The physical properties of cellular membranes are influenced by protein and lipid interactions. In situ proximity labeling interactomic methods are well suited to characterize these dynamic and often fleeting interactions. Yet, available methods require distinct chemistries for proteins and lipids. Here we establish a singlet oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids using cell-penetrant photosensitizer reagents. Cholesterol-directed POCA captured known and unprecedented cholesterol-binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by physiologically relevant lipoprotein uptake. Protein-directed POCA accurately mapped intracellular membrane complexes, defined sterol-dependent changes to the interactome of the cholesterol transport protein Aster-B and revealed singlet oxygen-mediated domain-specific Aster crosslinking. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, fulfilling unmet needs in intracellular singlet oxygen-based proximity labeling proteomics.

Discovery of novel structural imidazolylvinylquinolones exerting excellent broad-spectrum antibacterial efficacy with multitargeting potential.

The Major Facilitator Superfamily (MFS) comprises a large and diverse group of membrane transport proteins involved in the translocation of metabolites across cellular membranes. The genome of Toxoplasma gondii encodes approximately 60 putative MFS transporters, yet the functions of most remain poorly characterized. Conserved across the superphylum Alveolata, the inner membrane complex (IMC) is a specialized peripheral membrane system essential for parasite replication, structural integrity, motility, and host cell invasion. Here, we identify Toxoplasma gondii Daughter Cell Transporter 1 (TgDCT1), a previously uncharacterized MFS transporter, as a critical regulator of daughter cell formation. TgDCT1 localizes predominantly to the daughter cell IMC and contains a predicted spinster-like MFS domain. Phylogenetic and structural analyses reveal that TgDCT1 is conserved across Alveolata, shares a canonical MFS fold with its Plasmodium falciparum orthologue, and exhibits striking structural similarity to the human sphingosine-1-phosphate (S1P) transporter SPNS2, suggesting an evolutionarily conserved role in lipid transport. Conditional depletion of TgDCT1 results in severe defects in cytokinesis, including disrupted IMC architecture, aberrant daughter cell morphology, and failure of plasma membrane abscission. Although TgDCT1-depleted parasites retain the capacity for microneme secretion and egress, they display profoundly impaired motility and host cell invasion, ultimately leading to arrest of the lytic cycle. Notably, pharmacological inhibition of the S1P transporter SPNS2 using the compounds 11i and 33p phenocopies TgDCT1 depletion, impairing parasite morphogenesis, intracellular replication, and division synchrony. Furthermore, transgenic complementation demonstrates that the spinster-like domain of the P. falciparum DCT1 orthologue can functionally substitute for TgDCT1, indicating that these transporters likely recognize the same substrate. Together, these findings establish TgDCT1 as a central regulator of lipid homeostasis required for IMC maturation, endodyogeny, and parasite propagation in Toxoplasma gondii and likely other Apicomplexa.

The Major Facilitator Superfamily (MFS) comprises a large and diverse group of membrane transport proteins involved in the translocation of metabolites across cellular membranes. The genome of Toxoplasma gondii encodes approximately 60 putative MFS transporters, yet the functions of most remain poorly characterized. Conserved across the superphylum Alveolata, the inner membrane complex (IMC) is a specialized peripheral membrane system essential for parasite replication, structural integrity, motility, and host cell invasion. Here, we identify Toxoplasma gondii Daughter Cell Transporter 1 (TgDCT1), a previously uncharacterized MFS transporter, as a critical regulator of daughter cell formation. TgDCT1 localizes predominantly to the daughter cell IMC and contains a predicted spinster-like MFS domain. Phylogenetic and structural analyses reveal that TgDCT1 is conserved across Alveolata, shares a canonical MFS fold with its Plasmodium falciparum orthologue, and exhibits striking structural similarity to the human sphingosine-1-phosphate (S1P) transporter SPNS2, suggesting an evolutionarily conserved role in lipid transport. Conditional depletion of TgDCT1 results in severe defects in cytokinesis, including disrupted IMC architecture, aberrant daughter cell morphology, and failure of plasma membrane abscission. Although TgDCT1-depleted parasites retain the capacity for microneme secretion and egress, they display profoundly impaired motility and host cell invasion, ultimately leading to arrest of the lytic cycle. Notably, pharmacological inhibition of the S1P transporter SPNS2 using the compounds 11i and 33p phenocopies TgDCT1 depletion, impairing parasite morphogenesis, intracellular replication, and division synchrony. Furthermore, transgenic complementation demonstrates that the spinster-like domain of the P. falciparum DCT1 orthologue can functionally substitute for TgDCT1, indicating that these transporters likely recognize the same substrate. Together, these findings establish TgDCT1 as a central regulator of lipid homeostasis required for IMC maturation, endodyogeny, and parasite propagation in Toxoplasma gondii and likely other Apicomplexa.

ABSTRACT Freestanding complex oxide membranes enable the release and transfer of epitaxial films, offering new design freedoms for next‐generation electronics. While the LaAlO 3 /SrTiO 3 (LAO/STO) heterostructure exhibits remarkable tunable conductivity at its interface, the active interface remains buried beneath the substrate, limiting access to this functionality. Here, we demonstrate how the LAO/STO heterostructure, in membrane form, can be flipped and precisely positioned on silicon and other platforms using polymer‐free micromanipulation. The transferred membranes preserve atomically smooth surfaces, high crystallinity, and key electronic properties. Through the 44‐nm insulating STO layer, ultra‐low‐voltage electron‐beam lithography (ULV‐EBL) writes conductive nanostructures at the now‐accessible STO/LAO interface, offering the potential to function as programmable local gates that modulate charge carriers in the underlying silicon. The platform establishes a general strategy for integrating complex oxide heterostructures with semiconductors, quantum materials, and flexible substrates, enabling new architectures for reprogrammable nanoelectronic devices.

Sodium-pumping NADH: ubiquinone oxidoreductase (Na+-NQR) is an important component of the aerobic respiratory chain of Vibrio cholerae. It oxidizes NADH, reduces ubiquinone, and uses the free energy of this redox reaction to move sodium across the cell membrane. The enzyme is a membrane complex of six subunits, two 2Fe−2S centers, and four flavins. Both the oxidized and reduced forms of Na+-NQR exhibit EPR signals due to flavin semiquinone radicals. It has been shown that in the oxidized form of the enzyme, the radical is a neutral flavin, while in the NADH-reduced form, the radical is an anionic flavin. Electron Spin Echo Envelope Modulation Spectroscopy (ESEEM) was used to probe the presence of the magnetic nucleus 23Na in the immediate vicinity of the paramagnetic centers. The contribution of the 23Na nucleus was observed only in the ESEEM spectra of the anionic flavin semiquinone previously assigned to FMNNqrB. Analysis shows that the Na+ ion is within ~3–4 Å of the flavin radical. This distance is consistent with two models: (i) complexation of the Na+ ion with the carbonyl group of CO4; or alternatively, (ii) a “cation-π interaction,” between Na+ and the electron-rich π-system of the flavin aromatic rings.

To report a case of intrascleral crystal deposition in a patient with genetically confirmed Bietti Crystalline Dystrophy (BCD). Case report of a 51-year-old Asian male with high myopia, nyctalopia since early adulthood and progressive visual decline. Clinical examination, fundus photography, infrared reflectance, spectral-domain OCT, and ultra-widefield swept-source (SS) OCT were performed. Genetic testing was performed to evaluate for pathogenic mutations in CYP4V2. Pseudocolor fundus photography revealed yellow-white crystalline deposits primarily in the posterior pole with poor visualization of crystals more peripherally. Cross-sectional OCT localized hyperreflective deposits predominantly at the retinal pigment epithelium-Bruch's membrane complex, with additional foci in the outer and inner retina, accompanied by chorioretinal atrophy. Intrascleral crystals were also identified on spectral domain OCT. Ultra-widefield OCT extended the assessment beyond the vascular arcades, revealing peripheral zones of atrophy and scattered crystals not visible on standard field scans. Intrascleral crystal distribution may aid in broadening the phenotypic spectrum of BCD, supporting more accurate diagnosis and contributing to the understanding of its pathophysiology. UWF and multimodal imaging provide complementary insights into BCD extent and crystal localization. Infrared reflectance outperforms pseudocolor photography for crystal detection, while UWF-OCT expands structural evaluation into the periphery, potentially improving monitoring strategies.

The Dot/Icm type IV secretion system (T4SS) is essential for Legionella pneumophila infection, but its in situ architecture and mechanism remain incompletely understood. Using cryo-electron tomography, we performed subtomogram averaging and 3D classification to resolve structural heterogeneity within the complex. We identified multiple assembly states of the inner membrane complex, including a fully assembled form with a hexamer-of-dimers DotO ATPase and symmetry mismatches between subcomplexes. A composite in situ model revealed a central channel above the inner membrane, likely used for substrate secretion. Imaging of infected macrophages showed T4SSs tethered to host vacuoles and extracellular vesicle release, suggesting additional effector delivery routes. These findings provide insight into Dot/Icm T4SS structure and infection-related dynamics.

Parkinson's disease (PD) is a chronic, progressive neurodegenerative disorder characterized by severe motor symptoms. While the degeneration of dopaminergic neurons in the substantia nigra plays a central role, other neurotransmitter systems also contribute to PD symptoms. α-Synuclein (αSyn), normally expressed in neurons to support synaptic function and neurotransmitter release, becomes pathologically accumulated in PD, despite not being upregulated under physiological conditions. Intracellular aggregation of αSyn into Lewy bodies is a hallmark of synucleinopathies. A vital facet of both the onset and progression of PD involves mitochondrial dysfunction, which links αSyn misimport into mitochondria with neuronal death. The interaction of αSyn with mitochondrial membranes has been identified, yet the complex stepwise biological mechanisms of αSyn misimport into the mitochondrial compartments, followed by its aggregation, culminating in mitochondria-mediated apoptosis, remain unknown. The Translocase of the Outer Mitochondrial Membrane (TOM) complex, vital for unidirectional import of >1300 mitochondrial proteins from the cytosol, can additionally misimport αSyn into mitochondria. This TOM-αSyn interplay can alter calcium homeostasis, reduce ATP biogenesis, elevate reactive oxygen species generation, and compromise mitochondrial dynamics, resulting in mitochondrial dysfunction and triggering cell death in dopaminergic neurons. Detailed analyses of TOM complex function, interactome, and TOM-αSyn association could lead to treatment approaches that restore mitochondrial homeostasis by mitigating the effects of αSyn pathology in neurodegenerative conditions. This review details the most recent findings on independent regulators of αSyn and the TOM complex and discusses TOM-αSyn interaction mechanisms and their outcomes on mitochondrial dynamics toward promoting development of therapeutics for neurodegeneration.

A complete and thorough understanding of the complicated heterogeneous structure of polyamide separation membranes is crucial to improving their performance. Electron tomography has been used to study density variations in dense polymer membranes; however, the nonuniformity of membrane thickness and surface morphology present major challenges to the accuracy of that method. In this article, we show that nanoscale 2D electron energy loss spectroscopy (EELS) maps can be correlated with 3D scanning transmission electron microscopy (STEM) tomography to improve the quantitative mapping of density. We reveal quantitative nanoscale structural differences between commercial seawater and brackish water polyamide thin film composite reverse osmosis membranes and compare them to thin uniform printed membranes. To reduce electron beam damage, we employ a high-speed direct electron detector for low-dose EELS, which allows for membrane thickness and electron scattering measurements to be spatially correlated to improve the measurement of density. We resolve nanoscale differences between three polyamide membranes which have distinctly different separation performances. Our work provides a framework for the use of STEM and EELS to extract heterogeneous density variations in structurally complex membranes.

Carotenoid (Car) in photosynthesis plays an essential role in photoprotection by quenching chlorophyll triplet excitation (3Chl*) via a Chl-to-Car triplet energy transfer (TET). However, mechanistic studies on the Car triplet photoprotection (CTP) have been complicated by the involvement of the O2 quenching and the complexity of the thylakoid membrane. To clarify the interplay of the TET with the effects of O2 and lipid-protein interaction, we prepared nanodiscoidal lipid-protein assemblies of the light-harvesting complexes of photosystem II (LHCII) of Bryopsis corticulans and spinach and examined their triplet excitation dynamics in a broad temporal regime of 1-105 ns. For both kinds of LHCII complexes, besides the well-known ultrafast TET at both L1 and L2 sites, an O2 insensitive, slow TET reaction at the L1 (but not L2) site proceeding with a time constant of 11-25 ns was verified. Lipid membranes can substantially accelerate the slow TET reaction and prolong the 3Car* lifetime. On the other hand, both kinds of LHCII complexes bear a minor fraction of Car-unquenchable 3Chl*, i.e., 2.2% and 4.6% (1.5% and 2.2%) for Bry. corticulans (spinach) LHCII complexes in lipid and aqueous phases, respectively, which can be fully quenched by O2. In addition, the lipid membrane promotes the O2 accessibility of LHCII proteins and, compared to L2, the L1 site is (30-130)% more permissive to O2 access. The heterogeneous 3Chl* deactivation pathways and CTP potency of Bry. corticulans and spinach are compared.

KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K+ ions translocate through the inter-subunit tunnel as a queue of ions or individually within a hydrated environment. Using molecular dynamics simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K+ ions. Our results identify only one stable intermediate binding site for K+ within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA-KdpB interface, making spontaneous K+ translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K+ transport. These findings challenge previous models containing a continuous wire of K+ ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K+ movement into KdpB.

The interactome era: Integrating RNA-seq, proteomics, and network biology to decode cellular senescence.

Endoplasmic reticulum membrane complex 1 facilitates BK polyomavirus genotype III/IV replication for nephropathy.

Plant tetraspanins: dynamic membrane players for bolstering crop stress resilience.

Ssy1 in Saccharomyces cerevisiae is an amino acid receptor evolved from amino acid transporters. It is situated in the plasma membrane in the SPS complex, together with the WD40-repeat protein Ptr3 and the endoprotease Ssy5. Binding of extracellular amino acids to Ssy1 triggers liberation of the catalytic domain of Ssy5, which removes an inhibitory domain from the transcription factor Stp1, freeing it to activate genes encoding amino acid transporters. We mapped 7 constitutively signaling and hyper-responsive SSY1 mutations onto AlphaFold- and Phyre2-based 3D-models of Ssy1 to inform conformational steps involved in signaling. The predictions suggest a model in which an occluded, inward-facing conformation of Ssy1 leads to signaling. The mutations suggest a hinge in TM12 which, combined with a C-terminal 'latch', offers a mechanism for signaling. AlphaFold 3 modeling suggests that conserved sequence boxes in the N-terminal cytoplasmic domain of Ssy1 serve as interaction faces for binding of Ptr3, Ssy5 and casein kinases Yck1 and Yck2 (Yck). In addition, interaction faces between Ptr3 and Ssy5 were predicted. Antagonism between phosphorylation and dephosphorylation of Ptr3 and Ssy5 by Yck and Protein Phosphatase 2A (PP2A) is key in signaling. We found Yck phosphorylation motifs as well as binding motifs for regulatory subunit Rts1 of PP2A, in both Ptr3 and Ssy5. These motifs, together with sites of PTR3 and SSY5 gain-of-function mutations, were mapped onto AlphaFold models of Ptr3 and Ssy5. The results constitute a basis for predicting novel aspects of phosphorylation in the signaling mechanism.

PurposeTo present the clinical and multimodal imaging features of three patients diagnosed with stellate multiform amelanotic choroidopathy (SMACH), a recently described, rare chorioretinal entity.MethodsCase series.ResultsAll patients, aged 5, 10, and 38 years, presented with unilateral, yellowish choroidal lesions extending from the fovea toward the temporal macula. Multimodal imaging was used for diagnosis. Structural disorganization of the subfoveal choroid with irregular, undulating hyperreflective alterations of the retinal pigment epithelium/Bruch's membrane complex with focal protrusions was noted in all patients, while subretinal fluid (SRF) was present in two patients at presentation on optical coherence tomography (OCT). No patient had systemic findings. The first and the third cases received intravitreal bevacizumab injections and the SRF persisted with fluctuations in both patients. In the second case, although there was no SRF initially, it occurred with fluctuations during follow-up. Throughout the follow-up periods, visual acuities remained stable in all patients without any additional treatment.ConclusionSMACH should be considered in the differential diagnosis of choroidal structural disorganization, particularly in young patients. Notably, one patient was 5 years old, which represents the youngest age reported to date. Multimodal imaging modalities are essential for the recognition of the disease and a confirmed diagnosis of SMACH may prevent unnecessary treatments such as intravitreal injections and photodynamic therapy.

Superhydrophilic membranes hold significant promise for oil/water separation in effluent treatment due to their exceptional separation efficiency and inherent anti-oil properties. However, persistent challenges such as irreversible membrane fouling and complex cleaning procedures caused by oil ingress during operation remain unresolved. While extensive research has explored diverse substrates, such as stainless steel mesh, polysulfone ultrafiltration membranes, and copper mesh, conventional fabrication methods often rely on environmentally hazardous processes, such as chemical etching or intricate substrate engineering, to achieve robust adhesion between substrates and superhydrophilic coatings. Furthermore, existing strategies for emulsified oil separation frequently compromise flux efficiency. To address these limitations, this study introduces a laser-assisted morphogenetic fabrication technique to create an antifouling, scale-like substrate. A biodegradable, nontoxic chitosan modification layer is integrated via binding sites, enhancing interfacial adhesion while optimizing mechanical stability and coating porosity. The resultant membrane exhibits self-cleaning functionality alongside superhydrophilicity, achieving a sustained flux of 59 683.103Lm- 2h-1 and maintaining >99.996% separation efficiency over 100 cycles. The mild, eco-friendly synthesis process and the membrane's superior performance underscore its potential for scalable application in sustainable oil/water separation. This work advances the rational design of durable superhydrophilic coatings and offers a viable pathway for developing high-flux, antifouling separation technologies.

We describe a viral replicon-based CRISPR knockout (KO) screening approach to specifically identify host factors essential for viral replication which are often missed in live virus screens. We benchmark the replicon screening using a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line and successfully identify host genes known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components), along with additional genes that have not been reported in prior CRISPR KO screens with DENV-2. We extend this replicon screening approach to chikungunya virus (CHIKV), a positive-sense RNA virus, and Ebola virus (EBOV), a negative-sense RNA virus, and identify distinct sets of genes required for replication of each virus. Our findings indicate that viral replicon-based CRISPR screens are a useful approach to identify host factors essential for replication of diverse viruses and to elucidate potential novel targets for host-directed medical countermeasures.

Data suggest that antagonism between bacteria is prevalent within the gut microbiome. Such antagonism could have profound consequences on the fitness of species; however, the susceptibility determinants to even the most pervasive antagonistic factors in this ecosystem remain incompletely understood. Here, we screened for genetic factors that impact the susceptibility of Bacteroides to type VI secretion system (T6SS)-delivered toxins. This revealed that the Bte2 family of pore-forming toxins, which are widespread in B. fragilis and other human gut-associated Bacteroidales, strictly requires the H+/Na+-translocating ferredoxin:NAD+ reductase (Rnf) electron transport chain within target cells in order to intoxicate. In Bacteroides, the precise metabolic role of the conserved Rnf pathway has not been defined. We establish that the Rnf complex is important for redox balancing within cells utilizing sugars derived from dietary fiber and is critical for fitness in vivo. Surprisingly, we find that while the intact Rnf membrane complex is required for Bte2 intoxication, Rnf-catalyzed electron transport is dispensable. We propose that the Rnf complex facilitates Bte2 membrane insertion, leading to intoxication via membrane depolarization. Our data suggest that T6SS toxins may avoid collateral damage within a complex ecosystem by recognizing discriminatory features of competitor species.