Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster.

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Object-based attention during scene perception elicits boundary contraction in memory.

Boundary contraction and extension are two types of scene transformations that occur in memory. In extension, viewers extrapolate information beyond the edges of the image, whereas in contraction, viewers forget information near the edges. Recent work suggests that image composition influences the direction and magnitude of boundary transformation. We hypothesize that selective attention at encoding is an important driver of boundary transformation effects, selective attention to specific objects at encoding leading to boundary contraction. In this study, one group of participants (N = 36) memorized 15 scenes while searching for targets, while a separate group (N = 36) just memorized the scenes. Both groups then drew the scenes from memory with as much object and spatial detail as they could remember. We asked online workers to provide ratings of boundary transformations in the drawings, as well as how many objects they contained and the precision of remembered object size and location. We found that search condition drawings showed significantly greater boundary contraction than drawings of the same scenes in the memorize condition. Search drawings were significantly more likely to contain target objects, and the likelihood to recall other objects in the scene decreased as a function of their distance from the target. These findings suggest that selective attention to a specific object due to a search task at encoding will lead to significant boundary contraction.

PROTOCOL FOR HUMAN EXPOSURE TO OPIOIDS AND CONCENTRATED MEDETOMIDINE USED IN FIELD APPLICATIONS.

Wildlife professionals routinely use potent sedatives and anesthetics when chemically immobilizing wildlife and zoo species in remote environments. Accidental exposure to these prescription veterinary drugs is rare but could be rapidly fatal. Commonly used agents include opioids and α2 adrenoreceptor agonists. These drugs can be reversed with specific antagonists; however, they are often not approved for human use. The protocol created here can be used by wildlife health professionals in a field setting with basic human emergency medical response training in coordination with local Emergency Medical Services (EMS). Key components include, building local relationships between EMS and wildlife professionals, focused EMS training, administering opioid and α2 adrenergic antagonists off label, and local evacuation procedures. This framework could allow wildlife management agencies or zoos to mitigate the risk of human exposures to these commonly used drugs, significantly improving occupational safety in an otherwise high-risk environment.

Rapid kidney function decline and increased risk of heart failure in patients with type 2 diabetes: findings from the ACCORD cohort : Rapid kidney function decline and heart failure in T2D.

BACKGROUND: Impaired kidney function and albuminuria are associated with increased risk of heart failure (HF) in patients with type 2 diabetes (T2D). We investigated whether rapid kidney function decline over time is an additional determinant of increased HF risk in patients with T2D, independent of baseline kidney function, albuminuria, and other HF predictors. METHODS: Included in the study were 7,539 participants in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study with baseline urinary albumin-to-creatinine ratio (UACR) data, who had completed 4 years of follow-up and had ≥ 3 eGFR measurements during that period (median eGFR/year = 1.9, IQR 1.7-3.2). The association between rapid kidney function decline (eGFR loss ≥ 5 ml/min/1.73 m2/year) and odds of HF hospitalization or HF death during the first 4 years of follow-up was estimated by logistic regression. The improvement in risk discrimination provided by adding rapid kidney function decline to other HF risk factors was evaluated as the increment in the area under the Receiving Operating Characteristics curve (ROC AUC) and integrated discrimination improvement (IDI). RESULTS: Over 4 years of follow-up, 1,573 participants (20.9%) experienced rapid kidney function decline and 255 (3.4%) experienced a HF event. Rapid kidney function decline was associated with a ~ 3.2-fold increase in HF odds (3.23, 95% CI, 2.51-4.16, p < 0.0001), independent of baseline CVD history. This estimate was not attenuated by adjustment for potential confounders, including eGFR and UACR at baseline as well as at censoring (3.74; 95% CI 2.63-5.31). Adding rapid kidney function decline during follow-up to other clinical predictors (WATCH-DM score, eGFR, and UACR at study entry and end of follow-up) improved HF risk classification (ROC AUC = + 0.02, p = 0.027; relative IDI = + 38%, p < 0.0001). CONCLUSIONS: In patients with T2D, rapid kidney function decline is associated with a marked increase in HF risk, independent of starting kidney function and/or albuminuria. These findings highlight the importance of serial eGFR measurements over time to improve HF risk estimation in T2D.

Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

Working memory control predicts fixation duration in scene-viewing.

When viewing scenes, observers differ in how long they linger at each fixation location and how far they move their eyes between fixations. What factors drive these differences in eye-movement behaviors? Previous work suggests individual differences in working memory capacity may influence fixation durations and saccade amplitudes. In the present study, participants (N = 98) performed two scene-viewing tasks, aesthetic judgment and memorization, while viewing 100 photographs of real-world scenes. Working memory capacity, working memory processing ability, and fluid intelligence were assessed with an operation span task, a memory updating task, and Raven's Advanced Progressive Matrices, respectively. Across participants, we found significant effects of task on both fixation durations and saccade amplitudes. At the level of each individual participant, we also found a significant relationship between memory updating task performance and participants' fixation duration distributions. However, we found no effect of fluid intelligence and no effect of working memory capacity on fixation duration or saccade amplitude distributions, inconsistent with previous findings. These results suggest that the ability to flexibly maintain and update working memory is strongly related to fixation duration behavior.

A Prospective, Randomized Controlled, Single-Blinded Study to Assess the Effect of a 33-Gauge Needle Versus a 34-Gauge Needle on Pain Experienced During Injection of Local Anesthetic on the Face.

BACKGROUND: Most pain experienced by patients during Mohs micrographic surgery is associated with the initial injection. Previous studies have shown that a smaller gauge needle (33-gauge vs 30-gauge) is associated with less patient-reported pain. OBJECTIVES: To evaluate patient-reported pain levels following injection with a 33-gauge versus a 34-gauge needle.  Methods: During this prospective, randomized, controlled, single-blinded study, 480 patients were randomized into a 33-gauge versus a 34-gauge needle group. Pain levels following needle insertion were recorded using the validated numerical rating scale (VNRS)-11 scale.  Results: Injection of local anesthetic with a 34-gauge needle is associated with significantly less pain compared to a 33-gauge needle across all subgroups (P=0.007, average pain level 0.49 [34-gauge group] vs 0.79 [33-gauge group] rated on a 0-10 pain scale). Females, first-time Mohs patients, patients under age 65, patients with basal cell carcinoma, and those with tumor locations on the nose experienced the most pain reduction with the use of a 34-gauge needle.  Limitations: This was a single-blinded study; thus, the injector was able to see which needle was being used. This knowledge could have subconsciously affected the angle, speed, or force used to insert the needle. CONCLUSIONS: Injections with a 33-gauge and a 34-gauge needle are both tolerated well and associated with minimal pain. While the pain reduction associated with using a 34-gauge needle is statistically significant, the use of a 34-gauge needle may be most clinically relevant for certain patient subgroups. J Drugs Dermatol. 2023;22(11): doi:10.36849/JDD.7689.

Reactance to Healthy Eating and Physical Activity Messages: Face Threat and Face Management Strategies in Memorable Daily Conversations Among Couples.

Most adults in the United States (U.S.) fail to meet guidelines for physical activity (PA) and nutrition outlined by the Centers for Disease Control. One important predictor of engagement in healthy behavior is support from one's romantic partner. However, messages from partners may fail to motivate healthy behavior if they threaten recipients' face and cause reactance. The present study examines face-threatening acts (FTAs) and face management strategies (FMSs) in conversations between romantic couples and their associations with reactance, healthy eating, and PA behaviors. Cohabitating couples (N = 70) were recruited, and one member of the couple completed a 10-day diary survey in which they reported on daily memorable conversations they had with their partner about PA and/or healthy eating. Participants completed measures of positive and negative face threat, as well as the extent to which they engaged in healthy eating and PA that day. Trained raters assessed reported conversations for positive and negative face threat as well as positive and negative FMSs. Results indicate that both participant and trained raters' reports of positive face threat were associated with increased reactance, whereas only participants' reports of negative face threat were associated with increased reactance. Both positive and negative FMSs were associated with reduced reactance. Reactance was negatively associated with healthy eating and PA. Results are discussed in terms of implications for reactance and politeness theories, as well as pragmatic implications for the millions of partnered individuals in the U.S. seeking to improve their health.

Confirmation, Disconfirmation, and Communal Coping for Joint Physical Activity in Romantic Dyads.

Most people in the United States do not engage in sufficient physical activity (PA). However, certain communication behaviors from romantic partners can motivate PA. Research indicates that confirming communication and communal coping (CC) in romantic relationships can increase PA efforts, but less research has examined the role of explicitly disconfirming communication or relationships between confirmation, disconfirmation, and CC on PA outcomes. We examined models in which shared PA appraisals mediate relationships between (a) confirmation and (b) disconfirmation and joint PA behavior in heterosexual, romantic dyads. Sex differences in actor and partner effects were also considered. Partners (N = 144) in 72 dyads completed assessments of key constructs. Results indicated that shared PA appraisals were critical in the confirmation model, mediating relationships between perceptions of confirmation and reports of joint PA. Unexpectedly, both partners' reports of partner disconfirmation were positively associated with their partners' reports of joint PA. Only one statistically significant sex difference emerged. Theoretical and pragmatic implications are discussed.

Analysis and validation of silica-immobilised BST polymerase in loop-mediated isothermal amplification (LAMP) for malaria diagnosis.

Bacillus stearothermophilus large fragment (BSTLF) DNA polymerase is reported, isolated on silica via a fused R5 silica-affinity peptide and used in nucleic acid diagnostics. mCherry (mCh), included in the fusion construct, was shown as an efficient fluorescent label to follow the workflow from gene to diagnostic. The R5 immobilisation on silica from cell lysate was consistent with cooperative R5-specific binding of R52-mCh-FL-BSTLF or R52-mCh-H10-BSTLF fusion proteins followed by non-specific protein binding (including E. coli native proteins). Higher R5-binding could be achieved in the presence of phosphate, but phosphate residue reduced loop-mediated isothermal amplification (LAMP) performance, possibly blocking sites on the BSTLF for binding of ß- and γ-phosphates of the dNTPs. Quantitative assessment showed that cations (Mg2+ and Mn2+) that complex the PPi product optimised enzyme activity. In malaria testing, the limit of detection depended on Plasmodium species and primer set. For example, 1000 copies of P. knowlesi 18S rRNA could be detected with the P.KNO-LAU primer set with Si-R52-mCh-FL-BSTLF , but 10 copies of P. ovale 18S rRNA could be detected with the P.OVA-HAN primer set using the same enzyme. The Si-immobilised BSTLF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R52-mCh-FL-BSTLF production was transferred from Cambridge to Accra and set up de novo for a trial with clinical samples. Different detection limits were found, targeting the mitochondrial DNA or the 18S rRNA gene for P. falciparum. The results are discussed in comparison with qPCR and sampling protocol and show that the Si-BSTLF polymerase can be optimised to meet the WHO recommended guidelines.

Distinct Representational Structure and Localization for Visual Encoding and Recall during Visual Imagery.

During memory recall and visual imagery, reinstatement is thought to occur as an echoing of the neural patterns during encoding. However, the precise information in these recall traces is relatively unknown, with previous work primarily investigating either broad distinctions or specific images, rarely bridging these levels of information. Using ultra-high-field (7T) functional magnetic resonance imaging with an item-based visual recall task, we conducted an in-depth comparison of encoding and recall along a spectrum of granularity, from coarse (scenes, objects) to mid (e.g., natural, manmade scenes) to fine (e.g., living room, cupcake) levels. In the scanner, participants viewed a trial-unique item, and after a distractor task, visually imagined the initial item. During encoding, we observed decodable information at all levels of granularity in category-selective visual cortex. In contrast, information during recall was primarily at the coarse level with fine-level information in some areas; there was no evidence of mid-level information. A closer look revealed segregation between voxels showing the strongest effects during encoding and those during recall, and peaks of encoding-recall similarity extended anterior to category-selective cortex. Collectively, these results suggest visual recall is not merely a reactivation of encoding patterns, displaying a different representational structure and localization from encoding, despite some overlap.

Highly similar and competing visual scenes lead to diminished object but not spatial detail in memory drawings.

Drawings of scenes made from memory can be highly detailed and spatially accurate, with little information not found in the observed stimuli. While prior work has focused on studying memory for distinct scenes, less is known about the specific detail recalled when episodes are highly similar and competing. Here, participants (N = 30) were asked to study and recall eight complex scene images using a drawing task. Importantly, four of these images were exemplars of different scene categories, while the other four images were from the same scene category. The resulting 213 drawings were judged by 1764 online scorers for a comprehensive set of measures, including scene and object diagnosticity, spatial information, and fixation and pen movement behaviour. We observed that competition in memory resulted in diminished object detail, with drawings and objects that were less diagnostic of their original image. However, repeated exemplars of a category did not result in differences in spatial memory accuracy, and there were no differences in fixations during study or pen movements during recall. These results reveal that while drawings for distinct categories of scenes can be highly detailed and accurate, drawings for scenes from repeated categories, creating competition in memory, show reduced object detail.

I'm Lovin' It: How Fast Food Advertising Influences Meat-Eating Preferences.

Overconsumption of red and processed meat is associated with a multitude of negative health outcomes. Previous research shows exposure to advertising messaging can influence dietary behaviors but research on the influence of meat advertising on diet, specifically, is scant. Theoretically informed by the Reasoned Action Approach, the present experiment randomly assigned participants to view a version of a print McDonald's advertisement that included meat imagery (a Big Mac), non-meat imagery (French fries), or no food (just the McDonalds' logo and slogan), which acted as a control. An online survey in the United States included 514 U.S. adults (Mage = 51 years). Participants exposed to meat imagery compared to the non-meat imagery reported a higher desire to eat meat. The meat imagery and control conditions were also significantly associated with increased cognitive accessibility of meat concepts, compared to when respondents were shown the no-meat condition. Desire to eat meat, but not the cognitive accessibility of meat concepts, was significantly associated with attitude, normative pressure, and perceived behavioral control for avoiding eating meat one day per week; these constructs predicted intention and willingness to avoid meat. Results indicate that exposure to meat imagery in advertising does have the potential to influence meat consumption behavior and also has implications for the use of meat imagery in persuasive messaging for public health campaigns.

Nurse-led intervention to decrease drug use among LTBI positive homeless adults.

BACKGROUND: People experiencing homelessness (PEH) are disproportionately diagnosed with active tuberculosis. While promoting latent tuberculosis infection (LTBI) treatment has been a call to action, PEH engaging in substance use often experience challenges in completing LTBI treatment. METHODS: In this non-randomized single arm study, we tested an innovative, community-based, nurse-led community health worker (RN-CHW) model, on reducing drug use among 50 PEH, residing in homeless shelters or living on the streets in Los Angeles. Follow-up was at 3- and 6- months. RESULTS: Findings revealed significant and ongoing decrease in any drug use (odds ratio [OR] = 0.30; 95% confidence interval [CI] = 0.14-0.68); p = .004), amphetamine use (OR = 0.14; 95% CI = 0.02-0.81; p = .029), cannabis use (OR = 0.26; 95% CI = 0.12-0.57; p = .001) and methamphetamine use (OR = 0.30; 95% CI = 0.10-0.90; p = .031) at 6-month follow-up. CONCLUSIONS: To our knowledge, this pilot study is the first to evaluate the impact a RN-CHW delivered intervention on reduction in drug use among PEH enrolled in a LTBI intervention. LTBI interventions may serve as an entryway into reduction in drug use among this underserved population.

Family and Cultural Perceptions About Meat Consumption among Hispanic/Latino and White Adults in the United States.

The study examines the extent to which Hispanic/Latino Americans and non-Hispanic White Americans report that meat is an important aspect of their cultural beliefs and practices. An online national survey was completed using quota sampling to recruit approximately equal groups of U.S. Hispanic/Latino and non-Hispanic White Americans (n = 512). A path model predicting willingness to reduce meat consumption was estimated. Hispanic/Latino Americans viewed meat as a more integral aspect of their cultural food practices, and reported that meat dishes comprise a higher percentage of their culture's traditional foods. Cultural beliefs were associated with meat consumption and willingness to reduce meat consumption.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

Lidocaine infusions and reduced opioid consumption-Retrospective experience in pediatric hematology and oncology patients with refractory pain.

BACKGROUND: Despite a more robust experience with lidocaine infusions for pain management in adults and general pediatric population, there is limited evidence of efficacy of lidocaine infusions for pain management in patients with pediatric hematology and oncology diagnoses. METHODS: Data pertaining to continuous intravenous lidocaine infusions prescribed between January 2009 and June 2019 were reviewed, including patients' demographic characteristics, hematology/oncology and pain diagnoses, concurrent pain medications, and lidocaine infusion dose regimens and duration. Pain scores and opioid consumption calculations based on morphine equivalent doses (mg/kg/day) of patient-controlled analgesia were collected 1 day before infusion (D1), during infusion (D2), and 1 day after infusion (D3). RESULTS: The mean opioid consumption on D3 was significantly lower than that on D2 (p = .01). The pain scores on D3 were significantly lower than those on D1 when measured as average pain scores per 24 hours (p < .001) or as single pain scores immediately before and after infusions (p < .001). No significant associations were found between cumulative doses of lidocaine (loading dose plus total infusion dose) and either a decrease in the opioid consumption or a decrease in pain scores. CONCLUSIONS: In this retrospective series of pediatric hematology and oncology cases, we report positive outcomes in reducing opioid consumption and pain scores after lidocaine infusions. Prospective investigations designed in a collaborative, multi-institutional fashion, including a variety of pediatric populations are needed to further investigate the efficacy of lidocaine infusions.

The Mediating Role of Family and Cultural Food Beliefs on the Relationship between Family Communication Patterns and Diet and Health Issues across Racial/Ethnic Groups.

Consumption of red meat has been linked to a variety of health issues, yet Americans are resistant to reducing their meat consumption. Family communication environments shape beliefs about food and meat consumption, and therefore are locations for potential interventions to change the way people think about food. Families are embedded in cultures, and both family and cultural norms shape beliefs about what people should eat. This study (N = 773) is interested in understanding how family communication is associated with food beliefs, meat consumption, and health issues across three racial/ethnic groups: Black/African American (n = 256), Hispanic (n = 260), non-Hispanic White (n = 257). Structural equation modeling results showed that conversation orientation was consistently associated with stronger endorsement of family cultural food beliefs across race/ethnicity groups. Family food beliefs were associated with either more health issues or more meat consumption depending on race/ethnicity and mediated the association between conversation orientation and health issues/meat consumption. Conversation orientation moderated the association between conformity orientation and food beliefs for Hispanic and non-Hispanic White participants. Implications for family communication patterns theory and health scholars are discussed along with recommendations for culturally tailored family-focused health interventions.

Transitioning into the Community: Perceptions of Barriers and Facilitators Experienced By Formerly Incarcerated, Homeless Women During Reentry-A Qualitative Study.

Formerly incarcerated, homeless women on parole or probation experience individual-and structural-level barriers and facilitators as they prepare to transition into the community during reentry. A qualitative study was undertaken using focus group methods with formerly incarcerated, currently homeless women (N = 18, Mage = 37.67, SD 10.68, 23-53 years of age) exiting jail or prison. Major themes which emerged included the following: (1) access to resources-barriers and facilitators during community transition, (2) familial reconciliation and parenting during community transition, and (3) trauma and self-care support during community transition. These findings suggest a need to develop multi-level interventions at the individual, program and institutional/societal level with a gender-sensitive lens for women who are transitioning to community reentry. It is hoped that providing such resources will reduce the likelihood of homelessness and reincarceration.