Journal: bioRxiv

Article Title: Cryptochrome Loss Drives COPD-like Lung Pathology through Disrupted Alveolar Epithelial Proliferation and Immune Homeostasis

doi: 10.64898/2026.05.19.726266

Figure Lengend Snippet: MLE12 cells were transfected with 50 pmole/ml control, Cry1 , or Cry2 siRNA on day 0 and day 1. A. Western blot showed CRY1/CRY2 protein levels on day 2. B. The transcript levels of cytokines, including C-C motif chemokine ligand 2 ( Ccl2 ), C-X-C motif chemokine ligand 1 ( Cxcl1 ), Interleukin-6 ( Il6 ), Il8/Cxcl5 , matrix metalloproteinase 1 and 7 ( Mmp1 and Mmp7 ), and Tnfa were determined with RT-qPCR. C. Cell numbers were measured with the WST1 assay 1-, 2-, or 3-days post siRNA transfection. n=6. D-E. Primary alveolar ECs were isolated from WT or Cry1/2 dKO mice for RNA sequencing analysis. D. Heatmap image of up (501) and down (369) regulated genes in Cry1/2 dKO mice alveolar ECs compared to WT alveolar ECs. Each gene is represented as a horizontal line. E. GO Biological Process (BP) analysis of DEGs that were in Cry1/2 dKO lung ECs. F. GO Molecular Function (MF) analysis of DEGs. G. GO Cellular Component (CC) analysis of DEGs. The top 5 terms were shown for both up and down-regulated genes. H. Enriched pathways (Metascape) of overlapping genes from human COPD patient ECs DEGs with DEGs from (D). I. GO BP, J. MF and K . CC analysis of common genes between human COPD patient lungs with DEGs in Cry1/2 dKO ECs. Data are presented as mean ± SEM. ** p<0.1, *** p<0.001, and **** p<0.0001 Cry1/2 si vs si_con (Two-way ANOVA followed by Bonferroni adjusted multiple comparisons). For C , only the asterisks for si- Cry1/2 are shown.

Article Snippet: Human lung epithelial cell line A549 and mouse primary lung epithelial cell line MLE12 were procured from ATCC (Manassas, VA).

Techniques: Transfection, Control, Western Blot, Quantitative RT-PCR, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: Cryptochrome Loss Drives COPD-like Lung Pathology through Disrupted Alveolar Epithelial Proliferation and Immune Homeostasis

doi: 10.64898/2026.05.19.726266

Figure Lengend Snippet: A. Experimental schematic: Cry1/2 dKO mice were fed with regular (RD) or 1% NOB-containing diets starting at 1 month old and analyzed at 4 months. B. Representative images of H&E-stained lung sections collected from mice fed with a RD or NOB diet. C. MLI quantification of H&E-stained lung sections from male or female Cry1/2 dKO fed with RD or NOB diets. D. RNA-seq was performed using primary lung ECs isolated from the WT mice, or Cry1/2 dKO mice fed with RD or NOB diet. Heatmap of RNA-seq data of lung ECs from different treatment groups. E. GO BP analysis of DEGs that were rescued by NOB treatment in Cry1/2 dKO lung ECs. F. GO MF analysis of rescued DEGs. G. GO CC analysis of rescued DEGs. H. RT qPCR confirmed the upregulation and rescue of genes involved in cilium movement. I. The transcript expression changes of important differentially expressed chemokines, cytokines, Mmps , and surfactant proteins from RNA-seq data. Data were presented as fold changes to WT. J. MlE12 cells were transfected with control siRNA or siRNA targeting Cry1 and Cry2 . 36 h after transfection, cells were treated with 5 ng/ml TNF-α for 15 min. Phosphorylated P-65 and P65 levels were determined with Western blots. K. MLE12 cells were treated with 20 µM NOB for 24 h, followed by 5 ng/ml TNF-α for 0, 15, or 30 mins. Phosphorylated P-65 and P65 levels were determined with Western blots. L. MLE12 cells were transfected with control siRNA, or siRNA targeting Cry1 and Cry2 , and treated with 20 µM NOB for 24 h before TNF-α stimulation (15 mins). Representative confocal images of p65 (green) are shown (×400 magnification; scale bar = 10 µm). M. The percentage of cells with p65 localized in the cytoplasm (C), both cytoplasm and nucleus (C+N), or nucleus (N) was quantified for each treatment condition. N. Western blot of CRY1/2 and COPD-associated proteins in primary lung ECs from different treatment groups. Data are presented as mean ± SEM. P-value was calculated from a two-tailed t-test for H and I, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 in RD vs in Cry1/2 dKO samples.

Article Snippet: Human lung epithelial cell line A549 and mouse primary lung epithelial cell line MLE12 were procured from ATCC (Manassas, VA).

Techniques: Staining, RNA Sequencing, Isolation, Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Two Tailed Test

Journal: iScience

Article Title: The Drosophila connectome reveals axo-axonic synapses on descending neurons

doi: 10.1016/j.isci.2026.115624

Figure Lengend Snippet: Axcs make cholinergic axo-axonic synapses on the GFs as predicted by the MANC connectome (A) AN08B098-type neurons (green) form axo-axonic connections at distinct locations along giant fiber (red, GF) axons. The scale bars shown are 20 μm. (B) The AN08B098-GF connections reconstructed in Neuroglancer (Neuroglancer scale 530.45 μm/vh) mirror the morphology seen with fluorescence in A . (C) We validated AN08B098-to-GF connectivity using anti-bruchpilot to stain for T-bars in active zones (white) along AN08B098-type neurons and colocalized the resulting fluorescence with the GFs (red), which were filled with tetramethylrhodamine. The scale bars shown are 20 μm. (D) Neuroglancer reveals presynaptic sites at similar locations seen in colocalized fluorescent image (Neuroglancer scale 530.45 μm/vh). (E and F) XY and XZ plane views of the preparation shown in (A) and (B). The zoomed-in 6 μm (scale bars 3 μm) inlays show AN08B098 forming a single synapse with the GF in (E). The yellow arrows detail the precise location AN08B098 forms the Brp-positive chemical synapse (white) to the GFs in (F). Scale bars shown are 5 μm. (G–O) EM images showing monosynaptic connections between single GF (green) and AN08B098 neurons identified by the following MANC id: (G and H) 21041, (I) 21589, (J) 23949, (K) 152261, (L) 16900, (M) 20444, (N) 22275, and (O) 24038. Pre-and postsynaptic sites are detected in EM slices using a 3D convolutional neural network to identify T-bars (cyan dots) and postsynaptic densities (PSDs, magenta dots). (P–S) We expressed anti-choline acetyltransferase (anti-ChAT) and anti-GFP in AN08B098 neurons. Anti-ChAT colocalizes to AN08B098 cells, with particularly bright staining in the cell bodies. This finding suggests acetylcholine synthesis is present within these cells, validating connectome transmitter predictions for AN08B098. Scale bars shown are (P) 20 μm and (Q–S) 5 μm.

Article Snippet: A mouse primary antibody against bruchpilot (1:50, DSHB, NC82) was used to label presynaptic chemical active zones and coupled to secondary Goat anti-mouse Alexa Fluor 647 (1:500, 115-605-003).

Techniques: Fluorescence, Staining

Journal: The Journal of General Physiology

Article Title: Beat-locked ATP microdomains in the sinoatrial node map a Ca 2+ -timed energetic hierarchy and regional pacemaker roles

doi: 10.1085/jgp.202513874

Figure Lengend Snippet: Superior SA node myocytes exhibit elevated diastolic ATP and metabolic flux compared with the inferior region. (A) 3D segmented maximum-intensity projection of a whole-mount SA node immunolabeled for CD31 (vasculature, red) and cyto-iATP (myocytes, green). The dashed line denotes the boundary between superior and inferior regions. (B) Image-processing workflow illustrating merged maximum-intensity projections, binary segmentation masks, and extraction of grayscale cyto-iATP signals used for quantitative analysis. (C) Mean cyto-iATP fluorescence intensity per myocyte, grouped by region ( N = 5 mice per region), reporting expression levels of the EGFP-tagged cyto-iATP sensor. (D) Live confocal imaging of cyto-iATP signals showing representative line-scan images and corresponding normalized fluorescence traces (F/F 0 ) from superior and inferior regions. (E and F) Summary quantification of cyto-iATP signal mass rate (E) and estimated diastolic [ATP] i (F). P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.

Article Snippet: For immunolabeling, SA nodes were incubated for 48 h at 4°C with a goat anti-mouse CD31 primary antibody (1:50, AF3628; R&D Systems).

Techniques: Immunolabeling, Extraction, Fluorescence, Expressing, Imaging