Analysis of the Expression of LSF Transcription Factor in the Regulation of Transcription and TSG101 during the Neoplastic Transformation of Endometrial Cells.

Previous research indicates that carcinogenesis involves disrupting the functions of numerous genes, including factors involved in the regulation of transcription and cell proliferation. For these reasons, in endometrial carcinogenesis, we decided to investigate the expression of TSG101 (a suppressor of tumor transformation) and LSF (a transcription factor involved in numerous cellular processes, such as cell cycle regulation, cell growth, development, and apoptosis). LSF may be involved in the regulation of TSG101 expression. The research material consisted of endometrial cancer samples from 60 patients. The control group consisted of normal endometrium samples donated by 60 women undergoing surgery for benign diseases of the female reproductive organs. The samples were subjected to immunohistochemical staining with antibodies specific to TSG101 and LSF. Specific antibodies were used to identify TSG101 and LSF in the examined histopathological preparations. An approximately 14-fold lower risk of endometrial cancer development was observed in patients with TSG expression in more than 75% of the assessed cells (4% vs. 36%; OR = 0.07; p = 0.0182). There was a four-fold lower risk of endometrial cancer development in patients with LSF expression in more than 50% of the assessed cells (32% vs. 64%; OR = 0.26; p = 0.0262). A more than three-fold lower risk of endometrial cancer development was observed in patients with LSF expression in more than 75% of the assessed cells (24% vs. 52%; OR = 0.29; p = 0.0454). Endometrial cancer was diagnosed in those with a lower level of TSG101 expression than in those with a cancer-free endometrium. Decreased expression of TSG101 may be a marker of endometrial cancer, and increased expression of LSF when diagnosed with endometrial cancer may indicate greater advancement of the disease. These markers might be used as diagnostic and prognostic markers-however, there is a lack of a correlation between them.

Comparative Analysis of the Placental Microbiome in Pregnancies with Late Fetal Growth Restriction versus Physiological Pregnancies.

A comparative analysis of the placental microbiome in pregnancies with late fetal growth restriction (FGR) was performed with normal pregnancies to assess the impact of bacteria on placental development and function. The presence of microorganisms in the placenta, amniotic fluid, fetal membranes and umbilical cord blood throughout pregnancy disproves the theory of the "sterile uterus". FGR occurs when the fetus is unable to follow a biophysically determined growth path. Bacterial infections have been linked to maternal overproduction of pro-inflammatory cytokines, as well as various short- and long-term problems. Proteomics and bioinformatics studies of placental biomass allowed the development of new diagnostic options. In this study, the microbiome of normal and FGR placentas was analyzed by LC-ESI-MS/MS mass spectrometry, and the bacteria present in both placentas were identified by analysis of a set of bacterial proteins. Thirty-six pregnant Caucasian women participated in the study, including 18 women with normal pregnancy and eutrophic fetuses (EFW > 10th percentile) and 18 women with late FGR diagnosed after 32 weeks of gestation. Based on the analysis of the proteinogram, 166 bacterial proteins were detected in the material taken from the placentas in the study group. Of these, 21 proteins had an exponentially modified protein abundance index (emPAI) value of 0 and were not included in further analysis. Of the remaining 145 proteins, 52 were also present in the material from the control group. The remaining 93 proteins were present only in the material collected from the study group. Based on the proteinogram analysis, 732 bacterial proteins were detected in the material taken from the control group. Of these, 104 proteins had an emPAI value of 0 and were not included in further analysis. Of the remaining 628 proteins, 52 were also present in the material from the study group. The remaining 576 proteins were present only in the material taken from the control group. In both groups, we considered the result of ns prot ≥ 60 as the cut-off value for the agreement of the detected protein with its theoretical counterpart. Our study found significantly higher emPAI values of proteins representative of the following bacteria: Actinopolyspora erythraea, Listeria costaricensis, E. coli, Methylobacterium, Acidobacteria bacterium, Bacteroidetes bacterium, Paenisporsarcina sp., Thiodiazotropha endol oripes and Clostridiales bacterium. On the other hand, in the control group statistically more frequently, based on proteomic data, the following were found: Flavobacterial bacterium, Aureimonas sp. and Bacillus cereus. Our study showed that placental dysbiosis may be an important factor in the etiology of FGR. The presence of numerous bacterial proteins present in the control material may indicate their protective role, while the presence of bacterial proteins detected only in the material taken from the placentas of the study group may indicate their potentially pathogenic nature. This phenomenon is probably important in the development of the immune system in early life, and the placental microbiota and its metabolites may have great potential in the screening, prevention, diagnosis and treatment of FGR.

Mitochondrial DNA Polymorphism in HV1 and HV2 Regions and 12S rDNA in Perimenopausal Hypertensive Women.

Estrogens enhance cellular mitochondrial activity. The diminution of female hormones during menopause may have an effect on the mitochondrial genome and the expression of mitochondrial proteins. Hence, oxidative stress and the pro-inflammatory state contribute to the formation of systemic illnesses including arterial hypertension (AH). This study aimed to determine the types and frequency of mutations in the mitochondrial DNA (mtDNA) nucleotide sequence in the hypervariable regions 1 and 2 (HV1 and HV2) and the 12S RNA coding sequence of the D-loop in postmenopausal women with hypertension. In our study, 100 women were investigated, 53 of whom were postmenopausal and 47 of whom were premenopausal (53.9 ± 3.7 years vs. 47.7 ± 4.2 years, respectively). Of those studied, 35 premenopausal and 40 postmenopausal women were diagnosed with AH. A medical checkup with 24 h monitoring of blood pressure (RR) and heart rate was undertaken (HR). The polymorphism of the D-loop and 12S rDNA region of mtDNA was examined. Changes in the nucleotide sequence of mtDNA were observed in 23% of the group of 100 women. The changes were identified in 91.3% of HV1 and HV2 regions, 60.9% of HV1 segments, 47.5% of HV2 regions, and 43.5% of 12S rDNA regions. The frequency of nucleotide sequence alterations in mtDNA was substantially higher in postmenopausal women (34%) than in premenopausal women (10.6%), p = 0.016. A higher frequency of changes in HV1 + HV2 sections in postmenopausal women (30.2%) compared to the premenopausal group (10.6%) was detected, p = 0.011. Only postmenopausal women were found to have modifications to the HV2 segment and the 12S rDNA region. After menopause, polymorphism in the mtDNA region was substantially more frequent in women with arterial hypertension than before menopause (p = 0.030; 37.5% vs. 11.5%). Comparable findings were observed in the HV2 and HV1 regions of the AH group (35% vs. 11.5%), p = 0.015, in the HV1 segment (25% vs. 11.5%), p = 0.529, and in the HV2 segment, 12S rDNA (25% vs. 0%). More than 80% of all changes in nucleotide sequence were homoplasmic. The mtDNA polymorphisms of the nucleotide sequence in the HV1 and HV2 regions, the HV2 region alone, and the 12S RNA coding sequence were associated with estrogen deficiency and a more severe course of arterial hypertension, accompanied by symptoms of adrenergic stimulation.

Placental proteome in late­onset of fetal growth restriction.

Fetal growth restriction (FGR) occurs when the fetus does not reach its genetically programmed intrauterine potential for growth and affects ~5­10% of pregnancies. This condition is one of the leading causes of perinatal mortality and morbidity associated with obstetric and neonatal complications. Placental dysfunction in FGR causes an impairment in the transfer of nutrients and oxygen from the mother to the developing fetus. Maternal adaptations to placental insufficiency may also play a role in the pathophysiology of FGR. The present study aimed to compare the proteome of the placentas of 18 women with the physiological course of pregnancy and eutrophic fetus [estimated fetal weight (EFW) >10th percentile; control group] and 18 women with late FGR (EFW <10th percentile) diagnosed after 32 weeks of pregnancy, according to the Delphi consensus (study group). The U. Mann­Whitney test was used to compare two independent groups. The R. Spearman correlation coefficient significance test was used to assess the existence of a relationship between the analyzed measurable parameters. P<0.05 was considered to indicate a statistically significant difference. The tests showed the presence of 356 different proteins which were responsible for the regulation of gene transcription control, inhibiting the activity of proteolytic enzymes, regulation of trophoblast proliferation and angiogenesis and inflammatory response. In the FGR placental proteome, other detected proteins were mostly involved in response to oxidative stress, cellular oxidation and detoxication, apoptosis, hemostatic and catabolic processes, energy transduction protein interactions, cell proliferation, differentiation and intracellular signaling. The present study used chromatographic mass­spectrometry to compare the placental proteome profiles in pregnancies complicated by late­onset FGR and normal pregnancy. Comparative analysis of proteomes from normal and FGR placentas showed significant differences. Further research is needed to clarify maternal and fetal adaptations to FGR.

The Activity of Chelidonium majus L. Latex and Its Components on HPV Reveal Insights into the Antiviral Molecular Mechanism.

Yellow-orange latex of Chelidonium majus L. has been used in folk medicine as a therapeutic agent against warts and other visible symptoms of human papillomavirus (HPV) infections for centuries. The observed antiviral and antitumor properties of C. majus latex are often attributed to alkaloids contained therein, but recent studies indicate that latex proteins may also play an important role in its pharmacological activities. Therefore, the aim of the study was to investigate the effect of the crude C. majus latex and its protein and alkaloid-rich fractions on different stages of the HPV replication cycle. The results showed that the latex components, such as alkaloids and proteins, decrease HPV infectivity and inhibit the expression of viral oncogenes (E6, E7) on mRNA and protein levels. However, the crude latex and its fractions do not affect the stability of structural proteins in HPV pseudovirions and they do not inhibit the virus from attaching to the cell surface. In addition, the protein fraction causes increased TNFα secretion, which may indicate the induction of an inflammatory response. These findings indicate that the antiviral properties of C. majus latex arise both from alkaloids and proteins contained therein, acting on different stages of the viral replication cycle.

Dual Role of YY1 in HPV Life Cycle and Cervical Cancer Development.

Human papillomaviruses (HPVs) are considered to be key etiological agents responsible for the induction and development of cervical cancer. However, it has been suggested that HPV infection alone may not be sufficient to promote cervical carcinogenesis, and other unknown factors might be required to establish the disease. One of the suggested proteins whose deregulation has been linked with oncogenesis is transcription factor Yin Yang 1 (YY1). YY1 is a multifunctional protein that is involved not only in the regulation of gene transcription and protein modification, but can also control important cell signaling pathways, such as cell growth, development, differentiation, and apoptosis. Vital functions of YY1 also indicate that the protein could be involved in tumorigenesis. The overexpression of this protein has been observed in different tumors, and its level has been correlated with poor prognoses of many types of cancers. YY1 can also regulate the transcription of viral genes. It has been documented that YY1 can bind to the HPV long control region and regulate the expression of viral oncogenes E6 and E7; however, its role in the HPV life cycle and cervical cancer development is different. In this review, we explore the role of YY1 in regulating the expression of cellular and viral genes and subsequently investigate how these changes inadvertently contribute toward the development of cervical malignancy.

P1 promoter IGF-1 polymorphism and IGF-1, IGF-R, LSF, and TSG 101 expression profile in endometriosis.

OBJECTIVES: The presence of the endometrium outside the uterine cavity affects about 10% of women of childbearing age. Studies of the progression of endometriosis to cancer have been supported by numerous evidences of gene expression or gene defect caused by oxidative stress and inflammation. We decided to check the expression of selected factors responsible for the proliferation, as in the stages of neoplasia. MATERIAL AND METHODS: A group of 80 women with ovary localization of endometriosis was qualified for research. The control group was 90 patients with ovarian simplex or follicular cysts. The DNA isolation, immunohistochemical analysis of IGF 1, IGF-R, TSG 101, and LSF expressions with a quantitative scoring of slides and electron microscopy was performed. RESULTS: The IGF-1-immunopositive cells in the reference group were in statistically significantly higher number compared to the cells forming the foci of endometriosis (p = 0.0282). However, the number of IGF-R-immunopositive cells was comparable to the endometriosis (p = 0.1264). In the control group, the number of LSF-immunopositive cells was statistically significantly higher in comparison to endometriosis foci (p = 0.000001), but the number of TSG 101-immunositive cells was comparable to endometriosis foci (p = 0.3834). A weak negative correlation between the number of cells expressing the TSG 101 factor and the IGF-1 receptor was found in the endometriosis group (r = -0.26, p = 0.0196). The analysis of CA single nucleotide polymorphism in the DNA isolated from both groups showed a comparable incidence of MSS and MSI-L genotypes (chi2 p = 0,9160). CONCLUSIONS: How these factors affect the development of endometriosis and whether they could be helpful in the diagnosis requires further research.

Combined Protein and Alkaloid Research of Chelidonium majus Latex Reveals CmMLP1 Accompanied by Alkaloids with Cytotoxic Potential to Human Cervical Carcinoma Cells.

Chelidonium majus L. is a latex-bearing plant used in traditional folk medicine to treat human papillomavirus (HPV)-caused warts, papillae, and condylomas. Its latex and extracts are rich in many low-molecular compounds and proteins, but there is little or no information on their potential interaction. We describe the isolation and identification of a novel major latex protein (CmMLP1) composed of 147 amino acids and present a model of its structure containing a conserved hydrophobic cavity with high affinity to berberine, 8-hydroxycheleritrine, and dihydroberberine. CmMLP1 and the accompanying three alkaloids were present in the eluted chromatographic fractions of latex. They decreased in vitro viability of human cervical cancer cells (HPV-negative and HPV-positive). We combined, for the first time, research on macromolecular and low-molecular-weight compounds of latex-bearing plants in contrast to other studies that investigated proteins and alkaloids separately. The observed interaction between latex protein and alkaloids may influence our knowledge on plant defense. The proposed toolbox may help in further understanding of plant disease resistance and in pharmacological research.

The Influence of Maternal Obesity on Cell-Free Fetal DNA and Blood Pressure Regulation in Pregnancies with Hypertensive Disorders.

Background and Objectives: obesity and blood pressure disorders are one of the main risk factors for antenatal, intra, postpartum, and neonatal complications. In preeclampsia (PE), the placental hypoxia leads to vascular endothelium dysfunction, cell necrosis, and apoptosis. This condition is associated with the release of free fetal DNA (cffDNA) circulating in plasma. The disturbance of the efficiency of vasodilatation and blood pressure regulation in PE can be confirmed by analyzing the apelin, salusin, and prosalusin. This study aimed to assess the influence of obesity on cffDNA, and the effectiveness of maintaining normal blood pressure in patients with preeclampsia and gestational hypertension. Material and Methods: the research material was blood serum and oral mucosa swabs, obtained from 168 patients. Pregnant women were divided into the following: a control group (C)-67 women; a gestational hypertension group (GH)-35 patients; a preeclampsia with obesity group (PE + O) (pre-gravid BMI > 30)-23 patients. The rest were lean preeclamptic women (PE)-66 patients-(pre-gravid BMI < 25 in 43 women). Results: the cffDNA was observed in 1.50% of women in the C group, in 2.45% in the GH group, but in 18.18% of lean patients with preeclampsia. The cffDNA was detected in 58% of obese pregnant women with PE. The greater the placental hypoxia was in preeclampsia, the less efficient the hypotensive mechanisms, according to an analysis of the studied adipokines. The prosalusin concentration was significantly lower in the PE group with cffDNA than in the PE group without it (p = 0.008). Apelin was higher in the PE group with cffDNA (p = 0.006) compared to other groups. The same results were also observed in the subgroup with obesity. Conclusion: in preeclamptic women, obesity seems to act as an additive factor of placental damage by means of the dysregulation of hypotensive mechanisms.

Characterization and expression of a novel thaumatin-like protein (CcTLP1) from papaveraceous plant Corydalis cava.

Thaumatin-like proteins (TLPs, osmotins) form a protein family which shares a significant sequence homology to the sweet-tasting thaumatin from the plant Thaumatococcus daniellii. TLPs are not sweet-tasting and are involved in response to biotic stresses and developmental processes. Recently it has been shown using a proteomic approach that the tuber extract from Corydalis cava (Papaveraceae) contains a TLP protein. The aim of this work was to characterize the structure and expression of TLP from C. cava tubers. The results obtained using a PCR approach with degenerate primers demonstrated a coding sequence of a novel protein, named CcTLP1. It consists of 225 aa, has a predicted molecular weight of 24.2 kDa (NCBI GenBank accession no. KJ513303) and has 16 strictly conserved cysteine residues, which form 8 disulfide bridges and stabilize the 3D structure. CcTLP1 may be classified into class IX of plant TLPs. The highest CcTLP1 expression levels were shown by qPCR in the stem of the plant compared to other organs and in the medium-size plants compared to other growth phases. The results confirm that CcTLP1 is expressed during plant growth and development until flowering, with a possible defensive function against different stress conditions.

Effect of Protoberberine-Rich Fraction of Chelidonium majus L. on Endometriosis Regression.

Endometriosis is a gynecological disease defined by the presence of endometrial tissue outside the uterus. To date, the effective treatment of this disease is still based on invasive surgery or laparoscopy. Chelidonium majus L. (Papaveraceae) belongs to medicinal, latex-bearing plants. Extracts from the plant are a rich source of pharmacologically active agents. Protoberberine compounds derived from C. majus possess anticancer and antiproliferative activities. In the present study of a rat model of endometriosis, we investigated the influence of the plant protoberberine-rich fraction (BBR) obtained from the medicinal plant C. majus on the development of endometriosis. To understand of BBR therapeutic potential for endometriosis, metabolomics has been applied to study. BBR was prepared from an ethanolic extract of dry plants C. majus. Rats (n = 16) with confirmed endometriosis were treated with BBR administered orally (1 g/kg) for 14 days. Blood serum samples were collected from all of the animals and metabolites were studied using the NMR method. The metabolomic pattern was compared before and after the protoberberine treatment. The performed analysis showed significant changes in the concentrations of metabolites that are involved in energy homeostasis, including glucose, glutamine, and lactate. Histopathological studies showed no recurrence of endometriosis loci after treatment with BBR. The results of the study found that BBR treatment prevents the recurrence of endometriosis in rats. Moreover, metabolomics profiling can be applied to better understand the mechanisms of action of these protoberberine secondary plant metabolites. Our findings provide new insights into the pharmaceutical activity of natural protoberberine plant compounds.

Alternations in mitochondrial genome in carcinogenesis of HPV positive cervix.

OBJECTIVE: It is well known that mitochondrial dysfunctions are involved in tumorigenesis. A special interest of scientists is mitochondrial ND1 gene (mtND1). Recently detected mutations in the mtND1 can disrupt the normal activity of complex I and affect oxidative phosphorylation, thus leading to increase reactive oxygen species production. This study was undertaken to determine the alternations in the mtND1 and evaluate their association with development of precancerous lesions and cervical cancer. METHODS: In the study 29 cervical cancer, 28 low grade squamous intraepithelial lesion (L-SIL) and 30 high grade squamous intraepithelial lesion (H-SIL) HPV positive fragments tissue were screened for the presence of mtND1 mutations. RESULTS: Our study showed that mutations in the mtND1 gene were detected in patients with precancerous stage, as well as cervical cancer. We have identified 12 point mutations in 116 analyzed precancerous and cancer samples HPV positive. Most detected missense mutations were previously described, except one (p. M156K) with Grantham value 95. The lower expression of mRNA ND1 was detected in cervical cancer cases and in all samples in which mtND1 mutations were identified. Our analyses revealed that level of ROS production was higher in cervical cancer tissues and all cases characterized by mtND1 mutations. CONCLUSIONS: We hypothesize that mutations in MT-ND1 observed in H-SIL and cancer could activate cervical carcinogenesis by increased ROS production.

Antiviral activity of berberine.

Plants are a rich source of new antiviral, pharmacologically active agents. The naturally occurring plant alkaloid berberine (BBR) is one of the phytochemicals with a broad range of biological activity, including anticancer, anti-inflammatory and antiviral activity. BBR targets different steps in the viral life cycle and is thus a good candidate for use in novel antiviral drugs and therapies. It has been shown that BBR reduces virus replication and targets specific interactions between the virus and its host. BBR intercalates into DNA and inhibits DNA synthesis and reverse transcriptase activity. It inhibits replication of herpes simplex virus (HSV), human cytomegalovirus (HCMV), human papillomavirus (HPV), and human immunodeficiency virus (HIV). This isoquinoline alkaloid has the ability to regulate the MEK-ERK, AMPK/mTOR, and NF-κB signaling pathways, which are necessary for viral replication. Furthermore, it has been reported that BBR supports the host immune response, thus leading to viral clearance. In this short review, we focus on the most recent studies on the antiviral properties of berberine and its derivatives, which might be promising agents to be considered in future studies in the fight against the current pandemic SARS-CoV-2, the virus that causes COVID-19.

Antiviral compounds isolated from plants / Przeciwwirusowe zwiazki izolowane z roslin.

Viruses are intracellular pathogens which utilize a number of host metabolic processes for virus replication in addition to proteins which are encoded for virus itself. Therefore, an effective antiviral drug must interfere with virus encoded proteins without affecting any cellular metabolic processes. Unfortunately, many antiviral drugs that have an inhibitory effect on virus replication, also have an inhibitory effect on molecular processes in infected, as well as uninfected, cells. There is currently no approved remedy for many viruses. Plants represent a large potential source of antiviral agents, such as: alkaloids, flavonoids, phenolic acids, phenylpropanoids, lignins, terpenoids, quinine, tannins, thiophenes, polyacetylenes or proteins. Some of them possess broad spectrum of antiviral activity.

Oncogenic viruses and cancer / Wirusy onkogenne a nowotwory.

Oncogenic viruses (oncoviruses) are implicated in approximately 12% of all human cancers. Currently, the viruses known to cause human cancer are: Hepatitis B and C viruses (HBV and HCV), Human Papillomaviruses (HPV), Merkel Cell Polyomavirus (MCV), Human Herpesvirus-8 (HHV-8), Epstein-Barr Virus (EBV) and Human T-cell lymphotropic virus-1 (HTLV-1). However, oncoviruses are not complete carcinogens, need additional factors andisplay different roles in transformation. Oncoviruses can directly disrupt important regulatory cell genes by inserting virus genom into the DNA of the host cell. They also contain their own genes that damage the regulation of the cell. Some viruses have v-onc that cause disregulation of cellular processes and can lead to cancerous growth.

Co-occurrence of human papillomavirus (HPV) in newborns and their parents.

BACKGROUND: The potential HPV transmission route includes horizontal transmission "in utero" and vertical transmission from parents. Less is known about the role of child's father as a potential source of HPV infection and involved in the pathogen's epidemic chain. A possible consequence of perinatal infection includes HPV-related childhood diseases and carrying the risk of cervical cancer development in female offspring. In view of the evidence, studies of HPV co-occurrence in one or both parents and their offspring seem vital for the implementation of respective preventive measures. Consequently, the aim of this study was to determine the incidence of common HPV 16/18 infections in newborns and their parents, and to assess its role of the periconceptional transmission. METHODS: To determine the incidence of common HPV infections in newborns from single pregnancies and their parents. The study included 146 pregnant women, as well as their partners, and newborns. They were tested for the presence of HPV 16/18 DNA using the PCR method. HPV types 16 and/or 18 were identified using type-specific PCR primers. The quality of the extracted DNA was evaluated by PCR using PC03/PC04 ß-globin-specific primers. The relationship between the presence of neonatal and parental HPV infection was analyzed using a multivariable regression model. Calculations were carried out with the Statistica 10. RESULTS: The presence of HPV DNA was detected in 19 (13,01%) newborns, 28 (19,18%) mothers, and 20 (13,7%) fathers. The viral DNA was detected in 14 newborns delivered by HPV-positive mothers (OR = 26,08; CI: 8,07-84,31, p < 0.001), 12 descendants of HPV-positive fathers (OR = 22,13; CI: 6,97-70,27, p < 0.001), and 10 children originating from two infected parents (OR = 24,20; CI: 6,84-85,57 p < 0.001). Those findings points to a increase risk of an acquired infection in newborns with HPV-positive parents. CONCLUSION: Our findings suggest the possible role of the periconceptional transmission in the mode of acquired HPV 16/18 infections.

Protoberberine compounds extracted from Chelidonium majus L. as novel natural photosensitizers for cancer therapy.

BACKGROUND: It has been shown that secondary metabolites occur in Chelidonium majus L. (C. majus) crude extract and milky sap (alkaloids such as berberine, coptisine, chelidonine, chelerythrine, sanguinarine, and protopine) are biologically active compounds with a wide spectrum of pharmacological functions. Berberine, an isoquinoline alkaloid extracted from plants, possesses a wide range of biological activities, including inhibition of growth of a variety of cancer cell lines. PURPOSE AND STUDY DESIGN: In the present study, we investigated the potential anticancer effect of a protoberberine alkaloidal fraction (BBR-F) isolated from the medicinal plant C. majus on HeLa and C33A cervical cancer cells after light irradiation (PDT treatment). METHODS: BBR-F was prepared from an ethanolic extract of stems of C. majus. Identification of alkaloidal compounds was performed using high-performance liquid chromatography - mass spectrometry (HPLC/ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. BBR-F was then biologically evaluated for its anticancer properties. Cytotoxic activity after PDT treatment and without light irradiation (dark cytotoxicity) was determined by colorimetric WST-1 assay. The impact of the protoberberine alkaloidal fraction on the morphology and function of the cells was assessed by fluorescence and confocal microscopy as well as by flow cytometric analysis. To investigate the proinflammatory effect of the extracted natural BBR-F, nitric oxide concentration was determined using the Griess method. RESULTS: An effective reduction in HeLa and C33A cell viability was observed after PDT treatment of BBR-F treated cells. Furthermore, microscopic analysis identified various morphological changes in the studied cells that occurred during apoptosis. Apoptosis of HeLa and C33A cells was also characterized by biochemical changes in cell membrane composition, activation of intracellular caspases, disruption of the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation. CONCLUSION: Our results strongly suggest that the components of the natural plant protoberberine fraction (BBR-F) extracted from C. majus may represent promising novel photosensitive agents and can be applied in cancer photodynamic therapy as natural photosensitizers.

Microbiota dysbiosis is associated with HPV-induced cervical carcinogenesis.

Cervical microbial communities serve a crucial role in the persistence and development of oncogenic human papilloma virus (HPV) infections. In the present study, the authors hypothesised that disturbed heterogeneity of microbial flora was associated with HPV-induced carcinogenesis. Swabs of the cervical microbiota were collected from 250 women and the 16S ribosomal DNA was sequenced using a high throughput assay. The swabs of cervical microbiota were grouped according to the community state types (CSTs) as follows: Healthy cervical swabs; swabs taken from low-grade squamous intra-epithelial lesions (LSIL) and swabs taken from high-grade squamous intra-epithelial lesions (HSIL). Analysis of the bacterial classes revealed that the CST cervical swabs of the volunteers were characterised by Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis, however, Gardnerella vaginalis and Lactobacillus acidophilus were absent. In the CST of patients with LSIL the predominant type of bacteria was Lactobacillus acidophilus and Lactobacillus iners, however Lactobacillus crispatus was not detected. Swabs from CST women diagnosed with HSIL exhibited abundant Gardnerella vaginalis and Lactobacillus acidophilus, however, lacked Lactobacillus taiwanensis, Lactobacillus iners and Lactobacillus crispatus. The abundance of Lactobacillus acidophilus in swabs from the healthy women was compared with the swabs from the women with LSIL. The results of the present study indicated that the development of HPV-induced cancer is associated with a high diversity of vaginal microbiota, which is involved in the control of viral persistence, and is therefore indicative of disease prognosis.

Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses.

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

Isolation and characterization of a non-specific lipid transfer protein from Chelidonium majus L. latex.

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins, which mostly play a role in intracellular lipid transport and antimicrobial defense. Recently it was shown using shotgun proteomic approach that the whole plant extract of Chelidonium majus L. (Papaveraceae) contains relatively abundant nsLTPs. Therefore the aim of the work was to isolate and characterize nsLTP from C. majus latex. Results obtained using PCR approach with degenerate primers showed the presence of nsLTP protein in C. majus root latex, named CmLTP 9.5. The protein consists of 93 aa with a molecular weight of 9.5kDa (NCBI GenBank accession no. ALT21495, coded by KP733898). The mature form of CmLTP 9.5 has a molecular weight of 7.147kDa and contains typical eight strictly conserved cysteine residues. A 3D model of CmLTP 9.5 displays a hydrophobic cavity. The isolated protein fraction tested using diffusion method and critical dilution assay showed strong antibacterial activity towards Gram-negative Campylobacter jejuni as well as Gram-positive Listeria greyi and Clostridium perfringens. Further studies using protein expression system are required to fully understand CmLTP 9.5 mode of action.