Journal: Military Medical Research

Article Title: USP18 exacerbates myocardial I/R injury by inhibiting Parkin mitophagy through the deubiquitinase PTEN-L

doi: 10.1016/j.mmr.2026.100004

Figure Lengend Snippet: Overexpression of USP18 in the heart exacerbates mitochondrial dysfunction, acute cardiac injury, and cardiac remodeling following I/R in mice. a TTC staining of heart tissue 24 h post-I/R in each group ( n= 5). Scale bar=0.5 cm. ⁎⁎⁎ P <0.001. b Mitochondrial DNA levels ( n= 6) and mitochondrial complexes I and II–III activity ( n= 5) 24 h post-I/R in each group. ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. c Representative electron microscopy images of heart sections 24 h post-I/R. Quantitative analysis of mitochondrial volume density and the percent of mitochondria with cristae loss in each group ( n= 5). Scale bar=10 μm (top) and 6 μm (bottom). Red arrows indicate mitochondria with cristae loss. ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. d Oxygen consumption rate (OCR) and quantitative statistical analysis of basal respiration, ATP-related respiration, maximal respiration, and spare respiratory capacity in mitochondria in the indicated groups ( n= 4). ⁎ P <0.05, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. e H&E staining, PSR staining, and quantitative statistical analysis of cell size and left ventricl e (LV) fibrotic area in AAV9-USP18-transfected mice at 4 weeks after I/R injury ( n= 5). Scale bar=1 mm (left), 50 μm (middle), and 100 μm (right). ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. f Representative B-mode and M-mode echocardiographic images of LV from AAV9-USP18-transfected mice 4 weeks after I/R injury. g Cardiac function of AAV9-USP18-transfected mice after I/R injury at the indicated time points ( n= 6). ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001 vs . AAV9-NC, ns non-significant. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; NC. Negative control; AAV9. Adeno-associated virus serotype 9; AAV9-USP18. Adeno-associated virus serotype 9 encoding USP18; TTC. 2,3,5-triphenyltetrazolium chloride; ATP. Adenosine triphosphate; H&E. Hematoxylin and eosin; PSR. picrosirius red; FCCP. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; LVIDd. Left ventricle internal diameter at diastole; LVISd. Left ventricle internal diameter at systole; LVEF. Left ventricle ejection fraction; LVFS. Left ventricle fractional shortening.

Article Snippet: To induce cardiac-specific USP18 overexpression, male C57BL/6 J mice received a single tail vein injection of adeno-associated virus serotype 9 (AAV9)-cardiac troponin T (cTnT)-USP18 or control AAV9-negative control [60–80 μl, (5.0–6.5)×1013 viral genome/ml, Vigene Bioscience, China] under 1.5%–2% isoflurane anesthesia.

Techniques: Over Expression, Staining, Activity Assay, Electron Microscopy, Transfection, Ubiquitin Proteomics, Negative Control, Virus

Journal: Military Medical Research

Article Title: USP18 exacerbates myocardial I/R injury by inhibiting Parkin mitophagy through the deubiquitinase PTEN-L

doi: 10.1016/j.mmr.2026.100004

Figure Lengend Snippet: Parkin knockdown counteracts the protection of USP18 deficiency in vivo. USP18-cKO mice were infected with AAV9-shParkin and subjected to I/R surgery. a Parkin protein levels in mouse hearts infected with AAV9-shParkin ( n= 4). b TTC staining of heart tissue 24 h post-I/R in each group ( n= 5). Scale bar=0.5 cm. c Serum levels of cTnI, CK-MB, and LDH in AAV9-shParkin-infected mice 4 h after I/R surgery ( n= 5). d DNA fragmentation and cleaved caspase-3 activity in heart tissue from AAV9-shParkin-infected mice 24 h after I/R injury ( n= 6). e Oxygen consumption rate (OCR) and quantitative statistical analysis of basal respiration, ATP-related respiration, maximal respiration, and spare respiratory capacity in mitochondria in the indicated groups ( n= 4). f Mitochondrial DNA ( n= 6) and complexes I and II–III activity ( n= 5) 24 h post-I/R in each group. g Representative electron microscopy images of heart sections 24 h post-I/R. The mitochondrial volume density and percent of mitochondria with cristae loss were measured in each group ( n= 5). Scale bar=10 μm (top) and 6 μm (bottom). h Protein levels of P62, ubiquitinated proteins (Ub), and LC3II in mitochondria from heart tissue 24 h after I/R injury ( n= 4). i H&E staining and quantitative statistical analysis of cell size ( n= 5) in USP18-cKO mice and AAV9-shParkin-infected mice 4 weeks after I/R injury. Scale bar=1 μm (top) and 50 μm (bottom). j Heart weight-to-tibia length ratio (HW/TL) ( n= 6) in each group. k Representative B-mode and M-mode echocardiographic images of the left ventricle from USP18-cKO mice and AAV9-shParkin-infected mice 4 weeks after I/R injury. l Cardiac function of USP18-cKO mice and AAV9-shParkin-infected mice 4 weeks after I/R injury ( n= 6). ⁎ P <0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; cTnI. Cardiac Troponin I; CK-MB. Creatine kinase-MB isoenzyme; LDH. Lactate dehydrogenase; NC. Negative control; AAV9. Adeno-associated virus serotype 9; AAV9-USP18. Adeno-associated virus serotype 9 encoding USP18; TTC. 2,3,5-triphenyltetrazolium chloride; ATP. Adenosine triphosphate; H&E. Hematoxylin and eosin; PSR. Picrosirius red; FCCP. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; LVIDd. Left ventricle internal diameter at diastole; LVISd. Left ventricle internal diameter at systole; LVEF. Left ventricle ejection fraction; LVFS. Left ventricle fractional shortening; HR. Heart rate.

Article Snippet: To induce cardiac-specific USP18 overexpression, male C57BL/6 J mice received a single tail vein injection of adeno-associated virus serotype 9 (AAV9)-cardiac troponin T (cTnT)-USP18 or control AAV9-negative control [60–80 μl, (5.0–6.5)×1013 viral genome/ml, Vigene Bioscience, China] under 1.5%–2% isoflurane anesthesia.

Techniques: Knockdown, In Vivo, Infection, Staining, Activity Assay, Electron Microscopy, Ubiquitin Proteomics, Negative Control, Virus

Journal: Military Medical Research

Article Title: USP18 exacerbates myocardial I/R injury by inhibiting Parkin mitophagy through the deubiquitinase PTEN-L

doi: 10.1016/j.mmr.2026.100004

Figure Lengend Snippet: USP18 inhibits mitophagy degradation and facilitates cardiac I/R injury through deubiquitinating and upregulating PTEN-L. a The protein levels of PTEN-L and PTEN in USP18-cKO mouse hearts 24 h after I/R injury ( n= 4). ⁎⁎⁎⁎ P <0.0001, ns non-significant. b The protein levels of PTEN-L and PTEN in AAV9-USP18-infected mouse hearts 24 h after I/R injury ( n= 4). ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001, ns non-significant. c Co-IP of USP18 and PTEN-L in NRVMs (left); NRVMs were transfected with HA-PTEN-L and Flag-USP18 (middle); Co-IP of Flag-USP18 and HA-PTEN-L in NRVMs (right). d Endogenous Co-IP of USP18 and PTEN-L in NRVMs subjected to H/R injury. e Endogenous Co-IP of USP18 and PTEN-L in hearts subjected to I/R injury. f PTEN-L ubiquitination (Ub) levels assessed by CO-IP in NRVMs subjected to H/R injury (top) and hearts subjected to I/R injury (bottom). g NRVMs were transfected with Ad-USP18 or USP18 siRNA or HA-PTEN-L and Myc-Ub and treated with MG132. Co-IP of Myc-Ub and HA-PTEN-L. h Schematic representations of t h e domains of PTEN-L involved in binding to USP18. Full-length PTEN-L or truncated PTEN-L was coexpressed with USP18 in HEK293T cells. Cells were subjected to immunoprecipitation with an anti-Myc antibody or an anti-HA antibody, followed by immunoblotting with the indicated antibodies. i Schematic representations of USP18 residues involved in binding to PTEN-L. Full-length USP18 or USP18 truncations were coexpressed with PTEN-L in HEK293T cells. Cells were subjected to immunoprecipitation with an anti-Myc antibody or an anti-HA antibody, followed by immunoblotting with the indicated antibodies. j Ub assay of PTEN-L in HEK293T cells cotransfected with Myc-USP18, Myc-USP18-mut, and Flag-PTEN-L and treated with 10 μmol/L MG132. k, l NRVMs were transfected with scRNA or USP18 siRNA ( k ), infected with Ad-NC or Ad-USP18 ( l ), and then treated with cycloheximide (CHX, 10 μmol/L) for the indicated time periods. Representative immunoblot analysis of PTEN-L protein levels in each group. ⁎⁎⁎⁎ P <0.0001 vs . scRNA or Ad-NC. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; H/R. Hypoxia-reoxygenation; PTEN. Phosphatase and tensin homolog; PTEN-L. Phosphatase and tensin homolog-long; AAV9. Adeno-associated virus serotype 9; NC. Negative control.

Article Snippet: To induce cardiac-specific USP18 overexpression, male C57BL/6 J mice received a single tail vein injection of adeno-associated virus serotype 9 (AAV9)-cardiac troponin T (cTnT)-USP18 or control AAV9-negative control [60–80 μl, (5.0–6.5)×1013 viral genome/ml, Vigene Bioscience, China] under 1.5%–2% isoflurane anesthesia.

Techniques: Infection, Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics, Binding Assay, Immunoprecipitation, Western Blot, Virus, Negative Control

Journal: Poultry Science

Article Title: Development of a recombinant chimeric Newcastle disease virus-vectored vaccine conferring single-dose, triple protection against genotype VII NDV, IBDV, and H9N2 AIV

doi: 10.1016/j.psj.2026.106993

Figure Lengend Snippet: Generation and characterization of the recombinant rLaS-VIIF/HN-HA-VP2. (A) Schematic representation of the construction strategy for rLaS-VIIF/HN-HA-VP2. The open reading frames (ORFs) of the H9N2 AIV HA gene and the very virulent IBDV VP2 gene, each flanked by the non-coding region (NCR) sequences of the NDV HN gene, were inserted between the P and M genes and the F and HN genes of the pNDFL-VII-F/HN plasmid, respectively, to generate the pNDFL-VII-F/HN-HA-VP2 construct. (B) Western blot analysis of rLaS-VIIF/HN-HA-VP2 expression. Allantoic fluid harvested from embryonated eggs infected with rLaS-VIIF/HN-HA-VP2 was subjected to immunoblotting using specific antisera. Three specific bands corresponding to the uncleaved HA precursor (HA0, ∼62 kDa) and the cleaved subunits HA1 (∼47 kDa) and HA2 (∼25 kDa) were detected. Additionally, a specific band corresponding to the VP2 protein (∼42 kDa) was detected in the rLaS-VIIF/HN-HA-VP2 samples.

Article Snippet: Antibodies against IBDV were measured using the ProFLOK PLUS Infectious Bursal Disease Virus (IBD) Antibody Test Kit (Zoetis, USA) according to the manufacturer's instructions.

Techniques: Recombinant, Plasmid Preparation, Construct, Western Blot, Expressing, Infection

Journal: Poultry Science

Article Title: Development of a recombinant chimeric Newcastle disease virus-vectored vaccine conferring single-dose, triple protection against genotype VII NDV, IBDV, and H9N2 AIV

doi: 10.1016/j.psj.2026.106993

Figure Lengend Snippet: Humoral immune responses in SPF chickens immunized with rLaS-VIIF/HN-HA-VP2 and rLaS-VIIF/HN vaccines. Chickens were immunized via the ocular and nasal routes (100 µL per route) with a total of 200 µL containing 10⁶ EID₅₀ of rLaS-VIIF/HN-HA-VP2 or parental virus rLaS-VIIF/HN. Sera samples were collected before immunization and at day 10 and 20 post-immunization. (A) Antibodies against IBDV were measured using a commercial ELISA kit. (B, C) Antibodies against genotype VII NDV and H9N2 AIV were measured by hemagglutination inhibition (HI) test, respectively. Data are presented as mean ± SD; significance is denoted as * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies against IBDV were measured using the ProFLOK PLUS Infectious Bursal Disease Virus (IBD) Antibody Test Kit (Zoetis, USA) according to the manufacturer's instructions.

Techniques: Vaccines, Virus, Enzyme-linked Immunosorbent Assay, HI Assay

Journal: Poultry Science

Article Title: Development of a recombinant chimeric Newcastle disease virus-vectored vaccine conferring single-dose, triple protection against genotype VII NDV, IBDV, and H9N2 AIV

doi: 10.1016/j.psj.2026.106993

Figure Lengend Snippet: Protective efficacy of the rLaS‑VIIF/HN‑HA-VP2 vaccine against challenge with a very virulent IBDV strain. (A) Survival rates of chickens challenged with IBDV and monitored daily for 7 days post-challenge (dpc; n = 10 per group). (B) Representative gross lesions in the bursa of Fabricius at necropsy (7 dpc). Severe atrophy, accompanied by internal hemorrhage and necrosis, was evident in the bursae of birds in the rLaS‑VIIF/HN control group. In contrast, no notable gross pathological changes were observed in the rLaS‑VIIF/HN‑HA-VP2 immunized group or the uninfected controls. (C) Bursa-to-body weight index (BBIX) in immunized chickens following IBDV challenge. The mean BBIX value was significantly higher in the rLaS‑VIIF/HN‑HA-VP2 immunized group compared to the rLaS‑VIIF/HN control group (****, P < 0.0001). (D) Representative histopathological lesions in the bursa of Fabricius at 7 dpc (H&E stain; original magnification, 400 ×). Bursal tissue from birds in the rLaS‑VIIF/HN control group exhibited severe lymphoid follicle atrophy and lymphoid cell degeneration. No significant histopathological changes were observed in the rLaS‑VIIF/HN‑HA-VP2 immunized group or the uninfected controls.

Article Snippet: Antibodies against IBDV were measured using the ProFLOK PLUS Infectious Bursal Disease Virus (IBD) Antibody Test Kit (Zoetis, USA) according to the manufacturer's instructions.

Techniques: Control, Staining