PPARγ) and neuropeptide-Y (NPY) expression was reduced to a greater extent in the NC line than the RBC2 line. At 33°C, expression of PPARγ, NPY, and CCAAT/enhancer-binding protein-β (C/EBPβ) was upregulated, but only in the RBC2 line. During differentiation, both lines showed greater changes in lipid accumulation and in C/EBPβ and NPY expression if the thermal challenge was initiated during proliferation. These data suggest that adipogenic gene expression is more responsive to thermal challenge in proliferating SCs than in differentiating SCs, and that growth-selection has increased temperature sensitivity of SCs, which may significantly affect breast muscle structure and composition.As multipotential stem cells, satellite cells (SCs) have the potential to express adipogenic genes resulting in lipid synthesis with thermal stress. The present study determined the effect of temperature on intracellular lipid synthesis and adipogenic gene expression in SCs isolated from the pectoralis major (p. major) muscle of 7-day-old fast-growing modern commercial (NC) turkeys compared to SCs from unselected slower-growing turkeys [Randombred Control Line 2 (RBC2)]. Since proliferating and differentiating SCs have different responses to thermal stress, three incubation strategies were used: (1) SCs proliferated at the control temperature of 38°C and differentiated at 43° or 33°C; (2) SCs proliferated at 43° or 33°C and differentiated at 38°C; or (3) SCs both proliferated and differentiated at 43°, 38°, or 33°C. During proliferation, lipid accumulation increased at 43°C and decreased at 33°C with the NC line showing greater variation than the RBC2 line. During proliferation at 43°C, peroxisome proliferator-activated receptor-γ ( In vitro studies with cultured pmSCs from both chickens (Satellite cells (SCs) are a heterogenous stem cell population , functiochickens  and turkchickens  have demPPARγ)   and somePPARγ)  as well PPARγ) . The pro pathway . The expntiation . The PPAyoblasts  and pre-yoblasts . After bt muscle . Furthert muscle  and turkt muscle  through  factors  consists factors . Some me factors  and KLF7 factors  are mainNPY promotes lipid deposition by promoting adipogenesis , functions as an extracellular signal regulating adipogenesis by interacting with NPY receptors . The expogenesis  and inhiogenesis . At the ntiation . With heey pmSCs . Becausepression , it may Birds are homotherms and maintain their body temperature in a limited range , 2015. FFaster-growing chickens produce more metabolic heat than shower-growing lines because of a higher metabolic rate . In addiin vitro. These findings have potential application in the development of management strategies to control lipid production by pmSCs which has been reported to affect the fiber organization and cellular properties of the breast muscle  turkeys compared to those from Randombred Control Line 2 (RBC2) turkeys. The commercial turkeys used in this study were selected for growth characteristics including high growth rate and heavy breast muscling, whereas the RBC2 turkeys represent commercial turkeys in the mid-1960s  and are t muscle .Satellite cells were isolated from the p. major muscle of 7-day-old RBC2 turkeys and 7-day-old NC turkeys based on the method of Three different experimental strategies were used in the present study: (1) pmSCs proliferated at 38°C (control) and differentiated at 43° or 33°C; (2) pmSCs proliferated at 43° or 33°C and differentiated at 38°C; or (3) pmSCs both proliferated and differentiated at 43°, 38°, or 33°C . Cell cu2PO4, and 7.81 mM of Na2HPO4, was used to rinse the plate twice. Third, another 1 mL of PBS containing 30 μL of AdipoRed was added to each well. One well with only 1 mL PBS without AdipoRed was used as a negative control. After incubating for 15 min, the AdipoRed levels were quantified based on the fluorescence absorbance at a wavelength of 485 nm in a plate reader . The AdipoRed assay was repeated in three independent cultures with four wells per treatment group per cell line.AdipoRed fluorochrome  was used to quantify intracellular lipid content in pmSCs every 24 h during 72 h of proliferation and every 24 h during 72 h of differentiation according to the manufacturer’s procedure. One plate from each treatment group was collected and was assayed at each sampling time. First, the medium was removed from each plate. Second, phosphate buffered saline (PBS) (pH 7.08), which contained 137 mM of NaCl, 2.68 mM of KCl,1.47 mM of KHTM ND-1000, Thermo Fisher Scientific). Reverse transcription was performed using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV)  to produce cDNA from total RNA. Real-Time Quantitative PCR (RT-qPCR) was conducted using DyNAmo Hot Start SYBR Green qPCR kit (Thermo Fisher Scientific) to quantify the expression of C/EBPβ, KLF1, KLF7, NPY, and PPARγ. Primers for KLF1 and KLF7 were designed using primer-BLAST tool on the website of National Center for Biotechnology Information. The amplification specificity of each primer was validated by sequencing the PCR product. Primers for C/EBPβ, NPY, PPARγ, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  were previously designed and specificity confirmed . Main effect of temperature, main effect of cell line, interaction effect between temperature and cell line, and random effect of repeat experiment were included in this model. The statement of Least Square Means in the MIXED procedure was used to determine each mean value and the standard error of the mean (SEM). Differences between each mean were separated with the Pdiff option. Line effect within each treatment group and temperature effect within each cell line was determined with the SLICE option at each sampling time. For the AdipoRed assay, the REG procedure was used to evaluate the linear relationship between sampling times and optical densities of AdipoRed per cell line per treatment group. Difference in the linear relationship was determined with the Contrast statement. P < 0.001) and 43°C (P = 0.003) than the RBC2 line. At 24 h, the NC line showed a significant increase (P < 0.001) in lipid content at 43°C than at 38°C. At 48 and 72 h, lipid content of both the RBC2 (P ≤ 0.001) and NC (P < 0.001) lines was greater at 43°C than at 38°C. Lipid content of the RBC2 and NC lines increased 6.35-fold (P < 0.001) and 4.73-fold (P < 0.001) at 43°C than at 38°C at 72 h of proliferation, respectively. A significant interaction effect between temperature and cell line was observed at 24 h (P = 0.002) and 72 h (P = 0.041).The effect of heat stress (43°C) on intracellular lipid accumulation in pmSCs from the RBC2 and NC turkeys was measured every 24 h during 72 h of proliferation . Lipid cP < 0.001) at 24 and 72 h than at 0 h at 33°C. At 48 and 72 h, both lines had lower lipid content (P < 0.001) at 33°C than at 38°C. Lipid content in the RBC2 and NC lines decreased 4.00-fold (P = 0.003) and 11.83-fold (P < 0.001) at 33°C compared to 38°C at 72 h of proliferation. There was a significant interaction effect between temperature and cell line at 24 h (P = 0.014), 48 h (P = 0.004), and 72 h (P < 0.001).The effect of cold stress (33°C) on intracellular lipid accumulation in pmSCs from both lines was measured every 24 h during 72 h of proliferation . Lipid cP = 0.007) than the 38°→38°C group, and no significant change in lipid content occurred in the RBC2 line (P = 0.329). Lipid content in the RBC2 and NC lines was 2.21-fold (P < 0.001) and 2.36-fold (P < 0.001) higher in the 43°→38°C group than the 38°→38°C group at 24 h. In the 43°→43°C group at 24 h, lipid content was 1.60-fold (P = 0.002) and 1.42-fold (P < 0.001) higher in the RBC2 and NC lines than the 38°→38°C group. Line effect was significant in the 38°→38°C (P = 0.005), 43°→38°C (P < 0.001), and 43°→43°C (P = 0.002) groups at 24 h. A significant interaction effect between temperature and cell line was observed at 24 h (P < 0.001). At 48 h, no significant difference in lipid content was observed in either line (P > 0.118) between the 38°→43°C and 38°→38°C groups. Lipid content was greater in the 43°→38°C group in both lines (P < 0.001) than the 38°→38°C group at 48 h. Greater lipid content was observed only in the NC line (P = 0.002) in the 43°→43°C group than the 38°→38°C group at 48 h. A significant line effect was observed in the 43°→38°C (P < 0.001) and 43°→43°C (P = 0.017) groups at 48 h. An interaction effect between temperature and cell line was not significant at 48 h (P = 0.179). At 72 h, no significant difference in lipid content was observed in either line in 38°→43°C (P > 0.643) and 43°→43°C (P > 0.154) groups than the 38°→38°C group. A slightly greater amount of lipid  was observed only in the RBC2 line in the 43°→38°C group than the 38°→38°C group at 72 h. Line effect was significant in the 38°→38°C (P = 0.005) and 38°→43°C (P = 0.003) groups at 72 h. No significant interaction effect was observed between temperature and cell line at 72 h (P = 0.698). From 24 to 72 h, a linear decrease in lipid content was observed in both lines in the 38°→38°C, 43°→38°C and 43°→43°C groups. In the 38°→38°C group, the NC line had a steeper linear reduction in lipid content than the RBC2 line . Lipid content in the NC line showed greater reduction than the RBC2 line in the 43°→38°C group . In the 43°→43°C group, both lines had a linear decrease in lipid content from 24 to 72 h , and there was no line difference (P = 0.067).A heat stress (43°C) was administrated during the 72 h of proliferation and/or during the 72 h of differentiation. Treatment groups were 38°→43°C, 43°→38°C, and 43°→43°C, and these groups were compared to the 38°→38°C control group. Lipid content in pmSCs from both the RBC2 and NC lines was measured at 24, 48, and 72 h of differentiation . At 24 hP = 0.035) than the 38°→38°C group. Lipid content in the RBC2 and NC lines was 2.75-fold (P < 0.001) and 5.44-fold (P < 0.001) lower in the 33°→38°C group than the 38°→38°C group at 24 h. In the 33°→33°C group, lipid content was 2.80-fold (P = 0.002) and 6.13-fold (P < 0.001) lower in RBC2 and NC lines than the 38°→38°C group at 24 h. Line effects were significant in the 38°→38°C (P < 0.001) and 38°→33°C (P < 0.001) groups at 24 h. An interaction effect between temperature and cell line was significant at 24 h (P < 0.001). At 48 h, a greater amount of lipid was measured only in the NC line  in the 38°→33°C group than the 38°→38°C group. Lipid content in the RBC2 and NC lines was 2.36-fold (P < 0.001) and 2.95-fold (P < 0.001) lower in the 33°→38°C group than the 38°→38°C group at 48 h. In the 33°→33°C group, lipid content was 2.51-fold (P = 0.002) and 3.20-fold (P < 0.001) lower in RBC2 and NC lines than the 38°→38°C group at 48 h. A significant line effect occurred in the 38°→38°C (P < 0.001) and 38°→33°C (P < 0.001) groups at 48 h. There was an interaction effect between temperature and cell line at 48 h (P < 0.001). At 72 h, a slightly greater amount of lipid content was observed only in the RBC2 line in the 38°→33°C group  than the 38°→38°C group. Lipid content in the RBC2 and NC lines was 2.66-fold (P < 0.001) and 3.00-fold (P < 0.001) lower in the 33°→38°C group than the 38°→38°C group at 72 h. In the 33°→33°C group, lipid content was 3.48-fold (P = 0.002) and 3.56-fold (P < 0.001) lower in RBC2 and NC lines than the 38°→38°C group at 72 h. A line effect was significant in the 38°→38°C (P = 0.001) and 38°→33°C (P < 0.001) groups at 72 h. There was an interaction between the effect of temperature and cell line at 72 h (P < 0.001). From 24 to 72 h, the amount of lipid content in both lines was very low throughout the duration of the experiment in the 38°→33°C, 33°→38°C, and 33°→33°C groups. A minor linear reduction in lipid content was only observed in the NC line (slope: −5.06) in the 38°→33°C group from 24 to 72 h.A cold stress (33°C) was administrated during the 72 h of proliferation and/or during the 72 h of differentiation. Treatment groups were 38°→33°C, 33°→38°C, and 33°→33°C, and these groups were compared to the 38°→38°C control group. Lipid content in pmSCs from both lines was measured at 24, 48, and 72 h of differentiation . At 24 hPPARγ expression was lower (P < 0.010) in the NC line than the RBC2 line at 38°C (PPARγ at 43°C was decreased 4.06-fold (P < 0.001) and 2.32-fold (P < 0.001) in the NC and RBC2 lines, respectively, compared to 38°C at 72 h of proliferation (PPARγ was increased 2.02-fold (P < 0.001) and 1.88-fold (P = 0.021) in both the RBC2 and NC lines compared to the 38°C at 72 h of proliferation  in the 38°→43°C group than the 38°→38°C group  and NC  lines showed a lower expression of PPARγ in the 43°→43°C group than the 38°→38°C group  and NC  lines in the 38°→33°C group than the 38°→38°C group  and 33°→33°C  groups than the 38°→38°C group at 48 h of differentiation  in the NC line than the RBC2 line at 38°C (C/EBPβ was reduced 2.23-fold in the NC line (P = 0.003) whereas no difference (P = 0.593) was observed in the RBC2 line at 43°C compared to the 38°C at 72 h of proliferation  compared to the 38°C treatment at 72 h of proliferation, and no significant change (P = 0.603) was observed in the NC line (P = 0.006) and 33°C  than the RBC2 line in the 38°→38°C group  than the 38°→38°C group  and 43°→43°C  groups than the 38°→38°C group at 48 h of differentiation  and 33°→33°C  groups than the 38°→38°C group  than the 38°→38°C group  in the NC line than the RBC2 line at 38°C  and NC  lines at 43°C compared to the 38°C at 72 h of proliferation (NPY was increased 2.67-fold (P < 0.001) in the RBC2 line while no significant change (P = 0.056) was observed in the NC line compared to the 38°C at 72 h of proliferation (P = 0.003) and 33°C  than the RBC2 line in the 38°→38°C group , 43°→38°C , and 43°→43°C  groups than the 38°→38°C group  only in the 43°→43°C group than the 38°→38°C group (NPY expression was lower in both the RBC2 (P < 0.001) and NC (P < 0.001) lines in the 38°→33°C , 33°→38°C , and 33°→33°C  groups than the 38°→38°C group  than the NC line at 38°C  line at 43°C compared to 38°C  while no significant (P = 0.659) difference was observed in the NC line compared to the 38°C at 72 h of proliferation (KLF1 expression between the two cell lines (P = 0.473) was observed in the 38°→38°C group  than the 38°→38°C group  in the 43°→43°C group than the 38°→38°C group  and 33°→38°C  groups than the 38°→33°C group (P < 0.001) and NC  lines had higher expression of KLF1 than the 38°→33°C group  than the RBC2 line at 38°C (KLF7 expression (P = 0.434) in SCs from both lines at 72 h of proliferation  but not in the NC line (P = 0.175) at 72 h of proliferation  was observed between the two cell lines in the 38°→38°C group  and 43°→43°C  groups than the 38°→38°C group  and the NC  lines in the 43°→38°C group than the 38°→38°C group  and 33°→38°C  groups than the 38°→38°C group  but decreased in the RBC2 line  than the 38°→38°C group , expression of NPY and C/EBPβ increased in both lines compared to other treatment groups , and the NC line showed the highest increase. Taken together with the results by NPY and C/EBPβ than the RBC2 line in the control group.During myogenic differentiation, most SCs are committed to a myogenic pathway, as reflected in the increased myogenic differentiation and enhanced expression of myogenic genes as previously shown . This maIn addition to the intrinsic difference in adipogenic potential, selection for growth may have altered the sensitivity of turkey breast muscle to temperature stimuli. Faster-growing poultry lines tend to have larger diameter myofibers from excessive hypertrophic SC-mediated muscle growth in the breast muscle than slower-growing lines . As an aNPY and PPARγ expression was downregulated in the NC line during proliferation than the RBC2 line at the control temperature, and the expression of NPY and PPARγ was decreased with heat treatment during proliferation in both lines. A reduction in PPARγ and C/EBPβ expression in the heat-treated proliferating pmSCs was observed by NPY, PPARγ, and C/EBPα expression in both abdominal and subcutaneous adipose tissue . Thus, when intracellular lipid was decreased with cold stress, pmSCs from the NC line may have difficulty regulating the lipid loss. This may explain why the cold-treated pmSCs from the NC line had a greater reduction in intracellular lipid than the RBC2 line during proliferation.Adipogenic regulatory genes mediate the response to a thermal stress. The expression of both e tissue . Furthernt model . In the  in mice  and chic in mice . In turkin vivo, resulting in prolonged effects on turkey breast muscle structure and composition. Future studies will need to focus on defining the signal transduction pathways involved in the regulation of adipogenesis and their response to heat and cold stress.In summary, the current study showed that thermal stress alters the adipogenic potential of turkey pmSCs at 7-days of age. During pmSC proliferation, hot temperatures increased the intracellular lipid content in pmSCs while cold temperatures had the opposite effect. Proliferating pmSCs were more sensitive to both heat and cold stress in the determination of intracellular lipid content than differentiating pmSCs. The pmSCs from the faster-growing NC turkeys showed a higher lipid synthesis potential and their cellular fate was more sensitive to both heat and cold stress than the slower-growing RBC2 turkeys. The increased intracellular lipid accumulation of the heat-treated pmSCs may eventually promote intramuscular fat deposition The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the animal study because the study did not use any birds. Cell lines used in the study were previously isolated. Only satellite cells lines were used.All authors have made a substantial and intellectual contribution to the work, and approved the submitted article. SV conceived and designed the experiments with GS and KR. JX performed the experiments and statistical analysis, and wrote the manuscript. SV, GS, and KR revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, only the anti-L1-16 antibodies were associated with the high-grade lesions and cervical cancer (CIN3/CC) group (OR = 12.18). In conclusion, our results suggest that anti-VLPs-16/18 antibodies are effective and type-specific to detect HPV-vaccinated women, but anti-L1-16 antibodies better differentiate the CIN3/CC group. However, a larger population study is needed to validate these results.Antibodies against the Human Papillomavirus (HPV) L1 protein are associated with past infections and related to the evolution of the disease, whereas antibodies against L1 Virus-Like Particles (VLPs) are used to follow the neutralizing antibody response in vaccinated women. In this study, serum antibodies against conformational (VLPs) and linear epitopes of HPV16/18 L1 protein were assessed to distinguish HPV-vaccinated women from those naturally infected or those with uterine cervical lesions. The VLPs-16/18 were generated in baculovirus, and L1 proteins were obtained from denatured VLPs. Serum antibodies against VLPs and L1 proteins were evaluated by ELISA. The ELISA-VLPs and ELISA-L1 16/18 assays were validated with a vaccinated women group by ROC analysis and the regression analysis to distinguish the different populations of female patients. The anti-VLPs-16/18 and anti-L1-16/18 antibodies effectively detect vaccinated women . The regression analysis showed that anti-VLPs-16/18 and anti-L1-16/18 antibodies were associated with the vaccinated group (OR = 2.11 × 10 Cervical cancer (CC) is the fourth-most common cancer worldwide . In deveThe use of neutralization assays such as the PBNA  is considered the gold standard to evaluate the protective potential of the antibodies induced by the HPV vaccines . HoweverAt first, the L1 protein was generated as a denatured protein in bacteria and used in an ELISA (Enzyme-Linked Immunosorbent Assay) to measure the anti-L1 antibodies. More recently, the Luminex immune assay has been used to evaluate several anti-L1 HPV types. Based on the results from these systems, several groups have reported a high prevalence of these antibodies in patients with low-grade lesions (~60%), which increases with the severity of the uterine cervical lesion ,17,18. AIn this study, we used a direct ELISA to determine the serum antibody levels against linear (L1) and conformational (VLPs) epitopes from HPV16 and HPV18 (HPV16/18) in women with uterine cervical lesions or HPV-vaccinated. In turn, we also evaluated the usefulness of these anti-VLPs and anti-L1 HPV16/18 antibodies as markers to distinguish different uterine cervical lesions, as well as to differentiate from HPV-vaccinated populations . Our results showed that the anti-VLPs antibodies 16/18 are type-specific and highly effective to detect vaccinated women, whereas anti-L1-16 antibodies were better to differentiate women with CIN3/CC from the study population. Our results are promising, but to validate the performance of these anti-L1-16 antibodies to differentiate high-grade lesions and CC from the general population, a larger female study group with different HPV exposure is needed.n = 68), who had a negative Papanicolaou (Pap) test; (2) women with different degrees of cervical lesions and cervical cancer (n = 62), diagnosed by histopathology ; (3) HPV-vaccinated women (n = 36). In the HPV-vaccinated group, 44.4% were immunized with the bivalent vaccine (n = 16), and 55.6% received the quadrivalent vaccine (n = 20). This group also self-reported a normal Pap test in the last year and no history of previous uterine cervical lesions.This is a retrospective study that used samples from three different serum banks corresponding to naturally infected women, women with uterine cervical lesions, and HPV-vaccinated women to characterize the antibody response against HPV16 and HPV18 VLPs  and denatured L1 (linear epitopes). The women included in the study who may have been exposed to HPV were divided into 3 groups: (1) normal adult women  from a serum bank were used to determine the cut-off points of the HPV16 and HPV18 ELISAs (VLPs and denatured L1 antigens).The HPV DNA typing was performed by polymerase chain reaction (PCR) amplification on biopsies from patients, as described elsewhere . BiologiThis research was conducted following the Declaration of Helsinki, and the protocol was evaluated and approved by the Ethical Committee at the National Institute of Public Health, Mexico (Projects No. 781 and CI 1518).7 High Five cells/mL were incubated for 1 h at 27 °C with 9 MOI (Multiplicity of Infection) of BaCU-L1-16 or 5 MOI of BaCU-L1-18. The cells were diluted with fresh Express FiveTM -SFM medium  at 1 × 106 cells/ mL and left to incubate with shaking for 72 h. Finally, the cells were harvested by centrifugation at 154× g for 5 min and washed with cold PBS  to proceed with lysis and purification through a cesium chloride (CsCl) gradient, as described elsewhere [Baculovirus HPV18-L1 (BaCU-L1-18) to produce VLPs was generated using the Bac-to-Bac Baculovirus Expression System  by PCR amplification of the HPV18-L1 gene 1728 bp). Baculovirus HPV16-L1 (BaCU-L1-16) was provided by Dr. Martin Müller . To produce VLPs, 1 × 10 bp. BacuThe CsCl gradient fractions were examined for the presence of L1 protein by 10% sodium dodecyl sulfate-polyacrylamate gel electrophoresis (SDS-PAGE) and then either Coomassie-stained to verify the presence and purity of the L1 protein from HPV16/18 or treated for Western blot to corroborate the identity of L1 proteins in the preparations . ProteinAliquots of gradient fractions containing the VLPs from HPV16 or 18 were adsorbed onto copper grids covered with formvar-carbon for 1 min, contrasted with uranyl acetate for 1 min, and the excess stain removed. The grids let to air-dry for 5 min and samples analyzed in a Carl Zeiss Libra 120 Transmission Electron Microscope (TEM)  in search for VLP structures and microphotographs taken to evaluate the size and shape. The TEM preparations from CsCl fractions contained adequately structured and sized (~53 nm) VLPs from HPV16 or HPV18 . Denatur2O2 in 0.1-M sodium acetate, pH 6.0) for 30 min at room temperature. The reaction was stopped with 1 M H2SO4 and read at 450 nm.The ELISA assay was carried out with complete VLPs (diluted in PBS) to look for antibodies to conformational epitopes. In parallel, an ELISA-L1 was carried out to look for antibodies to linear L1 epitopes (VLPs irreversibly denatured with 0.2-M carbonate buffer) . ELISA pThe ELISA-VLPs 16/18 assay specificity was tested with a set of sera-positive for antibodies against VLPs 16 or 18 from women HPV DNA-positive for only one of the HPV types and negative against L1 protein by Western blot (denatured conditions). No antibody crossreaction between these two ELISA-VLPs 16/18 assays was observed when sera positive for anti-VLPs-16 was tested with VLPs-18 and vice versa.For the ELISA-L1 16/18 assay specificity, we used the K1H8 monoclonal antibody (mAb) , which recognizes a linear L1 epitope from several HPV types, including 16 and 18, by Western blot and in the ELISA-L1 but does not recognize the conformational epitopes in the ELISA-VLPs. We used this mAb as a positive control to detect the L1 protein in ELISA-L1 and as a negative control for ELISA-VLPs, and this also allows us to evaluate the presence of the L1 protein in the VLP preparations. The anti-L1 antibody crossreaction (10–20%) to linear epitopes between different HPV types have been reported , and thiThe sensitivity and specificity of the ELISA-VLPs and ELISA-L1 HPV16/18 systems, set up in our laboratory, were validated by the receiver operating characteristic curve (ROC) analysis by using sera from HPV-vaccinated women , and forThe antibody titers in the ELISA system were calculated by using a four-parameter logistic equation from IVD Tools AssayFit V1.6 (Excell compatible) and expressed as ELISA units (EU)/mL. The reference curves for the anti-VLPs and anti-L1 for HPV16/18 antibodies were prepared as described by Harper et al. . BrieflyThe statistical analysis was performed with the STATA 13.0 software . Dispersion of the anti-VLPs and anti-L1 antibody levels was carried out by boxplot analyses of the different groups of females studied, and the difference between the medians (M) was calculated with Kruskal–Wallis and adjusted with the Dunnet test. Sensitivity, specificity, and areas under the ROC curve (AUC) for the two types of antibodies were calculated in the group of vaccinated women to validate the performance of the ELISA-VLPs and ELISA-L1 assays used in this study. Logistic regression analysis was used to estimate the odds ratio (OR) and 95% CIs for the association of the anti-L1 and anti-VLPs antibodies with precancerous lesions, CC, and vaccinated women, as well as with different risk factors for CC.n = 68) were from adult women that reported normal Pap tests ; 40.8% (n = 62) were from women with different cervical lesions ; and 21.6% (n = 36) were from women who received the complete three-dose schedule of an HPV vaccine , although 77.8% of the vaccinated women were in the range of 18–27 years of age, corresponding to the main age group that has received HPV vaccination in Mexico. Most of the vaccinated women were single (77.8%) and had an educational level of high school or more (100%); 41.7% were smokers. In the case of the NL women group and women that presented cervical lesions, most of them reported being married or having a steady partner  and have a basic educational level (>75%); the majority were nonsmokers (>85%). Regarding the sexual behavior characteristics, 72.2% of the vaccinated women reported having less than seven years of sexual activity, and 50% reported having had at least two sexual partners in the last year. In contrast, the majority of the NL group and the women with lesions reported having more than 18 years of sexual activity , but the number of sexual partners reported in the last year was different for these two groups . The HPVp < 0.0001) (p < 0.05)  than the NL group (p < 0.05)  and anti-L1-18 antibody levels (M = 193.9 EU/mL) than the NL group  (p ≤ 0.001) and, to a lesser extent, with the CIN3/CC group , albeit without statistical significance (p = 0.032), whereas smoking was associated with anti-VLPs-16 (OR = 3.99) and anti-VLPs-18 antibodies (OR = 3.88), and this was statistically significant   and anti-L1 (linear epitopes) antibodies, a logistic regression analysis with these antibodies and other behavioral characteristics was carried out . The anaificance . On the ctively) .p = 0.000). Besides, an association between these antibodies and the group of women with CIN3/CC was only observed with anti-L1-16 (OR = 12.18), and this was statistically significant (p = 0.023) (p = 0.032). In the case of women that smoked, high associations were observed for anti-L1-16 and -18 antibodies , and those were highly significant  ; these associations were statistically significant (= 0.023) . The assctively) .Since a positive association was observed between anti-L1-16 antibodies and the CIN3/CC group, we carried out a ROC analysis by using the regression model for the HPV-exposed women groups only (not considering HPV-vaccinated women) to evaluate the performances of the anti-L1-16 antibodies to distinguish the cases from the general population. The results showed that the anti-L1-16 antibodies presented a moderate performance to distinguish the CIN3/CC group (AUC = 0.63) with high specificity (98.5%) and low sensitivity (15.4%), but the assay correctly classified 93.5% of the subjects (data not shown). The low sensitivity of the anti-L1-16 assay could be due to the small number of subjects in the group and because the analysis was not restricted to HPV DNA status.It was not possible to carry out the association analysis for the different 16/18 antibodies with the variables of age and time of sexual activity, because the youngest age group (18–27 years old) was used as the reference group for the analysis, and 72.8% of the HPV-vaccinated women corresponded to this group. In this study, we aim to understand why women with persistent HPV infection progressed to CC, even though they generated antibodies against the L1 protein, and if any of these antibodies could be useful as markers of stage disease. To achieve this, we looked for antibodies against conformational epitopes (VLPs) as an indirect measure of neutralizing activity, as has been previously demonstrated by Dessy et al. , and forFrom different studies, contradictory results have been reported when anti-VLPs and anti-L1 antibodies were evaluated in different populations. For instance, in the Wang SS and colleagues study , it was Moreover, our combined ELISA-VLP and –L1 assays also showed a differential antibody response against conformational and linear epitopes , as the antibody levels against HPV16/18 VLPs were higher  among vaccinated women than among naturally infected women, and this is in agreement with what has been reported previously for anti-VLPs antibodies . HoweverThe performance of anti-VLPs antibodies to detect uterine cervical lesions has been analyzed previously. Coseo and colleagues  reportedAnother explanation for the discrepancy with the Coseo results  could beOn the other hand, it has been reported that lower levels of anti-VLPs-18 antibodies compared to anti-VLPs-16 antibodies are commonly found among vaccinated women . This loThere are still limitations for the ELISA test, especially regarding its sensitivity to detect VLPs or L1 antigens. This is partly because the seroconversion period during a natural HPV infection differs between women (from six to more than 12 months), and the antibody titers do as well. Several research groups have identified a heterogeneous anti-VLPs and anti-L1 antibody prevalence in different female populations. For instance, by using and ELISA-VLPs for HPV16, the prevalence range was from 7% to 43% in healthy women, from 25% to 68% among women with CIN lesions, and from 28% to 100% among women with CC ,21,23,40Therefore, a standardized procedure to carried out the ELISA assays for VLPs and L1 needs to be established to correctly evaluate the protective immune responses of the HPV vaccine  and, also, to clarify the association of the anti-L1-16 antibodies as surrogate markers of high-grade uterine cervical lesions.Overall, our results suggest that the anti-VLPs 16/18 antibodies are highly effective to detect vaccinated women , but the anti-L1 16 antibodies are better markers to distinguish women with CIN3/CC in the general population. However, to validate the performance of this ELISA-L1 for HPV16, the HPV status of the women should be included, and a larger population study should be carried out.


Programmed death-ligand 1 (PD-L1) regulates immune homeostasis by promoting T-cell exhaustion. It is involved in chronic infections and tumor progression. Nuclear imaging using radiolabeled anti-PD-L1 antibodies can monitor PD-L1 tissue expression and antibody distribution. However, physiological PD-L1 can cause rapid antibody clearance from blood at imaging doses. Therefore, we hypothesized that inflammatory responses, which can induce PD-L1 expression, affect anti-PD-L1 antibody distribution. Here, we investigated the effects of three different infectious stimuli on the pharmacokinetics and tumor targeting of radiolabeled anti-PD-L1 antibodies in tumor-bearing mice.111In]In-DTPA-anti-mPD-L1 and [111In]In-DTPA-IgG2a, respectively). We evaluated the effect of inflammatory responses on the pharmacokinetics of [111In]In-DTPA-anti-mPD-L1 in RenCa tumor-bearing BALB/c mice in three conditions: lipopolysaccharide (LPS), local Staphylococcus aureus, and heat-killed Candida albicans. After intravenous injection of 30 or 100 µg of [111In]In-DTPA-anti-mPD-L1 or [111In]In-DTPA-IgG2a, blood samples were collected 1, 4, and 24 h p.i. followed by microSPECT/CT and ex vivo biodistribution analyses. PD-L1 expression, neutrophil, and macrophage infiltration in relevant tissues were evaluated immunohistochemically.Anti-mouse-PD-L1 and isotype control antibodies were labelled with indium-111 , resulting in lower blood levels  and reduced tumor targeting . Local S. aureus infections showed high PD-L1+ neutrophil influx resulting in significantly increased [111In]In-DTPA-anti-mPD-L1 uptake in affected muscles . Heat-killed Candida albicans  challenge did not affect pharmacokinetics. Increasing [111In]In-DTPA-anti-mPD-L1 dose to 100 µg normalized blood clearance and tumor uptake in LPS-challenged mice, although lymphoid organ uptake remained higher. Infectious stimuli did not affect [111In]In-DTPA-IgG2a pharmacokinetics.In 30 µg of [This study shows that anti-PD-L1 antibody pharmacokinetics and tumor targeting can be significantly altered by severe inflammatory responses, which can be compensated for by increasing the tracer dose. This has implications for developing clinical PD-L1 imaging protocols in onco-immunology. We further demonstrate that radiolabeled anti-PD-L1 antibodies can be used to evaluate PD-L1 expression changes in a range of infectious diseases. This supports the exploration of using these techniques to assess hosts’ responses to infectious stimuli. Immune checkpoint molecules play a vital role in immune homeostasis; they prevent autoimmune disease and maintain tissue integrity during inflammatory responses. Programmed death-ligand 1  is a key immune checkpoint, with a dynamic expression profile on a broad range of tissues . Blockinin vivo  in tumor-bearing mice. Analysis of blood clearance and in vivo biodistribution of different doses of indium-111-labeled anti-mPD-L1 antibodies showed that inflammatory responses can significantly alter the physiological PD-L1 expression in lymphoid organs, both locally and systemically. This resulted in accelerated blood clearance and decreased tumor targeting of radiolabeled PD-L1 antibodies.The aim of our study was to assess changes in PD-L1 expression in response to a range of inflammatory stimuli and how these would influence imaging of PD-L1 expression in the tumor. Severe-to-mild bacterial infection was mimicked by inducing systemic inflammatory responses with lipopolysaccharide (LPS), or local responses with S. aureus intramuscular, 3) Hk-C. albicans intraperitoneal, 4) vehicle intramuscular, or 5) vehicle intraperitoneal. Subsequently, mice were injected intravenously with 30 µg of [111In]In-DTPA-anti-mPD-L1 mAb  /CT and ex vivo biodistribution analysis. PD-L1 tissue expression was investigated immunohistochemically. All in vivo experiments were approved by the Animal Welfare Body of the Radboud University, Nijmegen, and the Central Authority for Scientific Procedures on Animals and were conducted in accordance with the principles laid out by the Dutch Act on Animal Experiments (2014).The effect of three clinically relevant infection models on the pharmacokinetics of anti-mPD-L1 antibodies was investigated. For this purpose, RenCa tumor-bearing female BALB/c mice were injected with 1) LPS intraperitoneal, 2) imaging) . Next, t2. Cells were tested and found negative for mycoplasma and mouse pathogens. The maximum number of passages between thawing and tumor cell inoculation was five.RenCa murine renal cell carcinoma cells  were cultured in Roswell Park Memorial Institute (RPMI) 1640 , supplemented with 10% fetal calf serum  and 2 mmol/L glutamine  at 37°C in a humidified atmosphere containing 5% COad libitum. Mice (n = 121) were injected subcutaneously (right flank) with 0.5 × 106 RenCa cells in 200 µl of RPMI 1640. Tumor size was measured using a caliper twice weekly. When mean tumor volume reached approximately 0.2 cm3, mice were block-randomized into the following groups; 1) LPS intraperitoneal, 2) S. aureus intramuscular, 3) Hk-C. albicans intraperitoneal, 4) vehicle control intraperitoneal, or 5) vehicle control intramuscular. A total of 21 mice were excluded from analysis for the following reasons: no discernable tumor present at the time of dissection (n = 16), a humane endpoint was reached (n = 3), the animal deceased before the end of the experiment (n = 1), or presence of a large concavity in the tumor (n = 1).Female BALB/c mice  were housed under specific-pathogen-free conditions in individually ventilated cages with a filter top  in which cage enrichment was present and food and water were available LPS  was dissolved in physiological saline  and stored at –20°C at a concentration of 1 mg/ml. Solutions were thawed, and physiological saline was added to a final concentration of 0.075 mg/ml. LPS was injected intraperitoneally (0.6 mg/kg body weight) 24 h before radiolabeled antibody injection, the previously determined optimal time point  were seeded in Columbia III agar containing 5% sheep blood  and incubated overnight at 37°C. Next, in duplicate, 3 to 5 colonies S. aureus were transferred to 4.5 ml of brain heart infusion (BHI) broth (BD) in 15-ml falcon tubes and incubated overnight at 37°C. The following day, the suspension was spun down at 1,000g, and the pellet was resuspended in 5 ml of NaCl 0.9% ; subsequently, the content of both tubes were pooled /ml). This suspension was transported at –80°C on dry ice and thawed 1 h later right before use. The bacterial suspension was mixed 1:1 v/v with heparinized homologous blood collected by heart puncture from isoflurane-anesthetized donor mice. The exact CFU count was calculated from a titration series of the sample, which was incubated overnight on agar at 37°C. Mice were anesthetized using isoflurane, and S. aureus  was injected in the left calf muscle 48 h before radiolabeled antibody injection for the infection and subsequent inflammatory response to develop sufficiently  were incubated overnight in Sabouraud broth at 30°C and subsequently killed by heating at 56°C for 30 min, and the preparation was kindly provided by M. Jaeger and M. Netea, Radboudumc Nijmegen  24 h before radiolabeled antibody injection for antifungal immune responses to develop  and injected 24 h before radiolabeled antibody injection. For the intramuscular S. aureus group, vehicle control was a mixture of autologous blood:physiological saline 1:1 v/v (prepared according to the protocol for the S. aureus infection). The mixture was injected 48 h before radiolabeled antibody injection.For the groups receiving intraperitoneal injection, physiological saline (0.9% NaCl) was used as a vehicle control  as follows: DTPA-anti-mPD-L1 or DTPA-IgG2a were incubated with 111InCl3 in a ratio of 1 MBq per µg antibody in 0.5 M of MES buffer (pH 5.4) at room temperature (RT) for 20 min. Thereafter, unincorporated 111In was chelated by adding 1/10th of the labeling volume of 50 mM of ethylenediaminetetraacetic (EDTA) in PBS to reach a final concentration of 5 mM of EDTA. The labeling efficiencies were evaluated using instant thin-layer chromatography (iTLC), using silica gel impregnated glass microfiber membrane chromatography strips  and 0.1 M, pH 6.0, of citrate buffer (Sigma-Aldrich) as a mobile phase. The activity profiles were captured with photosensitive plates  and analyzed using a phosphor imager . When labeling efficiency was ≥92.5%, pH was adjusted by adding 1/10th volume 10× PBS (pH 7.4). When labeling efficiency was below 92.5%, non-labeled 111In was removed using PD10 buffer exchange columns (Cytiva) and PBS as eluant following the manufacturer’s protocol. Moreover, to lower the salt concentration of all IgG2a labeling reactions and subsequent injection fluids, buffers were exchanged to PBS using PD10 columns directly after the radiolabeling procedure. Subsequently, the desired concentration of either 30 or 100 µg of antibody per 200 µl was reached by adding the appropriate amount of unlabeled antibody and volume of sterile pyrogen-free PBS. The radiochemical purity of the injected products exceeded 92.5% for all experiments. For each infection model groups, mice were injected in the lateral caudal vein with 200 µl of PBS containing one of the following; a) 30 µg of [111In]In-DTPA-anti-mPD-L1 mAb : 0.34–0.57 MBq/µg), b) 100 µg of 111In-mPD-L1 (As: 0.102–0.172 MBq/µg), c) 30 µg of [111In]In-DTPA-IgG2a , or d) 100 µg of 111In-IgG2a (As: 0.0055 MBq/µg). Heat lamps warmed the mice prior to i.v. injections. The in vitro and in vivo characterization of 111In-mPD-L1 and 111In-rIgG2a has been reported previously  and rat IgG2a non-biological affine control  antibodies were conjugated with isothiocyanatobenzyl-diethylenetriaminepentaacetic acid , as described previously , 24. DTPeviously , 24. Dur111In-mPD-L1 over time, blood samples of all mice were collected using 25-µl capillary tubes  following lateral caudal venipuncture, at 1, 4, and 24 h post tracer injection. When lateral caudal venipuncture proved unsuccessful, blood samples were obtained by saphenous venipuncture. At 24 h post tracer injection, all mice were euthanized by CO2/O2 asphyxiation. In each group, 2 mice were used for SPECT/CT imaging to visualize the in vivo biodistribution of 111In-mPD-L1 with SPECT/CT  using the following acquisition settings: 25-min acquisition time, 1.0-mm-diameter pinhole mouse high sensitivity collimator, and CT parameters of 160-µm spatial resolution, 615 µA, and 65 kV. Data were reconstructed using the MILabs software (version 2.04) using the following settings: energy windows at 171 keV (range 154 to 188 keV) and 245 keV (range 220 to 270 keV), 1 iteration, 16 subsets, and a 0.4-mm voxel size. The Inveon Research Workplace software package (version 4.1) was used to create maximum intensity projections (MIPs). For all mice, directly following euthanasia or SPECT/CT scanning, tumor and other tissues of interest  were collected for ex vivo biodistribution analysis. Blood and tissue samples were weighed  and measured with a well-type gamma-counter . For reference, aliquots of injection fluid were measured concomitantly. Antibody accumulation was calculated as a fraction of the injected dose and normalized for tissue weight [percentage injected dose per gram tissue (%ID/g)]. Tumor, the spleen, and S. aureus-infected and vehicle-injected muscle were stored in 4% formalin in PBS for subsequent immunohistochemical evaluation.To assess the blood clearance of S. aureus-infected and vehicle control muscle were evaluated by immunohistochemistry for PD-L1 expression and PD-L1, F4/80, and Ly6G expression respectively. All sections were deparaffinized and rehydrated. For F4/80, sections were post fixated with 4% formalin in PBS for 10 min at RT. Antigen retrieval (AR) was performed in either 10 mM of sodium citrate buffer (pH 6.0) at 96°C for 10 min for both PD-L1 and Ly6G, or at 37°C for 2 h followed by 0.075% trypsin in PBS for 7 min at 37°C for F4/80. Endogenous peroxidases were blocked before AR with 3% H2O2  in methanol for 20 min for F4/80, or after AR with 3% H2O2 in PBS for 10 min for both PD-L1 and Ly6G. For PD-L1, endogenous biotin was blocked using a biotin/avidin blocking set . Next, for PD-L1 and F4/80, blocking was performed with 10% normal rabbit serum  and for Ly6G with 20% normal goat serum  in PBS for 30 min. Primary antibody incubation was performed with either goat-a-murine PD-L1  diluted 1:500 in PBS/1% bovine serum albumin  overnight at 4°C, rat-anti-F4/80  diluted 1:1,000 in PBS/2% NrabS and incubated overnight at 4°C, or rat-anti-mouse-Ly6G  diluted 1:1,000 in PBS+1% BSA and incubated for 2 h at RT. Thereafter, tissues were incubated for 30 min at RT with the following secondary antibodies: biotinylated-rabbit-anti-goat  diluted 1:400 in PBS/1% BSA, goat-a-Rat-peroxidase  diluted 1:100 in PBS/1% BSA, or biotinylated rabbit-anti-rat-IgG  1:200 dilution in PBS-2% NrabS, for the PD-L1, Ly6G, or F4/80, respectively. Next, for PD-L1 and F4/80, horseradish peroxidase-Biotin/Streptavidin Complex  diluted 1:250 in PBS/1% BSA for 30 min at RT was used. Finally, all tissues were incubated with bright DAB  for 8 min, followed by nuclei blueing with hematoxylin , and dehydrated with ethanol  and xylene . Sections were mounted with a coverslip using Permount . Consecutive sections to the PD-L1-, F4/80-, and Ly6G-stained sections were stained for morphological reference with H&E Y .Formalin-fixed paraffin-embedded tissue sections (5 µm) of tumor and the spleen, and 111In-mPD-L1 and 111In-IgG2a were tested for significance using two-way ANOVAs with a Bonferroni post-hoc test. Values are presented as mean ± SD. Statistical significance was defined as a p-value below 0.05. Statistical analyses were performed using GraphPad Prism version 5.03 for Windows.Differences in tissue uptake and blood concentrations of both doses 111In-mPD-L1 in tumor-bearing mice is presented in 111In-mPD-L1, compared with control mice . Increased PD-L1 antibody uptake was also observed in lymph nodes , bone marrow , and, to a lesser extent, other tissues with physiological PD-L1 expression, such as the lungs, heart, duodenum, and brown adipose tissue. The increased uptake of 111In-mPD-L1 in primary and secondary lymphoid organs occurred at the expense of tumor uptake, which was only 8.1 ± 4.5%ID/g in LPS-challenged mice versus 25.2 ± 5.2%ID/g in control mice (p < 0.001).The effect of LPS on the pharmacokinetics of 111In-mPD-L1 restored blood levels and tumor uptake in LPS-challenged mice to similar levels as in control mice (111In-mPD-L1 in lymphoid organs remained elevated but was less pronounced as compared with the lower antibody dose. Spleen uptake in mice injected with 100 µg of 111In-mPD-L1 was 21.0 ± 5.0%ID/g versus 12.6 ± 1.9%ID/g (p < 0.01), for LPS-challenged versus control mice. SPECT/CT imaging confirmed the ex vivo biodistribution analyses for both antibody doses , using the same experimental design. In contrast to 111In-mPD-L1, blood clearance and in vivo biodistribution of both the 30 µg and 100 µg dose levels of 111In-IgG2a were not affected by LPS challenge  did not affect blood clearance of 111In-mPD-L1 . SPECT/CT imaging confirmed these results as, apart from the accumulation of 111In-mPD-L1 in the S. aureus-infected muscle, no differences in antibody distribution were observed , indicating that uptake was predominantly PD-L1 mediated. Similarly, as for 111In-mPD-L1, S. aureus infection did not alter blood clearance, and tumor and normal tissue targeting of 111In-IgG2a.Whether the S. aureus-infected muscle was predominantly PD-L1 specific, we performed immunohistochemical analysis of the S. aureus-infected muscle. We Observed a high number of PD-L1 positive cells in and around sites of S. aureus infections, whereas only a few PD-L1 expressing cells were detected in the control site  or PD-L1 positive immune cells. Furthermore, we showed that the localized increase in PD-L1 expression is caused by monocytes and high numbers of neutrophils. These data are consistent with the immunological response to local S. aureus infections with induction of PD-L1 expression  and the Dutch Cancer Society .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Supplemental Digital Content is available in the text. The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome  and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients.Tbx5G125R) and performed electrophysiologic analyses , transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling .We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome  that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties.We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers. A pathogenic variant in the fifth exon of TBX5 ([T-box transcription factor 5] p.G125R) found in a Dutch, atypical Holt–Oram syndrome family with early onset atrial fibrillation was modeled in mice.This is the first human pathogenic variant based on a patient family in this key cardiac transcription factor explored in vivo.We identify widespread electrophysiologic, transcriptional, and epigenetic changes  in the atria of Tbx5-p.G125R heterozygous mice distinct from the changes in atria of Tbx5 insufficiency models.The characterization of the Tbx5 p.G125R mouse model indicates that a patient-specific pathogenic variant in TBX5 induces changes in regulatory element activity, altered balance in the regulatory network of atrial cardiomyocytes, and clinically relevant changes in cardiomyocyte function.These findings shed light on the target and off-target genes of Tbx5-p.G125R because of its altered properties, which are at the basis of distinct electrophysiologic changes observed in a subset of Holt–Oram syndrome patients with atrial fibrillation.This work may provide insight into the epigenetic changes and transcriptional underpinning of arrhythmia in the general population caused by small increases in TBX5-expression caused by common variants predisposing to atrial fibrillation.TBX5 (T-box transcription factor 5) have been linked to Holt–Oram syndrome (HOS), which is typically characterized by varying degrees of limb and cardiac malformations and conduction defects (OMIM [Online Mendelian Inheritance in Man] #142900).3 Tbx5 is dose-dependently required for heart development and specification and function of the conduction system, and determines the working myocardial phenotype of the atrium.8 Tbx5 has been put forward as key regulator of ion-handling protein-encoding gene expression and rhythm control in vivo.13 Genome-wide association studies uncovered several variants near TBX5 associated with atrial fibrillation (AF). Moreover, transcriptome-wide analysis of human heart tissue identified an association of increased TBX5 expression with AF risk.14Pathogenic variants in T-box transcription factor (TF) TBX5 causing a glycine to arginine substitution  in the T-box domain was found in a Dutch, atypical HOS family.15 In this family, affected members show HOS symptoms such as mild skeletal abnormalities and septal defects, as well as atypical early onset paroxysmal AF. The function of missense variants in this key TF and the impact on gene regulation in the context of heart function in vivo are poorly understood. Therefore, we engineered the p.G125R variant in the highly conserved T-box of mouse Tbx5, and used this model to gain insight into the molecular mechanisms underlying structural and electrophysiological changes predisposing to AF in TBX5 missense variant carriers.A pathogenic variant in the fifth exon of Supplemental Material.Detailed Methods are provided in the Whole-tissue RNA-sequencing , cardiomyocyte (CM) nucleus H3K27ac (histone H3 lysine 27 acetylation) CUT&RUN (cleavage under targets and release under nuclease), CM nucleus assay for transposase-accessible chromatin using sequencing (ATAC-seq), and single-nuclei RNA-seq are available on Gene Expression Omnibus (GSE167082). All other data and supporting materials have been provided with the published article.The study was conducted according to the guidelines of the Declaration of Helsinki. Written informed consent was obtained from the families presented in the paper.Animal care and experiments conform to the Directive 2010/63/EU of the European Parliament. All animal work was approved by the Animal Experimental Committee of the Amsterdam University Medical Centers , was performed in compliance with the Dutch government guidelines, and was approved by the Central Committee Animal Experiments.Supplemental Material which also describes the specific statistical tests used to analyze large data sets. Results of statistical analysis are given in the text or the figure legends.A detailed description of the statistics can be found in the 15 Here, we reanalyzed in detail the available ECG traces (as obtained by 24-h Holter or 12-lead ECG) of the patients previously analyzed.15 We found a range of additional abnormalities—including irregular RR interval, right bundle branch block, atrioventricular (AV) junctional escape, AV junctional rhythm, sinus arrest, left ventricular noncompaction, atrial extras, and sick sinus syndrome—that were not checked for previously . This shows that these patients suffer from other types of supraventricular arrhythmia that indicate dysfunctionality of the atrial muscle, including the sinoatrial and AV nodes.Heterozygous TBX5-p.G125R carriers show atypical HOS, including cardiac septal defects, mild skeletal abnormalities, and early onset AF . Interbreeding of Tbx5G125R/+ animals showed that from embryonic day (E) 12.5 to E16.5, homozygous Tbx5G125R/G125R fetuses were necrotic or dead (Table S2), revealing that homozygosity of the mutation is incompatible with life. Tbx5G125R/G125R fetuses showed ventricular and atrial septal defects (E14.5–E16.5), as well as enlarged right atria at E16.5 , which were also present in patients heterozygous for this pathogenic variant . We did not observe differences in CM proliferation rates in embryonic (E12.5) right atria or left ventricles between different genotypes , nor differences in the heart weight–tibia length ratio between control (n=6) and Tbx5G125R/+ mice (n=10) at 20 days after birth (Table S3).To investigate how the p.G125R pathogenic variant in TBX5 affects heart function, we generated a mouse model with this point mutation in Tbx5G125R/+ mice  and Wenckebach cycle lengths compared with controls . Hearts isolated from Tbx5G125R/+ mice also showed longer and more variable RR intervals, as well as shorter PR intervals, than controls . In addition, these experiments show that the atrial ectopic beats occurred during or after the QRS complex, and occurred in all cases in response to a premature ventricular beat  and Tbx5G125R/+ mice were susceptible to atrial arrhythmias, we performed programmed atrial stimulation in vivo using a transesophageal catheter . Similarly, recorded action potentials in isolated atrial CMs  and at all action potential repolarization phases .To assess whether r Figure A. We wers Figure D reveales Figure E. ActionTbx5G125R/+ nuclei were obtained . Only the CM cluster showed some degree of segregation between genotypes . CMs showed 59 genes that were significantly differentially expressed; endothelial cells showed 17 genes and fibroblasts showed 16 . Analyses of the Gene Ontology terms for the differentially expressed genes in CMs showed mostly terms involving muscle contraction regulation and ion transport (Table S7). The fraction of CMs and fibroblasts differed between genotypes . Picrosirius red–staining sections of 8-month-old control (n=4) and Tbx5G125R/+ (n=5) atria revealed a trend toward a smaller fibrotic area in the atria of Tbx5G125R/+ mice compared with controls; however, it was not significant . Together, these findings suggest that the Tbx5G125R/+ atria do not show elevated fibrosis even though fibrosis has been associated with AF.To gain insight into the cell types affected by Tbx5-p.G125R, we performed single-nucleus RNA-seq on right atrial tissue. Profiles from 3475 wild-type nuclei and 2933 Tbx5G125R/+ (n=5) animals . The point mutation may affect mRNA levels of Tbx5G125R . Using RNA-seq reads and Varscan,16 we quantified the number of Tbx5 transcripts tags containing the wild-type allele (G) and the number containing the mutant allele (C). In control animals, the wild-type allele had a frequency of 100%. In Tbx5G125R/+ animals, the variant allele had a frequency of 54% , indicating both the wild-type and Tbx5G125R/+ transcripts are present at equal levels in Tbx5G125R/+ animals.To determine at greater depth the transcriptional responses to heterozygosity of Tbx5-p.G125R, RNA-seq was performed on whole right atria of adult female control (n=5) and Padj < 0.05; Table S8; Figure Tbx5G125R/+ atria, 610 were upregulated. While the cardiac function of many of the most significantly differentially expressed genes (Table S8) has not been defined, Lgals3  and Padi2 (peptidyl arginine deiminase-2) have been implicated in cardiac dysfunction.18 We observed differential expression in 33 of 300 analyzed gene-encoding proteins involved in ion handling/electrophysiology, among which were several intracellular Ca2+-modulating and gap junction genes , as well as K+ channel genes such as Kcnj15 (potassium inwardly rectifying channel subfamily J member 15) and Kcnn2 . Reduced expression of Scn5a (sodium voltage-gated channel α subunit 5), Ryr2 (ryanodine receptor 2), and Atp2a2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2) was linked to conduction slowing and altered intracellular Ca2+ concentrations in Tbx5 haploinsufficient mice.11 However, these and other genes that were previously found to respond to Tbx5 insufficiency  were not responsive to Tbx5-p.G125R. Gene Ontology term analysis yielded general terms for genes downregulated in the Tbx5G125R/+, but pointed to actin filament and muscle process, as well as ion transport, for upregulated genes . Transcriptional profiles of left atria of mutants and controls were in accordance with those of the right atria (Table S8).We found 1316 genes differentially expressed  in whole-tissue RNA-seq of right and left atria, of which only Postn (activated fibroblast marker) was decreased in expression in right atria of Tbx5G125R/+ animals . These data further support the absence of fibrosis and activated fibroblasts in Tbx5G125R/+ atria. Furthermore, as the right atrial samples all contained sinoatrial node tissue, we explored the expression of sinoatrial node–specific genes23 in the whole-tissue RNA-seq data. Of 30 markers tested, 14 showed differential expression. Notably, transcripts for key TFs Tbx3, Isl1 (insulin gene enhancer protein ISL-1), and Shox2 (short stature homeobox 2), and key ion channels Hcn4 (hyperpolarization activated cyclic nucleotide gated potassium channel 4), Hcn1 (hyperpolarization activated cyclic nucleotide gated potassium channel 1), and Cacna2d2  were not differentially expressed between genotypes. However, Cacna1d, , Cacna2d3, calcium voltage-gated channel auxiliary subunit α2 δ3), Cacna1g , Cacna1h , and Ryr3 (ryanodine receptor 3), as well bone morphogenetic protein (BMP)–signaling components , showed significant differential abundance in the data sets .We explored fibrotic tissue markers  using ATAC-seq  and reduced accessibility  compared with sites of unchanging accessibility . TBX5 was reported to interact and cooperatively regulate cardiac target gene expression with KLF13 (Krüppel-like factor 13).26 These data suggest that Tbx5-p.G125R may have reduced interaction with KLF/SP factors, leading to reduced accessibility of sites normally co-occupied by Tbx5 and KLF/SP factors.To gain insight into the changes in epigenetic state and regulatory element deployment in response to Tbx5-p.G125R, we assessed the chromatin accessibility profile of control and q Figure A. There Tbx5G125R/+ (n=6) pooled left and right atria . We detected 127 286 regions enriched for H3K27ac association and 1653 sites with significantly differential H3K27ac regions , of which 1335 regions have increased signal in Tbx5G125R/+ and 318 regions have reduced signal . Of the 127 286 H3K27ac regions, 18% (22 911/127 286) overlapped with Tbx5 binding sites in the fetal mouse heart24 (Table S16), whereas in the regions with increased H3K27ac association, 40%  overlapped Tbx5 binding sites . For example, accessibility and H3K27ac signal of Tbx5-bound regulatory elements in Padi2 and Wdr3 (WD repeat-containing protein 3) are strongly increased in Tbx5G125R/+ atria . Interestingly, the H3K27ac regions that are reduced in the Tbx5G125R/+ atria all overlap Tbx5 binding sites . These data are in agreement with the chromatin accessibility analysis, and together indicate that Tbx5-p.G125R induces altered epigenetic states of sites (including putative enhancers) normally occupied by Tbx5 and—to a lesser extent—of sites not occupied by Tbx5.To further examine the impact of Tbx5-p.G125R on genome-wide regulatory element activity, we examined the genomic localization of H3K27ac association by CUT&RUN in isolated PCM1+  CM nuclei of 8-week-old control (n=5) and 12 By crossing our RNA-seq data with the ATAC-seq and CUT&RUN data sets , we detected 128 enhancer-derived transcripts  that were differentially expressed between control and Tbx5G125R/+ animals in the right atria , of which 42 were more abundant in the control and 86 more abundant in the Tbx5G125R/+ animals . Out of the 128 thus identified elements, 71 showed Tbx5 occupation (Table S17).24 The remaining 57 ncRNA-associated elements often showed Tbx5 occupation of accessible/H3K27ac-marked elements at a few-kbp distance, but these were considered to not colocalize. An example of a differentially expressed ncRNA with a transcriptional start site overlapping differential accessibility and H3K27ac is shown in Figure Bmp6 (bone morphogenetic protein 6). Of these differentially active regulatory elements, we identified 166 candidate target genes that were differently expressed between genotypes (Table S17). Highly interesting candidates include Tbx20 (T-box transcription factor 20), Sema3a (semaphorin 3A), Kcna4 (potassium voltage-gated channel subfamily A member 4), Slc8a1, and Tgfb2 (transforming growth factor β2), which are likely to impact atrial function in Tbx5G125R/+ animals 31 Differentially transcribed ncRNAs, in combination with differential accessibility and differential target gene expression, are shown for the Tgfb2, Sema3a, and Kcnj15 loci in Figure S8. Interestingly, previously identified prominent Tbx5-dependent ncRNAs (close to Ryr2 and to Atp2a2 [Serca2a (sarcoplasmic/endoplasmic reticulum CA2+ ATPase 2a])12 and their putative target genes  were not affected in Tbx5G125R/+ atria .Previously, Tbx5-dependent enhancers and noncoding RNA (ncRNA) were identified by analyzing Tbx5-dependent enhancer transcription.15 Here, we show that these patients suffer from other types of supraventricular arrhythmias that indicate dysfunctionality of the atrial muscle, including the sinoatrial node and AV node, that were not identified previously (Table S1). Typical HOS is usually associated with loss-of-function variants leading to haploinsufficiency; the atypical HOS phenotype of TBX5-p.G125R carriers suggest the pathogenic missense variant causes TBX5 to gain specific function(s). In the mouse model with this variant, the quantity of Tbx5 (wild-type) and Tbx5-p.G125R mRNA is equal in the adult Tbx5G125R/+ mouse atria, indicating that loss of expression  causing haploinsufficiency does not occur. We did identify broadly changed expression profiles and epigenetic states induced by heterozygosity for Tbx5-p.G125R. However, important genes and enhancers previously found to respond to Tbx5 insufficiency 12 did not respond to Tbx5-p.G125R. This indicates Tbx5-p.G125R has gained functions invoking molecular mechanisms different from those invoked by reduced Tbx5 dose. Intriguingly, expression quantitative trait loci analysis revealed that AF risk variant carriers in the human population express significantly more, not less, TBX5 in the heart.14 It will be interesting to investigate whether the Tbx5-p.G125R TF and increased TBX5 dose in risk variant carriers share transcriptional responses underlying AF propensity.Previous characterization of a Dutch family with the pathogenic TBX5-p.G125R missense variant revealed atypical HOS, with mild skeletal abnormalities and lower penetrance of congenital heart defects but a high prevalence of AF.33 The right atrial RNA-seq data sets indicated differential expression in 14 of 30 selected sinoatrial node markers. While these findings require validation, they suggest that the sinoatrial node may be transcriptionally affected in Tbx5G125R/+ mice. The differentially expressed genes included genes encoding calcium channels and BMP-signaling components important for sinoatrial node development and function,22 which may contribute to dysfunction of the sinoatrial node in Tbx5G125R/+ mice. Moreover, the balance between Tbx5 and the transcriptional repressor Tbx3 was found to determine the function of conduction system components.35 The altered DNA binding properties of Tbx5-p.G125R could affect both the regulation of important regulators in the nodes, and change the competitive balance with Tbx3. Further studies are required to explore the impact and underlying mechanisms of Tbx5-p.G125R on the sinoatrial and AV nodes.The patients displayed AV junctional escape beats, atrial extras, sinus bradycardia, and sick sinus syndrome in addition to AF. These arrhythmias were reproduced in the Tbx5-p.G125R mouse model, indicating Tbx5-p.G125R function and impact on cardiac electrophysiology in vivo are conserved between human and mice. Tbx5 is critical for the development and gene regulation in the sinoatrial and the AV nodes.Tbx5-haploinsufficient and -deficient mouse models implicate reduced expression of Ryr2 and Atp2a2 in AF propensity,36 these genes are not deregulated in atria of heterozygous Tbx5-p.G125R mice. However, we observed changes in expression of many other ion-handling protein-encoding genes, and in particular, genes associated with atrial function. Notably, the transcriptional profiling and differential regulatory element usage analysis identified Tbx5-p.G125R–mediated increased atrial expression of Tbx20, Sema3a, Kcna4, and Pitx2c , and decreased atrial expression of Tgfb2. Each of these genes has been causally linked to heart function and rhythm control, further indicating that multiple complex mechanisms underlie the arrhythmia phenotype. Tbx20 is an essential cardiac TF, controlling CM function, ion channel gene expression, and atrial development.28 Because T-box TF function is highly dose-sensitive, increased Tbx20 expression in Tbx5+/G125R atria is likely to broadly affect cardiac gene expression and function. Sema3a, produced by CMs, controls cardiac sympathetic innervation and is associated with arrhythmia when disrupted or overexpressed in mice.29 Moreover, Sema3a was identified as a naturally occurring Kv4.3 (potassium voltage-gated channel) inhibitor,37 suggesting its induction could affect Kv4.3. An induction of Kv1.4 (encoded by Kcna4) has been observed in pathophysiologic conditions in the human heart . An increase of Kv1.4 (partly replacing Kv4.3) in atria was suggested to prolong ADP and to alter its dynamics, causing AF predisposition.31Transcriptional profiling of the atria showed that heterozygosity of Tbx5-p.G125R leads to extensive dysregulation of gene expression in the atria, indicating multiple and complex mechanisms underlie the arrhythmia phenotype. While studies of 40 In the formed left atrium, Pitx2c was shown to interact with Tbx5 to regulate ion channel gene expression and electrophysiology.10 Ectopic induction of Pitx2c in the right atrium could potentially affect sinoatrial node development and function, consistent with the observation that both human and mouse TBX5-pG125R carriers display sinoatrial node dysfunction. Furthermore, the atrial TF network around Tbx5 is likely affected by ectopic presence of Pitx2c in the right atrium, leading to altered ion channel gene expression.10 The decreased expression of Tgfb2 in the atria of heterozygous Tbx5-p.G125R mice may lead to reduced TGF-β signaling and diminished fibrosis,30 as is observed in the atria of these mice.Pitx2c is specifically expressed in the left atrium, where it imposes left identity on the atrium, initiates pulmonary vein myocardium formation, and suppresses sinoatrial node development during embryogenesis.Tbx5G125R/+ CMs show a prolongation of the APD. While APD is often shortened in AF patients, mainly in the setting of sustained arrhythmias,42 APD prolongation is associated with increased risk of developing AF in patients.45 In atria of Tbx5G125R/+ mice, APD was prolonged as well, which is consistent with the patient clinical picture as well as with the increase in atrial arrhythmias we observed. The decreased Ca2+ amplitude and intracellular Ca2+ concentrations may be a result of longer Ca2+ currents because of reduced Ca2+-induced Ca2+ current inactivation, as well as decreased activation of Ca2+-activated K+ channels, and both mechanisms may contribute to the prolonged ADP that was observed. Moreover, deregulation of genes encoding channel subunits for various voltage-gated potassium currents ], Kcna4 [Kv1.4]; see Figure 2+-modulating proteins such as Cacna1c and Cacna1g could be at the basis of the decreased of Ca2+ transient amplitude observed in the mutant mice. Another explanation could involve Mrvi1 (murine retrovirus integration site 1 homolog), which uniquely responds to Tbx5-p.G125R and is decreased in the Tbx5G125R/+ atria. Mrvi1 is associated with Pln and plays a key role in cardiac contractility by modulating nitric oxide–induced inhibition of Ca2+ signaling.47Patch clamp measurements of isolated atrial 24 suggesting TBX5-p.G125R may display increased interaction with genuine Tbx5-occupied sites in vivo, in keeping with its increased DNA-binding affinity in vitro.15 However, sites normally not occupied by Tbx5 also showed increased accessibility, and the sites displaying decreased accessibility were enriched for Tbx5-binding and T-box motifs. Together, these observations suggest Tbx5-p.G125R has acquired properties that both quantitatively and qualitatively influence chromatin interaction. Additional analyses  are required to define these altered interaction properties of Tbx5-p.G125R. The changed chromatin interaction may involve a shift in affinity for binding partner TFs or cofactors, as was previously also reported for missense mutations in GATA4 (GATA-binding protein 4) and NKX2-5.49 The sites of reduced accessibility in heterozygous mutants were highly enriched for motifs of Sp/KLF, MEF2, and T-box families of TFs, pointing to candidate TFs that normally interact with Tbx5, and to a lesser extent, Tbx5-p.G125R. Mef2c and Klf13 are reported functional interaction partners of Tbx5 in the regulation of cardiac development and gene regulation.26 The Sp4 TF is expressed in the heart, including the atria and AV node, and Sp4-deficient mice display a high incidence of sinus bradycardia and AV block.50 Future studies could explore whether the Tbx5-p.G125R interactions with Mef2c—as well as the Sp and Klf TF family members expressed in the atria—are affected.A relatively large number of sites with increased accessibility and with increased H3K27ac association were observed in heterozygous Tbx5-p.G125R atrial CMs. These sites were enriched for Tbx5 binding in the normal heart,In conclusion, Tbx5-p.G125R displays altered properties that induce changes in the epigenetic state, such as chromatin accessibility and H3K27ac, that underlie deregulated expression of both Tbx5 target genes and off-target genes, which is at the basis of distinct electrophysiologic changes that recapitulate clinical observations in patients heterozygous for TBX5-p.G125R.We thank Berend de Jonge, Aho Ilgun, and Corrie de Gier-de Vries for their contributions. We thank Jan Ruijter for assisting with statistical analysis and presentations.This work was supported by CardioVasculair Onderzoek Nederland , Leducq Foundation (14CVD01 to V.M.C.), and Dutch CardioVascular Alliance (OUTREACH to V.M.C.).None.Supplemental MethodsFigures S1–S9Table S1–S18References 51–72


Local control of gene expression provides critical mechanisms for regulating development, maintenance and plasticity in the nervous system. Among the strategies known to govern gene expression locally, mRNA transport and translation have emerged as essential for a neuron’s ability to navigate developmental cues, and to establish, strengthen and remove synaptic connections throughout lifespan. Substantiating the role of RNA processing in the nervous system, several RNA binding proteins have been implicated in both developmental and age dependent neurodegenerative disorders. Of these, TDP-43 is an RNA binding protein that has emerged as a common denominator in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and related disorders due to the identification of causative mutations altering its function and its accumulation in cytoplasmic aggregates observed in a significant fraction of ALS/FTD cases, regardless of etiology. TDP-43 is involved in multiple aspects of RNA processing including splicing, transport and translation. Given that one of the early events in disease pathogenesis is mislocalization from the nucleus to the cytoplasm, several studies have focused on elucidating the pathogenic role of TDP-43 in cytoplasmic translation. Here we review recent findings describing TDP-43 translational targets and potential mechanisms of translation dysregulation in TDP-43 proteinopathies across multiple experimental models including cultured cells, flies, mice and patient derived neurons. One of the most unique features of a neuron is its dramatic morphology. Aside from the requisite cell body, which contains the nucleus and as little as 1% of the cytoplasm, neurons typically wield a robust network of processes including dendrites and a single axon , Fused in Sarcoma (FUS) and Senataxin (SETX) has been linked to neurodegenerative disease pathogenesis. TDP-43 is of particular interest as it participates in numerous RNA processing steps and has been identified as a major component of pathological cytoplasmic inclusions in ALS and frontotemporal dementia (FTD) . These cRecent reports have identified TDP-43 dependent alterations in the translatome . Whetherin vitro and reduce neurotoxicity  with a nuclear localization signal , two RNA-recognition motifs , and an intrinsically disordered C-terminal low-complexity domain  see . Insighttoxicity . Anothercificity . DisruptRecently, a short TDP-43 (sTDP-43) isoform upregulated by neuronal hyperactivity has been identified . It resuUnder physiological conditions, TDP-43 resides predominantly in the nucleus where it modulates transcription, splicing and miRNA biogenesis . Due to As an RNA binding protein, TDP-43 has a broad influence on gene expression, however, its impact on translation in particular is not well understood. Aside from direct alterations to the translation machinery, disturbances in granule dynamics and mRNA localization may contribute considerably to changes in the translatome. As new transcripts are generated in canonical cap-dependent translation , initially discovered within viral RNAs and subsequently identified within mRNAs with certain structural features . Cap indin vivo, overexpression of cytoplasmic TDP-43 in the forebrains of transgenic mice (ΔNLS-TDP-43) results in decreased global translation as evidenced by both SUnSET and polysome profiling  . To mech neurons . These enscripts . Taken tRac1 mRNA into dendritic spines and premature translation as evidenced by an elegant TRICK RNA reporter that allows real time visualization of mRNA transport and translation  demonstrated that RNAs bound by TDP-43 contained G-quadruplexes and were transported to distal neurites along with TDP-43 for local translation, consistent with G-quadruplexes being novel structural motifs within RNAs associated with TDP-43  were confirmed by immunofluorescence in mouse spinal cord motor neurons. Immunohistochemistry experiments in patient derived spinal cords confirmed that DDX58 and MTHFSD, both of which are RNA binding proteins, are differentially expressed in ALS compared to controls , translational changes specifically associated with disease could be distinguished. Candidate mRNAs Syngr4 and Plekhb1 were up- and downregulated, respectively, at the transition from asymptomatic to early symptomatic motor dysfunction, as identified by TRAP and RNA sequencing approaches in spinal cord motor neurons (A315T and TDP-43Q331K), suggesting that they may play critical role in the progression of mutant TDP-43 driven ALS  in motor neuron-like cells and in primary cortical neurons enhanced the translation of he 3′UTR . Importa disease .dlp mRNA in TDP-43 complexes and depletion from ribosomes in the context of TDP-43 proteinopathy (RNAi) was sufficient to deplete Dlp from the NMJ, but not induce significant ventral cord neuropil puncta, highlighting that TDP-43 nuclear depletion and cytoplasmic accumulation have distinct contributions to disease pathomechanism. Restoring Dlp by overexpression, in motor neurons specifically, restores Dlp expression at the synaptic terminal and mitigates TDP-43-dependent locomotor deficits. Importantly, these findings align with ALS patient data indicating that GPC6 protein, a human homolog of Dlp, accumulates in puncta within the spinal cord, mimicking the Dlp accumulations in the Drosophila ventral cord neuropil  as a novel TDP-43 candidate target, based on the enrichment of inopathy . This stneuropil . Additio neurons .Interestingly, several recent studies have identified mRNA targets of TDP-43 that encode distinct populations of proteins, such as ribosomal proteins (RPs), mitochondrial proteins, and translation factors . For insGrowing evidence of alterations to the translatome in TDP-43 proteinopathies supports a multifaceted role for TDP-43 involvement in translation, highlighting both physiological and pathogenic functions. TDP-43 knock-down studies demonstrate that TDP-43 is necessary for the RNP-mediated transport of mRNAs into axons  and dendAlthough TDP-43 dependent alterations in the global translatome are well documented , and TDPRegardless of mechanism, it is clear that TDP-43 accumulation in the cytoplasm is driving translation dysregulation see . The ideQuestions remain on the potential connections between TDP-43’s role in transcription, splicing, RNA stability and translation that could help identify additional strategies for restoring more downstream translation deficits. A significant caveat is the limited knowledge regarding the physiological role of TDP-43 in RNA processing within the cytoplasm. The discovery of the cytoplasmically localized sTDP-43 , the incRTB, NPM, and DCZ wrote the manuscript. SL, RTB, and NPM made the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Based on the experimental results, the optimized three layers of graphene TENG (3L-Gr-TENG) with CTL showed a 30-fold enhancement in output power compared to its counterpart, 3L-Gr-TENG without CTL. This remarkably enhanced performance can be ascribed to the synergistic effect between the optimized graphene layers with high dielectric CTL. Moreover, the device exhibited outstanding stability after continuous operation of >2000 cycles. Additionally, the device was capable of powering 20 green LEDs and sufficient to power an electronic timer with rectifying circuits. This research provides a new insight to improve the charge density of Gr-TENGs as energy harvesters for next-generation flexible electronics.The continuous quest to enhance the output performance of triboelectric nanogenerators (TENGs) based on the surface charge density of the tribolayer has motivated researchers to harvest mechanical energy efficiently. Most of the previous work focused on the enhancement of negative triboelectric charges. The enhancement of charge density over positive tribolayer has been less investigated. In this work, we developed a layer-by-layer assembled multilayer graphene-based TENG to enhance the charge density by creatively introducing a charge trapping layer (CTL) Al With the advent of the fourth industrial revolution, the demand for flexible, portable and wearable electronic devices has increased dramatically. However, powering them in a stable manner remains a challenge due to the ongoing energy crisis worldwide . In this2-bonded carbon atoms, can be a promising material for TENGs owing to its unique properties such as high electrical conductivity, excellent mechanical flexibility, optical transparency and environmental stability [The triboelectrification process involves charge generation, charge storing and charge decay of triboelectric materials . Therefotability ,17. The tability ,19. Kim tability ,21. Furttability ,11,12,22tability ,24. Extetability . Furthertability . Howevertability . Based o2O3 CTL in between the positive triboelectric layer and the bottom conducting electrode. The Gr-TENG with Al2O3 introduces a mechanism of surface charge enhancement in conduction domains. Relying on the synergistic effect of optimized graphene layers and high dielectric Al2O3 CTL, there is a large triboelectric charge yield. The optimized flexible 3L-Gr-TENG with Al2O3 exhibits an enhanced output voltage and current of ~55 V and 0.78 µA, respectively. These values were nearly 5-fold higher than those of the counterpart pristine 3L-Gr-TENG (without Al2O3 CTL). The output power of the 3L-Gr-TENG is increased from 0.77 to 25 µW (~30 times higher) with the Al2O3 CTL under ambient conditions. Importantly, by taking advantage of multilayer graphene as a positive triboelectric layer, the bottom graphene layers can act as a charge transport bridge between Al2O3 and the top graphene layer due to its high electrical conductivity, thus accumulating more positive charge on the graphene surface and facilitating the electron flow from graphene to the opposite triboelectric layer. Moreover, the device shows high stability and durability after continuous operation of >2000 cycles. Furthermore, the generated power can light up more than 20 commercial green LEDs and charge various capacitors to power an electronic timer through the rectifying circuits. Most importantly, this work demonstrates a novel and cost-effective method to improve the performance of flexible Gr-TENGs, which can be a power candidate for next-generation flexible energy harvesting systems.In this work, we report a new approach to achieve high output performance of layer-by-layer assembled multilayer graphene-based TENG by introducing Al2O3 as the CTL over a PET substrate. In this study, the conductor-to-dielectric contact mode was used. To design the vertical contact–separation mode TENG device, the positive friction layer of the TENG was fabricated as follows: (i) First, a 3 × 3 cm2, 80 μm thick commercial aluminum (Al) foil was taken as the conducting electrode, which was attached over the polyethylene terephthalate (PET) substrate (~188 μm) with double-sided tape. Then, the Al-foil/PET substrate was cleaned by ethanol and dried in a stream of N2 gas. (ii) Thereafter, a thin layer of Al2O3 (~10 nm), as the CTL, was thermally deposited over the Al-foil/PET substrate by the thermal evaporation method [2O3/Al-foil/PET substrate using the PMMA transfer method, as shown in 2O3/Al-foil/PET substrate. The positive charge trapping nature and higher relative permittivity of Al2O3 helped to enhance the surface charge over graphene for high output performance [2O3 provided extra support for the enhancement in the electrical output of the Gr-TENG. Subsequently, a commercially available polytetrafluoroethylene (PTFE) film (3 × 3 cm2) served as the negative friction layer due to its high electronegativity, which could accept more electrons when rubbed against a positive friction layer [n method . (iii) Fn method , as a poformance . In addion layer . A condu2O3/Al-foil/PET substrate. −1) and the 2D peak (at ~2700 cm−1) are the characteristics of the sp2-hybridized C–C bonds in graphene. Basically, these two bands are used to determine the number of layers in graphene. In addition, a negligible D peak (at ~1359 cm−1) corresponds to atomic-scale defects or lattice disorder in graphene [2D/IG of ~2.18 of CVD-grown graphene indicates high-quality monolayer graphene and gradually decreases with an increase in the number of layers, as shown in 2O3/Al-foil/PET substrate, as shown in 2O3 over the Al-foil/PET substrate, which is beneficial for enhancing the output performance because the effective contact increases during the triboelectrification process.Raman spectroscopy is a powerful and nondestructive tool to analyze the quality of graphene. graphene . Here, tgraphene , as showgraphene . Moreove2O3/Al-foil/PET substrates, we performed XRD, which is shown in 2O3/Al-foil/PET substrates retain the same peak position, indicating that the crystalline structure of graphene is restored after transfer over the Al2O3/Al-foil/PET substrates, which is helpful for the enhancement of the electrical output of the TENG.To further confirm the quality of graphene  on the Al-foil/PET and Al2O3 CTL, FESEM analysis was conducted, as illustrated in 2 was used. As clearly seen from 2O3/Al-foil/PET substrate exhibits a rougher wrinkled surface morphology in comparison with the graphene over Al-foil/PET substrate  to quantify the atomic composition of the graphene sample. The graphene samples contain carbon (C), oxygen (O) and aluminum (Al), as depicted in 2O3/Al-foil/PET substrate. This increase in oxygen will help to enhance the output performance because oxygen has excellent electron-donating ability due to its high Lewis basicity, which makes the graphene layer more tribopositive [Further, to investigate the surface morphology of graphene after the insertions of Alubstrate a due to positive .2O3/Al-foil/PET substrates. The surface roughness values of graphene  on the Al-foil/PET substrate were 7.78, 9.57 and 11.2 nm, respectively  at zero transferred charge can be expressed according to the following equation [x(t) represent the vacuum permittivity, surface charge density and distance between graphene and PTFE triboelectric layers, respectively. According to Equation (1), the OCV is strongly related to the A is the surface area and d is the thickness of the triboelectric layer. According to Equation (2), a thin film with high relative permittivity can exhibit high output performance. Therefore, for the enhanced output performance, the optimized number of graphene layers needs to be investigated.Prior to performing the electrical measurement and examining the relationship between the output performance of the flexible pristine Gr-TENG and Gr-TENG with Ald in 2O3  as the Cequation :(1)VOC=σ follows :(2)Cmax2O3 CTL were systematically investigated, as shown in 2O3 CTL is depicted in SC of ~155.9 nA and VOC of ~12 V compared to those of 1L-Gr-based (ISC ~43.1 nA and VOC ~7.5 V), 2L-Gr-based (ISC ~66.1 nA and VOC ~9 V (SC ~113.9 nA and VOC ~11 V (SC ~108 nA and VOC ~9.5 V) TENGs. The experimental results indicate that with the increase in the number of graphene layers, the conductivity gradually increases due to the decrease in sheet resistance, as shown in SC and VOC of the 3L-Gr-TENG. However, there is a decrease in electrical output for the 4L- and 5L-Gr-based TENGs compared with the 3L-Gr-based TENG. It should be noted that the increase in output ISC and VOC not only depends on the conductivity but also has a relationship with the work function of the triboelectric layer . The work function of graphene increases with an increase in the number of graphene layers, as discussed in The electrical output current and voltage of the Gr-TENG  with and without AlVOC ~9 V ), 4L-Gr-OC ~11 V ) and 5L-2O3) under the graphene will be an effective method to further enhance the output performance of Gr-TENGs because the electrical output of TENG strongly depends on the surface charge density [2O3 as the CTL structure was systematically investigated. Apparently, the output ISC and VOC of the Gr-TENG with Al2O3 as the CTL are remarkably enhanced compared with those of the pristine Gr-TENG, as shown in 2O3 possess output ISC and VOC of 0.33 μA and 32 V, 0.43 μA and 37 V (SC and VOC is mainly attributed to the following: (i) The increase in surface charge density over the graphene surface is due to the high charge storage capacity of Al2O3 [2O3 compared with graphene is also one of the dominant factors [2O3 increases the contact area between PTFE and the graphene layer during the triboelectrification process, providing additional support for the enhancement in output performance compared with the flexible Gr-TENG without CTL. Furthermore, SC and VOC of Gr-TENG on the Al2O3 CTL. Although the outputs increase with the CTL and lower work function of triboelectric material, the 3L-Gr-TENG with Al2O3 exhibits a maximum electrical output compared to the 1L-, 2L-, 4L- and 5L-Gr-TENGs with Al2O3. This result indicates that not only the work function but also the higher surface roughness of 3L-Gr-TENG with Al2O3  of Gr-TENG  with Al2O3 are comparatively higher than that of Gr-TENG without CTL, as shown in 2O3 CTL. However, with the increase in the number of layers (from 1L-Gr to 3L-Gr) with Al2O3 CTL, the surface charge density increases, but the further increase in the number of layers (5L-Gr) does not contribute to the increase in surface charge density of Gr-TENG, which supports the electrical analysis as demonstrated in 2O3 as a function of external load resistance ranging from 10 MΩ to 1 GΩ. The TENG output voltage gradually increases with increasing load resistance, while the output current value follows the opposite trend due to ohmic loss. 2O3 as charge trapping layer as a function of external load resistance. This output power was calculated by using Equation (3):V and I correspond to the output peak voltage and current value at various load resistances. The maximum value of the output power reaches 25 μW at a loading resistance of 300 MΩ, which is 30 times larger than that of the pristine 3L-Gr-TENG, as shown in 2O3 device shows a maximum power density of 6.25 μW/cm2 at a load resistance of ~300 MΩ, which is higher than that of a previously reported graphene-based TENG [To experimentally investigate the effect of the charge trapping layer in Gr-TENGs, we fabricated a metal–insulator–metal (MIM) device structure in which 3L graphene over Aln effect . Furthersed TENG ,43,46. T2O3 as the CTL, we continuously applied >2000 cycles  and conducting electrode (Al-foil) contributes to an enhancement in the surface charge density. The remarkable key mechanism for this enhancement can be explained as follows: (i) The high positive charge storage capacity of high-k Al2O3 [2O3 CTL and top graphene layer due to its high electrical conductivity, which helps to improve the surface charge density , as shown in 2O3, it can be used to drive the electronic timer for a long time.To further demonstrate the application of a Gr-TENG with Al2O3 CTL is summarized in 2O3 as CTL shows higher output performance than the Gr-TENG without CTL. These results indicate that the CTL in between the friction layer and conducting electrode is an effective path to improve the triboelectric property of Gr-TENGs. Thus, it is an effective strategy to fabricate the high-performance flexible Gr-TENG with Al2O3 CTL as an energy harvester.According to the aforementioned experimental results, we hereby conclude that the optimized structure, proper selection of material and surface modification of the tribolayer without degrading its inherent property such as the addition of a CTL to Gr-TENGs promotes the ability to maintain a high surface charge density, resulting in enhanced output power. A brief comparison of the electrical output performance of Gr-based TENGs with and without Al2O3 as a CTL between a positive triboelectric material (graphene) and a bottom contact electrode (Al foil). The strong tendency to repel electrons and the positive charge trapping nature of Al2O3 help to enhance the charge density on the graphene layer. By varying the number of graphene layers  and evaluating the electrical performance, we found the optimal layered structure (3L-Gr) of a flexible Gr-TENG. Maximum VOC and ISC values of ~55 V and 0.78 µA were achieved by the 3L-Gr-TENG with an Al2O3 CTL. Additionally, this TENG exhibited a maximum power of ~25 μW at a load resistance of ~300 MΩ, which was 30 times higher than that of the pristine 3L-Gr-TENG. Finally, the generated output power was capable of driving 20 commercially available green LEDs connected in series and able to turn on an electronic timer by using a rectifier circuit. Therefore, based on the above results, we believe that our proposed structure holds high promise for enhancing the surface charge density of Gr-TENGs by fusion of CTL, which possesses promising applications for future flexible and portable energy harvesting systems.In summary, we demonstrated a novel and simple fabrication methodology for enhanced triboelectric performance by introducing Al