Intracellular Vimentin Regulates the Formation of Classical Swine Fever Virus Replication Complex through Interaction with NS5A Protein.

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV.

Prophage Induction Causes Geobacter Electroactive Biofilm Decay.

Sustaining a metabolically active electroactive biofilm (EAB) is essential for the high efficiency and durable operation of microbial fuel cells (MFCs). However, EABs usually decay during long-term operation, and, until now, the causes remain unknown. Here, we report that lysogenic phages can cause EAB decay in Geobacter sulfurreducens fuel cells. A cross-streak agar assay and bioinformatic analysis revealed the presence of prophages on the G. sulfurreducens genome, and a mitomycin C induction assay revealed the lysogenic to lytic transition of those prophages, resulting in a progressive decay in both current generation and the EAB. Furthermore, the addition of phages purified from decayed EAB resulted in accelerated decay of the EAB, thereafter contributing to a faster decline in current generation; otherwise, deleting prophage-related genes rescued the decay process. Our study provides the first evidence of an interaction between phages and electroactive bacteria and suggests that attack by phages is a primary cause of EAB decay, having significant implications in bioelectrochemical systems.

Sulfur-decorated Fe/C composite synthesized from MIL-88A(Fe) for peroxymonosulfate activation towards tetracycline degradation: Multiple active sites and non-radical pathway dominated mechanism.

Peroxymonosulfate (PMS)-mediated advanced oxidation processes gain growing attention in degrading antibiotics (e.g., tetracycline (TC)) in wastewater for their high capacity and relatively low cost, while designing efficient catalysts for PMS activation remains a challenge. In this study, a sulfur-doped Fe/C catalyst (Fe@C-S) synthesized from iron metal-organic frameworks (Fe-MOFs) was developed for PMS activation towards TC removal. Under optimal conditions, the TC removal efficiency of Fe@C-S150/PMS system within 40 min was 91.2%. Meanwhile, the k value for Fe@C-S150/PMS system (0.2038 min-1) was 3.36-fold as high as the S-free Fe@C-based PMS system. Also, Fe@C-S150/PMS system showed high robustness in different water matrices. Further studies found that the TC degradation mechanism was mainly ascribed to the non-radical pathway (1O2 and electron transfer). Fe nanoparticles, S and CO groups on the catalyst all participated in the generation of reactive oxygen species (ROS). Besides, S species could enhance the Fe2+/Fe3+ redox cycle and accelerate the electron transfer process. This work highlights the critical role of S in enhancing the catalytic performance of Fe/C-based catalysts for PMS activation, which would provide meaningful insights into the design of high-performance PMS activators for the sustainable remediation of emerging contaminants-polluted water bodies.

Evidence of aircraft activity impact on local air quality: A study in the context of uncommon airport operation.

Wuhan Tianhe International Airport (WUH) was suspended to contain the spread of COVID-19, while Shanghai Hongqiao International Airport (SHA) saw a tremendous flight reduction. Closure of a major international airport is extremely rare and thus represents a unique opportunity to straightforwardly observe the impact of airport emissions on local air quality. In this study, a series of statistical tools were applied to analyze the variations in air pollutant levels in the vicinity of WUH and SHA. The results of bivariate polar plots show that airport SHA and WUH are a major source of nitrogen oxides. NOx, NO2 and NO diminished by 55.8%, 44.1%, 76.9%, and 40.4%, 33.3% and 59.4% during the COVID-19 lockdown compared to those in the same period of 2018 and 2019, under a reduction in aircraft activities by 58.6% and 61.4%. The concentration of NO2, SO2 and PM2.5 decreased by 77.3%, 8.2%, 29.5%, right after the closure of airport WUH on 23 January 2020. The average concentrations of NO, NO2 and NOx scatter plots at downwind of SHA after the lockdown were 78.0%, 47.9%, 57.4% and 62.3%, 34.8%, 41.8% lower than those during the same period in 2018 and 2019. However, a significant increase in O3 levels by 50.0% and 25.9% at WUH and SHA was observed, respectively. These results evidently show decreased nitrogen oxides concentrations in the airport vicinity due to reduced aircraft activities, while amplified O3 pollution due to a lower titration by NO under strong reduction in NOx emissions.

Clinical study on the use of advanced magnetic resonance imaging in lupus nephritis.

OBJECTIVES: To investigate the correlation between the histopathology of the kidney and clinical indicators in patients with lupus nephritis (LN) using magnetic resonance imaging (MRI). METHODS: A total 50 female participants were enrolled in the study. Thirty patients with LN were divided into types 2, 3, 4, and 5, according to their pathological features. The control group consisted of 20 healthy female volunteers. Serum creatinine, C3, C1q, and anti-ds-DNA were measured. Conventional MRI, DTI, DWI, and BOLD scanning was performed to obtain the FA, ADC, and R2* values for the kidney. RESULTS: Compared with the control group, FA and the ADC were decreased in patients with LN, while the R2* value was increased (P < 0.05). The overall comparison of the SLEDAI (Activity index of systemic lupus erythematosus) score, total pathological score, AI, and serum creatinine C3 showed that these were significantly different between the two groups (P < 0.05). FA and the ADC were negatively correlated with urinary, blood ds-DNA, and serum creatinine and positively correlated with C1q (P < 0.05). The R2* value was positively correlated with urinary NGAL, blood ds-DNA, and serum creatinine (P < 0.05). FA and the ADC were negatively correlated with the SLEDAI score, total pathological score, AI, CI, nephridial tissue C3, and C1q. The R2* value was positively correlated with the SLEDAI score, total pathological score, AI, CI, nephridial tissue C3, and C1q (P < 0.05). CONCLUSIONS: MRI examination in female patients with LN was correlated with pathologic test results, which may have clinical significance in determining the disease's severity, treatment, and outcome.

Effects of Cinnamomum camphora Leaves Extracts-Flocculants Composite Algaecide on Microcystis aeruginosa Growth and Microcystins Release.

In this study, a composite algaecide containing flocculants and Cinnamomum. camphora leaves extracts (CCCLE) were synthesized. The inhibition and flocculation effects on Microcystis aeruginosa (M. aeruginosa) were investigated, and the release of microcystin-LR (MC-LR) was determined. Results showed that the CCLEC composite algaecide was effective for the inhibition and flocculation of M. aeruginosa, and the optimal dose of CCLEC composite algaecide was 1.8%, which resulted in an algae inhibition ratio of 98.00% and a flocculation efficiency of 99.44% within 5 days of M. aeruginosa culturing. Besides, the total amount of MC-LR decreased by 80.04% on day 20 compared with the control group, while the concentration of intracellular MC-LR on day 5 was 36.69 µg L-1, which was related to a portion of cells underwent apoptosis-like cell death under CCLEC composite algaecide stress. The results of this study may improve our understanding of the M. aeruginosa control by CCCLE composite algaecide.

Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments.

The electrically conductive pili (e-pili) of Geobacter species enable extracellular electron transfer to insoluble metallic minerals, electrodes and other microbial species, which confers biogeochemical significance and global prevalence on Geobacter in diverse anaerobic environments. E-pili are constructed by truncated PilA which is considered to have evolved from full-length pilin by gene fission under positive evolutionary selection. However, this hypothesis is based on phylogenetic analysis and has not yet been experimentally confirmed. Here, we reconstructed an ancestral strain of G. sulfurreducens (designated COMB) carrying full-length PilA by combining genes GSU1496 and GSU1497. The results demonstrated that strain COMB expressed and assembled the full-length fused PilA and exhibited an outer membrane c-type cytochrome profile similar to the wild-type strain. Surprisingly, the generated COMB-pili were also conductive, indicating the evolution of truncated PilA did not occur for conductivity. Moreover, strain COMB minimally reduced Fe(III) oxides but maintained its ability to respire electrodes, demonstrating the truncation of pilin enables iron respiration. This study provides the first experimental evidence that the truncation of pilin in Geobacter species confers adaption to Fe(III)-mineral-mediated selective pressures, and suggests an evolutionary event during which the separation of the GSU1497 gene helped Geobacter survive and thrive in natural environments.

Brevibacillus composti sp. nov., isolated from hyperthermophilic compost.

Two aerobic, Gram-stain-positive, rod-shaped, endospore-forming, thermophilic bacterial strains, designated FJAT-54423T and FJAT-54424, were isolated from hyperthermophilic compost sampled in Shanxi Province, PR China. Growth was observed at 30-60 °C (optimum, 50 °C) and pH 6.0-9.0 (optimum, pH 7.0), with up to 2.0 % (w/v) NaCl (optimum, 0 % NaCl). The 16S rRNA gene sequence similarity between FJAT-54423T and FJAT-54424 was 99.9%, and the maximum similarity to a valid taxon was observed with Brevibacillus borstelensis (98.3%). Further, in phylogenetic and phylogenomic trees, strains FJAT-54423T and FJAT-54424 branched with members of the genus Brevibacillus. The menaquinone was MK-7, and the major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The main polar lipids included phosphatidylmethylethanolamine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content of strains FJAT-54423T and FJAT-54424 were 54.3 and 54.4 mol%, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strain FJAT-54423T and its most closely related reference strain B. borstelensis DSM 6347T were 77.7 and 21.5 %, respectively, which were lower than the recommended species delineation thresholds of ANI (95%) and dDDH (70%). Based on the observed physiological properties, chemotaxonomic characteristics and ANI and dDDH values, FJAT-54423T and FJAT-54424 belong to a novel species of the genus Brevibacillus, for which the name Brevibacillus composti sp. nov. is proposed. The type strain is FJAT-54423T (=GDMCC 1.2054T=KCTC 43273T).

SLX4IP acts with SLX4 and XPF-ERCC1 to promote interstrand crosslink repair.

Interstrand crosslinks (ICLs) are highly toxic DNA lesions that are repaired via a complex process requiring the coordination of several DNA repair pathways. Defects in ICL repair result in Fanconi anemia, which is characterized by bone marrow failure, developmental abnormalities, and a high incidence of malignancies. SLX4, also known as FANCP, acts as a scaffold protein and coordinates multiple endonucleases that unhook ICLs, resolve homologous recombination intermediates, and perhaps remove unhooked ICLs. In this study, we explored the role of SLX4IP, a constitutive factor in the SLX4 complex, in ICL repair. We found that SLX4IP is a novel regulatory factor; its depletion sensitized cells to treatment with ICL-inducing agents and led to accumulation of cells in the G2/M phase. We further discovered that SLX4IP binds to SLX4 and XPF-ERCC1 simultaneously and that disruption of one interaction also disrupts the other. The binding of SLX4IP to both SLX4 and XPF-ERCC1 not only is vital for maintaining the stability of SLX4IP protein, but also promotes the interaction between SLX4 and XPF-ERCC1, especially after DNA damage. Collectively, these results demonstrate a new regulatory role for SLX4IP in maintaining an efficient SLX4-XPF-ERCC1 complex in ICL repair.

Study on the Optimization of Hyperspectral Characteristic Bands Combined with Monitoring and Visualization of Pepper Leaf SPAD Value.

Chlorophyll content is an important indicator of plant photosynthesis, which directly affects the growth and yield of crops. Using hyperspectral imaging technology to quickly and non-destructively estimate the soil plant analysis development (SPAD) value of pepper leaf and its distribution inversion is of great significance for agricultural monitoring and precise fertilization during pepper growth. In this study, 150 samples of pepper leaves with different leaf positions were selected, and the hyperspectral image data and SPAD value were collected for the sampled leaves. The correlation coefficient, stability competitive adaptive reweighted sampling (sCARS), and iteratively retaining informative variables (IRIV) methods were used to screen characteristic bands. These were combined with partial least-squares regression (PLSR), extreme gradient boosting (XGBoost), random forest regression (RFR), and gradient boosting decision tree (GBDT) to build regression models. The developed model was then used to build the inversion map of pepper leaf chlorophyll distribution. The research results show that: (1) The IRIV-XGBoost model demonstrates the most comprehensive performance in the modeling and inversion stages, and its Rcv2, RMSEcv, and MAEcv are 0.81, 2.76, and 2.30, respectively; (2) The IRIV-XGBoost model was used to calculate the SPAD value of each pixel of pepper leaves, and to subsequently invert the chlorophyll distribution map of pepper leaves at different leaf positions, which can provide support for the intuitive monitoring of crop growth and lay the foundation for the development of hyperspectral field dynamic monitoring sensors.

Evidence for the coexistence of direct and riboflavin-mediated interspecies electron transfer in Geobacter co-culture.

Geobacter species can secrete free redox-active flavins, but the role of these flavins in the interspecies electron transfer (IET) of Geobacter direct interspecies electron transfer (DIET) co-culture is unknown. Here, we report the presence of a new riboflavin-mediated interspecies electron transfer (RMIET) process in a traditional Geobacter DIET co-culture; in this process, riboflavin contributes to IET by acting as a free-form electron shuttle between free Geobacter species and serving as a bound cofactor of some cytochromes in Geobacter co-culture aggregates. Multiple lines of evidence indicate that RMIET facilitates the primary initiation of syntrophic growth between Geobacter species before establishing the DIET co-culture and provides additional ways alongside the DIET to transfer electrons to achieve electric syntrophy between Geobacter species. Redox kinetic analysis of riboflavin on either Geobacter species demonstrated that the Gmet_2896 cytochrome acts as the key riboflavin reduction site, while riboflavin oxidation by Geobacter sulfurreducens is the rate-limiting step in RMIET, and the RMIET makes only a minor contribution to IET in Geobacter DIET co-culture. The discovery of a new RMIET process in Geobacter DIET co-culture suggests the complexity of IET in syntrophic bacterial communities and provides suggestions for the careful examination of the IET of other syntrophic co-cultures.

Human BCR/ABL1 induces chronic myeloid leukemia-like disease in zebrafish.

Chronic myeloid leukemia (CML) is induced by the BCR/ABL1 oncogene, which encodes a protein tyrosine kinase. We examined the effect of direct overexpression of the human p210 BCR/ABL1 oncoprotein in zebrafish. Humanized p210 BCR/ABL1 protein was detectable in Tg(hsp70: p210BCR/ABL1 ) transgenic zebrafish embryos and adult kidney marrow. Transgenic zebrafish developed CML, which could be induced via cells transplanted into recipients. The expression of human BCR/ABL1 promoted myeloid lineages in Tg(hsp70:p210BCR/ABL1) transgenic embryos. A total of 77 of 101 (76.24%) Tg(hsp70:p210BCR/ABL1) adult transgenic zebrafish (age 6 months-1 year) developed CML. CML in zebrafish showed a triphasic phenotype, similar to that in humans, involving a chronic phase predominantly characterized by neutrophils in various degrees of maturation, an accelerated phase with an increase in blasts and immature myeloid elements, and a blast phase with >90% blasts in both the peripheral blood and kidney marrow. Tyrosine kinase inhibitors, as the standard drug treatment for human CML, effectively reduced the expanded myeloid population in Tg(hsp70:p210BCR/ABL1) transgenic embryos. Moreover, we screened a library of 171 compounds and identified ten new drugs against BCR/ABL1 kinase-dependent or -independent pathways that could also reduce lcp1+ myeloid cell numbers in Tg(hsp70:p210BCR/ABL1) transgenic embryos. In summary, we generated the first humanized zebrafish CML model that recapitulates many characteristics of human CML. This novel in vivo model will help to elucidate the mechanisms of CML disease progression and allow high-throughput drug screening of possible treatments for this disease.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability.

Assessment of the predictive role of pretreatment Ki-67 and Ki-67 changes in breast cancer patients receiving neoadjuvant chemotherapy according to the molecular classification: a retrospective study of 1010 patients.

PURPOSE: To assess the predictive role of pretreatment ki67 and Ki67 changes in breast cancer (BC) patients treated with neoadjuvant chemotherapy (NAC) in various molecular subtypes. METHODS: 1010 BC patients who had undergone anthracycline and taxane-based NAC from January 2012 to July 2017 were retrospectively analyzed. Clinical and pathological parameters of the patients were retrieved and the predictive factors for NAC response were evaluated. RESULTS: 705 patients showed clinical response (cRes), and 131 patients acquired pathologic complete response (pCR). Patients with higher pretreatment Ki67 (≥ 14%), tumor size ≥ 4 cm, and positive clinical nodal had better clinical therapy response, while patients with negative ER and PR, higher pretreatment Ki67 (≥ 14%), and tumor size < 4 cm were more probable to attain pCR. The pretreatment Ki67 could be used as a predictor of NAC only in luminal subtypes, and 25.5% were identified as an ideal cut-off point to differentiate the cRes from non-cRes cases. Although a decrease in Ki67 had been found in almost all molecular subtypes after NAC, no statistically significant differences were found in the decrease of Ki67 were validated between the cRes and non-cRes group in HER2-rich and triple-negative subtypes (P = 0.488 and P = 0.111, respectively). CONCLUSIONS: The best cut-off for pretreatment Ki67 in predicting the connection with the tumor size lessening was 25.5% in luminal subtype. Aggressive adjuvant systemic treatments should be considered for patients with HER2-rich and triple-negative subtype who exhibit tumor shrinkage in NAC but still have high levels of Ki67.

Effect of piperine on the bioavailability and pharmacokinetics of rosmarinic acid in rat plasma using UPLC-MS/MS.

1. The purpose of the present study was to investigate the effect of piperine (PP) on the pharmacokinetics of rosmarinic acid (RA) in rat plasma and to determine whether PP could enhance the oral bioavailability of RA via inhibition of its glucuronidation. 2. The pharmacokinetic profiles of RA between oral administration of RA (50 mg/kg) alone and in combination with different oral dose PP (20, 40, 60, and 80 mg/kg) to rats were investigated via a validated UPLC/MS/MS method. 3. The AUC and Cmax of RA were significantly increased in combination with different dose PP dose dependently, especially in the presence of 60 and 80 mg/kg PP (p < 0.01). The relative bioavailability of RA in the presence of 20, 40, 60, and 80 mg/kg PP was 1.24-, 1.32-, 2.02-, and 2.26-folds higher, respectively, compared with the control group given RA alone. Compared with RA, the pharmacokinetic modulations of RA glucuronide were even more apparent, and the glucuronidation of RA was remarkedly inhibited. 4. This study demonstrated that PP significantly improved the in vivo bioavailability of RA partly attributing to the inhibition of gut and hepatic metabolism enzymes of RA.

A Long-Cycle-Life Self-Doped Polyaniline Cathode for Rechargeable Aqueous Zinc Batteries.

Rechargeable aqueous zinc batteries are promising energy-storage systems for grid applications. Highly conductive polyaniline (PANI) is a potential cathode, but it tends to deactivate in electrolytes with low acidity (i.e. pH >1) owing to deprotonation of the polymer. In this study, we synthesized a sulfo-self-doped PANI electrode by a facile electrochemical copolymerization process. The -SO3 - self-dopant functions as an internal proton reservoir to ensure a highly acidic local environment and facilitate the redox process in the weakly acidic ZnSO4 electrolyte. In a full zinc cell, the self-doped PANI cathode provided a high capacity of 180 mAh g-1 , excellent rate performance of 70 % capacity retention with a 50-fold current-density increase, and a long cycle life of over 2000 cycles with coulombic efficiency close to 100 %. Our study opens a door for the use of conducting polymers as cathode materials for high-performance rechargeable zinc batteries.

Pumping through porous hydrophobic/oleophilic materials: an alternative technology for oil spill remediation.

Recently, porous hydrophobic/oleophilic materials (PHOMs) have been shown to be the most promising candidates for cleaning up oil spills; however, due to their limited absorption capacity, a large quantity of PHOMs would be consumed in oil spill remediation, causing serious economic problems. In addition, the complicated and time-consuming process of oil recovery from these sorbents is also an obstacle to their practical application. To solve the above problems, we apply external pumping on PHOMs to realize the continuous collection of oil spills in situ from the water surface with high speed and efficiency. Based on this novel design, oil/water separation and oil collection can be simultaneously achieved in the remediation of oil spills, and the oil sorption capacity is no longer limited to the volume and weight of the sorption material. This novel external pumping technique may bring PHOMs a step closer to practical application in oil spill remediation.

Tumor Dormancy Within the Lymphovascular Embolus Is Regulated by Multiple Metabolism-signaling Pathways.

BACKGROUND/AIM: Recently, we demonstrated that cancer dormancy is initiated within the lymphovascular tumor embolus and consists of decreased proliferation and lower mammalian target of rapamycin (mTOR) activity. In the present study, we investigated other intersecting metabolism-signaling pathways that may ultimately determine whether the lymphovascular tumor embolus remains dormant or undergoes cell death. MATERIALS AND METHODS: The present study exploited a singular patient-derived xenograft (PDX) of inflammatory breast cancer (Mary-X) that spontaneously forms high density spheroids, the in vitro equivalent of emboli. The AMPK metabolic checkpoint pathway, the mTOR nutrient-responsive cell growth pathway, the P13K/Akt intracellular quiescence regulating pathway, and the calpain-mediated E-cadherin proteolytic pathway responsible for spontaneous spheroid-genesis were also investigated, to determine their relative contributions to dormancy. RESULTS: The levels of phosphorylated AMPK proteins (AMPKα and ß subunits) decreased gradually with the formation of MARY-X spheroids in vitro. Rapamycin down-regulated mTOR activity, yet dormancy persisted. LY294002, a PI3K/Akt inhibitor, completely abolished mTOR and induced spheroid disadherence and apoptosis. Compound C (AMPK inhibitor) up-regulated mTOR and induced spheroid disadherence and apoptosis. Increasing cellular metabolism led to cell death, even in enriched medium, whereas growing the spheroids in serum-free media (starvation) did not result in further mTOR inhibition, and dormancy was maintained. CONCLUSION: An increase in our understanding of dormancy from the standpoint of internal signaling pathways might ultimately provide clues to the external stimuli (starvation, hypoxia or other not yet understood phenomena) that act through these pathways to maintain or disrupt dormancy.