Data_Sheet_1_Identification of bacteria and fungi responsible for litter decomposition in desert steppes via combined DNA stable isotope probing.docx

IntroductionSoil microorganisms play crucial roles in determining the fate of litter in desert steppes because their activities constitute a major component of the global carbon (C) cycle. Human activities lead to increased ecosystem nitrogen (N) deposition, which has unpredictable impacts on soil microorganism diversity and functions. Nowadays, it is necessary to further study the succession of these microorganisms in the process of litter decomposition in desert steppe, and explore the effect of N deposition on this process. This issue is particularly important to resolve because it contributes to the broader understanding of nutrient cycling processes in desert steppes.MethodsIn this study, DNA stable isotope probing (DNA-SIP) was used to study changes in soil bacterial and fungal community composition and function during 8 weeks of culture of 13C-labeled litter in desert steppes.ResultsThe results were as follows: (1) Actinomycetota, Pseudomonadota, and Ascomycota are the main microorganisms involved in litter decomposition in desert steppes; (2) N deposition (50 kg ha−1 year−1) significantly increased the relative abundance of some microorganisms involved in the decomposition process; and (3) N deposition likely promotes litter decomposition in desert steppes by increasing the abundances of N cycles bacteria (usually carrying GH family functional genes).DiscussionThese findings contribute to a deeper understanding of the C assimilation mechanisms associated with litter residue production, emphasizing the importance of extensive C utilization.</p

Additional file 2: Figure S2. of Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Neither ThioS positive structures nor neurodegeneration are observed in AAV-αsyn animals. (a-i) Sagittal brain sections were incubated with anti human asyn antibody followed by 5 min in 1% thioS solution. Thalamus (a-c) and cortex (d-f) of AAV-asyn animal show strong asyn immunoreactivity (a, d) that is not thioS- positive (b, e). As a control, human DLBD brain was co-stained in parallel. Cortical Lewy bodies positive for human asyn (g) are reactive to thioS (h, i). Representative images of NeuN-labeled cells in the cortex of AAV-asyn (n = 9) and AAV-venus (n = 7) at 6 months of age (k). Quantification of NeuN-positive cells in the whole cortex (area delineated in blue). Data are presented as as mean ± S.E.M means. Scale bars in i = 40 μm and applied to a-h; Scale bars in k = 2 mm. Abbreviation: DLBD; Diffuse Lewy Body Disease. (PDF 1541 kb

Additional file 1: Figure S1. of Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Representative intensity of Human αsyn immunostaining (a) Photomicrographs representative of the variability of expression observed in the different group of animals at 1, 3 and 6 months of age (b) Level of expression of the transgene was assessed by western blot in AAV-αsyn at 3 months of age and compared to transgenic mice overexpressing αsyn under Thy1 promoter (line 61) at the same age. Antibody recognizing human and mouse αsyn was used (clone 42). (c) Quantification of the western blot shows αsyn level increase of 2.93 ± 0.33 fold in the AAV-αsyn animals and 3.23 ± 0.12 fold in the line 61. .The data are expressed as the amount of total level of αsyn normalized to actin (*, p < 0.05) and are from 3 repeated experiments. (PDF 1678 kb

Rabbit mAbs against pS409/410-TDP-43 detect TDP-43 pathology in brain tissues from FTLD and ALS patients and rNLS8 mice.

(A‒C) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the frontal cortex of normal controls (A), FTLD-TDP type A patients (B), and FTLD-TDP type B patients (C), and in the motor cortex of ALS patients (C). Black arrows indicate neuronal cytoplasmic inclusions (NCI), and red arrows mark dystrophic neurites (DN). Inserts in B are higher magnifications of neuronal intranuclear inclusions (NCII). (D) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the cortex of non-transgenic (nTg) and rNLS8 mice. Inserts are higher magnifications of NCI. (E) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the cortex of AAV-2R and AAV-149R mice. Inserts are higher magnifications of inclusions. For all panels, scale bars are 20 μm.</p

Schematic representation of the methods and procedures to generate, screen and characterize monoclonal antibodies against pS409/410-TDP-43.

Schematic representation of the methods and procedures to generate, screen and characterize monoclonal antibodies against pS409/410-TDP-43.</p

26H10 rabbit mAb exhibits high specificity to pS409/410-TDP-43.

26H10 rabbit mAb exhibits high specificity to pS409/410-TDP-43.</p

Immunoreactivity of B cell clones against TDP-43 species as measured by ELISA immunoassay.

Immunoreactivity of B cell clones against TDP-43 species as measured by ELISA immunoassay.</p

26H10 rabbit mAb detects TDP-43 pathology in FTLD-TDP brain tissues.

26H10 rabbit mAb detects TDP-43 pathology in FTLD-TDP brain tissues.</p

Characteristics of patients with FTD/ALS.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.</div

Rabbit mAbs against pS409/410-TDP-43 detect the pathological accumulation of phosphorylated TDP-43 in FTLD-TDP brain tissues.

(A) Immunoblot analysis of urea-soluble fractions from the frontal cortex of FTLD-TDP patients and normal controls using the indicated rabbit mAbs. (B) MSD analysis of phosphorylated TDP-43 protein levels in urea-soluble fractions from the frontal cortex of FTLD-TDP patients and normal controls using the indicated rabbit mAbs (n = 4−5 per group). Data shown as the mean ± SEM. ** (left to right) P  =  0.0050, 0.0091 and 0.0075, unpaired two-tailed t-test.</p