Factors associated with engraftment success of patient-derived xenografts of breast cancer.

BACKGROUND: Patient-derived xenograft (PDX) models serve as a valuable tool for the preclinical evaluation of novel therapies. They closely replicate the genetic, phenotypic, and histopathological characteristics of primary breast tumors. Despite their promise, the rate of successful PDX engraftment is various in the literature. This study aimed to identify the key factors associated with successful PDX engraftment of primary breast cancer. METHODS: We integrated clinicopathological data with morphological attributes quantified using a trained artificial intelligence (AI) model to identify the principal factors affecting PDX engraftment. RESULTS: Multivariate logistic regression analyses demonstrated that several factors, including a high Ki-67 labeling index (Ki-67LI) (p < 0.001), younger age at diagnosis (p = 0.032), post neoadjuvant chemotherapy (NAC) (p = 0.006), higher histologic grade (p = 0.039), larger tumor size (p = 0.029), and AI-assessed higher intratumoral necrosis (p = 0.027) and intratumoral invasive carcinoma (p = 0.040) proportions, were significant factors for successful PDX engraftment (area under the curve [AUC] 0.905). In the NAC group, a higher Ki-67LI (p < 0.001), lower Miller-Payne grade (p < 0.001), and reduced proportion of intratumoral normal breast glands as assessed by AI (p = 0.06) collectively provided excellent prediction accuracy for successful PDX engraftment (AUC 0.89). CONCLUSIONS: We found that high Ki-67LI, younger age, post-NAC status, higher histologic grade, larger tumor size, and specific morphological attributes were significant factors for predicting successful PDX engraftment of primary breast cancer.

Proteomic analysis of breast cancer based on immune subtypes.

BACKGROUND: Immunotherapy is applied to breast cancer to resolve the limitations of survival gain in existing treatment modalities. With immunotherapy, a tumor can be classified into immune-inflamed, excluded and desert based on the distribution of immune cells. We assessed the clinicopathological features, each subtype's prognostic value and differentially expressed proteins between immune subtypes. METHODS: Immune subtyping and proteomic analysis were performed on 56 breast cancer cases with neoadjuvant chemotherapy. The immune subtyping was based on the level of tumor-infiltrating lymphocytes (TILs) and Klintrup criteria. If the level of TILs was ≥ 10%, it was classified as immune-inflamed type without consideration of the Klintrup criteria. In cases of 1-9% TIL, Klintrup criteria 1-3 were classified as the immune-excluded subtype and Klintrup criteria not available (NA) was classified as NA. Cases of 1% TILs and Klintrup 0 were classified as the immune-desert subtype. Mass spectrometry was used to identify differentially expressed proteins in formalin-fixed paraffin-embedded biopsy tissues. RESULTS: Of the 56 cases, 31 (55%) were immune-inflamed, 21 (38%) were immune-excluded, 2 (4%) were immune-desert and 2 (4%) were NA. Welch's t-test revealed two differentially expressed proteins between immune-inflamed and immune-excluded/desert subtypes. Coronin-1A was upregulated in immune-inflamed tumors (adjusted p = 0.008) and α-1-antitrypsin was upregulated in immune-excluded/desert tumors (adjusted p = 0.008). Titin was upregulated in pathologic complete response (pCR) than non-pCR among immune-inflamed tumors (adjusted p = 0.036). CONCLUSIONS: Coronin-1A and α-1-antitrypsin were upregulated in immune-inflamed and immune-excluded/desert subtypes, respectively. Titin's elevated expression in pCR within the immune-inflamed subtype may indicate a favorable prognosis. Further studies involving large representative cohorts are necessary to validate these findings.

Pseudoknot-targeting Cas13b combats SARS-CoV-2 infection by suppressing viral replication.

CRISPR-Cas13-mediated viral genome targeting is a novel strategy for defending against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we generated mRNA-encoded Cas13b targeting the open reading frame 1b (ORF1b) region to effectively degrade the RNA-dependent RNA polymerase gene. Of the 12 designed CRISPR RNAs (crRNAs), those targeting the pseudoknot site upstream of ORF1b were found to be the most effective in suppressing SARS-CoV-2 propagation. Pseudoknot-targeting Cas13b reduced expression of the spike protein and attenuated viral replication by 99%. It also inhibited the replication of multiple SARS-CoV-2 variants, exhibiting broad potency. We validated the therapeutic efficacy of this system in SARS-CoV-2-infected hACE2 transgenic mice, demonstrating that crRNA treatment significantly reduced viral titers. Our findings suggest that the pseudoknot region is a strategic site for targeted genomic degradation of SARS-CoV-2. Hence, pseudoknot-targeting Cas13b could be a breakthrough therapy for overcoming infections by SARS-CoV-2 or other RNA viruses.

Aberration Estimation for Synthetic Aperture Digital Holographic Microscope Using Deep Neural Network.

Digital holographic microscopy (DHM) is a valuable technique for investigating the optical properties of samples through the measurement of intensity and phase of diffracted beams. However, DHMs are constrained by Lagrange invariance, compromising the spatial bandwidth product (SBP) which relates resolution and field of view. Synthetic aperture DHM (SA-DHM) was introduced to overcome this limitation, but it faces significant challenges such as aberrations in synthesizing the optical information corresponding to the steering angle of incident wave. This paper proposes a novel approach utilizing deep neural networks (DNNs) for compensating aberrations in SA-DHM, extending the compensation scope beyond the numerical aperture (NA) of the objective lens. The method involves training a DNN from diffraction patterns and Zernike coefficients through a circular aperture, enabling effective aberration compensation in the illumination beam. This method makes it possible to estimate aberration coefficients from the only part of the diffracted beam cutoff by the circular aperture mask. With the proposed technique, the simulation results present improved resolution and quality of sample images. The integration of deep neural networks with SA-DHM holds promise for advancing microscopy capabilities and overcoming existing limitations.

Prediction of indoor PM2.5 concentrations and reduction strategies for cooking events through various IAQ management methods in an apartment of South Korea.

Indoor PM2.5 in apartments must be effectively managed to minimize adverse impacts on human health. Cooking is the one of the main PM2.5 sources in apartments, and indoor air quality (IAQ) management methods (natural ventilation, mechanical ventilations, range hoods, and air purifiers) are typically used to reduce PM2.5 generated during cooking. For effective control of indoor PM2.5 , prediction of PM2.5 reduction for various IAQ management methods is necessary. This study carefully predicted indoor PM2.5 concentrations in an apartment when IAQ management methods were applied separately and/or in combination during cooking. The infiltration and exfiltration were verified by comparing the experimental results of CO2 concentration with those predicted with or without mechanical ventilation. The deposition rate for PM2.5 generated by cooking was also derived by comparing the experimental PM2.5 changes with the predicted values for PM2.5 natural decay. Through this method, effective PM2.5 control ways during cooking in apartments can be proposed, such as natural ventilation with a range hood for 30 min and then the operation of an air purifier for 30 min. Additionally, if this prediction is combined with energy consumption, it will be possible to propose the most energy-efficient indoor PM2.5 control methods for various seasons and outdoor conditions.

Evaluation of Antiviral Effect against SARS-CoV-2 Propagation by Crude Polysaccharides from Seaweed and Abalone Viscera In Vitro.

Crude polysaccharides, extracted from two seaweed species (Hizikia fusiforme and Sargassum horneri) and Haliotis discus hannai (abalone) viscera, were evaluated for their inhibitory effect against SARS-CoV-2 propagation. Plaque titration revealed that these crude polysaccharides efficiently inhibited SARS-CoV-2 propagation with IC50 values ranging from 0.35 to 4.37 µg/mL. The crude polysaccharide of H. fusiforme showed the strongest antiviral effect, with IC50 of 0.35 µg/mL, followed by S. horneri and abalone viscera with IC50 of 0.56 and 4.37 µg/mL, respectively. In addition, immunofluorescence assay, western blot, and quantitative RT-PCR analysis verified that these polysaccharides could inhibit SARS-CoV-2 replication. In Vero E6 cells, treatment with these crude polysaccharides before or after viral infection strongly inhibited the expression level of SARS-CoV-2 spikes, nucleocapsid proteins, and RNA copies of RNA-dependent RNA-polymerase and nucleocapsid. These results show that these crude marine polysaccharides effectively inhibit SARS-CoV-2 propagation by interference with viral entry.

PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins.

PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the Cripto-1/FRL1/Cryptic domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4, and contactin-1, in addition to CRIPTO, are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.

Sulfated syndecan 1 is critical to preventing cellular senescence by modulating fibroblast growth factor receptor endocytosis.

Cellular senescence can be triggered by various intrinsic and extrinsic stimuli. We previously reported that silencing of 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) induces cellular senescence through augmented fibroblast growth factor receptor 1 (FGFR1) signaling. However, the exact molecular mechanism connecting heparan sulfation and cellular senescence remains unclear. Here, we investigated the potential involvement of heparan sulfate proteoglycans (HSPGs) in augmented FGFR1 signaling and cellular senescence. Depletion of several types of HSPGs revealed that cells depleted of syndecan 1 (SDC1) exhibited typical senescence phenotypes, and those depleted of PAPSS2-, SDC1-, or heparan sulfate 2-O sulfotransferase 1 (HS2ST1) showed decreased FGFR1 internalization along with hyperresponsiveness to and prolonged activation of fibroblast growth factor 2 (FGF2)-stimulated FGFR1- v-akt murine thymoma viral oncogene homolog (AKT) signaling. Clathrin- and caveolin-mediated FGFR1 endocytosis contributed to cellular senescence through the FGFR1-AKT-p53-p21 signaling pathway. Dynasore treatment triggered senescence phenotypes, augmented FGFR1-AKT-p53-p21 signaling, and decreased SDC1 expression. Finally, the replicatively and prematurely senescent cells were characterized by decreases of SDC1 expression and FGFR1 internalization, and an increase in FGFR1-AKT-p53-p21 signaling. Together, our results demonstrate that properly sulfated SDC1 plays a critical role in preventing cellular senescence through the regulation of FGFR1 endocytosis.

Fixation of Dinitrogen at an Asymmetric Binuclear Titanium Complex.

A new type of dititanium dinitrogen complex supported by a triphenolamine (TPA) ligand is reported. Analysis by single-crystal X-ray diffraction and Raman and NMR spectroscopy reveals different coordination geometries for the two titanium centers. Hence, coordination of TPA and a nitrogen ligand results in trigonal-bipyramidal geometry, while an octahedral titanium center is obtained upon additional coordination of an ethoxide generated upon C-O bond cleavage in a diethyl ether solvent molecule. The titanium complex successfully generates ammonia in the presence of an excess amount of PCy3HI and KC8 in 154% yield (per titanium atom). A titanium complex with a bulkier TPA does not form a dinitrogen complex, and mononuclear titanium dinitrogen complexes were not accessible, presumably because of the high tendency of early transition metals to form binuclear dinitrogen complexes.

The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.

Highly Reproducible Large-Area Perovskite Solar Cell Fabrication via Continuous Megasonic Spray Coating of CH3 NH3 PbI3.

A simple, low-cost, large area, and continuous scalable coating method is proposed for the fabrication of hybrid organic-inorganic perovskite solar cells. A megasonic spray-coating method utilizing a 1.7 MHz megasonic nebulizer that could fabricate reproducible large-area planar efficient perovskite films is developed. The coating method fabricates uniform large-area perovskite film with large-sized grain since smaller and narrower sized mist droplets than those generated by existing ultrasonic spray methods could be generated by megasonic spraying. The volume flow rate of the CH3 NH3 PbI3 precursor solution and the reaction temperature are controlled, to obtain a high quality perovskite active layer. The devices reach a maximum efficiency of 16.9%, with an average efficiency of 16.4% from 21 samples. The applicability of megasonic spray coating to the fabrication of large-area solar cells (1 cm2 ), with a power conversion efficiency of 14.2%, is also demonstrated. This is a record high efficiency for large-area perovskite solar cells fabricated by continuous spray coating.

Seizure-Induced Regulations of Amyloid-ß, STEP61, and STEP61 Substrates Involved in Hippocampal Synaptic Plasticity.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Pathologic accumulation of soluble amyloid-ß (Aß) oligomers impairs synaptic plasticity and causes epileptic seizures, both of which contribute to cognitive dysfunction in AD. However, whether seizures could regulate Aß-induced synaptic weakening remains unclear. Here we show that a single episode of electroconvulsive seizures (ECS) increased protein expression of membrane-associated STriatal-Enriched protein tyrosine Phosphatase (STEP61) and decreased tyrosine-phosphorylation of its substrates N-methyl D-aspartate receptor (NMDAR) subunit GluN2B and extracellular signal regulated kinase 1/2 (ERK1/2) in the rat hippocampus at 2 days following a single ECS. Interestingly, a significant decrease in ERK1/2 expression and an increase in APP and Aß levels were observed at 3-4 days following a single ECS when STEP61 level returned to the baseline. Given that pathologic levels of Aß increase STEP61 activity and STEP61-mediated dephosphorylation of GluN2B and ERK1/2 leads to NMDAR internalization and ERK1/2 inactivation, we propose that upregulation of STEP61 and downregulation of GluN2B and ERK1/2 phosphorylation mediate compensatory weakening of synaptic strength in response to acute enhancement of hippocampal network activity, whereas delayed decrease in ERK1/2 expression and increase in APP and Aß expression may contribute to the maintenance of this synaptic weakening.

Evaluation of vaccine efficacy with 2B/T epitope conjugated porcine IgG-Fc recombinants against foot-and-mouth disease virus.

The inactivated vaccine is effective in controlling foot-and-mouth disease (FMD), but it has drawbacks such as the need for a biosafety level 3 laboratory facility to handle live foot-and-mouth disease virus (FMDV), high production costs, and biological safety risks. In response to these challenges, we developed a new recombinant protein vaccine (2BT-pIgG-Fc) containing porcine IgG-Fc to enhance protein stability in the body. This vaccine incorporates two-repeat B-cell and one-single T-cell epitope derived from O/Jincheon/SKR/2014. Our study confirmed that 2BT-pIgG-Fc and a commercial FMDV vaccine induced FMDV-specific antibodies in guinea pigs at 28 days post-vaccination. The percentage inhibition (PI) value of 2BT-pIgG-Fc was 90.43%, and the commercial FMDV vaccine was 81.75%. The PI value of 2BT-pIgG-Fc was 8.68% higher than that of commercial FMDV vaccine. In pigs, the primary target animals for FMDV, all five individuals produced FMDV-specific antibodies 42 days after vaccination with 2BT-pIgG-Fc. Furthermore, serum from 2BT-pIgG-Fc-vaccinated pigs exhibited neutralizing ability against FMDV infection. Intriguingly, the 2BT-pIgG-Fc recombinant demonstrated FMDV-specific antibody production rates and neutralization efficiency similar to commercial inactivated vaccines. This study illustrates the potential to enhance vaccine efficacy by strategically combining well-known antigenic domains in the development of recombinant protein-based vaccines.

Masked Kinematic Continuity-aware Hierarchical Attention Network for pose estimation in videos.

Existing methods for estimating human poses from video content exploit the temporal features of the video sequences and have shown impressive results. However, most methods address spatiotemporal issues separately. They compromise on accuracy to reduce jitter, or require high-resolution images to deal with occlusion, preventing full consideration of temporal features. Unfortunately, these two issues are interrelated. For example, occlusion causes uncertainty between successive frames, leading to unsmoothed results. To address these issues, we propose the Masked Kinematic Continuity-aware Hierarchical Attention Network (M-HANet) as a novel framework that exploits masked kinematic keypoint features by extending our framework HANet framework. First, we randomly select and mask a keypoint to treat the masked keypoint as it is occluded, which allows us to make the network resilient to occlusion. We also use the velocity and acceleration of each individual keypoint to effectively capture temporal features. Second, the proposed hierarchical transformer encoder refines a 2D or 3D input pose derived from existing estimators by aggregating the masked continuity of the spatiotemporal dependencies of human motion. Finally, to facilitate collaborative optimization, we perform an online cross-supervision between the final pose from our decoder and the refined input pose produced by our encoder. We validate the effectiveness of our model demonstrating that our proposed approach improves PCK@0.05 by 14.1% and MPJPE by 8.7 mm compared to the existing method on a variety of tasks, including 2D and 3D pose estimation, body mesh recovery, and sparsely annotated multi-human pose estimation.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

BACKGROUND/AIM: The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. MATERIALS AND METHODS: We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. RESULTS: CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. CONCLUSION: This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

Towards a Customizable, SLA 3D-Printed Biliary Stent: Optimizing a Commercially Available Resin and Predicting Stent Behavior with Accurate In Silico Testing.

Inflammation of the bile ducts and surrounding tissues can impede bile flow from the liver into the intestines. If this occurs, a plastic or self-expanding metal (SEM) stent is placed to restore bile drainage. United States (US) Food and Drug Administration (FDA)-approved plastic biliary stents are less expensive than SEMs but have limited patency and can occlude bile flow if placed spanning a duct juncture. Recently, we investigated the effects of variations to post-processing and autoclaving on a commercially available stereolithography (SLA) resin in an effort to produce a suitable material for use in a biliary stent, an FDA Class II medical device. We tested six variations from the manufacturer's recommended post-processing and found that tripling the isopropanol (IPA) wash time to 60 min and reducing the time and temperature of the UV cure to 10 min at 40 °C, followed by a 30 min gravity autoclave cycle, yielded a polymer that was flexible and non-cytotoxic. In turn, we designed and fabricated customizable, SLA 3D-printed polymeric biliary stents that permit bile flow at a duct juncture and can be deployed via catheter. Next, we generated an in silico stent 3-point bend test to predict displacements and peak stresses in the stent designs. We confirmed our simulation accuracy with experimental data from 3-point bend tests on SLA 3D-printed stents. Unfortunately, our 3-point bend test simulation indicates that, when bent to the degree needed for placement via catheter (~30°), the peak stress the stents are predicted to experience would exceed the yield stress of the polymer. Thus, the risk of permanent deformation or damage during placement via catheter to a stent printed and post-processed as we have described would be significant. Moving forward, we will test alternative resins and post-processing parameters that have increased elasticity but would still be compatible with use in a Class II medical device.

Suspected Human-to-Cat Spillover of SARS-CoV-2 Omicron Variant in South Korea.

This retrospective study reports the isolation and characterization of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) from a household cat in South Korea. The cat, which was presented with respiratory symptoms, was identified during a retrospective analysis of samples collected between April 2021 and March 2022. Genomic sequencing revealed that the isolated virus belonged to the Omicron variant (BA.1), coinciding with its global emergence in early 2022. This case study provides evidence for the potential of direct human-to-cat transmission of the Omicron variant in South Korea during its period of widespread circulation. Our findings underscore the importance of continuous monitoring of SARS-CoV-2 in both human and animal populations to track viral evolution and potential spillover events.

Effect of activated carbon electrode material characteristics on hardness control performance of membrane capacitive deionization.

Capacitive deionization (CDI) is an electrochemical-based water treatment technology that has attracted attention as an effective hardness-control process. However, few systematic studies have reported the criteria for the selection of suitable electrode materials for membrane capacitive deionization (MCDI) to control hardness. In this study, the effect of electrode material characteristics on the MCDI performance for hardness control was quantitatively analyzed. The results showed that the deionization capacity and the deionization rate were affected by the specific capacitance and BET-specific surface area of the activated carbon electrode. In addition, the deionization rate also showed significant relationship with the BET specific surface area. Furthermore, it was observed that the deionization capacity and the deionization rate have a highly significant relationship with the BET specific surface area divided by the wettability performance expressed as the minimum wetting rate (MWR). These findings highlighted that the electrode material should have a large surface area and good wettability to increase the deionization capacity and the deionization rate of MCDI for hardness control. The results of this study are expected to provide effective criteria for selecting MCDI electrode materials aiming hardness control.

Strategies for Effective Management of Indoor Air Quality in a Kindergarten: CO2 and Fine Particulate Matter Concentrations.

The educational and play-related activities of children proceed mainly indoors in a kindergarten. High concentrations of indoor PM2.5 and CO2 have been linked to various harmful effects on children, considerably impacting their educational outcomes in kindergarten. In this study, we explore different scenarios involving the operation of mechanical ventilation systems and air purifiers in kindergartens. Using numerical models to analyze indoor CO2 and PM2.5 concentration, we aim to optimize strategies that effectively reduce these harmful pollutants. We found that the amount of ventilation required to maintain good air quality, per child, was approximately 20.4 m3/h. However, we also found that as the amount of ventilation increased, so did the concentration of indoor PM2.5; we found that this issue can be resolved using a high-grade filter (i.e., a MERV 13 grade filter with a collection efficiency of 75%). This study provides a scientific basis for reducing PM2.5 concentrations in kindergartens, while keeping CO2 levels low.