Machine learning-based motion tracking reveals an inverse correlation between adhesivity and surface motility of the leptospirosis spirochete.

Bacterial motility is often a crucial virulence factor for pathogenic species. A common approach to study bacterial motility is fluorescent labeling, which allows detection of individual bacterial cells in a population or in host tissues. However, the use of fluorescent labeling can be hampered by protein expression stability and/or interference with bacterial physiology. Here, we apply machine learning to microscopic image analysis for label-free motion tracking of the zoonotic bacterium Leptospira interrogans on cultured animal cells. We use various leptospiral strains isolated from a human patient or animals, as well as mutant strains. Strains associated with severe disease, and mutant strains lacking outer membrane proteins (OMPs), tend to display fast mobility and reduced adherence on cultured kidney cells. Our method does not require fluorescent labeling or genetic manipulation, and thus could be applied to study motility of many other bacterial species.

Force Measurement of Bacterial Swimming Using Optical Tweezers.

Velocity is a physical parameter most commonly used to quantify bacterial swimming. In the steady-state motion at a low Reynolds number, the swimming force can be estimated from the swimming velocity and the drag coefficient based on the assumption that the swimming force balances with the drag force exerted on the bacterium. Though the velocity-force relation provides a significant clue to understand the swimming mechanism, the odd configuration of bacteria could develop problems with the accuracy of the force estimation. This chapter describes the force measurement using optical tweezers. The method uses parameters obtained from the shape and movement of a microsphere attached to the bacteria, improving the quantitativeness of force measurement.

Implications of back-and-forth motion and powerful propulsion for spirochetal invasion.

The spirochete Leptospira spp. can move in liquid and on a solid surface using two periplasmic flagella (PFs), and its motility is an essential virulence factor for the pathogenic species. Mammals are infected with the spirochete through the wounded dermis, which implies the importance of behaviors on the boundary with such viscoelastic milieu; however, the leptospiral pathogenicity involving motility remains unclear. We used a glass chamber containing a gel area adjoining the leptospiral suspension to resemble host dermis exposed to contaminated water and analyzed the motility of individual cells at the liquid-gel border. Insertion of one end of the cell body to the gel increased switching of the swimming direction. Moreover, the swimming force of Leptospira was also measured by trapping single cells using an optical tweezer. It was found that they can generate [Formula: see text] 17 pN of force, which is [Formula: see text] 30 times of the swimming force of Escherichia coli. The force-speed relationship suggested the load-dependent force enhancement and showed that the power (the work per unit time) for the propulsion is [Formula: see text] 3.1 × 10-16 W, which is two-order of magnitudes larger than the propulsive power of E. coli. The powerful and efficient propulsion of Leptospira using back-and-forth movements could facilitate their invasion.

A Pseudopolyrotaxane for Glucose-Responsive Insulin Release: The Effect of Binding Ability and Spatial Arrangement of Phenylboronic Acid Group.

A pseudopolyrotaxane (PPRX) comprising 3-carboxy-5-nitrophenylboronic acid modified γ-cyclodextrin (NPBA-γ-CyD) and naphthalene modified polyethylene glycol (Naph-PEG) as a sugar-responsive supramolecular structure is prepared. The binding of sugar by the NPBA group induced disintegration of the Naph-PEG/NPBA-γ-CyD PPRX, allowing the components to be dissolved. The Naph-PEG/NPBA-γ-CyD PPRX exhibited better sensitivity compared to that of a PPRX based on 4-carboxyphenylboronic acid modified γ-cyclodextrin (PBA-γ-CyD). We have previously reported the unique structure of Naph-PEG/PBA-γ-CyD PPRX, which formed an inclusion complex with a single-stranded PEG chain being threaded through the γ-CyD rings, with the remaining internal space being occupied by the sugar-sensing PBA moiety from a neighboring ring, thus shielding it from sugar molecules and reducing the sugar sensitivity of the PPRX. In contrast, structural analyses in this study revealed that the sugar-sensing NPBA moiety in the Naph-PEG/NPBA-γ-CyD PPRX is not included in the neighboring NPBA-γ-CyD. This spatial arrangement and the high affinity of NPBA for sugar contributed to the improved sugar responsivity. The enhanced NPBA-γ-CyD was then applied to a PPRX containing Naph-PEG-appended insulin (Naph-PEG-Ins) that showed an improved response for glucose-induced insulin release.

Correlation between salivary interleukin-6 levels and early locoregional recurrence in patients with oral squamous cell carcinoma: preliminary study.

BACKGROUND: We have demonstrated that salivary interleukin-6 (IL-6) concentrations change during the treatment in patients with oral squamous cell carcinoma (OSCC). We sought to elucidate the correlations between salivary IL-6 concentration and early locoregional recurrence in OSCC. METHODS: Stimulated saliva was collected before and after surgery from 27 consecutive patients with OSCC. Recurrence-free survival (RFS) curves were plotted using the Kaplan-Meier method. RESULTS: Of the 27 patients, 11 (41%) were diagnosed with locoregional recurrence within 24 months postsurgery. The median concentrations of IL-6 presurgery and postsurgery were 2.8 pg/mL and 2.1 pg/mL, respectively. The median postsurgery concentration of IL-6 was significantly higher in patients with than without locoregional recurrence (p = .02). Multivariate analysis revealed that postsurgery salivary IL-6 concentration was an independent risk factor for locoregional recurrence (p = .03; risk ratio, 0.14). CONCLUSIONS: Posttreatment concentration of salivary IL-6 may predict early locoregional recurrence in OSCC.

Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells.

Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.