Assessing eating ability and mealtime behaviors of persons living with dementia: A systematic review of instruments.

This systematic review aimed to describe the characteristics of instruments that assess eating ability and/or mealtime behaviors in persons living with dementia, and evaluate their psychometric properties. Five databases were searched for relevant records between 1/1/1980 and 5/25/2023. Records included instruments assessing eating ability and/or mealtime behaviors of people with dementia. The psychometric quality of the instruments was evaluated using the Psychometric Assessment for Self-report and Observational Tools (PAT). 45 eligible instruments were identified from 115 records. While 38 instruments were scored as having low psychometric quality, 7 had moderate quality. Edinburgh Feeding Evaluation in Dementia (EdFED), Mealtime Difficulty Scale for older adults with Dementia (MDSD), and Dementia Hyperphagic Behavior Scale (DHBS) were scored as having the highest quality (total PAT score = 9). Further refinement of existing instruments and additional psychometric testing in larger, diverse samples will improve pragmatic use in dementia mealtime care research and practice.

Multi-System-Level Analysis Reveals Differential Expression of Stress Response-Associated Genes in Inflammatory Solar Lentigo.

Although the pathogenesis of solar lentigo (SL) involves chronic ultraviolet (UV) exposure, cellular senescence, and upregulated melanogenesis, underlying molecular-level mechanisms associated with SL remain unclear. The aim of this study was to investigate the gene regulatory mechanisms intimately linked to inflammation in SL. Skin samples from patients with SL with or without histological inflammatory features were obtained. RNA-seq data from the samples were analyzed via multiple analysis approaches, including exploration of core inflammatory gene alterations, identifying functional pathways at both transcription and protein levels, comparison of inflammatory module (gene clusters) activation levels, and analyzing correlations between modules. These analyses disclosed specific core genes implicated in oxidative stress, especially the upregulation of nuclear factor kappa B in the inflammatory SLs, while genes associated with protective mechanisms, such as SLC6A9, were highly expressed in the non-inflammatory SLs. For inflammatory modules, Extracellular Immunity and Mitochondrial Innate Immunity were exclusively upregulated in the inflammatory SL. Analysis of protein-protein interactions revealed the significance of CXCR3 upregulation in the pathogenesis of inflammatory SL. In conclusion, the upregulation of stress response-associated genes and inflammatory pathways in response to UV-induced oxidative stress implies their involvement in the pathogenesis of inflammatory SL.

Effect of illite pretreatment on germinated Brown rice with Special Reference to amino acids, antioxidants, texture, and mineral elements.

The pretreatment process of various foods has been reported to improve their nutritional properties. The soaking of brown rice improves the texture and nutrients, which are crucial for cooking and maintaining its high functional value. Illite, a clay mineral, has recently been discovered to improve the nutritional value of seeds. Based on these findings, we soaked brown rice with different concentrations of illite solution for different durations and allowed the germination to perform analyses. Soaking the brown rice for 6 h with a germination period of 48 h was determined to be the optimal condition because of its higher sprout length. In addition, this optimal condition had improved textural characteristics such as reduced hardness, gumminess, chewiness, and cohesiveness, and it also had increased adhesiveness and stabilized resilience and springiness. The treatment solutions were free from heavy metal contaminants, whereas the mineral contents such as K, Ca, Fe, Mg, and Na were significantly increased with the increase in illite concentration. Moreover, our results showed that illite treatment could preserve the color appearance and seed germination. The ratio of essential amino acids to non-essential amino acids and antioxidants (phenolic contentγ-oryzanol, and flavonoid) of germinated brown rice was considerably increased with illite treatment. In germinated brown rice, an increase in DPPH and superoxide dismutase levels, a slight decrease in flavonoids, and no difference in polyphenol content were observed. These findings suggest that pre-soaking brown rice seeds with the appropriate concentration of illite could enhance their nutritional properties, which might attract consumers' interest to include this in their daily diet.

Introduction of extraperitoneal tunneling method: a way to secure the drain tube in the pelvic cavity after proctectomy.

The effectiveness of closed drainage tube insertion after low anterior resection has been controversial. We believe that drain tube displacement, which occurs up to 35% in real clinical practice, reduces the effectiveness of the drain tube. We report in this video a simple way to secure the drain tube in the pelvic cavity after low anterior resection and introduce a case that used the drain fixation method and treated anastomotic leakage without interventional procedure.

Evaluation of the reliability and validity of the upright head roll test for lateral semicircular canal benign paroxysmal positional vertigo.

BACKGROUND: The upright head roll test (UHRT) is a recently introduced diagnostic maneuver for lateral semicircular canal benign paroxysmal positional vertigo (LSC-BPPV). OBJECTIVE: This study aimed to evaluate the reliability and validity of the UHRT. METHODS: Two separate studies were conducted. Study 1 analyzed 827 results of videonystagmography (VNG) to assess UHRT reliability, and Study 2 analyzed 130 LSC-BPPV cases to evaluate UHRT validity. RESULTS: The inter-test reliability between UHRT and the supine head roll test (SHRT) showed substantial agreement (Cohen's kappa = 0.753) in direction-changing positional nystagmus (DCPN) and almost perfect agreement (Cohen's kappa = 0.836) in distinguishing the direction of DCPN. The validity assessment of UHRT showed high accuracy in diagnosing LSC-BPPV (80.0%) and in differentiating the variant types (74.6%). UHRT was highly accurate in diagnosing the canalolithiasis type in LSC-BPPV patients (Cohen's kappa = 0.835); however, it showed only moderate accuracy in diagnosing the cupulolithiasis type (Cohen's kappa = 0.415). The intensity of nystagmus in UHRT was relatively weaker than that in SHRT (P < 0.05). CONCLUSION: UHRT is a reliable test for diagnosing LSC-BPPV and distinguishing subtypes. However, UHRT has a limitation in discriminating the affected side owing to a weaker intensity of nystagmus than SHRT.

Assessing Amounts of Genetic Variability in Key Horticultural Traits Underlying Core Korean Breeding Lines of Cut Chrysanthemums.

The cut chrysanthemum holds one of the most substantial segments of the global floriculture market, particularly in Korea. We conducted a detailed assessment of the genetic structures across the cut chrysanthemum breeding lines in Korea. Using standard and spray chrysanthemum breeding lines from leading Korean research institutes, we first compared the variability of 12 horticultural traits, revealing a wide range of variation for most traits. We found that the overall flower diameter (OFD) and ray floret length (RFL) showed a solid positive relationship, regardless of the type. From a multivariate approach, OFD, RFL, and ray floret width (RFW) show consistently high association. Genotypic and phenotypic coefficients of variation analyses further indicated the significant genetic control over most traits. However, certain traits, like the volume of flowers (VF) in standard types, are more influenced by environments. Lastly, our analysis demonstrated substantial variability in broad-sense heritability (H); plant height (PH) consistently showed high H in both types. But the number of side branches (NOSB) and VF exhibited inconsistent H scores. These findings highlight the need for type-specific breeding strategies and modulating environmental management to optimize the trait expressions depending on the H scores, which offers significant implications for future breeding strategies.

Communication Patterns and Characteristics of Family Caregivers and Persons Living With Dementia: Secondary Analysis of Video Observation.

BACKGROUND: It is essential to characterize communication patterns for better health outcomes for family caregivers and persons living with dementia. OBJECTIVE: This study aimed to examine the relationships between communication patterns and the characteristics of dyads of family caregivers and persons living with dementia. METHODS: A secondary analysis was conducted using 75 video-recorded home care observations from 19 dyads. Participant characteristics and caregiver burden, depression, and sense of competence were collected from the parent study. The video-recorded dyadic communication patterns were assessed using a coding scheme developed based on Communication Accommodation Theory and Classical Test Theory. The relative frequency of the communication patterns was compared between groups. RESULTS: Overall, 8311 caregiver and 8024 care recipient communication behaviors were observed. Caregiver communication patterns were categorized as facilitative, disabling, and neutral. Care recipient communication patterns were categorized as engaging, challenging, and neutral. Caregiver gender, care recipient gender, care recipient education level, dementia diagnosis length, types of dementia, dyadic gender difference, burden, depression, and competence of caregiver, and types of communication were significantly associated with caregiver communication. Dementia diagnosis length, caregiver competence, dyadic gender difference, and types of communication were significantly associated with care recipient communication. CONCLUSIONS: The findings demonstrated different communication patterns depending on individual and dyad characteristics and evidence for dyadic communication support to promote meaningful interaction for persons living with dementia. Further analysis is needed to identify mediating factors and causal relationships.

Changes in the expression of satellite glial cell-specific markers during postnatal development of rat sympathetic ganglia.

The sympathetic ganglia represent a final motor pathway that mediates homeostatic "fight and flight" responses in the visceral organs. Satellite glial cells (SGCs) form a thin envelope close to the neuronal cell body and synapses in the sympathetic ganglia. This unique morphological feature suggests that neurons and SGCs form functional units for regulation of sympathetic output. In the present study, we addressed whether SGC-specific markers undergo age-dependent changes in the postnatal development of rat sympathetic ganglia. We found that fatty acid-binding protein 7 (FABP7) is an early SGC marker, whereas the S100B calcium-binding protein, inwardly rectifying potassium channel, Kir4.1 and small conductance calcium-activated potassium channel, SK3 are late SGC markers in the postnatal development of sympathetic ganglia. Unlike in sensory ganglia, FABP7 + SGC was barely detectable in adult sympathetic ganglia. The expression of connexin 43, a gap junction channel gradually increased with age, although it was detected in both SGCs and neurons in sympathetic ganglia. Glutamine synthetase was expressed in sensory, but not sympathetic SGCs. Unexpectedly, the sympathetic SGCs expressed a water-selective channel, aquaporin 1 instead of aquaporin 4, a pan-glial marker. However, aquaporin 1 was not detected in the SGCs encircling large neurons. Nerve injury and inflammation induced the upregulation of glial fibrillary acidic protein, suggesting that this protein is a hall marker of glial activation in the sympathetic ganglia. In conclusion, our findings provide basic information on the in vivo profiles of specific markers for identifying sympathetic SGCs at different stages of postnatal development in both healthy and diseased states.

Comparison of posttreatment stability after total mandibular arch distalization with mini-implants and mandibular setback surgery.

OBJECTIVES: To compare posttreatment stability in skeletal Class III patients between those treated by total mandibular arch distalization (TMAD) with buccal mini-implants and those by mandibular setback surgery (MSS). MATERIALS AND METHODS: The samples included 40 Class III adults, 20 treated by TMAD using buccal interradicular mini-implants and 20 treated with MSS. Lateral cephalograms were taken at pretreatment, posttreatment, and at least 1-year follow-up, and 24 variables were compared using statistical analysis. RESULTS: Mandibular first molars moved distally 1.9 mm with intrusion of 1.1 mm after treatment in the TMAD group. The mandibular incisors moved distally by 2.3 mm. The MSS group exhibited a significant skeletal change of the mandible, whereas the TMAD group did not. During retention, there were no skeletal or dental changes other than 0.6 mm labial movement of the mandibular incisors (P < .05) in the MSS group. There was 1.4° of mesial tipping (P < .01) and 0.4 mm of mesial movement of the mandibular molars and 1.9° of labial tipping (P < .001) and 0.8 mm of mesial movement of the mandibular incisors in the TMAD group. These dental changes were not significantly different between the two groups. CONCLUSIONS: The TMAD group showed a slightly decreased overjet with labial tipping of the mandibular incisors and mesial tipping of the first molars during retention. Posttreatment stability of the mandibular dentition was not significantly different between the groups. It can be useful to plan camouflage treatment by TMAD with mini-implants in mild-to-moderate Class III patients.

Towards a dynamic, comprehensive conceptualization of dyslexia.

Here we build from the central strength of the existing definition of dyslexia-its emphasis on neurobiological origins-and proffer a set of seven core principles for a new, more comprehensive conceptualization of dyslexia. These principles derive from two major research directions: (1) the still evolving history of attempts to explain dyslexia, including in varied writing systems; and (2) the study of the reading brain circuit, its development, and its genetic and environmental influences. What emerges from connecting these two directions is a dynamic conceptualization of dyslexia that incorporates the extensive research on the heterogeneity of dyslexia and the interdependent contributions of multiple biological and socio-cultural risk and preventive factors. A new definition of dyslexia, therefore, needs to transcend both past unitary characterizations and past assumptions based largely on the English orthography. Such a conceptualization references the ways that different languages interact with the reading brain circuit to produce different sources of reading failure. Similarly, the characteristics and consequences of dyslexia that have been considered as secondary sequela (e.g., reduced reading comprehension, social-emotional issues) should be part of a more comprehensive narrative. Of critical importance, any definition of dyslexia should clarify persisting misconceptions that associate dyslexia with a lack of intelligence, potential to learn, or talents. Thus, the overall purpose of such a definition should serve as an instrument of knowledge and an enduring reason for pursuing growth in reading for the individual, the educator, and the public.

Molecular Masking of Synthetic Immunomodulator Evokes Antitumor Immunity With Reduced Immune Tolerance and Systemic Toxicity by Temporal Activity Recovery and Sustained Stimulation.

Activation of the innate immune system counteracts tumor-induced immunosuppression. Hence, small molecule-based toll-like receptor 7/8 agonists (TLR7/8a), which can modulate immunosuppression in the tumor microenvironment along with the activation of innate immunity, are emerging as essential components of cancer immunotherapy. However, the clinical application of synthetic TLR7/8a therapies is limited by systemic immune-associated toxicity and immune tolerance induced by uncontrolled stimulatory activities and repeated treatments. To address these limitations, a dynamic immunomodulation strategy incorporating masking and temporal recovery of the activity of TLR7/8a through prodrug-like TLR7/8a (pro-TLR7/8a) at the molecular level and a sustained and controlled release of active TLR7/8a from nanoliposome (pro-TLR7/8a) (NL(pro-TLR7/8)) in a macroscale depot are designed. Immunization with cationic NL(pro-TLR7/8) and anionic antigens triggers robust activation of innate immune cells as well as antigen-specific T cell responses, eliciting reprogramming of immunosuppressive cells into tumor-suppressive cells, with decreased systemic adverse effects and immune tolerance. Combination treatment with NL(pro-TLR7/8a) and immune checkpoint inhibitors (anti-CTLA-4 plus anti-PD-L1) or nanoliposomes (Doxorubicin) has synergistic effects on antitumor immunity in various tumor models. The concept of pro-TLR7/8a suggested herein may facilitate the advancement of small-molecule-based immunomodulators for clinical translation and safe and effective cancer immunotherapy.

Cav3.2 T-Type Calcium Channel Mediates Acute Itch and Contributes to Chronic Itch and Inflammation in Experimental Atopic Dermatitis.

Voltage-gated calcium channels regulate neuronal excitability. The Cav3.2 isoform of the T-type voltage-activated calcium channel is expressed in sensory neurons and is implicated in pain transmission. However, its role in itch remains unclear. In this study, we demonstrated that Cav3.2 is expressed by mechanosensory and peptidergic subsets of mouse dorsal root ganglion neurons and colocalized with TRPV1 and receptors for type 2 cytokines. Cav3.2-positive neurons innervate human skin. A deficiency of Cav3.2 reduces histamine, IL-4/IL-13, and TSLP-induced itch in mice. Cav3.2 channels were upregulated in the dorsal root ganglia of an atopic dermatitis (AD)-like mouse model and mediated neuronal excitability. Genetic knockout of Cav3.2 or T-type calcium channel blocker mibefradil treatment reduced spontaneous and mechanically induced scratching behaviors and skin inflammation in an AD-like mouse model. Substance P and vasoactive intestinal polypeptide levels were increased in the trigeminal ganglia from AD-like mouse model, and genetic ablation or pharmacological inhibition of Cav3.2 reduced their gene expression. Cav3.2 knockout also attenuated the pathologic changes in ex vivo skin explants cocultured with trigeminal ganglia neurons from AD-induced mice. Our study identifies the role of Cav3.2 in both histaminergic and nonhistaminergic acute itch. Cav3.2 channel also contributes to AD-related chronic itch and neuroinflammation.

Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

Store-operated calcium entry in the satellite glial cells of rat sympathetic ganglia.

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca2+ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca2+ entry (SOCE) which is critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca2+ stores were depleted in the absence of extracellular Ca2+, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca2+ entry channels. Intracellular Ca2+ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca2+ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

Regulation of Flowering Time by Environmental Factors in Plants.

The timing of floral transition is determined by both endogenous molecular pathways and external environmental conditions. Among these environmental conditions, photoperiod acts as a cue to regulate the timing of flowering in response to seasonal changes. Additionally, it has become clear that various environmental factors also control the timing of floral transition. Environmental factor acts as either a positive or negative signal to modulate the timing of flowering, thereby establishing the optimal flowering time to maximize the reproductive success of plants. This review aims to summarize the effects of environmental factors such as photoperiod, light intensity, temperature changes, vernalization, drought, and salinity on the regulation of flowering time in plants, as well as to further explain the molecular mechanisms that link environmental factors to the internal flowering time regulation pathway.

The effect of surface treatment and low-temperature degradation on flexural strength of additive manufactured zirconia.

This study aimed to assess the effect of low-temperature degradation (LTD) and surface treatment on the flexural strength of additive-manufactured (AM) zirconia by comparison to subtractive-manufactured (SM) zirconia. Disc-shaped zirconia specimens were fabricated using AM and SM technology, and each group was assigned to 3 subgroups according to the type of surface treatment: control, sandblasting (SB), and 9% hydrofluoric acid etching (HF). The groups were then further divided into 2 subgroups: unaged and aged. Biaxial flexural strength, crystal phase, surface topography, and surface roughness were measured to evaluate the mechanical properties. Statistical analyses were performed with 3-way ANOVA, followed by the comparison of means with Bonferroni post hoc analyses. The means and standard deviations of the biaxial flexural strength and Weibull parameters were calculated with descriptive statistics. All SM groups showed significantly greater flexural strength than the AM groups (p < .05), and LTD did not affect flexural strength except for the SMHF group (p < .05). After LTD, monoclinic phases (m-phase) were found in all groups, and SEM images showed grain pullout due to zirconia volume expansion in both control groups. Sandblasting significantly affected flexural strength (p < .05), whereas the HF group did not affect flexural strength except in the SMHF group after LTD (p < .05). No significant difference was observed in the surface roughness of AM compared to SM groups conditioned with the same surface treatment regardless of LTD. AM zirconia has comparable mechanical properties to SM zirconia, regardless of low-temperature degradation and surface treatment, which indicates the potential of the AM technique for clinical applications.

Korean Immigrant Mothers and the Journey to Autism Diagnosis and Services for Their Child in the United States.

Since autism diagnosis is directly linked to the availability of supportive services, identifying best practices for early diagnosis of autism has long been a concern of professionals and families. Meanwhile, studies show persistent racial disparities in autism diagnosis. Although numerous clinical diagnostic guidelines have been published, there is not enough discussion of diagnostic procedures through the lens of culturally diverse families. PURPOSE: This study focuses on the autism diagnostic experiences that Korean immigrant mothers had with their children. METHODS: Eleven first-generation Korean-American mothers of children with autism were included in the study. The data was collected using semi-structured interviews in Korean. RESULTS: The main five factors (i.e., cultural beliefs and values, language barriers, complex emotions, immigration and navigating systems, and facilitators and assets) that mainly influence the diagnosis process were identified through thematic analysis. CONCLUSION: Dynamics are interactive within and between the factors, influencing the entire diagnostic process by either delaying or facilitating the identification of a child's autism and the provision of treatment.

Detection of thiocyanate in postmortem blood of a fatal potassium thiocyanate-poisoning patient using gas chromatography-mass spectrometry.

Thiocyanate is an inorganic compound used in industrial applications. Here, we report a case of suicidal death due to acute thiocyanate overdose. A 44-year-old man who consumed an unknown amount of thiocyanate solution was transferred to the emergency room and died 2 h after admission. An autopsy was performed 2 days after death. General toxicological analysis of blood using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry found no drug or alcohol. Quantification using GC-MS post-derivatization with pentafluorobenzyl bromide revealed 2,290 and 1,920 mg/L of thiocyanate in the heart and femoral blood samples, respectively. Thus, the cause of death was attributed to thiocyanate overdose. This study provides useful information for the interpretation of thiocyanate-related fatalities.

The effectiveness of negative-pressure wound therapy for wound healing after stoma reversal: a randomized control study.

Purpose: The purse-string closure (PSC) method is used for skin closure after stoma reversal to reduce surgical site infections (SSIs). However, PSC requires a longer healing period than primary closure. The application of negative-pressure wound therapy (NPWT) may reduce the healing period of many wound types. This study aimed to investigate whether the application of NPWT promotes wound healing after the PSC method for stoma reversal. Methods: This study was a randomized controlled study. Patients who had undergone stoma reversal surgery were randomized to receive NPWT or simple dressing after surgery. The primary outcome was a complete wound-healing period. We also investigated SSI, hospital stay, total cost, and the patient and observer scar assessment scale (POSAS) as secondary outcomes. Results: A total of 36 patients were randomized into 2 groups. The median wound healing period was shorter in the NPWT group than in the control group (17.5 days [range, 11-24 days] vs. 21.5 days [range, 14-41 days], P = 0.006). SSI rate and hospital stay did not differ between the groups. However, the number of dressings was lower in the NPWT group than in the control group (5 [range, 3-7] vs. 17 [range, 10-30], P < 0.001). The total cost for dressing was comparable between the groups. The POSAS was not different between the groups. Conclusion: This study revealed that NPWT application after PSC for stoma reversal site is effective in reducing the wound healing period compared to simple dressing, without increasing SSI and total cost.