Partial Inhibition Research Articles

TYK2 regulates tau levels, phosphorylation and aggregation in a tauopathy mouse model.

Alzheimer's disease is one of at least 26 diseases characterized by tau-positive accumulation in neurons, glia or both. However, it is still unclear what modifications cause soluble tau to transform into insoluble aggregates. We previously performed genetic screens that identified tyrosine kinase 2 (TYK2) as a candidate regulator of tau levels. Here we verified this finding and found that TYK2 phosphorylates tau at tyrosine 29 (Tyr29) leading to its stabilization and promoting its aggregation in human cells. We discovered that TYK2-mediated Tyr29 phosphorylation interferes with autophagic clearance of tau. We also show that TYK2-mediated phosphorylation of Tyr29 facilitates pathological tau accumulation in P301S tau-transgenic mice. Furthermore, knockdown of Tyk2 reduced total tau and pathogenic tau levels and rescued gliosis in a tauopathy mouse model. Collectively, these data suggest that partial inhibition of TYK2 could thus be a strategy to reduce tau levels and toxicity.

Modulation of cardiac metabolism in heart failure

Heart failure is associated with altered cardiac metabolism, in part, due to maladaptive mechanisms, in part secondary to comorbidities such as diabetes and ischemic heart disease. The metabolic derangements taking place in heart failure are not limited to the cardiac myocytes but extend to skeletal muscles and the vasculature causing changes that contribute to the worsening of exercise capacity. Modulation of cardiac metabolism with partial inhibition of free fatty acid oxidation has been shown to be beneficial in patients with heart failure. At the present, the bulk of evidence for this class of drugs comes from trimetazidine. Newer compounds partially inhibiting free fatty acid oxidation or facilitating the electron transport on the mitochondrial cristae are in early phase of their clinical development.

Suppression of non-muscle myosin II boosts T cell cytotoxicity against tumors.

Increasing evidence highlights the importance of immune mechanoregulation in establishing and sustaining tumor-specific cytotoxicity required for desirable immunotherapeutic outcomes. However, the molecular connections between mechanobiological inputs and outputs and the designated immune activities remain largely unclear. Here, we show that partial inhibition of non-muscle myosin II (NM II) augmented the traction force exerted by T cells and potentiated T cell cytotoxicity against tumors. By using T cells from mice and patients with cancer, we found that NM II is required for the activity of NKX3-2 in maintaining the expression of ADGRB3, which shapes the filamentous actin (F-actin) organization and ultimately attributes to the reduced traction force of T cells in the tumor microenvironment. In animal models, suppressing the NM II-NKX3-2-ADGRB3 pathway in T cells effectively suppressed tumor growth and improved the efficacy of the checkpoint-specific immunotherapy. Overall, this work provides insights into the biomechanical regulation of T cell cytotoxicity that can be exploited to optimize clinical immunotherapies.

Long-term reprogramming of primed microglia after moderate inhibition of CSF1R signaling.

In acute neuroinflammation, microglia activate transiently, and return to a resting state later on. However, they may retain immune memory of such event, namely priming. Primed microglia are more sensitive to new stimuli and develop exacerbated responses, representing a risk factor for neurological disorders with an inflammatory component. Strategies to control the hyperactivation of microglia are, hence, of great interest. The receptor for colony stimulating factor 1 (CSF1R), expressed in myeloid cells, is essential for microglia viability, so its blockade with specific inhibitors (e.g. PLX5622) results in significant depletion of microglial population. Interestingly, upon inhibitor withdrawal, new naïve microglia repopulate the brain. Depletion-repopulation has been proposed as a strategy to reprogram microglia. However, substantial elimination of microglia is inadvisable in human therapy. To overcome such drawback, we aimed to reprogram long-term primed microglia by CSF1R partial inhibition. Microglial priming was induced in mice by acute neuroinflammation, provoked by intracerebroventricular injection of neuraminidase. After 3-weeks recovery, low-dose PLX5622 treatment was administrated for 12 days, followed by a withdrawal period of 7 weeks. Twelve hours before euthanasia, mice received a peripheral lipopolysaccharide (LPS) immune challenge, and the subsequent microglial inflammatory response was evaluated. PLX5622 provoked a 40%-50% decrease in microglial population, but basal levels were restored 7 weeks later. In the brain regions studied, hippocampus and hypothalamus, LPS induced enhanced microgliosis and inflammatory activation in neuraminidase-injected mice, while PLX5622 treatment prevented these changes. Our results suggest that PLX5622 used at low doses reverts microglial priming and, remarkably, prevents broad microglial depletion.

Spatiotemporal analysis of F-actin polymerization with micropillar arrays reveals synchronization between adhesion sites.

We repurposed micropillar-arrays to quantify spatiotemporal inter-adhesion communication. Following the observation that integrin adhesions formed around pillar tops we relied on the precise repetitive spatial control of the pillars to reliably monitor F-actin dynamics in mouse embryonic fibroblasts as a model for spatiotemporal adhesion-related intracellular signaling. Using correlation-based analyses we revealed localized information-flows propagating between adjacent pillars that were integrated over space and time to synchronize the adhesion dynamics within the entire cell. Probing the mechanical regulation, we discovered that stiffer pillars or partial actomyosin contractility inhibition enhances inter-adhesion F-actin synchronization, and that inhibition of Arp2/3, but not formin, reduces synchronization. Our results suggest that adhesions can communicate and highlight the potential of using micropillar arrays as a tool to measure spatiotemporal intracellular signaling. [Media: see text].

Hyperactive mTORC1/4EBP1 signaling dysregulates proteostasis and accelerates cardiac aging.

The mechanistic target of rapamycin complex 1 (mTORC1) has a major impact on aging by regulation of proteostasis. It is well established that mTORC1 signaling is hyperactivated with aging and age-related diseases. Previous studies have shown that partial inhibition of mTOR signaling by rapamycin reverses age-related deteriorations in cardiac function and structure in old mice. However, the downstream signaling pathways involved in this protection against cardiac aging have not been established. mTORC1 phosphorylates 4E-binding protein 1 (4EBP1) to promote the initiation of cap-dependent translation. The objective of this project is to examine the role of the mTORC1/4EBP1 axis in age-related cardiac dysfunction. We used a whole-body 4EBP1 KO mouse model, which mimics a hyperactive mTORC1/4EBP1/eIF4E axis, to investigate the effects of hyperactive mTORC1/4EBP1 axis in cardiac aging. Echocardiographic measurements of middle-aged 4EBP1 KO mice show impaired diastolic function and myocardial performance compared to age-matched WT mice and these parameters are at similar levels as old WT mice, suggesting that 4EBP1 KO mice experience accelerated cardiac aging. Old 4EBP1 KO mice show further decline in systolic and diastolic function compared to middle-aged counterparts and have worse systolic and diastolic function than age-matched WT mice. Gene expression levels of heart failure markers are not different between 4EBP1 KO and WT hearts. However, ribosomal biogenesis and protein ubiquitination are significantly increased in 4EBP1 KO hearts when compared to WT controls, suggesting dysregulated proteostasis in 4EBP1 KO hearts. Together, these results show that a hyperactive mTORC1/4EBP1 axis accelerates cardiac aging, potentially by dysregulating proteostasis.

High temperature and hyperkalemia increase vulnerability of navaga cod (Eleginus nawaga) cardiomyocytes to the ecotoxicant 3-methyl-phenanthrene

Oil and gas mining and transportation in the Arctic can lead to release of polycyclic aromatic hydrocarbons (PAHs) in the ocean and freshwater basins. PAHs are known for their toxic effects in fish hearts, including the inhibition of main ionic currents (IKr, INa and ICaL) in fish cardiac myocytes. The present study is the first one to assess the effect of a particular PAH abundant in crude oil and diesel, namely 3-methyl-phenanthrene (3-MP), on the electrical excitability (EE) of cardiomyocytes from navaga cod (Eleginus nawaga), commercial fish species from the Arctic. Action potentials (APs) were elicited in current-clamp experiments at 9, 15 and 21 °C, and AP characteristics and the current needed to elicit APs were examined. Also, the effects of 3 μM 3-MP were tested at 3 temperatures and in normal (3.5 mM) and high (8 mM) extracellular K+ concentrations.Elevation of temperature leads to hyperpolarization of resting membrane potential and AP shortening, but does not decrease EE. 3-MP was found to suppress EE in cardiomyocytes at 9 and 15 °C, but not at 21 °C. High extracellular K+ itself drastically decreases EE, although it does not worsen the effect of 3-MP. However, combination of hyperthermia and high K+ leads to augmentation of depressive effect of 3-MP on EE. We hypothesize that hyperthermia rescues Na+ channels from inactivation due to membrane hyperpolarization, thereby compensating for the partial inhibition of INa by 3-MP. However, elevation of extracellular K+ nullifies this protective mechanism by depolarizing the resting potential and aggravates the effect of 3-MP.

7778 Factors Predicting Recovery of Hypogonadism in Men with Prolactinoma Treated with Dopamine Agonists

Abstract Disclosure: S. Constantinescu: None. O. Alexopoulou: None. D.M. Maiter: None. Introduction: Recovery of hypogonadism is an important objective of dopamine agonist (DA) treatment in men with prolactinoma. However, the extent, timeline, and predictive factors of gonadotrope axis recovery are still unclear. Methods: We report data from a large cohort of 89 men with prolactinoma, looking at the evolution of testosterone levels after 6 and 12 months of treatment with DA. We excluded patients with peripheral hypogonadism, surgical treatment performed less than 12 months after onset of DA treatment, and patients with small tumors (≤ 5 mm and prolactin ≤ 50 µg/l) who did not achieve tumor shrinkage during treatment. We defined low testosterone as a morning value < 10 nmol/l. Patients who had started testosterone supplementation were considered as having persistent hypogonadism. Results: Among the 89 men (mean age ± SD: 44 ± 14 years; 65/89 with a macroprolactinoma), 14 (16%) had normal initial testosterone levels (NT) and 75 (84%) had low testosterone at diagnosis (LT). Compared to LT, NT patients had significantly lower PRL (median 53 µg/l versus 490 µg/l, p=0.009) and smaller tumors (median 6.5 mm versus 21.2 mm, p=0.013). In the NT group, after 6 (n=11) and 12 months (n=10), testosterone rose from a median of 13.4 to 16.6 (p=0.266) and 17.9 nmol/l (p=0.012), respectively. In LT patients, after 6 (n=56) and 12 months (n=42), testosterone rose significantly from a median of 5.8 to 10.7 (p<0.001) and 12.5 nmol/l (p<0.001).In the LT group, 45% (32/71) had recovered eugonadism at 6 months and 57% (40/70) at 12 months. These proportions rose to 61% (22/36) and 67% (n=26/39) at 6 and 12 months when prolactin concentrations were normalized. Factors associated with persistent hypogonadism after one year were chiasmal compression at diagnosis (p=0.05), cavernous sinus invasion (p=0.042), cystic tumoral component (p=0.038), a greater tumoral diameter (mean 25 mm vs 15 mm, p<0.01), lower testosterone concentrations at diagnosis (mean 3.6 nmol/l vs 6.8 nmol/l, p<0.001) and at 6 months (mean 6.0 nmol/l vs 13.0 nmol/l, p<0.03), and higher prolactin levels at one year (median 17.15 µg/l vs 8.4 µg/l, p=0.064). Testosterone < 6.7 nmol/l at 6 months predicted the long-term persistence of hypogonadism with 72% sensitivity and 92% specificity. Conclusion: The sensitivity of the gonadotrope axis to high prolactin levels is highly variable in men with prolactinoma and 16% of them retain normal testosterone concentrations. However, this finding does not exclude partial inhibition of the gonadotrope axis that will improve with DA treatment. In patients with low testosterone levels, the recovery rate of hypogonadism is 45% at 6 months and 57% at 12 months, increasing to 67% if prolactin is normalized. Testosterone supplementation could be started early in patients with large, invasive, cystic and/or compressive tumors, with a testosterone concentration lower than 6.7 nmol/L after 6 months of treatment. Presentation: 6/3/2024

Improved hydrogen production in immobilized Chlamydomonas reinhardtii cells with inhibited inter-photosystem electron transfer

The production of molecular hydrogen (H2) by microalgae holds great promise, and immobilization techniques offer potential for further advancement in this field. The current study focuses on investigating the positive impact of immobilization on maintaining the stability and activity of photosystem II (PSII) over incubation time, with the aim of enhancing H2 production potential in green microalgae Chlamydomonas reinhardtii. For this purpose, immobilized cells within alginate beads were treated with small concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) inhibitor to induce the partial inhibition of inter-photosystem electron transport, recently reported as a novel approach for sustaining microalgal H2 production. A comparative analysis of fluorescence decay kinetic changes and EPR spectroscopy of the cell beads revealed the superior capacity of immobilization for sustaining PSII stability and activity in batch culture over time. Treatment of the cell beads with 3.5 μM DBMIB led to sustained H2 production yielding over 200 μmol H2/mg Chl within 3 weeks, with an average H2 production rate of approximately 10 μmol/mg Chl per day, both of which were roughly twice as high as those observed in free cells treated with DBMIB. Our findings underscore the significance of integrating immobilization with a proven and effective method for H2 production, thereby enhancing its sustainability and productivity.

Chitosan coatings reinforced with cellulose crystals and oregano essential oil as antimicrobial protection against the microbiological contamination of stone sculptures

The proliferation of microorganisms in outdoor stone sculptures and cultural objects can damage the structure and aesthetics of the materials through biodeterioration mechanisms. Biocides and synthetic products are often used to prevent this phenomenon, despite their negative impact on the environment and human health. Less toxic alternatives with reduced environmental impact can be an option for the preventive conservation of stone sculptures to reduce the environmental impact. In this work, chitosan formulations reinforced with two types of cellulose crystals (microcrystalline cellulose (MCC) or cellulose nanocrystals (CNCs)) and with or without citric acid and sodium tripolyphosphate were prepared. The films obtained with these formulations showed low solubility, and those only containing MCC or CNCs had the lowest wettability. The formulation containing 2% (w/v) MCC was selected for further analysis and supplemented with oregano essential oil (OEO) at 1% (v/v) and 2% (v/v), exhibiting low solubility, swelling and wettability when polymerised in film form. Inoculation of the films with Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa and Rhodotorula spp. resulted in total or partial inhibition of their growth, as well as a 60–100% reduction in Penicillium chrysogenum growth, depending on the concentration of OEO. The formulation with 2% (v/v) OEO was applied to samples of granite, marble and limestone, forming a protective, yet irregular coating on their surfaces. The wettability of the stones’ surfaces was reduced without becoming completely water-repellent and the coating did not cause visible colour changes.

Kinesins regulate the heterogeneity in centrosome clustering after whole-genome duplication.

After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.

Partial chemogenetic inhibition of the locus coeruleus due to heterogeneous transduction of noradrenergic neurons preserved auditory salience processing in wild-type rats.

The acoustic startle reflex (ASR) and prepulse inhibition of the ASR (PPI) assess the efficiency of salience processing, a fundamental brain function that is impaired in many psychiatric conditions. Both ASR and PPI depend on noradrenergic transmission, yet the modulatory role of the locus coeruleus (LC) remains controversial. Clonidine (0.05 mg/kg, i.p.), an alpha2-adrenoreceptor agonist, strongly reduced the ASR amplitude. In contrast, chemogenetic LC inhibition only mildly suppressed the ASR and did affect the PPI in virus-transduced rats. The canine adenovirus type 2 (CAV2)-based vector carrying a gene cassette for the expression of inhibitory receptors (hM4Di) and noradrenergic cell-specific promoter (PRSx8) had high cell-type specificity (94.4±3.1%) but resulted in heterogeneous virus transduction of DbH-positive LC neurons (range: 9.2-94.4%). Clozapine-N-oxide (CNO; 1mg/kg, i.p.), a hM4Di actuator, caused the firing cessation of hM4Di-expressing LC neurons, yet complete inhibition of the entire population of LC neurons was not achieved. Case-based immunohistochemistry revealed that virus injections distal (> 150μm) to the LC core resulted in partial LC transduction, while proximal (< 50 μm) injections caused neuronal loss due to virus neurotoxicity. Neither the ASR nor PPI differed between the intact and virus-transduced rats. Our results suggest that a residual activity of virus-non-transduced LC neurons might have been sufficient for mediating an unaltered ASR and PPI. Our study highlights the importance of a case-based assessment of the virus efficiency, specificity, and neurotoxicity for targeted cell populations and of considering these factors when interpreting behavioral effects in experiments employing chemogenetic modulation.

The Possibility of Using Waste from Dye Sorption for Methane Production

The aim of this study was to determine the effect of sorption of Basic Red 46 (BR46) dye by lignocellulosic biomass on the susceptibility of the sorbed waste to anaerobic decomposition by anaerobic digestion. The research material used in the experiment consisted of two types of biomass: stalks with leaves and inflorescences after mowing Canadian goldenrod (Solidago canadensis L.) (GB), and rapeseed hulls (RHs) after oil pressing. During the anaerobic decomposition of RHs, 732.30 NmL/gVS and 646.63 NmL/gVS of methane were obtained from the non-sorbed substrate and the plant material after dye sorption, respectively. Similarly, in the variants using Canadian goldenrod, the production was 220.70 NmL/gVS and 183.20 NmL/gVS. The GB sorbent sorbed 34% more BR46 dye than the RH sorbent, which is likely to have resulted in the accumulation of VFA and contributed to the partial inhibition of methane production. In light of the obtained results and the literature data, it is concluded that there is a possibility of effective use of dye sorption waste for methane production.

Evaluation of the antibacterial activity of selenium nanoparticles and those conjugated with lysozyme against Bacillus cereus spiked in pasteurized skim milk

Selenium nanoparticles (SeNPs) and SeNPs conjugated with lysozyme (SeNPs-lysozyme) were synthesized via sodium selenite reduction with ascorbic acid and polysorbate-80 stabilization. These were physiochemically characterized to impart antibacterial properties. X-ray diffraction analysis (XRD) revealed that both formulations had hexagonal crystalline structures. Fourier transform infrared (FT-IR) confirmed the successful conjugation of lysozyme to SeNPs through the emergence of amine peaks at 1542 cm−1. Dynamic light scattering (DLS) showed lysozyme conjugation increased nanoparticle size from 76.6 to 92.4 nm and charge from − 12.6 to + 17.6 mV. Transmission electron microscopy (TEM) images confirmed that spherical morphologies and size increased from 73–79 nm for SeNPs to 84–98 nm for SeNPs-lysozyme after lysozyme capping. Both formulations exhibited identical minimum inhibitory concentrations (MIC) of 125 μg/mL. However, lysozyme conjugation reduced SeNPs cytotoxicity by 2.4-fold based on IC50 values from an MTT (Thiazolyl Blue Tetrazolium Bromide) assay. When tested in phosphate-buffered saline (PBS), SeNPs and SeNPs-lysozyme fully inhibited Bacillus cereus (B. cereus) proliferation up to 6 log10 CFU/mL inoculums over 24 h of incubation. However, at (7 log10 CFU/mL) inoculum, only partial inhibition occurred, with (2.5 ± 0.3 log10 CFU/mL) growth still detected for SeNPs and (2.2 ± 0.2 log10 CFU/mL) for SeNPs-lysozyme. At all inoculum levels (5, 6, and 7) log10 CFU/mL, B. cereus grew a lot in milk (more than 8 log10 CFU/mL). This was probably because SeNPs or SeNPs-lysozyme tend to aggregate around milk components, making them less effective.

Single molecule Lipid Biosensors Mitigate Inhibition of Endogenous Effector Proteins.

Genetically encoded lipid biosensors are essential cell biological tools. They are the only technique that provide real time, spatially resolved kinetic data for lipid dynamics in living cells. Despite clear strengths, these tools also carry significant drawbacks; most notably, lipid molecules bound to biosensors cannot engage with their effectors, causing inhibition. Here, we show that although PI 3-kinase (PI3K)-mediated activation of Akt is not significantly reduced in a cell population transfected with a PH-Akt1 PIP3/PI(3,4)P2 biosensor, single cells expressing the PH-Akt at visible levels (used for live-cell imaging) have no activated Akt at all. Tagging endogenous AKT1 with neonGreen at its genomic locus reveals its EGF-mediated translocation to the plasma membrane, accumulating at densities of ~0.3 molecules/μm2. Co-transfection with the PH-Akt biosensor or other PIP3 biosensors completely blocks this translocation, despite robust recruitment of the biosensors. A partial inhibition is even observed with PI(3,4)P2-selective biosensor. However, we found that expressing lipid biosensors at low levels, comparable with those of endogenous AKT, produced no such inhibition. Helpfully, these single-molecule biosensors revealed improved dynamic range and kinetic fidelity compared with over-expressed biosensor. This approach represents a less invasive way to probe spatiotemporal dynamics of the PI3K pathway in living cells.

Unlocking the potential: Cinnamomum cassia (L.) D. Don aqueous extracts as natural defenders against MSRV in largemouth bass aquaculture

Aquatic viral diseases pose significant threats to aquaculture, necessitating the exploration of effective preventive measures. In this study, we evaluated the antiviral potential of Cinnamomum cassia (L.) D. Don (CCD) aqueous extracts against Micropterus salmoides rhabdovirus (MSRV) in largemouth bass aquaculture. Initially, twelve plant extracts were screened for antiviral activity against MSRV, revealing that CCD extracts at 100 mg/L concentration significantly inhibited MSRV proliferation in vitro. Further dose-response analysis revealed a concentration-dependent antiviral effect of CCD extracts, with higher concentrations demonstrating increased efficacy against MSRV. Subsequent investigations focused on the protective effects of CCD extracts on MSRV-infected cells. At a concentration of 63 mg/L, CCD extracts significantly reduced MSRV titers, preventing the onset of cytopathic effects (CPE) in cells. Fluorescence and transmission electron microscopy further illustrated the protective effects of CCD extracts on cellular morphology. MSRV-infected cells treated with CCD exhibited preserved cell morphology akin to normal cells, contrasting with the typical apoptotic features observed in untreated infected cells. Additionally, dietary supplementation of CCD extracts in largemouth bass demonstrated notable benefits. Fish fed with CCD extracts exhibited increased survival rates when infected with MSRV, with a 26 % higher survival rate compared to the virus-only group. Furthermore, CCD supplementation enhanced the expression of interferon-gamma (IFNγ), indicating improved immune responses in largemouth bass. Moreover, immersion of CCD extracts in water showed partial inhibition of horizontal MSRV transmission, effectively reducing viral spread and mitigating disease risks in fish populations. These findings highlight the potential of CCD aqueous extracts as feed additives for combating MSRV in aquaculture, showcasing their promising application prospects in disease prevention and control strategies.

Abstract 09: Leucine Rich Repeat Containing 8A Anion Channels Modulate Vascular Reactivity in ApoE Knockout Mice

Volume-regulated anion channels (VRACs) are comprised of Leucine Rich Repeat Containing 8 (LRRC8) family proteins which include LRRC8A, B, C, D and E. We have proposed that these channels may allow influx of extracellular superoxide to drive inflammation and modulate cultured vascular smooth muscle cell (VSMC) inflammation. We previously demonstrated that LRRC8A knockdown or inhibition inhibits tumor necrosis factor alpha (TNFα)-induced inflammation. Mesenteric arteries from male smooth muscle-specific LRRC8A knockout (KO) mice displayed enhanced responsiveness to both endothelium-dependent (acetylcholine, ACh), and independent (sodium nitroprusside, SNP) vasodilators compared to vessels from WT mice. KO vessels were also protected from vascular dysfunction induced by TNFα in tissue culture (48hr exposure). We hypothesized that reduced VSMC inflammation in LRRC8A KO mice would protect vascular function in ApoE knockout (ApoE -/- ). We created ApoE -/- mice lacking LRRC8A only in VSMCs and assessed vascular reactivity of isolated mesenteric arteries by wire myography in female and male WT, Heterozygous (HET) and KO tissues after 14 weeks of high fat diet. WT vessels displayed enhanced in contraction compared to LRRC8A KO or HET (male PE max contraction of KCL (%), WT 177 ± 4.6 vs. KO 152 ± 3.3 or HET 153 ± 3.0, p &lt; 0.05, n=6-7). Relaxation responses to ACh were increased in female KO or HET compared to WT (ACh LogEC 50 , WT -7.2 ± 0.09 vs. KO -7.5 ± 0.09 or HET -7.4 ± 0.07, p &lt; 0.05, n=5-6). SNP-mediated relaxation were increased in male KO vessels compared to WT (SNP LogEC 50 , WT -8.1 ± 0.06 vs. KO -8.3 ± 0.06, p &lt; 0.05, n=7). These results demonstrate that loss (KO) or reduction (HET) of LRRC8A expression preserves vascular function in the face of the inflammatory response induced by hypercholesterolemia in ApoE -/- mice. These channels may represent a novel therapeutic target for preservation of vascular function and this may only require partial inhibition of current.

Abstract P197: Identification and Characterization of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Proteins in Placenta

Preeclampsia is life threatening condition affecting about 10% of all pregnancies. A potential mechanism is via malfunctioning syncytiotrophoblasts in the placenta. RhoBTB1 is an adapter protein for the CUL3 E3 ubiquitin ligase which delivers protein substrates for proteasomal degradation. An analysis of gene expression databases revealed that the highest level of RhoBTB1 expression is in the placenta. RNAscope in situ hybridization data reveals that RhoBTB1 expression is specifically localized to syncytiotrophoblasts. In blood vessels, RhoBTB1 controls the state of nitric oxide mediated vasodilation because it acts as a substrate adaptor for Phosphodiesterase 5. Thus, we wanted to identify potential protein targets of RhoBTB1 in the placenta to assess if RhoBTB1 plays a role in placental function. To simplify the first identification of RhoBTB1 binding proteins, we employed HTR-8 /SVneo (HTR-8) cells as a model of placental trophoblast cells. We biotinylated the HTR-8 proteome utilizing ascorbate peroxidase (APEX2) proximity labelling system followed by mass spectrometry to identify RhoBTB1 targets. We employed a fusion protein consisting of the C-terminal half of RhoBTB1 (B1B2C), the region required for the binding of other protein substrates, and a control lacking the most C-terminal region (B1B2). We next chose proteins with binding to B1B2C &gt; B1B2 of 2-fold or greater that were expressed in the placenta based on the Human Protein Atlas. Rho GTPase Activating Protein 29 (ARHGAP29), which controls the regulation of small GTP binding proteins, and S-phase Kinase associated Protein 2 (SKP2), a regulator of cell cycle progression was studied further. SKP2 is interesting because it exhibits a tissue specific tropism for placental expression with its highest expression in extravillous trophoblasts, cytotrophoblasts, and syncytiotrophoblasts. Expression of ARHGAP29 and SKP2 in HTR-8 cells were markedly upregulated by MLN4924 suggesting they are regulated by the CUL pathway. They were also modestly upregulated after partial inhibition of RhoBTB1 by siRNA suggesting they may be RhoBTB1-CUL3 targets. Studies are ongoing to validate the physical interaction of ARHGAP29 and SKP2 with RhoBTB1 by co-immunoprecipitation. We are also using RNAscope to assess the co-expression of RhoBTB1, ARHGAP29 and SKP2 in the human placenta from normal and preeclamptic pregnancies.

Exploring and Re-Assessing Reverse Anomeric Effect in 2-Iminoaldoses Derived from Mono- and Polynuclear Aromatic Aldehydes.

A curious and noticeable structural feature in Schiff bases from 2-aminoaldoses is the fact that imino tautomers arranged equatorially in the most stable ring conformation exhibit a counterintuitive reverse anomeric effect (RAE) in the mutarotational equilibrium, i.e., the most stable and abundant anomer is the equatorial one (β). As shown by our very recent research, this effect arises from the total or partial inhibition of the exo-anomeric effect due to the presence of an intramolecular hydrogen bond between the anomeric hydroxyl and the iminic nitrogen in the axial anomer (α). When the Schiff base adopts either an enamine structure or the imino group is protonated, the exo-anomeric effect is restored, and the axial α-anomer becomes the most stable species. Although the intramolecular H-bonding should appropriately be interpreted as a genuine stereoelectronic effect, the magnitude of the RAE could be affected by other structural parameters. Herein and through a comprehensive analysis of benzylidene, cinnamylidene, naphthalene, phenanthrene, and anthracene aldehydes, we show the robustness of the RAE effect, which is similar in extent to simple aldehydes screened so far, irrespective of the size and/or hydrophobicity of the substituent at the nitrogen atom.

Light-Responsive conjugated polymer nanoparticles with Spatial-Controlled camptothecin release via π − π stacking for improved Combinatorial therapy of breast cancer

The treatment of breast cancer (BC) remains a significant challenge, exemplified by the limitations of chemotherapy due to its high side effects. Light-responsive drug delivery systems (DDS) offer a promising approach to spatially and temporally release drugs, minimizing chemotherapy side effects and enhancing efficacy. However, achieving precise delivery and site-specific drug release poses complex challenges. Here, We present a novel nano-system combining light-responsive photothermal therapy (PTT) with photothermal-responsive chemotherapy via π − π Stacking. Conjugated polymer nanoparticles (cRGD-PTer N25/CPT NPs) is designed for spatiotemporal targeted PTT/chemotherapy. Research shows that the strong near-infrared absorption of PTer N25 endows cRGD-PTer N25/CPT NPs with excellent photothermal capabilities(57.4 %) and near-infrared laser-triggered drug release performance. The cRGD-PTer N25/CPT NPs enhanced BC-specific cellular accumulation, prolonged blood circulation, exhibited good biocompatibility, and improved PTT and chemotherapy efficacy in BC cell lines and mouse models while reducing systemic toxicity. The photothermal effect triggered the on-demand release of camptothecin (CPT), producing therapeutic effects and downregulating heat shock protein 70 (HSP 70), reducing cancer cell thermoresistance, and enhancing combined treatment efficacy. Under spatial and temporal control, the tumor growth inhibition rate of the cRGD-PTer N25/CPT NPs group reached 93.6 %, nearly eradicating tumor presence, whereas the control group exhibited only partial inhibition. Minimal cardiotoxicity and metastatic side effects are not observed. This work presents a viable strategy for designing novel controlled-release drug systems to improve breast cancer treatment efficiency.