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Frontiers in Veterinary Science 2023Arthrodesis, performed as a salvage surgical procedure to treat intractable joint conditions in dogs and cats, is associated with a high incidence of complications intra...
INTRODUCTION
Arthrodesis, performed as a salvage surgical procedure to treat intractable joint conditions in dogs and cats, is associated with a high incidence of complications intra and postoperative, proving the need for improved and new techniques in arthrodesis surgery. Adding a new resorbable bone glue to the arthrodesis could potentially add fixation strength and lower complications. The objectives of this experimental biomechanical study were therefore to develop a biomechanical test model of partial tarsal arthrodesis and to determine whether the new resorbable bone glue (phosphoserine modified cement) produced measurable fixation strength in canine calcaneoquartal arthrodesis, without orthopedic implants.
METHODS
Four biomechanical test models with a total of 35 canine tarsal joints were used. Soft tissues were dissected to 4 different test models with variable contributions from soft tissues. The calcaneoquartal joint was prepared as arthrodesis and the glue was applied to joint surfaces as a liquid/putty (0.4 cc). After curing for 24 h, a shear force was applied to the joint (1 mm per minute) and the failure strength was recorded.
RESULTS
Calcaneoquartal joints, where all soft tissues had been completely resected and fixated with glue (1-1.5 cm joint surface), withstood 2-5 mm of displacement and an average of 100 ± 58 N/cm of shear force (Model 1). Similar adhesive fixation strengths were obtained in Model 2 and 3 with increasing contributions from soft tissues (80 ± 44 and 63 ± 23 N/cm, = 0.39, ANOVA).
CONCLUSION
The developed biomechanical model was sensitive enough to measure differences in fixation strengths between different glue formulations. The average fixation strength (60-100 N/cm) should be strong enough to support short-term load bearing in medium sized canines (20 kg). The developed cadaver biomechanical test model is of potential use for other arthrodesis studies. The new resorbable glue can potentially contribute to stability at arthrodesis surgery, acting as a complement to today's standard fixation, metal implants.
PubMed: 37799403
DOI: 10.3389/fvets.2023.1250147 -
The Journal of Biological Chemistry Nov 2023Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18...
Rad18 interacts with the SMC5/6 localization factor 1 (SLF1) to recruit the SMC5/6 complex to DNA damage sites for repair. The mechanism of the specific Rad18 recognition by SLF1 is unclear. Here, we present the crystal structure of the tandem BRCT repeat (tBRCT) in SLF1 (SLF1) bound with the interacting Rad18 peptide. Our structure and biochemical studies demonstrate that SLF1 interacts with two phosphoserines and adjacent residues in Rad18 for high-affinity and specificity Rad18 recognition. We found that SLF1 utilizes mechanisms common among tBRCTs as well as unique ones for Rad18 binding, the latter include interactions with an α-helical structure in Rad18 that has not been observed in other tBRCT-bound ligand proteins. Our work provides structural insights into Rad18 targeting by SLF1 and expands the understanding of BRCT-mediated complex assembly.
Topics: Protein Binding; Protein Domains; DNA Damage; Ubiquitin-Protein Ligases; Peptides; DNA Repair
PubMed: 37748650
DOI: 10.1016/j.jbc.2023.105288 -
The Oncologist Mar 2024To date, no study has systematically explored the potential role of serum metabolites and lipids in the diagnosis of small cell lung cancer (SCLC). Therefore, we aimed...
BACKGROUND
To date, no study has systematically explored the potential role of serum metabolites and lipids in the diagnosis of small cell lung cancer (SCLC). Therefore, we aimed to conduct a case-cohort study that included 191 cases of SCLC, 91 patients with lung adenocarcinoma, 82 patients with squamous cell carcinoma, and 97 healthy controls.
METHODS
Metabolomics and lipidomics were applied to analyze different metabolites and lipids in the serum of these patients. The SCLC diagnosis model (d-model) was constructed using an integrated machine learning technology and a training cohort (n = 323) and was validated in a testing cohort (n=138).
RESULTS
Eight metabolites, including 1-mristoyl-sn-glycero-3-phosphocholine, 16b-hydroxyestradiol, 3-phosphoserine, cholesteryl sulfate, D-lyxose, dioctyl phthalate, DL-lactate and Leu-Phe, were successfully selected to distinguish SCLC from controls. The d-model was constructed based on these 8 metabolites and showed improved diagnostic performance for SCLC, with the area under curve (AUC) of 0.933 in the training cohort and 0.922 in the testing cohort. Importantly, the d-model still had an excellent diagnostic performance after adjusting the stage and related clinical variables and, combined with the progastrin-releasing peptide (ProGRP), showed the best diagnostic performance with 0.975 of AUC for limited-stage patients.
CONCLUSION
This study is the first to analyze the difference between metabolomics and lipidomics and to construct a d-model to detect SCLC using integrated machine learning. This study may be of great significance for the screening and early diagnosis of SCLC patients.
Topics: Humans; Small Cell Lung Carcinoma; Lung Neoplasms; Carcinoma, Non-Small-Cell Lung; Cohort Studies; Biomarkers, Tumor; Lipids
PubMed: 37706531
DOI: 10.1093/oncolo/oyad261 -
RSC Advances Sep 2023Chemodynamic therapy (CDT) has received more and more attention as an emerging therapeutic strategy, especially transition metals with Fenton or Fenton-like activity...
Chemodynamic therapy (CDT) has received more and more attention as an emerging therapeutic strategy, especially transition metals with Fenton or Fenton-like activity have good effects in CDT research, manganese dioxide nanosheets (MnO NSs) and their complexes have become one of the most favored nanomaterials in CDT of tumors. CDT is mainly based on the role of reactive oxygen species (ROS) in tumor treatment, which have clear chemical properties and produce clear chemical reactions. However, their mechanism of interaction with cells has not been fully elucidated. Here, we performed CDT on mouse breast cancer cells (4T1) based on MnO NSs, extracted the metabolites from the 4T1 cells during the treatment, and analyzed the differences in metabolites by using high-resolution liquid chromatography-mass spectrometry (LC-MS). Untargeted metabolomics studies were conducted using the relevant data. This study mainly explored the changes in MnO NSs on the metabolite profile of 4T1 cells and their potential impact on tumor therapy, in order to determine the mechanism of action of MnO NSs in the treatment of breast cancer. The results of the study showed the presence of 11 different metabolites in MnO NSs CDT for 4T1 tumor cells, including phosphoserine, sphingine, phosphocholine, and stearoylcarnitine. These findings provide a deeper understanding of breast cancer treatment, and are beneficial for the further research and clinical application of CDT.
PubMed: 37681048
DOI: 10.1039/d3ra03992g -
Biomolecules Aug 2023In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate...
In humans, the phosphorylated pathway (PP) converts the glycolytic intermediate D-3-phosphoglycerate (3-PG) into L-serine through the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase (PSAT) and phosphoserine phosphatase. From the pathogenic point of view, the PP in the brain is particularly relevant, as genetic defects of any of the three enzymes are associated with a group of neurometabolic disorders known as serine deficiency disorders (SDDs). We recombinantly expressed and characterized eight variants of PSAT associated with SDDs and two non-SDD associated variants. We show that the pathogenetic mechanisms in SDDs are extremely diverse, including low affinity of the cofactor pyridoxal 5'-phosphate and thermal instability for S179L and G79W PSAT, loss of activity of the holo form for R342W PSAT, aggregation for D100A PSAT, increased K for one of the substrates with invariant ks for S43R PSAT, and a combination of increased K and decreased k for C245R PSAT. Finally, we show that the flux through the in vitro reconstructed PP at physiological concentrations of substrates and enzymes is extremely sensitive to alterations of the functional properties of PSAT variants, confirming PSAT dysfunctions as a cause of SSDs.
Topics: Humans; Transaminases; Brain; Pyridoxal Phosphate; Serine
PubMed: 37627284
DOI: 10.3390/biom13081219 -
NPJ Vaccines Aug 2023In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and...
In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.
PubMed: 37573422
DOI: 10.1038/s41541-023-00711-0 -
Archives of Biochemistry and Biophysics Aug 2023Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase...
Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase family. It is a pseudophosphatase, which lacks the essential amino acids histidine and cysteine in the catalytic active signature motif (HCXR). We previously reported that MK-STYX interacts with G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding-1] and reduces stress granules, stalled mRNA. To determine how MK-STYX reduces stress granules, truncated domains, CH2 (cell division cycle 25 phosphatase homology 2) and DUSP, of MK-STYX were used. Wild-type MK-STYX and the DUSP domain significantly decreased stressed granules that were induced by sodium arsenite, in which G3BP1 (a stress granule nucleator) was used as the marker. In addition, HEK/293 and HeLa cells co-expressing G3BP1-GFP and mCherry-MK-STYX, mCherry-MK-STYX-CH2, mCherry-MK-STYX-DUSP or mCherry showed that stress granules were significantly decreased in the presence of wild-type MK-STYX and the DUSP domain of MK-STYX. Further characterization of these dynamics in HeLa cells showed that the CH2 domain increased the number of stress granules within a cell, relative to wild-type and DUSP domain of MK-STYX. To further analyze the interaction of G3BP1 and the domains of MK-STYX, coimmunoprecipitation experiments were performed. Cells co-expressing G3BP1-GFP and mCherry, mCherry-MK-STYX, mCherry-MK-STYX-CH2, or mCherry-MK-STYX-DUSP demonstrated that the DUSP domain of MK-STYX interacts with both G3BP1-GFP and endogenous G3BP1, whereas the CH2 domain of MK-STYX did not coimmunoprecipitate with G3BP1. In addition, G3BP1 tyrosine phosphorylation, which is required for stress granule formation, was decreased in the presence of wild-type MK-STYX or the DUSP domain but increased in the presence of CH2. These data highlight a model for how MK-STYX decreases G3BP1-induced stress granules. The DUSP domain of MK-STYX interacts with G3BP1 and negatively alters its tyrosine phosphorylation- decreasing stress granule formation.
Topics: Humans; DNA Helicases; HeLa Cells; Intracellular Signaling Peptides and Proteins; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; Stress Granules; Tyrosine
PubMed: 37516290
DOI: 10.1016/j.abb.2023.109702 -
Molecular Systems Biology Aug 2023Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site-specifically incorporate biologically...
Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site-specifically incorporate biologically relevant non-standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post-translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems-level biology of OTS development and utilization remains underexplored. In this study, we employ a phosphoserine OTS (pSerOTS) as a model to systematically investigate global interactions between OTS components and the cellular environment, aiming to improve OTS performance. Based on this analysis, we design OTS variants to enhance orthogonality by minimizing host process interactions and reducing stress response activation. Our findings advance understanding of system-wide OTS:host interactions, enabling informed design practices that circumvent deleterious interactions with host physiology while improving OTS performance and stability. Furthermore, our study emphasizes the importance of establishing a pipeline for systematically profiling OTS:host interactions to enhance orthogonality and mitigate mechanisms underlying OTS-mediated host toxicity.
Topics: Amino Acids; Phosphorylation; Protein Processing, Post-Translational; Amines
PubMed: 37477096
DOI: 10.15252/msb.202110591 -
Acta Neuropathologica Communications Jul 2023Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis...
Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1β, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.
Topics: Animals; Mice; Antibodies, Monoclonal; Epitopes; Immunization; Intermediate Filaments; NF-kappa B
PubMed: 37434215
DOI: 10.1186/s40478-023-01592-z -
MBio Aug 2023HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK...
HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.
Topics: Humans; Down-Regulation; HIV-1; Ligands; Antibody-Dependent Cell Cytotoxicity; Killer Cells, Natural; Signaling Lymphocytic Activation Molecule Family; HIV Infections
PubMed: 37404017
DOI: 10.1128/mbio.00789-23