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Internalized pools of membrane attack complexes (MACs) promote NF-kB and dysregulated tissue inflammation. Here, we show that C9, a MAC-associated protein, promotes loss of proteostasis to become intrinsically immunogenic. Surface-bound C9 is internalized into Rab5 + endosomes whose intraluminal acidification promotes C9 aggregates. A region within the MACPF/CDC domain of C9 stimulates aggrephagy to induce NF-kB, inflammatory genes, and EC activation. This process requires ZFYVE21, a Rab5 effector, which links LC3A/B on aggresome membranes to RNF34-P62 complexes to mediate C9 aggrephagy. C9 aggregates form in human tissues, C9-associated signaling responses occur in three mouse models, and ZFYVE21 stabilizes RNF34 to promote C9 aggrephagy in vivo. Gene-deficient mice lacking ZFYVE21 in ECs showed reduced MAC-induced tissue injury in a skin model of chronic rejection. While classically defined as cytotoxic effectors, MACs may impair proteostasis, forming aggregates that behave as intracellular alarmins.

Background: The assessment of heavy metals' effects on human health is frequently limited to investigating one metal or a group of related metals. The effect of heavy metals mixture on heart attack is unknown.

Methods: This study applied the Bayesian kernel machine regression model (BKMR) to the 2011-2016 National Health and Nutrition Examination Survey (NHANES) data to investigate the association between heavy metal mixture exposure with heart attack. 2972 participants over the age of 20 were included in the study.

Results: Results indicate that heart attack patients have higher levels of cadmium and lead in the blood and cadmium, cobalt, and tin in the urine, while having lower levels of mercury, manganese, and selenium in the blood and manganese, barium, tungsten, and strontium in the urine. The estimated risk of heart attack showed a negative association of 0.0030 units when all the metals were at their 25^th percentile compared to their 50^th percentile and a positive association of 0.0285 units when all the metals were at their 75^th percentile compared to their 50^th percentile. The results suggest that heavy metal exposure, especially cadmium and lead, may increase the risk of heart attacks.

Conclusions: This study suggests a possible association between heavy metal mixture exposure and heart attack and, additionally, demonstrates how the BKMR model can be used to investigate new combinations of exposures in future studies.

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

Extending the dosing interval of a primary series of mRNA COVID-19 vaccination has been employed to reduce myocarditis risk in adolescents, but previous evaluation of impact on vaccine effectiveness (VE) is limited to risk after second dose. Here, we quantified the impact of the dosing interval based on case notifications and vaccination uptake in Hong Kong from January to April 2022. We estimated that the hazard ratio (HR) and odds ratio (OR) of infections after the second dose for extended (28 days or more) versus regular (21-27 days) dosing intervals ranged from 0.86 to 0.99 from calendar-time proportional hazards models, and from 0.85 to 0.87 from matching approaches, respectively. Adolescents in the extended dosing groups (including those who did not receive a second dose in the study period) had a higher hazard of infection than those with a regular dosing interval during the intra-dose period (HR: 1.66; 95% CI: 1.07, 2.59; p = 0.02) after the first dose. Implementing an extended dosing interval should consider multiple factors including the degree of myocarditis risk, the degree of protection afforded by each dose, and the extra protection achievable using an extended dosing interval.

Objective: Premature infants frequently face feeding challenges due to disrupted coordination of sucking, swallowing, and breathing, increasing their risk of dysphagia. There are few effective treatment options available for these infants. In adults experiencing dysphagia, consuming cold foods or liquids can be an effective strategy. This method stimulates the sensory receptors in the pharyngeal mucosa, promoting safer and more effective swallowing. We have previously demonstrated that short-duration feeding (5 swallows) with cold liquid significantly reduces dysphagia in preterm infants; however, the impact of extended cold milk feeding remains unexplored. This study aims to assess the safety of cold milk feedings in preterm infants diagnosed with uncoordinated feeding patterns and its effect on feeding performance.

Study design: Preterm infants with uncoordinated feeding patterns (n=26) were randomized to be fed milk at either room or cold temperatures using an experimental, randomized crossover design. We monitored axillary and gastric content temperatures, mesenteric blood flow, and feeding performance.

Result: The findings suggest that preterm infants can safely tolerate cold milk without any clinically significant changes in temperature or mesenteric blood flow, and it may enhance certain aspects of feeding performance.

Conclusion: These results suggest that cold milk feeding could be a safe therapeutic option for preterm infants. These results highlight the potential for further comprehensive studies to explore the use of cold milk as an effective therapeutic approach for addressing feeding and swallowing difficulties in preterm infants. Registered at clinicaltrials.org #NCT04421482.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations (VM) - including small telangiectasias and large arteriovenous malformations (AVMs) - focally develop in multiple organs. There are few treatment options and no cure for HHT. Most HHT patients are heterozygous for loss-of-function mutations affecting Endoglin (ENG) or Alk1 (ACVRL1); however, why loss of these genes manifests as VMs remains poorly understood. To complement ongoing work in animal models, we have developed a fully human, cell-based microphysiological model based on our Vascularized Micro-organ (VMO) platform (the HHT-VMO) that recapitulates HHT patient VMs. Using inducible ACVRL1 -knockdown, we control timing and extent of endogenous Alk1 expression in primary human endothelial cells (EC). Resulting HHT-VMO VMs develop over several days. Interestingly, in chimera experiments AVM-like lesions can be comprised of both Alk1-intact and Alk1-deficient EC, suggesting possible cell non-autonomous effects. Single cell RNA sequencing data are consistent with microvessel pruning/regression as contributing to AVM formation, while loss of PDGFB implicates mural cell recruitment. Finally, lesion formation is blocked by the VEGFR inhibitor pazopanib, mirroring positive effects of this drug in patients. In summary, we have developed a novel HHT-on-a-chip model that faithfully reproduces HHT patient lesions and that can be used to better understand HHT disease biology and identify potential new HHT drugs.

Objective: The aim of this study was to develop a predictive model for uncorrected/actual fluid intelligence scores in 9-10 year old children using magnetic resonance T1-weighted imaging. Explore the predictive performance of an autoencoder model based on reconstruction regularization for fluid intelligence in adolescents.

Methods: We collected actual fluid intelligence scores and T1-weighted MRIs of 11,534 adolescents who completed baseline tasks from ABCD Data Release 3.0. A total of 148 ROIs were selected and 604 features were proposed by FreeSurfer segmentation. The training and testing sets were divided in a ratio of 7:3. To predict fluid intelligence scores, we used AE, MLP and classic machine learning models, and compared their performance on the test set. In addition, we explored their performance across gender subpopulations. Moreover, we evaluated the importance of features using the SHapley Additive Explain method. Results: The proposed model achieves optimal performance on the test set for predicting actual fluid intelligence scores (PCC = 0.209 ± 0.02, MSE = 105.212 ± 2.53). Results show that autoencoders with refactoring regularization are significantly more effective than MLPs and classical machine learning models. In addition, all models performed better on female adolescents than on male adolescents. Further analysis of relevant characteristics in different populations revealed that this may be related to gender differences in underlying fluid intelligence mechanisms.

Conclusions: We construct a weak but stable correlation between brain structural features and raw fluid intelligence using autoencoders. Future research may need to explore ensemble regression strategies utilizing multiple machine learning algorithms on multimodal data in order to improve the predictive performance of fluid intelligence based on neuroimaging features.

Background: Cervical fibrosis (CF) as a late consequence in patients after radiotherapy significantly impacts the long-term symptoms, functionality, and quality of life of these cancer patients due to a hardening process of different histological tissues. Modern Shear Wave Ultrasound Elastography now enables a differentiated analysis of the changes in various tissue types. In this study, tissue-specific changes in CF induced by radiation therapy in head and neck (ENT) cancer patients were quantified and correlated with cervical range of motion (CROM).

Materials and methods: 16 patients after radiation of the cervical lymphatic drainage were selected as the observation group (OG). Further, 16 people without radiation in the head and neck region were matched by gender, age, and BMI as the control group (CG). Stiffness measurements in kilopascal (kPa; 1 Pa = 1 N m^-2) were performed using shear wave elastography (SWE) to assess the elasticity of muscle, fascia, and subcutaneous tissue within and surrounding the sternocleidomastoid muscle (SCM). Specific parameters of the OG were compared to the CG and correlated with functional parameters and quality of life (QoL).

Results: The OG exhibited significantly higher stiffness values (Emean, Emax, Emin) across all tissue types than the CG, suggesting a tangible effect of radiation therapy on tissue stiffness. Muscle compartment analysis revealed the most significant stiffness differences. Thickness measurements indicated changes in the muscle and skin but not in the subcutaneous tissue. CROM measurements within the OG fell within normal ranges, suggesting a possible homogenizing effect of radiation treatment on CROM variability. Strong correlations were observed between age and specific stiffness measures, particularly in the OG group, indicating a broader impact of aging or radiation therapy on physiological measures. Significant correlations between tissue stiffness and CROM were found.

Conclusion: CF after radiotherapy occurs primarily in the muscle tissue and its fascia, with the hardening being about twice as pronounced as in the average population and becoming more pronounced with increasing age and correlates with CROM.

Background: Vancomycin, an antibiotic with activity against Methicillin-resistant Staphylococcus aureus (MRSA), is frequently included in empiric treatment for community-acquired pneumonia (CAP) despite the fact that MRSA is rarely implicated in CAP. Conducting polymerase chain reaction (PCR) testing on nasal swabs to identify the presence of MRSA colonization has been proposed as an antimicrobial stewardship intervention to reduce the use of vancomycin. Observational studies have shown reductions in vancomycin use after implementation of MRSA colonization testing, and this approach has been adopted by CAP guidelines. However, the ability of this intervention to safely reduce vancomycin use has yet to be tested in a randomized controlled trial.

Methods: STOP-Vanc is a pragmatic, prospective, single center, non-blinded randomized trial. Adult patients with suspicion for CAP who are receiving vancomycin and admitted to the Medical Intensive Care Unit at Vanderbilt University Medical Center will be screened for eligibility. Eligible patients will be enrolled and randomized in a 1:1 ratio to either receive MRSA nasal swab PCR testing in addition to usual care (intervention group), or usual care alone (control group). PCR testing results will be transmitted through the electronic health record to the treating clinicians. Primary providers of intervention group patients with negative swab results will also receive a page providing clinical guidance recommending discontinuation of vancomycin. The primary outcome will be vancomycin-free hours alive, defined as the number of hours alive and free of the use of vancomycin within the first seven days following trial enrollment estimated using a proportional odds ratio model. Secondary outcomes include 30-day all-cause mortality and time alive off vancomycin.

Discussion: STOP-Vanc will provide the first randomized controlled trial data regarding the use of MRSA nasal swab PCR testing to guide antibiotic de-escalation. This study will provide important information regarding the effect of MRSA PCR testing and antimicrobial stewardship guidance on clinical outcomes in an intensive care unit setting.

Trial registration: This trial was registered on ClinicalTrials.gov on February 22, 2024. (ClinicalTrials.gov identifier: NCT06272994).

Background-: Glaucoma is a complex multifactorial disease where apoptosis and inflammation represent two key pathogenic mechanisms. However, the relative contribution of apoptosis versus inflammation in axon degeneration and death of retinal ganglion cells (RGCs) is not well understood. In glaucoma, caspase-8 is linked to RGC apoptosis, as well as glial activation and neuroinflammation. To uncouple these two pathways and determine the extent to which caspase-8-mediated inflammation and/or apoptosis contributes to the death of RGCs, we used the caspase-8 D387A mutant mouse (Casp8 ^DA/DA ) in which a point mutation in the auto-cleavage site blocks caspase-8-mediated apoptosis but does not block caspase-8-mediated inflammation.

Methods-: Intracameral injection of magnetic microbeads was used to elevate the intraocular pressure (IOP) in wild-type, Fas deficient Fas^lpr, and Casp8 ^DA/DA mice. IOP was monitored by rebound tonometry. Two weeks post microbead injection, retinas were collected for microglia activation analysis. Five weeks post microbead injection, visual acuity and RGC function were assessed by optometer reflex (OMR) and pattern electroretinogram (pERG), respectively. Retina and optic nerves were processed for RGC and axon quantification. Two- and five-weeks post microbead injection, expression of the necrosis marker, RIPK3, was assessed by qPCR.

Results-: Wild-type, Fas^lpr, and Casp8 ^DA/DA mice showed similar IOP elevation as compared to saline controls. A significant reduction in both visual acuity and pERG that correlated with a significant loss of RGCs and axons was observed in wild-type but not in Fas^lpr mice. The Casp8 ^DA/DA mice displayed a significant reduction in visual acuity and pERG amplitude and loss of RGCs and axons similar to that in wild-type mice. Immunostaining revealed equal numbers of activated microglia, double positive for P2ry12 and IB4, in the retinas from microbead-injected wild-type and Casp8 ^DA/DA mutant mice. qPCR analysis revealed no induction of RIPK3 in wild-type or Casp8 ^DA/DA mice at two- or five-weeks post microbead injection.

Conclusions-: Our results demonstrate that caspase-8-mediated extrinsic apoptosis is not involved in the death of RGCs in the microbead-induced mouse model of glaucoma implicating caspase-8-mediated inflammation, but not apoptosis, as the driving force in glaucoma progression. Taken together, these results identify the caspase-8-mediated inflammatory pathway as a potential target for neuroprotection in glaucoma.