Controlling network topology and mechanical properties of co‐assembling peptide hydrogels

Oligopeptides are well‐known to self‐assemble into a wide array of nanostructures including β‐sheet‐rich fibers that when present above a critical concentration become entangled and form self‐supporting hydrogels. The length, quantity, and interactions between fibers influence the mechanical properties of the hydrogel formed and this is typically achieved by varying the peptide concentration, pH, ionic strength, or the addition of a second species or chemical cross‐linking agent. Here, we outline an alternative, facile route to control the mechanical properties of the self‐assembling octa‐peptide, FEFEFKFK (FEKII); simply doping with controlled quantities of its double length peptide, FEFEFKFK‐GG‐FKFKFEFE (FEKII18). The structure and properties of a series of samples were studied here (0–100 M% of FEKII18) using Fourier transform infrared, small angle X‐ray scattering, transmission electron microscopy, and oscillatory rheology. All samples were found to contain elongated, flexible fibers and all mixed samples contained Y‐shaped branch points and parallel fibers which is attributed to the longer peptide self‐assembling within two fibers, thus creating a cross‐link in the network structure. Such behavior was reflected in an increase in the elasticity of the mixed samples with increasing quantity of double peptide. Interestingly the elastic modulus increased up to 30 times the pure FEKII value simply by adding 28 M% of FEKII18. These observations provide an easy, off‐the‐shelf method for an end‐user to control the cross‐linked network structure of the peptide hydrogel, and consequently strength of the hydrogel simply by physically mixing pre‐determined quantities of two similar peptide molecules. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 669–680, 2014.     

Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal

The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L⁻¹. Decreasing the cultivation temperature from 25 °C to 16 °C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL⁻¹, and the yield of SSX2NORD was 21.6 mg L⁻¹.     

The anticancer drug perillyl alcohol is a Na/K-ATPase inhibitor

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that suppresses adaptive T-cell immunity by catabolizing tryptophan from the cellular microenvironment. Inhibition of IDO pathway might enhance the efficacy of immunotherapeutic strategies for cancer. We synthesized 1-alkyl-tryptophan targeted IDO inhibitors and compared their effects on IDO expression and activity in dendritic cells (DCs) with the common IDO inhibitor 1-methyl-dl-tryptophan (1-MT). The IDO gene expression was examined by RT-PCR and realtime PCR. The toxicity of these analogs on the proliferation of DCs was detected by MTT assay. All of these analogs inhibited IDO expression and activity induced by IFN-γ and showed no cytotoxicity to DCs at 100 μM. 1-MT intensively suppressed IDO1 expression and activity in DCs, and 1-propyl-tryptophan (1-PT) and 1-isopropyl-tryptophan (1-isoPT) moderately inhibited them. 1-Butyl-tryptophan (1-BT) and 1-ethyl-tryptophan (1-ET) mainly inhibited IDO2 expression. Our results suggest that those analogs differed in their inhibitory activity on IDO expression may give us a clue for developing active IDO inhibitors.     

Bone Marrow Involvement in a Patient with Paracoccidioidomycosis: A Rare Presentation of Juvenile Form

Paracoccidioides brasiliensis rarely shows bone marrow involvement and its response to treatment with itraconazole in children needs further assessment. We describe here a child with a juvenile disseminated form of paracoccidioidomycosis, which showed reticuloendothelial system involvement and the presence of Paracoccidioides brasiliensis in the bone marrow. The patient showed an effective and rapid response to itraconazole therapy.     

<Emphasis Type="Italic">Medicago truncatula</Emphasis> contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

Background  

Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula.     

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.