Journal: Frontiers in Immunology

Article Title: Single-cell sequencing reveals reversible glial remodeling in the visual cortex during visual deprivation and recovery

doi: 10.3389/fimmu.2026.1730619

Figure Lengend Snippet: Microglia: Enrichment, Pathways & Differential Genes (FDM/REC/NC). (A) Top 10 enriched Gene Ontology (GO) terms for genes upregulated in FDM microglia compared to NC (adjusted P < 0.05). (B) Top 15 enriched GO terms for genes downregulated in REC microglia compared to FDM (adjusted P < 0.05). (C) Top 15 enriched KEGG pathways for genes upregulated in FDM microglia (excluding disease-related terms). (D) Top 15 enriched KEGG pathways for genes downregulated in REC microglia. (E) Venn diagram showing overlap of genes upregulated in FDM and downregulated in REC. (F) Volcano plot of differentially expressed genes (DEGs) in microglia: NC vs. FDM. Red dots: upregulated in FDM (log2FC > 2, adjusted P < 0.001). Blue dots: downregulated in FDM. Key genes are labeled, including ZFP36, ITPA, and the oligodendrocyte marker Plp1 (downregulated in FDM). (G) Volcano plot of DEGs in microglia: FDM vs. REC. Red dots: upregulated in FDM (log2FC > 2, adjusted P < 0.001). Blue dots: downregulated in FDM (i.e., upregulated in REC). Key genes are labeled, including ZFP36, ITPA, and Plp1 (upregulated in REC).

Article Snippet: The following primary antibodies were used: The following primary antibodies were used: ITPA (1:50, 16134-1-AP, Proteintech), ZFP36 (1:50, 12737-1-AP, Proteintech), Zbtb16 (1:50, F2699, Selleck), and β-actin (1:50, 66009-1-Ig, Proteintech).

Techniques: Labeling, Marker

Journal: Frontiers in Immunology

Article Title: Single-cell sequencing reveals reversible glial remodeling in the visual cortex during visual deprivation and recovery

doi: 10.3389/fimmu.2026.1730619

Figure Lengend Snippet: Expression of microglial markers and ITPA/ZFP36 in NC, FDM, and REC groups. (A) Representative immunofluorescence images of Iba1+ microglia (green) and DAPI (blue) in V1. Scale bar: 20 μm. Three non-overlapping fields per animal (n = 3 animals per group) were randomly selected for imaging; all acquisition parameters were kept constant. (B) Reconstructed microglial skeletons showing morphological changes: ramified in NC, activated (hypertrophied) in FDM, and restored ramification in REC. (C) Quantification of Iba1+ cell density. Data are mean ± SEM with individual data points (n = 3 animals per group, 3 fields per animal). ***P < 0.001, one-way ANOVA with Tukey’s HSD test. (D, E) qPCR validation of ITPA (D) and ZFP36 (E) mRNA expression (n = 3 per group). Data are normalized to β-actin and presented as fold change relative to NC. ***P < 0.001, one-way ANOVA with Tukey’s HSD test. (F) Representative capillary Western blot images for ITPA, ZFP36, and β-actin (loading control). (G, H) Densitometric quantification of ITPA (G) and ZFP36 (H) protein levels normalized to β-actin (n = 3 per group). Data are mean ± SEM with individual points. ***P < 0.001, one-way ANOVA with Tukey’s HSD test.

Article Snippet: The following primary antibodies were used: The following primary antibodies were used: ITPA (1:50, 16134-1-AP, Proteintech), ZFP36 (1:50, 12737-1-AP, Proteintech), Zbtb16 (1:50, F2699, Selleck), and β-actin (1:50, 66009-1-Ig, Proteintech).

Techniques: Expressing, Immunofluorescence, Imaging, Biomarker Discovery, Western Blot, Control

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Phylogenetic tree of 83 Eurotiale order fungal IMPDHs, including 62 IMPDH-As and 21 IMPDH-Bs constructed using BALi-Phy . Branches with ≥80% support are shown. Five major subclades are found: Byssochlamys and Talaromyces (olive green), Aspergillus (purple), Penicillium IMPDH-As (blue), IMPDH-Bs (red), and outgroups composed closely related Trichophyton , Microsporum , and Coccoidiodes. Bi-partition branch supports are listed to the left of their node of origin. PbreA, PnalA, PbreB and PnalB are highlighted to illustrate the incongruence of the IMPH-A and IMPDH-B phylogeny. Resurrected ancestors are denoted by the number along the branches. MPA producers are denoted with stars. Branch length scale is shown at the bottom. Abbreviations: Acan , Aspergillus candidus ; Apse , Aspergillus pseudoglaucus ; BsAF , Byssochlamys sp AF001B; Pani , Paecilomyces nivea ; Pant , Penicillium antarcticum ; Pbre , P. brevicompactum ; Pbra, P. brasilianum ; Pcam , P. camemberti ; Pcar, P. carneum ; Pcop, P. coprophilum ; Pdig, P. digitatum ; Pexp , P. expansum ; Pfre , P. freii ; Pgris , P. griseofulvum ; Pita , P. italicum ; Pnal , P. nalgiovense ; Pnor, P. nordicum ; Ppar, P. parvum ; Ppol, P. polonicum ; Proq , P. roqueforti ; PruW, P. rubens Wisconsin ; Psam , P. samsonianum ; Psol , P. solitum ; Pste , P. steckii ; Pvul , P. vulpinum . B-G. The values of K iapp for MPA ( B ), k cat ( C ), K IMP ( D ), K NAD ( E ), k cat /K IMP ( F ) and k cat /K NAD ( G ) are shown for IMPDH-Bs from producers (Prod, magenta) and nonproducers (Non, teal) and ancestors (Anc, colored as noted), as well as MPA-sensitive IMPDHs from the literature (MPA S , black, defined as K iapp ≤ 30 nM), See , and and and for data and parameters for each enzyme. **, p ≤ 0.01. **, p ≤ 0.01. B. IMPDH-Bs and Anc3-Anc6 are MPA-resistant while Anc1 and Anc2 are MPA S . C. The values of k cat are similar for IMPDH-Bs and MPA S enzymes. D. The values of K IMP are larger for IMPDH-Bs than MPA S enzymes. E. The values of K NAD are comparable among IMPDH-Bs and MPA S enzymes. F. The values of k cat /K IMP of MPA S IMPDHs are greater than IMPDH-Bs. Producer IMPDH-Bs tend to be more active than nonproducer enzymes (p = 0.18; p= 0.02 if the thermolabile Pani B value is omitted). G. The values of k cat /K NAD of MPA S IMPDHs are greater than nonproducer IMPDH-Bs.

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Construct

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: Enzyme concentrations are listed in the Methods section. Reactions were with fixed substrate concentrations as listed and at 25°C unless otherwise specified. The average and range of duplicate reactions are plotted. Data was analyzed in GraphPad Prism. The values of K IMP were determined by fits to the Michaelis-Menten equation, values of K NAD were determined by fits to Michealis-Menten or substrate inhibition equation as appropriate. The values of K iapp for MPA were determined as described in the Methods. CI, 95% confidence intervals. Values are listed in .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Inhibition

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A . Clustal Omega sequence alignment of ancestral IMPDHs . Ancestors listed in order of appearance in the tree in . First change at a given position denoted in yellow, second change in green, third change in magenta. B. AlphaFold model of Anc1 colored by pLDDT . Inset shows PAE. C. Anc1 is shown in purple ribbon, substitutions in Anc2-7 are shown colored as in the key. K + , pink ball. IMP, gray sticks. MPA, olive sticks. These ligands were positioned by aligning Anc1 with the C. griseus E-XMP*•MPA complex (PDB: 1JR1, ).

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Sequencing

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Timelines for the changes in resistance (values of K iapp for MPA and RVP) and catalytic efficiency ( k cat /K IMP and k cat /K NAD ) for the resurrected ancestors. B. Ancestor substitutions near the MPA binding site. The structure of Anc1 (purple) was predicted using AlphaFold2 ( ; ). Substitutions in Anc2-7 are shown colored as in the key in panel A . K + , pink ball; IMP, gray sticks; MPA, olive sticks. These ligands were positioned by aligning Anc1 with the C. griseus E-XMP*•MPA complex (PDB: 1JR1, ). C-E. Timelines for the changes in resistance (values of K iapp for MPA and RVP) and catalytic efficiency ( k cat /K IMP and k cat /K NAD ) for the ancestors and their extant descendants. MPA producers are magenta, nonproducers are teal, ancestors are colored as in the key in panel A .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Linear free energy relationship between the values of k cat /K IMP and MPA resistance ( K iapp ). MPA S , K iapp ≤ 30 nM; MPA-moderately resistant (MPA M ), 1 μM ≥ K iapp > 30 nM; MPA R , K iapp > 1 μM. See Tables S1 and S2 for all values. B. Linear free energy relationship between k cat /K NAD and MPA resistance ( K iapp ). Key as in panel A . C. E•RVP mimics E-XMP*. The structures of E-XMP*•MPA (cornflower blue, PDB: 1JR1) and E•RVP•MAD (olive, PDB: 1NF7; MAD = mycophenolate adenine dinucleotide) are overlaid. E-XMP* is shown in cyan, MPA in spring green, RVP in magenta, and MAD in dark magenta. Figure rendered in UCSF Chimera .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques:

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: Enzyme concentrations are listed in Methods section. Reactions performed in duplicate at 0.5* K IMP and 1.8* K NAD and 1 mM RVP at 25°C unless otherwise noted. Anc6, Pani B and Pfre B were assayed at 18°C. Pcam B was assayed at 11.5°C. Gray denotes representative eukaryotic IMPDHs, magenta denotes enzymes from MPA producers, teal denotes enzymes from nonproducers and ancestors are colored as in - .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: