Interplay between the RNA Chaperone Hfq, Small RNAs and Transcriptional Regulator OmpR Modulates Iron Homeostasis in the Enteropathogen Yersinia enterocolitica.

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica.

Videodermoscopy in the Assessment of Patients With Ocular Demodicosis.

INTRODUCTION: There is growing evidence of the potential uses of dermoscopy in diagnostics of demodicosis. No previous studies have analyzed dermoscopic features in patients with ocular demodicosis. OBJECTIVES: To evaluate the potential usefulness of videodermoscopy in diagnostics of ocular demodicosis. METHODS: It was a single-center prospective observational study in which results of videodermoscopic examination of the eyelids were compared to the results of classic microscopic examination in patients with suspected ocular demodicosis and healthy volunteers. RESULTS: Study group included 16 women and 15 men. In fifteen (48.4%) patients, microbiological examination of epilated eyelashes was positive. The results of forms filled by the patients concerning known subjective clinical symptoms of ocular demodicosis revealed no significant differences between the group with positive and negative results of microscopic examination. The presence of Demodex tails and madarosis observed during dermoscopic assessment correlated positively with positive results of microscopic examination. At least one Demodex tail was found in 86.7% (13/15) cases with positive results of microscopic examination. In the two remaining cases microscopic evaluation showed the presence of Demodex brevis. In 37.5% (6/16) of patients with negative results of microscopic examination, videodermoscopy showed the presence of Demodex tails. CONCLUSIONS: Videodermoscopy may facilitate the diagnostics of ocular demodicosis. Patients reporting clinical symptoms suggesting ocular demodicosis but negative results of videodermoscopic examination should be referred to classical microscopic examination to exclude the presence of Demodex brevis. In patients with negative microscopic examination results and symptoms suggesting ocular demodicosis, dermoscopy-guided microscopic re-evaluation could be considered.

Inactivation of lmo0946 (sif) induces the SOS response and MGEs mobilization and silences the general stress response and virulence program in Listeria monocytogenes.

Bacteria have evolved numerous regulatory pathways to survive in changing environments. The SOS response is an inducible DNA damage repair system that plays an indispensable role in bacterial adaptation and pathogenesis. Here we report a discovery of the previously uncharacterized protein Lmo0946 as an SOS response interfering factor (Sif) in the human pathogen Listeria monocytogenes. Functional genetic studies demonstrated that sif is indispensable for normal growth of L. monocytogenes in stress-free as well as multi-stress conditions, and sif contributes to susceptibility to ß-lactam antibiotics, biofilm formation and virulence. Absence of Sif promoted the SOS response and elevated expression of mobilome genes accompanied by mobilization of the A118 prophage and ICELm-1 mobile genetic elements (MGEs). These changes were found to be associated with decreased expression of general stress response genes from the σB regulon as well as virulence genes, including the PrfA regulon. Together, this study uncovers an unexpected role of a previously uncharacterized factor, Sif, as an inhibitor of the SOS response in L. monocytogenes.

Dermoscopic features of eyelid margin tumors: A single-center retrospective study.

Despite rapid growth in the significance of dermoscopy in dermatological oncology, relatively little is yet known about the dermoscopic patterns of eyelid margin tumors. The aim of the study was to analyze the dermoscopic features of eyelid margin tumors. This was a retrospective, single-center, consecutive study which included clinical and dermoscopic analysis of eyelid margin tumors diagnosed at the Department of Dermatology, Venereology and Allergology at the Medical University of Gdansk from 1 June 2016 to 31 December 2020. Dermoscopic features significantly more prevalent in malignant non-melanocytic lesions compared to benign ones were alteration in eyelash growth, structureless pink areas, starry milia-like cysts, and perpendicular vessels. In contrast, there were no dermoscopic features that occurred significantly more frequently in malignant melanocytic lesions when compared to benign ones. Basal cell carcinoma, in comparison to hidrocystoma, more commonly presented with ulceration and structureless pink areas. The main features differentiating basal cell carcinoma from dermal nevus were the presence of ulceration, alteration in eyelash growth, structureless pink and structureless white areas, and perpendicular vessels within the tumor with each of these features observed more commonly in basal cell carcinoma. Blue nevus, hemangioma, or pigmented hidrocystoma presenting exclusively with blue structureless areas may be difficult to differentiate based on dermoscopy. The study offers additional dermoscopic clues in the assessment of eyelid margin tumors. Some observations reported previously to be typical of basal cell carcinoma (e.g., linear vessels arranged perpendicularly to the eyelid margin) were documented also within the normal eyelid margin accompanying other cases, and according to our study, cannot be useful as a pathognomonic feature. In contrast, it seems that yellow structures (half-moon sign, starry milia-like cysts) may be important dermoscopic features, though further studies are needed to confirm our observations.

The Regulatory Circuit Underlying Downregulation of a Type III Secretion System in Yersinia enterocolitica by Transcription Factor OmpR.

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS.

The Regulator OmpR in Yersinia enterocolitica Participates in Iron Homeostasis by Modulating Fur Level and Affecting the Expression of Genes Involved in Iron Uptake.

In this study, we found that the loss of OmpR, the response regulator of the two-component EnvZ/OmpR system, increases the cellular level of Fur, the master regulator of iron homeostasis in Y. enterocolitica. Furthermore, we demonstrated that transcription of the fur gene from the YePfur promoter is subject to negative OmpR-dependent regulation. Four putative OmpR-binding sites (OBSs) were indicated by in silico analysis of the fur promoter region, and their removal affected OmpR-dependent fur expression. Moreover, OmpR binds specifically to the predicted OBSs which exhibit a distinct hierarchy of binding affinity. Finally, the data demonstrate that OmpR, by direct binding to the promoters of the fecA, fepA and feoA genes, involved in the iron transport and being under Fur repressor activity, modulates their expression. It seems that the negative effect of OmpR on fecA and fepA transcription is sufficient to counteract the indirect, positive effect of OmpR resulting from decreasing the Fur repressor level. The expression of feoA was positively regulated by OmpR and this mode of action seems to be direct and indirect. Together, the expression of fecA, fepA and feoA in Y. enterocolitica has been proposed to be under a complex mode of regulation involving OmpR and Fur regulators.

Urease Expression in Pathogenic Yersinia enterocolitica Strains of Bio-Serotypes 2/O:9 and 1B/O:8 Is Differentially Regulated by the OmpR Regulator.

Yersinia enterocolitica exhibits a dual lifestyle, existing as both a saprophyte and a pathogen colonizing different niches within a host organism. OmpR has been recognized as a regulator that controls the expression of genes involved in many different cellular processes and the virulence of pathogenic bacteria. Here, we have examined the influence of OmpR and varying temperature (26°C vs. 37°C) on the cytoplasmic proteome of Y. enterocolitica Ye9N (bio-serotype 2/O:9, low pathogenicity). Differential label-free quantitative proteomic analysis indicated that OmpR affects the cellular abundance of a number of proteins including subunits of urease, an enzyme that plays a significant role in acid tolerance and the pathogenicity of Y. enterocolitica. The impact of OmpR on the expression of urease under different growth conditions was studied in more detail by comparing urease activity and the transcription of ure genes in Y. enterocolitica strains Ye9N and Ye8N (highly pathogenic bio-serotype 1B/O:8). Urease expression was higher in strain Ye9N than in Ye8N and in cells grown at 26°C compared to 37°C. However, low pH, high osmolarity and the presence of urea did not have a clear effect on urease expression in either strain. Further analysis showed that OmpR participates in the positive regulation of three transcriptional units encoding the multi-subunit urease (ureABC, ureEF, and ureGD) in strain Ye9N, but this was not the case in strain Ye8N. Binding of OmpR to the ureABC and ureEF promoter regions was confirmed using an electrophoretic mobility shift assay, suggesting that this factor plays a direct role in regulating the transcription of these operons. In addition, we determined that OmpR modulates the expression of a ureR-like gene encoding a putative regulator of the ure gene cluster, but in the opposite manner, i.e., positively in Ye9N and negatively in Ye8N. These findings provide some novel insights into the function of OmpR in adaptation strategies of Y. enterocolitica.

Clinical manifestations of Wilson disease in organs other than the liver and brain.

Wilson disease (WD) is an inherited genetic disorder that is caused by copper metabolism disturbances with main hepatic, neurological, and psychiatric presentation. Deposits of copper accumulate in different organs and may cause a broad range of clinical manifestations. Patients with WD may present with ophthalmological symptoms, or renal, cardiac and osteoarticular involvement. The most common ophthalmological sign as a result of copper accumulation is the Kayser-Fleischer corneal ring, whereas sunflower cataracts are observed rarely. Retinal degeneration, present in WD patients, may serve as a marker of neurodegeneration. Osteoarticular involvement is quite common and includes osteopenia, osteoporosis and arthropathy, which may lead to bone fractures and joint problems mainly affecting knees and wrists. Renal disturbances include tubular dysfunction and renal calculi. A recent cardiac study has shown a higher risk of atrial fibrillation and heart failure in WD patients than in non-WD patients. Autonomic system dysfunction is also observed, but involvement is subclinical in most cases. Another manifestation of WD concerns endocrine system disturbances, which can lead to recurrent abortions, infertility, growth disruption, and parathyroid failure. However, it is possible to become pregnant for females with mild WD symptoms and for those who are compliant with therapy. Hematologic disturbances are frequent and may include acute hemolytic anemia, leucopenia, anemia and low platelet count. Other observed symptoms include lipomas and characteristic of WD skin changes like hyperpigmentation of the legs, xerosis or azure lunulae of the nails. In this paper, we present some of the less common, but nevertheless, important manifestations of WD.

Difficulties in diagnosis and treatment of Wilson disease-a case series of five patients.

Wilson disease (WD) may present symptomatically at any age. There is great variability in the neurological symptoms present, in the clinical state of WD patients, and in the response to decoppering therapy. Early diagnosis and compliance with anti-copper therapy are essential. Here we present five different WD cases to illustrate different problems encountered during diagnosis and treatment. The first case demonstrates that decoppering therapy may be very effective even with severe neurological symptoms. In addition, we see the importance of family screening, especially among the proband's siblings. Case 2 shows that we must be very careful during diagnosis. In the reported family, WD was diagnosed in the father of the proband although her brother had liver pathology but not caused by WD. Other cases teach us that decoppering therapy with d-penicillamine must be introduced slowly because of the high risk of neurological deterioration, especially in patients with typical WD brain changes even without neurological signs. We also have to consider concomitant therapies in WD patients. Neuroleptics may cause exacerbation and should be used at a low dose and for the shortest period possible. A full consideration for the issues surrounding the diagnosis and treatment of WD can lead to optimised care with reduced risk of progression and disability.

The application of an in vitro micronucleus test in mouse fibroblast L929 cells.

The MNa (in vitro the micronucleus assay) is recommended for studying genotoxicity of chemicals. However, no protocol is currently available for experiments with mouse fibroblast L929 cells. The aim of this study was to improve the scope of CBMNb (cytokinesis-block micronucleus) test. Optimization consisted of: selection of a non-cytotoxic concentration of cytokinesis blocker - cytoBc (cytochalasin B) and type and definition of the positive controls, verification of the efficacy of phenobarbital/5,6-benzoflavone as an S9 enzyme inducer as well as the identification of an optimal staining method. The compounds were tested in three exposure regimens: 6 h exposure with S9 activation followed by a 24 h recovery period, 6 h exposure followed by a 24 h recovery without metabolic activation of S9 and 30 h continuous exposure without S9. Different parameters, such as internal and interlaboratory reproducibility were investigated and criteria for test correctness were proposed. Higher MN rates were achieved using 1 µg/mL cytoBc as a cytokinesis blocker, and MMSd (methyl methanesulfonate), (250 µM), Cole (colchicine), (0.5 µM) and CPf (cyclophosphamide), (30 µM) as positive controls. In regard to the recommended S9 inducer, phenobarbital/5,6-benzoflavone was more effective as Aroclor 1254. Giemsa and acridine orange stains were optimal for the evaluation of MN formation. The protocol described in this study with L929 cells produced the reliable results and is suitable for performing the CBMNb experiments according to the current OECD Guideline #487.

OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9.

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem-1 and weaker phem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.

The Role of OmpR in the Expression of Genes of the KdgR Regulon Involved in the Uptake and Depolymerization of Oligogalacturonides in Yersinia enterocolitica.

Oligogalacturonide (OGA)-specific porins of the KdgM family have previously been identified and characterized in enterobacterial plant pathogens. We found that deletion of the gene encoding response regulator OmpR causes the porin KdgM2 to become one of the most abundant proteins in the outer membrane of the human enteropathogen Yersinia enterocolitica. Reporter gene fusion and real-time PCR analysis confirmed that the expression of kdgM2 is repressed by OmpR. We also found that kdgM2 expression is subject to negative regulation by KdgR, a specific repressor of genes involved in the uptake and metabolism of pectin derivatives in plant pathogens. The additive effect of kdgR and ompR mutations suggested that KdgR and OmpR regulate kdgM2 expression independently. We confirmed that kdgM2 occurs in an operon with the pelP gene, encoding the periplasmic pectate lyase PelP. A pectinolytic assay showed strong upregulation of PelP production/activity in a Y. enterocolitica strain lacking OmpR and KdgR, which corroborates the repression exerted by these regulators on kdgM2. In addition, our data showed that OmpR is responsible for up regulation of the kdgM1 gene encoding the second specific oligogalacturonide porin KdgM1. This indicates the involvement of OmpR in the reciprocal regulation of both KdgM1 and KdgM2. Moreover, we demonstrated the negative impact of OmpR on kdgR transcription, which might positively affect the expression of genes of the KdgR regulon. Binding of OmpR to the promoter regions of the kdgM2-pelP-sghX operon, and kdgM1 and kdgR genes was confirmed using the electrophoretic mobility shift assay, suggesting that OmpR can directly regulate their transcription. We also found that the overexpression of porin KdgM2 increases outer membrane permeability. Thus, OmpR-mediated regulation of the KdgM porins may contribute to the fitness of Y. enterocolitica in particular local environments.

Perception of Lower Urinary Tract Symptoms by psychiatrists in mentally affected patients. / Percepcja objawów z dolnego odcinka dróg moczowych (Lower Urinary Tract Symptoms ­ LUTS) przez lekarzy psychiatrów.

OBJECTIVES: Lower urinary tract symptoms (LUTS) are highly prevalent and costly condition worldwide. Numerous studies have demonstrated their negative impact on health-related quality of life (HRQL), as well as on physical and mental health. The co-existence of LUTS and psychiatric symptoms is common and has been described by psychiatrists, urologists and gynecologists. However, data are lacking regarding the perception of urological symptoms by psychiatrists in their day-to-day clinical practice. METHODS: 31-question survey was designed to learn what is the perception of LUTS among psychiatrists. Survey link was sent by email to all psychiatrists registered to the Polish Association of Psychiatry via the association's email lists. The SurveyMonkey website was used as a platform where responses were collected and stored. RESULTS: 953 physicians completed the questionnaire. Majority of investigated psychiatrists only 'occasionally'ask their patients about voiding dysfunctions. Respondents estimated the frequency of voiding dysfunctions in their patients as 'moderately frequent'with a '10-30%' prevalence. However, discrepancies between different subgroups of psychiatrists have been noted. Furthermore, psychiatrists may not be fully aware of the effects of psychiatric treatment (psychotherapy/pharmacotherapy) on LUTS improvement, as well as possible deteriorations of voiding dysfunctions with psychiatric disorder progression. CONCLUSIONS: This survey showed that the perception of urological symptoms by psychiatrists in their patients may be limited. Therefore, it is necessary to adequately inform and educate psychiatrists in terms of the impact of urological symptoms on patients'management, prognosis and quality of life.

Simultaneous analysis of steviol and steviol glycosides by liquid chromatography with ultraviolet detection on a mixed-mode column: application to Stevia plant material and Stevia-containing dietary supplements.

Simultaneous separation of steviol and steviol glycosides is challenging because of differences in their polarity and chemical structure. In this study, simultaneous analysis of steviol and steviol glycosides was achieved by LC with UV detection using a mixed-mode RP weak anion exchange chromatography column. Steviol and seven steviol glycosides were analyzed on an Acclaim Mixed-Mode Wax-1 (Dionex) column with a linear gradient of deionized water adjusted to pH 3.00 with phosphoric acid and acetonitrile. The extraction was performed by sonicating dry plant material at 40 degreesC in acetonitrile-water (30 + 70, v/v). LOQ values (mg/g dry weight of plant material) were rebaudioside B, 0.50; steviol, 0.70, dulcoside A, 1.0; steviolbioside, 1.2; stevioside and rebaudioside C, 2.0; rebaudioside D, 3.3; and rebaudioside A, 5.0. The method demonstrated suitable performance for all analytes tested with respect to accuracy (mean recoveries 95-99%), intraday and interday precision for retention times (0.070-0.28% and 0.33-1.0% RSD, respectively), and linearity. The method was used to authenticate steviol glycosides in several samples of Stevia plant material as well as to quantitate steviol glycosides in dietary supplements containing Stevia.

Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts.

Stevia rebaudiana extracts and plant materials are increasingly used as natural sweeteners. Polyphenolic and stevioside compounds contained in S. rebaudiana extracts were separated by comprehensive LC. A polyamine column operated in normal phase mode was used for the first dimension separation (D1), and a UHPLC C18 column operated in reversed phase mode was used for the second dimension separation (D2). The sub-2 µm column (2.1 mm × 30 mm, maintained at 70°C) and the UHPLC pump employed for D2 elution allowed a separation/cycle time of 20 s, with a backpressure oscillating between 805 and 922 bar at 3.4 mL/min. The reduced D2 cycle time allowed 3-12 D2 samplings for each peak eluted by D1. Polyphenolic and stevioside compounds were identified by combining the information coming from the position of the compounds in the 2D plot and UV spectra with that of reference materials.