United States

Background
The pathogenesis of allergic asthma is classically understood to involve allergens crosslinking Immunoglobulin E (IgE) bound to mast cells and basophils, leading to the release of inflammatory mediators. However, omalizumab, an anti-IgE antibody, reduces exacerbation rates in only about 40% of adult patients with severe asthma. These findings highlight the need for further research to fully address the role of IgE in allergic diseases like asthma. This study aims to explore which allergen induces Th2-associated airway inflammation through an IgE-dependent mechanism. 

Methods
We used IgE deficient mice (IgE KO) to generate asthma mouse models induced by two highly clinically relevant allergens house dust mite extract (HDM), cockroach extract (CRE) and one classic allergen ovalbumin (OVA). Hematoxylin and Eosin (HE) staining and Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining was performed on lung tissue to evaluate inflammatory responses and mucus production, respectively. The count of inflammatory cells in Bronchial Alveolar Lavage Fluids (BALF) was determined using flow cytometry. Passive Cutaneous Anaphylaxis (PCA) was conducted to quantify IgE involvement in the anaphylactic reaction, and Toluidine Blue (TB) staining of dorsal skin tissue was used to visualize mast cell recruitment.

Results
All mice treated with allergens exhibited significant signs of airway inflammation, as evidenced by the recruitment of inflammatory cells to the lung parenchyma and goblet cell hyperplasia. However, IgE KO mice treated with different allergens demonstrated significantly variable degrees of inflammation compared to the WT control. Of these, HDM-treated IgE KO mice showed an attenuated inflammatory profile with reduced inflammatory cells, particularly eosinophils. These mice had lower serum levels of HDM-specific IgE1 (sIgE1) and IgG1 (sIgG1) and reduced levels of Th2 cytokines IL-4 and IL-5 in BALF. While CRE-treated IgE KO mice also displayed a reduced inflammation compared to WT mice, the only statistically significant difference was found for eosinophils. No significant differences were observed between OVA-treated IgE KO and WT mice. These findings were further supported by our well-established PCA models. Both HDM and CRE-induced PCA model showed significantly less Evan’s blue dye leakage and mast cell recruitment in the ears of IgE KO mice compared to WT mice. No differences were observed for OVA-induced PCA and mast cell recruitment.

Conclusion
The results highlight the variability in IgE pathway dependence among different allergens, emphasizing the need for further research is to delineate the mechanisms and develop more effective therapeutic strategies for allergic diseases.

AMA Research Challenge 2024 

Allergic Asthma and Anaphylaxis Induced by Different Allergens Show Distinctive IgE Dependence

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AAPT SM 20

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Abstract Title:
Antimicrobial Effects of Inhaled Nitric Oxide Therapy on Pseudomonas Aeruginosa Lung Colonization in a Swine Model of Independent Lung Ventilation.

Background:
Pseudomonas Aeruginosa is the most common cause of gram-negative nosocomial pneumonia, and up to 26% of ventilator-associated pneumonia globally. Recent findings indicate that inhaled nitric oxide (iNO) at 300 parts-per-million (ppm) effectively reduces lung bacterial colonization of Klebsiella pneumonia and Pseudomonas aeruginosa (P. aeruginosa). Unlike inhaled antibiotics, iNO300 therapy might enhance the immune response in non-ventilated lung regions due to consolidated parenchyma. 

Methods:
Twelve swine were kept under general anesthesia and mechanical ventilation for 72 hours. All pigs were challenged with P. aeruginosa via bronchoscopy and twice via nebulization at 24 and 48 hours. The control group (n=6) received standard ventilation through a single-lumen endotracheal tube. The iNO300 group (n=6) was ventilated using a double-lumen endotracheal tube (DLT), with one lung receiving iNO300 and the other standard ventilation. Lung isolation was confirmed through respiratory mechanics, bronchoscopy, and lung imaging. iNO300 was administered for 30 minutes every 4 hours, to right (n=3) and left (n=3) lungs in different animals. After 72 hours, lung tissue samples were collected. Measurements of Colony Forming Units (CFUs)/gram of P. aeruginosa in each lung lobe, and pathohistological scoring of infection and inflammation (0=none/minimal, 1=focal/mild, 2=moderate, 3=diffuse/severe), were obtained. Percent methemoglobin (MetHb), mean arterial pressure, and PaO2/FiO2 were measured during treatments.

Results:
Lung tissue samples demonstrated that animals treated with iNO300 had statistically lower bacterial loads in both lungs compared to the control group (2.3±1.0 vs. 4.4±0.6 Log10 CFU/g, p-value=0.045). In the iNO group there was no difference between the treated and non-treated lungs (2.3±1.0 vs 2.3±1.4 Log10 CFU/g, p-value=0.98).

Compared to controls, iNO300-treated swine showed less severe signs of acute bronchopneumonia (0.59±0.91, vs 1.91±0.72, p-value <0.001), interstitial inflammation (0.54±0.63 vs 1.19±0.69, p-value <0.001) and airway inflammation (0.67±0.7 vs 1.46±0.69, p-value <0.001) in both lungs. In the iNO group there was no difference in acute bronchopneumonia (p-value=0.71) or interstitial inflammation (p-value=0.76) between treated and non-treated lungs.

Baseline MetHb levels were measured (1.73±1.02%), and levels peaked 30 minutes (3.67±1.82%) during iNO treatments but returned to baseline within 30 minutes post-treatment (1.13±0.76%). Mean arterial pressure (p-value=0.97) and PaO2/FiO2 (p-value=0.22) remained stable during treatments

Conclusion:
In a swine model of P. aeruginosa pneumonia, iNO300 delivered to only one lung reduces bacterial load and lung tissue inflammation in both treated and untreated lungs, suggesting antimicrobial mechanisms beyond direct contact. The transient rise in MetHb levels and stable hemodynamic parameters indicate iNO300 does not adversely affect cardiovascular health.

Antimicrobial Effects of Inhaled Nitric Oxide Therapy on Pseudomonas Aeruginosa Lung Colonization



WHOLE TESTES CRYOPRESERVATION FOR FUTURE AUTO-TRANSPLANTATION: COMPARISON OF DIFFERENT FREEZING METHODS 



Chenchu Nagarakanti, MD1; Robert R.A. Wilson, Bsc1; Elizabeth D Greene, LATG.2; Ibrahim Hacibey, MD1; Megan Escott, MD1,4; Yalcin Kulahci1; Vijay Gorantla1; Anthony Atala, MD1,3; Kelvin G.M. Brockbank, PhD2,3; Hooman Sadri-Ardekani, MD, PhD1,3 

1Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC. 

2Tissue Testing Technologies LLC, North Charleston, SC 

3Department of Bioengineering, Clemson University, Clemson, SC 

4Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC. 



Background: 

In the past decades, survival rates of cancer patients have increased significantly, though infertility remains a long-term complication of cancer therapy. Whole testes (WT) cryopreservation for successive transplantation is one of the potential fertility-sparing strategies in childhood cancer patients with solid tumors. 

Methods: 

The study involved eight rats, each were sacrificed and submitted to orchiectomy. WT organs from each rat were randomly assigned to four groups: Group A, fresh tissue (control); Group B, slow freezing with 10% DMSO and rewarmed at 37°C; Group C, slow freezing with 10% DMSO and rewarmed on ice, and Group D, vitrified using VS55, a complex mixture containing DMSO, formamide, and 1,2-propanedio. Diluted VS55 (4°C) was added sequentially to achieve full-strength VS55. The samples were rapidly cooled to -100°C in 2-methylbutane and then transferred to vapor phase nitrogen for slower cooling and storage below -135°C for >24 h before testing (vitrification). Vitrified testes were slowly warmed to -100°C followed by rapid warming to melting. Each group had two replicates from different rats. Each testicle was measured metrically and with an orchidometer. Histological examination was conducted to assess morphological changes in the testicular tissues. Immunohistochemical staining techniques, specifically targeting PGP, Calretinin, Vimentin and Smooth Muscle Actine (SMA) were employed to identify undifferentiated spermatogonia, Leydig cells, Sertoli cells and peritubular cells. Additionally, micro-CT scanning was used to evaluate the distribution and effectiveness of DMSO within the testicular tissue across different loading times (from 6 hours to 4 days). 

Results: 

Slight morphological alterations were observed in the experimental groups compared to the control, including testicle tubule tissue shrinkage in vitrified samples. Frozen tissue demonstrated more significant tubule tissue shrinkage, minor disruption of the epididymal tissues, and more significant disruption of the endothelial layers of the blood vessels in the vascular pedicle. Arterial vasospasm was present in all four groups. PGP 9.5 IHC identified undifferentiated spermatogonial cells. 1-way ANOVA test showed a statistically significantly lower number of cells in the three experimental groups than in the control. It found that 10% DMSO gave 10% concentration in the testes after 12 hours of loading time, which is close to optimum. 

Conclusion: 

Slow freezing with loading 10% DMSO for 12 hours could preserve WT structure cell-specific sensitivity to the freeze-thaw process has been detected. All methods revealed significantly decreased PGP9.5 and calretinin expression, and further studies are necessary to optimize PGP 9.5 and calretinin expression and confirm testicular tissue function for possible auto-transplantation

WHOLE TESTES CRYOPRESERVATION FOR FUTURE AUTO-TRANSPLANTATION:
Comparison of Different Freezing Methods

Abstract Title
Retrospective Study of the Relationship between Daily Oral Multivitamin Intake and Sleep Complaints in Post-Menopausal Women – An Analysis of NHANES Data (1999-2020)

Background
This study investigates whether daily oral multivitamin intake affects sleep patterns and sleep complaints in post-menopausal women. The hypothesis is that daily multivitamin intake may improve sleep quality in this demographic.

Methods
Data from the NHANES surveys (1999-2020) were analyzed to study the relationship between multivitamin use and sleep complaints. Dietary supplement usage, menopause status, and sleep data were extracted and pre-processed. Participants were categorized into two groups: those who took daily multivitamins and those who did not. Post-menopausal status was identified, and relevant sleep data from 2005-2020 was included. The final sample comprised 1,647 post-menopausal women who took daily multivitamins and 2,643 who did not.

The primary variable of interest was hours of sleep per night (SLD010/12). Outliers were removed, and the Shapiro-Wilk test was conducted to assess normality. Due to non-normal distributions (p < 0.05), the Mann-Whitney U test was used to compare the sleep duration between groups.

The secondary variable was sleep complaints (SLQ050), evaluated using a Chi-Square test of independence to assess the association between multivitamin use and reported sleep troubles. A post-hoc Fisher’s Exact test was also performed to confirm the significance of the findings.

Results
The analysis revealed that post-menopausal women who took daily multivitamins had a significantly higher mean sleep duration (7.289 hours) compared to those who did not take multivitamins (6.946 hours), with a median of 7.0 hours for both groups. The Mann-Whitney U test indicated a significant difference in sleep duration between the groups (Z = -6.754, p < 0.001). Additionally, there was a significant association between multivitamin use and reported sleep troubles, with the Chi-Square test showing (Χ² (1) = 4.167, p = .041) and the Fisher’s Exact test confirming a statistically significant difference (p < 0.001). These findings suggest that while multivitamin intake may increase sleep duration, it is also associated with a higher incidence of reported sleep troubles among post-menopausal women.

Conclusion
Daily multivitamin use may increase the number of sleep hours per night but is also associated with reported sleep troubles. These findings highlight the importance of discussing multivitamin use with healthcare providers for post-menopausal women. Further research should consider additional confounding factors and the broader implications of multivitamin use on sleep quality.

Retrospective Study of the Relationship between Daily Oral Multivitamin Intake and Sleep Complaints

Background
Endothelial Notch signaling is a key player in regulating inflammation. While Notch1 has been extensively studied in the context of vascular inflammation, the role of endothelial Notch4, remains relatively unexplored. Growing evidence suggests that Notch4 plays critical roles in human endothelial inflammatory disorders. Polymorphisms in Notch4 are associated with endothelial dysfunction in preeclampsia, HIV-triggered glomerular injury, and severity of COVID-19 endothelial inflammation. Our recent work has shown that pharmacologic inhibition of endothelial Notch4 signaling using an anti-Notch4 antibody alters macrophage number and phenotype in mouse models of breast cancer, further supporting a distinct role for Notch4 in regulation of inflammatory targets. Our preliminary work has determined that Notch4 regulates an endothelial transcriptome distinct from Notch1, which is enriched for inflammatory genes such as CCL2 and IL6. However, all Notch proteins bind DNA via a common transcriptional co-factor, CSL, and the mechanism of how different Notch proteins regulate distinct transcriptional targets remains unclear. Better methods for detection of real-time and gene-specific activation of Notch signaling are critical for dissecting the roles of different Notch proteins in endothelium.

Methods
To examine gene-specific Notch transcriptional activity, we used the NanoBiT protein-protein-interaction system, a complementation-based luciferase reporter system composed of two split luciferase fragments, LgBiT and SmBiT. The NanoBiT PPI system allows for detection of protein:protein interactions through two subunits, Large BiT (LgBit) and Small BiT (SmBit), fused to two proteins of interest. Interaction between target proteins allows the subunits to come together resulting in luminescent signal that can be quantified in live cells. Real-time quantification allows for dynamic monitoring, capturing transient interactions and providing temporal resolution that homogenate testing lacks. We designed fusions of the active intracellular domain of Notch4 (ICN4) or Notch1 (ICN1) with SmBiT, and CSL with LgBiT and expressed the fusion proteins in HeLa cells. The resulting interactions between fusion proteins resulted in luminescent signal that was quantified using the SpectraMax L Microplate Reader. 

Results
We validated that NanoBiT CSL and ICN fusion proteins were expressed at high levels and retained their ability to induce Notch pathway targets. Co-expression of CSL-LgBiT with either ICN4-SmBiT or ICN1-SmBiT resulted in robust induction of luminescent signal. Interaction between CSL and ICN4 consistently yielded a higher luminescent signal than ICN1, suggesting that Notch4 may more strongly interact with CSL. 

Conclusion
We have designed and tested a novel method for real time measurement of gene-specific Notch pathway activation. Our preliminary findings suggest that Notch4 plays a distinct role in inflammatory signaling, possibly due to its robust interaction with CSL. Our NanoBiT-based assay may be used to determine protective targets in inflammatory diseases. 


Endothelial Notch4 regulates inflammatory signaling in a manner distinct from Notch1

AUTHORS: Dana Wang (2), Victor A. Lopez (1), Jessica Gannon (1), Sheryl Coutermarsh-Ott (3), Jeremy A. Brown (4), Adam Maxwell (1), Joanne Tuohy (5), Eli Vlaisavljevich (1)

(1) Biomedical Engineering and Mechanics, Virginia Tech 
(2) Virginia Tech Carilion School of Medicine, Virginia Tech 
(3) Biological Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine 
(4) School of Biomedical Engineering, Dalhousie University
(5) Department of Small Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine 

BACKGROUND: Oral tumors are frequently diagnosed in both humans and dogs and have potential for malignancy. Current standard of care treatments like surgery, radiation, and chemotherapy are invasive, often leading to facial disfigurements and other adverse effects that decrease quality of life. There is a pressing need for less invasive treatments that preserve oral function and appearance.

Histotripsy is a non-invasive, non-thermal, and non-ionizing image-guided focused ultrasound technique. It utilizes acoustic cavitation to break down tissue into acellular debris by producing high-pressure ultrasound pulses that converge at the transducer focus to generate a cavitation bubble cloud for mechanical tissue ablation. While current clinical histotripsy systems are large, cart-based devices operating between 500kHz to 1MHz for deep tissue treatments like the liver, this study aims to develop miniaturized high-frequency histotripsy transducers for targeting smaller, superficial structures like oral tumors.

METHODS: CT images of canines with oral tumors were segmented and analyzed in MATLAB to identify accessible acoustic windows with histotripsy treatment. A ray-sphere intersection approach was used to guide transducer designs based on predicted acoustic windows. 2MHz and 6.3MHz single-element transducers were developed and tested on excised canine oral tumors, producing bubble cloud diameters of approximately 2mm and 0.25mm, respectively. Excised canine oral tumors were degassed, embedded in 1% agarose gel, and underwent treatment with both transducers using a pulse repetition frequency of 1kHz and 2,000 pulses per point. Real-time ultrasound imaging was used for treatment monitoring and post-treatment ablation was quantified histologically with hematoxylin and eosin stain. 

RESULTS: CT acoustic window analysis produced a matrix showing where ultrasound pulses could reach the tumor unimpeded by the skull. This visual representation illustrated the spatial relationship between the acoustic window, skull, and oral tumor. During ex-vivo treatments, real-time ultrasound imaging indicated that both 2MHz and 6.3MHz transducers generated well-confined bubble clouds. After treatment, hypoechoic regions observed within the tissue suggested tumor ablation and liquefaction. Histological analysis confirmed complete, delineated ablation and decellularization of the tissue within the treated area. The 2MHz transducer demonstrated faster ablation rates, approximately 7.4 times quicker than the 6.3MHz transducer.

CONCLUSIONS: Preliminary acoustic window analysis informed optimal transducer positioning for targeting oral tumors effectively with a smaller transducer system. Both the 2MHz and 6.3MHz transducers successfully ablated excised canine oral tumors. Overall, this study shows the feasibility of ablating oral tumors with histotripsy for the first time and provides insight on optimizing histotripsy devices for oral tumor treatment.

Histotripsy for the Treatment of Oral Tumors: A Canine Ex-Vivo Feasibility Study

Abstract Title
Hodgkin-Huxley Algorithm to Diagnose Channelopathies from Empirical Action Potentials

Background
Human channelopathies are diagnosed by symptom profile or genetic testing which fail to provide a mechanistic basis for patients’ experiences. This project aims to develop an algorithm that decomposes a patient’s electrophysiology data into clinically relevant values that would better focus treatment efforts. Our model system for variation in action potential pathology are garter snakes (Thamnophis spp.) that have evolved resistance to their neurotoxic prey (Taricha spp.). The toxin, tetrodotoxin, occludes the pore of voltage-gated sodium channels that conduct sensory and motor impulses. However, resistance mutations observed in snake skeletal muscle voltage-gated sodium channels significantly reduce channel function by limiting sodium conductance. Our model aims to reproduce that level of mechanistic detail from action potentials recorded in vivo directly in the target tissue rather than by complicated whole-cell electrophysiology techniques.

Methods
To observe sodium channel conductivity deficits, we collected action potentials from known healthy and pathological samples and then trained a model based on this dataset. We recorded from muscles carrying tetrodotoxin-sensitive sodium channels (NaV1.4⁺, N=9) from Thamnophis atratus as well as from snakes with mildly resistant channels (A1281P, N=11) suffering mild deficits. We then tailored the Hodgkin-Huxley model of the action potentials to fit skeletal muscle recordings. We deployed the model to decompose action potentials into distinct, molecularly-based mathematical parameters, including sodium channel conductance (GNa), sodium channel activation (m), and inactivation (h). We generated representative action potentials based on averages of each group. Then, R was used to fit a parameter set to each group to assess differences in molecularly-based parameters between groups (e.g. GNa).

Results
The model reproduced the expected negative correlation between sodium channel mutations and channel conductance (e.g. reduced GNa). Furthermore, it revealed significant decreases in two kinetic subparameters, m and h, providing an additional explanation of reduced conductance. These data validate our model against peer-reviewed research (del Carlo et al., 2024)

Conclusion
This model offers the first estimate of sodium channel function in native snake skeletal muscle. Our model produced results congruent with findings of peer-reviewed reports using gold-standard methods. However, the model currently lacks sufficient specificity to evaluate patient-level data. Ongoing work aims to expand this model as a diagnostic tool by training it on even more extreme snake channelopathies. With further refinement, this model could be a clinically useful tool for the differential diagnosis of human channelopathies, advancing personalized medicine for inherited disorders.

Hodgkin-Huxley Algorithm to Diagnose Channelopathies from Empirical Action Potentials

The nicotinic acetylcholine receptor (nAChR) has been shown to play a functionally distinct role in the development of the adolescent brain through the modulation of neurotransmitter release across various neurodevelopmental milestones. CHRNA2 encodes for the α2 nicotinic acetylcholine receptor associated with CA1 oriens lacunosum moleculare GABAergic interneurons and is associated with learning and memory. Prior literature consistently supports the finding that chronic nicotine exposure during adolescence leads to an impairment in learning in adulthood. Previously, we found that adolescent male hypersensitive (CHRNA2 L9'S/L9'S) mice had impairments in learning and memory during a pre-exposure-dependent contextual fear conditioning task that could be rescued by low-dose nicotine exposure. In this study, female adolescent CHRNA2 L9'S/L9'S mice and wild-type (WT) littermate controls were exposed to saline or nicotine (0.09 mg/kg) using a hippocampus-dependent task of pre-exposure-dependent contextual fear conditioning. Alterations in freezing behavior—an indication of learning—were compared between CHRNA2 L9'S/L9'S and WT female adolescent mice as well as against adolescent male CHRNA2 L9'S/L9'S and WT mice. We found that nicotine-treated adolescent WT female mice had significantly greater freezing behavior than both saline-treated WT mice and nicotine-treated CHRNA2 L9'S/L9'S female mice. Nicotine-treated adolescent CHRNA2 L9'S/L9'S female mice did not have enhanced freezing behavior on context test (CT) day when compared with saline-treated CHRNA2 L9'S/L9'S female mice. These results indicate that hypersensitivity of the CHRNA2 gene in female adolescent mice produces deficits in nicotine-dependent learning and memory in the hippocampus. These results are contrasted with the results obtained in the previously published male data where we found that male hypersensitive (CHRNA2 L9'S/L9'S) mice had impairments in learning and memory that could be rescued by low-dose nicotine exposure. Thus, nicotine exposure mediates a sexually dimorphic pattern of learning and memory in WT and CHRNA2 L9'S/L9'S adolescent mice exposed to a pre-exposure-dependent contextual fear conditioning paradigm. At present, the mechanism driving sexual dimorphisms between nicotine-exposed adolescent CHRNA2 L9'S/L9'S mice is unknown. Further research must be conducted in both in-vitro and in-vivo studies examining the role of α2- expressing nAChR signaling mediating sex-dependent effects.

Hypersensitivity of the nicotinic acetylcholine receptor subunit (CHRNA2 L9’S) in female adolescent mice produces deficits in learning and memory that are not rescued by nicotine


Background 

Tumor cells in the tumor microenvironment (TME) are known to upregulate endoplasmic reticulum (ER) stress as a survival mechanism and ER stress transmission to the mitochondria is associated with tumorigenesis. We studied the mitochondrial unfolded protein response (mtUPR) associated Lon protease 1 (LONP1) in tumor cells undergoing ER stress. Our hypothesis is LONP1 inhibition during ER stress promotes immunogenic cell death (ICD) in melanoma cells.

Methods 

To simulate the TME, we used murine B16 melanoma cells that were treated with thapsigargin, a compound known to induce ER stress. Then, some cells were co-treated with both thapsigargin and CDDO-Me, a pharmacological inhibitor of LONP1. RT-PCR was used to assess potential targets associated with mt-UPR. TIMER is a database we used to assess the clinical significance of LONP1 in skin cutaneous melanoma patients. MitoProbe DIOC assay was used to assess mitochondrial membrane potential. Annexin V/PI assay was used to determine the proportion of apoptotic cells. Calreticulin exposure assay was used to determine the expression level of cell-surface calreticulin. CellTiter-Glo assay was used to determine both the intracellular and extracellular concentrations of ATP.

Results

RT-PCR showed that Lonp1 is expressed in response to ER stress. TIMER showed that expression of LONP1 is negatively correlated with the infiltration of cytotoxic CD8+ T cells, CD4+ T cells and activated NK cells. MitoProbe DIOC assay of co-treated cells showed the lowest mitochondrial membrane potential. Annexin V/PI assay of those cells showed an increase in apoptosis versus all other conditions. Calreticulin exposure assay of co-treated cells showed an increase in cell-surface calreticulin exposure versus all other conditions. CellTiterGlo assay showed both decreased intracellular ATP and increased extracellular ATP in cotreated cells only.

Conclusion

B16 melanoma cells undergoing ER stress in the absence of LONP1 function showed higher cell-surface calreticulin, higher extracellular ATP release, increased entry into late apoptosis and their mitochondrial membrane potential was significantly reduced. We conclude that inhibition of LONP1 in the presence of ER stress induced ICD in our cells, suggesting LONP1 as a potential target for cancer immunotherapy.

Inhibiting LONP1 Promotes Immunogenic Cell Death in Melanoma Cells Undergoing Endoplasmic Reticulum Stress

Background
Laser powder bed fusion (LPBF) is a 3D printing technique that rapidly and selectively melts metal powder via laser irradiation which then solidifies in the prescribed pattern before a new layer of powder is spread across the surface and the process repeats. LPBF is the most common 3D printing method used with stainless 316L alloys. It offers advantages in the biomedical field by improving mechanical properties such as fracture toughness and strength, enabling complex, patient-specific geometries and reducing material waste, crucial for applications like stents and implants. Nevertheless, consideration of corrosion behavior is crucial in assessing the longevity of these materials in aqueous environments. 

Methods
Various corrosion tests were conducted on LPBF-produced stainless steel alloys. These tests included polarization scans in acidic environments to assess corrosion resistance, mass loss testing in ferric chloride and sodium chloride solutions to measure susceptibility to localized corrosion, and galvanostatic etching in ammonium persulfate to identify microstructural sites prone to selective attack. Transmission electron microscopy was used to analyze local chemical composition differences at a microscale. Scanning electron microscopy and optical microscopy characterized the microstructure, focusing on features susceptible to localized corrosion.

Results
Corrosion testing showed lower rates for LPBF-produced material compared to conventionally processed stainless steel, suggesting enhanced corrosion resistance. However, post-test analysis revealed various localized corrosion forms, including dissolution through cellular structures formed during the rapid solidification inherent to LPBF. Transmission electron microscopy uncovered local variations in elements critical to corrosion resistance like chromium and molybdenum, impacting corrosion behavior. These findings emphasize the complexity of corrosion mechanisms in LPBF materials and the importance of detailed microstructural analysis.

Conclusion
The study identified lower corrosion rates in LPBF-produced stainless steel alloys but also highlighted localized corrosion pathways through cellular structures, pores formed due to inadequate metal fusion, and other microstructural features inherent to the LPBF process. Advanced microscopy techniques revealed elemental variations influencing corrosion behavior. Future research should explore diverse environments, long-term corrosion performance, and predictive models linking microstructure to corrosion behavior. Addressing these challenges is vital for developing corrosion-resistant materials for biomedical applications and enhancing device performance in clinical settings.

Investigating Corrosion Behavior in 3D Printed Laser Powder Bed Fusion Stainless Steel 316L Alloys - Insights into Material Behavior Relevant for Biomedical Applications

P21-activated kinase 1 (PAK1) is a serine/threonine protein kinase that plays a crucial role in cardiomyocyte survival in response to various stresses. However, the underlying cardioprotective mechanisms of PAK1 remain unclear. Autophagy is a lysosome degradation pathway essential for cellular homeostasis. In the present study, we showed the ability of PAK1 to regulate autophagy in cardiomyocytes and identified p62 as a potential downstream mediator. PAK1 is silenced in H9c2 cardiac myoblasts by siRNA or overexpressed by adenovirus. Autophagy flux was determined by LC3-II protein levels and a fluorescent autophagy reporter with and without the lysosomal protease inhibitors Pepstatin A (pepA) and Aloxistatin (E64d). Downregulation of PAK1 reduced LC3-II levels in whole cell lysates, which was not affected by pepA and E64d, suggesting that PAK1 knockdown reduced autophagy flux. Conversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 is sufficient to promote autophagy. Interestingly, the protein expression of p62, a ubiquitin-binding autophagy adaptor, was increased by PAK1 overexpression and reduced by PAK1 knockdown. Importantly, overexpression of p62 rescued the defective autophagy flux in the absence of PAK1, suggesting that p62 may mediate PAK1-dependent autophagy. RT-qPCR assay showed that the p62 mRNA levels were unchanged by either overexpression or knockdown of PAK1 in vitro and in vivo. The cells were treated with the protein synthesis inhibitor cycloheximide (CHX) and p62 protein levels were measured over time in both PAK1-silencing and overexpressing conditions. The results showed that p62 degradation was mitigated by PAK1 overexpression and accelerated by PAK1 knockdown. Together, these results suggest that PAK1 is sufficient and necessary to maintain autophagy in cardiomyocytes, which is likely mediated by its ability to affect p62 protein stability.

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Antimicrobial Effects of Inhaled Nitric Oxide Therapy on Pseudomonas Aeruginosa Lung Colonization

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