Integrative phylogenetic, phylogeographic and morphological characterisation of the Unio crassus species complex reveals cryptic diversity with important conservation implications.

The global decline of freshwater mussels and their crucial ecological services highlight the need to understand their phylogeny, phylogeography and patterns of genetic diversity to guide conservation efforts. Such knowledge is urgently needed for Unio crassus, a highly imperilled species originally widespread throughout Europe and southwest Asia. Recent studies have resurrected several species from synonymy based on mitochondrial data, revealing U. crassus to be a complex of cryptic species. To address long-standing taxonomic uncertainties hindering effective conservation, we integrate morphometric, phylogenetic, and phylogeographic analyses to examine species diversity within the U. crassus complex across its entire range. Phylogenetic analyses were performed using cytochrome c oxidase subunit I (815 specimens from 182 populations) and, for selected specimens, whole mitogenome sequences and Anchored Hybrid Enrichment (AHE) data on âˆ¼ 600 nuclear loci. Mito-nuclear discordance was detected, consistent with mitochondrial DNA gene flow between some species during the Pliocene and Pleistocene. Fossil-calibrated phylogenies based on AHE data support a Mediterranean origin for the U. crassus complex in the Early Miocene. The results of our integrative approach support 12 species in the group: the previously recognised Unio bruguierianus, Unio carneus, Unio crassus, Unio damascensis, Unio ionicus, Unio sesirmensis, and Unio tumidiformis, and the reinstatement of five nominal taxa: Unio desectusstat. rev., Unio gontieriistat. rev., Unio mardinensisstat. rev., Unio nanusstat. rev., and Unio vicariusstat. rev. Morphometric analyses of shell contours reveal important morphospace overlaps among these species, highlighting cryptic, but geographically structured, diversity. The distribution, taxonomy, phylogeography, and conservation of each species are succinctly described.

Frequency and reporting of complications after Dupuytren's contracture interventions: A systematic review and meta-analysis.

INTRODUCTION: Numerous complications are reported following interventions for Dupuytren's contracture; however, their incidence, management, and outcomes remain poorly reported. The aims of this review were to report the proportions of complications, compare likelihood of complications between interventions, and evaluate reporting of complications, including assessment, grading, management, and subsequent reporting of their impact on patient outcomes. METHODS: Extracted data included patient demographics, intervention details, complications, their management, and final outcomes. Analysis of descriptive data enabled review of complications reporting. Meta-analysis(MA) of non-comparative datasets enabled estimation of proportions of patients experiencing complications. Network meta-analysis(NMA) of comparative studies estimated the relative occurrence of complications between interventions. Risk of bias analysis was performed. RESULTS: 26 studies, comprising 10,831 patients, were included. Interventions included collagenase injection, percutaneous needle fasciotomy(PNF), limited fasciectomy(LF), open fasciotomy(OF), and dermofasciectomy(DF). Overall quality and consistency of outcomes reporting was poor. MA enabled estimates of probabilities for three common complications(infection, nerve injury, complex regional pain syndrome(CRPS)) across all interventions; the reported rates for LF were 4.5% for infection, 3% for nerve injury, and 3.3% for CRPS. As the commonest intervention, LF was used as the reference intervention for comparison of the commonest complications via NMA, including haematoma [OF OR 0.450(0.277, 0.695); PNF OR 0.245(0.114, 0.457)], infection [PNF OR 0.2(0.0287, 0.690); DF OR 2.02(1.02, 3.74)], and neuropraxia [PNF OR 0.0926(0.00553, 0.737)]. We noted that the complication incidence was higher the more invasive the intervention. CONCLUSIONS: There was limited reporting of complication occurrence, management, and outcomes following interventions, contributing to a gap in information for informed patient consent. MA was possible for reporting of proportions for infection, nerve injury, and CRPS across interventions. NMA enabled direct comparison of the six commonest complications between interventions. These findings can guide intervention selection. Improving consistency and quality in complications reporting is essential to aid counselling of patients regarding the true rates and consequences of the risks of interventions. TYPE OF STUDY/LEVEL OF EVIDENCE: 2.

Curcumin abrogates cobalt-induced neuroinflammation by suppressing proinflammatory cytokines release, inhibiting microgliosis and modulation of ERK/MAPK signaling pathway.

Curcumin, a bioactive polyphenol derived from turmeric, has been reported to have anti-inflammatory properties. The current study investigated the anti-inflammatory effect of curcumin in the hippocampal subfields (CA1 and CA3) after exposure to cobalt (Co) and the impact of ERK protein. Twenty-eight albino Wistar rats were divided into four groups, each with seven randomly selected rats as follows: Control (distilled water), Cobalt (Co) only (40 mg/kg), 120 mg/kg or 240 mg/kg curcumin + Co (40 mg/kg). Treatment was via oral gavage for 28 days. We performed a biochemical investigation to determine the levels of proinflammatory cytokines (TNFα and IL-1ß). Furthermore, we conducted an immunohistochemical evaluation to assess the expression of IBA1 by microglial cells and the immunoexpression of ERK protein in the hippocampus. Results revealed a significant (p<0.05) elevation in the tissue level of TNFα and IL-1ß, an increase in the number of IBA1-positive microglia, and upregulation of ERK protein in the hippocampal subfields of the rats after exposure to cobalt-only. Nevertheless, pretreatment with curcumin restored these parameters to levels comparable to control. In conclusion, our results showed that curcumin abrogated the Co-induced neuroinflammation by suppressing the release of proinflammatory biomarkers, reducing microgliosis, and modulating the ERK/MAPK pathway.

Collective chemomechanical oscillations in active hydrogels.

We report on the collective response of an assembly of chemomechanical Belousov-Zhabotinsky (BZ) hydrogel beads. We first demonstrate that a single isolated spherical BZ hydrogel bead with a radius below a critical value does not oscillate, whereas an assembly of the same BZ hydrogel beads presents chemical oscillation. A BZ chemical model with an additional flux of chemicals out of the BZ hydrogel captures the experimentally observed transition from oxidized nonoscillating to oscillating BZ hydrogels and shows this transition is due to a flux of inhibitors out of the BZ hydrogel. The model also captures the role of neighboring BZ hydrogel beads in decreasing the critical size for an assembly of BZ hydrogel beads to oscillate. We finally leverage the quorum sensing behavior of the collective to trigger their chemomechanical oscillation and discuss how this collective effect can be used to enhance the oscillatory strain of these active BZ hydrogels. These findings could help guide the eventual fabrication of a swarm of autonomous, communicating, and motile hydrogels.

Intraoperative computed tomography for orbital reconstruction: a systematic review.

Orbital reconstruction is a common procedure with inherent challenges and important consequences. Intraoperative use of computed tomography (CT) is an emerging application that facilitates accurate intraoperative evaluation to improve clinical outcomes. This review aims to investigate the intraoperative and postoperative outcomes of intraoperative CT use in orbital reconstruction. PubMed and Scopus databases were systematically searched. Inclusion criteria were: clinical studies investigating intraoperative CT use in orbital reconstruction. Exclusion criteria were: duplicates; non-English publications; non-full-text publications; studies with insufficient data. Of the 1022 articles identified, seven eligible articles representing 256 cases were included. The mean age was 39 years. Most cases were male (69.9%). With regards to intraoperative outcomes, the mean revision rate was 34.1%, with plate repositioning being the most common type (51.1%). Intraoperative time was variably reported. With regards to postoperative outcomes, there were no revisions, and only one case that had a complication (transient exophthalmos). Mean volumetric difference between the repaired and contralateral orbits was reported in two studies. The findings of this review present an updated evidence-based summary of the intraoperative and postoperative outcomes of intraoperative CT use in orbital reconstruction. Robust longitudinal comparisons of clinical outcomes between intraoperative and non-intraoperative CT cases are required.

[Transition, fertility and options for preservation of fertility]. / Transition, fertilité et options de préservation de fertilité.

Oftentimes ignored or infrequently expressed, some transgender persons harbor a desire for parenthood. Given the evolution of medical techniques and the enacting of legislative reforms, it is henceforth possible to propose fertility preservation strategies in the overall context of gender transidentity. During the "female to male" (FtM) transition pathway, androgen therapy has an impact on gonadic function, generally inducing blockage of the ovarian function, with amenorrhea. Even though these events may be reversed on cessation of treatment, the possible long-term effects on future fertility and on the health of children yet to be born are little known. Moreover, transition surgeries definitively compromise the possibility of pregnancy insofar as they involve bilateral adnexectomy and/or hysterectomy. Options for fertility preservation in the framework of FtM transition are premised on cryopreservation of oocytes and/or ovarian tissue. In a comparable manner, even though relevant documentation is lacking, hormonal treatments for persons transitioning from male to female (MtF) can have an impact on future fertility. In the event of surgery involving bilateral orchidectomy in which spermatozoid cryopreservation has not been carried out, fertility is definitively impossible. In both cases and under present-day legislation, numerous legal and regulatory barriers render highly problematic the reutilization of cryopreserved gametes. Given these different constraints, it is indispensable to closely supervise these types of treatment by proposing psychological support.

Barriers and enablers of vigorous intermittent lifestyle physical activity (VILPA) in physically inactive adults: a focus group study.

BACKGROUND: Vigorous Intermittent Lifestyle Physical Activity (VILPA) refers to brief bouts of vigorous intensity physical activity performed as part of daily living. VILPA has been proposed as a novel concept to expand physical activity options among the least active. As a nascent area of research, factors which impede or encourage VILPA in physically inactive adults are yet to be explored. Such information is pertinent in the design of future interventions. We examined the barriers and enablers of VILPA among physically inactive adults using the Capability, Opportunity, Motivation, Behavior (COM-B) model as a conceptual framework. METHODS: We recruited a sample of self-identified physically inactive middle-aged and older adults (N = 78) based in Australia to take part in 19 online focus groups across three age groups: young-middle (age 35-44), middle (age 45-59) and old (age 60-76). We analyzed interviews using a critical realist approach to thematic analysis. Identified barriers and enablers were subsequently mapped onto the COM-B model components. RESULTS: The data generated 6 barriers and 10 enablers of VILPA that corresponded to COM-B concepts. Barriers included physical limitations (physical capability), perceptions of aging, need for knowledge (psychological capability), environmental constraints (physical opportunity), perceptions of effort and energy, and fear (automatic motivation). Enablers included convenience, reframing physical activity as purposeful movement, use of prompts and reminders (physical opportunity), normalization of taking the active option, gamification (social opportunity), sense of achievement, health improvements, personally salient rewards (reflective motivation), identity fit, and changing from effortful deliberation to habitual action (automatic motivation). CONCLUSION: The barriers and enablers of VILPA span capability, opportunity, and motivation beliefs. Promoting the time-efficient nature and simplicity of VILPA requiring no equipment or special gym sessions, the use of prompts and reminders at opportune times, and habit formation strategies could capitalize on the enablers. Addressing the suitability of the small bouts, the development of specific guidelines, addressing safety concerns, and explicating the potential benefits of, and opportunities to do, VILPA could ameliorate some of the barriers identified. Future VILPA interventions may require limited age customization, speaking to the potential for such interventions to be delivered at scale.

Surface Functionalization of 4D Printed Substrates Using Polymeric and Metallic Wrinkles.

Wrinkle topographies have been studied as simple, versatile, and in some cases biomimetic surface functionalization strategies. To fabricate surface wrinkles, one material phenomenon employed is the mechanical-instability-driven wrinkling of thin films, which occurs when a deforming substrate produces sufficient compressive strain to buckle a surface thin film. Although thin-film wrinkling has been studied on shape-changing functional materials, including shape-memory polymers (SMPs), work to date has been primarily limited to simple geometries, such as flat, uniaxially-contracting substrates. Thus, there is a need for a strategy that would allow deformation of complex substrates or 3D parts to generate wrinkles on surfaces throughout that complex substrate or part. Here, 4D printing of SMPs is combined with polymeric and metallic thin films to develop and study an approach for fiber-level topographic functionalization suitable for use in printing of arbitrarily complex shape-changing substrates or parts. The effect of nozzle temperature, substrate architecture, and film thickness on wrinkles has been characterized, as well as wrinkle topography on nuclear alignment using scanning electron microscopy, atomic force microscopy, and fluorescent imaging. As nozzle temperature increased, wrinkle wavelength increased while strain trapping and nuclear alignment decreased. Moreover, with increasing film thickness, the wavelength increased as well.

E-scooters and maxillofacial fractures: a seven-year multi-centre retrospective review.

BACKGROUND: Standing electric scooters (e-scooters) are a cost-effective and environmentally-friendly transport alternative, but also elicit substantial concern regarding associated craniofacial injuries. This study aims to describe the patient factors, procedural factors and post-operative outcomes of maxillofacial fractures caused by e-scooter accidents. METHODS: Retrospective chart review of patients aged 18 years or older who were surgically treated for these injuries in 2014-2020 at two Australian tertiary hospitals. RESULTS: There were 18 cases included. Most cases were male (66%). The mean age was 35 years. Common risk factors were alcohol use (86%) and lack of helmet use (62%). The most common fracture pattern was zygomatico-maxillary complex (ZMC) fractures (50%). There were no associated systemic injuries. Mean operation timing was 12 days post-injury for ZMC fractures and 3 days post-injury for condyle fractures. For ZMC fractures, the most common method of fixation was 2-point fixation (66%). For condyle fractures, the most common surgical approach was arch bars only (83%). Post-operative complications were reported in six cases, with malocclusion being the most common (n = 3). Revision surgeries were performed in two cases. CONCLUSIONS: Maxillofacial fractures associated with e-scooter accidents appear to be increasing in incidence. Robust longitudinal evaluations with larger sample sizes are required to better understand associated presentations, surgical approaches and post-operative complications.

Dihydroartemisinin-induced ferroptosis in acute myeloid leukemia: links to iron metabolism and metallothionein.

Artemisinin is an anti-malarial drug that has shown anticancer properties. Recently, ferroptosis was reported to be induced by dihydroartemisinin (DHA) and linked to iron increase. In the current study, we determined the effect of DHA in leukemic cell lines on ferroptosis induction and iron metabolism and the cytoprotective effect triggered in leukemic cells. We found that treatment of DHA induces early ferroptosis by promoting ferritinophagy and subsequent iron increase. Furthermore, our study demonstrated that DHA activated zinc metabolism signaling, especially the upregulation of metallothionein (MT). Supportingly, we showed that inhibition MT2A and MT1M isoforms enhanced DHA-induced ferroptosis. Finally, we demonstrated that DHA-induced ferroptosis alters glutathione pool, which is highly dependent on MTs-driven antioxidant response. Taken together, our study indicated that DHA activates ferritinophagy and subsequent ferroptosis in AML and that MTs are involved in glutathione regenerating and antioxidant response.

A high-fidelity quantum matter-link between ion-trap microchip modules.

System scalability is fundamental for large-scale quantum computers (QCs) and is being pursued over a variety of hardware platforms. For QCs based on trapped ions, architectures such as the quantum charge-coupled device (QCCD) are used to scale the number of qubits on a single device. However, the number of ions that can be hosted on a single quantum computing module is limited by the size of the chip being used. Therefore, a modular approach is of critical importance and requires quantum connections between individual modules. Here, we present the demonstration of a quantum matter-link in which ion qubits are transferred between adjacent QC modules. Ion transport between adjacent modules is realised at a rate of 2424 s-1 and with an infidelity associated with ion loss during transport below 7 × 10-8. Furthermore, we show that the link does not measurably impact the phase coherence of the qubit. The quantum matter-link constitutes a practical mechanism for the interconnection of QCCD devices. Our work will facilitate the implementation of modular QCs capable of fault-tolerant utility-scale quantum computation.

Modulatory Role of Curcumin on Cobalt-Induced Memory Deficit, Hippocampal Oxidative Damage, Astrocytosis, and Nrf2 Expression.

Chemical overexposure is a growing environmental risk factor for many medical issues. Cobalt toxicity from environmental, industrial, and medical exposure has previously been linked to neurological impairment. Hence, the current study looked into the neuroprotective potential of curcumin, a natural polyphenol contained in the spice turmeric, against cobalt-induced neurotoxicity. Adult rats were randomly divided into six groups as follows: control, 40 mg/kg cobalt chloride (CoCl2) only, 240 mg/kg curcumin only, 120 mg/kg or 240 mg/kg curcumin, or 100 mg/kg vitamin C co-administered with CoCl2. The administration was via oral route daily for 4 weeks. After that, neurobehavioral tests were undertaken to evaluate short-term spatial memory. Biochemical investigation was performed to determine the hippocampal levels of status via measures of SOD, CAT, GST, and LPO. Furthermore, immunohistochemical assessment of the expression of GFAP and Nrf2 in the hippocampus was carried out. In the CoCl2 group, the results showed altered behavioral responses, a decrease in antioxidant activities, increased expression of GFAP and the number of activated astrocytes, and decreased immunoexpression of Nrf2. These effects were mitigated in the curcumin- and vitamin C-treated groups. These results collectively imply that curcumin enhances memory functions in rats exposed to cobalt possibly by attenuating oxidative responses, mitigating astrocytosis, and modulating Nrf2 signaling.

[Fertility preservation for transmen]. / Place de la préservation de la fertilité dans le parcours des hommes transgenres.

The evolution of medical techniques as well as legislative changes currently allow to propose fertility preservation strategies in the context of transidentity. During "female to male" transition, androgen therapy has an impact on gonadal function since it usually induces a blockage of ovulation with amenorrhea. Although this effect is reversible when treatment is stopped, the possible long-term effects of testosterone treatment on future fertility or health of future children are poorly known. In addition, transitional surgeries definitely compromise fecundity when they include bilateral ovariectomy and/or hysterectomy. Yet, although long ignored or poorly expressed, the desire for parenthood is a reality in transgender men. Fertility preservation options in FtM transition rely on oocyte or ovarian tissue cryopreservation. The purpose of this review is to provide an overview of the literature regarding fertility preservation in transgender men. Although series remain limited, the increase in the number of recently published articles reflects the interest in improving the management of fertility issues in transgender men.

Stark differences in cancer epidemiological data between GLOBOCAN and GBD: Emphasis on oral cancer and wider implications.

Background: GLOBOCAN 2020 and Global Burden of Disease (GBD) 2019 are the two most established global online cancer databases. It is important to examine the differences between the two platforms, to attempt to explain these differences, and to appraise the quality of the data. There are stark differences for lip and oral cancers (LOC) and we attempt to explain these by detailed analysis of ten countries at the extremes of differences. Methods: Age-standardised incidence rates (ASIR) of LOC were obtained from GLOBOCAN 2020 and GBD 2019. Five countries with the greatest and smallest fold differences were selected. A systematic search of PubMed and Embase electronic databases was then performed to identify publications reporting the incidence of LOC in the selected countries between 2015 and 2022. Specifically, data sources of the articles were examined and evaluated. Findings: For LOC, greatest differences were found in Papua New Guinea, Vietnam, China, Pakistan, and Indonesia (group A). In contrast, the United States of America (USA), Brazil, France, Germany, and India (group B) had the least differences between the two databases. Interpretation: It is not surprising that when GLOBOCAN and GBD could not obtain high-quality or accessible LOC data from national or local cancer registries, as in group A, discrepancies would be seen between the two online databases. In contrast, where only minor differences were seen between GLOBOCAN and GBD, as in group B, presumptively due to those countries having well-established cancer registries and healthcare administrative systems, the literature is more consistent. Moreover, many studies have grouped lip and oral cavity with pharynx and categorised outputs as "oral and oropharyngeal cancer" or "oral cavity and pharynx cancer". Those categorisations lacked subsite accuracy and failed to realise that oral cancer and oropharyngeal cancer have completely different etiological factors, pathogeneses, prognosis, and treatment outcomes. Funding: This research received no specific grant or funding from any funding agency in the public, commercial, or not-for-profit sectors, and the authors received no financial support for the research, authorship, and/or publication of this article.

Natural and Anthropogenic Processes Affecting Domestic Groundwater Quality within the Northwestern Appalachian Basin.

Domestic wells serve as the primary drinking-water source for rural residents in the northern Appalachian Basin (NAB), despite a limited understanding of contaminant distributions in groundwater sources. We employ a newly collected dataset of 216 water samples from domestic wells in Ohio and West Virginia and an integrated contaminant-source attribution method to describe water quality in the western NAB and characterize key agents influencing contaminant distributions. Our results reveal arsenic and nitrate concentrations above federal maximum contaminant levels (MCLs) in 6.8 and 1.3% of samples and manganese concentrations above health advisory in 7.3% of samples. Recently recharged groundwaters beneath upland regions appear vulnerable to surface-related impacts, including nitrate pollution from agricultural activities and salinization from road salting and domestic sewage sources. Valley regions serve as terminal discharge points for long-residence-time groundwaters, where mixing with basin brines is possible. Arsenic impairments occurred in alkaline groundwaters with major-ion compositions altered by ion exchange and in low-oxygen metal-rich groundwaters. Mixing with as much as 4-10% of mine discharge-like waters was observed near coal mining operations. Our study provides new insights into key agents of groundwater impairment in an understudied region of the NAB and presents an integrated approach for contaminant-source attribution applicable to other regions of intensive resource extraction.

Detection of Borrelia and Babesia species in Haemaphysalis punctata ticks sampled in Southern England.

The distribution and population size of the red sheep tick (Haemaphysalis punctata) are increasing in Northern Europe, and in the United Kingdom reports of human biting by this species have increased in recent years. To assess the risk of tick-borne disease (TBD) transmission to humans and livestock by H. punctata, ticks sampled from sites in Southern England were screened using PCR for either Borrelia species or piroplasms over a three year period, 2018-2020. A total of 302 H. punctata were collected from eight locations. From these, two Babesia species associated with TBD infections in livestock, Babesia major and Babesia motasi, and the human pathogen Borrelia miyamotoi were detected, predominantly from a single location in Sussex. Consequently, the range expansion of this tick across Southern England may impact public and livestock health.

Endometriosis classification, staging and reporting systems: a review on the road to a universally accepted endometriosis classification.

BACKGROUND: In the field of endometriosis, several classification, staging and reporting systems have been developed. However, endometriosis classification, staging and reporting systems that have been published and validated for use in clinical practice have not been systematically reviewed up to now. OBJECTIVES: The aim of the current review is to provide a historical overview of these different systems based on an assessment of published studies. MATERIALS AND METHODS: A systematic Pubmed literature search was performed. Data were extracted and summarised. RESULTS: Twenty-two endometriosis classification, staging and reporting systems have been published between 1973 and 2021, each developed for specific and different purposes. There is still no international agreement on how to describe the disease. Studies evaluating different systems are summarised showing a discrepancy between the intended and the evaluated purpose, and a general lack of validation data confirming a correlation with pain symptoms or quality of life for any of the current systems. A few studies confirm the value of the Enzian system for surgical description of deep endometriosis. With regards to infertility, the endometriosis fertility index has been confirmed valid for its intended purpose. CONCLUSIONS: Of the 22 endometriosis classification, staging and reporting systems identified in this historical overview, only a few have been evaluated, in 46 studies, for the purpose for which they were developed. It can be concluded that there is no international agreement on how to describe endometriosis or how to classify it, and that most classification/staging systems show no or very little correlation with patient outcomes. WHAT IS NEW?: This overview of existing systems is a first step in working towards a universally accepted endometriosis classification.

An International Terminology for Endometriosis, 2021.

BACKGROUND: Different classification systems have been developed for endometriosis, using different definitions for the disease, the different subtypes, symptoms and treatments. In addition, an International Glossary on Infertility and Fertility Care was published in 2017 by the International Committee for Monitoring Assisted Reproductive Technologies (ICMART) in collaboration with other organisations. An international working group convened over the development of a classification or descriptive system for endometriosis. As a basis for such system, a terminology for endometriosis was considered a condition sine qua non. OBJECTIVES: The aim of the current paper is to develop a set of terms and definitions on endometriosis that would be the basis for standardisation in disease description, classification and research. MATERIALS AND METHODS: The working group listed a number of terms relevant to be included in the terminology, documented currently used and published definitions, and discussed and adapted them until consensus was reached within the working group. Following stakeholder review, further terms were added, and definitions further clarified. Although definitions were collected through published literature, the final set of terms and definitions is to be considered consensus-based. After finalisation of the first draft, the members of the international societies and other stakeholders were consulted for feedback and comments, which led to further adaptations. RESULTS: A list of 49 terms and definitions in the field of endometriosis is presented, including a definition for endometriosis and its subtypes, different locations, interventions, symptoms and outcomes. Endometriosis is defined as a disease characterised by the presence of endometrium-like epithelium and/or stroma outside the endometrium and myometrium, usually with an associated inflammatory process. CONCLUSIONS: The current paper outlines a list of 49 terms and definitions in the field of endometriosis. The application of the defined terms aims to facilitate harmonisation in endometriosis research and clinical practice. Future research may require further refinement of the presented definitions. WHAT IS NEW?: A consensus based international terminology for endometriosis for clinical and research use.