Usher syndrome proteins ADGRV1 (USH2C) and CIB2 (USH1J) interact and share a common interactome containing TRiC/CCT-BBS chaperonins.

The human Usher syndrome (USH) is the most common form of a sensory hereditary ciliopathy characterized by progressive vision and hearing loss. Mutations in the genes ADGRV1 and CIB2 have been associated with two distinct sub-types of USH, namely, USH2C and USH1J. The proteins encoded by the two genes belong to very distinct protein families: the adhesion G protein-coupled receptor ADGRV1 also known as the very large G protein-coupled receptor 1 (VLGR1) and the Ca2+- and integrin-binding protein 2 (CIB2), respectively. In the absence of tangible knowledge of the molecular function of ADGRV1 and CIB2, pathomechanisms underlying USH2C and USH1J are still unknown. Here, we aimed to enlighten the cellular functions of CIB2 and ADGRV1 by the identification of interacting proteins, a knowledge that is commonly indicative of cellular functions. Applying affinity proteomics by tandem affinity purification in combination with mass spectrometry, we identified novel potential binding partners of the CIB2 protein and compared these with the data set we previously obtained for ADGRV1. Surprisingly, the interactomes of both USH proteins showed a high degree of overlap indicating their integration in common networks, cellular pathways and functional modules which we confirmed by GO term analysis. Validation of protein interactions revealed that ADGRV1 and CIB2 mutually interact. In addition, we showed that the USH proteins also interact with the TRiC/CCT chaperonin complex and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Immunohistochemistry on retinal sections demonstrated the co-localization of the interacting partners at the photoreceptor cilia, supporting the role of USH proteins ADGRV1 and CIB2 in primary cilia function. The interconnection of protein networks involved in the pathogenesis of both syndromic retinal dystrophies BBS and USH suggest shared pathomechanisms for both syndromes on the molecular level.

The adhesion G protein-coupled receptor VLGR1/ADGRV1 controls autophagy.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest known adhesion G protein-coupled receptor. Mutations in VLGR1/ADGRV1 cause Usher syndrome (USH), the most common form of hereditary deaf-blindness, and have been additionally linked to epilepsy. Although VLGR1/ADGRV1 is almost ubiquitously expressed, little is known about the subcellular function and signalling of the VLGR1 protein and thus about mechanisms underlying the development of diseases. Using affinity proteomics, we identified key components of autophagosomes as putative interacting proteins of VLGR1. In addition, whole transcriptome sequencing of the retinae of the Vlgr1/del7TM mouse model revealed altered expression profiles of gene-related autophagy. Monitoring autophagy by immunoblotting and immunocytochemistry of the LC3 and p62 as autophagy marker proteins revealed evoked autophagy in VLGR1-deficient hTERT-RPE1 cells and USH2C patient-derived fibroblasts. Our data demonstrate the molecular and functional interaction of VLGR1 with key components of the autophagy process and point to an essential role of VLGR1 in the regulation of autophagy at internal membranes. The close association of VLGR1 with autophagy helps to explain the pathomechanisms underlying human USH and epilepsy related to VLGR1 defects.

Monitoring paxillin in astrocytes reveals the significance of the adhesion G protein coupled receptor VLGR1/ADGRV1 for focal adhesion assembly.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest adhesion G protein-coupled receptor (aGPCR). Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf-blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FAs) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin-DsRed2 consistently showed that FA disassembly was not altered, but de novo assembly of FA was significantly delayed in Vlgr1-deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments, recovery rates were significantly reduced in Vlgr1-deficient FAs, indicating reduced turnover kinetics in VLGR1-deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1.

Abdominal ultrasound findings in adult patients with cystic fibrosis.

INTRODUCTION AND AIMS: Cystic fibrosis (CF) is a genetic disease whose gastrointestinal compromise mainly involves the pancreas, bile ducts, and liver. Our aim was to analyze abdominal ultrasound findings. METHODS: A retrospective, descriptive study was conducted on adults (patients ≥ 16 years of age) diagnosed with CF, within the time frame of 2006-2019. Clinical and genetic parameters, body mass index, forced expiratory volume in one second, pancreatic insufficiency, CF-related diabetes, cirrhosis secondary to CF, and abdominal ultrasound images were analyzed. RESULTS: Seventy patients, 39 of whom were men (55.8%), had a mean age of 27 years and a mean body mass index of 21.3 ± 2.8 kg/m2 (r: 17-30.9). Forty-seven (67.1%) presented with pancreatic insufficiency, 6 (8.5%) with cirrhosis secondary to CF, and 21 (30%) had CF-related diabetes. Median forced expiratory volume in one second was 47% and the F508del mutation was found in 56.1%. Images of the pancreas: no pathologic findings in 49 (70%), increased echogenicity in 18 (25.7%), and cysts in 3 (4.3%). Gallbladder images: microgallbladder in 3 (4.2%), biliary sludge in 2 (2.8%), gallstones in 7 (10%), and a history of cholecystectomy in 4 (5.8%). Liver and spleen images: no pathologic findings in 47 (67.2%), homogeneous hepatomegaly with splenomegaly in 2 (2.8%), a heterogeneous pattern of the parenchyma in 11 (15.8%), increased echogenicity in 4 (5.7%), and heterogeneous echo patterns, lobulated liver contour, and splenomegaly in 6 (8.5%). CONCLUSION: Abdominal ultrasound is a safe, low-cost technique that enables the identification of some degree of chronic liver and pancreatic diseases, improving the approach and follow-up decisions in adult patients with CF.

Affinity Proteomics Identifies Interaction Partners and Defines Novel Insights into the Function of the Adhesion GPCR VLGR1/ADGRV1.

The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer's disease.

Isolation and culturing of primary mouse astrocytes for the analysis of focal adhesion dynamics.

Primary astrocytes have gained attention as an important model for in vitro biological and biochemical research in the last decades. In this protocol, we describe a fast and cost-effective technique for isolating, culturing, and maintaining primary mouse astrocytes at ∼ 80% purity levels, which can be used in in vitro studies for migration and focal adhesion dynamics. In addition, we present an optimized transfection and manual quantification approach for focal adhesion analysis in fixed and living cells. For complete details on the use and execution of this protocol, please refer to Kusuluri et al. (2021).

Adhesion G protein-coupled receptor VLGR1/ADGRV1 regulates cell spreading and migration by mechanosensing at focal adhesions.

VLGR1 (very large G protein-coupled receptor-1) is by far the largest adhesion G protein-coupled receptor in humans. Homozygous pathologic variants of VLGR1 cause hereditary deaf blindness in Usher syndrome 2C and haploinsufficiency of VLGR1 is associated with epilepsy. However, its molecular function remains elusive. Herein, we used affinity proteomics to identify many components of focal adhesions (FAs) in the VLGR1 interactome. VLGR1 is localized in FAs and assembles in FA protein complexes in situ. Depletion or loss of VLGR1 decreases the number and length of FAs in hTERT-RPE1 cells and in astrocytes of Vlgr1 mutant mice. VLGR1 depletion reduces cell spread and migration kinetics as well as the response to mechanical stretch characterizing VLGR1 as a metabotropic mechanosensor in FAs. Our data reveal a critical role of VLGR1 in the FA function and enlighten potential pathomechanisms in diseases related to VLGR1.

Antifungal susceptibility, species distribution and risk factors associated with mortality of invasive candidiasis in children in Turkey: A six-year retrospective, single-centre study.

Invasive candidiasis (IC) is a life-threatening fungal infection with high morbidity and mortality. In this study, we aimed to investigate the Candida species distribution and antifungal drug susceptibility and to identify the risk factors associated with IC mortality in children. We conducted a retrospective, single-centre study of paediatric IC in patients from a tertiary care hospital in Turkey between January 2013 and February 2019. A total of 56 Candida isolates underwent antifungal susceptibility testing performed by Sensititre YeastOne YO10 panel, and the demographic and clinical data of 65 patients were examined during the study period. The most commonly isolated species was Candida albicans in 30 patients (46%), followed by C. parapsilosis in 25 patients (38%) and C. tropicalis in three patients (5%). According to the antifungal drug susceptibility testing, C. albicans was fully susceptible to fluconazole and the other antifungal agents (100%). None of the isolates displayed resistance to anidulafungin, micafungin, flucytosine, posaconazole, voriconazole or itraconazole. There were low rates of resistance to fluconazole (1.8%), caspofungin (1.8%) and micafungin (1.8%). In addition, 5.3% of the Candida isolates were susceptible in a dose-dependent manner to itraconazole, 3.6% were susceptible to voriconazole and fluconazole and 1.8% were susceptible to anidulafungin. The mortality rate of IC was 15.4%. Thrombocytopenia after IC treatment was significantly associated with mortality in the multivariate analysis. These results, which help determine the species distribution, antifungal susceptibility patterns and risk factors for mortality, could make a significant contribution to the management of these challenging infections, including choosing appropriate empirical antifungal therapy.

Spontaneous enanti-omorphism in poly-phased alkaline salts of tris-(oxalato)ferrate(III): crystal structure of cubic NaRb5[Fe(C2O4)3]2.

We show here that the phenomenon of spontaneous resolution of enanti-omers occurs during the crystallization of the sodium and rubidium double salts of the transition metal complex tris-(oxalato)ferrate(III), namely sodium penta-rubidium bis-[tris-(oxalato)ferrate(III)], NaRb5[Fe(C2O4)3]2. One enanti-omer of the salt crystallizes in the cubic space group P4332 with Z = 4 and a Flack absolute structure parameter x = -0.01 (1) and its chiral counterpart in the space group P4132 with x = -0.00 (1). All metal ions are at crystallographic special positions: the iron(III) ion is on a threefold axis, coordinated by three oxalate dianions in a propeller-like conformation. One of the two independent rubidium ions is on a twofold axis in an eightfold coordination with neighbouring oxalate oxygen atoms, and the other one on a threefold axis in a sixfold RbO6 coordination. The sodium ion is at a site of D3 point group symmetry in a trigonal-anti-prismatic NaO6 coordination.

Electronic Cigarettes and Awareness of Their Health Effects.

The use of electronic cigarettes or e-cigarettes is strongly on the rise. The literature confirms that in the process of quitting smoking using an electronic device dispensing nicotine should be a transitional stage before the complete cessation of smoking. The aim of the present study was to assess the popularity of e-cigarettes, the underlying reasons for use of such nicotine products, and the level of awareness of health hazards associated with e-cigarettes. The study is of a survey type. The material consisted of data collected from an anonymous survey distributed among 46 female and 23 male users of e-cigarettes in 2015. We used a questionnaire of our own design. The findings demonstrate that the main reason for a recourse to e-cigarettes is a desire to use fashionable technological innovations, and the conviction that such cigarettes are less harmful than the traditional tobacco products. Some respondents used e-cigarettes to quit smoking; others to minimize the harmful effects of smoking. Most respondents acquired information about e-cigarettes from friends or from the Internet. There was a high awareness of the chemical composition of substances contained in e-cigarettes. An interest in e-cigarettes is caused by an increased knowledge on the negative effects of traditional smoking. Currently, the e-cigarettes remains a technological novelty, so that the exact health effects of their long-term use are open to conjecture.

Insecticidal effects of extracts of Humulus lupulus (hops) L. cones and its principal component, xanthohumol.

Insecticidal effects of the dichloromethane, ethyl acetate, acetone, ethanol and methanol extracts of Humulus lupulus (hops) L. cones and its principal components, xanthohumol was investigated on five stored pests, Sitophilus granarius (L.), Sitophilus oryzae (L.), Acanthoscelides obtectus (Say.), Tribolium castaneum (Herbst) and Lasioderma serricorne (F.). The mortality of adults of the insects treated with 2, 5, 5, 10 and 20 mg mlÌ -1 concentrations of the extracts and xanthuhumol was counted after 24, 48, 72, 96 and 120 h. In order to determine the toxic effects of the substances tested against all tested insects, durations for 50% mortality of the adults, and LD50 values were also determined in the first 48 h by probit analysis. Our results also showed that xanthohumol was more toxic against the pests in comparison with the extracts applications. LD50 values for xanthohumol were found to be low dose as compared with the extracts. Xanthohumol was more toxic against S. granarius (L.) with 6.8 µg of LD50 value. Among the extracts, methanol extract was less effective than other extracts against all tested insects. The ethyl acetate extract of H. lupulus cones was the most effective extract against the tested pests. The quantitative amounts of xanthohumol in the extracts were determined using a high-performance liquid chromatography. The quantitative data indicated that amount of xanthohumol in the extracts increased with increase of polarity of the solvents used from methanol to dichloromethane. The methanol extract contained the high amount of xanthohumol with 5.74 g/100 g extract (0.46 g/100 g plant sample).

Concha Bullosa in Paleoanthropological Material.

Concha bullosa is a variant of the sinonasal anatomy in which the middle nasal turbinate contains pneumatized cells, which leads to turbinate enlargement. The reason for concha bullosa formation is unclear, but the variant is seen in up to half the modern population and it may predispose to paranasal sinusitis. The variant has hitherto featured little in paleopathology. Therefore, in the present study we seek to determine the presence of concha bullosa, with the coexisting hypertrophy of the middle turbinate and signs of sinusitis or other pathology of the paranasal complex, in a population living in Tomersdorf-Toporow in the Upper Lausatia, a historical region in Germany and Poland, presently Zgorzelec County in the Lower Silesian voivodeship, at the turn of the nineteenth and twentieth century. The material consisted of 32 skeletons (24 males, 8 females). The gender, age, and stress indicators and the presence of pathological signs were assessed, followed by CT of the skulls. We found 2 skulls (6.3 %) with concha bullosa. In one case septal nasal deviation was present. We conclude that the incidence of concha bullosa could be lower in the past times than at present. Wider research is necessary to settle whether concha bullosa is indeed a rare respiratory paleopathology or a missed, and thus underreported observation.

Concha Bullosa in Paleoanthropological Material.

Concha bullosa is a variant of the sinonasal anatomy in which the middle nasal turbinate contains pneumatized cells, which leads to turbinate enlargement. The reason for concha bullosa formation is unclear, but the variant is seen in up to half the modern population and it may predispose to paranasal sinusitis. The variant has hitherto featured little in paleopathology. Therefore, in the present study we seek to determine the presence of concha bullosa, with the coexisting hypertrophy of the middle turbinate and signs of sinusitis or other pathology of the paranasal complex, in a population living in Tomersdorf-Toporow in the Upper Lausatia, a historical region in Germany and Poland, presently Zgorzelec County in the Lower Silesian voivodeship, at the turn of the nineteenth and twentieth century. The material consisted of 32 skeletons (24 males, 8 females). The gender, age, and stress indicators and the presence of pathological signs were assessed, followed by CT of the skulls. We found 2 skulls (6.3 %) with concha bullosa. In one case septal nasal deviation was present. We conclude that the incidence of concha bullosa could be lower in the past times than at present. Wider research is necessary to settle whether concha bullosa is indeed a rare respiratory paleopathology or a missed, and thus underreported observation.

Relationships Between Tobacco Abuse and Self-Assessment of Health.

Smoking cigarettes negatively influences the functioning of the body. Among other effects, it has an important impact on the respiratory system, circulation, and behavior. It leads to morphological and physiological changes in organs and tissues, so it can change mood. The aim of this study was to assess the relationships between tobacco abuse and self-assessment of health. The survey was conducted among Polish (243) and foreign (80) medical students at the Pomeranian Medical University in Szczecin, Poland. The study was based on a survey questionnaire of the authors' own design, comprising open and multi-choice questions. Our questionnaire was based on the international standard questionnaire from the Health Behavior in School-Aged Children study (Currie et al. 2009). 80 % of students surveyed were free of any chronic diseases. The results showed that only 23 % of the women and 20 % of the men assessed their health as very good, over 60 % as good, and the remaining at lower levels. We did not observe significant differences between smokers and non-smokers. Physical activity in both groups was generally assessed as good or sufficient. We did not observe significant differences between groups in the incidence of headache, abdominal pain, or vertigo. Significant differences were found in the intake of painkillers.

Diseases of the Upper Respiratory Tract in Preschool and School Age Children in Ambulatory Ear Nose Throat Practice.

The most common diseases of the upper respiratory tract in children treated by ear-nose-throat (ENT) specialists in ambulatory practice are infections, such as colds, rhinitis, sinusitis and pharyngitis, very frequently accompanied and promoted by chronic nasal obstructions of various etiology. These diseases, when treated incorrectly or for too long, cause frequent school absenteeism and may also lead to hearing disorders linked with acute or suppurative otitis. They may also habitually perpetuate abnormal breathing and result in occlusal disorders. The aim of this study was to assess the incidence and type of upper respiratory tract diseases in children, depending on age and sex of patients and on the seasons. We also discussed the role of the ENT specialist in the diagnosis and treatment of certain diseases. In the study we analyzed the medical records of patients of preschool and school age treated in the ENT outpatient clinic over one calendar year. It was found that the largest group of patients comprised children of 3-7 years of age, and most children visited the outpatient clinic in the period March-May. The most common main disorder, according to ICD-10, was acute nasopharyngitis (J00) and vasomotor and allergic rhinitis (J30). Among the comorbid disorders H65 and H66 were the most frequent. No significant gender differences were noted in the frequency of particular types of disease.

Vertigo with a Vestibular Dysfunction in Children During Respiratory Tract Infections.

Sudden balance disorders with violent vegetative symptoms (nausea and vomiting) pose a diagnostic and therapeutic problem. In children vertigo/dizziness with symptoms of vestibular dysfunction is rare, but as vascular etiology is unlikely in children such symptoms arouse concern. This article presents two cases of this type of vertigo. The patients were two boys (6 and 9 years old). They came down with similar symptoms: sudden dizziness, disabled walking, nausea and vomiting, spontaneous nystagmus, and a positive Romberg test. The onset of the balance disorder was preceded by respiratory infection: common cold with symptoms of inflammation of the mucous membrane in the nose and throat. Laboratory tests revealed increased levels of C-reactive protein only in the older boy. Neuroinfection and a displacement process were ruled out. Videonystagmography revealed vestibular dysfunction and vestibular neuronitis on the left side.

Vanadium and cancer treatment: antitumoral mechanisms of three oxidovanadium(IV) complexes on a human osteosarcoma cell line.

We report herein the antitumor actions of three oxidovanadium(IV) complexes on MG-63 human osteosarcoma cell line. The three complexes: VO(oda), VO(oda)bipy and VO(oda)phen (oda=oxodiacetate), caused a concentration dependent inhibition of cell viability. The antiproliferative action of VO(oda)phen could be observed in the whole range of concentrations (at 2.5 µM), while VO(oda)bipy and VO(oda) showed a decrease of cell viability only at higher concentrations (at 50 and 75 µM, respectively) (p<0.01). Moreover, VO(oda)phen caused a decrease of lysosomal and mitochondrial activities at 2.5 µM, while VO(oda) and VO(oda)bipy affected neutral red uptake and mitochondrial metabolism at 50 µM (p<0.01). On the other hand, no DNA damage studied by the Comet assay could be observed in MG-63 cells treated with VO(oda) at 2.5-10 µM. Nevertheless, VO(oda)phen and VO(oda)bipy induced DNA damage at 2.5 and 10 µM, respectively (p<0.01). The generation of reactive oxygen species increased at 10 µM of VO(oda)phen and only at 100 µM of VO(oda) and VO(oda)bipy (p<0.01). Besides, VO(oda)phen and VO(oda)bipy triggered apoptosis as determined by externalization of the phosphatidylserine. The determination of DNA cleavage by agarose gel electrophoresis showed that the ability of VO(oda)(bipy) is similar to that of VO(oda), while VO(oda)(phen) showed the highest nuclease activity in this series. Overall, our results showed a good relationship between the bioactivity of the complexes and their structures since VO(oda)phen presented the most potent antitumor action in human osteosarcoma cells followed by VO(oda)bipy and then by VO(oda) according to the number of intercalating heterocyclic moieties.

Respiratory diseases in the late middle ages.

Respiratory diseases have beset humanity from the earliest times. The aim of this study was to evaluate the prevalence of respiratory diseases in the inhabitants of a large medieval city. We analyzed 110 skeletons, coming from 12 to 17th century of individuals of either gender. In order to assess pneumatisation of paranasal sinuses and nasal septum curvature, radiographs were performed in the PA projection. Nasal septum deviation was found in 50% of individuals, asymmetry of the frontal sinuses and their aplasia in 11% of individuals. There was no significant relationship between the curvature of the nasal septum and frontal sinus aplasia. One case of tuberculosis and one case of periradicular abscess causing perforation of the maxillary sinus were noted. Developmental variation of the sternum was present in two individuals. In another two individuals, rib fractures with bone union were observed. Analysis of the presence of respiratory disease in bone material is particularly difficult, but the use of modern methods: X-rays, CT scans and endoscopy significantly extends the capability to unravel the underlying causes of bygone diseases.

Fabrication of phospholipid-xanthan microcapsules by combining microfluidics with self-assembly.

We report the synthesis of an amphiphilic polysaccharide, a phospholipid (1,2-dioleoyl-sn-glycero-phosphoetilamine, DOPE) conjugated with the anionic xanthan gum, and its ability to spontaneously self-assemble under mild aqueous conditions. This work also aimed to apply a microfluidic platform that can precisely fabricate microsized and monodispersed capsules for cell encapsulation. Stable hollow capsular structures were obtained by the generation of homogeneous spherical droplets of the self-assembled polymer in the microfluidic device through the formation of a water-in-oil emulsion, followed by the stabilization of the polymer aggregates in a separate collection vessel containing phosphate-buffered saline (physiological ionic strength and pH). The properties (size, morphology, permeability) and performance (stability) of the obtained microcapsules were studied, as well their ability to support the viability, function and proliferation of encapsulated cells. ATDC5 cells were encapsulated within the capsules and shown to remain viable, evidencing increased cellular metabolic activity over 21 days of in vitro culture. By combining microfluidic droplet generation and self-assembly of xanthan-DOPE, we were able to fabricate microcapsules that provided an adequate environment for cells to survive and proliferate.