Clinical Outcomes after Median Arcuate Ligament Release in Patients Responsive to Celiac Plexus Block.

PURPOSE: To determine if symptom relief with celiac plexus block (CPB) is associated with favorable clinical outcomes after median arcuate ligament release (MALR) surgery. MATERIALS AND METHODS: A retrospective review was performed from January 2000 to December 2021. Fifty-seven patients (42 women, 15 men; mean age, 43 years [range, 18-84 years]) with clinical and radiographic features suggestive of median arcuate ligament syndrome (MALS) underwent computed tomography (CT)-guided percutaneous CPB for suspected MALS. Clinical outcomes of CPB and MALR surgery were correlated. Adverse events were classified according to the Society of Interventional Radiology (SIR) guidelines. RESULTS: CT-guided percutaneous CPB was successfully performed in all 57 (100%) patients with suspected MALS. A cohort of 38 (67%) patients showed clinical improvement with CPB. A subset of 28 (74%) patients in this group subsequently underwent open MALR surgery; 27 (96%) responders to CPB showed favorable clinical outcomes with surgery. There was 1 (4%) CPB-related mild adverse event. There were no moderate, severe, or life-threatening adverse events. CONCLUSIONS: Patients who responded to CPB were selected to undergo surgery, and 96% of them improved after surgery.

Long-Term Acetylcholinesterase Depletion Alters the Levels of Key Synaptic Proteins while Maintaining Neuronal Markers in the Aging Zebrafish (Danio rerio) Brain.

INTRODUCTION: Interventions targeting cholinergic neurotransmission like acetylcholinesterase (AChE) inhibition distinguish potential mechanisms to delay age-related impairments and attenuate deficits related to neurodegenerative diseases. However, the chronic effects of these interventions are not well described. METHODS: In the current study, global levels of cholinergic, cellular, synaptic, and inflammation-mediating proteins were assessed within the context of aging and chronic reduction of AChE activity. Long-term depletion of AChE activity was induced by using a mutant zebrafish line, and they were compared with the wildtype group at young and old ages. RESULTS: Results demonstrated that AChE activity was lower in both young and old mutants, and this decrease coincided with a reduction in ACh content. Additionally, an overall age-related reduction in AChE activity and the AChE/ACh ratio was observed, and this decline was more prominent in wildtype groups. The levels of an immature neuronal marker were upregulated in mutants, while a glial marker showed an overall reduction. Mutants had preserved levels of inhibitory and presynaptic elements with aging, whereas glutamate receptor subunit levels declined. CONCLUSION: Long-term AChE activity depletion induces synaptic and cellular alterations. These data provide further insights into molecular targets and adaptive responses following the long-term reduction of AChE activity that was also targeted pharmacologically to treat neurodegenerative diseases in human subjects.

Comparative analysis of the contribution of municipal waste management policies to GHG reductions in China.

Waste generation and disposal have been a global issue for decades. The total global greenhouse gas (GHG) emissions in 2019 were 49,758 MtCO2e with waste disposal accounting for 3.2%. With rapid urbanization trends, municipal solid waste (MSW) has become a global challenge which needs to be addressed. A large fraction of MSW such as food wastes, e-waste among others still ends up with unregulated dumps or openly burned in low-income countries. As a response, China initiated the 'zero-waste' pilot program which has been running since 2019. To investigate the potential contribution of MSW management to GHG reductions, this study selected four 'zero-waste' cities in China, namely Shenzhen, Panjin, Xining and Tongling, as case studies to assess the impacts of different MSW management policies on GHG reductions from 2015 to 2019. Results demonstrated that Shenzhen city achieved progress in reducing GHGs, which decreased by more than 40% between 2015 and 2019. This study provides policy recommendations and waste management approaches and practices to optimize MSW management and reduction of GHGs.

TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas.

Telomerase is reactivated in the majority of cancers. For instance, in gliomas, it is common that the TERT promoter is mutated. Research on telomere promoter GC islands have been focused primarily on proximal TERT promoter but little is known about the distal promoter. Therefore, in this study, we investigated the proximal and distal TERT promoter, in terms of DNA methylation. We did bisulfite sequencing in zebrafish tissue samples for the distal tert promoter. In the zebrafish brain tissues, we identified a hypomethylation site in the tert promoter, and found that this hypomethylation was associated with aging and shortened telomeres. Through site directed mutagenesis in glioma cell lines, we changed 10 GC spots individually, cloned into a reporter vector, and measured promoter activity. Finally, we silenced DNMT3B and measured telomerase activity along with vidaza and adriamycin treatments. Site directed mutagenesis of glioma cell lines revealed that each of the 10 GC spots are critical for telomerase activity. Changing GC to AT abolished promoter activity in all spots when transfected into glioma cell lines. Then, through silencing of DNMT3B, we observed a reduction in hTERT expression levels, while hTR remained the same, and a major increase in senescence-associated beta-galactosidase activity. Finally, we propose a model regarding the efficacy of two chemotherapeutic drugs, adriamycin and azacytidine, on gliomas. Here, we show that distal TERT promoter is critical; changing even one GC to AT abolishes TERT promoter activity. DNMT3B, a de novo methyltransferase, together with GC islands in distal TERT promoter plays an important role in regulation of telomerase expression and senescence.

Silky dogwood (Swida amomum) seed dispersal by freshwater turtles.

PREMISE: Effective seed dispersal is essential to the success of plant species. Swida amomum (silky dogwood) has a seed-dispersal syndrome characteristic of autumn-ripening shrubs with fleshy fruits; attached fruits are ingested and defecated by birds, while fallen fruits are consumed by ground-foraging birds and mammals. METHODS: We documented that fallen fruits of this shrub were consumed by two aquatic turtle species (eastern painted turtle [Chrysemys picta] and red-eared slider [Trachemys scripta]) and that their seeds were defecated. We compared germination success (percentage of seeds germinated) of defecated seeds, seeds collected from a pond surface, and seeds removed from shrubs. RESULTS: While four seed species were identified in fecal samples, seeds of S. amomum were the most frequent (93%) among samples and the most numerous (106 seeds) in any sample. Average proportion of fecal seeds germinated (85.99%) exceeded that of seeds from the pond surface (82.76%) and from shrubs (60.24%), albeit the difference in germination success was insignificant. When analyzed using fecal samples from painted turtles only, the difference in germination success between fecal seeds and those collected from pond or shrub became significant. CONCLUSIONS: Our findings represent the first report of S. amomum seeds being dispersed by turtle gut passage and suggest aquatic turtles could be an important part of a secondary seed dispersal process influencing woody plant community composition in temperate wetland ecosystems.

How does the global plastic waste trade contribute to environmental benefits: Implication for reductions of greenhouse gas emissions?

Plastic waste pollution has been identified as a serious global issue, posing environmental risks in terms of massive waste generation, ocean pollution, and increases in greenhouse gas (GHG) emissions. Despite documented environmental impacts, it remains debatable whether the global plastic waste trade (GPWT) for reutilization and recycling, as part of the global circular economy (CE), has historically contributed to environmental benefits. To investigate if historical GPWT has contributed to environmental benefits in terms of reductions of GHG emissions, this study analyzed GPWT between China and trading countries through their trajectories, characteristics and driving forces of reductions of GHG emissions between 1992 and 2017. Results indicated an increasing trend of reductions of GHG emissions in GPWT between China and trading countries over 25 years. A net reduction of 8.27 million metric tons carbon dioxide equivalent (CO2e) was observed in 2012, nearly 84 times levels observed in 1992. Policy implications after China's recent ban of imports of GPWT in December 2017 and recent changes of GPWT to other Asian countries are discussed. Dramatic changes in sustainable approaches to GPWT for reutilization and recycling are required.

The mental health of Australian medical practitioners during Covid-19.

OBJECTIVES: To ascertain whether doctors were experiencing higher rates of distress during Covid-19 and whether this was impacted by demographic factors. Our hypotheses were that being a junior doctor, having a previous mental health diagnosis and treating Covid-19 positive patients would predict higher rates of distress. METHODS: Cross-sectional survey conducted via Survey Monkey. Voluntary participants were recruited from the mailing list of a national-based referral service for doctors to psychiatrists. Distress was measured using the Kessler Psychological Distress Scale (K10). Demographic factors were analysed for predictive value of a higher rating on the K10. Areas of concern in relation to Covid-19 and preference for support services were measured on a Likert scale and compared to levels of distress. RESULTS: The rate of very high distress was 15%. Being a junior doctor and having a previous mental health diagnosis were predictive factors of a higher K10 score. K10 was not affected by likelihood of contact with Covid-19-positive patients. Social isolation had a larger impact on mental health in the context of a previous psychiatric diagnosis. Face-to-face assessments were preferred. CONCLUSIONS: Rates of distress in doctors have been higher than baseline during Covid-19. Some groups have been particularly vulnerable.

Sexual Dimorphism in Cardiovascular Disease Risk and Risk Factors Among Vegetarians: an Exploration of the Potential Mechanisms.

PURPOSE OF THE REVIEW: The goal of this review is to explore the potential mechanisms behind the observed phenomenon of sexual dimorphism in cardiovascular disease (CVD) risk among vegetarians. To date, research has primarily focused on the association between vegetarian diets and CVD risk factors and events. However, few publications have explored gender differences in the point estimates of risk of CVD in vegetarians. Current data suggest that following a vegetarian diet is associated with a larger CVD risk reduction in males. RECENT FINDINGS: Although not a completely novel observation, recent systematic reviews have reported sexual dimorphism in CVD outcomes among vegetarians. Data regarding other conditions of which metabolic dysfunction is a fundamental component, such as metabolic syndrome and diabetes, provide insight into potential mechanisms that may explain differences in CVD risk among male and female vegetarians. Evidence from observational studies suggests that a vegetarian diet is cardioprotective for both men and women. Differences in the point estimates of relative risk of major CVD outcomes for both genders when compared with their respective omnivorous counterparts raise the question of whether or not the benefits may be attenuated in women and whether this difference may be due to a combination of several behavioral and biological factors. Differences in dietary intake patterns and hormonal shifts which affect inflammation, body fat distribution, and metabolic syndrome risk have adverse effects on women as they age. Clinical trials comparing CVD outcomes in men and women who follow a vegetarian diet are needed to confirm the observed dimorphism in cohort studies and to elucidate the potential mechanisms that may explain this phenomenon. Currently, studies assessing the impact of other cardioprotective dietary interventions, such as the Mediterranean diet, indicate that there may be a physiological basis for the divergent outcomes of men and women who are given the same dietary intervention. Further research investigating the dietary habits of males and females, as well as other potential confounding lifestyle factors, is needed.

Characterizing Gestational Weight Gain According to Institute of Medicine Guidelines in Women with Type 1 Diabetes Mellitus: Association with Maternal and Perinatal Outcome.

Objective This study aims to evaluate the association between gestational weight gain (GWG) defined by the current Institute of Medicine (IOM) guidelines and pregnancy outcomes in women with type 1 diabetes mellitus (DM). Study design This is a secondary analysis of a cohort of 293 pregnancies of women with type 1 DM between 24 and 41 weeks' gestation. Women were categorized according to GWG per week over the second and third trimester: below, within, and above the IOM guidelines. Adjusted odds ratios (aORs) with 95% confidence intervals (CIs) were calculated for maternal and neonatal outcomes, controlling for covariates and confounders (referent: GWG within the IOM guidelines). Results Of the 293 women, there were 49 women (16.7%) with the GWG below the IOM guidelines, 86 women (29.4%) with the GWG within the IOM guidelines, and 158 women (53.9%) with the GWG above the IOM guidelines. Women with the GWG above the IOM guidelines had a higher risk of macrosomia and neonatal hyperbilirubinemia (aOR: 2.78; 95% CI: 1.23-6.30 and 2.31; 1.22-4.35, respectively). Conclusion GWG above the IOM guidelines is associated with an increased risk of macrosomia and neonatal hyperbilirubinemia. Maintaining GWG within the IOM guidelines may decrease the risk of excessive fetal growth and neonatal hyperbilirubinemia in infants of women with type 1 DM.

Characterization of a novel zebrafish (Danio rerio) gene, wdr81, associated with cerebellar ataxia, mental retardation and dysequilibrium syndrome (CAMRQ).

BACKGROUND: WDR81 (WD repeat-containing protein 81) is associated with cerebellar ataxia, mental retardation and disequilibrium syndrome (CAMRQ2, [MIM 610185]). Human and mouse studies suggest that it might be a gene of importance during neurodevelopment. This study aimed at fully characterizing the structure of the wdr81 transcript, detecting the possible transcript variants and revealing its expression profile in zebrafish, a powerful model organism for studying development and disease. RESULTS: As expected in human and mouse orthologous proteins, zebrafish wdr81 is predicted to possess a BEACH (Beige and Chediak-Higashi) domain, a major facilitator superfamily domain and WD40-repeats, which indicates a conserved function in these species. We observed that zebrafish wdr81 encodes one open reading frame while the transcript has one 5' untranslated region (UTR) and the prediction of the 3' UTR was mainly confirmed along with a detected insertion site in the embryo and adult brain. This insertion site was also found in testis, heart, liver, eye, tail and muscle, however, there was no amplicon in kidney, intestine and gills, which might be the result of possible alternative polyadenylation processes among tissues. The 5 and 18 hpf were critical timepoints of development regarding wdr81 expression. Furthermore, the signal of the RNA probe was stronger in the eye and brain at 18 and 48 hpf, then decreased at 72 hpf. Finally, expression of wdr81 was detected in the adult brain and eye tissues, including but not restricted to photoreceptors of the retina, presumptive Purkinje cells and some neurogenic brains regions. CONCLUSIONS: Taken together these data emphasize the importance of this gene during neurodevelopment and a possible role for neuronal proliferation. Our data provide a basis for further studies to fully understand the function of wdr81.

Gene expression changes in aging zebrafish (Danio rerio) brains are sexually dimorphic.

BACKGROUND: Brain aging is a multi-factorial process due to both genetic and environmental factors. The zebrafish has recently become a popular model organism for examining aging and age-related diseases because as in humans they age gradually and exhibit cognitive decline. Few studies have examined the biological changes in the aging brain that may contribute to these declines and none have examined them within individuals with respect to gender. Our aim was to identify the main genetic pathways associated with zebrafish brain aging across gender. We chose males and females from specific age groups (young, 7.5-8.5 months and old, 31-36 months) based on the progression of cognitive decline in zebrafish. RNA was isolated from individual brains and subjected to microarray and qPCR analysis. Statistical analyses were performed using a two-way ANOVA and the relevant post-hoc tests. RESULTS: Our results demonstrated that in the brains of young and old male and female zebrafish there were over 500 differentially expressed genes associated with multiple pathways but most notably were those related to neurogenesis and cell differentiation, as well as brain and nervous system development. CONCLUSIONS: The gene expression of multiple pathways is altered with age and differentially expressed in males and females. Future studies will be aimed at determining the causal relationships of age-related changes in gene expression in individual male and female brains, as well as possible interventions that counteract these alterations.

Passive exposure to visual motion leads to short-term changes in the optomotor response of aging zebrafish.

Numerous studies have shown that prior visual experiences play an important role in sensory processing and adapting behavior in a dynamic environment. A repeated and passive presentation of visual stimulus is one of the simplest procedures to manipulate acquired experiences. Using this approach, we aimed to investigate exposure-based visual learning of aging zebrafish and how cholinergic intervention is involved in exposure-induced changes. Our measurements included younger and older wild-type zebrafish and achesb55/+ mutants with decreased acetylcholinesterase activity. We examined both within-session and across-day changes in the zebrafish optomotor responses to repeated and passive exposure to visual motion. Our findings revealed short-term (within-session) changes in the magnitude of optomotor response (i.e., the amount of position shift by fish as a response to visual motion) rather than long-term and persistent effects across days. Moreover, the observed short-term changes were age- and genotype-dependent. Compared to the initial presentations of motion within a session, the magnitude of optomotor response to terminal presentations decreased in the older zebrafish. There was a similar robust decrease specific to achesb55/+ mutants. Taken together, these results point to short-term (within-session) alterations in the motion detection of adult zebrafish and suggest differential effects of neural aging and cholinergic system on the observed changes. These findings further provide important insights into adult zebrafish optomotor response to visual motion and contribute to understanding this reflexive behavior in the short- and long-term stimulation profiles.

A toxicology study of Csf2ra complementation and pulmonary macrophage transplantation therapy of hereditary PAP in mice.

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP), a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα+Mϕs) or saline-control intervention in Csf2raKO or wild-type (WT) mice including single ascending dose and repeat ascending dose studies evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα+Mϕs. Following PMT, mGM-Rα+Mϕs engrafted, remained within the lungs, and did not undergo uncontrolled proliferation or result in bronchospasm, pulmonary function abnormalities, pulmonary or systemic inflammation, anti-transgene product antibodies, or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient, minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14 days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα+Mϕs, respectively) and only in Csf2raKO mice but not in WT mice. PMT reduced lung disease severity in Csf2raKO mice. Results indicate PMT of mGM-Rα+Mϕs was safe, well tolerated, and therapeutically efficacious in Csf2raKO mice, and established a no adverse effect level and 10-fold safety margin.

Irreversible Electroporation as a Valid Treatment Option for Hepatic Epithelioid Hemangioendothelioma: An International Multicenter Experience.

PURPOSE: Hepatic epithelioid hemangioendothelioma (HEHE) is a rare tumor with currently no established standard of care. This international multicenter retrospective study assesses the use of percutaneous irreversible electroporation (IRE) as an ablative tool to treat HEHE and provides a clinical overview of the current management and role of IRE in HEHE treatment. MATERIAL AND METHODS: Between 2017 and 2023, 14 patients with 47 HEHE tumors were treated with percutaneous IRE using CT-scan guidance in 23 procedures. Baseline patient and tumor characteristics were evaluated. Primary outcome measures included safety and effectiveness, analyzed using Common Terminology Criteria for Adverse Events (CTCAE) and treatment response by mRECIST criteria. Secondary outcome measures included technical success, post-treatment tumor sizes and length of hospital stay. Technical success was defined as complete ablation with an adequate ablative margin (intentional tumor free ablation margin > 5 mm). RESULTS: IRE treatment resulted in technical success in all tumors. Following a median follow-up of 15 months, 30 tumors demonstrated a complete response according to mRECIST criteria. The average tumor size pre-treatment was 25.8 mm, accompanied by an average reduction in tumor size by 7.5 mm. In 38 out of 47 tumors, there was no evidence of local recurrence. In nine tumors, residual tumor was present. There were no cases of progressive disease. Median length of hospital stay was one day. Only one grade 3 CTCAE event occurred, a pneumothorax requiring chest tube placement. CONCLUSION: The current study provides evidence that IRE is a safe and efficacious minimally invasive treatment option for HEHE.

Review of clinical medicine and religious practice.

The purpose was to evaluate faith-based studies within the medical literature to determine whether there are ways to help physicians understand how religion affects patients' lives and diseases. We reviewed articles that assessed the influence of religious practices on medicine as a primary or secondary variable in clinical practice. This review evaluated 49 articles and found that religious faith is important to many patients, particularly those with serious disease, and patients depend on it as a positive coping mechanism. The findings of this review can suggest that patients frequently practice religion and interact with God about their disease state. This spiritual interaction may benefit the patient by providing comfort, increasing knowledge about their disease, greater treatment adherence, and quality of life. The results of prayer on specific disease states appear inconsistent with cardiovascular disease but stronger in other disease states.

Viability and In Vitro Gastrointestinal Transit Tolerance of Multispecies Probiotic Combinations Incorporated into Orange Juice and Drinking Water.

Little is known about how combining probiotics affects the storage survival and functional performance of individual probiotics when incorporated into non-dairy drinks. Viability of Lacticaseibacillus rhamnosus GG (LG), Limosilactobacillus reuteri ATCC 55730 (LR), Bifidobacterium animalis subsp. lactis BB-12 (Bb), and Propionibacterium jensenii 702 (PJ), either alone or in multi-species combinations included in orange juice (OJ), were assessed during storage in refrigerated conditions and compared with bottled water (BW). The tolerance of probiotics included in refrigerated OJ to simulated gastrointestinal conditions was also examined. LG and LR viabilities were significantly higher in OJ than in BW (p ≤ 0.001), while the reverse was evident for PJ. Bb maintained high viability in both drinks. LG-PJ in both drinks and Bb-PJ in BW resulted in greater viabilities among the paired combinations compared to their respective monocultures when incorporated separately (p ≤ 0.001). The viability of LG in the LG-Bb-PJ combination improved significantly in BW compared with LG alone (p ≤ 0.001). OJ did not alter bacterial tolerance to simulated gastric juice but diminished tolerance to simulated intestinal juice (SIJ). In all combinations, tolerance of LG and LR to SIJ was improved, whereas tolerance of PJ declined significantly compared with respective monocultures (p ≤ 0.001). In conclusion, probiotic storage stability and gastrointestinal transit tolerance were species-dependent and affected by carrier type and combinations. These effects should be considered when formulating probiotic products.

Caloric restriction reinforces the stem cell pool in the aged brain without affecting overall proliferation status.

Overfeeding (OF) and obesity increase the risk for brain aging and neurodegenerative diseases due to increased oxidative stress and neuroinflammation, which likely contribute to cellular dysfunction. In contrast, caloric restriction (CR) is an intervention known for its effects on extending both life- and health-span. In the current study, the effects on the aging brain of two short-term feeding regimens, OF and CR, were investigated. We applied these diets for 12 weeks to both young and aged zebrafish. We performed protein and mRNA level analysis to examine diet-mediated effects on any potential age-related alterations in the brain. Markers implicated in the regulation of brain aging, cell cycle, proliferation, inflammation, and cytoskeleton were analyzed. The most prominent result observed was a downregulation in the expression levels of the stem cell marker, Sox2, in CR-fed animals as compared to OF-fed fish. Furthermore, our data highlighted significant age-related downregulations in Tp53, Myca, and L-plastin levels. The multivariate analyses of all datasets suggested that as opposed to OF, the adaptive mechanisms increasing lifespan via CR are likely exerting their effects by reinforcing the stem cell pool and downregulating inflammation. The data reveal important therapeutic targets with respect to the state of nutrient uptake for the slowing down of the detrimental effects of aging, resulting in a healthy and extended lifespan, as well as lowering the risk for neurodegenerative disease.

Zebrafish optomotor response to second-order motion illustrates that age-related changes in motion detection depend on the activated motion system.

Various aspects of visual functioning, including motion perception, change with age. Yet, there is a lack of comprehensive understanding of age-related alterations at different stages of motion processing and in each motion system. To understand the effects of aging on second-order motion processing, we investigated optomotor responses (OMR) in younger and older wild-type (AB-strain) and acetylcholinesterase (achesb55/+) mutant zebrafish. The mutant fish with decreased levels of acetylcholinesterase have been shown to have delayed age-related cognitive decline. Compared to previous results on first-order motion, we found distinct changes in OMR to second-order motion. The polarity of OMR was dependent on age, such that second-order stimulation led to mainly negative OMR in the younger group while older zebrafish had positive responses. Hence, these findings revealed an overall aging effect on the detection of second-order motion. Moreover, neither the genotype of zebrafish nor the spatial frequency of motion significantly changed the response magnitude. Our findings support the view that age-related changes in motion detection depend on the activated motion system.

Synthesis and preclinical evaluation of QS-21 variants leading to simplified vaccine adjuvants and mechanistic probes.

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine.

The importance of Niemann-Pick C1 Like-1 (NPC1L1) protein in intestinal absorption of dietary sterols, including both cholesterol and phytosterols, is well documented. However, the exact mechanism by which NPC1L1 facilitates cholesterol transport remains controversial. This study administered 22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3ß-ol (NBD-cholesterol) and [(3)H]cholesterol to Npc1l1(+/+) and Npc1l1(-/-) mice to determine whether NPC1L1 facilitates dietary sterol uptake by enterocytes and/or participates in intracellular sterol delivery to the endoplasmic reticulum (ER) for lipoprotein assembly before secretion into plasma circulation. Results showed that [(3)H]cholesterol absorption was reduced but not abolished in Npc1l1(-/-) mice compared with Npc1l1(+/+) mice. In the presence of Pluronic L-81 to block pre-chylomicron exit from the ER, significant amounts of [(3)H]cholesterol were found to be associated with lipid droplets in the intestinal mucosa of both Npc1l1(+/+) and Npc1l1(-/-) mice, and the intracellular [(3)H]cholesterol can be esterified to cholesteryl esters. These results provided evidence indicating that the main function of NPC1L1 is to promote cholesterol uptake from the intestinal lumen but that it is not necessary for intracellular cholesterol transport to the ER. Surprisingly, NBD-cholesterol was taken up by intestinal mucosa, esterified to NBD-cholesteryl esters, and transported to plasma circulation to similar extent between Npc1l1(+/+) and Npc1l1(-/-) mice. Ezetimibe treatment also had no impact on NBD-cholesterol absorption by Npc1l1(+/+) mice. Thus, NBD-cholesterol absorption proceeds through an NPC1L1-independent and ezetimibe-insensitive sterol absorption mechanism. Taken together, these results indicate that NBD-cholesterol can be used to trace the alternative cholesterol absorption pathway but is not suitable for tracking NPC1L1-mediated cholesterol absorption.