Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast-associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied

The prognostic utility of prehospital qSOFA in addition to emergency department qSOFA for sepsis in patients with suspected infection: A retrospective cohort study.

BackgroundThe quick sequential organ failure assessment (qSOFA) was widely used to estimate the risks of sepsis in patients with suspected infection in the prehospital and emergency department (ED) settings. Due to the insufficient sensitivity of qSOFA on arrival at the ED (ED qSOFA), the Surviving Sepsis Campaign 2021 recommended against using qSOFA as a single screening tool for sepsis. However, it remains unclear whether the combined use of prehospital and ED qSOFA improves its sensitivity for identifying patients at a higher risk of sepsis at the ED.MethodsWe retrospectively analyzed the data from the ED of a tertiary medical center in Japan from April 2018 through March 2021. Among all adult patients (aged ≥18 years) transported by ambulance to the ED with suspected infection, we identified patients who were subsequently diagnosed with sepsis based on the Sepsis-3 criteria. We compared the predictive abilities of prehospital qSOFA, ED qSOFA, and the sum of prehospital and ED qSOFA (combined qSOFA) for sepsis in patients with suspected infection at the ED.ResultsAmong 2,407 patients with suspected infection transported to the ED by ambulance, 369 (15%) patients were subsequently diagnosed with sepsis, and 217 (9%) died during hospitalization. The sensitivity of prehospital qSOFA ≥2 and ED qSOFA ≥2 were comparable (c-statistics for sepsis [95%CI], 0.57 [0.52-0.62] vs. 0.55 [0.50-0.60]). However, combined qSOFA (cutoff, ≥3 [max 6]) was more sensitive than ED qSOFA (cutoff, ≥2) for identifying sepsis (0.67 [95%CI, 0.62-0.72] vs. 0.55 [95%CI, 0.50-0.60]). Using combined qSOFA, we identified 44 (12%) out of 369 patients who were subsequently diagnosed with sepsis, which would have been missed using ED qSOFA alone.ConclusionsUsing both prehospital and ED qSOFA could improve the screening ability of sepsis among patients with suspected infection at the ED

Risk factors of sepsis among patients with qSOFA<2 in the emergency department

Objective: Studies have suggested that qSOFA can be used for early detection of sepsis immediately upon arrival at the emergency department (ED). Despite this, little is known about the risk factors associated with the subsequent diagnosis of sepsis among patients with qSOFA<2 in the ED. Methods: This is a retrospective cohort study using ED data from a large tertiary medical center in Japan, 2018–2020. We included adult patients (aged ≥18 years) presenting to the ED with suspected infection (e.g., having a fever) and qSOFA<2. We identified patients who developed sepsis based on the Sepsis-3 criteria, and compared patient characteristics (e.g., demographics, vital signs upon the initial triage, chief complaint, and comorbidities) between patients who developed sepsis or not. Additionally, we identified the potential risk factors of sepsis among patients with qSOFA<2 using a multivariable logistic regression model. Results: We identified 151 (7%) patients who developed sepsis among 2025 adult patients with suspected infection and qSOFA<2. Compared with patients who did not develop sepsis, patients who developed sepsis were likely to be older and have vital signs suggestive of imminent sepsis (e.g., high respiratory rate). In the multivariable logistic regression model, the potential risk factors of sepsis among patients with qSOFA<2 were older age (adjusted OR, 1.92 [95%CI 1.19–3.19]), vital signs suggestive of imminent sepsis (e.g., adjusted OR of altered mental status, 3.50 [95%CI 2.25–5.50]), receipt of oxygen therapy upon arrival at the ED (adjusted OR, 1.91 [95%CI 1.38–2.26]), chief complaint of sore throat (adjusted OR, 2.15 [95%CI 1.08–4.13]), and the presence of comorbid diabetes mellitus, ischemic heart disease, and chronic kidney disease (e.g., adjusted OR of diabetes mellitus, 1.47 [95%CI 1.10–1.96]). On the contrary, chief complaint of abdominal and chest pain were associated with a lower risk of sepsis (e.g., adjusted OR of abdominal pain, 0.26 [95%CI 0.14–0.45]). Conclusions: We found that older age, vital signs prognosticating sepsis, and the presence of some comorbidities were the potential risk factors of sepsis in patients with qSOFA<2. These potential risk factors could be useful to efficiently recognize patients who might develop sepsis in the ED.12 month embargo; available online: 27 September 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Saireito (TJ-114), a Japanese traditional herbal medicine, reduces 5-fluorouracil-induced intestinal mucositis in mice by inhibiting cytokine-mediated apoptosis in intestinal crypt cells.

Clinical chemotherapy frequently causes intestinal mucositis as a side effect, which is accompanied by severe diarrhea. We recently showed that the cytokine-mediated apoptotic pathway might be important for the development of intestinal mucositis induced by 5-fluorouracil (5-FU). Saireito, the traditional Japanese herbal (Kampo) medicine, is widely used to treat diarrhea and various inflammatory diseases in Japan. In the present study, we investigated the effect of saireito on 5-FU-induced intestinal mucositis in mice, especially in relation to apoptosis in the intestinal crypt. Male C57BL/6 mice were given 5-FU (50 mg/kg), i.p. once daily for 6 days. Intestinal mucositis was evaluated histochemically. Saireito (100-1000 mg/kg) was administered p.o. twice daily for 6 days. Repeated 5-FU treatment caused severe intestinal mucositis including morphological damage, which was accompanied by body weight loss and diarrhea. Daily administration of saireito reduced the severity of intestinal mucositis in a dose-dependent manner. Body weight loss and diarrhea during 5-FU treatment were also significantly attenuated by saireito administration. The number of apoptotic and caspase-3-activated cells in the intestinal crypt was increased, and was accompanied by up-regulated tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA within 24 h of the first 5-FU injection. However, all of these measures were significantly lower after saireito administration. These results suggest that saireito attenuates 5-FU-induced intestinal mucositis. This action may come from the reduction of apoptosis in the intestinal crypt via suppression of the up-regulation of inflammatory cytokines. Therefore, saireito may be clinically useful for the prevention of intestinal mucositis during cancer chemotherapy

Identification of Nuclear Localization Signals in the Human Homeoprotein, MSX1

MSX1 is one of the homeoproteins with the homeodomain (HD) sequence, which regulates proliferation and differentiation of mesenchymal cells. In this study, we investigated the nuclear localization signal (NLS) in the MSX1 HD by deletion and amino acid substitution analyses. The web-based tool NLStradamus predicted two putative basic motifs in the N- and C-termini of the MSX1 HD. Green fluorescent protein (GFP) chimera studies revealed that NLS1 (161RKHKTNRKPR170) and NLS2 (216NRRAKAKR223) were independently insufficient for robust nuclear localization. However, they can work cooperatively to promote nuclear localization of MSX1, as was shown by the two tandem NLS motifs partially restoring functional NLS, leading to a significant nuclear accumulation of the GFP chimera. These results demonstrate a unique NLS motif in MSX1, which consists of an essential single core motif in helix-I, with weak potency, and an auxiliary subdomain in helix-III, which alone does not have nuclear localization potency. Additionally, other peptide sequences, other than predicted two motifs in the spacer, may be necessary for complete nuclear localization in MSX1 HD.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single -guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets