The quest to map STIM1 activation in granular detail.

The conformational change in STIM1 that communicates sensing of ER calcium-store depletion from the STIM ER-luminal domain to the STIM cytoplasmic region and ultimately to ORAI channels in the plasma membrane is broadly understood. However, the structural basis for the STIM luminal-domain dimerization that drives the conformational change has proven elusive. A recently published study has approached this question via molecular dynamics simulations. The report pinpoints STIM residues that may be part of a luminal-domain dimerization interface, and provides unexpected insight into how torsional movements of the STIM luminal domains might trigger release of the cytoplasmic SOAR/CAD domain from its resting tethers to the STIM CC1 segments.

Adaptation of STIM1 structure-function relationships for optogenetic control of calcium signaling.

In cellular contexts, the oscillation of calcium ions (Ca2+) is intricately linked to various physiological processes, such as cell proliferation, metabolism, and survival. Stromal interaction molecule 1 (STIM1) proteins form a crucial regulatory component in the store-operated calcium entry process. The structural attributes of STIM1 are vital for its functionality, encompassing distinct domains situated in the endoplasmic reticulum lumen and the cytoplasm. The intraluminal domain enables the timely detection of diminishing Ca2+ concentrations, prompting structural modifications that activate the cytoplasmic domain. This activated cytoplasmic domain undergoes conformational alterations and engages with membrane components, opening a channel that facilitates the influx of Ca2+ from the extracellular environment. Given its multiple domains and interaction mechanisms, STIM1 plays a foundational role in cellular biology. This review focuses on the design of optogenetic tools inspired by the structure and function of STIM1. These tools offer a groundbreaking approach for studying and manipulating intracellular Ca2+ signaling with precise spatiotemporal control. We further explore the practical applications of these tools, spanning fundamental scientific research, clinical studies, and their potential for translational research.

Multitargeted molecular docking and dynamics simulation studies of 1,3,4-thiadiazoles synthesised from (R)-carvone against specific tumour protein markers: An In-silico study of two diastereoisomers.

In the present work, we describe the synthesis of new 1,3,4-thiadiazole derivatives from natural (R)-carvone in three steps including, dichloro-cyclopropanation, a condensation with thiosemicarbazide and then a 1,3-dipolar cycloaddition reaction with various nitrilimines. the targeted compounds were structurally identified by 1H & 13C NMR and HRMS analyses. The cytotoxic assay demonstrated that some synthesized novel compounds were potent on certain cancer cell lines. Molecular modeling studies were undertaken to rationalize the wet lab study results. Furthermore, molecular docking was performed to unveil the binding potential of the most active derivatives, 3a and 6c, to caspase-3 and COX-2. The stabilities of the protein-compound complexes obtained from the docking were evaluated using MD simulation. Furthermore, FMO and related parameters of the active compounds and their stereoisomers were examined through DFT studies. The docking study showed compound 6c had a higher binding potential than caspase-3. However, the binding strength of 6c was found to be less than that of the standard drug, doxorubicin, as it formed lower conventional hydrogen bonds. On the other hand, compound 3a had a higher binding potential to COX-2. However, the binding potential 3a was much lower than that of the standard COX-2 inhibitor, celecoxib. The MD simulation demonstrated that the caspase-3-6c complex was less stable than the caspase-3-doxorubicin complex. In contrast, the COX-2-3a complex was stable, and 3a was anticipated to remain inside the protein's binding pocket. The DFT study showed that 3a had higher chemical stability than 6c. The electron exchange capacity, chemical stability, and molecular orbital distributions of the stereoisomers of the active compounds were also found to be alike.

Unraveling druggable cancer-driving proteins and targeted drugs using artificial intelligence and multi-omics analyses.

The druggable proteome refers to proteins that can bind to small molecules with appropriate chemical affinity, inducing a favorable clinical response. Predicting druggable proteins through screening and in silico modeling is imperative for drug design. To contribute to this field, we developed an accurate predictive classifier for druggable cancer-driving proteins using amino acid composition descriptors of protein sequences and 13 machine learning linear and non-linear classifiers. The optimal classifier was achieved with the support vector machine method, utilizing 200 tri-amino acid composition descriptors. The high performance of the model is evident from an area under the receiver operating characteristics (AUROC) of 0.975 ± 0.003 and an accuracy of 0.929 ± 0.006 (threefold cross-validation). The machine learning prediction model was enhanced with multi-omics approaches, including the target-disease evidence score, the shortest pathways to cancer hallmarks, structure-based ligandability assessment, unfavorable prognostic protein analysis, and the oncogenic variome. Additionally, we performed a drug repurposing analysis to identify drugs with the highest affinity capable of targeting the best predicted proteins. As a result, we identified 79 key druggable cancer-driving proteins with the highest ligandability, and 23 of them demonstrated unfavorable prognostic significance across 16 TCGA PanCancer types: CDKN2A, BCL10, ACVR1, CASP8, JAG1, TSC1, NBN, PREX2, PPP2R1A, DNM2, VAV1, ASXL1, TPR, HRAS, BUB1B, ATG7, MARK3, SETD2, CCNE1, MUTYH, CDKN2C, RB1, and SMARCA4. Moreover, we prioritized 11 clinically relevant drugs targeting these proteins. This strategy effectively predicts and prioritizes biomarkers, therapeutic targets, and drugs for in-depth studies in clinical trials. Scripts are available at https://github.com/muntisa/machine-learning-for-druggable-proteins .

Development of Ligands and Degraders Targeting MAGE-A3.

Type I melanoma antigen (MAGE) family members are detected in numerous tumor types, and expression is correlated with poor prognosis, high tumor grade, and increased metastasis. Type I MAGE proteins are typically restricted to reproductive tissues, but expression can recur during tumorigenesis. Several biochemical functions have been elucidated for them, and notably, MAGEs regulate proteostasis by serving as substrate recognition modules for E3 ligase complexes. The repertoire of E3 ligase complexes that can be hijacked for targeted protein degradation continues to expand, and MAGE-E3 complexes are an especially attractive platform given their cancer-selective expression. Additionally, type I MAGE-derived peptides are presented on cancer cell surfaces, so targeted MAGE degradation may increase antigen presentation and improve immunotherapy outcomes. Motivated by these applications, we developed novel, small-molecule ligands for MAGE-A3, a type I MAGE that is widely expressed in tumors and associates with TRIM28, a RING E3 ligase. Chemical matter was identified through DNA-encoded library (DEL) screening, and hit compounds were validated for in vitro binding to MAGE-A3. We obtained a cocrystal structure with a DEL analog and hypothesize that the small molecule binds at a dimer interface. We utilized this ligand to develop PROTAC molecules that induce MAGE-A3 degradation through VHL recruitment and inhibit the proliferation of MAGE-A3 positive cell lines. These ligands and degraders may serve as valuable probes for investigating MAGE-A3 biology and as foundations for the ongoing development of tumor-specific PROTACs.

New nano-complexes targeting protein 3S7S in breast cancer and protein 4OO6 in liver cancer investigated in cell line.

Cancer, a lethal ailment, possesses a multitude of therapeutic alternatives to combat its presence, metal complexes have emerged as significant classes of medicinal compounds, exhibiting considerable biological efficacy, especially as anticancer agents. The utilization of cis-platin in the treatment of various cancer types, including breast cancer, has served as inspiration to devise novel nanostructured metal complexes for breast cancer therapy. Notably, homo- and hetero-octahedral bimetallic complexes of an innovative multifunctional ether ligand (comprising Mn(II), Ni(II), Cu(II), Zn(II), Hg(II), and Ag(I) ions) have been synthesized. To ascertain their structural characteristics, elemental and spectral analyses, encompassing IR, UV-Vis, 1H-NMR, mass and electron spin resonance (ESR) spectra, magnetic moments, molar conductance, thermal analysis, and electron microscopy, were employed. The molar conductance of these complexes in DMF demonstrated a non-electrolytic nature. Nanostructured forms of the complexes were identified through electron microscopic data. At ambient temperature, the ESR spectra of the solid complexes exhibited anisotropic and isotropic variants, indicative of covalent bonding. The ligand and several of its metal complexes were subjected to cytotoxicity testing against breast cancer protein 3S7S and liver cancer protein 4OO6, with the Ag(I) complex (7) evincing the most potent effect, followed by the Cu(II) with ligand (complex (2)), Cis-platin, the ligand itself, and the Cu(II)/Zn(II) complex (8). Molecular docking data unveiled the inhibitory order of several complexes.

Structural and theoretical basis for drug development targeting TMEM16A: Inhibition mechanism of tracheloside analogs.

Ion channels play a crucial role in the electrophysiological activities of organisms. The calcium-activated chloride channel TMEM16A is involved in various physiological processes. Therefore, inhibitors of TMEM16A are used to treat diseases caused by TMEM16A dysfunction. However, the unclear inhibition mechanism hinders the progress of drug development. Based on our previous study, we found that the molecular structures of TMEM16A inhibitors tracheloside, matairesinoside and arctigenin are similar. In this study, we conducted a structure-based virtual screening of tracheloside analogs from the PubChem database. The six tracheloside analogs with the highest affinity to TMEM16A were selected, and their inhibitory effects were detected by fluorescence and electrophysiological experiments. Subsequently, the interaction between the tracheloside analogs and TMEM16A was investigated through molecular docking and site-directed mutagenesis. Based on the above results, the mechanism of inhibition of TMEM16A gated conformation by tracheloside analogs was proposed. These findings provide a structural and theoretical basis for drug development targeting TMEM16A.

The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation.

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

Study of individual domains contributing to MALT1 dimerization in BCL10-independent and dependent assembly.

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.

Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Proteolytic cleavage of the TGFß co-receptor CD109 changes its conformation, resulting in protease inhibition via activation of its thiol ester, and dissociation from the cell membrane.

The glycosylphosphatidylinositol (GPI)-anchored protein cluster of differentiation 109 (CD109) is expressed on many human cell types and modulates the transforming growth factor ß (TGF-ß) signaling network. CD109 belongs to the alpha-macroglobulin family of proteins, known for their protease-triggered conformational changes. However, the effect of proteolysis on CD109 and its conformation are unknown. Here, we investigated the interactions of CD109 with proteases. We found that a diverse selection of proteases cleaved peptide bonds within the predicted bait region of CD109, inducing a conformational change that activated the thiol ester of CD109. We show CD109 was able to conjugate proteases with this thiol ester and decrease their activity toward protein substrates, demonstrating that CD109 is a protease inhibitor. We additionally found that CD109 has a unique mechanism whereby its GPI-anchored macroglobulin 8 (MG8) domain dissociates during its conformational change, allowing proteases to release CD109 from the cell surface by a precise mechanism and not unspecific shedding. We conclude that proteolysis of the CD109 bait region affects both its structure and location, and that interactions between CD109 and proteases may be important to understanding its functions, for example, as a TGF-ß co-receptor.

Molecular mechanism of bovine Gasdermin D-mediated pyroptosis.

Pyroptosis is a form of programmed cell death characterized by cell swelling, pore formation in the plasma membrane, lysis, and releases of cytoplasmic contents. To date, the molecular mechanism of human and murine Gasdermin D-mediated pyroptosis have been fully investigated. However, studies focusing on molecular mechanism of bovine Gasdermin D (bGSDMD)-mediated pyroptosis and its function against pathogenic infection were unclear. In the present study, we demonstrate that bovine caspase-1 (bCaspase-1) cleaves bGSDMD at amino acid residue D277 to produce an N-terminal fragment (bGSDMD-p30) which leads to pyroptosis. The amino acid residues T238 and F239 are critical for bGSDMD-p30-mediated pyroptosis. The loop aa 278-299, L293 and A380 are the key sites for autoinhibitory structure of the full length of bGSDMD. In addition, bCaspase-3 also cleaves bGSDMD at residue Asp86 without inducing cell death. Therefore, our study provides the first detailed elucidation of the mechanism of bovine GSDMD-mediated pyroptosis. The results will establish a significant foundation for future research on the role of pyroptosis in bovine infectious diseases.

Decoding the dynamics of BCL9 triazole stapled peptide.

BCL9 is a key protein in Wnt signaling pathway. It acts as a transcriptional co-activator to ß-catenin, and dysregulation in this pathway leads to tumor growth. Inhibiting such a protein-protein interaction is considered as a therapeutic challenge. The interaction between ß-catenin and BCL9 is facilitated by a 23-residue helical domain from BCL9 and a hydrophobic groove of ß-catenin. To prevent this interaction, a peptide that mimics the alpha-helical domain of BCL9 can be designed. Stapling is considered a successful strategy in the pursuit of designing such peptides in which amino acids side are stitched together using chemical moieties. Among the various types of cross-linkers, triazole is the most rapid and effective one synthesized via click reaction. However, the underlying interactions behind maintaining the secondary structure of stapled peptides remain less explored. In the current work, we employed the molecular dynamics simulation to study the conformational behavior of the experimentally synthesized single and double triazole stapled BCL9 peptide. Upon the addition of a triazole staple, there is a significant reduction in the conformational space of BCL9. The helical character of the stapled peptide increases with an increase in separation between the triazole cross-linkers. Also, we encompassed the Replica Exchange with Solute Tempering (REST2) simulation to validate the high-temperature response of the stapled peptide. From REST2, the PCA and t-SNE show the reduction in distinct cluster formation on the addition of triazole staple. Our study infers further development of these triazole-stapled BCL9 peptides into effective inhibitors to target the interaction between ß-catenin and BCL9.

KH-like Domains in PARP9/DTX3L and PARP14 Coordinate Protein-Protein Interactions to Promote Cancer Cell Survival.

Certain members of the ADP-ribosyltransferase superfamily (ARTD or PARP enzymes) catalyse ADP-ribosylation in response to cellular stress, DNA damage and viral infection and are upregulated in various tumours. PARP9, its binding partner DTX3L and PARP14 protein levels are significantly correlated in head and neck squamous cell carcinoma (HNSCC) and other tumour types though a mechanism where PARP9/DTX3L regulates PARP14 post-transcriptionally. Depleting PARP9, DTX3L or PARP14 expression in HNSCC or HeLa cell lines decreases cell survival through a reduction of proliferation and an increase in apoptosis. A partial rescue of survival was achieved by expressing a PARP14 truncation containing a predicted eukaryotic type I KH domain. KH-like domains were also found in PARP9 and in DTX3L and contributed to protein-protein interactions between PARP9-DTX3L and PARP14-DTX3L. Homodimerization of DTX3L was also coordinated by a KH-like domain and was disrupted by site-specific mutation. Although, cell survival promoted by PARP14 did not require ADP-ribosyltransferase activity, interaction of DTX3L in vitro suppressed PARP14 auto-ADP-ribosylation and promoted trans-ADP-ribosylation of PARP9 and DTX3L. In summary, we characterised PARP9-DTX3L-PARP14 interactions important to pro-survival signalling in HNSCC cells, albeit in PARP14 catalytically independent fashion.

An overview of the co-transcription factor NACC1: Beyond its pro-tumor effects.

Nucleus accumbens-associated protein 1 (NACC1) is a member of the broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) protein families, mainly exerting its biological functions as a transcription co-regulator. NACC1 forms homo- or hetero-dimers through the BTB/POZ or BANP, E5R, and NACC1 (BEN) domain with other transcriptional regulators to regulate downstream signals. Recently, the overexpression of NACC1 has been observed in various tumors and is positively associated with tumor progression, high recurrence rate, indicating poor prognosis. NACC1 also regulates biological processes such as embryonic development, stem cell pluripotency, innate immunity, and related diseases. Our review combines recent research to summarize advancements in the structure, biological functions, and relative molecular mechanisms of NACC1. The future development of NACC1 clinical appliances is also discussed.

Chemical modulation of gasdermin D activity: Therapeutic implications and consequences.

The gasdermin family of proteins are central effectors of the inflammatory, lytic cell death modality known as pyroptosis. Characterized in 2015, the most well-studied member gasdermin D can be proteolyzed, typically by caspases, to generate an active pore-forming N-terminal domain. At least well-studied three pharmacological inhibitors (necrosulfonamide, disulfiram, dimethyl fumarate) since 2018 have been shown to affect gasdermin D activity either through modulation of processing or interference with pore formation. A multitude of murine in vivo studies have since followed. Here, we discuss the current state of research surrounding these three inhibitors, caveats to their use, and a set of guiding principles that researchers should consider when pursuing further studies of gasdermin D inhibition.

Crystal structures of the DExH-box RNA helicase DHX9.

DHX9 is a DExH-box RNA helicase with versatile functions in transcription, translation, RNA processing and regulation of DNA replication. DHX9 has recently emerged as a promising target for oncology, but to date no mammalian structures have been published. Here, crystal structures of human, dog and cat DHX9 bound to ADP are reported. The three mammalian DHX9 structures share identical structural folds. Additionally, the overall architecture and the individual domain structures of DHX9 are highly conserved with those of MLE, the Drosophila orthologue of DHX9 previously solved in complex with RNA and a transition-state analogue of ATP. Due to differences in the bound substrates and global domain orientations, the localized loop conformations and occupancy of dsRNA-binding domain 2 (dsRBD2) differ between the mammalian DHX9 and MLE structures. The combined effects of the structural changes considerably alter the RNA-binding channel, providing an opportunity to compare active and inactive states of the helicase. Finally, the mammalian DHX9 structures provide a potential tool for structure-based drug-design efforts.

The Pyroptotic and Nonpyroptotic Roles of Gasdermins in Modulating Cancer Progression and Their Perspectives on Cancer Therapeutics.

Gasdermins (GSDMs) are a protein family encoded by six paralogous genes in humans, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME (also known as DFNA5), and DFNB59 (also known as pejvakin). Structurally, members of the GSDM family possess a C-terminus (an autoinhibitory domain) and a positively charged N-terminus (a pore-forming domain) linked with divergent peptide linkers. Recently, GSDMs have been identified as key executors of pyroptosis (an immunogenic programmed cell death) due to their pore-forming activities on the plasma membrane when proteolytically cleaved by caspases or serine proteases. Accumulating studies suggest that chemoresistance is attributed to dysregulation of apoptotic machinery and that inducing pyroptosis to bypass aberrant apoptosis can potently resensitize apoptosis-resistant cancer to chemotherapeutics. Pyroptosis is initiated by pore formation and culminates with plasma membrane rupture; these processes enable the release of proinflammatory cytokines (e.g., IL-1ß and IL-18) and damage-associated molecular patterns, which further modulate antitumor immunity within the tumor microenvironment. Although pyroptosis is considered a promising strategy to boost antitumor effects, it is also reported to cause unwanted tissue damage (e.g., gut damage and nephrotoxicity). Intriguingly, mounting evidence has uncovered nonpyroptotic roles of GSDMs in tumorigenesis, such as proliferation, invasion, metastasis, and drug resistance. Thus, this provides a rationale for GSDMs as potential therapeutic targets. Taken together, we shed unbiased light on the pyroptosis-dependent roles of GSDMs in cancer progression and highlighted how GSDMs modulate tumorigenesis in a pyroptosis-independent manner. It is evident that targeting GSDMs seems profound in cancer management; however, several problems require further investigation to target GSDMs from bench to bedside, which is elucidated in the discussion section.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.