Role of interleukin-17 in a murine community-associated methicillin-resistant Staphylococcus aureus pneumonia model

Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.     

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser⁶⁵ by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.     

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.     

Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site

Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo.     

Are black flies of the subgenus Wilhelmia (Diptera: Simuliidae) multiple species or a single geographical generalist? Insights from the macrogenome

Organisms with vast distributions often represent geographical mosaics of cryptic species. The black fly Simulium (Wilhelmia) lineatum is among the most widely distributed members of the family Simuliidae, ranging from the British Isles to eastern China. Rather than viewing S. lineatum as a possible aggregate of multiple species, taxonomists have suggested a more inclusive taxon with additional synonyms. Accordingly, S. lineatum is an ideal candidate for testing the hypothesis that a wide geographical distribution signals the presence of more than one species. A cytogenetic approach was used to probe the macrogenome of S. lineatum and other taxa proposed by taxonomists as conspecific. The banding patterns in the polytene chromosomes of 480 larvae from 15 countries across the Palearctic Region revealed 128 rearrangements of the complement. All rearrangements were autosomal and 89% were inversions nonrandomly distributed among species and among chromosome arms. The analyses clarify long‐standing confusion over previously proposed names and reveal a longitudinal succession of four species sequentially replacing one another from west to east: Simulium lineatum s.s., Simulium balcanicum, Simulium turgaicum, and Simulium takahasii. Thus, S. turgaicum is recalled from synonymy and the other three species are validated. Within the most‐represented species, S. balcanicum, the frequency of inversions follows a longitudinal gradient with a north–south bias; as the distance between the sites increases along this north‐west–south‐east axis, the similarity of inversion frequencies between sites decreases. Validation of the concept that broadly distributed black flies are composites of structurally similar species provides a framework for guiding discovery of additional biodiversity. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 163–183.     

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8⁺ T-cell responses in convalescent individuals, the role of virus-specific CD8⁺ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8⁺ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 10⁵ or 10⁶ TCID₅₀ of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8⁺ T cells were undetectable on day 7 and thereafter, while virus-specific CD8⁺ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10–17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8⁺ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8⁺ T cells, implying that CD8⁺ T-cell dysfunction may not solely lead to viral control failure.     

Social buffering reduces male rats' behavioral and corticosterone responses to a conditioned stimulus

In social species, the presence of an affiliative same-sex conspecific ameliorates acute stress responses in threatening conditions. We previously found that the presence of an unfamiliar male rat separated by a wire mesh barrier blocks the behavioral responses and Fos expression in the paraventricular nucleus of the hypothalamus (PVN) in a male subject rat that had previously been exposed to an auditory conditioned stimulus (CS) paired with foot shocks. Based on the Fos expression in the PVN, we hypothesized that the presence of a conspecific ameliorated the hypothalamic–pituitary–adrenal (HPA) axis activation and induced social buffering of conditioned fear responses. The direct evidence for this hypothesis, however, is still lacking. To clarify this point, we exposed fear-conditioned and non-conditioned subjects to the CS either alone or with a conspecific separated by a wire mesh barrier. When the fear-conditioned subject alone was re-exposed to the CS, it exhibited increased freezing, decreased sniffing, and elevated corticosterone levels. In contrast, the presence of the conspecific suppressed these behavioral and HPA axis responses to a level similar to those observed in the non-conditioned subjects. These results suggest that the presence of a conspecific suppressed the behavioral responses and HPA axis activation to the CS. The present results provide direct evidence for the existence of social buffering of conditioned fear responses in male rats.