Journal: Cell Discovery

Article Title: The accessory protein ORF3a hijacks the CLCC1 chloride channel to disrupt ER homeostasis in betacoronavirus pathogenesis

doi: 10.1038/s41421-026-00918-0

Figure Lengend Snippet: a AAV-PHP.eB-based transgene delivery strategy involving retro-orbital injection in mice. EGFP-tagged SARS-CoV-2 ORF3a and EGFP were driven by a CAG promoter. b – d SARS-CoV-2 ORF3a expression leads to weight loss and mortality in mice. The wild-type mice were injected with AAV-PHP.eB at postnatal day 22 (P22) and weighed at P40 ( b ). The mice were sacrificed when substantial weight loss ( c ) and movement impairment ( d ) were observed. The dose of AAV-PHP. eB, 1 × 10 11 vg/20 g mice. e, f SARS-CoV-2 ORF3a interacts with endogenous CLCC1 and induces LC3 lipidation, ER-phagy, nucleophagy, and apoptosis in mouse brains. Four mice were sacrificed 28 days post AAV-PHP.eB injection. Brain lysates were prepared for co-IP experiments ( e ) and immunoblot analysis ( f ). In e , GFP-trap beads captured the EGFP-tagged SARS-CoV-2 ORF3a and EGFP. Red arrows indicate EGFP-tagged SARS-CoV-2 ORF3a as input. The relative expression level of endogenous CLCC1 was calculated and normalized ( e ). In f , antibodies against LC3, FAM134B, LMNB1, and caspase-3 were used to detect LC3-I/LC3-II conversion, ER-phagy, nucleophagy, and caspase-3-dependent apoptosis, respectively. g TEM images of hippocampal regions expressing SARS-CoV-2 ORF3a-EGFP or EGFP. Red arrow, the bleb amid NE; red asterisk, the autolysosome; blue arrow, ER whorl-like membrane stacks. N nucleus, M mitochondria, NE nuclear envelope, G Golgi apparatus; E endoplasmic reticulum, L lysosome. h , i Immunofluorescence analysis of mouse brains expressing SARS-CoV-2 ORF3a-EGFP and EGFP. The mice were sacrificed 20 days post AAV-PHP.eB administration. ER stress, misfolded protein accumulation, and protein degradation were assessed using staining for Bip, ubiquitin, and K48-linked ubiquitin, respectively, in the dentate gyrus and olfactory bulbs. j TUNEL assay of the hippocampal dentate gyrus in mice expressing SARS-CoV-2 ORF3a-EGFP vs EGFP control. k Neuroinflammatory profiling in thalamic regions of ORF3a-EGFP-expressing mice. Microglial activation (Iba1) and astrogliosis (GFAP) were assessed by immunostaining. Scale bar, 200 nm in g ; 5 μm in h and i ; 20 μm in j ; 50 μm in k . In b – d , n = 5. The values are presented as the mean ± SD. ** P < 0.01, by Student’s t -test ( c ) and the Mantel‒Cox test ( d ).

Article Snippet: AAV-PHP.eB virus harboring a GFP and SARS-CoV-2 ORF3a cassette (1 × 10 13 vg/mL) was generated by Packgene Biotech and diluted to a final concentration of 50 × 10 10 vg/600 μL in sterile PBS.

Techniques: Injection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Membrane, Immunofluorescence, Staining, Ubiquitin Proteomics, Olfactory, TUNEL Assay, Control, Activation Assay, Immunostaining

Journal: bioRxiv

Article Title: Streamlined Production of Recombinant Adeno-Associated Viruses Using Gateway Cloning Technology for Neural Circuit All-Optical Interrogation

doi: 10.64898/2026.09.22.752944

Figure Lengend Snippet: (A) Schematic of the viral injection strategies used to selectively express fluorescent indicators in Purkinje cells, all generated using the Gateway cloning pipeline. Numbered circles (①–③) indicate the color-coded strategies referenced throughout: ① GCaMP6f (green), ② jRGECO1a (red), ③ JEDI2P-Kv (black). For JEDI2P-Kv, two strategies were designed — a single-recombinase CRE/lox approach analogous to the calcium indicator strategies, and a dual-recombinase approach combining TRE-driven expression amplification with FLP-dependent conditionality to enhance sparse labelling. (B) Representative two-photon fluorescence images of Purkinje cell dendrites labelled using strategy ① at three CRE-driver dilutions (1×10¹², 5×10¹⁰, and 1×10¹⁰ GC/ml co-injected against a fixed 1×10¹¹ GC/ml indicator virus). Reducing the CRE-driver titer progressively decreases labelling density; sparse individual arbors can be resolved 500 µm from the injection site. Images displayed on a photon-count scale (0 to max; max = 12 except for the 1×10¹⁰ GC/ml condition, where max = 5). Scale bars: 20 µm. (C) Three-dimensional reconstruction of a Purkinje cell (grayscale) with two representative ULoVE excitation volumes (red) positioned on the dendritic arbor. Scale bar: 10 µm. (D) Representative in vivo calcium transients from Purkinje cell dendrites expressing GCaMP6f (strategy ①, top, green) and jRGECO1a (strategy ②, bottom, red), showing normalized ΔF/F over time. Repeated stereotyped events correspond to climbing fiber-evoked dendritic calcium transients. Traces smoothed with a 3 ms Gaussian kernel for display. (E) Representative two-photon fluorescence image of Purkinje cell dendrites expressing JEDI2P-Kv (strategy ③). Scale bar: 20 µm. (F) Voltage trace recorded from a JEDI2P-Kv-expressing Purkinje cell dendrite showing spontaneous dendritic events (labelled i–v), displayed as –ΔF/F so that membrane depolarizations appear as positive deflections. Trace smoothed with a 0.5 ms Gaussian kernel. Bottom: expanded views of individual events i–v from the raw trace, illustrating the diversity of event morphology, including two- and three-peak spikelets. (G) Average normalized waveforms for GCaMP6f (n = 4639 events, 42 dendrites), jRGECO1a (n = 1333 events, 6 dendrites), and JEDI2P-Kv (n = 863 events, 6 dendrites), aligned to event onset. Shaded areas: ±SEM. Note the markedly faster kinetics of the voltage indicator. (H) Distributions of exponential rise time constants (τ rise , left) and decay time constants (τ decay , right) estimated per dendrite from average waveforms of temporally isolated onset-aligned events. Solid green curves: Gaussian fits to summed distributions. (I) Distribution of inter-peak intervals for JEDI2P-Kv dendritic events classified as 2-peaks (dark grey, n = 521) or 3-peaks (light grey, n = 229). Log-normal fit (red curve) reveals a skewed distribution with excess around 2.5 and 6 ms.. (J) Left: average normalized waveforms for 3-peaks (orange, n = 16), 2-separate-peak (blue, n = 27), and 2-closed-peaks (grey, n = 19) event categories, selected for temporal homogeneity. Shaded areas: ±SEM. An afterhyperpolarization (AHP, arrow) is present in 3-peak events and absent from 2-peak events. Right: time-frequency spectrograms (Morlet wavelet analysis) for each category, revealing oscillatory components at 290 Hz (2-closed-peaks), 190 Hz (2-separate-peaks), and 260/120 Hz (3-peaks).

Article Snippet: The first, AAV1.eF1α.DiO.nls.FLPo (virus ENS 008, produced in-house from Addgene 87306), provides nuclear-localized FLPo recombinase.

Techniques: Injection, Generated, Cloning, Expressing, Amplification, Fluorescence, Virus, In Vivo, Membrane, Morphology, Isolation, Analysis

Journal: bioRxiv

Article Title: Streamlined Production of Recombinant Adeno-Associated Viruses Using Gateway Cloning Technology for Neural Circuit All-Optical Interrogation

doi: 10.64898/2026.09.22.752944

Figure Lengend Snippet: Schematic of experimental design. In a vGlut2::CRE;GlyT2::eGFP double-transgenic mouse, CRE recombinase expressed in vGlut2+ excitatory DCN neurons drives expression of the two co-injected CRE-dependent viruses: AAV1.eF1α.DiO.nls-FLPo and AAV1.ihSyn1.DiO.tTA. A third Gateway-cloned virus, AAV1.TRE.fDiO.tdTomato (virus ENS 068, pEXPR A-183), assembled by MultiSite LR recombination of a TRE promoter entry clone with a fDiO-tdTomato transgene entry clone, requires both tTA-driven TRE activation and FLPo-mediated inversion of the fDiO cassette for tdTomato expression, restricting labelling to vGlut2+ neurons in which all three conditions are satisfied. Limiting the dilution of the CRE-dependent FLPo-expressing virus further restricts the labelled population to a sparse subset. (Right) Representative fluorescence image showing sparse but strong tdTomato expression in vGlut2+ excitatory neurons of the medial deep cerebellar nucleus (DCN), with individual axonal projections clearly resolvable for anatomical tracing ( see Supplement to ), alongside GlyT2::eGFP-labelled inhibitory DCN neurons (green). GlyT2::eGFP also labels glycinergic Golgi and Lugaro cells of the cerebellar cortex, providing anatomical context within and around the nucleus.

Article Snippet: The first, AAV1.eF1α.DiO.nls.FLPo (virus ENS 008, produced in-house from Addgene 87306), provides nuclear-localized FLPo recombinase.

Techniques: Transgenic Assay, Expressing, Injection, Clone Assay, Virus, Activation Assay, Fluorescence

Journal: bioRxiv

Article Title: Streamlined Production of Recombinant Adeno-Associated Viruses Using Gateway Cloning Technology for Neural Circuit All-Optical Interrogation

doi: 10.64898/2026.09.22.752944

Figure Lengend Snippet: (A) Experimental timeline. Mice were injected with a CRE-dependent ChR2(H134R)-YFP construct together with the c-kit CRE driver (D0), and acute cerebellar slices were prepared at D15 for whole-cell voltage-clamp recordings combined with two-photon photostimulation. Full injection and viral parameters are described in Methods (“Cerebellar slice preparation and electrophysiology”). (B) Top: two-photon fluorescence images, shown rotated by 45° relative to the indicated plane (left, xy; right, xz), of the scanned doughnut excitation patterns. Scale bars: 10 µm. Bottom: representative two-photon fluorescence image of a ChR2(H134R)-YFP-expressing molecular layer interneuron (basket cell subtype). Scale bar: 10 µm. (C) Left: schematic of the multi-doughnut excitation pattern (20 doughnuts, 70 µs per doughnut, 1.4 ms total per cell). Right: representative ChR2(H134R) photocurrents recorded in whole-cell voltage clamp at increasing squared laser powers (11.3, 42.5, 149, and 215 mW²), with expanded timescale inset. Note that photocurrents were initiated before the full 20-doughnut sequence was completed, confirming sub-millisecond photoexcitation efficiency. (D) Input–output curve of normalized photocurrent amplitude as a function of squared laser power. Half-saturation power √P₀ 2 = 8 mW. Data shown as mean ± SEM. (E) Spatial resolution of two-photon doughnut optogenetic activation. Photocurrent amplitude as a function of lateral (left) or axial (right) displacement of the ULoVE focus relative to the MLI soma. Lateral resolution constant: 0.72 cell diameters; axial resolution: 8.3 µm. (F) Top: two-photon fluorescence image of ChR2(H134R)-YFP-expressing MLIs; numbered cells (1–9) indicate the nine sequentially photostimulated cells. Red arrow indicates the presynaptically connected cell (MLI #8). Bottom: infrared Dodt contrast image showing the recorded Purkinje cell at the tip of the patch pipette. Scale bar: 50 µm. Photocurrents were probed at a revisit cycle frequency of 710 Hz, with each MLI receiving a 1.4 ms ULoVE excitation per cycle. (G) Evoked IPSCs recorded at the Purkinje cell soma from each of the nine sequentially photostimulated MLIs shown in (F). Only MLI #8 (red arrow; right inset) generated a reliable postsynaptic response, with a mean IPSC amplitude of 46 pA across 10 trials and one failure.

Article Snippet: The first, AAV1.eF1α.DiO.nls.FLPo (virus ENS 008, produced in-house from Addgene 87306), provides nuclear-localized FLPo recombinase.

Techniques: Injection, Construct, Slice Preparation, Fluorescence, Expressing, Sequencing, Activation Assay, Infrared, Transferring, Generated

Journal: Science Advances

Article Title: mTORC1-TFEB/TFE3 signaling is associated with bile acid diversification during hepatic metabolic adaptation

doi: 10.1126/sciadv.aee1905

Figure Lengend Snippet: ( A ) Gross liver appearance of WT and Ltor2-LKO mice. Scale bars, 10 mm. ( B ) Representative hematoxylin and eosin (H&E)–stained liver sections from WT and Ltor2-LKO mice. Scale bars, 1 mm and 100 μm in zoomed images. ( C to E ) Body weight, relative liver weight, and serum ALT/AP levels in 10- to 16-week-old WT and Ltor2-LKO mice fed normal chow and euthanized ad libitum ( n = 5 to 7). ( F to I ) Hepatic glycogen, serum glucose, serum insulin, and hepatic Pck1 / G6pase expression in male WT and Ltor2-LKO mice ( n = 5 to 7). ( J ) Hepatic Lamtor2 mRNA expression in WT and Ltor2-LKO mice ( n = 5 to 7). ( K ) Hepatic LAMTOR2 protein expression in WT and Ltor2-LKO mice. ( L ) pS6, LAMTOR2, and HNF4α protein expression in primary mouse hepatocytes and nonparenchymal cells isolated from WT and Ltor2-LKO livers ( n = 2 mice per genotype). ( M ) Experimental scheme for AAV-mediated adult hepatocyte Lamtor2 deletion. Created in BioRender. A. Zaufel (2026); https://BioRender.com/vd8qjiy . ( N ) Body-weight change after injection of AAV8-TBG-Cre or control virus in male and female Lamtor2 flox/flox mice. ( O and P ) Terminal body weight and relative liver weight four weeks after virus injection ( n = 3 to 6). ( Q ) Representative H&E-stained liver sections from Ltor2/AAV-Ctrl and Ltor2/AAV-Cre mice. Scale bars, 100 μm. ( R ) pS6 and LAMTOR2 protein expression in bulk liver extracts. ( S ) Serum ALT/AP levels four weeks after virus injection ( n = 3 to 6). ( T to W ) Serum glucose, serum insulin, hepatic glycogen, and hepatic Lamtor2 , Pck1 , and G6pase expression four weeks after virus injection ( n = 3 to 6). Data are means ± SEM. Statistical significance was calculated by two-tailed unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To induce acute gene KO in adult differentiated hepatocytes of Lamtor2 flox/flox , Raptor flox/flox , Tsc1 flox/flox , and tdTomato reporter mice, Cre-mediated recombination was induced with AAV.TBG.PI.Cre.rBG (a gift from J. M. Wilson; Addgene viral prep #107787-AAV8; http://n2t.net/addgene:107787 ; RRID:Addgene_107787).

Techniques: Staining, Expressing, Isolation, Injection, Control, Virus, Two Tailed Test

Journal: Science Advances

Article Title: mTORC1-TFEB/TFE3 signaling is associated with bile acid diversification during hepatic metabolic adaptation

doi: 10.1126/sciadv.aee1905

Figure Lengend Snippet: ( A ) Lineage tracing after chronic Alb-Cre-mediated or acute AAV-Cre-mediated Lamtor2 deletion. Confocal images of liver sections from the four indicated tdTomato reporter genotypes show native tdTomato fluorescence (tdTom) and anti-tdTomato staining (RFP). In chronic Ltor2-LKO livers, native tdTom fluorescence is selectively reduced in the PP small-hepatocyte population, defined morphologically by reduced cell size, pyknotic nuclei, and clear glycogenated cytoplasm, whereas anti-RFP staining remains detectable. Asterisks indicate representative RFP-positive hepatocytes with reduced or absent native tdTom fluorescence. In contrast, tdTom signal remains uniform after acute AAV-Cre-mediated Lamtor2 deletion. Homogeneous recombination/gene knockout was observed in >99% of hepatocytes in the tested genotypes. Images are representative of at least three mice per condition. Scale bars, 100 μm. ( B ) Uniform manifold approximation and projection (UMAP) visualization of 23,590 liver nuclei isolated from four genotypes. Unsupervised clustering identified 10 manually annotated liver cell populations. For the snRNA-seq experiment, mice were approximately 16 to 20 weeks old at tissue collection; AAV-treated mice were analyzed 4 weeks after virus injection. ( C ) Cluster composition in each genotype. Pie charts show the relative contribution of each annotated population to total nuclei. Hepatocyte subpopulations are labeled Hep_1 to Hep_4. ( D to F ) Feature plots showing marker genes for hepatocytes, cholangiocytes, and LPCs. ( G ) Hepatocyte differentiation/reprogramming trajectory visualized by UMAP and colored by pseudotime. ( H ) Feature plot showing tdTomato expression.

Article Snippet: To induce acute gene KO in adult differentiated hepatocytes of Lamtor2 flox/flox , Raptor flox/flox , Tsc1 flox/flox , and tdTomato reporter mice, Cre-mediated recombination was induced with AAV.TBG.PI.Cre.rBG (a gift from J. M. Wilson; Addgene viral prep #107787-AAV8; http://n2t.net/addgene:107787 ; RRID:Addgene_107787).

Techniques: Confocal, Fluorescence, Staining, Gene Knockout, Isolation, Tissue, Virus, Injection, Labeling, Marker, Expressing

Journal: Science Advances

Article Title: mTORC1-TFEB/TFE3 signaling is associated with bile acid diversification during hepatic metabolic adaptation

doi: 10.1126/sciadv.aee1905

Figure Lengend Snippet: ( A ) mRNA expression of hepatobiliary marker genes and genes related to canonical and noncanonical mTORC1 signaling in primary mouse hepatocytes isolated from Lamtor2 flox/flox mice and treated in vitro with control or Adeno-CMV-Cre virus ( n = 12 from at least two mice per condition). ( B ) Representative confocal images showing tdTomato with TFEB, TFE3, SOX9, HNF4α, or pS6 in Ltor2/AAV-Ctrl and Ltor2/AAV-Cre livers four weeks after virus injection. Images are representative of at least three male and female mice per condition. Scale bars, 100 μm. ( C and D ) Hepatic expression and immunoblot analysis of hepatobiliary and lysosome-associated markers in Ltor2/AAV-Ctrl and Ltor2/AAV-Cre mice ( n = 6 to 9). ( E ) Hepatic lipid profiles measured by LC-MS and immunoblot analysis of lipogenic proteins in the same mice ( n = 3). Lipid species were normalized to the average of all mice. ( F ) Hepatic expression of lipogenic genes and TF isoforms in male Ltor2/AAV-Ctrl and Ltor2/AAV-Cre mice ( n = 6). ( G ) Expression of lipogenic genes and TF isoforms in control- and Cre-treated Lamtor2 flox/flox primary hepatocytes ( n = 12 from at least two mice per condition). For (F) and (G), mean Ct values of the control group are shown in parentheses below selected transcript names. ( H ) Expression of lipogenic genes and TF isoforms in nonparenchymal cells from control and Ltor2-LKO mice ( n = 12 from at least two mice per condition). ( I ) Developmental expression of lipogenic genes and TF isoforms from postnatal days 1 to 105 ( n = 3 to 5 per time point). nd, not detectable. Data are means ± SEM. Statistical significance was calculated by two-tailed unpaired Student’s t test [(A), (C), (F), (G), and (H)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To induce acute gene KO in adult differentiated hepatocytes of Lamtor2 flox/flox , Raptor flox/flox , Tsc1 flox/flox , and tdTomato reporter mice, Cre-mediated recombination was induced with AAV.TBG.PI.Cre.rBG (a gift from J. M. Wilson; Addgene viral prep #107787-AAV8; http://n2t.net/addgene:107787 ; RRID:Addgene_107787).

Techniques: Expressing, Marker, Isolation, In Vitro, Control, Virus, Confocal, Injection, Western Blot, Analysis, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Science Advances

Article Title: mTORC1-TFEB/TFE3 signaling is associated with bile acid diversification during hepatic metabolic adaptation

doi: 10.1126/sciadv.aee1905

Figure Lengend Snippet: ( A and B ) Hepatic mRNA expression of hepatobiliary markers, non-canonical mTORC1 targets, and BA biosynthetic enzymes, and immunoblot analysis of BA biosynthetic proteins, pS6, and LAMTOR2 in control, Flcn-LKO (FKO), Tfe3-KO (3KO), and DKO mice ( n = 3 to 5). ( C and D ) Serum BA composition, 12-OH/non–12-OH and 6-OH/non–6-OH ratios, and total BAs in the same genotypes ( n = 5). ( E ) Heatmap showing Spearman correlations between hepatic Rragd , Tfe3 , Tfeb , Cyp7a1 , Cyp8b1 , and Cyp2c70 expression and the indicated parameters in fructose, palmitate, and cholesterol (FPC) diet and choline-deficient, methionine-reduced high-fat diet (CDAHFD) mouse MASH datasets. ( F and G ) Serum BA composition, 12-OH/non–12-OH and 6-OH/non–6-OH ratios, and total BAs in Rptor/AAV-Cre, Tsc1/AAV-Cre, and control mice four weeks after virus injection ( n = 4 to 11). ( H to J ) Serum BA composition, 12-OH/non–12-OH and 6-OH/non–6-OH ratios, total BAs, and serum ALT in vehicle-treated Tsc1-LKO mice, and rapamycin-treated Tsc1-LKO mice ( n = 4 to 9). ( K ) Immunoblot analysis of TSC1, BA biosynthetic enzymes, and pS6 in vehicle- and rapamycin-treated Tsc1-LKO livers. Data are means ± SEM. Statistical significance was calculated by two-tailed unpaired Student’s t test [(I) and (J)] or one-way ANOVA with Holm-Šídák post hoc testing [(A), (D), and (G)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: To induce acute gene KO in adult differentiated hepatocytes of Lamtor2 flox/flox , Raptor flox/flox , Tsc1 flox/flox , and tdTomato reporter mice, Cre-mediated recombination was induced with AAV.TBG.PI.Cre.rBG (a gift from J. M. Wilson; Addgene viral prep #107787-AAV8; http://n2t.net/addgene:107787 ; RRID:Addgene_107787).

Techniques: Expressing, Western Blot, Analysis, Control, Virus, Injection, Two Tailed Test