Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non‐motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup‐like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup‐like structure composed of two membranes.     

Distinct signaling mechanisms regulate migration in unconfined versus confined spaces

Using a microchannel assay, we demonstrate that cells adopt distinct signaling strategies to modulate cell migration in different physical microenvironments. We studied α4β1 integrin–mediated signaling, which regulates cell migration pertinent to embryonic development, leukocyte trafficking, and melanoma invasion. We show that α4β1 integrin promotes cell migration through both unconfined and confined spaces. However, unlike unconfined (2D) migration, which depends on enhanced Rac1 activity achieved by preventing α4/paxillin binding, confined migration requires myosin II–driven contractility, which is increased when Rac1 is inhibited by α4/paxillin binding. This Rac1–myosin II cross talk mechanism also controls migration of fibroblast-like cells lacking α4β1 integrin, in which Rac1 and myosin II modulate unconfined and confined migration, respectively. We further demonstrate the distinct roles of myosin II isoforms, MIIA and MIIB, which are primarily required for confined and unconfined migration, respectively. This work provides a paradigm for the plasticity of cells migrating through different physical microenvironments.     

Diethylmaleate and iodoacetate in combination caused profound cell death in astrocytes

Energy failure and oxidative stress have been implicated in the pathogenesis of ischemia. Here, we report a potential link between cytosolic phospholipase A₂ (cPLA₂) activation and energy failure/oxidative stress‐induced astrocyte damage involving reactive oxygen species (ROS), protein kinase C‐α (PKC‐α), Src, Raf, and extracellular signal‐regulated kinase (ERK) signaling and concurrent elevation of endogenous chelatable zinc. Energy failure and oxidative stress were produced by treating astrocytes with glycolytic inhibitor iodoacetate and glutathione chelator diethylmaleate, respectively. Diethylmaleate and iodoacetate in combination caused augmented damage to astrocytes in a time‐ and concentration‐dependent manner. The cell death caused by diethylmaleate/iodoacetate was accompanied by increased ROS generation, PKC‐α membrane translocation, Src, Raf, ERK, and cPLA₂ phosphorylation. Pharmacological studies revealed that these activations all contributed to diethylmaleate/iodoacetate‐induced astrocyte death. Intriguingly, the mobilization of endogenous chelatable zinc was observed in diethylmaleate/iodoacetate‐treated astrocytes. Zinc appears to act as a downstream mediator in response to diethylmaleate/iodoacetate treatment because of the attenuating effects of its chelator N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine. These observations indicate that ROS/PKC‐α, Src/Raf/ERK signaling and cPLA₂ are active participants in diethylmaleate/iodoacetate‐induced astrocyte death and contribute to a vicious cycle between the depletion of ATP/glutathione and the mobilization of chelatable zinc as critical upstream effectors in initiating cytotoxic cascades.

     

Association of a d-Alanyl-d-Alanine Carboxypeptidase Gene with the Formation of Aberrantly Shaped Cells during the Induction of Viable but Nonculturable Vibrio parahaemolyticus

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containing d-cycloserine (50 μg/ml) but was lower in a medium containing cephalosporin C (10 μg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodes d-alanyl-d-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in the dacB mutant strain than in the parent strain, but the proportion was restored in the presence of the complementary dacB gene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNC V. parahaemolyticus cells under specific conditions.     

Inhibitory effect on NO production of triterpenes from the fruiting bodies of Ganoderma lucidum

Four new lanostane triterpenes, butyl lucidenate P (1), butyl lucidenate D₂ (2), butyl lucidenate E₂ (3) and butyl lucidenate Q (4) along with 11 known compounds (5–15) were isolated from the fruiting bodies of Ganoderma lucidum. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW 264.7 cells. Compounds 1, 3, 4, 9, 10 and 15 showed inhibitory potency with IC₅₀ values of 7.4, 6.4, 4.3, 9.4, 9.2 and 4.5 μM, respectively. Compounds 1, 3 and 15 dose-dependently reduced the LPS-induced iNOS expressions. Preincubation of cell with 1, 3 and 15 significantly suppressed LPS-induced expression of COX-2 protein.     

Risk Factors for Progressive Visual Field Loss in Primary Angle-Closure Glaucoma: A Retrospective Cohort Study

Purpose

To investigate risk factors associated with progressive visual field (VF) loss in primary angle closure glaucoma (PACG).

Methods

We retrospectively reviewed medical record of PACG patients who had ≥5 reliable VF examinations (central 24-2 threshold test, Humphrey Field Analyzer) and ≥2 years of follow-up. Each VF was scored using Collaborative Initial Glaucoma Treatment Study system. Progression was defined if 3 consecutive follow-up VF tests had an increased score of ≥3 above the mean of the first 2 VF scores. Factors associated with VF progression were evaluated by Cox proportional hazards models.

Results

A total of 89 eyes from 89 patients (mean age, 69.8 ± 7.9 years), who received a mean of 6.9 ± 2.3 VF tests (mean deviation at initial, -8.1 ± 4.4 dB) with a mean follow-up of 63.9 ± 23.9 months were included. VF progression was detected in 9 eyes (10%). The axial length (AL), anterior chamber depth, and intraocular pressure (IOP) in patients with and without progression were 22.5 ± 0.6 and 23.1 ± 0.9 mm, 2.5 ± 0.3 and 2.5 ± 0.3 mm, 14.8 ± 2.4 and 14.3 ± 2.3 mm Hg, respectively. AL was the only factor associated with progression in both Cox proportional hazards univariate (p = 0.031) and multivariate models (p = 0.023).

Conclusion

When taking into account age, IOP, follow-up period, and number of VF tests, a shorter AL is the only factor associated with VF progression in this cohort of Chinese patients with PACG. Further studies are warranted to verify the role of AL in progressive VF loss in PACG.     

A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP^32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP^32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP^32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP^32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP^32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP^32–41 internalization routes. ECP^32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.     

The De Novo Assembly of Mitochondrial Genomes of the Extinct Passenger Pigeon (Ectopistes migratorius) with Next Generation Sequencing

The information from ancient DNA (aDNA) provides an unparalleled opportunity to infer phylogenetic relationships and population history of extinct species and to investigate genetic evolution directly. However, the degraded and fragmented nature of aDNA has posed technical challenges for studies based on conventional PCR amplification. In this study, we present an approach based on next generation sequencing to efficiently sequence the complete mitochondrial genome (mitogenome) of two extinct passenger pigeons (Ectopistes migratorius) using de novo assembly of massive short (90 bp), paired-end or single-end reads. Although varying levels of human contamination and low levels of postmortem nucleotide lesion were observed, they did not impact sequencing accuracy. Our results demonstrated that the de novo assembly of shotgun sequence reads could be a potent approach to sequence mitogenomes, and offered an efficient way to infer evolutionary history of extinct species.