IL-6 Revisited: From Rheumatoid Arthritis to CAR T Cell Therapy and COVID-19.

The diverse biological activity of interleukin-6 (IL-6) contributes to the maintenance of homeostasis. Emergent infection or tissue injury induces rapid production of IL-6 and activates host defense through augmentation of acute-phase proteins and immune responses. However, excessive IL-6 production and uncontrolled IL-6 receptor signaling are critical to pathogenesis. Over the years, therapeutic agents targeting IL-6 signaling, such as tocilizumab, a humanized anti-IL-6 receptor antibody, have shown remarkable efficacy for rheumatoid arthritis, Castleman disease, and juvenile idiopathic arthritis, and their efficacy in other diseases is continually being reported. Emerging evidence has demonstrated the benefit of tocilizumab for several types of acute inflammatory diseases, including cytokine storms induced by chimeric antigen receptor T cell therapy and coronavirus disease 2019 (COVID-19). Here, we refocus attention on the biology of IL-6 and summarize the distinct pathological roles of IL-6 signaling in several acute and chronic inflammatory diseases.

Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization.

Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements, with mechanistic target of rapamycin (mTOR) kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here we show that an mTOR-Semaphorin 6D (Sema6D)-Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1hi macrophages and prevents development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity, and metabolism via PPARγ.

Targeting Interleukin-6 Signaling in Clinic.

Interleukin-6 (IL-6) is a pleiotropic cytokine with roles in immunity, tissue regeneration, and metabolism. Rapid production of IL-6 contributes to host defense during infection and tissue injury, but excessive synthesis of IL-6 and dysregulation of IL-6 receptor signaling is involved in disease pathology. Therapeutic agents targeting the IL-6 axis are effective in rheumatoid arthritis, and applications are being extended to other settings of acute and chronic inflammation. Recent studies reveal that selective blockade of different modes of IL-6 receptor signaling has different outcomes on disease pathology, suggesting novel strategies for therapeutic intervention. However, some inflammatory diseases do not seem to respond to IL-6 blockade. Here, we review the current state of IL-6-targeting approaches in the clinic and discuss how to apply the growing understanding of the immunobiology of IL-6 to clinical decisions.

Gp130-HIF1α axis-induced vascular damage is prevented by the short-term inhibition of IL-6 receptor signaling.

Protection against endothelial damage is recognized as a frontline approach to preventing the progression of cytokine release syndrome (CRS). Accumulating evidence has demonstrated that interleukin-6 (IL-6) promotes vascular endothelial damage during CRS, although the molecular mechanisms remain to be fully elucidated. Targeting IL-6 receptor signaling delays CRS progression; however, current options are limited by persistent inhibition of the immune system. Here, we show that endothelial IL-6 trans-signaling promoted vascular damage and inflammatory responses via hypoxia-inducible factor-1α (HIF1α)-induced glycolysis. Using pharmacological inhibitors targeting HIF1α activity or mice with the genetic ablation of gp130 in the endothelium, we found that inhibition of IL-6R (IL-6 receptor)-HIF1α signaling in endothelial cells protected against vascular injury caused by septic damage and provided survival benefit in a mouse model of sepsis. In addition, we developed a short half-life anti-IL-6R antibody (silent anti-IL-6R antibody) and found that it was highly effective at augmenting survival for sepsis and severe burn by strengthening the endothelial glycocalyx and reducing cytokine storm, and vascular leakage. Together, our data advance the role of endothelial IL-6 trans-signaling in the progression of CRS and indicate a potential therapeutic approach for burns and sepsis.

T1-VIBE and STIR MRI of lumbar pars interarticularis injuries in elite athletes: fracture characterisation and potential prognostic indicators.

OBJECTIVES: To assess how pars interarticularis fracture characteristics on T1-VIBE and STIR MRI relate to healing and identify anatomical parameters that may impact healing. MATERIALS AND METHODS: A retrospective review of an MRI series of lumbar pars interarticularis injuries in elite athletes over a 3-year period. Fracture configurations, signal intensities and anatomical parameters were recorded by two radiologists. Statistical analysis employed multilevel mixed-effects linear regressions, adjusted for repeated measures and baseline covariates. RESULTS: Forty-seven lumbar pars interarticularis injuries among 31 athletes were assessed. On final scans for each athlete, 15% (7/47) injuries had worsened, 23% (11/47) remained stable, 43% (20/47) partially healed and 19% (9/47) healed completely. Healing times varied, quickest was 49 days for a chronic fracture in a footballer. Bone marrow oedema signal was highest in worsened fractures, followed by improved, and lowest in stable fractures. As healing progressed, T1-VIBE signal at the fracture line decreased. Bone marrow oedema and fracture line signal peaked at 90-120 days before decreasing until 210-240 days. Fractures with smaller dimensions, more vertical orientation and a longer superior articular facet beneath were significantly associated with better healing (p < 0.05). CONCLUSION: Most diagnosed athletic pars interarticularis injuries improve. Normalising T1-VIBE signal at the fracture line is a novel measurable indicator of bony healing. Contrastingly, bone marrow oedema signal is higher in active fractures irrespective of healing or deterioration. Injuries initially perceived as worsening may be exhibiting the normal osteoclastic phase of healing. Better outcomes favour smaller, vertical fractures with a longer superior articular facet beneath.

Disparities in utilization of preventive health services among Asian young adults in the United States.

Despite the favorable health impacts of preventive services use, young adults remain at a higher risk of not using these services compared with older adults. This study seeks to identify barriers to receiving recommended preventive services among Asian young adults compared to other racial/ethnic young adults. Using 2016-2018 National Health Interview Survey data, this study examined barriers to recommended preventive services among non-Hispanic (NH) Asian young adults aged 18-39 years compared with other racial/ethnic groups in the United States (Total = 25,430; NH Asians = 6.3%). General prevention included fasting blood sugar, cholesterol, blood pressure, and Human Immunodeficiency Virus checkups. We documented information on vaccinations for influenza, pneumonia, tetanus, hepatitis A/B, and female-specific preventive care measures. NH Asian young adults reported blood pressure checkups less often than NH Whites (72.88% vs. 79.92%, p < 0.001). NH Asian young adults were also less likely to report HIV testing than all other racial/ethnic groups (p < 0.001). After controlling for covariates, NH Whites (odds ratio [OR] = 2.00, 95% confidence interval [CI] = 1.60, 2.50), NH Blacks (OR = 1.55, 95% CI = 1.18, 2.02), and other races (OR = 2.40, 95% CI = 1.60, 3.58) were more likely to receive any preventive services than NH Asians. Among those receiving any preventive services, there were no differences between NH Asians and all other racial/ethnic groups in whether they reported receiving relatively more preventive services. Our findings demonstrate that the rates of certain recommended preventive services use were lower among NH Asian young adults. Targeted public health strategies are needed to increase the use of preventive healthcare for racial/ethnic minority young adults.

IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome.

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.

Using Single-Crystal Graphene to Form Arrays of Nanocapsules Enabling the Observation of Light Elements in Liquid Cell Transmission Electron Microscopy.

We have designed and fabricated a TEM (transmission electron microscopy) liquid cell with hundreds of graphene nanocapsules arranged in a stack of two Si3N4-x membranes. These graphene nanocapsules are formed on arrays of nanoholes patterned on the Si3N4-x membrane by focused ion beam milling, allowing for better resolution than for the conventional graphene liquid cells, which enables the observation of light elements, such as atomic structures of silicon. We suggest that multiple nanocapsules provide opportunities for consecutive imaging under the same conditions in a single liquid cell. The use of single-crystal graphene windows offers an excellent signal-to-noise ratio and high spatial resolution. The motion of silicon nanoparticles (a low atomic number (Z) material) interacting with nanobubbles was observed, and analyzed, in detail. Our approach will help advance liquid-phase TEM observations by providing a straightforward method to encapsulate liquid between monolayers of various 2-dimensional materials.

Chemical Conformation of the Essential Glutamate Site of the c-Ring within Thermophilic Bacillus FoF1-ATP Synthase Determined by Solid-State NMR Based on its Isolated c-Ring Structure.

Proton translocation through the membrane-embedded Fo component of F-type ATP synthase (FoF1) is facilitated by the rotation of the Fo c-subunit ring (c-ring), carrying protons at essential acidic amino acid residues. Cryo-electron microscopy (Cryo-EM) structures of FoF1 suggest a unique proton translocation mechanism. To elucidate it based on the chemical conformation of the essential acidic residues of the c-ring in FoF1, we determined the structure of the isolated thermophilic Bacillus Fo (tFo) c-ring, consisting of 10 subunits, in membranes by solid-state NMR. This structure contains a distinct proton-locking conformation, wherein Asn23 (cN23) CγO and Glu56 (cE56) CδOH form a hydrogen bond in a closed form. We introduced stereo-array-isotope-labeled (SAIL) Glu and Asn into the tFoc-ring to clarify the chemical conformation of these residues in tFoF1-ATP synthase (tFoF1). Two well-separated 13C signals could be detected for cN23 and cE56 in a 505 kDa membrane protein complex, respectively, thereby suggesting the presence of two distinct chemical conformations. Based on the signal intensity and structure of the tFoc-ring and tFoF1, six pairs of cN23 and cE56 surrounded by membrane lipids take the closed form, whereas the other four in the a-c interface employ the deprotonated open form at a proportion of 87%. This indicates that the a-c interface is highly hydrophilic. The pKa values of the four cE56 residues in the a-c interface were estimated from the cN23 signal intensity in the open and closed forms and distribution of polar residues around each cE56. The results favor a rotation of the c-ring for ATP synthesis.

The structural and functional investigation of the VapBC43 complex from Mycobacterium tuberculosis.

Toxin - Antitoxin systems are crucial for bacterial survival against harsh circumstances such as antibiotic treatment. The VapBC systems are the most abundant Toxin-Antitoxin systems among the Toxin - Antitoxin systems in the Mycobacterium tuberculosis. The VapBC43 system is one of them, which is related to the response to the vancomycin treatment. However, the structure of the VapBC43 complex remained unknown. Here, we present the crystal structure of the VapBC43 complex in which a single VapB43 molecule binds to the VapC43 dimer. The electrophoretic mobility shift assay shows that the VapB43 can bind to its promoter DNA. In addition, this structure reveals that the VapC43 contains a PIN (PilT N-terminus) domain motif which is essential for ribonuclease activity but has less conserved acidic residues than other homologs. The results of ribonuclease assays show that the VapC43 exhibits ribonuclease activity despite the lack of acidic residues which are well conserved in a PIN domain superfamily. Based on the previous finding that the VapBC43 contributes to the survival of Mycobacterium tuberculosis under vancomycin treatment, the structural information of the VapBC43 complex may enable the development of the inhibitor of VapC43 that can be used as an adjuvant for vancomycin therapy against M. tuberculosis.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

Neural guidance factors as hubs of immunometabolic cross-talk.

Semaphorins were originally identified as axon-guidance molecules essential for neural development. In addition to their functions in the neural system, members of the semaphorin family have critical functions in many pathophysiological processes, including immune responses, bone homeostasis, cancer and metabolic disorders. In particular, several lines of evidence indicate that mammalian/mechanistic target of rapamycin (mTOR), a central regulator of cell metabolism, regulates the functions of semaphorins in various types of cells, revealing a novel link between semaphorins and cell metabolism. In this review, we discuss recent advances in the immunometabolic functions of semaphorins, with a particular focus on mTOR signaling.

Feedback regulation of Arid5a and Ppar-γ2 maintains adipose tissue homeostasis.

Immune cells infiltrate adipose tissues and provide a framework to regulate energy homeostasis. However, the precise underlying mechanisms and signaling by which the immune system regulates energy homeostasis in metabolic tissues remain poorly understood. Here, we show that the AT-rich interactive domain 5A (Arid5a), a cytokine-induced nucleic acid binding protein, is important for the maintenance of adipose tissue homeostasis. Long-term deficiency of Arid5a in mice results in adult-onset severe obesity. In contrast, transgenic mice overexpressing Arid5a are highly resistant to high-fat diet-induced obesity. Inhibition of Arid5a facilitates the in vitro differentiation of 3T3-L1 cells and fibroblasts to adipocytes, whereas its induction substantially inhibits their differentiation. Molecular studies reveal that Arid5a represses the transcription of peroxisome proliferator activated receptor gamma 2 (Ppar-γ2) due to which, in the absence of Arid5a, Ppar-γ2 is persistently expressed in fibroblasts. This phenomenon is accompanied by enhanced fatty acid uptake in Arid5a-deficient cells, which shifts metabolic homeostasis toward prolipid metabolism. Furthermore, we show that Arid5a and Ppar-γ2 are dynamically counterregulated by each other, hence maintaining adipogenic homeostasis. Thus, we show that Arid5a is an important negative regulator of energy metabolism and can be a potential target for metabolic disorders.

The spectrum of macrophage activation by immunometabolism.

Macrophages are heterogeneous and plastic, and play several diverse functions in immune responses. Emerging data provide evidence of multiple roles for metabolic pathways in the control of macrophage effector functions. The diverse functions of macrophages are categorized into two main subsets: classical activated macrophages (M1) and alternative activated macrophages (M2). M1 macrophages secrete pro-inflammatory cytokines and reactive oxygen species and migrate into inflamed sites as a part of host defenses. On the other hand, M2 macrophages are involved in immune homeostasis by producing anti-inflammatory cytokines and phagocytosing apoptotic cells. Metabolic reprogramming of environmental or cellular nutrients such as glucose, lipids and amino acids supports this diversity. Mechanistically, the mammalian target of rapamycin (mTOR) network plays important roles in the effector functions of macrophages by modulating cellular metabolism and regulating gene expression at the transcriptional and translational levels. In this review, we outline immunometabolism and provide insights into metabolic regulation by mTOR in macrophages.

Humanized cereblon mice revealed two distinct therapeutic pathways of immunomodulatory drugs.

Immunomodulatory drugs (IMiDs), including thalidomide derivatives such as lenalidomide and pomalidomide, offer therapeutic benefit in several hematopoietic malignancies and autoimmune/inflammatory diseases. However, it is difficult to study the IMiD mechanism of action in murine disease models because murine cereblon (CRBN), the substrate receptor for IMiD action, is resistant to some of IMiDs therapeutic effects. To overcome this difficulty, we generated humanized cereblon (CRBNI391V) mice thereby providing an animal model to unravel complex mechanisms of action in a murine physiological setup. In our current study, we investigated the degradative effect toward IKZF1 and CK-1α, a target substrate of IMiDs. Unlike WT mice which were resistant to lenalidomide and pomalidomide, T lymphocytes from CRBNI391V mice responded with a higher degree of IKZF1 and CK-1α protein degradation. Furthermore, IMiDs resulted in an increase in IL-2 among CRBNI391V mice but not in the WT group. We have also tested a thalidomide derivative, FPFT-2216, which showed an inhibitory effect toward IKZF1 protein level. As opposed to pomalidomide, FPFT-2216 and lenalidomide degrades CK-1α. Additionally, we assessed the potential therapeutic effects of IMiDs in dextran sodium sulfate (DSS)-induced colitis. In both WT and humanized mice, lenalidomide showed a significant therapeutic effect in the DSS model of colitis, while the effect of pomalidomide was less pronounced. Thus, while IMiDs' degradative effect on IKZF1 and CK-1α, and up-regulation of IL-2, is dependent on CRBN, the therapeutic benefit of IMiDs in a mouse model of inflammatory bowel disease occurs through a CRBN-IMiD binding region independent pathway.

Tailoring Solution-Processable Li Argyrodites Li6+xP1-xMxS5I (M = Ge, Sn) and Their Microstructural Evolution Revealed by Cryo-TEM for All-Solid-State Batteries.

Owing to their high Li+ conductivities, mechanical sinterability, and solution processability, sulfide Li argyrodites have attracted much attention as enablers in the development of high-performance all-solid-state batteries with practicability. However, solution-processable Li argyrodites have been developed only for a composition of Li6PS5X (X = Cl, Br, I) with insufficiently high Li+ conductivities (∼10-4 S cm-1). Herein, we report the highest Li+ conductivity of 0.54 mS cm-1 at 30 °C (Li6.5P0.5Ge0.5S5I) for solution-processable iodine-based Li argyrodites. A comparative investigation of three iodine-based argyrodites of unsubstituted and Ge- and Sn-substituted solution-processed Li6PS5I with varied heat-treatment temperature elucidates the effect of microstructural evolution on Li+ conductivity. Notably, local nanostructures consisting of argyrodite nanocrystallites in solution-processed Li6.5P0.5Ge0.5S5I have been directly captured by cryogenic transmission electron microscopy, which is a first for sulfide solid electrolyte materials. Specifically, the promising electrochemical performances of all-solid-state batteries at 30 °C employing LiCoO2 electrodes tailored by the infiltration of Li6.5P0.5Ge0.5S5I-ethanol solutions are successfully demonstrated.

Comparison of ABO isoagglutinin titres by three different methods: tube haemagglutination, micro-column agglutination and automated immunohematology analyzer based on erythrocyte-magnetized technology.

BACKGROUND AND OBJECTIVES: ABO isoagglutinin titre is important for evaluating and monitoring ABO-incompatible (ABOi) stem cells or solid organ transplantations. There are several methods to measure the titre level, including the tube haemagglutination method, micro-column agglutination and erythrocyte-magnetized technology (EMT). However, few studies have reported isoagglutinin measured by EMT. Here, we compared the isoagglutinin titre of normal individuals obtained by an automated instrument with that obtained by conventional manual methods to evaluate the feasibility of replacing the manual method with the automated instrument. MATERIALS AND METHODS: The ABO isoagglutinin titre was measured on residual samples of healthy individuals who visited the health promotion centre of the National Cancer Center, Korea, from April to October 2015. Samples from 120 patients were collected, which included 20 males and 20 females for each blood group (A, B and O). IgM and IgG ABO isoagglutinin titres of each blood group were measured by the tube haemagglutination, micro-column agglutination and EMT techniques. The median (minimum-maximum) titres were compared, and the concordance between two methods was evaluated with the rate of results showing within one titre difference. RESULTS: The median ABO IgM and IgG titres of all blood groups obtained by the EMT method were higher than that obtained by the conventional tube haemagglutination and micro-column agglutination. CONCLUSION: The agreement between the two methods was comparable in case of IgM but low in IgG.

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.

Gallbladder-derived surfactant protein D regulates gut commensal bacteria for maintaining intestinal homeostasis.

The commensal microbiota within the gastrointestinal tract is essential in maintaining homeostasis. Indeed, dysregulation in the repertoire of microbiota can result in the development of intestinal immune-inflammatory diseases. Further, this immune regulation by gut microbiota is important systemically, impacting health and disease of organ systems beyond the local environment of the gut. What has not been explored is how distant organs might in turn shape the microbiota via microbe-targeted molecules. Here, we provide evidence that surfactant protein D (SP-D) synthesized in the gallbladder and delivered into intestinal lumen binds selectively to species of gut commensal bacteria. SP-D-deficient mice manifest intestinal dysbiosis and show a susceptibility to dextran sulfate sodium-induced colitis. Further, fecal transfer from SP-D-deficient mice to wild-type, germ-free mice conveyed colitis susceptibility. Interestingly, colitis caused a notable increase in Sftpd gene expression in the gallbladder, but not in the lung, via the activity of glucocorticoids produced in the liver. These findings describe a unique mechanism of interorgan regulation of intestinal immune homeostasis by SP-D with potential clinical implications such as cholecystectomy.

Native Void Space for Maximum Volumetric Capacity in Silicon-Based Anodes.

Volumetric energy density is considered a primary factor in developing high-energy batteries. Despite its significance, less efforts have been devoted to its improvement. Silicon-based materials have emerged as next-generation anodes for lithium-ion batteries due to their high specific capacity. However, their volumetric capacities are limited by the volume expansion rate of silicon, which restricts mass loading in the electrodes. To address this challenge, we introduce porous silicon templated from earth-abundant minerals with native internal voids, capable of alleviating volumetric expansion during repeated cycles. In situ transmission electron microscopy analysis allows the precise determination of the expansion rate of silicon, thus presenting an analytical model for finding the optimal content in silicon/graphite composites. The inner pores in silicon reduce problems associated with its expansion and allow higher silicon loading of 42% beyond the conventional limitations of 13-14%. Consequently, the anode designed in this work can deliver a volumetric capacity of 978 mAh cc-1. Thus, suppressing volume expansion with natural abundant template-assisted materials opens new avenues for cost-effective fabrication of high volumetric capacity batteries.