The role of the antennal glands and gills in acid-base regulation and ammonia excretion of a marine osmoconforming brachyuran.

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister - an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH4+ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na+/K+-ATPase detection was observed. The V-type H+-ATPase - a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.

Identification of different physiological functions within the gills and epipodites of the American lobster: Differences in metabolism, transbranchial transport, and mRNA expression.

Transbranchial transport processes are responsible for the homeostatic regulation of most essential physiological functions in aquatic crustaceans. Due to their widespread use as laboratory models, brachyuran crabs are commonly used to predict how other decapod crustaceans respond to environmental stressors including ocean acidification and warming waters. Non-brachyuran species such as the economically-valuable American lobster, Homarus americanus, possess trichobranchiate gills and epipodites that are known to be anatomically distinct from the phyllobranchiate gills of brachyurans; however, studies have yet to define their potential physiological differences. Our results indicate that the pleuro-, arthro-, and podobranch gills of the lobster are functionally homogenous and similar to the respiratory gills of brachyurans as indicated by equivalent rates of H+Eq., CO2, HCO3-, and ammonia transport and mRNA expression of related transporters and enzymes. The epipodites were found to be functionally distinct, being capable of greater individual rates of H+Eq., CO2, and ammonia transport despite mRNA transcript levels of related transporters and enzymes being only a fraction found in the gills. Collectively, mathematical estimates infer that the gills are responsible for 91% of the lobster's branchial HCO3- accumulation whereas the epipodites are responsible for 66% of branchial ammonia excretion suggesting different mechanisms exist in these tissues. Furthermore, the greater metabolic rate and amino acid catabolism in the epipodites suggest that the tissue much of the CO2 and ammonia excreted by this tissue originates intracellularly rather than systemically. These results provide evidence that non-brachyuran species must be carefully compared to brachyuran models.

Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation.

Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.

Protocol and establishment of a Queensland renal biopsy registry in Australia.

BACKGROUND: Renal biopsy is often required to obtain information for diagnosis, management and prognosis of kidney disease that can be broadly classified into acute kidney injury (AKI) and chronic kidney disease (CKD). The most common conditions identified on renal biopsy are glomerulonephritis and tubulo-interstitial disorders. There is a paucity of information on management strategies and therapeutic outcomes in AKI and CKD patients. A renal biopsy registry will provide information on biopsy-proven kidney disorders to improve disease understanding and tracking, healthcare planning, patient care and outcomes. METHODS: A registry of patients, that includes biopsy-proven kidney disease, was established through the collaboration of nephrologists from Queensland Hospital and Health Services and pathologists from Pathology Queensland services. The registry is in keeping with directions of the Advancing Kidney Care 2026 Collaborative, established in September 2018 as a Queensland Health initiative. Phase 1 of the registry entailed retrospective acquisition of data from all adult native kidney biopsies performed in Queensland, Australia, from 2002 to 2018. Data were also linked with the existing CKD.QLD patient registry. From 2019 onwards, phase 2 of the registry involves prospective collection of all incident consenting patients referred to Queensland public hospitals and having a renal biopsy. Annual reports on patient outcomes will be generated and disseminated. DISCUSSION: Establishment of the Queensland Renal Biopsy Registry (QRBR) aims to provide a profile of patients with biopsy-proven kidney disease that will lead to better understanding of clinico-pathological association and facilitate future research. It is expected to improve patient care and outcomes.

A DELPHI study on aspects of study design to overcome knowledge gaps on the burden of disease caused by serogroup B invasive meningococcal disease.

BACKGROUND: Value assessment of vaccination programs against serogroup B invasive meningococcal disease (IMD) is on the agenda of public health authorities. Current evidence on the burden due to IMD is unfit for pinning down the nature and magnitude of the full social and economic costs of IMD for two reasons. First, the concepts and components that need to be studied are not agreed, and second, measures of the concepts that have been studied are weak and inconsistent. Thus, the economic evaluation of the available serogroup B meningococcal (MenB) vaccines is difficult. The aims of this DELPHI study are to: (1) agree on the concepts and components determining the burden of MenB diseases that need to be studied; and (2) seek consensus on appropriate methods and study designs to measure quality of life (QoL) associated with MenB induced long-term sequelae in future studies. METHODS: We designed a DELPHI questionnaire based on the findings of a recent systematic review on the QoL associated with IMD-induced long-term sequelae, and iteratively interviewed a panel of international experts, including physicians, health economists, and patient representatives. Experts were provided with a controlled feedback based on the results of the previous round. RESULTS: Experts reached consensus on all questions after two DELPHI rounds. Major gaps in the literature relate (i) to the classification of sequelae, which allows differentiation of severity levels, (ii) to the choice of QoL measures, and (iii) to appropriate data sources to examine long-term changes and deficits in patients' QoL. CONCLUSIONS: Better conceptualisation of the structure of IMD-specific sequelae and of how their diverse forms of severity might impact the QoL of survivors of IMD as well as their family network and care-providers is needed to generate relevant, reliable and generalisable data on QoL in the future. The results of this DELPHI panel provide useful guidance on how to choose the study design, target population and appropriate QoL measures for future research and hence, help promote the appropriateness and consistency in study methodology and sample characteristics.

Autophagy inhibition in endogenous and nutrient-deprived conditions reduces dorsal root ganglia neuron survival and neurite growth in vitro.

Peripheral neuropathies can result in cytoskeletal changes in axons, ultimately leading to Wallerian degeneration and cell death. Recently, autophagy has been studied as a potential target for improving axonal survival and growth during peripheral nerve damage. This study investigates the influence of autophagy on adult dorsal root ganglia (DRG) neuron survival and axonal growth under control and nutrient deprivation conditions. Constitutive autophagy was modulated with pharmacological activators (rapamycin; Rapa) and inhibitors (3-methyladenine, bafilomycin A1) in conjunction with either a nutrient-stable environment (standard culture medium) or a nutrient-deprived environment (Hank's balanced salt solution + Ca(2+) /Mg(2+) ). The results demonstrated that autophagy inhibition decreased cell viability and reduced neurite growth and branching complexity. Although autophagy was upregulated with nutrient deprivation compared with the control, it was not further activated by rapamycin, suggesting a threshold level of autophagy. Overall, both cellular and biochemical approaches combined to show the influence of autophagy on adult DRG neuron survival and growth. © 2016 Wiley Periodicals, Inc.

Cotyledon-Generated Auxin Is Required for Shade-Induced Hypocotyl Growth in Brassica rapa.

Plant architecture is optimized for the local light environment. In response to foliar shade or neighbor proximity (low red to far-red light), some plant species exhibit shade-avoiding phenotypes, including increased stem and hypocotyl growth, which increases the likelihood of outgrowing competitor plants. If shade persists, early flowering and the reallocation of growth resources to stem elongation ultimately affect the yield of harvestable tissues in crop species. Previous studies have shown that hypocotyl growth in low red to far-red shade is largely dependent on the photoreceptor phytochrome B and the phytohormone auxin. However, where shade is perceived in the plant and how auxin regulates growth spatially are less well understood. Using the oilseed and vegetable crop species Brassica rapa, we show that the perception of low red to far-red shade by the cotyledons triggers hypocotyl cell elongation and auxin target gene expression. Furthermore, we find that following shade perception, elevated auxin levels occur in a basipetal gradient away from the cotyledons and that this is coincident with a gradient of auxin target gene induction. These results show that cotyledon-generated auxin regulates hypocotyl elongation. In addition, we find in mature B. rapa plants that simulated shade does not affect seed oil composition but may affect seed yield. This suggests that in field settings where mutual shading between plants may occur, a balance between plant density and seed yield per plant needs to be achieved for maximum oil yield, while oil composition might remain constant.

Impact of different privacy conditions and incentives on survey response rate, participant representativeness, and disclosure of sensitive information: a randomized controlled trial.

BACKGROUND: Anonymous survey methods appear to promote greater disclosure of sensitive or stigmatizing information compared to non-anonymous methods. Higher disclosure rates have traditionally been interpreted as being more accurate than lower rates. We examined the impact of 3 increasingly private mailed survey conditions-ranging from potentially identifiable to completely anonymous-on survey response and on respondents' representativeness of the underlying sampling frame, completeness in answering sensitive survey items, and disclosure of sensitive information. We also examined the impact of 2 incentives ($10 versus $20) on these outcomes. METHODS: A 3X2 factorial, randomized controlled trial of 324 representatively selected, male Gulf War I era veterans who had applied for United States Department of Veterans Affairs (VA) disability benefits. Men were asked about past sexual assault experiences, childhood abuse, combat, other traumas, mental health symptoms, and sexual orientation. We used a novel technique, the pre-merged questionnaire, to link anonymous responses to administrative data. RESULTS: Response rates ranged from 56.0% to 63.3% across privacy conditions (p = 0.49) and from 52.8% to 68.1% across incentives (p = 0.007). Respondents' characteristics differed by privacy and by incentive assignments, with completely anonymous respondents and $20 respondents appearing least different from their non-respondent counterparts. Survey completeness did not differ by privacy or by incentive. No clear pattern of disclosing sensitive information by privacy condition or by incentive emerged. For example, although all respondents came from the same sampling frame, estimates of sexual abuse ranged from 13.6% to 33.3% across privacy conditions, with the highest estimate coming from the intermediate privacy condition (p = 0.007). CONCLUSION: Greater privacy and larger incentives do not necessarily result in higher disclosure rates of sensitive information than lesser privacy and lower incentives. Furthermore, disclosure of sensitive or stigmatizing information under differing privacy conditions may have less to do with promoting or impeding participants' "honesty" or "accuracy" than with selectively recruiting or attracting subpopulations that are higher or lower in such experiences. Pre-merged questionnaires bypassed many historical limitations of anonymous surveys and hold promise for exploring non-response issues in future research.

Source identification of PM2.5 in Steubenville, Ohio using a hybrid method for highly time-resolved data.

A new source-type identification method, Reduction and Species Clustering Using Episodes (ReSCUE), was developed to exploit the temporal synchronicity typically observed between ambient species in high time resolution fine particulate matter (PM2.5) data to form clusters that vary together. High time-resolution (30 min) PM2.5 sampling was conducted for a month during the summer of 2006 in Steubenville, OH, an EPA designated nonattainment area for the U.S. National Ambient Air Quality Standards (NAAQS). When the data were evaluated, the species clusters from ReSCUE matched extremely well with the source types identified by EPA Unmix demonstrating that ReSCUE is a valuable tool in identifying source types. Results from EPA Unmix show that contributions to PM2.5 are mostly from iron/steel manufacturing (36% ± 9%), crustal matter (33% ± 11%), and coal combustion (11% ± 19%). More importantly, ReSCUE was useful in (i) providing objective data driven guidance for the number of source factors and key fitting species for EPA Unmix, and (ii) detecting tenuous associations between some species and source types in the results derived by EPA Unmix.

Unveiling the Multifaceted Roles of ISG15: From Immunomodulation to Therapeutic Frontiers.

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.

The Combined Effects of Nicotine and Cannabis on Cortical Thickness Estimates in Adolescents and Emerging Adults.

Early life substance use, including cannabis and nicotine, may result in deleterious effects on the maturation of brain tissue and gray matter cortical development. The current study employed linear regression models to investigate the main and interactive effects of past-year nicotine and cannabis use on gray matter cortical thickness estimates in 11 bilateral independent frontal cortical regions in 223 16-22-year-olds. As the frontal cortex develops throughout late adolescence and young adulthood, this period becomes crucial for studying the impact of substance use on brain structure. The distinct effects of nicotine and cannabis use status on cortical thickness were found bilaterally, as cannabis and nicotine users both had thinner cortices than non-users. Interactions between nicotine and cannabis were also observed, in which cannabis use was associated with thicker cortices for those with a history of nicotine and tobacco product (NTP) use in three left frontal regions. This study sheds light on the intricate relationship between substance use and brain structure, suggesting a potential modulation of cannabis' impact on cortical thickness by nicotine exposure, and emphasizing the need for further longitudinal research to characterize these interactions and their implications for brain health and development.

hnRNP A1 dysfunction alters RNA splicing and drives neurodegeneration in multiple sclerosis (MS).

Neurodegeneration is the primary driver of disease progression in multiple sclerosis (MS) resulting in permanent disability, creating an urgent need to discover its underlying mechanisms. Herein, we establish that dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) results in differential of binding to RNA targets causing alternative RNA splicing, which contributes to neurodegeneration in MS and its models. Using RNAseq of MS brains, we discovered differential expression and aberrant splicing of hnRNP A1 target RNAs involved in neuronal function and RNA homeostasis. We confirmed this in vivo in experimental autoimmune encephalomyelitis employing CLIPseq specific for hnRNP A1, where hnRNP A1 differentially binds and regulates RNA, including aberrantly spliced targets identified in human samples. Additionally, dysfunctional hnRNP A1 expression in neurons caused neurite loss and identical changes in splicing, corroborating hnRNP A1 dysfunction as a cause of neurodegeneration. Collectively, these data indicate hnRNP A1 dysfunction causes altered neuronal RNA splicing, resulting in neurodegeneration in MS.

The stage-specific toxicity of per- and polyfluoroalkyl substances (PFAS) in nematode Caenorhabditis elegans.

Per- and Polyfluoroalkyl Substances (PFAS) are a diverse class of industrial chemicals that have been used for decades in industrial and commercial applications. Due to their widespread usages, persistence in the environment, and bioaccumulation in animals and humans, great public health concerns have been raised on adverse health risks of PFAS. In this study, ten PFAS were selected according to their occurrence in different water bodies. The wild-type worms were exposed to individual PFAS at 0, 0.1, 1,10, 100, and 200 µM, and the toxic effects of PFAS on growth, development, fecundity, and behavior at different life stages were investigated using a high-throughput screening (HTS) platform. Our results showed that perfluorooctanesulfonic acid (PFOS), 1H,1H, 2H, 2H-perfluorooctanesulfonamidoacetic acid (NEtFOSAA), perfluorobutanesulfonic (PFBS), and perfluorohexanesulfonic acid (PFHxS) exhibited significant inhibitive effects on the growth in the L4 larva and later stages of worms with concentrations ranging from 0.1 to 200 µmol/L. PFOS and PFBS significantly decreased the brood size of worms across all tested concentrations (p < 0.05), and the most potent PFAS is PFOS with BMC of 0.02013 µM (BMCL, 1.6e-06 µM). During adulthood, all PFAS induced a significant reduction in motility (p < 0.01), while only PFOS can significantly induce behavior alteration at the early larvae stage. Furthermore, the adverse effects occurred in larval stages were found to be the most susceptible to the PFAS exposure. These findings provide valuable insights into the potential adverse effects associated with PFAS exposure and show the importance of considering developmental stages in toxicity assessments.

An MVA-based vector expressing cell-free ISG15 increases IFN-I production and improves HIV-1-specific CD8 T cell immune responses.

The human immunodeficiency virus (HIV), responsible of the Acquired Immune Deficiency Syndrome (AIDS), continues to be a major global public health issue with any cure or vaccine available. The Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is induced by interferons and plays a critical role in the immune response. ISG15 is a modifier protein that covalently binds to its targets via a reversible bond, a process known as ISGylation, which is the best-characterized activity of this protein to date. However, ISG15 can also interact with intracellular proteins via non-covalent binding or act as a cytokine in the extracellular space after secretion. In previous studies we proved the adjuvant effect of ISG15 when delivered by a DNA-vector in heterologous prime-boost combination with a Modified Vaccinia virus Ankara (MVA)-based recombinant virus expressing HIV-1 antigens Env/Gag-Pol-Nef (MVA-B). Here we extended these results evaluating the adjuvant effect of ISG15 when expressed by an MVA vector. For this, we generated and characterized two novel MVA recombinants expressing different forms of ISG15, the wild-type ISG15GG (able to perform ISGylation) or the mutated ISG15AA (unable to perform ISGylation). In mice immunized with the heterologous DNA prime/MVA boost regimen, the expression of the mutant ISG15AA from MVA-Δ3-ISG15AA vector in combination with MVA-B induced an increase in the magnitude and quality of HIV-1-specific CD8 T cells as well as in the levels of IFN-I released, providing a better immunostimulatory activity than the wild-type ISG15GG. Our results confirm the importance of ISG15 as an immune adjuvant in the vaccine field and highlights its role as a potential relevant component in HIV-1 immunization protocols.

Targeted Molecular Profiling of Circulating Cell-Free DNA in Patients With Advanced Hepatocellular Carcinoma.

PURPOSE: Next-generation sequencing (NGS) of tumor-derived, circulating cell-free DNA (cfDNA) may aid in diagnosis, prognostication, and treatment of patients with hepatocellular carcinoma (HCC). The operating characteristics of cfDNA mutational profiling must be determined before routine clinical implementation. METHODS: This was a single-center, retrospective study with the primary objective of defining genomic alterations in circulating cfDNA along with plasma-tissue genotype agreement between NGS of matched tumor samples in patients with advanced HCC. cfDNA was analyzed using a clinically validated 129-gene NGS assay; matched tissue-based NGS was analyzed with a US Food and Drug Administration-authorized NGS tumor assay. RESULTS: Fifty-three plasma samples from 51 patients with histologically confirmed HCC underwent NGS-based cfDNA analysis. Genomic alterations were detected in 92.2% of patients, with the most commonly mutated genes including TERT promoter (57%), TP53 (47%), CTNNB1 (37%), ARID1A (18%), and TSC2 (14%). In total, 37 (73%) patients underwent paired tumor NGS, and concordance was high for mutations observed in patient-matched plasma samples: TERT (83%), TP53 (94%), CTNNB1 (92%), ARID1A (100%), and TSC2 (71%). In 10 (27%) of 37 tumor-plasma samples, alterations were detected by cfDNA analysis that were not detected in the patient-matched tumors. Potentially actionable mutations were identified in 37% of all cases including oncogenic/likely oncogenic alterations in TSC1/2 (18%), BRCA1/2 (8%), and PIK3CA (8%). Higher average variant allele fraction was associated with elevated alpha-fetoprotein, increased tumor volume, and no previous systemic therapy, but did not correlate with overall survival in treatment-naïve patients. CONCLUSION: Tumor mutation profiling of cfDNA in HCC represents an alternative to tissue-based genomic profiling, given the high degree of tumor-plasma NGS concordance; however, genotyping of both blood and tumor may be required to detect all clinically actionable genomic alterations.

Inpatient Diabetes Management.

Management of diabetes in hospitalized patients requires interdisciplinary, coordinated care that includes communication between physicians in the hospital and primary care providers. As the clinical condition of hospitalized patients can change quickly, insulin dosing must be altered in a timely manner to avoid adverse events.

Gender disparities in the National Institutes of Health funding for hematologic malignancies and cellular therapies.

We investigated gender inequality in the National Institutes of Health (NIH) funding for hematologic malignancies and cellular therapies (HMCT). The data were retrieved from the NIH Research Portfolio Online Reporting Tools (RePORT). In 2018-2019, 1834 grants totaling $799 million were awarded (men 71% vs. women 29%) to 975 principal investigators (PIs), including 680 (70%) male PIs and 295 (30%) female PIs. There was no significant gender difference in the mean grant amount per PI. Male PIs as compared to female PIs had a higher h-index (44 vs 31, p < 0.001), a higher number of publications (159.5 vs 94, p < 0.001), and higher years of active research (26 vs 21, p < 0.001). In multivariate analyses, a higher h-index independently predicted a higher mean grant amount per PI (p = 0.010), and female PIs were independently less likely to have a higher h-index (p < 0.001). Our study shows significant gender disparity in the NIH funding for HMCT research.