Semisynthetic modification of mycothiazole (1) with Meerwein's salt to generate a more stable and/or potent analog than 8-O-acetylmycothiazole (2) unexpectedly yielded diastereomers devoid of the diene. Analysis of NMR, HR-LCMS, and optical rotation confirmed the structures as (-)-4,4-hydroxy-methyl-(5Z)-(8S)-(14Z)-mycothiazole (5a, 5b: dr 1:1.1). Both compounds demonstrated reduced stability versus 1 or 2. Cytotoxicity evaluation of 5a versus 5b indicated 5-fold differences in potency against pancreatic (IC50 = 7.81, 1.34 μM; PANC-1) and glioblastoma (IC50 = 8.53, 1.63 μM; U251N) cancer cells, respectively. In vivo evaluation of 5a and 5b, using Caenorhabditis elegans in aging studies, indicated 5b not 5a inhibited mitochondrial function, while neither affected lifespan compared to 1. These results demonstrate the diene of 1 may be required for its picomolar cytotoxic potency to cancer cells or effects on lifespan in C. elegans, and minor variations in the stereochemistry of this chemotype merit further investigation to modulate its bioactivity.

The actin cytoskeleton is a fundamental and highly conserved structure that functions in diverse cellular processes, yet its direct contribution to organismal aging remains unclear. Here, we systematically interrogated how genetic and pharmacologic perturbations of actin structure and function influence lifespan and various hallmarks of aging in Caenorhabditis elegans . Whole-animal and tissue-specific knockdown of actin and key actin-binding proteins (ABPs) - arx-2 (Arp2/3), unc-60 (cofilin), and lev-11 (tropomyosin) - led to premature disruption of filament organization, reduced lifespan, and tissue-specific physiological defects. Bulk and single-nucleus RNA-sequencing revealed that ABP knockdowns elicited a strongly “aged” transcriptome. Actin dysfunction broadly exacerbated many age-associated phenotypes, including mitochondrial dysfunction, lipid dysregulation, loss of proteostasis, impaired autophagy, and intestinal barrier failure. Pharmacological destabilization with Latrunculin A mirrored genetic knockdowns, while mild stabilization with Jasplakinolide modestly extended lifespan, emphasizing that optimal and finely-tuned actin function is critical for healthy aging. Finally, analysis of human genome-wide association data revealed that common ACTB polymorphisms correlate with differences in age-related decline in gait speed, suggesting evolutionary conservation of actin’s role in healthy aging. Taken together, our results provide a comprehensive and publicly accessible resource that maps, for the first time, how actin integrity intersects with diverse aging pathways across tissues and scales. This descriptive framework is intended to enable future mechanistic discovery by offering a deep, unbiased dataset that can be integrated with emerging studies to define how actin dynamics contribute to aging.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), MTZ, a newly employed ETC complex I inhibitor, exhibited higher cytotoxicity against cancer cell lines compared to certain non-cancer cell lines. Interestingly, 8-OAc demonstrated greater selectivity for cancer cells when compared to both MTZ and Rote, which has promising potential for anticancer therapeutic development. Furthermore, in vivo experiments with these small molecules utilizing a C. elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. We also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilizes the transcription factors ATFS-1 and HSF1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Scale-up isolation of (+)-(5Z)-(8S)-(14Z)-mycothiazole (1) from Vanuatu specimens of C. mycofijiensis to semisynthesize (+)-(5Z)-(8S)-8-O-acetyl-(14Z)-mycothiazole (2) revealed a new diastereomer, (-)-(5E)-(8R)-(14Z)-mycothiazole (4). The structure of 4 was determined using HRMS, NMR, and comparing optical rotation to (-)-(5Z)-(8R)-(14Z)-mycothiazole (3) and 2. The maximum tolerated dose of 2 in mice was 0.1 mg/kg. The IC50 of 4 in PANC-1 and HepG2 cancer cell lines was 111.6 and 115.0 nM. Evaluation of 4 in C. elegans showed similar oxygen consumption compared to 1-2, and all compounds significantly increased the lifespan. The Z orientation at Δ5,6 is crucial for picomolar cytotoxicity but not for mitochondrial inhibition.

Childhood/pre-pubertal obesity (PPO) is implicated in the development of chronic kidney disease (CKD), and there is evidence that PPO also increases the risk of developing Alzheimer disease (AD) later in life. In the current study, we aim to investigate the correlation between CKD and the development of AD-like pathology in a rat model of CKD associated with PPO. We hypothesize that systemic inflammation due to PPO exacerbates CKD and induces AD-like pathology in the brain at older age. In our preliminary studies, we utilized the Dahl salt sensitive leptin receptor mutant rat (SSLepRmutant), which is a novel model of PPO. 6-week-old Dahl salt-sensitive (SS) (control, n=3) and SSLepRmutant rats (n=5) were monitored for proteinuria every 3 weeks, for a 12-week period. Throughout the study, we observed significantly elevated proteinuria in SSLepRmutant rats (Week-6: 337±81 mg/day, Week-9: 371±147 mg/day, Week-18: 1920±318 mg/day) compared to SS rats (Week-6: 25±7 mg/day, Week-9: 35±39 mg/day, Week-18: 35±33 mg/day). At 18-week, creatinine levels were significantly higher in SSLepRmutant rats (1.5±0.5 mg/dL) versus SS rats (0.4±0.03 mg/dL) indicating CKD. Additionally, we found significant elevation of triglycerides in SSLepRmutant rats (5134±1211 mg/dL) compared to SS rats (79±21 mg/dL). To investigate the brain pathology, we performed immunochemical analysis (dot-blot assay) using A11 antibody to determine the levels of Amyloid-beta oligomers (Aβo). Our results show that there was tendency for Aβo levels to be increased in the brains from obese SSLepRmutant rats compared to SS. Finally, we tested the permeability of the blood-brain barrier to Evans blue dye and observed that the brain of SSLepRmutant rats was more permeable to the dye compared to SS rats as early as 8 weeks of age. Overall, these results indicate that PPO obesity not only increases the susceptibility to develop CKD but also causes Alzheimer-like pathology during the prepubescent stage. NIDDK (DK109133) and AG (AG057842) awarded to Jan M. Williams. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Childhood/prepubertal obesity (PPO) is a world-wide epidemic and is considered a risk factor for renal injury. Therefore, there is a need to control this trend to decrease the future risk of chronic kidney disease. Recently, we reported that the SS LepR mutant rat is a novel model to study the mechanisms of renal injury associated with PPO. The SS LepR mutant rat develops progressive renal injury in the absence of hyperglycemia before puberty. SGLT2 inhibitors have been demonstrated to exert renoprotective effects independent of lowering blood glucose. Therefore, the objective of this study was to determine the effects of SGLT2 blockade with empagliflozin on the early progression of proteinuria in SS LepR mutant rats, during PPO. Four-week-old SS and SS LepR mutant rats (n = 5/group) were either treated with vehicle (diet) or empagliflozin (30 mg/kg/day, orally) for four weeks. While the SS LepR mutant rats had significantly higher body weights vs SS rats, there were no detectable differences in body weight or blood glucose levels in response to empagliflozin treatment in SS LepR mutant rats. We observed a significant increase in plasma insulin in control SS LepR mutant rats vs SS rats (7±1 vs 1±0.3 ng/mL, respectively, p<0.05), and treatment with empagliflozin did not have any effect on plasma insulin in SS LepR mutant rats (8±2 ng/mL). At baseline, we did not observe any significant difference in urine flow rate between control SS LepR mutant vs SS rats. After two weeks of treatment, while there was no observed difference in urine flow rate between SS LepR mutant and SS rats (17±3 ml/day vs 18±2 ml/day, respectively), empagliflozin significantly increased urine flow rate in SS LepR mutant rats (29±4 ml/day, p<0.05), but not in SS rats (17±3 ml/day). At baseline, we observed a difference in proteinuria between control SS LepR mutant vs SS rats (68±11 vs 6±3 mg/day, respectively, p<0.05), which remained elevated over the course of the study (471±75 vs 32±5 mg/day, respectively, p<0.05). Treatment with empagliflozin significantly decreased proteinuria in SS LepR mutant rats (223±52 mg/day, p<0.05) without affecting SS rats (27±8 mg/day). At the end of the study, we observed a significant increase in GFR in control SS LepR mutant rats vs SS rats (2.4±0.6 vs 0.5 mL/min/gkwt, respectively, p<0.05). Treatment with empagliflozin only significantly reduced GFR in SS LepR mutant rats (0.9±0.2 mL/min/gkwt, p<0.05). In conclusion, these data suggest that empagliflozin attenuates renal hyperfiltration and confers renoprotective effects during the early progression of renal disease associated with PPO. DK109133 and HL151407 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

To the Editor: Therapies for actinic keratosis (AK) include photodynamic therapy (PDT), in which blue light activates photosensitizing compounds, like 5-aminolevulinic acid (5-ALA).1 The approved incubation time for 5-ALA before light irradiation is 14-18 hours, with prior studies reporting 0-18 hours.2,3 Nearly 10% of patients cannot complete therapy due to pain.4 This randomized controlled trial compared the effectiveness of BLU-U and Levulan Kerastick (ALA HCL, DUSA Pharmaceuticals) with 1-hour incubation to that without incubation.

Prepubertal obesity (PPO) is a public health problem that is now associated with renal injury. Recently, we reported that the obese SSLepRmutant rat develops inflammation and renal injury before puberty. Moreover, the progression of renal disease in the SSLepRmutant rat is associated with the increased renal expression of the mammalian target of rapamycin (mTOR). Previous studies have shown that the mTOR pathway contributes to inflammation and renal disease. Therefore, this study tested the hypothesis that rapamycin (a mTOR inhibitor) will attenuate renal inflammation and early progressive renal injury in SSLepRmutant rats. Four-week-old SS and SSLepRmutant rats were treated with either vehicle (saline) or rapamycin (1.5 mg/kg/day, ip) for 4 weeks. At baseline, body weight was significantly higher in SSLepRmutant vs SS rats and remained elevated throughout the study. Treatment with rapamycin significantly decreased body weight in SSLepRmutant rats without affecting SS rats. While blood glucose levels were in the normal physiological range throughout the study in SSLepRmutant and SS rats (≤120 mg/dL), treatment with rapamycin only increased blood glucose in SSLepRmutant rats (561±54 mg/dL) without affecting SS rats (116±23 mg/dL). At the end of the study, we observed significant elevations in plasma insulin in SSLepRmutant vs SS rats, and treatment with rapamycin significantly decreased insulin levels by >50% in SSLepRmutant rats. At baseline proteinuria was significantly higher in SSLepRmutant vs SS rats (72±52 vs. 6±6 mg/day), and it remained higher throughout the study (500±125 vs. 26±12 mg/day). Interestingly, treatment with rapamycin only decreased proteinuria in SSLepRmutant rats (62±8 mg/day). Glomerular injury and renal fibrosis were markedly greater in SSLepRmutant vs SS rats, and treatment with rapamycin ameliorated these histological changes. At the end of the study, we observed increased GFR (via creatinine clearance) in SSLepRmutant vs SS rats (3.93±0.5 vs. 0.56±0.2 mL/min/gkwt), and treatment with rapamycin reduced GFR in SSLepRmutant rats (0.47±0.1 mL/min/gkwt). While there were no detectable differences in the renal expression of IL-4 in SSLepRmutant vs SS rats, there was a significant decrease in the renal expression of IL-10 in SSLepRmutant vs SS rats. Treatment with rapamycin significantly increased the renal expression of IL-4 and IL-10 in SSLepRmutant rats, while not affecting SS rats. In conclusion, although rapamycin causes hyperglycemia, it prevents early progressive proteinuria by reducing GFR and increasing renal anti-inflammatory cytokines during PPO. Funding: DK109133 and HL151407 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Small molecule inhibitors of the mitochondrial electron transport chain (ETC) hold significant promise to provide valuable insights to the field of mitochondrial research and aging biology. In this study, we investigated two molecules: mycothiazole (MTZ) - from the marine sponge C. mycofijiensis and its more stable semisynthetic analog 8-O-acetylmycothiazole (8-OAc) as potent and selective chemical probes based on their high efficiency to inhibit ETC complex I function. Similar to rotenone (Rote), a widely used ETC complex I inhibitor, these two molecules showed cytotoxicity to cancer cells but strikingly demonstrate a lack of toxicity to non-cancer cells, a highly beneficial feature in the development of anti-cancer therapeutics. Furthermore, in vivo experiments with these small molecules utilizing C.elegans model demonstrate their unexplored potential to investigate aging studies. We observed that both molecules have the ability to induce a mitochondria-specific unfolded protein response (UPRMT) pathway, that extends lifespan of worms when applied in their adult stage. Interestingly, we also found that these two molecules employ different pathways to extend lifespan in worms. Whereas MTZ utilize the transcription factors ATFS-1 and HSF-1, which are involved in the UPRMT and heat shock response (HSR) pathways respectively, 8-OAc only required HSF-1 and not ATFS-1 to mediate its effects. This observation underscores the value of applying stable, potent, and selective next generation chemical probes to elucidate an important insight into the functional roles of various protein subunits of ETC complexes and their regulatory mechanisms associated with aging.

Particle mass analyzers (PMAs) classify particles by their mass-to-charge ratio. PMAs are often paired with bipolar chargers, which produce charge distributions that consist of positively-, negatively, and neutrally-charged particles, which greatly limits the transmission efficiency of PMAs and allows small, uncharged particles to be classified erroneously. These limitations provide an opportunity for unipolar chargers, which impart several charges per particle. However, the PMA setpoint (mass-to-charge ratio) loses physical meaning when the average number of charges per particle mass is unknown. The average charge of the PMA classified particles can be inferred from downstream size distribution measurements or by running detailed models. However, downstream measurements are often practically infeasible and detailed models require knowledge of the unclassified aerosol size and density. These challenges motivate a need for simplified approaches. Using simulations, we show that the unipolar charger-PMA average charge can be estimated to within ±20% for a reasonable range of parameters for typical aerosol scenarios. The most significant uncertainties behind the simplified approach are related to particle morphology (e.g., fractal versus spheroidal particles). Our simplified approach relies on the derivation of an empirical power-law relationship between the particle average charge and particle diameter, which is consistent with several previous experimental studies and existing models. The approaches described in this work improve the capabilities of unipolar charger-PMA systems to classify a known average mass (rather than a known mass-to-charge ratio) which is valuable for characterizing and calibrating the response of optical and mass instruments.

Background: Routine follow-up of newborns who screened positive for cystic fibrosis (CF) with an inconclusive diagnosis (CFSPID) is considered standard of care to observe for indications of clinical features of disease [1].Standard measures of lung function may not be sensitive enough to pick up early signs of disease.The lung clearance index (LCI), as measured by multiple-breath washout (MBW), has been found to be a sensitive measure of early lung disease and progression in cystic fibrosis and could be a useful tool for tracking the CFSPID population over time.Our previous analysis of this cohort was limited primarily to preschool-aged children [2]; with the availability of school-aged data in this group, this study aims to describe changes in LCI over time in CFSPID individuals.Methods: Children identified as CFSPID were prospectively followed annually with routine sweat chloride testing and clinical assessments, as outlined previously [1].A subset of performed MBW during their annual visits, using the Exhalyzer D (Eco Medics AG, Switzerland) with nitrogen as a tracer gas, and results were generated out of Spiroware v3.3.1 (Eco Medics AG).Visits were classified as stable or symptomatic if the individual had respiratory symptoms at the time of testing based on physician assessment.Spirometry and sweat chloride results were collected for each visit.Results: Fifty-eight MBW measurements were collected in 20 children (55% female).CFSPID children had a median of 3 (interquartile range 2-5) MBW measurements.Mean ± SD age at first MBW measurement was 5.4 ± 1.1.Overall, the mean ± SD LCI for all visits was 6.34 ± 0.67.The LCI was 0.48 units lower in school-aged (≥6) than preschool-aged (<6) children (95% CI, -0.83 to -0.12; p = 0.009).The LCI was 1.05 units higher for symptomatic visits than for stable visits (95% CI, 0.60-1.51;p < 0.001), with six of the seven symptomatic visits occurring in children younger than 7, reflecting the higher incidence of viral infections in younger children.All school-aged children with previously high LCI for whom repeated measures were available demonstrated decline (improvement) in LCI on follow-up, and LCI remained within the range defined for healthy children (Figure 1) [3].

Herein, we report on naturally derived microtubule stabilizers with activity against triple negative breast cancer (TNBC) cell lines, including paclitaxel, fijianolide B/laulimalide (3), fijianolide B di-acetate (4), and two new semisynthetic analogs of 3, which include fijianolide J (5) and fijianolide L (6). Similar to paclitaxel, compound 3 demonstrated classic microtubule stabilizing activity with potent (GI50 = 0.7-17 nM) antiproliferative efficacy among the five molecularly distinct TNBC cell lines. Alternatively, compounds 5 or 6, generated from oxidation of C-20 or C-15 and C-20 respectively, resulted in a unique profile with reduced potency (GI50 = 4-9 μM), but improved efficacy in some lines, suggesting a distinct mechanism of action. The C-15, C-20 di-acetate, and dioxo modifications on 4 and 6 resulted in compounds devoid of classic microtubule stabilizing activity in biochemical assays. While 4 also had no detectable effect on cellular microtubules, 6 promoted a reorganization of the cytoskeleton resulting in an accumulation of microtubules at the cell periphery. Compound 5, with a single C-20 oxo substitution, displayed a mixed phenotype, sharing properties of 3 and 6. These results demonstrate the importance of the C-15/C-20 chiral centers, which appear to be required for the potent microtubule stabilizing activity of this chemotype and that oxidation of these sites promotes unanticipated cytoskeletal alterations that are distinct from classic microtubule stabilization, likely through a distinct mechanism of action.

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (−)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (−)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (−)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms.

OBJECTIVE:To assess the effectiveness and safety of combined pulsed-dye laser (PDL) and NAFL for treatment of surgical scars.SUMMARY BACKGROUND DATA:PDL and NAFL have not been compared to healing by time alone.METHODS:Randomized controlled, single-blinded clinical trial at an urban, university hospital. Healthy adults' status post skin surgery with primary closure were randomized to either 3 sessions of combination PDL and NAFL every 2 to 8 weeks, or control of no treatment. At baseline and 36-week follow-up, Patient and observer Scar Assessment Scale and Scar Cosmesis Assessment and Rating were completed by participants and blinded physicians. The primary outcome was scar improvement, as measured by the score difference over time.RESULTS:Of 76 participants, 52 completed the study (July 2017 to June 2019). No severe adverse events were reported. Patient and observer Scar Assessment Scale assessments demonstrated improvement in total score in the laser group compared to controls, as reported by patients [mean difference (standard deviation), laser: 12.86 (6.91) vs control: 7.25 (6.34); P = 0.004] and blinded physicians [18.32 (8.69) vs 13.08 (9.63); P = 0.044]. Patients observed a greater improvement in scar thickness [3.68 (2.04) vs 1.88 (1.85); P = 0.002] and stiffness [3.57 (2.78) vs 1.50 (2.11); P = 0.004] with lasers, and physicians reported greater improvement in vascularity [3.71 (1.98) vs 1.71 (1.52); P = 0.0002]. The live Scar Cosmesis Assessment and Rating subscore for erythema improved significantly with lasers [1.04 (0.79) vs 0.42 (0.50); P = 0.001].CONCLUSIONS AND RELEVANCE:Combined PDL and NAFL resulted in scar improvement. Scar thickness, stiffness, and erythema were improved.TRIAL REGISTRATION:ClinicalTrials.gov (NCT03057964).

Purpose: Physical education teachers often report feeling isolated due to being the only specialist in a building. Professional learning communities (PLCs) are spaces where individuals who hold shared goals come together to build connections, allowing educators to feel connected, valued, and empowered. The authors sought to explore the shared values created by a PLC and the process through which that value emerged. Method: The authors facilitated a PLC for eight physical education teachers within one midsized school district and collected interview and meeting audio data to explore the process and outcomes of the PLC. Results: Coding using a Value Creation Framework revealed themes of building common ground, support for big and small problems of practice, and an increased connection with other school faculty. Discussion: Teachers were able to build a supportive network where ideas could be traded, partner support was provided, and problems of practice were discussed.

We recently reported that adoptive transfer of cytolytic Natural Killer cells (cNKs) from the Reduced Uterine Perfusion Pressure (RUPP) rat induces a preeclampsia (PE)-like phenotype in pregnant rats, accompanied by increased TNF-alpha. The purpose of this study was to investigate a role for increased TNF-alpha to induce oxidative stress (ROS), decrease nitric oxide (NO) bioavailability, and induce vascular dysfunction as mechanisms of hypertension (HTN) and intrauterine growth restriction (IUGR) in RUPPs. Pregnant Sprague Dawley rats underwent the RUPP or a Sham procedure on gestation day (GD) 14. On GDs 15 and 18, a subset of Sham and RUPP rats received i.p.injections of vehicle or 0.4 mg/kg of Etanercept (ETA), a soluble TNF-alpha receptor (n = 10/group). On GD18, Uterine Artery Resistance Index (UARI) was measured, and on GD19, mean arterial pressure (MAP), fetal and placental weights were measured, and blood and tissues were processed for analysis. TNF-alpha blockade normalized the elevated MAP observed RUPP. Additionally, both fetal and placental weights were decreased in RUPP compared to Sham, and were normalized in RUPP + ETA. Placental ROS was also increased in RUPP rats compared to Sham, and remained elevated in RUPP + ETA. Compared to Sham, UARI was elevated in RUPPs while plasma total nitrate was reduced, and these were normalized in ETA treated RUPPs. In conclusion, TNF-alpha blockade in RUPPs reduced MAP and UARI, improved fetal growth, and increased NO bioavailability. These data suggest that TNF-alpha regulation of NO bioavailability is a potential mechanism that contributes to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes and fetal growth.

Preeclampsia is a multi-system disorder of pregnancy characterized by hypertension (HTN), intrauterine growth restriction (IUGR), organ dysfunction, and inflammation. The NLRP3 inflammasome is a multi-protein complex that initiates the inflammatory response, and has been implicated in chronic kidney disease, AKI, and heart failure. The purpose of the present study was to determine if NLRP3 activation plays a causative role in preeclampsia pathophysiology. We hypothesized that inhibition of NLRP3 activation would attenuate hypertension and IUGR in response to placental ischemia. Pregnant Sprague Dawley rats underwent Sham or Reduced Uterine Perfusion Pressure (RUPP; model of preeclampsia) procedure on Gestation Day (GD) 14. A subset of Sham and RUPP rats received i.p. injections of the specific NLRP3 inhibitor MCC950 (20mg/kg/day) from GD14-GD19. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound. On GD19, mean arterial pressure (MAP), mean fetal and placental weight were measured, and blood and tissues were processed for additional analysis. MAP (mmHg) was significantly elevated in RUPP (130±1, n=8) compared to Sham (105±1, n=8; p<0.05). Treatment with MCC950 decreased MAP in RUPP+MCC950 (113±2, n=8; p<0.05 vs RUPP). Fetal and placental weights were significantly reduced in RUPP compared to Sham, and treatment with MCC950 had no effect. UARI was significantly increased in RUPP versus Sham (0.70±0.03 vs. 0.56±0.01, respectively; p<0.05) and was normalized to 0.52±0.03 in RUPP+ MCC950. T-helper 17 cells (TH17s) and cytolytic NK cells (cNKs) in the circulation, placenta, and kidney were evaluated by flow cytometry. Circulating TH17s (% gated) were significantly increased in RUPP (2.1±1) vs Sham (0.5±0.2; p<0.05 vs RUPP). Inhibition of NLRP3 significantly reduced circulating TH17s in RUPP+MCC950 (0.4±0.1; p<0.05 vs RUPP). Similar trends were also observed in TH17 populations in placentas and kidneys from each group. Circulating cNKs (% gated) increased from 2.3±0.3 in Sham to 6.4±0.8 in RUPP (p<0.05), and was significantly reduced to 2.3±0.6 in RUPP+MCC950 (p<0.05 vs RUPP). In a separate set of animals, renal function studies were performed to study the impact of MCC950 treatment on renal blood flow (RBF), glomerular filtration rate (GFR), and renal vascular resistance (RVR) in all groups. RBF (mL/min/g) was markedly reduced in RUPP (n=4) compared to Sham (n=5) (4±1 vs. 7±1, respectively). Importantly, treatment with MCC950 normalized RBF in RUPP+MCC950 (8±3, n=3) while having no effect in Sham (9±2, n=3). GFR (μL/min/g) was reduced from 1320±177 in Sham and 1117±240 in Sham+MCC950 to 829±124 in RUPP and remained low at 744±180 in RUPP+MCC950. RVR (mmHg/mL/min/g) was markedly increased in RUPP compared to values measured in Sham (42±8 vs. 23±4, respectively) and was normalized to 26±3 in RUPP after treatment with MCC950. The results of this study implicate NLRP3 in directly mediating inflammation, immune cell activation, vascular and renal dysfunction to cause maternal HTN in RUPP. These data suggest that NLRP3 is a potential therapeutic target for treatment in PE.

Women with preeclampsia (PE) have increased cytolytic NK cells (cNKs), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in the circulation and placenta. In previous studies, adoptive transfer of placental cNKs from the Reduced Uterine Perfusion Pressure (RUPP) model of PE into normal pregnant Sprague Dawley (SD) rats caused increased IFNγ and TNFα, and induced vascular dysfunction, hypertension (HTN), and restricted fetal growth (IUGR) similar to what is observed in PE patients. The purpose of this study was to examine the roles of IFNγ and TNFα in RUPP and PE pathophysiology. We hypothesized that neutralization of IFNγ or TNFα would attenuate PE‐associated pathophysiology in RUPP rats. Pregnant SD rats underwent RUPP or Sham procedure on Gestation Day (GD) 14. On GD 15 and 18, a subset of Sham and RUPP rats received vehicle or 10μg/kg anti‐IFNγ monoclonal antibody (IFNy‐ab; n=9/group) i.p. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound and on GD19, mean arterial pressure (MAP), fetal and placental weight were measured, and blood and tissues were processed for analysis. MAP was 105±2 mmHg and 108±2 mmHg in Sham and Sham+IFNγ‐ab respectively, was elevated to 125±1 mmHg in RUPP (p<0.05 vs Sham), and reduced to 112±2 mmHg in RUPP+ IFNγ‐ab (p<0.05 vs RUPP). Fetal and placental weights were significantly reduced in RUPP and remained so in RUPP+IFNγ‐ab (p<0.05 vs Sham). UARI was 0.6±0.02 in Sham and Sham+ IFNγ‐ab, was elevated to 0.73±0.02 in RUPP (p<0.05 vs Sham), and was normalized to 0.6±0.02 in RUPP+ IFNγ‐ab (p<0.05 vs RUPP). Both placental ROS and plasma soluble Fms‐like Tyrosine kinase 1 (sFlt‐1) were elevated in RUPP (p<0.05 vs Sham) and were normalized in RUPP+IFNγ‐ab (p<0.05 vs RUPP). In separate experiments, pregnant SD rats underwent RUPP or Sham procedure on GD 14. On GD 15 and 18, a subset of Sham and RUPP rats received vehicle or 0.4 mg/kg of Etanercept (ETA), a soluble TNFα receptor (n=10/group) i.p. Analyses were performed as described above. MAP was elevated in RUPP (127±2 mmHg) compared to Sham (105±3 mmHg) and Sham+ETA (109±2 mmHg, p<0.05) and was normalized to 109±3 mmHg in RUPP+ETA (p<0.05 vs RUPP). Both fetal and placental weights were significantly reduced in RUPP rats (p<0.05 vs Sham) and were normalized in RUPP+ETA (p<0.05 vs RUPP). UARI was 0.6±0.02 in Sham and 0.6±0.01 in Sham+ETA. UARI was elevated in RUPP (0.72±0.02, p<0.05 vs Sham) and was normalized to 0.61±0.02 in RUPP+ETA (p<0.05 vs RUPP). Plasma total nitrate was significantly reduced in RUPP (p<0.05 vs Sham) and was normalized in RUPP+ETA (p<0.05 vs RUPP). Placental ROS was elevated in RUPP (p<0.05 vs Sham) and remained so in RUPP+ETA (p>0.05 vs RUPP). Plasma sFlt‐1 was 92±34 pg/mL in Sham and 53±9 pg/mL in Sham+ETA, was elevated to 1039±211 pg/mL in RUPP (p<0.05 vs Sham), and was normalized to 145±33 pg/mL in RUPP+ETA (p<0.05 vs RUPP). The results of this study suggest that increased secretion of IFNγ and TNFα by cNKs mediate vascular dysfunction to cause maternal HTN and IUGR in RUPP and PE pathophysiology. IFNγ and TNFα are potential therapeutic targets for treatment in PE.

Reinvestigation of mycothiazole (1) revealed picomolar potency (IC50 = 0.00016, 0.00027, 0.00035 μM) against pancreatic, (PANC-1), liver (HepG2), and colon (HCT-116) tumor cell lines. Reevaluation of 1 provided [α]D data indicating Vanuatu specimens of C. mycofijiensis contain the 8S enantiomer of 1 and not the 8R configuration previously reported. Semisynthesis provided 8-O-acetylmycothiazole (2), 8-oxomycothiazole (8), mycothiazole nitrosobenzene derivatives (MND1, MND2: 9a, 9b), and MND3 (10) with IC50 = 0.00129, >1.0, >1.0, >1.0, >1.0 μM, respectively, against PANC-1 cell lines. These results highlight the significance of the penta-2,4-dien-1-ol residue as a key structural feature of 1 required for its cytotoxicty against tumor cell lines.

Recently, we reported that obese Dahl salt-sensitive (SS) leptin receptor mutant (SS LepR mutant) rats display progressive renal injury. The present study demonstrated that the early development of renal injury in the SS LepR mutant strain is associated with an increase in the renal infiltration of macrophages compared with lean SS rats. We also examined whether depletion of macrophages with clodronate would reduce the early progression of renal injury in the SS LepR mutant strain. Four-week-old SS and SS LepR mutant rats were treated with either vehicle (PBS) or clodronate (50 mg/kg ip, 2 times/wk) for 4 wk. While the administration of clodronate did not reduce renal macrophage infiltration in SS rats, clodronate decreased macrophages in the kidneys of SS LepR mutant rats by >50%. Interestingly, clodronate significantly reduced plasma glucose, insulin, and triglyceride levels and markedly improved glucose tolerance in SS LepR mutant rats. Treatment with clodronate had no effect on the progression of proteinuria or renal histopathology in SS rats. In the SS LepR mutant strain, proteinuria was markedly reduced during the first 2 wk of treatment (159 ± 32 vs. 303 ± 52 mg/day, respectively). However, after 4 wk of treatment, the effect of clodronate was no longer observed in the SS LepR mutant strain (346 ± 195 vs. 399 ± 50 mg/day, respectively). The kidneys from SS LepR mutant rats displayed glomerular injury with increased mesangial expansion and renal fibrosis versus SS rats. Treatment with clodronate significantly decreased glomerular injury and renal fibrosis in the SS LepR mutant strain. Overall, these data indicate that the depletion of macrophages improves metabolic disease and slows the early progression of renal injury in SS LepR mutant rats.