT-PCR), whereas the protein level of PARP1 was evaluated by fluorescence confocal microscopy and then confirmed by Western Blotting analysis. The expression of PARP1 was also assessed by immunohistochemistry in formalin-fixed paraffin-embedded tissues of 128 primary cutaneous melanoma patients and correlated with follow-up and clinicopathologic features. (3) Results: The in vitro study showed that melanoma cells exhibited significantly higher PARP1 expression at mRNA and protein levels than normal melanocytes. High PARP1 expression was also associated with the invasiveness of tumor cells. Elevated nuclear PARP1 expression in patients without nodal metastases strongly correlated with significantly shorter disease-free survival (p = 0.0015) and revealed a trend with shorter cancer-specific overall survival (p = 0.05). High PARP1 immunoreactivity in the lymph node-negative group of patients was significantly associated with higher Breslow tumor thickness, presence of ulceration, and a higher mitotic index (p = 0.0016, p = 0.023, and p < 0.001, respectively). In patients with nodal metastases, high PARP1 expression significantly correlated with the presence of microsatellitosis (p = 0.034), but we did not confirm the prognostic significance of PARP1 expression in these patients. In the entire analyzed group of patients (with and without nodal metastases at the time of diagnosis), PARP1 expression was associated with a high mitotic index (p = 0.001) and the presence of ulceration (p = 0.036). Moreover, in patients with elevated PARP1 expression, melanoma was more frequently located in the skin of the head and neck region (p = 0.015). In multivariate analysis, high PARP1 expression was an independent unfavorable prognosticator in lymph node-negative cutaneous melanoma patients. (4) Conclusions: In vitro molecular biology approaches demonstrated enhanced PARP1 expression in cutaneous melanoma. These results were confirmed by the immunohistochemical study with clinical parameter analysis, which showed that a high level of PARP1 correlated with unfavorable clinical outcome. These observations raise the potential role of PARP1 inhibitor-based therapy in cutaneous melanoma.

The influence of conjugated linoleic acid on the expression of peroxisome proliferator-activated receptor-γ and selected apoptotic genes in non-small cell lung cancer.

In recent years, peroxisome proliferator-activated receptor-γ (PPARγ) has been intensively studied. Because its activation is often associated with changes in the expression level of various apoptotic genes, many studies have emphasized the role of PPARγ as an important anticancer agent. However, in different types of cancer, different genes are influenced by PPARγ action. Previous studies showed that conjugated linoleic acid (CLA) was able to induce apoptosis, upregulate PPARG gene expression and activate PPARγ protein in certain human cancer cell lines. Moreover, some PPARγ agonists inhibited the growth of human lung cancer cells through the induction of apoptosis. Nevertheless, the impact of CLA on PPARγ mRNA and protein levels in non-small cell lung cancer (NSCLC) cell lines has not been investigated thus far. Therefore, in our study, we analysed the influence of the c9,t11 linoleic acid isomer on the expression of PPARG and other genes involved in the apoptotic response (BCL-2, BAX, and CDKN1A) in two NSCLC cell lines of different histological origin (A549 and Calu-1) and in normal human bronchial epithelial Beas-2B cells. Cells were treated with several doses of c9,t11 CLA, followed by RNA and protein isolation, cDNA synthesis, real-time quantitative PCR (RT-qPCR) and Western blot analysis. We showed that the investigated CLA isomer was able to enhance the expression of PPARγ in the examined cell lines and alter the mRNA and protein levels of genes involved in apoptosis. Fluorescent staining and MMT assay revealed the antiproliferative potential of CLA as well as its ability to activate pathways that lead to cell death.

Decreased expression of cytochrome p450 1B1 in non-small cell lung cancer.

Recent studies have associated oestrogen metabolism and cigarette smoking with their carcinogenic impact on the lungs. Compounds commonly found in tobacco smoke induce the activity of CYP1B1, the enzyme responsible for the synthesis of catecholic derivatives of oestrogens. During their redox transformations, these structures can release large amounts of reactive oxygen species or can form DNA adducts, which lead to the decomposition of genetic material. This process may illustrate the synergistic effect of oestrogenic activity and tobacco combustion on oestrogen-dependant lung cancer development. There is considerable evidence suggesting that the level of oestrogen in lung tumours is elevated. Therefore, by using reverse transcription, real-time PCR and Western Blot analysis, we evaluated the CYP1B1 status in tissues from 76 patients diagnosed with non-small cell lung cancer (NSCLC) to confirm whether potential overexpression of CYP1B1 may impact lung cancerogenesis induced by oestrogens. We found significantly lower levels of CYP1B1 transcripts (p=0.00001) and proteins (p=0.000085) in lung tumour material compared to corresponding, histopathologically unchanged tissues. We also analysed the association of CYP1B1 expression with gender, age and clinicopathological data of NSCLC patients. We observed lower amounts of CYP1B1 occurring in the middle stages of LC, regardless of gender, age or histological type of lung cancer.

Exploring estrogenic activity in lung cancer.

It is well known that a connection between xenobiotics inhalation, especially tobacco combustion and Lung Cancer development is strongly significant and indisputable. However, recent studies provide evidence indicating that another factors such as, estrogens are also involved in lung carcinoma biology and metabolism. Although the status of estrogen receptors (ER), in both cancerous and healthy lung tissue has been well documented, there is still inconclusive data with respect of which isoform of the receptor is present in the lungs. However according to several studies, ERß appears to be predominant form. Apart from ERs, estrogens can work through a recently discovered G-coupled estrogen receptor. Binding with both types of the receptors causes a signal, which leads to i.e. enhanced cell proliferation. There are many published reports which suggest that estrogen can be synthesized in situ in lung cancer. Some disturbances in the activity and expression levels of enzymes involved in estrogen synthesis were proved. This suggests that increased amounts of sex-steroid hormones can affect cells biology and be the reason of the accelerated development and pathogenesis of lung cancer. There also exist phenomena which associate estrogenic metabolism and tobacco combustion and its carcinogenic influence on the lungs. Compounds present in cigarette smoke induce the activity of CYP1B1, the enzyme responsible for estrogenic metabolism and synthesis of their cateholic derivatives. These structures during their redox cycle are able to release reactive oxygen species or form DNA adduct, which generally leads to destruction of genetic material. This process may explain the synergistic effect of smoking and estrogens on estrogen-dependent lung cancer development.

Selection of reliable reference genes in eutopic and ectopic endometrium for quantitative expression studies.

PURPOSE: Physiological changes during menstrual cycle cause the endometrium and endometriosis to develop specific kind of tissues, especially in regard to the gene expression profiles, which may include also housekeeping genes, commonly used as reference genes (RGs) in quantitative studies. Reverse transcription, followed by quantitative polymerase chain reaction (RT-qPCR) is the most precise and commonly used method in gene expression studies. In order to reduce effects of technical approaches and biological variability of gene's expression level, the studies often employ RGs in experimental data normalization. However, the expression of RGs is not always stable and depends on several variables. Thus, the selection of appropriate RG is one of the most significant steps to obtain reliable results in RT-qPCR-based methods. MATERIAL AND METHODS: With the usage of RT-qPCR, we researched the expression of seven genes (ACTB, B2M, G6PD, GAPD, GUSB, HPRT and PPIA) as reliable reference genes in eutopic and ectopic endometrial tissue specimens obtained during standard surgery of women of reproductive age. Stability of expression level was analyzed by the most universal MS Excel plug-ins including: geNorm, NormFinder and BestKeeper. The descriptive statistics were evaluated using Statistica software. RESULTS: The distribution of threshold (Ct) values was not equal. We identified genes with higher expression level (referring to Ct values) such as ACTB and B2M, medium e.g., GAPD and low expression level, e.g., G6PD and HPRT. We demonstrated that the stability of the analyzed reference genes was not homogenous, and different algorithms pointed to PPIA, GAPD and B2M as the most stable ones in eutopic and ectopic endometrium. On the contrary to these, GUSB and G6PD were the most unstable ones. CONCLUSIONS: In RT-qPCR-based analyses of gene expression level in eutopic and ectopic endometrium, we strongly recommend that a minimum of two reference genes are to be used and we determined that the most suitable seem to be PPIA and GAPD.

Increased expression of proline-, glutamic acid- and leucine-rich protein PELP1 in non-small cell lung cancer.

It has been demonstrated that estrogens are able to enhance lung tumorigenesis by estrogen receptor (ER) pathway. ER signaling is a highly complex process that requires a number of different coactivators, including proline-, glutamic acid- and leucine-rich protein-1 (PELP1). We studied PELP1 transcript and protein levels in cancerous and histopathologically unchanged lung tissues obtained from 73 patients diagnosed with non-small cell lung cancer (NSCLC). We observed increased levels of PELP1 transcript (P=0.00001) and protein (P=0.00001) in tumor tissues compared to adjacent histopathologically unchanged tissues. Significant increase of PELP1 transcript/protein level was found in all patients, regardless of gender (males: P=0.0003/P=0.000003; females: P=0.0005/P=0.02), age (≤ 60 patients: P=0.042/P=0.016; >60 patients: P=0.00001/P=0.00001) or histopathological type of tumor (adenocarcinoma [ADC]: P=0.004/P=0.0006; squamous cell carcinoma [SSC]: P=0.0009/P=0.0008). Increased PELP1 transcript/protein levels were also correlated with some lung cancer stage (1a: P=0.07/P=0.02; 1b: P=0.001/P=0.03; 2a: P=0.012/P=0.001), tumor size (T2a: P=0.0006/P=0.001) and lymph node metastasis (N0: P=0.0003/P=0.0006; N1: P=0.017/P=0.003). Moreover, significant increase in PELP1 transcript level in cancer stage 1a (P=0.02) was observed. PELP1 protein content was higher in tumor tissues of patients with cancer stage 3a (P=0.04) and in T1a tumor size (P=0.03). Our studies demonstrate significantly higher amounts of PELP1 transcript and protein in tumor tissues in patients with NSCLC. Moreover, we also determined the association of PELP1 transcript and protein level with some clinicopathological features of NSCLC.