Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ _max=390 nm) is converted to a red product (λ _max=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A ₃₉₀/A ₄₈₆ ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L⁻¹ and 50 μg L⁻¹ to 10 mg L⁻¹, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.     

Biomechanism of adhesion in gecko setae

The study of the adhesion of millions of setae on the toes of geckos has been advanced in recent years with the emergence of new technology and measurement methods. The theory of the mechanism of adhesion by van der Waals forces is now accepted and broadly understood. However, this paper presents limitations of this theory and gives a new hypothesis of the biomechanism of gecko adhesion. The findings are obtained through measurements of the magnitude of the adhesion of setae under three different conditions, to show the close relationship between adhesion and status of the setae. They are reinforced by demonstrating two setal structures, follicle cells and hair, the former making the setae capable of producing bioelectrical charges, which play an important role in attachment and detachment processes. It is shown that the abundant muscular tissues at the base of the setae cells, which are controlled by peripheral nerves, are instrumental in producing the foot movement involved in attachment and detachment. Our study will further uncover the adhesion mechanism of geckos, and provide new ideas for designing and fabricating synthetic setae.     

Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China

Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.     

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers

Common inflammatome gene signatures as well as disease‐specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co‐expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue‐specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response‐related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non‐drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.     

A transient parabiosis skin transplantation model in mice

Parabiosis-conjoined surgery to provide a shared circulation between two mice-has been previously developed to study the hematopoietic system. This protocol describes the use of parabiosis for efficient transplantation of skin from a transgenic to a wild-type mouse. It can be used to study the role of stromal cells in a spontaneous model of distant cancer dissemination (metastasis). We have recently shown that primary tumor-derived stromal cells may facilitate metastasis by providing a provisional stroma at the secondary site. Studying the role of primary tumor-derived stroma cells requires methods for distinguishing and targeting stromal cells originating from the primary tumor versus their counterparts in the metastatic site. Parabiosis may also be used, taking advantage of the shared circulation between the parabiosed mice, to study tumor metastasis from one parabiont to another, or to investigate the role of circulating inflammatory cells or stem cells. Studying the role of stromal cells in metastasis using this model typically takes up to 11 weeks.     

Host tree-recurrence of wood-decaying Dacrymycetes

Host-recurrence of wood-decaying polypores has been substantiated and some species of the class Dacrymycetes, consisting of wood-decaying jelly fungi, occur recurrently on coniferous and broad-leaved tree wood. However, there has been no quantitative analysis for Dacrymycetes. To evaluate their host-recurrence, the fruit body occurrence of Dacrymycetes was investigated at 60 sites, including 13 core sampling sites. A total of 577 samples of 28 species were collected. Principal component analysis revealed characteristic communities on conifers and broad-leaved trees. Binomial tests showed that 12 and four Dacrymycetes species occurred significantly more frequently on conifers and broad-leaved trees, respectively. Of these host-recurrent species, eight and two species exclusively occurred on conifers and broad-leaved trees, respectively, suggesting that some Dacrymycetes species have a strict host-recurrence on these tree groups.     

DNA barcoding analysis of Coleoidea (Mollusca: Cephalopoda) from Chinese waters

Coleoids are part of the Cephalopoda class, which occupy an important position in most oceans both at an ecological level and at a commercial level. Nevertheless, some coleoid species are difficult to distinguish with traditional morphological identification in cases when specimens are heavily damaged during collection or when closely related taxa are existent. As a useful tool for rapid species assignment, DNA barcoding may offer significant potential for coleoid identification. Here, we used two mitochondrial fragments, cytochrome c oxidase I and the large ribosomal subunit (16S rRNA), to assess whether 34 coleoids accounting for about one-third of the Chinese coleoid fauna could be identified by DNA barcoding technique. The pairwise intra- and interspecific distances were assessed, and relationships among species were estimated by NJ and bayesian analyses. High levels of genetic differentiation within Loliolus beka led to an overlap between intra- and interspecific distances. All remaining species forming well-differentiated clades in the NJ and bayesian trees were identical for both fragments. Loliolus beka possessed two mitochondrial lineages with high levels of intraspecific distances, suggesting the occurrence of cryptic species. This study confirms the efficacy of DNA barcoding for identifying species as well as discovering cryptic diversity of Chinese coleoids. It also lays a foundation for other ecological and biological studies of Coleoidea.     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.     

Altered Regulation of Akt Signaling with Murine Cerebral Malaria,Effects on Long-Term Neuro-Cognitive Function,Restoration with Lithium Treatment

Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling likely underlies long-term neurological sequelae observed in ECM and may yield adjunctive therapeutic targets for the management of CM.     

Parapapillary Atrophy: Histological Gamma Zone and Delta Zone

Background

To examine histomorphometrically the parapapillary region in human eyes.

Methodology/Principal Findings

The histomorphometric study included 65 human globes (axial length:21–37 mm). On anterior-posterior histological sections, we measured the distance Bruch''s membrane end (BME)-optic nerve margin (“Gamma zone”), BME-retinal pigment epithelium (RPE) (“Beta zone”), BME-beginning of non-occluded choriocapillaris, and BME-beginning of photoreceptor layer. “Delta zone” was defined as part of gamma zone in which blood vessels of at least 50 µm diameter were not present over a length of >300 µm. Beta zone (mean length:0.35±0.52 mm) was significantly (P = 0.01) larger in the glaucoma group than in the non-glaucomatous group. It was not significantly (P = 0.28) associated with axial length. Beta zone was significantly (P = 0.004) larger than the region with occluded choriocapillaris. Gamma zone (mean length:0.63±1.25 mm) was associated with axial length (P<0.001;r² = 0.73) with an increase starting at an axial length of 26.5 mm. It was not significantly (P = 0.24) associated with glaucomatous optic neuropathy. Delta zone (present only in eyes with axial length of ≥27 mm) was associated with axial length (P = 0.001) and scleral flange length (P<0.001) but not with glaucoma (P = 0.73).

Conclusions/Significance

Parapapillary gamma zone (peripapillary sclera without overlying choroid, Bruch''s membrane and deep retinal layers) was related with axial globe elongation and was independent of glaucoma. Delta zone (no blood vessels >50 µm diameter within gamma zone) was present only in highly axially elongated globes and was not related with glaucoma. Beta zone (Bruch''s membrane without RPE) was correlated with glaucoma but not with globe elongation. Since the region with occluded choriocapillaris was smaller than beta zone, complete loss of RPE may have occurred before complete choriocapillaris closure.