Inflammasome diversity: exploring novel frontiers in the innate immune response.

Pathogens elicit complex mammalian immune responses by activating multiple sensors within inflammasomes, which recognize diverse pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). This simultaneous activation induces the formation of protein complexes referred to as multiple inflammasomes, that orchestrate a spectrum of programmed cell death pathways, including pyroptosis, apoptosis, and necroptosis. This concept is crucial for comprehending the complexity of the innate immune system's response to diverse pathogens and its implications for various diseases. Novel contributions here include emphasizing simultaneous sensor activation by pathogens, proposing the existence of multiple inflammasome complexes, and advocating for further exploration of their structural basis. Understanding these mechanisms may offer insights into disease pathogenesis, paving the way for potential therapeutic interventions targeting inflammasome-mediated immune responses.

AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence.

Inflammasomes are important sentinels of innate immune defence, sensing pathogens and inducing cell death in infected cells1. There are several inflammasome sensors that each detect and respond to a specific pathogen- or damage-associated molecular pattern (PAMP or DAMP, respectively)1. During infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which can simultaneously engage multiple inflammasome sensors2-5. Here we found that AIM2 regulates the innate immune sensors pyrin and ZBP1 to drive inflammatory signalling and a form of inflammatory cell death known as PANoptosis, and provide host protection during infections with herpes simplex virus 1 and Francisella novicida. We also observed that AIM2, pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD, that drove inflammatory cell death (PANoptosis). Collectively, our findings define a previously unknown regulatory and molecular interaction between AIM2, pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that we term the AIM2 PANoptosome and comprising multiple inflammasome sensors and cell death regulators. These results advance the understanding of the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and pyrin-mediated diseases.

Ultrasound-guided genitofemoral nerve block for femoral arterial access gain and closure: a randomized controlled trial.

OBJECTIVES: This study aimed to compare the analgesic efficacy and safety of the femoral branch block of the genitofemoral nerve (FBB) versus local infiltration anesthesia (LIA) for femoral arterial access gain and closure. METHODS: Eighty-two patients (age, 64.8 ± 10.9 years; female, 30.5%) undergoing endovascular procedures using 5-Fr femoral sheath were assigned to either FBB (n = 41) or LIA (n = 41). In both groups, 2% lidocaine HCL with 1:100,000 epinephrine was used as an anesthetic solution. Pain scores during access gain and closure were evaluated using a visual analog scale (score 0-10), patient satisfaction levels with the quality of anesthesia were scored on a 7-point Likert scale, and adverse events were recorded. RESULTS: The primary endpoint, pain scores during access closure, was significantly lower in the FBB group than in the LIA group (0.1 ± 0.37 vs 1.73 ± 0.92; p < 0.001). The FBB group also had significantly lower pain scores during access gain compared to the LIA group (0.83 ± 0.83 vs 2.78 ± 1.26; p < 0.001). There was an inverse relationship between pain scores and FBB after adjustment for age, gender, and body mass index (p < 0.001). FBB group reported significantly higher satisfaction with anesthesia quality compared to the LIA group (6.49 ± 0.64 vs 4.05 ± 1.05; p < 0.001). No complications were recognized in either group. CONCLUSIONS: Ultrasound-guided genitofemoral nerve blocks offered better acute pain relief and higher patient satisfaction than LIA during femoral arterial access gain and closure. CLINICAL RELEVANCE STATEMENT: In this prospective randomized controlled trial, ultrasound-guided genitofemoral nerve blocks offered better acute pain relief than local infiltration anesthesia, resulting in enhanced patient satisfaction. KEY POINTS: • FBB provided better pain relief during access gain and closure than LIA. • FBB offered higher patient satisfaction with the quality of anesthesia than LIA. • No anesthesia-related or access site complications were recognized in either treatment group.

Coronaviruses: Innate Immunity, Inflammasome Activation, Inflammatory Cell Death, and Cytokines.

The innate immune system acts as the first line of defense against pathogens, including coronaviruses (CoVs). Severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV are epidemic zoonotic CoVs that emerged at the beginning of the 21st century. The recently emerged virus SARS-CoV-2 is a novel strain of CoV that has caused the coronavirus 2019 (COVID-19) pandemic. Scientific advancements made by studying the SARS-CoV and MERS-CoV outbreaks have provided a foundation for understanding pathogenesis and innate immunity against SARS-CoV-2. In this review, we focus on our present understanding of innate immune responses, inflammasome activation, inflammatory cell death pathways, and cytokine secretion during SARS-CoV, MERS-CoV, and SARS-CoV-2 infection. We also discuss how the pathogenesis of these viruses influences these biological processes.

Early versus delayed bronchial artery embolization for non-massive hemoptysis.

OBJECTIVES: The aims of this study were to compare clinical outcomes of early versus delayed bronchial artery embolization (BAE) for non-massive hemoptysis and to investigate predictors of recurrent hemoptysis. METHODS: From March 2018 to February 2021, 138 consecutive patients (age, 65.5 ± 12.4 years; male, 67.4%) with non-massive hemoptysis underwent BAE. The enrolled patients were divided into an early embolization (EE) group (within the first 24 h, n = 79) and a delayed embolization (DE) group (n = 59). RESULTS: The time to embolization ranged between 0 and 15 days and was shorter in the EE group (0.47 ± 0.5 days) than in the DE group (4.02 ± 2.8 days, p < 0.001). The in-hospital clinical outcomes were not different between the two groups, except for hospital stay and post-embolization hospital stay. The recurrence-free survival in the EE group was significantly better than that in the DE group (p = 0.018). The time to embolization (hazard ratio (HR), 1.21; 95% confidence interval (CI), 1.04-1.42; p = 0.015) and aspergilloma (HR, 6.89; 95% CI, 2.08-22.86; p = 0.002) were predictive factors for recurrent hemoptysis. CONCLUSIONS: BAE is an effective and safe treatment modality for non-massive hemoptysis. An early interventional strategy should be considered in patients presenting with non-massive hemoptysis to reduce the length of hospital stay and early recurrence. A delayed time to embolization and the presence of aspergilloma were independent risk factors for recurrent hemoptysis. KEY POINTS: • Bronchial artery embolization afforded good clinical improvement for treating non-massive hemoptysis without significant complications. • An early interventional strategy should be considered in patients presenting with non-massive hemoptysis to reduce the length of hospital stay and early recurrence. • A delayed time to embolization and the presence of aspergilloma were independent risk factors for recurrent hemoptysis.

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection.

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8 DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.

Machine-learning prediction of thermal expansion coefficient for perovskite oxides with experimental validation.

Perovskite oxides have been of high-interest and relatively well studied over the last 20 years due to their various applications, specifically for solid oxide fuel cells (SOFCs) and solid oxide electrolysis cells (SOECs). One of the key properties for a perovskite to perform well as a component in SOFCs, SOECs, and other high-temperature applications is its thermal expansion coefficient (TEC). The use of machine learning (ML) to predict material properties has greatly increased over the years and has proven to be a very useful tool for materials screening. The process of synthesizing and testing perovskite oxides is laborious and costly, and the use of physics-based models is often highly computationally expensive. Due to the amount of elements able to be accommodated in the ABO3 structure and the ability for crystallographic mixing in both the A and B-sites, there are a massive amount of possible ABO3 perovskites. In this paper, a ML model for the prediction of the TECs of AA'BB'O3 perovskites is produced and applied to millions of potential compositions resulting in reliable TEC predictions for 150 451 of the compositions.

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method.

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

Value of multiphase computed tomography for gastrointestinal bleeding before endovascular treatment in hemodynamically unstable patients.

BACKGROUND: There has been no practice-based study regarding the multiphase computed tomography (CT) before endovascular treatment in hemodynamically unstable gastrointestinal bleeding (GIB) and concerns exist regarding the time delay. PURPOSE: To evaluate the clinical efficacy of multiphase CT before endovascular treatment in hemodynamically unstable GIB and to investigate the predictors of angiographic localization and recurrent bleeding. MATERIAL AND METHODS: The multicenter retrospective study included 93 consecutive hemodynamically unstable patients who underwent conventional angiography for non-variceal GIB after failed endoscopic localization. Enrolled patients were divided into a CT group (n = 61) and a non-CT group (n = 32). RESULTS: The clinical characteristics did not differ between the two groups except for the time to angiography (CT group, 14.8±15.1 h; non-CT group, 9.2±11.7 h, P = 0.022). The rate of angiographic localization was significantly higher in the CT group than in the non-CT group only for lower GIB (P = 0.049). Indirect sign was significantly more frequent in the CT group than in the non-CT group (P = 0.014). CT localization was positive predictor (odd ratio [OR] = 7.66; 95% confidence interval [CI] = 2.1-27.94; P = 0.002) and prolonged time to angiography was negative predictor (OR = 0.94; 95% CI = 0.9- 0.98; P = 0.001) for angiographic localization. A higher systolic blood pressure until index angiography (OR = 0.95; 95% CI = 0.91-1; P = 0.044) was associated with a reduced risk of recurrent bleeding. CONCLUSION: In hemodynamically unstable patients, multiphase CT is particularly useful for angiographic localization of lower GIB. It should be considered immediately after failed endoscopic hemostasis to reduce time to angiography.

Double-Pigtail Drainage Catheter: A New Design for Efficient Pleural Drainage.

Background and Objectives: The novel double-pigtail catheter (DPC) has an additional pigtail coiling at the mid-shaft with multiple centripetal side holes. The present study aimed to investigate the advantages and efficacy of DPC in overcoming the complications of conventional single-pigtail catheters (SPC) used to drain pleural effusion. Materials and Methods: Between July 2018 and December 2019, 382 pleural effusion drainage procedures were reviewed retrospectively (DPC, n = 156; SPC without multiple side holes, n = 110; SPC with multiple side holes (SPC + M), n = 116). All patients showed shifting pleural effusions in the decubitus view of the chest radiography. All catheters were 10.2 Fr in diameter. One interventional radiologist performed all procedures and used the same anchoring technique. Complications (dysfunctional retraction, complete dislodgement, blockage, and atraumatic pneumothorax) were compared among the catheters using chi-square and Fisher's exact tests. Clinical success was defined as an improvement in pleural effusion within three days without additional procedures. Survival analysis was performed to calculate the indwelling time. Results: The dysfunctional retraction rate of DPC was significantly lower than that of the other catheters (p < 0.001). Complete dislodgement did not occur in any of the DPC cases. The clinical success rate of DPC (90.1%) was the highest. The estimated indwelling times were nine (95% confidence interval (CI): 7.3-10.7), eight (95% CI: 6.6-9.4), and seven (95% CI: 6.3-7.7) days for SPC, SPC + M, and DPC, respectively, with DPC showing a significant difference (p < 0.05). Conclusions: DPC had a lower dysfunctional retraction rate compared to conventional drainage catheters. Furthermore, DPC was efficient for pleural effusion drainage with a shorter indwelling time.

TMEM16A expression in cholinergic neurons of the medial habenula mediates anxiety-related behaviors.

TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.

Transcholecystic management of extrahepatic duct stones in poor candidates for endoscopic or transhepatic approaches.

OBJECTIVES: To investigate transcholecystic management of extrahepatic duct (EHD) stones using balloon ampulloplasty in patients who are poor candidates for endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous transhepatic biliary drainage (PTBD) and assess its efficacy and safety. METHODS: Forty-one patients who were unable to undergo ERCP or had failed ERCP with non-dilated intrahepatic ducts (IHD) between February 2019 and October 2020 were retrospectively enrolled. After clinical improvement with percutaneous cholecystostomy (PC), EHD stones were managed through cystic duct passage, guidewire unwinding, sheath insertion, and EHD stone removal using balloon ampulloplasty. If the transcholecystic route failed, a transhepatic approach was used according to the pre-existing cholangiogram obtained via PC. We evaluated the technical success rate and complications of each step. RESULTS: The technical success rate for the transcholecystic-only approach was 80.5%. The remaining cases were successfully managed with transhepatic conversion. Multiple stone removal sessions were required in 22% of the cases. One patient with combined IHD stones was initially converted to a transhepatic approach without any transcholecystic removal trial. The technical success rates for each step were as follows: cystic duct passage (38/40, 95%), guidewire unwinding (36/38, 94.7%), sheath insertion (36/36, 100%), and stone removal using balloon ampulloplasty (33/36, 91.7%). The overall clinical success was 97.6% (40/41) without major procedure-related complications. Thereafter, cholecystectomy was successfully performed in patients with concomitant gallstones (n = 20). No postprocedural complications occurred during the follow-up (1-70 days). CONCLUSIONS: Percutaneous EHD stone removal through transcholecystic and transhepatic routes after PC is effective and safe in poor candidates for PTBD or ERCP. KEY POINTS: • This study shows the safety and efficacy of extrahepatic duct (EHD) stones in patients who are poor candidates for initial percutaneous transhepatic biliary drainage and endoscopic retrograde cholangiopancreatography. • The overall technical success for the transcholecystic-only approach was 80.5% (33/41). Including transhepatic conversions, it was 100% (41/41). Stone removal was successful in one session in 78% (32/41) of the patients and in multiple sessions in 28.1% (9/41) of the patients. • Balloon ampulloplasty with stone expulsion using an occlusion balloon catheter is also a safe and effective method for removing EHD stones.

Imaging-Guided Deployment of a Balloon-Based Vascular Closure Device in Routine Clinical Practice.

This study evaluated the feasibility and safety of imaging-guided deployment of a Mynx Control device (Cordis, Miami Lakes, Florida) at the femoral access. A total of 201 Mynx devices were used under imaging guidance in 137 patients (mean age, 64.18 years ± 13.8; male, 68.6%) between June 2020 and February 2022. A 5-F sheath was used in 176 (87.6%) procedures, a 6-F sheath was used in 17 (8.5%) procedures, and a 7-F sheath was used in 8 (4%) procedures. Indicator errors occurred in 9 (4.5%) procedures. However, technical success was achieved via manipulation under imaging guidance in all procedures. The mean time to hemostasis was 3.3 minutes ± 0.5, and the clinical success rate was 100%. The mean length of hospital stay was 7 days ± 7.5. After 4 (2%) procedures, small hematomas reabsorbed spontaneously. Imaging-guided deployment of a Mynx Control device is a feasible and safe option for access site closure. Even after an indicator error, successful hemostasis was achieved via real-time interaction.

Association between ARID2 and RAS-MAPK pathway in intellectual disability and short stature.

BACKGROUND: ARID2 belongs to the Switch/sucrose non-fermenting complex, in which the genetic defects have been found in patients with dysmorphism, short stature and intellectual disability (ID). As the phenotypes of patients with ARID2 mutations partially overlap with those of RASopathy, this study evaluated the biochemical association between ARID2 and RAS-MAPK pathway. METHODS: The phenotypes of 22 patients with either an ARID2 heterozygous mutation or haploinsufficiency were reviewed. Comprehensive molecular analyses were performed using somatic and induced pluripotent stem cells (iPSCs) of a patient with ARID2 haploinsufficiency as well as using the mouse model of Arid2 haploinsufficiency by CRISPR/Cas9 gene editing. RESULTS: The phenotypic characteristics of ARID2 deficiency include RASopathy, Coffin-Lowy syndrome or Coffin-Siris syndrome or undefined syndromic ID. Transient ARID2 knockout HeLa cells using an shRNA increased ERK1 and ERK2 phosphorylation. Impaired neuronal differentiation with enhanced RAS-MAPK activity was observed in patient-iPSCs. In addition, Arid2 haploinsufficient mice exhibited reduced body size and learning/memory deficit. ARID2 haploinsufficiency was associated with reduced IFITM1 expression, which interacts with caveolin-1 (CAV-1) and inhibits ERK activation. DISCUSSION: ARID2 haploinsufficiency is associated with enhanced RAS-MAPK activity, leading to reduced IFITM1 and CAV-1 expression, thereby increasing ERK activity. This altered interaction might lead to abnormal neuronal development and a short stature.

Nicotine Affects Murine Aortic Stiffness and Fatigue Response During Supraphysiological Cycling.

Nicotine exposure is a major risk factor for several cardiovascular diseases. Although the deleterious effects of nicotine on aortic remodeling processes have been studied to some extent, the biophysical consequences are not fully elucidated. In this investigation, we applied quasi-static and dynamic loading to quantify ways in which exposure to nicotine affects the mechanical behavior of murine arterial tissue. Segments of thoracic aortas from C57BL/6 mice exposed to 25 mg/kg/day of subcutaneous nicotine for 28 days were subjected to uniaxial tensile loading in an open-circumferential configuration. Comparing aorta segments from nicotine-treated mice relative to an equal number of control counterparts, stiffness in the circumferential direction was nearly twofold higher (377 kPa ± 165 kPa versus 191 kPa ± 65 kPa, n = 5, p = 0.03) at 50% strain. Using a degradative power-law fit to fatigue data at supraphysiological loading, we observed that nicotine-treated aortas exhibited significantly higher peak stress, greater loss of tension, and wider oscillation band than control aortas (p ≤ 0.01 for all three variables). Compared to simple stress relaxation tests, fatigue cycling is shown to be more sensitive and versatile in discerning nicotine-induced changes in mechanical behavior over many cycles. Supraphysiological fatigue cycling thus may have broader potential to reveal subtle changes in vascular mechanics caused by other exogenous toxins or pathological conditions.

Correction: Koh et al. Targeting MicroRNA-485-3p Blocks Alzheimer's Disease Progression. Int. J. Mol. Sci. 2021, 22, 13136.

The authors wish to make the following corrections to this paper [...].

Radiologic and clinical results of transarterial ethanol embolization for renal angiomyolipoma.

OBJECTIVE: We sought to determine the efficacy and safety of selective arterial embolization for renal angiomyolipoma (AML) using ethanol alone or ethanol with additional embolic materials and to analyze the factors influencing safety and efficacy. METHODS: One hundred nineteen AMLs treated with embolization were included retrospectively during a 15-year period. Technical, radiologic, and clinical success were recorded and risk factor analysis was performed. Complications on follow-up images, post-embolization syndrome (PES), major complications, and changes in renal function were also evaluated. RESULTS: Technical success was achieved in 106 of 119 tumors. Tumor size significantly decreased after treatment (reduction rate: 55%). Significant risk factors for tumor reduction included tumor enhancement on preprocedural CT and residual tumor staining. Radiologic success was achieved in 114 of 119 tumors (risk factor: residual tumor staining), and clinical success was achieved in 22 of 23 patients. Complications on follow-up images occurred in 40 of 119 tumors, and PES occurred in 53 of 104 patients. No major complications occurred. There were no cases of renal function impairment. CONCLUSION: Selective transarterial embolization using ethanol alone or ethanol with additional embolic materials reduced AML size, alleviated symptoms, and can be performed safely without permanent impairment. KEY POINTS: • Percutaneous transarterial ethanol embolization reduces AML size and alleviates symptoms. • Embolization can be performed safely without permanent impairment of renal function.

Targeting MicroRNA-485-3p Blocks Alzheimer's Disease Progression.

Alzheimer's disease (AD) is a form of dementia characterized by progressive memory decline and cognitive dysfunction. With only one FDA-approved therapy, effective treatment strategies for AD are urgently needed. In this study, we found that microRNA-485-3p (miR-485-3p) was overexpressed in the brain tissues, cerebrospinal fluid, and plasma of patients with AD, and its antisense oligonucleotide (ASO) reduced Aß plaque accumulation, tau pathology development, neuroinflammation, and cognitive decline in a transgenic mouse model of AD. Mechanistically, miR-485-3p ASO enhanced Aß clearance via CD36-mediated phagocytosis of Aß in vitro and in vivo. Furthermore, miR-485-3p ASO administration reduced apoptosis, thereby effectively decreasing truncated tau levels. Moreover, miR-485-3p ASO treatment reduced secretion of proinflammatory cytokines, including IL-1ß and TNF-α, and eventually relieved cognitive impairment. Collectively, our findings suggest that miR-485-3p is a useful biomarker of the inflammatory pathophysiology of AD and that miR-485-3p ASO represents a potential therapeutic candidate for managing AD pathology and cognitive decline.

Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis.

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

A novel approach to investigate hypoxic microenvironment during rice colonization by Magnaporthe oryzae.

Because molecular oxygen functions as the final acceptor of electrons during aerobic respiration and a substrate for diverse enzymatic reactions, eukaryotes employ various mechanisms to maintain cellular homeostasis under varying oxygen concentration. Human fungal pathogens change the expression of genes involved in virulence and oxygen-required metabolisms such as ergosterol (ERG) synthesis when they encounter oxygen limitation (hypoxia) during infection. The oxygen level in plant tissues also fluctuates, potentially creating hypoxic stress to pathogens during infection. However, little is known about how in planta oxygen dynamics impact pathogenesis. In this study, we investigated oxygen dynamics in rice during infection by Magnaporthe oryzae via two approaches. First, rice leaves infected by M. oryzae were noninvasively probed using a microscopic oxygen sensor. Second, an immunofluorescence assay based on a chemical probe, pimonidazole, was used. Both methods showed that oxygen concentration in rice decreased after fungal penetration. We also functionally characterized five hypoxia-responsive genes participating in ERG biosynthesis for their role in pathogenesis. Resulting insights and tools will help study the nature of in planta oxygen dynamics in other pathosystems.