Prediction of efficiencies for diverse prime editing systems in multiple cell types.

Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.

Phosphorus-Ligand Redox Cooperative Catalysis: Unraveling Four-Electron Dioxygen Reduction Pathways and Reactive Intermediates.

The reduction of dioxygen to water is crucial in biology and energy technologies, but it is challenging due to the inertness of triplet oxygen and complex mechanisms. Nature leverages high-spin transition metal complexes for this, whereas main-group compounds with their singlet state and limited redox capabilities exhibit subdued reactivity. We present a novel phosphorus complex capable of four-electron dioxygen reduction, facilitated by unique phosphorus-ligand redox cooperativity. Spectroscopic and computational investigations attribute this cooperative reactivity to the unique electronic structure arising from the geometry of the phosphorus complex bestowed by the ligand. Mechanistic study via spectroscopic and kinetic experiments revealed the involvement of elusive phosphorus intermediates resembling those in metalloenzymes. Our result highlights the multielectron reactivity of phosphorus compound emerging from a carefully designed ligand platform with redox cooperativity. We anticipate that the work described expands the strategies in developing main-group catalytic reactions, especially in small molecule fixations demanding multielectron redox processes.

USP21 Deubiquitinase Regulates AIM2 Inflammasome Activation.

Innate immune sensing of cytosolic DNA via absent in melanoma 2 (AIM2) is a key mechanism leading to inflammatory responses. As aberrant immune responses by dysregulated AIM2 are associated with autoinflammatory diseases, activation of the AIM2 inflammasome should be tightly controlled. In this study, we discovered that ubiquitination and deubiquitination of AIM2 are critical events that regulate AIM2 inflammasome activation. In resting human macrophage cells, AIM2 is constitutively ubiquitinated and undergoes proteasomal degradation to avoid autoinflammation. Upon DNA stimulation, USP21 binds to AIM2 and deubiquitinates it, thereby increasing its protein stability. In addition to the role of USP21 in regulating AIM2 turnover, we uncovered that USP21-mediated deubiquitination of AIM2 is required for the assembly of the AIM2 inflammasome. Depletion of USP21 does not affect the DNA-binding ability of AIM2 but inhibits the formation of the AIM2-ASC complex. Our findings establish that fine-tuning of AIM2 by the ubiquitin system is important for regulating AIM2 inflammasome activation.

Sleep parameter characteristics of patients with OSA who have retropalatal circumferential narrowing and the clinical significance of lateral pharyngeal wall collapse during sleep.

BACKGROUND: The lateral pharyngeal wall (LPW) is a critical anatomic structure in patients with obstructive sleep apnea (OSA). Resolving the retropalatal circumferential (RC) narrowing caused by combination of both LPW collapse and antero-posterior (AP) narrowing holds promise for surgical treatment of OSA. We sought to determine the clinical characteristics and distinctive alterations in sleep parameters of patients with OSA who have RC narrowing and LPW collapse. METHODS: Drug-induced sleep endoscopy (DISE), polysomnography findings, and sleep questionnaires were reviewed retrospectively in patients with OSA. RESULTS: Of the 106 OSA patients examined, 48% showed RC narrowing and 44% showed AP narrowing at the oropharynx level during sleep while 8% of the patients showed only LPW collapse. Patients with RC narrowing with LPW collapse exhibited a higher BMI than those with AP narrowing only. In addition, patients with RC narrowing showed more aggravated sleep parameters including apneic events than patients with AP narrowing alone. The degree of RC narrowing correlated significantly with the severity of OSA as shown by a higher apnea index and lower oxygen desaturations. CONCLUSIONS: Our clinical findings suggest that the presence of RC narrowing with LPW collapse in OSA is closely related to increased apneic and oxygen desaturation events. RC narrowing with LPW collapse may be targets for surgical correction in patients with OSA to improve therapeutic outcomes.

DEP-induced ZEB2 promotes nasal polyp formation via epithelial-to-mesenchymal transition.

BACKGROUND: Diesel exhaust particles (DEPs) are associated with the prevalence and exacerbation of allergic respiratory diseases, including allergic rhinitis and allergic asthma. However, DEP-induced mechanistic pathways promoting upper airway disease and their clinical implications remain unclear. OBJECTIVE: We sought to investigate the mechanisms by which DEP exposure contributes to nasal polyposis using human-derived epithelial cells and a murine nasal polyp (NP) model. METHODS: Gene set enrichment and weighted gene coexpression network analyses were performed. Cytotoxicity, epithelial-to-mesenchymal transition (EMT) markers, and nasal polyposis were assessed. Effects of DEP exposure on EMT were determined using epithelial cells from normal people or patients with chronic rhinosinusitis with or without NPs. BALB/c mice were exposed to DEP through either a nose-only exposure system or nasal instillation, with or without house dust mite, followed by zinc finger E-box-binding homeobox (ZEB)2 small hairpin RNA delivery. RESULTS: Bioinformatics analyses revealed that DEP exposure triggered EMT features in airway epithelial cells. Similarly, DEP-exposed human nasal epithelial cells exhibited EMT characteristics, which were dependent on ZEB2 expression. Human nasal epithelial cells derived from patients with chronic rhinosinusitis presented more prominent EMT features after DEP treatment, when compared with those from control subjects and patients with NPs. Coexposure to DEP and house dust mite synergistically increased the number of NPs, epithelial disruptions, and ZEB2 expression. Most importantly, ZEB2 inhibition prevented DEP-induced EMT, thereby alleviating NP formation in mice. CONCLUSIONS: Our data show that DEP facilitated NP formation, possibly via the promotion of ZEB2-induced EMT. ZEB2 may be a therapeutic target for DEP-induced epithelial damage and related airway diseases, including NPs.

Cobalt-Carbon Bonding in a Salen-Supported Cobalt(IV) Alkyl Complex Postulated in Oxidative MHAT Catalysis.

The catalytic hydrofunctionalization of alkenes through radical-polar crossover metal hydrogen atom transfer (MHAT) offers a mild pathway for the introduction of functional groups in sterically congested environments. For M = Co, this reaction is often proposed to proceed through secondary alkylcobalt(IV) intermediates, which have not been characterized unambiguously. Here, we characterize a metastable (salen)Co(isopropyl) cation, which is capable of forming C-O bonds with alcohols as proposed in the catalytic reaction. Electron nuclear double resonance (ENDOR) spectroscopy of this formally cobalt(IV) species establishes the presence of the cobalt-carbon bond, and accompanying DFT calculations indicate that the unpaired electron is localized on the cobalt center. Both experimental and computational studies show that the cobalt(IV)-carbon bond is stronger than the analogous bond in its cobalt(III) analogue, which is opposite of the usual oxidation state trend of bond energies. This phenomenon is attributable to an inverted ligand field that gives the bond Coδ--Cδ+ character and explains its electrophilic reactivity at the alkyl group. The inverted Co-C bond polarity also stabilizes the formally cobalt(IV) alkyl complex so that it is accessible at unusually low potentials. Even another cobalt(III) complex, [(salen)CoIII]+, is capable of oxidizing (salen)CoIII(iPr) to the formally cobalt(IV) state. These results give insight into the electronic structure, energetics, and reactivity of a key reactive intermediate in oxidative MHAT catalysis.

Optimal application of soft-palate webbing flap pharyngoplasty combined with nasal surgery for surgical treatment of primary snoring and obstructive sleep apnea.

BACKGROUND: Excessive collapse of the soft palate and lateral pharyngeal wall narrowing are established causes of loud snoring and sleep apnea in subjects with obstructive sleep apnea (OSA). Therefore, delicate surgical techniques are needed to reshape the soft palate and create sufficient tension in the lateral pharyngeal wall. This study aimed to determine the therapeutic outcome and favorable indications of soft-palate webbing flap pharyngoplasty in subjects with OSA and primary snoring. METHODS: A total of 174 subjects who underwent soft-palate webbing flap pharyngoplasty combined with uvulopalatal flap and septoturbinoplasty from August 2015 to February 2020 were included in this study. Medical records, including pre- and postoperative sleep parameters, were retrospectively reviewed. The primary outcome measure was the degree of improvement in AHI after surgery. Other outcomes were differences in surgical response rates, subjective visual analog score (VAS) for snoring, sleep quality, and complications. RESULTS: Polysomnographic results showed that apnea-hypopnea index (AHI) scores were significantly reduced from 39.6 ± 6.1 to 22.9 ± 3.6 following soft-palate webbing flap pharyngoplasty in 59 subjects, and overall success and response rates of this technique were analyzed with 71%. We found that the successful outcomes were observed in 50% of mild (n = 12) and 56% of moderate (n = 16) subjects with OSA subjects due to lateral pharyngeal wall collapse. The success rate of soft-palate webbing flap pharyngoplasty was relatively higher in subjects with mild and moderate OSA than those with severe OSA. Additionally, the mean VAS snoring scale was 4.7 and subjects' primary snoring intensity significantly improved to 2.9 after soft-palate webbing flap pharyngoplasty. Subjective symptoms such as daytime sleepiness and sleep quality also showed improvement. Most complications were found to be minimal and improved by 1 month after the operation. CONCLUSION: Our data demonstrate that soft-palate webbing flap pharyngoplasty is an effective treatment for OSA and primary snoring and may be a promising technique to reduce lateral pharyngeal wall collapse.

An Innovative Way to Turn Catalyst into Substrate for Highly Efficient Water Splitting.

The energy-efficiency loss with high overpotential during hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), as well as economic inefficiencies including high-cost materials and complicated processes, is considered the major challenge to the implementation of electrochemical water splitting applications. The authors present a new platform for electrocatalysts that functions in an unprecedented way to turn a catalyst into substrate. The NiFe alloy catalyzed substrate (NiFe-CS) described herein is substantially active and stable electrocatalyst for both HER and OER, with low overpotential of 33 and 191 mV at 10 mA cm-2 for HER and OER, respectively. This structure enables not only the maximization of electrochemically active sites, but also the formation of hydroxyl species on the surface as the active phase. These outstanding results provide a new pathway for the development of electrocatalysts used in energy conversion technology.

Correlation of site of obstruction between two dynamic evaluation modalities in obstructive sleep apnea patients: drug-induced sleep endoscopy and sleep videofluoroscopy.

PURPOSE: Drug-induced sleep endoscopy (DISE) and sleep videofluoroscopy (SVF) are two dynamic modalities for evaluating the upper airway in patients with obstructive sleep apnea (OSA). We evaluated the correlation of obstructive sites determined by DISE and SVF in OSA patients and elucidate findings that can improve the accuracy of upper airway assessment. METHODS: A consecutive series of 63 patients with OSA who underwent DISE and SVF were the subjects of this study. The DISE and SVF findings were divided according to the anatomical structure responsible for the collapse, including the soft palate (SP), oropharyngeal lateral walls (LW), tongue base (TB), and larynx (LX). The obstruction was graded on the three-point scale: 0, no obstruction; 1, partial obstruction; or 2, complete obstruction. Additionally, grade 1.5 TB obstruction was designated when the posterior displacement of the anterior tongue was detected during simultaneous retropalatal obstruction. The agreement rate and Cohen's kappa test between the two modalities were also assessed. RESULTS: The agreement rate between the two modalities was highest in LX (88.9%) followed by SP (85.7%), TB (76.1%), and LW (74.6%) (Cohen's kappa value = 0.757 in LX, 0.642 in SP, 0.637 in TB, 0.612 in LW, respectively). When grade 1.5 and 2 TB obstructions were combined, the agreement rate increased to 88.9% (Cohen's kappa value = 0.757). CONCLUSIONS: We found a good overall agreement between the two dynamic airway evaluation modalities during drug-induced sleep, and this correlation may be improved if the posterior displacement of the anterior tongue during DISE is used as a sign of TB obstruction.

EV-Ident: Identifying Tumor-Specific Extracellular Vesicles by Size Fractionation and Single-Vesicle Analysis.

Tumor-derived extracellular vesicles (EVs) have emerged as a promising source of circulating biomarkers for liquid biopsies. However, understanding the heterogeneous physical and biochemical properties of EVs originating from multiple complex biogenesis pathways remains a major challenge. Here, we introduce EV-Ident for preparation of subpopulations of EVs in three different size fractions: large EVs (EV200 nm; 200-1 000 nm), medium EVs (EV100 nm; 100-200 nm), and small EVs (EV20 nm; 20-100 nm). Furthermore, this technology enables the in situ labeling of fluorescence markers for the protein profiling of individual EVs. As a proof-of-concept, we analyzed the presence of human epidermal growth factor receptor 2 (HER2) and prostate-specific membrane antigen (PSMA) in breast cancer and prostate cancer cell-derived EVs, respectively, using three different size fractions at the single-EV level. By reducing the complexity of EV heterogeneity in each size fraction, we found that HER2-positive breast cancer cells showed the greatest expression of HER2 in EV20 nm, whereas PSMA expression was the highest in EV200 nm derived from PSMA-expressing prostate cancer cells. This increase in HER2 expression in EV20 nm and PSMA expression in EV200 nm was further confirmed in plasma-derived nanoparticles (PNPs) obtained from breast and prostate cancer patients, respectively. Our study demonstrates that single-EV analysis using EV-Ident provides a practical way to understand EV heterogeneity and to successfully identify potent subpopulation of EVs for breast and prostate cancer, which has promising translational implications for cancer theranostics. Furthermore, these findings have the potential to address fundamental questions surrounding the biology and clinical applications of EVs.

Time-gated iterative phase conjugation for efficient light energy delivery in scattering media.

Light waves propagating through complex biological tissues are spatially spread by multiple light scattering, and this spread limits the working depth in optical bioimaging, phototherapy, and optogenetics. Here, we propose the iterative phase conjugation of time-gated backscattered waves for enhancing the light energy delivered to a target object embedded in a scattering medium. We demonstrate the enhancement of light energy delivered to a target object hidden behind a 200-µm-thick mouse skull by more than ten times in comparison with the initial random input. The maximum enhancement was reached in only 10 iterations, more than a hundred times smaller than existing methods based on either a time-gated reflection matrix or iterative feedback optimization of the time-gated reflection intensity. Consequently, the proposed method is less sensitive to sample perturbations. Furthermore, the number of images required for optimization remained almost unchanged with an increase in the illumination area, unlike existing methods, where the convergence time scales with the illumination area. The proposed method provides high operation speed over a wide illumination area, which can facilitate the use of wavefront shaping in practical applications.

Development of pyrazolo[3,4-d]pyrimidine-6-amine-based TRAP1 inhibitors that demonstrate in vivo anticancer activity in mouse xenograft models.

TNF Receptor Associated Protein 1 (TRAP1) is a mitochondrial paralog of Hsp90 related to the promotion of tumorigenesis in various cancers via maintaining mitochondrial integrity, reducing the production of reactive oxygen species, and reprogramming cellular metabolism. Consequently, Hsp90 and TRAP1 have been targeted to develop cancer therapeutics. Herein, we report a series of pyrazolo[3,4-d]pyrimidine derivatives that are mitochondria-permeable TRAP1 inhibitors. Structure-based drug design guided the optimization of potency, leading to the identification of compounds 47 and 48 as potent TRAP1 and Hsp90 inhibitors with good metabolic and plasma stability as well as acceptable CYP and hERG inhibition. X-ray co-crystallization studies confirmed both 47 and 48 interact with the ATP binding pocket in the TRAP1 protein. Compounds 47 and 48 demonstrated excellent anticancer efficiency in various cancer cells, with limited toxicity over normal hepatocyte and prostate cells. Mouse PC3 xenograft studies showed 47 and 48 significantly reduced tumor growth.

WHO implementation workshop on guidelines on procedures and data requirements for changes to approved biotherapeutic products, Seoul, Republic of Korea, 25-26 June 2019.

The first global workshop on implementation of the WHO guidelines on procedures and data requirements for changes to approved biotherapeutic products adopted by the WHO Expert Committee in 2018 was held in June 2019. The workshop participants recognized that the principles based on sound science and the potential for risk, as described in the WHO Guidelines on post-approval changes, which constitute the global standard for product life-cycle management are providing clarity and helping national regulatory authorities in establishing guidance while improving time-lines for an efficient regulation of products. Consequently, the regulatory situation for post-approval changes and guideline implementation is changing but there is a disparity between different countries. While the guidelines are gradually being implemented in some countries and also being considered in other countries, the need for regional workshops and further training on post-approval changes was a common theme reiterated by many participants. Given the complexities relating to post-approval changes in different regions/countries, there was a clear understanding among all participants that an efficient approach for product life-cycle management at a national level is needed to ensure faster availability of high standard, safe and efficacious medicines to patients as per the World Health Assembly Resolution 67.21.

Roles of Iron Complexes in Catalytic Radical Alkene Cross-Coupling: A Computational and Mechanistic Study.

A growing and useful class of alkene coupling reactions involve hydrogen atom transfer (HAT) from a metal-hydride species to an alkene to form a free radical, which is responsible for subsequent bond formation. Here, we use a combination of experimental and computational investigations to map out the mechanistic details of iron-catalyzed reductive alkene cross-coupling, an important representative of the HAT alkene reactions. We are able to explain several observations that were previously mysterious. First, the rate-limiting step in the catalytic cycle is the formation of the reactive Fe-H intermediate, elucidating the importance of the choice of reductant. Second, the success of the catalytic system is attributable to the exceptionally weak (17 kcal/mol) Fe-H bond, which performs irreversible HAT to alkenes in contrast to previous studies on isolable hydride complexes where this addition was reversible. Third, the organic radical intermediates can reversibly form organometallic species, which helps to protect the free radicals from side reactions. Fourth, the previously accepted quenching of the postcoupling radical through stepwise electron transfer/proton transfer is not as favorable as alternative mechanisms. We find that there are two feasible pathways. One uses concerted proton-coupled electron transfer (PCET) from an iron(II) ethanol complex, which is facilitated because the O-H bond dissociation free energy is lowered by 30 kcal/mol upon metal binding. In an alternative pathway, an O-bound enolate-iron(III) complex undergoes proton shuttling from an iron-bound alcohol. These kinetic, spectroscopic, and computational studies identify key organometallic species and PCET steps that control selectivity and reactivity in metal-catalyzed HAT alkene coupling, and create a firm basis for elucidation of mechanisms in the growing class of HAT alkene cross-coupling reactions.

Growth and Fabrication of High-Quality Single Nanowire Devices with Radial p-i-n Junctions.

Nanowires (NWs) with radial p-i-n junction have advantages, such as large junction area and small influence from the surface states, which can lead to highly efficient material use and good device quantum efficiency. However, it is difficult to make high-quality core-shell NW devices, especially single NW devices. Here, the key factors during the growth and fabrication process that influence the quality of single core-shell p-i-n NW devices are studied using GaAs(P) NW photovoltaics as an example. By p-doping and annealing, good ohmic contact is achieved on NWs with a diameter as small as 50-60 nm. Single NW photovoltaics are subsequently developed and a record fill factor of 80.5% is shown. These results bring valuable information for making single NW devices, which can further benefit the development of high-density integration circuits.

Iterative optimization of time-gated reflectance for the efficient light energy delivery within scattering media.

In complex media, light waves are diffused both in space and time due to multiple light scattering, and its intensity is attenuated with the increase of propagation depth. In this paper, we propose an iterative wavefront shaping method for enhancing time-gated reflection intensity, which leads to efficient light energy delivery to a target object embedded in a highly scattering medium. We achieved an over 10 times enhancement of reflectance at the specific flight time and demonstrated the focusing of light energy to the target object. Since the proposed method does not require reflection matrix measurement, it will be highly suited to samples in mechanically dynamic conditions.

The orphan nuclear receptor NR4A1 promotes FcεRI-stimulated mast cell activation and anaphylaxis by counteracting the inhibitory LKB1/AMPK axis.

BACKGROUND: Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. OBJECTIVES: The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. METHODS: To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) in vitro and on anaphylactic responses in vivo were evaluated. RESULTS: Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses in vivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. CONCLUSIONS: Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

Identification of new aryl hydrocarbon receptor (AhR) antagonists using a zebrafish model.

A new series of 1,3-diketone, heterocyclic and α,ß-unsaturated derivatives were synthesized and evaluated for their AhR antagonist activity using zebrafish and mammalian cells. Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among them, compound 3b, and 5b were found to have excellent AhR antagonist activity with IC50 of 3.36 nM and 8.3 nM in a luciferase reporter gene assay. In stem cell proliferation assay, compound 5b elicited marked HSC expansion.

Doping of Self-Catalyzed Nanowires under the Influence of Droplets.

Controlled and reproducible doping is essential for nanowires (NWs) to realize their functions. However, for the widely used self-catalyzed vapor-liquid-solid (VLS) growth mode, the doping mechanism is far from clear, as the participation of the nanoscale liquid phase makes the doping environment highly complex and significantly different from that of the thin film growth. Here, the doping mechanism of self-catalyzed NWs and the influence of self-catalytic droplets on the doping process are systematically studied using beryllium (Be) doped GaAs NWs. Be atoms are found for the first time to be incorporated into NWs predominantly through the Ga droplet that is observed to be beneficial for setting up thermodynamic equilibrium at the growth front. Be dopants are thus substitutional on Ga sites and redundant Be atoms are accumulated inside the Ga droplets when NWs are saturated, leading to the change of the Ga droplet properties and causing the growth of phase-pure zincblende NWs. This study is an essential step toward the design and fabrication of nanowire devices.

Developmental endothelial locus-1 prevents development of peritoneal adhesions in mice.

Postoperative peritoneal adhesions, fibrous bands formed in the peritoneal cavity following surgery, represent a common, challenging and costly problem faced by surgeons and patients, for which effective therapeutic options are lacking. Since aberrant inflammation is one of the key mechanisms underlying peritoneal adhesion formation, here we set out to study the role of developmental endothelial locus-1 (Del-1), which has been recently identified as an endogenous inhibitor of inflammation, in the formation of postoperative peritoneal adhesions using a mouse model of peritoneal adhesions induced by ischemic buttons. Del-1-deficient mice had a higher incidence of adhesions, and their adhesions had higher quality and tenacity scores. Del-1 deficiency also led to enhanced inflammation mediators and collagen production. Finally, Del-1 supplementation decreased the incidence and severity of postoperative peritoneal adhesions. Taken together, these results indicate a protective role for Del-1 in postoperative peritoneal adhesion formation.