Auditory brainstem responses in Cope’s gray treefrog (Hyla chrysoscelis): effects of frequency,level, sex and size

Our knowledge of the hearing abilities of frogs and toads is largely defined by work with a few well-studied species. One way to further advance comparative work on anuran hearing would be greater use of minimally invasive electrophysiological measures, such as the auditory brainstem response (ABR). This study used the ABR evoked by tones and clicks to investigate hearing in Cope’s gray treefrog (Hyla chrysoscelis). The objectives were to characterize the effects of sound frequency, sound pressure level, and subject sex and body size on ABRs. The ABR in gray treefrogs bore striking resemblance to ABRs measured in other animals. As stimulus level increased, ABR amplitude increased and latency decreased, and for responses to tones, these effects depended on stimulus frequency. Frequency-dependent differences in ABRs were correlated with expected differences in the tuning of two sensory end organs in the anuran inner ear (the amphibian and basilar papillae). The ABR audiogram indicated two frequency regions of increased sensitivity corresponding to the expected tuning of the two papillae. Overall, there was no effect of subject size and only small effects related to subject sex. Together, these results indicate the ABR is an effective method to study audition in anurans.     

Influence of Starting Material Particle Size on Pellet Surface Roughness

The purpose of this study was to investigate the effect of pelletization aids, i.e., microcrystalline cellulose (MCC) and cross-linked polyvinyl pyrrolidone (XPVP), and filler, i.e., lactose, particle size on the surface roughness of pellets. Pellets were prepared from powder blends containing pelletization aid/lactose in 1:3 ratio by extrusion–spheronization. Surface roughness of pellets was assessed quantitatively and qualitatively using optical interferometry and scanning electron microscopy, respectively. Both quantitative and qualitative surface studies showed that surface roughness of pellets depended on the particle size of XPVP and lactose used in the formulation. Increase in XPVP or lactose particle size resulted in rougher pellets. Formulations containing MCC produced pellets with smoother surfaces than those containing XPVP. Furthermore, surface roughness of the resultant pellets did not appear to depend on MCC particle size. Starting material particle size was found to be a critical factor for determining the surface roughness of pellets produced by extrusion–spheronization. Smaller particles can pack well with lower peaks and valleys, resulting in pellets with smoother surfaces. Similar surface roughness of pellets containing different MCC grades could be due to the deaggregation of MCC particles into smaller subunits with more or less similar sizes during wet processing. Hence, for starting materials that deaggregate during the wet processing, pellet surface roughness is influenced by the particle size of the material upon deaggregation.     

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.

Methodology/Principal Findings

Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.

Conclusions/Significance

We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.     

Establishing the Thematic Framework for a Diabetes-Specific Health-Related Quality of Life Item Bank for Use in an English-Speaking Asian Population

Aims

To establish a thematic framework for a Diabetes Mellitus (DM)-specific health-related quality of life (HRQoL) item bank by identifying important HRQoL themes and content gaps in existing DM-specific HRQoL measures and determining whether Patient-Reported Outcomes Measurement Information System (PROMIS) item banks are useful as a starting point.

Methodology

English-speaking Type 2 DM patients were recruited from an outpatient specialist clinic in Singapore. Thematic analysis was performed through open coding and axial coding. Items from four existing DM-specific measures and PROMIS Version 1.0 and 2.0 item banks were compared with identified themes and sub-themes.

Results

42 patients participated (25 men and 17 women; 28 Chinese, 4 Malay, 8 Indians, 2 other ethnicities). Median age was 53.70 years (IQR45.82–56.97) and the median disease duration was 11.13 (SD9.77) years. 10 subthemes (neutral emotions, coping emotions, empowered to help others, support from family, spend more time with family, relationships, financial burden on family, improved relationship, social support and religion/spirituality) were not covered by existing DM-specific measures. PROMIS covered 5 of 6 themes, 15 of 30 subthemes and 19 of 35 codes identified. Emotional distress (frustration, fear and anxiety) was most frequently mentioned (200 times).

Conclusions

We had developed a thematic framework for assessing DM-specific HRQoL in a multi-ethnic Asian population, identified new items that needed to be written and confirmed that PROMIS was a useful starting point. We hope that better understanding and measurement of HRQoL of Asian DM patients will translate to better quality of care for them.     

The C-terminus of PRK2/PKNgamma is required for optimal activation by RhoA in a GTP-dependent manner

PRK2/PKNγ is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure-function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Δ958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.     

Monocot leaves are eaten less than dicot leaves in tropical lowland rain forests: correlations with toughness and leaf presentation

Background and Aims

In tropical lowland rain forest (TLRF) the leaves of most monocots differ from those of most dicots in two ways that may reduce attack by herbivores. Firstly, they are tougher. Secondly, the immature leaves are tightly folded or rolled until 50–100 % of their final length. It was hypothesized that (a) losses of leaf area to herbivorous invertebrates are generally greatest during leaf expansion and smaller for monocots than for dicots, and (b) where losses after expansion are appreciable any difference between monocots and dicots then is smaller than that found during expansion.

Methods

At six sites on four continents, estimates were made of lamina area loss from the four most recently mature leaves of focal monocots and of the nearest dicot shoot. Measurements of leaf mass per unit area, and the concentrations of water and nitrogen were made for many of the species. In Panama, the losses from monocots (palms) and dicots were also measured after placing fully expanded palm leaflets and whole dicot leaves on trails of leaf-cutter ants.

Key Results

At five of six sites monocots experienced significantly smaller leaf area loss than dicots. The results were not explicable in terms of leaf mass per unit area, or concentrations of water or nitrogen. At only one site was the increase in loss from first to fourth mature leaf significant (also large and the same in monocots and dicots), but the losses sustained during expansion were much smaller in the monocots. In the leaf-cutter ant experiment, losses were much smaller for palms than for dicots.

Conclusions

The relationship between toughness and herbivory is complex; despite the negative findings of some recent authors for dicots we hypothesize that either greater toughness or late folding can protect monocot leaves against herbivorous insects in tropical lowland rain forest, and that the relative importance varies widely with species. The difficulties of establishing unequivocally the roles of leaf toughness and leaf folding or rolling in a given case are discussed.Key words: anti-herbivore defences, dicots, herbivory, leaf folding, leaf rolling, leaf toughness, monocots, palms, tropical rain forest     

Distinct and combined roles of the MAP kinases of Cochliobolus heterostrophus in virulence and stress responses

Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways.     

Identification of an IgG CDR sequence contributing to co‐purification of the host cell protease cathepsin D

Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb‐1. The current work was focused on identification of a primary sequence in mAb‐1 responsible for the binding and consequent co‐purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb‐1 and mAb‐6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb‐1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb‐1 and cathepsin D was weaker than that of mAb‐6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb‐1 are replaced with neutral serine residues in mAb‐6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140–145, 2017     

Regulation of ripening and opportunities for control in tomato and other fruits

Fruits are an important part of a healthy diet. They provide essential vitamins and minerals, and their consumption is associated with a reduced risk of heart disease and certain cancers. These important plant products can, however, be expensive to purchase, may be of disappointing quality and often have a short shelf life. A major challenge for crop improvement in fleshy fruit species is the enhancement of their health‐promoting attributes while improving quality and reducing postharvest waste. To achieve these aims, a sound mechanistic understanding of the processes involved in fruit development and ripening is needed. In recent years, substantial insights have been made into the mechanistic basis of ethylene biosynthesis, perception and signalling and the identity of master regulators of ripening that operate upstream of, or in concert with a regulatory pathway mediated by this plant hormone. The role of other plant hormones in the ripening process has, however, remained elusive, and the links between regulators and downstream processes are still poorly understood. In this review, we focus on tomato as a model for fleshy fruit and provide an overview of the molecular circuits known to be involved in ripening, especially those controlling pigment accumulation and texture changes. We then discuss how this information can be used to understand ripening in other fleshy fruit‐bearing species. Recent developments in comparative genomics and systems biology approaches are discussed. The potential role of epigenetic changes in generating useful variation is highlighted along with opportunities for enhancing the level of metabolites that have a beneficial effect on human health.