ClusterMatch aligns single-cell RNA-sequencing data at the multi-scale cluster level via stable matching.

MOTIVATION: Unsupervised clustering of single-cell RNA sequencing (scRNA-seq) data holds the promise of characterizing known and novel cell type in various biological and clinical contexts. However, intrinsic multi-scale clustering resolutions poses challenges to deal with multiple sources of variability in the high-dimensional and noisy data. RESULTS: We present ClusterMatch, a stable match optimization model to align scRNA-seq data at the cluster level. In one hand, ClusterMatch leverages the mutual correspondence by canonical correlation analysis and multi-scale Louvain clustering algorithms to identify cluster with optimized resolutions. In the other hand, it utilizes stable matching framework to align scRNA-seq data in the latent space while maintaining interpretability with overlapped marker gene set. Through extensive experiments, we demonstrate the efficacy of ClusterMatch in data integration, cell type annotation, and cross-species/timepoint alignment scenarios. Our results show ClusterMatch's ability to utilize both global and local information of scRNA-seq data, sets the appropriate resolution of multi-scale clustering, and offers interpretability by utilizing marker genes. AVAILABILITY AND IMPLEMENTATION: The code of ClusterMatch software is freely available at https://github.com/AMSSwanglab/ClusterMatch.

Delayed pericarditis following ethanol ablation of the vein of Marshall in the treatment of atrial fibrillation: a case report.

In this case report, we will discuss a 74-year-old female who presented with a chief complaint of abdominal pain, bloating, anorexia, and nausea for four days which preceded after catheter ablation and anhydrous ethanol infusion vein of Marshall (VOM) one month prior. She was admitted and treated as a general patient in the general ward. After hospital admission, a pericardiocentesis was guided by B-scan ultrasonography, resulting in the extraction of 20ml of pericardial effusion, followed by catheterization for drainage. The key takeaway in this report is that anhydrous ethanol infusion VOM may not always be without risks. Hence, during the procedure, it is imperative to carefully administer the appropriate volume of anhydrous ethanol into the VOM to prevent vessel damage and associated complications.

PEDF inhibits VEGF-induced vascular leakage through binding to VEGFR2 in acute myocardial infarction.

Pigment epithelium-derived factor (PEDF) could bind to vascular endothelial growth factor receptor 2 (VEGFR2) and inhibit its activation induced by VEGF. But how PEDF affects VEGFR2 pathway is still poorly understood. In this study, we elucidated the precise mechanism underlying the interaction between PEDF and VEGFR2, and subsequently corroborated our findings using a rat AMI model. PEDF prevented endocytosis of VE-cadherin induced by hypoxia, thereby protecting the endothelium integrity. A three-dimensional model of the VEGFR2-PEDF complex was constructed by protein-protein docking method. The results showed that the VEGFR2-PEDF complex was stable during the simulation. Hydrogen bonds, binding energy and binding modes were analyzed during molecular dynamics simulations, which indicated that hydrogen bonds and hydrophobic interactions were important for the recognition of VEGFR2 with PEDF. In addition, the results from exudation of fibrinogen suggested that PEDF inhibits vascular leakage in acute myocardial infarction and confirmed the critical role of key amino acids in the regulation of endothelial cell permeability. This observation is also supported by echocardiography studies showing that the 34mer peptide sustained cardiac function during acute myocardial infarction. Besides, PEDF and 34mer could inhibit the aggregation of myofiber in the heart and promoted the formation of a dense cell layer in cardiomyocytes, which suggested that PEDF and 34mer peptide protect against AMI-induced cardiac dysfunction. These results suggest that PEDF inhibits the phosphorylation of downstream proteins, thereby preventing vascular leakage, which provides a new therapeutic direction for the treatment of acute myocardial infarction.Communicated by Ramaswamy H. Sarma.

Correlation between dietary inflammation and mortality among hyperlipidemics.

BACKGROUND AND OBJECTIVE: Although the the Dietary Inflammatory Index (DII) serves to be one of the reliable indicator for hyperlipidaemia, there is still uncertainty about its relationship to prognosis in the hyperlipidaemic population. In current study, the DII levels were analyzed in relation to the mortality risk among among the hyperlipidaemic individuals with the aim of determining any prospective correlation. METHODS: 14,460 subjects with hyperlipidaemia from the 10-year (2001-2010) National Health and Nutrition Examination Survey (NHANES) were chosen for this study. The endpoint event for follow-up was all-cause mortality, and subjects were tracked for up to December 31, 2019, or death, whichever occurred first. The tertiles of the DII levels were utilized for categorizing the study population into three groups. Survival curves, Cox proportional hazards regression models, restricted cubic spline (RCS), subgroup and interaction analyses, and sensitivity analyses were employed sequentially for the purpose of evaluating the association of the DII with mortality. RESULTS: 3170 (21.92%) all-cause deaths were recorded during an average 148-month follow-up period. Kaplan-Meier survival curves indicated that the survival rate of participants divided into the low DII group was substantially improved compared to that of those in the higher DII group (log-rank P < 0.001). After controlling for confounders, higher levels of DII were observed to be meaningfully linked to an elevated risk of death, no matter whether DII was specified for the continuous (hazard ratio (HR): 1.06; 95% confidence interval (CI): 1.04-1.08) or the categorical variable (HR: 1.22; 95% CI: 1.11-1.33). The DII and mortality displayed a linear association, according to the RCS. Stratified and sensitivity analyses reinforced the proof that these findings were reliable. CONCLUSION: Among patients with hyperlipidaemia, the risk of death was positively and linearly linked with DII levels.

Association between estimated pulse wave velocity and risk of diabetes: A large sample size cohort study.

BACKGROUND AND AIM: Estimated pulse wave velocity (ePWV) measurements have good agreement with PWV measurements. However, the relationship between ePWV and the risk of new-onset diabetes remains unclear. Therefore, this study aimed to investigate whether ePWV was associated with new-onset diabetes. METHODS AND RESULTS: Based on a secondary analysis of the Chinese Rich Health Care Group's cohort study, 211,809 participants who met the criteria were enrolled and divided into four groups based on the ePWV quartiles. Diabetes events are of interest as a result of the study. Over a mean follow-up of 3.12 years, 3000 male (1.41%) and 1173 female (0.55%) patients were diagnosed with new-onset diabetes. The cumulative incidence curves based on quartile subgroups showed that the Q4 group had a significantly higher overall incidence of diabetes than the other subgroups. A multivariate Cox regression analysis showed that ePWV was an independent predictor of new-onset diabetes (hazard ratio, 1.233; 95% confidence interval, 1.198-1.269; P < 0.001). The receiver operating characteristic curve showed that the predictive value was higher than for age and blood pressure. The ePWV was treated as a continuous variable using MaxStat, which identified that the best cut-off point for diabetes risk was 8.47 m/s. A stratified analysis showed that the association between ePWV and the risk of diabetes remained significant in multiple strata. CONCLUSIONS: An elevated ePWV was independently associated with an increased risk of developing diabetes in Chinese adults. Thus, ePWV may be a reliable indicator of the risk of early diabetes.

Shikonin Inhibits Candida albicans Biofilms via the Ras1-cAMP-Efg1 Signalling Pathway.

Objective: To investigate the influence of shikonin (SK) on the formation of Candida albicans biofilms and discuss the possible mechanism. Methods: The inhibition of the formation of C. albicans biofilms by SK was observed by scanning electron microscopy. A silicone film method and a water-hydrocarbon two-phase assay were performed to investigate the effects of SK on cell adhesion. Real-time reverse-transcription polymerase chain reaction was used to analyse the expression of genes related to cell adhesion and Ras1-cyclic adenosine monophosphate (cAMP) - enhanced filamentous growth protein 1 (Efg1) signalling pathway. Finally, the level of cAMP in C. albicans was detected and exogenous cAMP rescue experiment was conducted. Results: The results showed that SK could destroy the typical three-dimensional structure of the biofilms, inhibit cell surface hydrophobicity and cell adhesion, downregulate the expression of Ras1-cAMP-Efg1 signalling pathway-related genes (ECE1, HWP1, ALS3, RAS1, CYR1, EFG1 and TEC1) and effectively reduce the production of key messenger cAMP in the Ras1-cAMP-Efg1 pathway. Meanwhile, exogenous cAMP reversed the inhibitory effect of SK on biofilms formation. Conclusion: Our results suggest that SK exhibits potential anti-C. albicans biofilms effects related to the inhibition of Ras1-cAMP-Efg1 pathway.

Optimization of formulation and atomization of lipid nanoparticles for the inhalation of mRNA.

Lipid nanoparticles (LNPs) have demonstrated efficacy and safety for mRNA vaccine administration by intramuscular injection; however, the pulmonary delivery of mRNA encapsulated LNPs remains challenging. The atomization process of LNPs will cause shear stress due to dispersed air, air jets, ultrasonication, vibrating mesh etc., leading to the agglomeration or leakage of LNPs, which can be detrimental to transcellular transport and endosomal escape. In this study, the LNP formulation, atomization methods and buffer system were optimized to maintain the LNP stability and mRNA efficiency during the atomization process. Firstly, a suitable LNP formulation for atomization was optimized based on the in vitro results, and the optimized LNP formulation was AX4, DSPC, cholesterol and DMG-PEG2K at a 35/16/46.5/2.5 (%) molar ratio. Subsequently, different atomization methods were compared to find the most suitable method to deliver mRNA-LNP solution. Soft mist inhaler (SMI) was found to be the best for pulmonary delivery of mRNA encapsulated LNPs. The physico-chemical properties such as size and entrapment efficiency (EE) of the LNPs were further improved by adjusting the buffer system with trehalose. Lastly, the in vivo fluorescence imaging of mice demonstrated that SMI with proper LNPs design and buffer system hold promise for inhaled mRNA-LNP therapies.

In vitro antifungal activity of Shikonin against Candida albicans by inducing cellular apoptosis and necrosis.

BACKGROUND: Our previous studies showed that Shikonin (SK) had a strong anti-Candida albican (C. albicans) activity, especially against some fluconazole-resistant strains, which is probably due to the oxidative damage of SK to C. albicans. METHODS AND RESULTS: In this study, we expanded the antifungal spectrum and evaluate the toxicity of SK. The results indicated that SK also exhibited potent invitro antifungal activities against other pathogenic fungi such as other Candida, Aspergillus, Cryptococcus, and Dermatophytes, but did not display apparent toxicity to the mammalian cells, suggesting that SK is safe to be a potential antifungal drug. Furtherly, we analyze the exact mechanism of SK against C. albicans. We found that SK could induce a series of apoptosis characteristics, including phosphatidylserine externalization, chromatin condensation and fragmentation, decreased cytochrome c oxidase activity as well as caspase activation. CONCLUSIONS: In summary, this study highlighted the antifungal activity and mechanism of SK against C. albicans, providing a potential therapeutic strategy for C. albicans infection.

Syntheses, structures, and magnetic properties of the lanthanide complexes of imidazole-substituted nitronyl nitroxide biradicals.

Two new bis-bidentate imidazole-substituted nitronyl nitroxide biradicals, BNITIm-C2 (BNITIm-C2 = 1,1'-(1,2-ethanediyl)bis(1H-imidazole-2-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-1-oxy-3-oxide)) and BNITIm-C4 (BNITIm-C4 = 1,1'-(1,4-butanediyl)bis(1H-imidazole-2-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-1-oxy-3-oxide)), and two series of lanthanide complexes, namely [(BNITIm-C2)Ln(NO3)3](MeOH) (Ln = Gd (1Gd) and Tb (2Tb)), (BNITIm-C2)Dy(NO3)3 (3Dy) and (BNITIm-C4)[Ln(hfac)3]2(C7H8)2 (Ln = Gd (4Gd), Tb (5Tb) and Dy (6Dy), hfac = hexafluoroacetylacetonate), have been prepared and characterized structurally and magnetically. Single crystal X-ray crystallographic analyses revealed that complexes 1Gd-3Dy exhibit 1D chain structures where the Ln(NO3)3 units are bridged by the BNITIm-C2 bis-bidentate biradical, while complexes 4Gd-6Dy exhibit binuclear structures with two Ln(hfac)3 units bridged by the BNITIm-C4 biradical. The bulky hfac anions prohibit the further coordination of LnIII to another NIT ligand and the formation of a similar 1D chain structure. Due to the very long intra- and intermolecular distances of the spin centers, complexes 1Gd-3Dy can be magnetically regarded as an isolated 2p-4f-2p tri-spin system while complex 4Gd-6Dy can be regarded as an isolated 2p-4f bi-spin system. Magnetic analyses on the two GdIII compounds revealed the ferromagnetic GdIII-NIT interactions and antiferromagnetic NIT-NIT interactions through the GdIII ion in 1Gd. Alternating-current (ac) magnetic susceptibility investigations revealed that complex 5Tb exhibits the typical SMM behavior under a zero dc field while complex 6Dy was proved to be a field-induced SMM. Ab initio calculations were performed on complexes 2Tb and 5Tb to understand their magnetic anisotropy together with their different magnetic dynamics.

Remdesivir powders manufactured by jet milling for potential pulmonary treatment of COVID-19.

Remdesivir is one of the effective drugs proposed for the treatment of coronavirus disease 2019 (COVID-19). However, the study on inhalable regimen is currently limited though COVID-19 is respiratory diseases and infects lung area. This work aims to prepare inhalable remdesivir formulations and verify their effectiveness through in vitro evaluations. Formulations containing different ratios of jet-milled inhalable remdesivir (5, 10, 20,40, and 70%) with excipients were produced and characterized in terms of the particle size distribution, particle morphology, flowability, water content, crystallinity, the water sorption and desorption capabilities, and the aerodynamic performance. Results indicating that drug loading are a vital factor in facilitating the dispersion of remdesivir dry powder, and the ternary excipient plays a negligible role in improving aerosol performance. Besides, the 70% remdesivir with lactose carrier (70% RD-Lac) was physically stable and retain high aerosol performance after conditioned at 40 °C and 75% RH for a month. Therefore, formulation 70% RD-Lac might be recommended as a candidate product for the potential treatment of COVID-19.

Computational Pressure-Fluid Dynamics Applied to Index of Microcirculatory Resistance, Predicting the Prognosis of Drug-Coated Balloons Compared With Drug-Eluting Stents in STEMI Patients.

Background: The impairment of microvascular injury on prognosis has increasingly drawn extensive awareness along with the high morbidity and mortality of ST-segment elevation myocardial infarction (STEMI) over recent years. The prognostic significance of computational pressure-fluid dynamics applied to index of microcirculatory resistance, derived from coronary angiography (CPFD-caIMR) in microvascular injury evaluation of STEMI patients remained inconclusive. Methods: A total of 213 patients who met the inclusion criteria were selected retrospectively from 1003 STEMI patients from February 2018 to February 2020. Propensity score matching (PSM) was thereafter finished. CPFD-caIMR of all patients was obtained off-line using the software (FlashAngio, Rainmed Ltd., Suzhou, China) after PPCI. The primary endpoint was to compare the CPFD-caIMR and the incidence of major adverse cardiovascular events (MACEs) between drug-coated balloons (DCB) and drug-eluting stents (DES) groups. The correlation between CPFD-caIMR and MACEs was analyzed, and the prognosis of patients with STEMI was evaluated by CPFD-caIMR by multivariate regression analysis. Results: Totally 213 STEMI patients with successful primary percutaneous coronary intervention (PPCI) were included, of whom 84 patients accepted DCB and 129 patients accepted DES respectively. Baseline characteristics and CPFD-caIMR were comparable between DCB and DES groups after PSM (62 patients in each group). CPFD-caIMR was not significantly different between two groups (DES vs. DCB: mean difference: 2.26, 95% CI -4.05 to 8.57, p = 0.45), and so was it when re-grouped by whether CPFD-caIMR > 40U or not (DES vs. DCB: 34.17% vs. 27.16%, p = 0.29). After a follow-up of 1 year, more MACEs occurred in DES group than DCB group (relative risk: 2.50, 95% CI 1.04 to 6.02, p = 0.04). The predictors of MACEs by multi-variate analysis found that, only time from symptom to balloon (p = 0.03) and time from door to balloon (p < 0.01) were independent predictors of MACEs, independent of treatment with DCB or DES intervention. Furthermore, CPFD-caIMR > 40U became an independent predictor of the combined events including cardiovascular deaths or heart failure readmission irrespective of PSM (odds ratio: 4.07, 95% CI: 1.06 to 7.66, p = 0.04). Conclusion: CPFD-caIMR was a promising method for prognosis, which can predict CV death or heart failure readmission in STEMI patients. DCB was a possible strategy in PPCI of STEMI patients, not inferior to DES based on microvascular injury evaluated by CPFD-caIMR.

Polydopamine-Induced Multilevel Engineering of Regenerated Silk Fibroin Fiber for Photothermal Conversion.

Solid photothermal materials with favorable biocompatibility and modifiable mechanical properties demonstrate obvious superiority and growing demand. In this work, polydopamine (PDA) induced functionalization of regenerated silk fibroin (RSF) fibers has satisfactory photothermal conversion ability and flexibility. Based on multilevel engineering, RSF solution containing PDA nanoparticles is wet spun to PDA-incorporating RSF (PDA@RSF) fibers, and then the fibers are coated with PDA via oxidative self-polymerization of dopamine to form PDA@RSF-PDA (PRP) fibers. During the wet spinning process, PDA is to adjust the mechanical properties of RSF by affecting its hierarchical structure. Meanwhile, coated PDA gives the PRP fibers extensive absorption of near-infrared light and sunlight, which is further fabricated into PRP fibrous membranes. The temperature of PRP fibrous membranes can be adjusted and increases to about 50 °C within 360 s under 808 nm laser irradiation with a power density of 0.6 W cm-2 , and PRP fibrous membranes exhibit effective photothermal cytotoxicity both in vitro and in vivo. Under the simulated sunlight, the temperature of PRP fiber increases to more than 200 °C from room temperature and the material can generate 4.5 V voltage when assembled with a differential thermal battery, which means that the material also has the potential for flexible wearable electronic devices.

C/EBPß enhances efficacy of sorafenib in hepatoblastoma.

Hepatoblastoma (HB) is the predominant hepatic neoplasm in infants and young children. Sorafenib has been used to treat adult and pediatric hepatocellular carcinoma. However, efficacy of monotherapy of sorafenib in HB is not sustained. In this study, we tested a possible combinatory therapy of sorafenib with the CCAAT/enhancer-binding proteins (C/EBP) overexpression in HB cell line. Firstly, we evaluated the expression level of C/EBPß in the patients with HB by analyzing The Cancer Genome Atlas data. Lower level of C/EBPß was observed in tumor tissues in comparison with matched normal tissues. Next, we observed that combination of sorafenib and C/EBPß overexpression led to dramatic growth and migration inhibition of live tumor cells which implied promising probability for clinical trial. Mechanistically, C/EBPß which can be downregulated by Ras v12, augmented messenger RNA and protein levels of p53. These data suggested that a combination of sorafenib and C/EBPß overexpression inhibited tumor growth synergistically and provided a promising approach to treat HB.

Biomimetic Salinity Power Generation Based on Silk Fibroin Ion-Exchange Membranes.

Powering implanted medical devices (IMDs) is a long-term challenge since their use in biological environments requires a long-term and stable supply of power and a biocompatible and biodegradable battery system. Here, silk fibroin-based ion-exchange membranes are developed using bionics principles for reverse electrodialysis devices (REDs). Silk fibroin nanofibril (SNF) membranes are negatively and positively modified, resulting in strong cation and anion selectivity that regulates ion diffusion to generate electric power. These oppositely charged SNF membranes are assembled with Ag/AgCl electrodes into a multicompartment RED. By filling them with 10 and 0.001 mM NaCl solutions, a maximum output power density of 0.59 mW/m2 at an external loading resistance of 66 kΩ is obtained. In addition, 10 pairs of SNF membranes produce a considerable voltage of 1.58 V. This work is a proof of concept that key components of battery systems can be fabricated with protein materials. Combined with the emergence of water-based battery technologies, the findings in this study provide insights for the construction of tissue-integrated batteries for the next generation of IMDs.

Role of mitochondrial complex III/IV in the activation of transcription factor Rst2 in Schizosaccharomyces pombe.

Mitochondria play essential roles in eukaryotic cells for glucose metabolism to produce ATP. In Schizosaccharomyces pombe, transcription factor Rst2 can be activated upon glucose deprivation. However, the link between Rst2 and mitochondrial function remains elusive. Here, we monitored Rst2 transcriptional activity in living cells using a Renilla luciferase reporter system, and found that inhibition of mitochondrial complex III/IV caused cells to produce reactive oxygen species (ROS) and nitric oxide (NO), which in turn activated Rst2. Furthermore, Rst2-GFP was observed to translocate from cytoplasm to nucleus upon mitochondrial complex III/IV inhibitors treatment, and deletion of genes associated with complex III/IV resulted in delayed process of Rst2-GFP nuclear exportation under glucose-rich condition. In particular, we found that Rst2 was phosphorylated following the treatment of complex III/IV inhibitors or SNAP. Altogether, our findings suggest that mitochondrial complex III/IV participates in the activation of Rst2 through ROS and NO generation in Schizosaccharomyces pombe.

Tailoring NiCoAl layered double hydroxide nanosheets for assembly of high-performance asymmetric supercapacitors.

NiCoAl layered double hydroxide nanosheets (NiCoAl-LDHNs) were prepared by a one-step solvothermal method. The shape and size of the obtained nanosheets are optimized by adjusting the solvothermal time and the molar concentration ratio of Ni2+/Co2+ to obtain the electrode material with the best performance. When the solvothermal time is 9 h and the molar concentration ratio of Ni2+/Co2+ is 1:1, NiCoAl-LDHNs has the best morphology and electrochemical performance. When assembled into a supercapacitor, NiCoAl-LDHN-9 has a high specific capacitance of 1228.5 F g-1 at 1 A g-1. As the current density is increased to 20 A g-1, the specific capacitance is 1001.8 F g-1, which still has a high capacitance retention of 81.6%. When NiCoAl-LDHN-9 was assembled into an asymmetric supercapacitor, NiCoAl-LDHN-9//AC has a specific capacitance of 102.1 F g-1 at 0.5 A g-1. The asymmetric supercapacitor devices also show excellent electrochemical performance in terms of energy density (35.9 Wh kg-1 at 225.8 W kg-1), power density (4.8 kW kg-1 at 22.2 Wh kg-1) and cycle life (capacitance retention rate after 10,000 cycles is 87.1%). Those results indicate that NiCoAl-LDHN have the potential to be promising electrode materials for high performance supercapacitors.

In vivo odorant input induces distinct synaptic plasticity of GABAergic synapses in developing zebrafish olfactory bulb.

In the first station of central odor processing, the main olfactory bulb, signal processing is regulated by synaptic interactions between glutamatergic and GABAergic inputs of the mitral cells (MCs), the major projection neurons. Our previous study has found that repetitive postsynaptic spiking within a critical time window after presynaptic activation by natural odorant stimulation results in persistent enhancement of glutamatergic inputs of MCs in larval zebrafish. Here we observed a long-term depression of GABAergic synapses induced by the same protocol. This long-term depression was mediated by presynaptic NMDA receptors (NMDARs). Further dissecting GABAergic neurotransmission revealed that the STDP-induction protocol induced persistent modification in recurrent and lateral inhibition with opposite directions and distinct requirements on NMDARs. Thus, at the plasticity level, different types of GABAergic inhibition may utilize different mechanisms to cooperate or compete with excitatory inputs to optimize patterns of olfactory bulb output.

NADPH-Cytochrome P450 Reductase Ccr1 Is a Target of Tamoxifen and Participates in Its Antifungal Activity via Regulating Cell Wall Integrity in Fission Yeast.

Invasive fungal diseases are a leading cause of mortality among immunocompromised populations. Treatment is notoriously difficult due to the limited number of antifungal drugs as well as the emergence of drug resistance. Tamoxifen (TAM), a selective estrogen receptor modulator frequently used for the treatment of breast cancer, has been found to have antifungal activities and may be a useful addition to the agents used to treat fungal infectious diseases. However, the molecular mechanisms underlying its antifungal actions remain obscure. Here, we screened for mutations that confer sensitivity to azole antifungal drugs by using the fission yeast Schizosaccharomyces pombe as a model and isolated a mutant with a mutation in cls1 (ccr1), an allele of the gene encoding the NADPH-cytochrome P450 reductase Ccr1. We found that strains with a deletion of the ccr1+ gene exhibited hypersensitivities to various drugs, including antifungal drugs (azoles, terbinafine, micafungin), the immunosuppressor FK506, and the anticancer drugs TAM and 5-fluorouracil (5-FU). Unexpectedly, the overexpression of Ccr1 caused yeast cell resistance to TAM but not the other drugs tested here. Additionally, strains with a deletion of Ccr1 displayed pleiotropic phenotypes, including defects in cell wall integrity and vacuole fusion, enhanced calcineurin activity, as well as increased intracellular Ca2+ levels. Overexpression of the constitutively active calcineurin suppressed the drug-sensitive phenotypes of the Δccr1 cells. Notably, TAM treatment of wild-type cells resulted in pleiotropic phenotypes, similar to those of cells lacking Ccr1. Furthermore, TAM inhibited Ccr1 NADPH-cytochrome P450 reductase activities in a dose-dependent manner. Moreover, TAM treatment also inhibited the NADPH-cytochrome P450 reductase activities of Candida albicans and resulted in defective cell wall integrity. Collectively, our findings suggest that Ccr1 is a novel target of TAM and is involved in the antifungal activity of TAM by regulating cell wall integrity in fission yeast.

AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeast.

Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.

A family of lanthanide complexes with a bis-tridentate nitronyl nitroxide radical: syntheses, structures and magnetic properties.

Eleven new lanthanide complexes based on a bis-tridentate nitronyl nitroxide radical NIT-Pm2Py (2-(4,6-di(pyridin-2-yl)pyrimidin-2-yl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazol-1-oxy-3-oxide), namely (NIT-Pm2Py)Ln(hfac)3 (Ln = Gd (1Gd), Tb (2Tb), Dy (3Dy), Ho (4Ho), Er (5Er), Yb (6Yb)), [(NIT-Pm2Py)Ln2(hfac)6]·xH2O (Ln = Gd (7Gd), Tb (8Tb), Ho (10Ho), x = 0.5 for 7Gd and 1 for 8Tb and 10Ho) and (NIT-Pm2Py)Ln2(hfac)6 (Ln = Dy (9Dy), Er (11Er)) were prepared and characterized. These complexes can be selectively prepared by controlling the reaction ratio of Ln(hfac)3·2H2O to the radical ligand NIT-Pm2Py. Single crystal X-ray crystallographic analyses confirmed that 1Gd-6Yb are isostructural 2p-4f LnIII-radical complexes, in which the NIT-Pm2Py radical acts as a terminal tridentate ligand chelating to one LnIII ion. On the other hand, 7Gd-11Er are isostructural 4f-2p-4f LnIII-radical-LnIII complexes with the NIT-Pm2Py acting as a bridging ligand between two LnIII ions. 7Gd-11Er represent a rare family of complexes showing the NIT bridged 4f-2p-4f three-spin motif. Alternating-current (ac) magnetic susceptibility investigations revealed that complex 6Yb exhibits field-induced frequency dependence, suggesting a possible field-induced single-molecule magnet behavior. Ab initio calculations were performed on all these complexes. The fitting of the magnetic susceptibilities of these complexes indicates weak antiferromagnetic coupling between the LnIII and NIT radical.