Light management by algal aggregates in living photosynthetic hydrogels.

Rapid progress in algal biotechnology has triggered a growing interest in hydrogel-encapsulated microalgal cultivation, especially for the engineering of functional photosynthetic materials and biomass production. An overlooked characteristic of gel-encapsulated cultures is the emergence of cell aggregates, which are the result of the mechanical confinement of the cells. Such aggregates have a dramatic effect on the light management of gel-encapsulated photobioreactors and hence strongly affect the photosynthetic outcome. To evaluate such an effect, we experimentally studied the optical response of hydrogels containing algal aggregates and developed optical simulations to study the resultant light intensity profiles. The simulations are validated experimentally via transmittance measurements using an integrating sphere and aggregate volume analysis with confocal microscopy. Specifically, the heterogeneous distribution of cell aggregates in a hydrogel matrix can increase light penetration while alleviating photoinhibition more effectively than in a flat biofilm. Finally, we demonstrate that light harvesting efficiency can be further enhanced with the introduction of scattering particles within the hydrogel matrix, leading to a fourfold increase in biomass growth. Our study, therefore, highlights a strategy for the design of spatially efficient photosynthetic living materials that have important implications for the engineering of future algal cultivation systems.

Pathogen and Antibody Identification in Children with Encephalitis in Myanmar.

OBJECTIVE: Prospective studies of encephalitis are rare in regions where encephalitis is prevalent, such as low middle-income Southeast Asian countries. We compared the diagnostic yield of local and advanced tests in cases of pediatric encephalitis in Myanmar. METHODS: Children with suspected subacute or acute encephalitis at Yangon Children's Hospital, Yangon, Myanmar, were prospectively recruited from 2016-2018. Cohort 1 (n = 65) had locally available diagnostic testing, whereas cohort 2 (n = 38) had advanced tests for autoantibodies (ie, cell-based assays, tissue immunostaining, studies with cultured neurons) and infections (ie, BioFire FilmArray multiplex Meningitis/Encephalitis multiplex PCR panel, metagenomic sequencing, and pan-viral serologic testing [VirScan] of cerebrospinal fluid). RESULTS: A total of 20 cases (13 in cohort 1 and 7 in cohort 2) were found to have illnesses other than encephalitis. Of the 52 remaining cases in cohort 1, 43 (83%) had presumed infectious encephalitis, of which 2 cases (4%) had a confirmed infectious etiology. Nine cases (17%) had presumed autoimmune encephalitis. Of the 31 cases in cohort 2, 23 (74%) had presumed infectious encephalitis, of which one (3%) had confirmed infectious etiology using local tests only, whereas 8 (26%) had presumed autoimmune encephalitis. Advanced tests confirmed an additional 10 (32%) infections, 4 (13%) possible infections, and 5 (16%) cases of N-methyl-D-aspartate receptor antibody encephalitis. INTERPRETATION: Pediatric encephalitis is prevalent in Myanmar, and advanced technologies increase identification of treatable infectious and autoimmune causes. Developing affordable advanced tests to use globally represents a high clinical and research priority to improve the diagnosis and prognosis of encephalitis. ANN NEUROL 2023;93:615-628.

Living jewels: iterative evolution of iridescent blue leaves from helicoidal cell walls.

BACKGROUND AND AIMS: Structural colour is responsible for the remarkable metallic blue colour seen in the leaves of several plants. Species belonging to only ten genera have been investigated to date, revealing four photonic structures responsible for structurally coloured leaves. One of these is the helicoidal cell wall, known to create structural colour in the leaf cells of five taxa. Here we investigate a broad selection of land plants to understand the phylogenetic distribution of this photonic structure in leaves. METHODS: We identified helicoidal structures in the leaf epidermal cells of 19 species using transmission electron microscopy. Pitch measurements of the helicoids were compared with the reflectance spectra of circularly polarized light from the cells to confirm the structure-colour relationship. RESULTS: By incorporating species examined with a polarizing filter, our results increase the number of taxa with photonic helicoidal cell walls to species belonging to at least 35 genera. These include 19 monocot genera, from the orders Asparagales (Orchidaceae) and Poales (Cyperaceae, Eriocaulaceae, Rapateaceae) and 16 fern genera, from the orders Marattiales (Marattiaceae), Schizaeales (Anemiaceae) and Polypodiales (Blechnaceae, Dryopteridaceae, Lomariopsidaceae, Polypodiaceae, Pteridaceae, Tectariaceae). CONCLUSIONS: Our investigation adds considerably to the recorded diversity of plants with structurally coloured leaves. The iterative evolution of photonic helicoidal walls has resulted in a broad phylogenetic distribution, centred on ferns and monocots. We speculate that the primary function of the helicoidal wall is to provide strength and support, so structural colour could have evolved as a potentially beneficial chance function of this structure.

Late gestation fetal hypothyroidism alters cell cycle regulation across multiple organ systems.

BACKGROUND: Hypothyroidism is a common endocrine disruption observed in utero that adversely affects fetal growth and maturation leading to long-term impacts on health; however, the exact molecular mechanisms by which these deleterious effects occur are unknown. We hypothesize that fetal hypothyroidism during late gestation will disrupt cell cycle regulation in a tissue-specific manner. To evaluate this, eight pregnant gilts were dosed with either methimazole or an equivalent negative control during days 85-106 out of 114 days of gestation (n = 4/group). Following treatment, the gilts were humanely euthanized, and tissue samples of fetal heart, ileum, kidney, lung, liver, muscle, spleen, and thymus taken from two male and two female fetuses (n = 32) from each gilt. RESULTS: The relative expression of three cell cycle promoters (CDK1, CDK2, and CDK4), and one cell cycle inhibitor (CDKN1A) was compared in each tissue to determine the effect of hypothyroidism on the developing fetus. All of the eight tissues examined experienced at least one significant up- or downregulation in the expression of the aforementioned genes as a result of treatment with methimazole. Substantial changes were observed in the liver and muscle, with the latter experiencing significant downregulations of CDK1, CDK2, and CDK4 as a result of treatment. In addition, all tissues were examined for changes in protein content, which further elucidated the impact of hypothyroidism on the fetal liver by the observation of a marked increase in protein content in the methimazole-treated group. Finally, the heart and liver were histologically examined for evidence of cellular hyperplasia and hypertrophy by measuring average nuclei density and size in each tissue, with the results showing a significant decrease in average nuclei size in the liver of hypothyroid fetuses. CONCLUSIONS: Collectively, these findings indicate the occurrence of organ-specific disruptions in cell cycle progression as a result of in utero hypothyroidism, which may explain the long term and widespread effects of hypothyroidism on fetal development.

Political variations in pandemic lifestyles and COVID-19 vaccination by age cohort in the United States.

While recent scholarship suggests that political affiliation is a robust predictor of pandemic behaviors and COVID-19 vaccination status, research has yet to examine whether the impact of political affiliation on these outcomes vary by age. Drawing on health lifestyles theory, we contribute to the social epidemiology of infectious disease behaviors by testing whether the impact of political affiliation on risky pandemic health lifestyles and COVID vaccination varies by age cohort. We employ data collected from the 2021 Crime, Health, and Politics Survey (CHAPS), a national study of adults from the United States, to formally assess this understudied association. In all models, Democrats reported less risky pandemic lifestyles compared to their Republican counterparts. Moreover, Democrats displayed greater odds of being vaccinated than Republicans or Independents. Further, the impact of political affiliation on vaccination status varied by age cohort, such that the impact of political affiliation was stronger among the oldest adults in our sample. Our analyses contribute to the growing study of politics and health lifestyles by challenging theoretical perspectives and cultural narratives that claim that older adults are less swayed by political influence when it comes to healthcare decisions. Our results help better our understanding of the ways in which political discourse shapes adopting public health recommendations.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

Evaluation of US Medical Student Bias Toward Mental Health Before and After First-Year Pre-clinical Psychiatry Education.

OBJECTIVE: Much of mental health care is provided by non-psychiatric providers, and unfortunately, bias toward patients with mental health conditions leads to worsened outcomes. The authors endeavored to determine if pre-clinical medical student psychiatry education had an impact on these perceptions. METHODS: All 366 first-year medical students at Indiana University were invited to participate in a survey that consisted of the Mental Illness: Clinician's Attitudes version 2 (MICA-2) and six supplemental questions, pre- and post-course. RESULTS: One hundred seventeen students completed both surveys. The pre- and post-course means were 36.6 and 33.6, a change of - 2.9 (paired t-test p-value < 0.001), indicating a reduction in bias. CONCLUSIONS: These results suggest that pre-clinical education can lead to a measurable decrease in bias in medical students early in training. Unfortunately, individual question results and free responses continue to highlight significant bias in US medical students against mental illness and the field of psychiatry. Health care educators should be aware of these biases and their potential impact on patient outcomes so that these harmful perceptions can be targeted.

Racial-ethnic inequities in age at death among adults with/without intellectual and developmental disability in the United States.

To identify potential differences in racial-ethnic inequities in mortality between adults with/without intellectual and developmental disability, we compared patterns in age at death by race-ethnic status among adults who did/did not have intellectual and developmental disability reported on their death certificate in the United States. Data were from the 2005-2017 U.S. Multiple Cause-of-Death Mortality files. Average age at death by racial-ethnic status was compared between adults, age 18 and older, with/without different types of intellectual and developmental disability reported on their death certificate (N = 32,760,741). A multiple descent pattern was observed among adults without intellectual or developmental disability, with age at death highest among Whites, followed by Asians, Hispanics and Blacks, then American Indians. In contrast, a bifurcated pattern was observed among adults with intellectual disability, with age at death highest among Whites, but lower and similar among all racial-ethnic minority groups. The severity of racial-ethnic inequities in age at death was most pronounced among adults with cerebral palsy. Policy makers and public health experts should be aware that racial-ethnic inequities are different for adults with intellectual and developmental disability - all minorities with intellectual and developmental disability are at greater risk of premature death than their White counterparts.

On the relation between mind wandering, PTSD symptomology, and self-control.

Here we examined the association between mind wandering, post-traumatic stress disorder (PTSD) symptomology, and self-control. In a large undergraduate sample (N = 5,387), we assessed trait-levels of spontaneous and deliberate mind wandering, self-control, and PTSD symptomology. Results indicated that, while PTSD symptomology was uniquely positively associated with spontaneous mind wandering, it was negatively associated with deliberate mind wandering and self-control. These findings suggest that the mechanism(s) underlying everyday mind wandering may also underlie PTSD symptomology and traumatic intrusions. Moreover, the unique negative association between PTSD symptomatology and self-control suggests that PTSD is characterized not only by impairments in inhibiting unwanted thoughts (as indexed by mind wandering), but also by impairments in inhibiting other unwanted behaviors.

Re-examining the effect of motivation on intentional and unintentional task-unrelated thought: accounting for thought constraint produces novel results.

It has been proposed that motivating participants to perform well on a cognitive task ought to lead to decreases in rates of intentional, but not unintentional, task-unrelated thought (TUT; a commonly studied variety of mind wandering). However, at odds with this prediction, research has found that increasing motivation results in decreases in both intentional and unintentional TUTs. One possible explanation for this surprising finding is that standard assessments of TUT may inadvertently conflate TUTs with another variety of mind wandering: unconstrained thought. If so, then deconfounding task-unrelated and unconstrained varieties of mind wandering might produce the predicted effect of a decrease in intentional, but not unintentional, TUT when motivation is increased. To explore this possibility, in the present study, participants completed a sustained-attention task after receiving standard instructions (normal-motivation condition) or instructions informing them that they could leave the study early if they achieved a certain level of performance (motivated condition). Throughout the task, we assessed rates of TUT (both intentional and unintentional) and unconstrained thoughts. Consistent with prior work, the results indicated that motivated participants reported being on-task significantly more frequently than non-motivated participants. However, unlike previous work, we found that when deconfounding TUTs and unconstrained thoughts, participants in the motivation condition reported significantly fewer bouts of intentional TUT than those in the non-motivation condition, but no differences in rates of unintentional TUT were observed between groups. These results suggest that (a) motivation specifically targets intentional TUT and (b) standard assessments of TUT conflate task-relatedness and thought constraint.

Telehealth as an effective method of follow-up for pediatric post tonsillectomy patients.

PURPOSE: To determine if pediatric patients can be safely and effectively managed postoperatively with nurse led telehealth communication. METHODS: This is a retrospective case series conducted at a tertiary academic medical center pediatric otolaryngology practice. Retrospective chart review was conducted on patients <18 years old who underwent tonsillectomy from January 2017 to December 2019. Patients were managed postoperatively with a telehealth communication on postoperative day (POD) 3-5 and again at 4-6 weeks. Patient demographics, satisfaction with follow-up, number of office visits, and postoperative complications were recorded. RESULTS: 829 tonsillectomy patients were identified. Average patient age was 5.7 years (range 10 months-16 years). Successful contact was made with the patient's caregiver on POD 3-5 for 511 patients. 322 patients successfully completed 4-6 weeks telehealth follow-up. 292 patients (91 %) reported improvement in pre-operative symptoms at 4-6 weeks. Overall, 98 % of patients who completed telehealth follow-up were satisfied with this method and did not desire an additional office appointment. 62 patients (21 %) participated in an office follow-up in 2017, 54 patients in 2018 (19 %), and 36 patients (14 %) in 2019. Only 61 of these visits were routinely scheduled postoperative tonsillectomy office visits. 53 patients (6 %) had a postoperative tonsillectomy bleed and 31 patients (4 %) required return to the operating room for cauterization. CONCLUSION: Telehealth is successful in reducing the number of post-tonsillectomy office visits for pediatric patients without a subsequent increase in complications. Reduction in office visits can lead to cost reduction and increased availability of pediatric otolaryngology appointments.

Presenting Symptoms and Diagnosis of Vulvar Lichen Sclerosus in Premenopausal Women: A Cross-Sectional Study.

OBJECTIVES/PURPOSE: Presenting symptoms of vulvar lichen sclerosus (LS) specific to premenopausal women are not well reported in the literature and may differ from those in postmenopausal women. This study aimed to characterize the presentation of vulvar LS among premenopausal women. MATERIALS AND METHODS: An observational web-based study was conducted in premenopausal women with biopsy-confirmed vulvar LS between the ages of 18-50 years. Participants completed a 28-question survey evaluating characteristics of symptoms, timing of diagnosis, alternate diagnoses, and presence of concomitant autoimmune conditions. RESULTS: Of the 956 responses received, 503 met inclusion criteria of biopsy-confirmed LS and premenopausal status. Average age of symptom onset was 27 years, and average age of diagnosis was 32 years, with a 4-year delay in diagnosis. Symptoms most present were dyspareunia (68%) and tearing with intercourse or vaginal insertion (63%). Symptoms that affect the individual most were also dyspareunia (44%) and tearing with intercourse or vaginal insertion (39%). Symptoms that most frequently prompted patients to seek medical attention were dyspareunia (35%), pruritus (31%) and tearing with intercourse or vaginal insertion (26%). Most common skin changes included hypopigmentation (81%), vulvar fissures (72%), and labial resorption (60%), with fissures affecting the individual the most (48%). Sixty-six percent of the respondents initially received an alternative diagnosis, most commonly vulvovaginal yeast infection (49%). Hypothyroidism was the most common concurrent autoimmune condition (10%). CONCLUSIONS: Vulvar LS affects premenopausal women, commonly presenting with dyspareunia and tearing with intercourse. This condition should be considered and evaluated in premenopausal women presenting with vulvar symptoms and sexual pain.

Self-reported eating disorder psychopathology prevalence in community-based female and male Albertans: Gender and age group comparisons.

OBJECTIVE: We describe and compare eating disorder (ED) psychopathology prevalence in Canadian females and males across five age groups. METHOD: This secondary data analysis study used the Eating Disorder Examination Questionnaire (EDE-Q) to assess ED psychopathology in 1,316 Alberta community-based female and male participants, aged 15 to 71. RESULTS: Overall, 15.3% of female and 8.2% of male participants reported clinically-significant ED disturbances. Female participants reported greater global ED psychopathology than male participants, except older men and women aged 65-71, who reported similar, relatively low levels of global ED psychopathology. Women aged 65-71 endorsed less global ED psychopathology than younger females. More females than males reported regular binge eating and self-induced vomiting, whereas other ED behaviors were endorsed at similar rates. DISCUSSION: This large-scale study, the first involving a Canadian sample, contributes to a small literature on ED psychopathology in community-based samples. The finding that 15% of female and 8% of male Albertans aged 15 to 71 endorsed clinically-significant ED disturbances is concerning; however, women aged 65-71 reported lower ED psychopathology than younger females. Gender and age differences were observed in global ED psychopathology. Routine ED screening of non-clinical adolescents and adults is warranted to permit prevention and early intervention.

The relation between task-unrelated media multitasking and task-related motivation.

In two experiments, we explored the relation between participants' (a) levels of motivation to complete a task and (b) task-unrelated media multitasking. In Experiment 1, we examined the extent to which participants' levels of motivation to complete a task influenced their tendency to engage in task-unrelated media multitasking. Participants completed a 1-back task, while having the opportunity to turn on and off an unrelated, optional video. Results showed that participants who were told they would finish the experiment early if they achieved a sufficient level of performance (the motivated group) were significantly less likely to play the optional video during the 1-back task than those who were not given the opportunity to finish early (control condition). In Experiment 2, we examined the extent to which engaging in task-unrelated media multitasking affected task-related motivation. Three groups of participants completed a 1-back task, while (a) no video was presented, (b) a video was continuously played, or (c) participants could turn on and off a video at their leisure (as in Experiment 1). At both the beginning and the end of Experiment 2, participants were asked to indicate their level of motivation to complete the task. Interestingly, results revealed that continuously having the video playing helped sustain task-related motivation. Thus, although greater motivation to perform a task reduces the likelihood of engaging in task-unrelated media multitasking, such media multitasking also appears to increase levels of motivation.

Expression profiles and function of IL6 in polymorphonuclear myeloid-derived suppressor cells.

IL6 is an inflammatory cytokine with pleiotropic functions in both immune and nonimmune cells, and its expression level is inversely correlated with disease prognosis in patients with cancer. However, blocking IL6 alone has only yielded minimal efficacy in human cancer patients. We aimed at defining IL6 expression profiles under inflammatory conditions and cancer, and elucidating the mechanism underlying IL6 intrinsic signaling in colon carcinoma. We report here that colonic inflammation induces IL6 expression primarily in the CD11b+Ly6G+Ly6Clo polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in colon. Although both tumor cells, T cells and myeloid cells all express IL6, PMN-MDSCs are the primary cell type that express IL6 in colon carcinoma, suggesting that IL6 up-regulation is a response to inflammation in colon epithelium and tumor microenvironment. Furthermore, we determined that IL6 activates STAT3 to up-regulate DNMT1 and DNMT3b expression in colon tumor cells, thereby revealing an epigenetic mechanism that mediates the IL6-STAT3 signaling pathway in colon carcinoma. Surprisingly, knocking out IL6 in colon tumor cells did not significantly alter tumor growth in WT mice. Conversely, IL6-sufficient colon and pancreatic tumor grow at similar rate in WT and IL6-deficient mice. However, overexpression of IL6 in colon tumor cells significantly increases tumor growth in vivo. Our findings determine that a high tumor local IL6 threshold is essential for IL6 function in colon tumor promotion and targeting the IL6-expressing PMN-MDSCs is potentially an effective approach to suppress colon tumor growth in vivo.

Early Sibling Conflict May Ultimately Benefit the Family.

Relatives often interact differently with each other than with nonrelatives, and whether kin cooperate or compete has important consequences for the evolution of mating systems, seed size, dispersal, and competition. Previous research found that the larger of the size dimorphic seeds produced by the annual plant Aegilops triuncialis suppressed germination of their smaller sibs by 25%-60%. Here, we found evidence for kin recognition and sibling rivalry later in life among Aegilops seedlings that places seed-seed interactions in a broader context. In experiments with size dimorphic seeds, seedlings reduced the growth of sibling seedlings by ∼40% but that of nonsibling seedlings by ∼25%. These sequential antagonistic interactions between seeds and then seedlings provide insight into conflict and cooperation among kin. Kin-based conflict among seeds may maintain dormancy for some seeds until the coast is clear of more competitive siblings. If so, biotically induced seed dormancy may be a unique form of cooperation, which increases the inclusive fitness of maternal plants and offspring by minimizing competition among kin.

Exome-Wide Association Study of Pancreatic Cancer Risk.

We conducted a case-control exome-wide association study to discover germline variants in coding regions that affect risk for pancreatic cancer, combining data from 5 studies. We analyzed exome and genome sequencing data from 437 patients with pancreatic cancer (cases) and 1922 individuals not known to have cancer (controls). In the primary analysis, BRCA2 had the strongest enrichment for rare inactivating variants (17/437 cases vs 3/1922 controls) (P = 3.27x10-6; exome-wide statistical significance threshold P < 2.5x10-6). Cases had more rare inactivating variants in DNA repair genes than controls, even after excluding 13 genes known to predispose to pancreatic cancer (adjusted odds ratio, 1.35; P = .045). At the suggestive threshold (P < .001), 6 genes were enriched for rare damaging variants (UHMK1, AP1G2, DNTA, CHST6, FGFR3, and EPHA1) and 7 genes had associations with pancreatic cancer risk, based on the sequence-kernel association test. We confirmed variants in BRCA2 as the most common high-penetrant genetic factor associated with pancreatic cancer and we also identified candidate pancreatic cancer genes. Large collaborations and novel approaches are needed to overcome the genetic heterogeneity of pancreatic cancer predisposition.

Repeated restraint stress exposure during early withdrawal accelerates incubation of cue-induced cocaine craving.

A major challenge for treating cocaine addiction is the propensity for abstinent users to relapse. Two important triggers for relapse are cues associated with prior drug use and stressful life events. To study their interaction in promoting relapse during abstinence, we used the incubation model of craving and relapse in which cue-induced drug seeking progressively intensifies ('incubates') during withdrawal from extended-access cocaine self-administration. We tested rats for cue-induced cocaine seeking on withdrawal day (WD) 1. Rats were then subjected to repeated restraint stress or control conditions (seven sessions held between WD6 and WD14). All rats were tested again for cue-induced cocaine seeking on WD15, 1 day after the last stress or control session. Although controls showed a time-dependent increase in cue-induced cocaine seeking (incubation), rats exposed to repeated stress in early withdrawal exhibited a more robust increase in seeking behavior between WD1 and WD15. In separate stressed and control rats, equivalent cocaine seeking was observed on WD48. These results indicate that repeated stress in early withdrawal accelerates incubation of cocaine craving, although craving plateaus at the same level were observed in controls. However, 1 month after the WD48 test, rats subjected to repeated stress in early withdrawal showed enhanced cue-induced cocaine seeking following acute (24 hours) food deprivation stress. Together, these data indicate that chronic stress exposure enhances the initial rate of incubation of craving during early withdrawal, resulting in increased vulnerability to cue-induced relapse during this period, and may lead to a persistent increase in vulnerability to the relapse-promoting effects of stress.