Molecular mapping of virus-infected cells with immunogold and metal-tagging transmission electron microscopy.

Transmission electron microscopy (TEM) has been essential to study virus-cell interactions. The architecture of viral replication factories, the principles of virus assembly and the components of virus egress pathways are known thanks to the contribution of TEM methods. Specially, when studying viruses in cells, methodologies for labeling proteins and other macromolecules are important tools to correlate morphology with function. In this review, we present the most widely used labeling method for TEM, immunogold, together with a lesser known technique, metal-tagging transmission electron microscopy (METTEM) and how they can contribute to study viral infections. Immunogold uses the power of antibodies and electron dense, colloidal gold particles while METTEM uses metallothionein (MT), a metal-binding protein as a clonable tag. MT molecules build gold nano-clusters inside cells when these are incubated with gold salts. We describe the necessary controls to confirm that signals are specific, the advantages and limitations of both methods, and show some examples of immunogold and METTEM of cells infected with viruses.

Rare bleeding disorders: Real-world data from a Spanish tertiary hospital.

INTRODUCTION: Due to their low prevalence, rare bleeding disorders (RBDs) remain poorly characterized. AIM: To gain insight of RBDs through our clinical practice. METHODS: Retrospective study of the medical records of RBD patients followed up at the Central University Hospital of Asturias between January 2019 and December 2022. RESULTS: A total of 149 patients were included. Factor (F) VII (44 %) and FXI (40 %) deficiencies were the most common diagnosed coagulopathies. Most of the patients were asymptomatic (60.4 %) and the most frequent type of bleeding were mucocutaneous and after surgery. All replacement treatments were administered on demand and no patient was on a prophylaxis regimen. Currently available products were safe; allergic reactions after administration of plasma were the most frequent complication. Genetic analysis, carried out on 55 patients (37 %), showed that the most frequent mutations in RBDs are of missense type (71.9 %). We identified 11 different novel genetic alterations in affected genes. The c.802C > T (p.Arg268Cys) variant, previously described, was identified in 71 % (15 of 21) of the patients with FXI deficiency genotyped and none were related (probable founder effect). CONCLUSION: Our study on an unusual large single center cohort of RBD patients portrays location-dependent distinct genetic drives and clinical practice particularities.

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis.

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-ß-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles from endosomes into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Exploring older people's experiences of the interpersonal care relationship between nurses and patients during hospitalization in the pandemic period: A qualitative study.

AIM: This article presents the findings of a qualitative study focusing on the experiences of people aged over 70 years in the interpersonal nurse-patient care relationship in hospital settings during and after the COVID-19 pandemic. The aim was to analyse different aspects of this relationship, including nurses' behaviour and caring attitudes, the person-centred model of care, patients' involvement in their own care and communication in the hospital context. DESIGN: An exploratory qualitative study was conducted on the basis of an interpretative framework. METHODS: Six focus groups were conducted. A total of 34 participants were purposively recruited from three nursing homes in Spain, Italy and Portugal. Specific inclusion criteria were used to select participants. RESULTS: Five main categories were identified. Analysis revealed the importance of empathy and sensitivity in caring relationships, as well as the need for personalized and patient-centred care. The importance of effective communication and recognition of ageist behaviour by professionals was highlighted. In addition, independent of the pandemic, situational factors in the hospital environment were identified that influence the interpersonal care relationship. CONCLUSIONS: The study highlights the need to promote a person-centred model of care that takes into account the specific preferences and needs of older people. This is achieved by identifying elements of the interpersonal nurse-patient relationship. In addition to highlighting the disparity of opinion regarding an active or passive role in self-care and decision making, the importance of addressing ageism and improving communication is emphasized. WHAT PROBLEM DID THE STUDY ADDRESS?: The importance of the patient-nurse relationship in hospital care has been highlighted in previous studies. Critical elements of the patient care experience have been identified as empathy and effective communication. Ageism in healthcare has been recognized as a potential barrier to patient-centred care. WHAT WERE THE KEY FINDINGS?: The main findings highlight the importance of empathy and personalized care, emphasize the importance of effective communication and address ageist behaviours in the nurse-patient relationship. WHERE AND WHO WILL THE RESEARCH IMPACT?: By highlighting the need for person-centred care and improved communication strategies, particularly in the context of caring for older patients during and after the COVID-19 pandemic, this research will have an impact on healthcare professionals, policy makers and carers. REPORTING METHOD: The COREQ guideline was used. PATIENT OR PUBLIC CONTRIBUTION: This study adopted a collaborative approach to ensure that patient perspectives were integrated into the research process. We organized regular focus groups. Patients were actively involved in shaping the research questions, refining the study design and interpreting the emerging findings. Their valuable input helped us to understand the nuances of their experience and to prioritize the aspects that were critical to their well-being. In addition, their insights guided the development of practical recommendations aimed at improving the interpersonal care relationship between nurses and patients in hospital settings, ensuring that their voices were heard and reflected in the proposed interventions. This patient-centred approach fostered a sense of empowerment among the participants. It reinforced the notion that their experiences and opinions are integral to shaping healthcare practice.

hsa-miR-424-5p and hsa-miR-513c-3p dysregulation mediated by IFN-γ is associated with salivary gland dysfunction in Sjögren's syndrome patients.

Salivary secretory dysfunction in SS-patients is associated with altered proteostasis, upregulation of ATF6α and components of the ERAD complex, such as SEL1L, and downregulation of XBP-1s and GRP78. Hsa-miR-424-5p is downregulated and hsa-miR-513c-3p is overexpressed in salivary glands from SS-patients. These miRNAs emerged as candidates that could regulate ATF6/SEL1L and XBP-1s/GRP78 levels, respectively. This study aimed to evaluate the effect of IFN-γ on hsa-miR-424-5p and hsa-miR-513c-3p expression and how these miRNAs regulate their targets. In labial salivary glands (LSG) biopsies from 9 SS-patients and 7 control subjects and IFN-γ-stimulated 3D-acini were analyzed. hsa-miR-424-5p and hsa-miR-513c-3p levels were measured by TaqMan assays and their localization by ISH. mRNA, protein levels, and localization of ATF6, SEL1L, HERP, XBP-1s and GRP78 were determined by qPCR, Western blot, or immunofluorescence. Functional and interaction assays were also performed. In LSGs from SS-patients and IFN-γ-stimulated 3D-acini, hsa-miR-424-5p was downregulated and ATF6α and SEL1L were upregulated. ATF6α and SEL1L were decreased after hsa-miR-424-5p overexpression, while ATF6α, SEL1L and HERP increased after hsa-miR-424-5p silencing. Interaction assays revealed that hsa-miR-424-5p targets ATF6α directly. hsa-miR-513c-3p was upregulated and XBP-1s and GRP78 were downregulated. XBP-1s and GRP78 were decreased after hsa-miR-513c-3p overexpression, while increases in XBP-1s and GRP78 were observed after hsa-miR-513c-3p silencing. Furthermore, we determined that hsa-miR-513c-3p targets XBP-1s directly. Significant correlations were found between both miRNA levels and clinical parameters. In conclusion, IFN-γ-dependent hsa-miR-424-5p and hsa-miR-513c-3p levels affect the expression of important factors involved in cellular proteostasis that control secretory function in LSG from SS-patients.

Clinical and genetic characteristics of Dent's disease type 1 in Europe.

BACKGROUND: Dent's disease type 1 (DD1) is a rare X-linked nephropathy caused by CLCN5 mutations, characterized by proximal tubule dysfunction, including low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis-nephrocalcinosis, progressive chronic kidney disease (CKD) and kidney failure (KF). Current management is symptomatic and does not prevent disease progression. Here we describe the contemporary DD1 picture across Europe to highlight its unmet needs. METHODS: A physician-based anonymous international e-survey supported by several European nephrology networks/societies was conducted. Questions focused on DD1 clinical features, diagnostic procedure and mutation spectra. RESULTS: A total of 207 DD1 male patients were reported; clinical data were available for 163 with confirmed CLCN5 mutations. Proteinuria was the most common manifestation (49.1%). During follow-up, all patients showed LMWP, 66.4% nephrocalcinosis, 44.4% hypercalciuria and 26.4% nephrolithiasis. After 5.5 years, ≈50% of patients presented with renal dysfunction, 20.7% developed CKD stage ≥3 and 11.1% developed KF. At the last visit, hypercalciuria was more frequent in paediatric patients than in adults (73.4% versus 19.0%). Conversely, nephrolithiasis, nephrocalcinosis and renal dysfunction were more prominent in adults. Furthermore, CKD progressed with age. Despite no clear phenotype/genotype correlation, decreased glomerular filtration rate was more frequent in subjects with CLCN5 mutations affecting the pore or CBS domains compared with those with early-stop mutations. CONCLUSIONS: Results from this large DD1 cohort confirm previous findings and provide new insights regarding age and genotype impact on CKD progression. Our data strongly support that DD1 should be considered in male patients with CKD, nephrocalcinosis/hypercalciuria and non-nephrotic proteinuria and provide additional support for new research opportunities.

MCL1 alternative polyadenylation is essential for cell survival and mitochondria morphology.

Alternative polyadenylation in the 3' UTR (3' UTR-APA) is a mode of gene expression regulation, fundamental for mRNA stability, translation and localization. In the immune system, it was shown that upon T cell activation, there is an increase in the relative expression of mRNA isoforms with short 3' UTRs resulting from 3' UTR-APA. However, the functional significance of 3' UTR-APA remains largely unknown. Here, we studied the physiological function of 3' UTR-APA in the regulation of Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic member of the Bcl-2 family essential for T cell survival. We found that T cells produce two MCL1 mRNA isoforms (pA1 and pA2) by 3' UTR-APA. We show that upon T cell activation, there is an increase in both the shorter pA1 mRNA isoform and MCL1 protein levels. Moreover, the less efficiently translated pA2 isoform is downregulated by miR-17, which is also more expressed upon T cell activation. Therefore, by increasing the expression of the more efficiently translated pA1 mRNA isoform, which escapes regulation by miR-17, 3' UTR-APA fine tunes MCL1 protein levels, critical for activated T cells' survival. Furthermore, using CRISPR/Cas9-edited cells, we show that depletion of either pA1 or pA2 mRNA isoforms causes severe defects in mitochondria morphology, increases apoptosis and impacts cell proliferation. Collectively, our results show that MCL1 alternative polyadenylation has a key role in the regulation of MCL1 protein levels upon T cell activation and reveal an essential function for MCL1 3' UTR-APA in cell viability and mitochondria dynamics.

Molecular characterization of waterborne protozoa in surface water and sediment in Brazil: a taxonomic survey of ciliated protozoa and their correlation with Giardia duodenalis and Cryptosporidium spp.

The detection of Giardia duodenalis and Cryptosporidium spp. was performed, along with the identification of the ciliated protozoa biodiversity, to evaluate the correlation between these protozoa in freshwater quality monitoring. Water and sediment samples from two sites in the Atibaia River (Campinas, São Paulo, Brazil) were collected monthly for 2 years (n = 96). Pathogenic protozoa in water and sediment were detected by using immunomagnetic separation, followed by visualization by immunofluorescence assay (IFA). All positive aliquots in IFA were subjected to DNA extraction and subsequently nested PCR. Qualitative (in vivo observation and silver impregnation) and quantitative (in vivo enumeration) analyses were performed for the ciliated protozoa. Giardia cysts were detected in 62.5% of the surface water samples and Cryptosporidium spp. in 25.0%. In the sediment, cysts were detected in 35.4% samples and oocysts in 16.6%. A total of 57 samples positive for Giardia cysts were subjected to sequencing, 40 of which were harboring G. duodenalis (24 were characterized as sub-assemblage AII). For ciliated protozoa, 73 taxa belonging to 53 genera were identified over the period of the study. These results revealed a high degree of contamination by waterborne protozoa in the main water source which supplies drinking water for more than one million people in Campinas (São Paulo), highlighting the need for continuous monitoring of this catchment site. In addition, the present study provides important data regarding the sources of the water body degradation, i.e., fecal contamination of human origin, in addition to the survey of the ciliated protozoa.

Changes in nerve conduction studies predate clinical symptoms onset in early onset Val30Met hereditary ATTR amyloidosis.

BACKGROUND AND PURPOSE: Hereditary amyloidosis related to transthyretin (ATTR) is a rare and progressive disease that, despite the phenotypic heterogeneity, a length-dependent sensorimotor axonal neuropathy (ATTR-PN) is the classic hallmark. Timely diagnosis is paramount for early treatment implementation. METHODS: Sixty-nine asymptomatic gene carriers (Val30Met) were assessed during a 4-year period to identify those remaining asymptomatic versus those converting to ATTRV30M-PN. Conversion to symptomatic was defined as presenting with two definite symptoms of ATTRV30M-PN. Composite neurophysiological scores of sensory (SNS), motor (MNS), and sympathetic skin response (SSRS) amplitudes were used to assess neuropathy progression. We used mixed-effects modeling and ordinal logistic regression to assess neurophysiological evolution over time. RESULTS: Of all asymptomatic gene carriers, 55.1% (n = 38/69) converted over the period of this analysis. The progression of the SNS relative to baseline was different between groups (asymptomatic gene carriers vs. converters), the decline being greater in the converter group (time × group interaction p = 0.040), starting about 2 years before symptom onset. No significant change occurred regarding MNS or SSRS. Moreover, the percentage of cases with an annual decline on the SNS of at least 25%, gradually and significantly increased in the converter group, representing a 1.92 increase in risk of developing symptoms for those with such reduction on the last evaluation. CONCLUSIONS: A simple composite neurophysiological sum score can predict the onset of ATTRV30M-PN symptoms by as much as 2 years, highlighting the importance of a systematic follow-up of asymptomatic gene carriers, allowing a timely diagnosis, and management of symptomatic disease.

Tofacitinib counteracts IL-6 overexpression induced by deficient autophagy: implications in Sjögren's syndrome.

OBJECTIVE: Altered homeostasis of salivary gland (SG) epithelial cells in Sjögren's syndrome (SS) could be the initiating factor that leads to inflammation, secretory dysfunction and autoimmunity. Autophagy is an important homeostatic mechanism, whose deficiency is associated with inflammation and accumulation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) components. We aimed to evaluate whether autophagy is altered in labial SG (LSG) epithelial cells from primary SS (pSS) patients and whether this contributes to inflammation through the JAK-STAT pathway. Furthermore, we investigated the anti-inflammatory effect of the JAK inhibitor tofacitinib in autophagy-deficient (ATG5 knockdown) three-dimensional (3D)-acini. METHODS: We analysed LSG biopsies from 12 pSS patients with low focus score and 10 controls. ATG5-deficient 3D-acini were generated and incubated with IL-6 in the presence or absence of tofacitinib. Autophagy markers, pro-inflammatory cytokine expression, and JAK-STAT pathway activation were evaluated by PCR or western blot, along with correlation analyses between the evaluated markers and clinical parameters. RESULTS: LSG from pSS patients showed increased p62 and decreased ATG5 expression, correlating negatively with increased activation of JAK-STAT pathway components (pSTAT1 and pSTAT3). Increased expression of STAT1 and IL-6 correlated with EULAR Sjögren's syndrome disease activity index and the presence of anti-Ro antibodies. ATG5-deficient 3D-acini reproduced the findings observed in LSG from pSS patients, showing increased expression of pro-inflammatory markers such as IL-6, which was reversed by tofacitinib. CONCLUSION: Decreased expression of ATG5 in LSG epithelial cells from pSS patients possibly contributes to increased inflammation associated with JAK-STAT pathway activation, as evidenced in ATG5-deficient 3D-acini. Interestingly, these results suggest that tofacitinib could be used as an anti-inflammatory agent in pSS patients.

A Gadolinium(III) Complex Based on the Thymine Nucleobase with Properties Suitable for Magnetic Resonance Imaging.

The paramagnetic gadolinium(III) ion is used as contrast agent in magnetic resonance (MR) imaging to improve the lesion detection and characterization. It generates a signal by changing the relaxivity of protons from associated water molecules and creates a clearer physical distinction between the molecule and the surrounding tissues. New gadolinium-based contrast agents displaying larger relaxivity values and specifically targeted might provide higher resolution and better functional images. We have synthesized the gadolinium(III) complex of formula [Gd(thy)2(H2O)6](ClO4)3·2H2O (1) [thy = 5-methyl-1H-pyrimidine-2,4-dione or thymine], which is the first reported compound based on gadolinium and thymine nucleobase. 1 has been characterized through UV-vis, IR, SEM-EDAX, and single-crystal X-ray diffraction techniques, and its magnetic and relaxometric properties have been investigated by means of SQUID magnetometer and MR imaging phantom studies, respectively. On the basis of its high relaxivity values, this gadolinium(III) complex can be considered a suitable candidate for contrast-enhanced magnetic resonance imaging.

Ferro- and Antiferromagnetic Interactions in Oxalato-Centered Inverse Hexanuclear and Chain Copper(II) Complexes with Pyrazole Derivatives.

Two novel copper(II) complexes of formulas {[Cu(4-Hmpz)4][Cu(4-Hmpz)2(µ3-ox-κ2O1,O2:κO2':κO1')(ClO4)2]}n (1) and {[Cu(3,4,5-Htmpz)4]2[Cu(3,4,5-Htmpz)2(µ3-ox-κ2O1,O2:κO2':κO1')(H2O)(ClO4)]2[Cu2(3,4,5-Htmpz)4(µ-ox-κ2O1,O2:κ2O2',O1')]}(ClO4)4·6H2O (2) have been obtained by using 4-methyl-1H-pyrazole (4-Hmpz) and 3,4,5-trimethyl-1H-pyrazole (3,4,5-Htmpz) as terminal ligands and oxalate (ox) as the polyatomic inverse coordination center. The crystal structure of 1 consists of perchlorate counteranions and cationic copper(II) chains with alternating bis(pyrazole)(µ3-κ2O1,O2:κO2':κO1'-oxalato)copper(II) and tetrakis(pyrazole)copper(II) fragments. The crystal structure of 2 is made up of perchlorate counteranions and cationic centrosymmetric hexanuclear complexes where an inner tetrakis(pyrazole)(µ-κ2O1,O2:κ2O2',O1'-oxalato)dicopper(II) entity and two outer mononuclear tetrakis(pyrazole)copper(II) units are linked through two mononuclear aquabis(pyrazole)(µ3-κ2O1,O2:κO2':κO1'-oxalato)copper(II) units. The magnetic properties of 1 and 2 were investigated in the temperature range 2.0-300 K. Very weak intrachain antiferromagnetic interactions between the copper(II) ions through the µ3-ox-κ2O1,O2:κO2':κO1' center occur in 1 [J = -0.42(1) cm-1, the spin Hamiltonian being defined as H = -J∑S1,i · S2,i+1], whereas very weak intramolecular ferromagnetic [J = +0.28(2) cm-1] and strong antiferromagnetic [J' = -348(2) cm-1] couplings coexist in 2 which are mediated by the µ3-ox-κ2O1,O2:κO2':κO1' and µ-ox-κ2O1,O2:κ2O2',O1' centers, respectively. The variation in the nature and magnitude of the magnetic coupling for this pair of oxalato-centered inverse copper(II) complexes is discussed in the light of their different structural features, and a comparison with related oxalato-centered inverse copper(II)-pyrazole systems from the literature is carried out.

Aberrant MUC1 accumulation in salivary glands of Sjögren's syndrome patients is reversed by TUDCA in vitro.

OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.

Framing the process in the implementation of care for people with generalized anxiety disorder in primary care: a qualitative evidence synthesis.

BACKGROUND: Generalized anxiety disorder (GAD) is one of the most common mental disorders in primary care (PC). GAD has low remission and high relapse rates over long follow-up periods. Qualitative evidence was synthesized to understand the implementation of care and treatment options for people with GAD in PC. METHODS: Research published from 2008 to September 2020 was searched in five databases (MEDLINE, EMBASE, CINAHL, WOS and PsycArticles). Studies that used qualitative methods for data collection and analysis to investigate the implementation of care and treatment options for people with GAD in PC and outpatient settings were included. Non-qualitative studies, mixed methods studies that did not separately report qualitative findings and studies in languages other than English or Spanish were excluded. We used the Confidence in the Evidence from Reviews of Qualitative Research (CERQual) framework to assess the overall confidence in the findings. RESULTS: The results with a moderate level of confidence showed that the trajectory of care for people with GAD in PC and outpatient settings is long and fluctuates over time, involving multiple difficulties in accessing and maintaining initial treatment or successive treatment options. In addition, there are wide variations in the preferences for and acceptability of different treatment options. The results with a high level of confidence indicated that more information on GAD and its treatment options is needed for PC practitioners, GAD patients and their carers. The results with a low level of confidence suggested that patients use antidepressants for longer than recommended and that the interruption of treatment is not usually planned. CONCLUSIONS: Initial resistance to new treatments among people with GAD can make access and adherence to treatment difficult. Improving care may require patients to be informed of possible trajectories in stepped care pathways before the initiation of treatment so they are aware that they may need to try a number of options until the most effective treatment for them is found. Increased awareness of and information materials on GAD may facilitate both appropriate diagnosis and long-term care.

Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex.

Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds.

Synaptotagmin-1 overexpression under inflammatory conditions affects secretion in salivary glands from Sjögren's syndrome patients.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy associated with severe secretory alterations by disruption of the glandular architecture integrity, which is fundamental for a correct function and localization of the secretory machinery. Syt-1, PI(4,5)P2 and Ca2+ are significant factors controlling exocytosis in different secretory cells, the Ca2+ role being the most studied. Salivary acinar cells from SS-patients show a defective agonist-regulated intracellular Ca2+ release together with a decreased IP3R expression level, and this condition may explain a reduced water release. However, there are not reports where Syt-1, PI(4,5)P2 and Ca2+ in acinar cells of SS patients had been studied. In the present study, we analyzed the expression and/or localization of Syt-1 and PI(4,5)P2 in acinar cells of labial salivary gland biopsies from SS-patients and control individuals. Also, we evaluated whether the overexpression of Syt-1 and the loss of cell polarity induced by TNF-α or loss of interaction between acinar cell and basal lamina, alters directionality of the exocytosis process, Ca2+ signaling and α-amylase secretion in a 3D-acini model stimulated with cholinergic or ß-adrenergic agonists. In addition, the correlation between Syt-1 protein levels and clinical parameters was evaluated. The results showed an increase of Syt-1 mRNA and protein levels, and a high number of co-localization points of Syt-1/STX4 and PI(4,5)P2/Ezrin in the acinar basolateral region of LSG from SS-patients. With regard to 3D-acini, Syt-1 overexpression increased exocytosis in the apical pole compared to control acini. TNF-α stimulation increased exocytic events in the basal pole, which was further enhanced by Syt-1 overexpression. Additionally, altered acinar cell polarity affected Ca2+ signaling and amylase secretion. Overexpression of Syt-1 was associated with salivary gland alterations revealing that the secretory dysfunction in SS-patients is linked to altered expression and/or localization of secretory machinery components together with impaired epithelial cell polarity. These findings provide a novel insight on the pathological mechanism implicated in ectopic secretory products to the extracellular matrix of LSG from SS-patients, which might initiate inflammation.

How to walk carrying a huge egg? Trade-offs between locomotion and reproduction explain the special pelvis and leg anatomy in kiwi (Aves; Apteryx spp.).

Kiwi (Aves; genus Apteryx) are famous for laying an enormous egg in comparison with their relatively small body size. Considering the peculiar gait of this flightless bird, we suspected the existence of morpho-functional trade-offs between reproduction and locomotion. To understand how structural constraints, imposed by a large egg size, might influence the terrestrial locomotion of Apteryx, we analysed the anatomy of the limb osteomuscular system in two species of kiwi (Apteryx mantelli and Apteryx owenii). We performed detailed dissections and brought to light specific anatomical features of kiwi, in comparison with other ratites and neognathous birds. Our osteological study revealed a strongly curved pelvis, a rigid tail, and enlarged ribs. Our myology study showed an unusual location of the caudofemoralis muscle origin and insertion. The insertion of the pars pelvica along the entire caudal face of the femur, contrasts with the proximal insertion usually seen in other birds. Additionally, the pars caudalis originates along the entire tail, whereas it only inserts on the uropygium in the other birds. To interpret these specificities from a functional point of view, we built three-dimensional osteomuscular models based on computed tomography scans, radiographies and our dissections. We chose three postures associated with reproductive constraints: the standing position of a gravid compared with a non-gravid bird, as well as the brooding position. The 3D model of the brooding position suggested that the enlarged ribs could support the bodyweight when leaning on the huge egg in both males and females. Moreover, we found that in gravid females, the unusual shape of the pelvis and tail allowed the huge egg to sit ventrally below the pelvis, whereas it is held closer to the rachis in other birds. The specific conformation of the limb and the insertions of the two parses of the caudofemoralis help to maintain the tail flexed, and to keep the legs adducted when carrying the egg. The caudal location of the hip and its flexed position explains the long stance phase during the strange gait of kiwi, revealing the functional trade-off between reproduction and locomotion in this emblematic New Zealand bird.

Thymic epithelial cells require p53 to support their long-term function in thymopoiesis in mice.

Thymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.

Coligand Effects on the Field-Induced Double Slow Magnetic Relaxation in Six-Coordinate Cobalt(II) Single-Ion Magnets (SIMs) with Positive Magnetic Anisotropy.

Two mononuclear cobalt(II) compounds of formula [Co(dmphen)2(OOCPh)]ClO4·1/2H2O·1/2CH3OH (1) and [Co(dmbipy)2(OOCPh)]ClO4 (2) (dmphen = 2,9-dimethyl-1,10-phenanthroline, dmbipy = 6,6'-dimethyl-2,2'-bipyridine and HOOCPh = benzoic acid) are prepared and magnetostructurally investigated. Each cobalt(II) ion is six-coordinate with a distorted octahedral CoN4O2 environment. The complex cations are interlinked leading to supramolecular chains (1) and pairs (2) that grow along the crystallographic c-axis with racemic mixtures of (Δ,Λ)-Co units. FIRMS allowed us to directly measure the zero-field splitting between the two lowest Kramers doublets, which led to axial anisotropy values of 58.3 cm-1 ≤ D < 60.7 cm-1 (1) and 63.8 cm-1 ≤ D < 64.1 cm-1 (2). HFEPR spectra of polycrystalline samples of 1 and 2 at low temperatures confirm the positive sign of D and provide an estimate of the E/D quotient [0.147/0.187 (1) and 0.052 (2)]. Detailed ac and dc magnetic studies reveal that 1 and 2 are new examples of field-induced single-ion magnets (SIMs) with small transversal anisotropy. CASSCF/NEVPT2 calculations support these results. Two Orbach processes or one Orbach plus a direct relaxation mechanism provide similar agreements with the nonlinear experimental Arrhenius plots at Hdc = 500 and 2500 G for 1. Two independent relaxation processes occur in 2, but in contrast to 1, an observed linear dependence of ln(τ) vs 1/T substantiates Orbach processes against the most widely proposed Raman and direct mechanisms. The analysis of each relaxation process in 2 provided values for Ea and τ0 that are very close to those found for 1, validating the predominant role of the Orbach relaxations in both compounds and, probably, also in other cobalt(II) SIMs. A mechanism based on a spin-phonon coupling is proposed to account for the SIM behavior in 1 and 2 with any Raman or direct processes being discarded.