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The autoantigen La protein is an important component of telomerase and a predominantly nuclear phosphoprotein. As a telomerase subunit, La protein associates with the telomerase ribonucleoprotein and influences telomere length. In the reverse transcription, La protein stimulates enzymatic activity and increases repeated addition processivity of telomerase. As nuclear phosphoprotein, La protein binds the 3′ poly(U)‐rich elements of nascent RNA polymerase III transcripts to facilitate its correct folding and maturation. In this work, we identified a La protein homolog (TbLa) from Trypanosoma brucei (T. brucei). We revealed that TbLa interacts with ribosome‐associated protein P34/P37, 40S ribosomal protein SA, and 60S ribosomal subunit L5 in T. brucei. In the interactions between TbLa protein and (P34/P37)/L5/SA, RNA recognition motif (RRM) domain of TbLa was indicated to make the major contribution to the processes. We determined the solution structure of TbLa RRM domain. NMR chemical shift perturbations revealed that the positively charged RNA‐binding pocket of TbLa RRM domain is responsible for its interaction with ribosomal and ribosome‐associated proteins P37/L5/SA. Furthermore, depletion of TbLa affected the maturation process of 5S rRNA and ribosomal assembly, suggesting TbLa protein might play a significant role in the ribosomal biogenesis pathway in T. brucei. Taken together, our results provide a novel insight and structural basis for better understanding the roles of TbLa and RRM domain in ribosomal biogenesis in T. brucei. Database: Structural data are available in the PDB under the accession number 5ZUH. [ABSTRACT FROM AUTHOR]

Centrin is an evolutionarily conserved EF‐hand‐containing protein, which is present in eukaryotic organisms as diverse as algae, yeast, and humans. Centrins are associated with the microtubule‐organizing center and with centrosome‐related structures, such as basal bodies in flagellar and ciliated cells, and the spindle pole body in yeast. Five centrin genes have been identified in Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub‐Saharan Africa. In the present study, we identified that centrin5 of T. brucei (TbCentrin5) is localized throughout the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca2+ ions with a high affinity and constructed a model of TbCentrin5 bound by Ca2+ ions. Meanwhile, we observed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 and that the interactions are Ca2+‐dependent, suggesting that TbCentrin5 is able to form different complexes with other TbCentrins to participate in relevant cellular processes. Our study provides a foundation for better understanding of the biological roles of TbCentrin5. [ABSTRACT FROM AUTHOR]

DNAX activating protein of 12 kDa (DAP12)-deficiency mice showed impaired differentiation of oligodendrocytes and reduced myelin in the central nervous system. Whether DAP12 is expressed by Schwann cells and its roles in the peripheral nervous system (PNS) remains unknown. In this study, expression of DAP12 was detected in Schwann cells in vivo and in vitro. The DAP12-knockout (KO) mice showed age-related motor deficits and thinner myelin in the sciatic nerve than WT mice but significantly faster clinical recovery after sciatic nerve crush injury. In sciatic nerves of DAP12 KO and WT mice, proteomic profiles analysis identified 158 differentially expressed proteins (DEPs) at 8-week-old, 29 DEPs at 54-week-old and 33 DEPs at two weeks after crush injury. Typically, of the DEPs at 54-week-old, up-regulated Lgmn and down-regulated RecK and Yap1 were associated with myelin loss in the sciatic nerve of DAP12 KO mice. Upregulation of nicotinamide nucleotide transhydrogenase and haptoglobin were associated with the accumulation of macrophages in the crushed sciatic nerve of DAP12 KO mice. After crush injury, there were significantly more M1 macrophages at one-week and more M2 macrophages at two-week in sciatic nerve of DAP12 KO mice than WT mice, indicating that DAP12 deletion promotes the phenotype conversion of macrophages from M1 to M2. Collectively, our findings suggest that DAP12 may exert dual roles in the PNS including promoting the physiological myelin formation and maintenance of Schwann cells but delaying nerve repair after injury by modulating the recruitment of macrophages and phenotype conversion. [Display omitted] • We demonstrated the expression of DNAX activating protein of 12 kDa (DAP12) in Schwann cells. • DAP12 deletion causes age-related motor deficits in peripheral nerve. • DAP12 deletion promotes the clinical recovery of mutant mice from sciatic nerve crush injury. [ABSTRACT FROM AUTHOR]

Schwann cells (SCs) isolation is one of the basic techniques for study of peripheral nervous system and peripheral neuropathy. A combined and effective method of isolating SCs from sciatic nerves of newborn mice with high yield and purity is still lacking. Sciatic nerves from neonatal mice aged 3–5 days serve as the source of SCs. Removal of adjacent connective tissue and epineurium, treatment with arabinoside hydrochloride and differential cell detachment technique were applied to eliminate fibroblast contamination and increase the purity of SCs. Combined use of collagenase/dispase and trypsin was chosen to increase the yield of SCs. Culture dishes precoated with poly-l-lysine and laminin, culture medium supplemented with heregulin β-1 and forskolin, and reasonable cell seeding density were implemented to increase the growth and proliferation of cultured SCs. Immunostaining of S100β and p75 neurotrophin receptor was used to identify the purity of SCs. Our method is able to obtain high-yield SCs with a purity of 90% within five days and a purity more than 99% within seven days from sciatic nerves of neonatal mice. Previous SCs isolation mostly focused on rats or adult mice and have a few limitations due to fibroblasts contamination, low yield and time-consuming. Our method permits SCs isolation from neonatal mice with a high yield and purity of primary SCs within 7 days. We described a fast, efficient and step-by-step method of isolating SCs from sciatic nerves of neonatal mice with high yield and purity. [Display omitted] • A new method of isolating Schwann cells (SCs) from newborn mice was described. • A high yield and purity (> 99%) of SCs isolation were obtained. • The whole procedure can be completely accomplished within 7 days. [ABSTRACT FROM AUTHOR]

Solution structure of TbUfm1 and its binding surface to TbUba5 (marked in red). • TbUfm1 adopts a typical β-grasp fold and helixes that wrapped by the β-grasp fold. • TbUfm1 can bind to TbUba5 in vitro. • TbUfm1 interact with TbUba5 via the hydrophobic pocket formed by α1α2β1β2. Ubiquitin-like proteins are conserved in eukaryotes and involved in numerous cellular processes. Ufm1 is proved to play important roles in endoplasmic reticulum homeostasis, vesicle transportation and embryonic development. Enzyme cascade of Ufm1 is similar to that of ubiquitin. Mature Ufm1 is activated and conjugated to substrates by assistance of Ufm1 activating enzyme Uba5 (E1), Ufm1 conjugating enzyme Ufc1 (E2), and Ufm1 ligating enzyme Ufl1 (E3). Here, we determined the solution structure of TbUfm1 from Trypanosoma brucei (T. brucei) by NMR spectroscopy and explored the interactions between TbUfm1 and TbUba5/TbUfc1/TbUfl1. TbUfm1 adopts a typical β-grasp fold, which partially wraps a central α-helix and the other two helixes. NMR chemical shift perturbation indicated that TbUfm1 interacts with TbUba5 via a hydrophobic pocket formed by α1α2β1β2. Although the structure and Uba5-interaction mode of TbUfm1 are conserved in Ufm1 proteins, there are also some differences, which might reflect the potential diversity of Ufm1 in evolution and biological functions. [ABSTRACT FROM AUTHOR]

In acute inflammatory demyelinating polyneuropathy (AIDP), myelin vesiculation mediated by complement activation contributes to nerve injury. Macrophage infiltration of the spinal roots has been demonstrated in AIDP, but its pathological significance remains uncertain. The present study aimed to investigate the role of macrophages in the pathogenic sequence of AIDP. A rabbit model of AIDP was induced by immunization with galactocerebroside. Immunostaining was performed to localize the macrophages and myelin injury. The rabbit developed tetraparesis with electrophysiological and pathological features of peripheral nerve demyelination. Immunostaining demonstrated colocalization of IgG antibodies, complement deposition and myelin injury apart from macrophages. Immunostaining and electron microscopy showed myelin injury preceded macrophage infiltration. There was significant disruption of voltage-gated sodium channel clusters at the nodes of Ranvier in the spinal roots. Macrophages acted may as scavengers to remove myelin debris following complement activation-mediated demyelination in the AIDP rabbit. Lesions at the node of Ranvier contribute to conduction failure and muscle weakness., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Although COVID-19 pneumonia is spreading internationally, knowledge regarding the factors associated with the illness severity of patients remains limited. We aimed to identify the factors associated with the disease severity of patients with COVID-19 pneumonia induced by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We prospectively enrolled a single-center case series of adult patients with COVID-19 admitted to the Infectious Disease Hospital of Jining, Jining City, Shandong Province, China, from January 24 to March 1, 2020. Demographics, clinical characteristics, and laboratory findings were compared to investigate the risk factors related with the disease severity of COVID-19 pneumonia patients. Results: We included a total of 78 patients with COVID-19 pneumonia, of whom 6 had the severe type. As compared to a moderately ill cohort, our analysis showed that shortness of breath, fatigue, longer days from illness onset to diagnosis confirmed, neutrophil percentages > 70%, neutrophil counts > 6.3 × 10 9 /L, lymphocyte percentages < 20%, lymphocyte counts < 1.0 × 10 9 /L, platelet < 100 × 10 9 /L, C-reactive protein (CRP) > 10 mg/L, neutrophil to platelet ratio (NPR) > 2.3, neutrophil to lymphocyte ratio (NLR) > 3.9, aspartate aminotransferase (AST) > 40 U/L, albumin < 40 g/L, lactate dehydrogenase (LDH) > 245 U/L, and glucose > 6.1 mmol/L were predictors of disease severity in COVID-19 pneumonia. In the sex-, age-, and comorbid illness-matched case-control study, neutrophil percentages > 70%, neutrophil counts > 6.3 × 10 9 /L, lymphocyte percentages < 20%, NPR > 2.3, NLR > 3.9, albumin < 40 g/L, and LDH > 245 U/L remained associated with the early detection and identification of severe patients. Conclusion: We demonstrated that neutrophil percentages > 70%, neutrophil counts > 6.3 × 10 9 /L, lymphocyte percentages < 20%, NPR > 2.3, NLR > 3.9, albumin < 40 g/L, and LDH > 245 U/L might predict the severity of illness in patients with COVID-19 pneumonia., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Kinetochores are the macromolecular protein complex that drives chromosome segregation by interacting with centromeric DNA and spindle microtubules in eukaryotes. Kinetochores in well studied eukaryotes bind DNA through widely conserved components like Centromere Protein (CENP)-A and bind microtubules through the Ndc80 complex. However, unconventional type of kinetochore proteins (KKT1-20) were identified in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei (T. brucei), indicating that chromosome segregation is driven by a distinct set of proteins. KKT proteins are comprised of sequential α-helixes that tend to form coiled-coil structures, which will further lead to polymerization and misfolding of proteins, resulting in the formation of inclusion bodies., Results and Conclusion: We expressed and purified the stable KKT proteins with Maltose Binding Protein (MBP) fusion tag in E. coli or Protein A tag in Human Embryonic Kidney (HEK) 293T cells. Furthermore, we identified interactions among KKT proteins using yeast two-hybrid system. The study provides an important basis for further better understanding of the structure and function of KKT proteins., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Centrin is a conserved calcium-binding protein that plays an important role in diverse cellular biological processes such as ciliogenesis, gene expression, DNA repair and signal transduction. In Trypanosoma brucei , TbCentrin4 is mainly localized in basal bodies and bi-lobe structure, and is involved in the processes coordinating karyokinesis and cytokinesis. In the present study, we solved the solution structure of TbCentrin4 using NMR (nuclear magnetic resonance) spectroscopy. TbCentrin4 contains four EF-hand motifs consisting of eight α-helices. Isothermal titration calorimetry experiment showed that TbCentrin4 has a strong Ca 2+ binding ability. NMR chemical shift perturbation indicated that TbCentrin4 binds to Ca 2+ through its C-terminal domain composed of EF-hand 3 and 4. Meanwhile, we revealed that TbCentrin4 undergoes a conformational change and self-assembly induced by high concentration of Ca 2+ Intriguingly, localization of TbCentrin4 was dispersed or disappeared from basal bodies and the bi-lobe structure when the cells were treated with Ca 2+ in vivo , implying the influence of Ca 2+ on the cellular functions of TbCentrin4. Besides, we observed the interactions between TbCentrin4 and other Tbcentrins and revealed that the interactions are Ca 2+ dependent. Our findings provide a structural basis for better understanding the biological functions of TbCentrin4 in the relevant cellular processes., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)