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Lin Y., Stevens C., Hrstka R., Harrison B., Fourtouna A., Pathuri S., Vojtesek B., Hupp T. (2008) An alternative transcript from the death-associated protein kinase 1 locus encoding a small protein selectively mediates membrane blebbing. FEBS J, 275: 2574–2584. https://doi.org/10.1111/j.1742-4658.2008.06404.x.

During the preparation of Figure 5 in the manuscript by Lin et al. [1], the authors did not denote where irrelevant lanes were excised from the immunoblot images. The authors and editors concluded that this omission does not affect the work performed or its conclusions. This Corrigendum replaces the original Figure 5 and its legend with a version that includes this mark-up. The authors have checked the entire document and assert that they found no further errors.

The corrected Figure 5 and its legend are shown below.

Thyroxine (T4) plays a crucial role in regulating various physiological functions in the human body. As a key biomolecule for molecular recognition, single-chain variable fragments (scFvs) offer advantages, such as small molecular size, high specificity, and strong affinity, making them ideal alternatives to traditional antibodies for diagnostic applications. This study aimed to achieve in vitro affinity maturation of T4-specific scFvs through computational design, identify variants with enhanced affinity, and elucidate their interaction mechanisms with T4. A T4-specific scFv (T4-scFv) was constructed based on the previously reported anti-T4 Fab fragment (PDB ID: 5MHE) using homology modeling, followed by molecular dynamics optimization. Molecular docking and virtual mutagenesis of eight key residues (Ser91, Ser92, Pro94, Tyr180, Tyr181, Pro182, Pro186, and Ser226) were performed, yielding three affinity-enhanced variants. These variants were expressed in E. coli and evaluated experimentally using indirect competitive ELISA (IC-ELISA). The results showed that the IC₅₀ values decreased from 34.59 ng·mL^-1 (T4-scFv) to 18.30 ng·mL^-1 (scFv-M92), 15.26 ng·mL^-1 (scFv-MS), and 11.84 ng·mL^-1 (scFv-M226), demonstrating a 2.9-fold improvement in affinity for the best mutant. The optimized scFv-M226 also exhibited high specificity, with cross-reactivity to T3 and rT3 of less than 2.0%. A quantitative T4 detection method based on scFv-M226 was developed, which showed a wide linear range (1.0-200 ng·mL^-1) and a detection limit (IC₁₀) of 0.93 ng·mL^-1. These findings confirm the accuracy of the computationally guided affinity maturation strategy and highlight the potential of scFv-M226 for clinical detection and diagnostic monitoring of T4 in serum.

The FEBS Journal publishes primary papers and reviews relating to the molecules and mechanisms underpinning biological processes. Editor-in-Chief Seamus Martin discusses some of the challenges posed by the increasing use of generative AI tools in scientific publishing and some of the highlights of the past year at the journal.

Glutathione transferases (GSTs) constitute a widespread superfamily of multifunctional enzymes with roles in cellular detoxification and secondary metabolism. We report that the poorly characterized Iota-class enzymes (GSTIs) are mainly found in photosynthetic prokaryotes and eukaryotes, excluding Spermatophyta, and in a few fungi of the order Chytridiomycota. GSTIs are distinguished from other GSTs by the presence of N- and C-terminal extensions of unknown function flanking the central GST domain. Focusing on the GSTI enzyme (SynGSTI1) of the model cyanobacterium Synechocystis sp. PCC 6803 (S.6803), we showed that recombinant SynGSTI1 protein purified from Escherichia coli and S.6803 exhibited thiol-transferase and dehydroascorbate reductase activities consistent with the presence of a CPYC catalytic motif in its GST domain. SynGSTI1 was found to be monomeric and to exhibit a spectrophotometric signature between 300 and 500 nm, which was attributed to a flavin mononucleotide (FMN). The C-terminal domain of SynGSTI1 contained a conserved PRDM/L motif involved in the binding of an FMN ligand and showed a structure similar to that of the α-subunit of phycoerythrin components of the light-harvesting antenna of some cyanobacteria, most red algae and some cryptophytes. The deletion of the SynGSTI1 encoding gene in S.6803 (i) caused a slight decrease in the photosynthetic pigment content without impairing growth in standard photoautotrophic conditions; (ii) increased sensitivity to moderate and high light intensities; (iii) reduced glutathione levels and consistently; (iv) decreased tolerance to oxidative and metal stresses triggered by H₂O₂, diamide and cobalt. Thus, SynGSTI1 defines a unique GST subclass with critical roles in redox homeostasis and stress tolerance.

N⁵,N⁶-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15) is a recently identified mitochondrial uncoupler with antitumor, anti-inflammatory, antioxidant and antiobesity properties. Although it has been shown that BAM15 has a high targeting ability to the liver, its capacity to improve liver metabolic disorders and the underlying mechanisms are not well understood. This study examined how BAM15 works in high-fat-diet (HFD) induced obese mice. Our results showed that compared with 2,4-Dinitrophenol (DNP) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), BAM15 has a higher binding capacity and stronger activity in mediating proton uncoupling, and effectively promoted mitochondrial fusion, division, autophagy, and the tricarboxylic acid cycle. BAM15 improved hepatic lipid metabolism disorders by enhancing mitochondrial autophagy through activation of the 5'-AMP-activated protein kinase (AMPK) pathway. This indicates that BAM15 could be used to treat liver lipid metabolism issues and offers a solid theoretical foundation for managing lipid-related diseases.

SARS-CoV-2 papain-like protease (PLpro) is a key antiviral target as it plays a dual role in viral replication and modulation of innate immune responses by deubiquitinating or deISGylating host proteins. Therefore, therapeutically targeting PLpro may serve as a two-pronged approach to mitigate SARS-CoV-2 infection. Interestingly, PLpro shares structural and functional similarities with cellular deubiquitinating enzymes (DUBs), and this fact was leveraged in our study to identify DUB inhibitors that target the ubiquitin/ISG15-binding site and the known substrate-binding pocket of PLpro. Among the identified compounds, flupenthixol, lithocholic acid, teneligliptin, and linagliptin markedly inhibited the proteolytic activity of purified PLpro and demonstrated potent antiviral effects against SARS-CoV-2 infection in a dose-dependent manner. Treatment with lithocholic acid and linagliptin suppressed the expression of inflammatory mediators, thereby restoring immune responses. Here, crystal structures of SARS-CoV-2 PLpro in complex with linagliptin and with lithocholic acid were determined, revealing insights into the mechanism of inhibition and unique interactions within the ubiquitin/ISG15-binding site (S2 site; Phe69, His73, Asn128, and His175) and the substrate-binding cleft. Additionally, oral and intraperitoneal treatments with linagliptin increased survival, reduced lung viral load, and ameliorated histopathological damage in a mouse-adapted model of SARS-CoV-2 infection. This study demonstrates for the first time that using DUB inhibitors that target the proteolytic activity of PLpro can simultaneously reinstate the host's immune response against SARS-CoV-2, highlighting the potential of this two-pronged therapeutic approach.

Organoid formation is driven by poorly understood intrinsic cellular properties and transcriptional programs that govern plasticity and differentiation. Deciphering these regulatory networks is essential for understanding normal tissue homeostasis and tumor initiation. Using a 3D organotypic model, which better recapitulates cell-matrix interactions and biochemical cues, we performed a miRNA-based screening strategy to identify key regulators of organoid initiation from human primary mammary epithelial cells. Our findings reveal that miR-106a-3p acts as a central modulator of mammary epithelial plasticity, enriching for stem/progenitor-like cells (CD44^high/CD24^low phenotype), driving organoid expansion, fostering K14⁺/K19⁺ lineage intermixing, and promoting branching morphogenesis characteristic of early ductal development. Further analysis revealed a core transcriptional network involving CBFB, NF-YA, GATA3, and REST, which supports organoid-forming potential. This regulatory program also induces a hybrid epithelial-mesenchymal transition (EMT) state, enhancing cellular plasticity while preserving organoid structural integrity. Extending these findings to cancer, we demonstrate that enforced expression of miR-106a-3p significantly increases tumoroid formation, suggesting that the tumor microenvironment, as modeled by 3D culture, promotes miR-106a-3p expression and functional relevance in tumorigenic processes. Collectively, these data indicate that miR-106a-3p drives a transient expansion of progenitor-like states and orchestrates transcriptional reprogramming during organoid initiation, with broader implications for breast tissue homeostasis and pathophysiological remodeling in cancer.

Phycobiliproteins are essential components of the light-harvesting antennae in cyanobacteria and red algae, requiring covalently bound open-chain tetrapyrrole chromophores (bilins) for proper function. In the red alga Galdieria sulphuraria, the primary chromophore is phycocyanobilin (PCB), despite the apparent presence of only biosynthetic genes for phycoerythrobilin (PEB) biosynthesis (PEBA and PEBB). This observation suggests the presence of an alternative, atypical biosynthetic pathway for PCB. In this study, we confirmed the presence of PEB:PCB isomerase activity in an enriched protein fraction from G. sulphuraria. To further investigate this unusual pathway, we combined in silico analyses with biochemical assays. Phylogenetic analyses confirmed the placement of the G. sulphuraria ferredoxin-dependent bilin reductases within the PEBA and PEBB lineages, typically associated with PEB synthesis, whereas the related red alga Cyanidioschyzon merolae was found to contain only PCYA. This gene distribution presents a functional paradox. G. sulphuraria harbors PEB biosynthesis genes but no detectable PEB chromophores and lacks known PCB-synthesizing enzymes despite containing PCB. Functional characterization of recombinant GsPEBA (G. sulphuraria PEBA) and GsPEBB (G. sulphuraria PEBB) confirmed their roles in PEB synthesis, demonstrating that these enzymes have not evolved to synthesize PCB or act as isomerases despite their phylogenetic placement. In contrast, CmPCYA (C. merolae PCYA) catalyzed direct PCB formation from biliverdin. Together, these findings reveal an atypical isomerase-based pathway for PCB biosynthesis in G. sulphuraria, expanding our understanding of bilin metabolism and providing new insight into the evolutionary flexibility of photosynthetic pigment biosynthesis in Rhodophyta.

Age-related declines in skeletal muscle capillarization are well-documented, yet commonly used indices, such as the capillary-to-fiber perimeter exchange index (CFPE), assume uniform myofiber shape. Because fiber size and shape changes with age and fiber type, this assumption may lead to overestimation of true capillary rarefaction. This study aimed to refine capillarization metrics by accounting for fiber shape. Muscle biopsies from 12 young (23 ± 3 years) and 11 older (73 ± 2 years) women were analyzed for capillary-to-fiber ratio (C:F), capillary density (CD), and individual capillary-to-fiber ratio (C:Fi). Analyses were fiber type-specific and performed on both rested and previously exercised legs. C:Fi was normalized to fiber perimeter (CFPE), cross-sectional area (CFCE), and adjusted for the shape factor index (SFI: perimeter²/4π·CSA). Older participants exhibited smaller type II fibers (28%) and greater fiber shape irregularity (8% SFI) compared with young. C:Fi and CFPE showed consistent age-related (from 22% to 34%) and fiber type related (from 12% to 29%) reductions in capillarization. Adjusting CFPE for fiber shape (CFPE_SFIadjusted) reduced age-related differences from 22-25% down to 19-20% (~10-25% relative reduction) and fiber type differences from 12-15% down to 8-9% (~25-45% relative reduction). Normalizing C:Fi to fiber CSA (CFCE) further attenuated or eliminated most differences, with only an 18% difference remaining in type I fibers between age groups. These patterns were consistent in the exercised leg, supporting internal validity. Adjusting CFPE using SFI reduced apparent differences in capillarization between young and old muscle, as well as between fiber types. Shape-sensitive indices may provide a more physiologically accurate assessment of capillary supply in skeletal muscle.

Recruitment of the small ribosomal subunit (30S) to messenger RNA (mRNA) marks a key step in bacterial translation initiation. Recruitment begins with a 30S standby site binding single-stranded mRNA regions at or near the translation initiation region (TIR). Subsequently, the mRNA is accommodated into the 30S mRNA binding channel to initiate translation. An essential part of accommodation is the recognition and proper positioning of the start codon to establish the correct reading frame. This is often facilitated by the Shine-Dalgarno (SD) sequence. Recent structural and biochemical studies provided snapshots of the first steps of 30S recruitment to a nascent mRNA preceding accommodation, which likely reflect similar events for fully transcribed mRNAs. These studies suggest that a transcribing RNA polymerase (RNAP) promotes 30S recruitment to the nascent mRNA via two distinct pathways. In one, RNAP cooperates with ribosomal protein bS1, which captures the mRNA and channels it toward the anti-Shine-Dalgarno (aSD) sequence for base pairing. In the other, direct coupling between RNAP and the 30S, mediated by transcription factor NusG, facilitates an alternative mRNA delivery pathway. We explore the current understanding of mRNA delivery, highlighting different modes of 30S recruitment to the nascent mRNA, the role of bS1, and how this leads to the establishment of transcription-translation coupling.