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Hsp70 molecular chaperones are essential components for maintaining protein homeostasis within cells. They interact with substrate or client proteins in a well characterised fashion that is regulated by ATP and supported by co-chaperones. In eukaryotes there is a vast array of Hsp70 isoforms that may facilitate adaption to a particular cellular compartment and distinct biological role. Emerging data indicate a novel type of interaction between Hsp70 and client protein that does not fit with the classical Hsp70 ATP regulated substrate mechanism. In this review, we highlight Hsp70 ATPase domain interactions with binding partners from various biological systems that we refer to as Hsp70 ATPase alternative binding proteins or HAAB proteins. We identify common mechanistic features that may define how Hsp70 operates when associating with proteins in this alternative HAAB mode of action.

Hsp70 chaperones interact with substrate proteins in a coordinated fashion that is regulated by nucleotides and enhanced by assisting cochaperones. There are numerous homologues and isoforms of Hsp70 that participate in a wide variety of cellular functions. This diversity can facilitate adaption or specialization based on particular biological activity and location within the cell. In this review, we highlight two specialized binding partner proteins, Tim44 and IRE1, that interact with Hsp70 at the membrane in order to serve their respective roles in protein translocation and unfolded protein response signalling. Recent mechanistic data suggest analogy in the way the two Hsp70 homologues (BiP and mtHsp70) can bind and release from IRE1 and Tim44 upon substrate engagement. These shared mechanistic features may underlie how Hsp70 interacts with specialized binding partners and may extend our understanding of the mechanistic repertoire that Hsp70 chaperones possess.

The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.

Spatial prediction and variable selection for the study area are both important issues in geostatistics. If spatially varying means exist among different subareas, globally fitting a spatial regression model for observations over the study area may be not suitable. To alleviate deviations from spatial model assumptions, this paper proposes a methodology to locally select variables for each subarea based on a locally empirical conditional Akaike information criterion. In this situation, the global spatial dependence of observations is considered and the local characteristics of each subarea are also identified. It results in a composite spatial predictor which provides a more accurate spatial prediction for the response variables of interest in terms of the mean squared prediction errors. Further, the corresponding prediction variance is also evaluated based on a resampling method. Statistical inferences of the proposed methodology are justified both theoretically and numerically. Finally, an application of a mercury data set for lakes in Maine, USA is analyzed for illustration.

Miniaturized devices (lab-on-a-chip) for performing laboratory operations on microscale are appealing. Low sample requirement is one of the major advantages of these devices, therefore less costs is needed for running such platforms and also fewer wastes to be handled. Together with the unique behavior of liquids on microscale facilitating control of molecular diffusion and interaction makes miniaturized devices particularly useful in chemical synthesis as well as biological and/or chemical analysis. In this report, we designed a microfluidic platform with programmable microvalves capable to carry out routine operations. This platform was further optimized to contain universal sample-processing capabilities, using a three-layered hybrid PDMS–PDMS–glass structure. Precise programmable control of the volumetric flow rate can be achieved via the discrete digital control of fluids in pneumatically actuated microvalves. The specific protocols of the system are optical path platforms consisting of MEMS combined with SU-8 arrays in microfluidic reactors for parallel biological analysis. To demonstrate the programming capabilities for biomolecular assay integration, we developed an automated assay with streptavidin immobilized on the SU-8 patterned surface; these optical microfluidic platforms featured with a low sample requirement (0.5 μl per single assay) were then employed as a protein sensor, which has working concentrations ranged from 39.3 to 2500 nM for detecting biotin in the sample solution. The results suggest potential applications of these platforms in either routine assay purposes or specific applications such as high-throughput screening of protein–protein and protein–ligand interactions.

Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8D270A/D270A mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8D270A mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes. Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.

Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long-term in vivo imaging applications. This paper reports that FNDs with a size of approximately 140 nm can be covalently conjugated with folic acid (FA) for receptor-mediated targeting of cancer cells at the single-particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble-averaged measurements with flow cytometry indicate that more than 50% of the FA-conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor-mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 microg mL(-1) is in the range of 100 particles per cell. The receptor-mediated uptake process is further elucidated by single-particle tracking of 35-nm FNDs in three dimensions and real time during the endocytosis.

Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of alpha-l-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Brønsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(lg) = -0.93, but that for k(cat) is biphasic, with beta(lg) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of beta(lg) for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter beta(lg) value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.

Gelatin-methotrexate conjugates (G-MTX) with known molecular weight (MW), drug load, and charge were prepared and evaluated for growth inhibition on leukemia cells.Gelatin (34 to 171 kDa) was reacted with a carbodiimide to prepare G-MTX with high (G-MTX-H) and low (G-MTX-L) drug loads. Cationic conjugates were prepared by ethylenediamine modification. MTX:gelatin molar ratios were determined spectrophotometrically. Isoelectric focusing electrophoresis (IEF) and turbidity were used to measure isoelectric points (IEP). Growth inhibition profiles and IC50 values were determined on HL-60 cells using a modified MTT assay.IC50 values of anionic G-MTX-L (drug loads 0.5:1 to 2.2:1) increased linearly from 46 to 180 nM with MW. But, IC50 values for anionic G-MTX-H (drug loads 7.4:1 to 25:1) showed little, if any, MW dependence and were about two times higher. IC50 values for cationic G-MTX-L ranged from 770 to 2,900 nM and the relationship with MW was non-linear.The growth inhibition ranking was MTXanionic G-MTX-Lanionic G-MTX-Hcationic G-MTX-L. High drug load may hinder lysosomal enzyme degradation and drug release and contribute to suppression of the MW effect observed with G-MTX-L. A mechanism change is suggested as the cationic conjugates increase to the highest MW.

Macromolecular gelatin-methotrexate conjugates have potential therapeutic advantages over the free drug. Conjugates with MTX:gelatin molar ratios (MR) ranging from 1:1 to 27:1 were examined for cell growth inhibition, stability, degradation, and methotrexate (MTX) release. Conjugate growth inhibition was less than that of free MTX whose IC 50 value of 1.3 × 10 −8 M was about 10-fold less. Cell uptake of fluorescein labeled gelatin (145 kD) was observed by 24–30 h. Higher MR conjugates produced less growth inhibition, measurably greater stability at pH 7.4 based on MTX release, and had less gelatin degradation in the conjugate by the lysosomal enzyme Cathepsin B (Cat B) compared to low MR conjugates. Cat B conjugate degradation was greater at the in vitro lysosomal pH of 4.8 than the intra-tumor pH of 6.5. The presence of Cat B did not meaningfully affect MTX release, but less MTX was released at pH 4.8 than pH 6.5. The maximum MTX release was a relatively low 7% after 72 h at pH 6.5 for the low MR conjugate. Low molecular weight conjugate fragments were also produced and were also influenced by pH and MR. Reduced growth inhibition by high MR conjugates may be due to a hindered enzymatic degradation in the lysosomes. A strong peptide conjugate bond at lysosomal pH and a 24–30 h delayed gelatin uptake may contribute to reduced growth inhibition of the conjugate compared to free MTX. MTX release under these in vitro conditions occurs by aqueous hydrolysis, not by Cat B cleavage of the conjugate bond.

This paper utilizes the recently defined equivalent noise sheet resistance to study the noise performance of future sub-100nm MOSFETs using strain engineering, channel engineering with III–V materials, and quantum-well structures for low-noise applications. Experimental results for devices fabricated in UMC's five different CMOS technology nodes and other published data down to 20 nm technology node are demonstrated. Strategies for the future low-noise technologies are also discussed.

Background: Many critical cellular functions are performed by multisubunit circular protein oligomers whose internal geometry has evolved to meet functional requirements. The subunit number is arguably the most critical parameter of a circular protein assembly, affecting the internal and external diameters of the assembly and often impacting on the protein’s function. Although accurate structural information has been obtained for several circular proteins, a lack of accurate information on alternative oligomeric states has prevented engineering such transitions. In this study we used the bacterial transcription regulator TRAP as a model system to investigate the features that define the oligomeric state of a circular protein and to question how the subunit number could be manipulated. Methodology/Principal Findings: We find that while Bacillus subtilis and Bacillus stearothermophilus TRAP form 11-subunit oligomers, the Bacillus halodurans TRAP exclusively forms 12-subunit assemblies. Significantly, the two states of TRAP are related by a simple rigid body rotation of individual subunits around inter-subunit axes. We tested if such a rotation could be induced by insertion or deletion mutations at the subunit interface. Using wild type 11-subunit TRAP, we demonstrate that removal of five C-terminal residues at the outer side of the inter-subunit axis or extension of an amino acid side chain at the opposite, inner side, increased the subunit number from 11 to 12. Our findings are supported by crystal structures of TRAP oligomers and by native mass spectrometry data. Conclusions/Significance: The subunit number of the TRAP oligomer can be manipulated by introducing deletion or addition mutations at the subunit interface. An analysis of available and emerging structural data on alternative oligomeric states indicates that the same principles may also apply to the subunit number of other circular assemblies suggesting that the deletion/addition approach could be used generally to engineer transitions between different oligomeric states.

Fluorescent nanodiamonds (FNDs) with a size in the range of 10 – 100 nm have been produced by ion irradiation and annealing, and isolated by differential centrifugation. Single particle spectroscopic characterization with confocal fluorescence microscopy and fluorescence correlation spectroscopy indicates that they are photostable and useful as an alternative to far-red fluorescent proteins for bioimaging applications. We demonstrate the application by performingin vivoimaging of bare and bioconjugated FND particles (100 nm in diameter) inC. elegansand zebrafishes and exploring the interactions between this novel nanomaterial and the model organisms. Our results indicate that FNDs can be delivered to the embryos of both organisms by microinjection and eventually into the hatched larvae in the next generation. No deleterious effects have been observed for the carbon-based nanoparticlesin vivo. The high fluorescence brightness, excellent photostability, and nontoxic nature of the nanomaterial have allowed long-term imaging and tracking of embryogenesis in the organisms.

Nanoscale carbon materials hold great promise for biotechnological and biomedical applications. Fluorescent nanodiamond (FND) is a recent new addition to members of the nanocarbon family. Here, we report long-term in vivo imaging of FNDs in Caenorhabditis elegans (C. elegans) and explore the nano-biointeractions between this novel nanomaterial and the model organism. FNDs are introduced into wild-type C. elegans by either feeding them with colloidal FND solution or microinjecting FND suspension into the gonads of the worms. On feeding, bare FNDs stay in the intestinal lumen, while FNDs conjugated with biomolecules (such as dextran and bovine serum albumin) are absorbed into the intestinal cells. On microinjection, FNDs are dispersed in the gonad and delivered to the embryos and eventually into the hatched larvae in the next generation. The toxicity assessments, performed by employing longevity and reproductive potential as physiological indicators and measuring stress responses with use of reporter genes, show that FNDs are stable and nontoxic and do not cause any detectable stress to the worms. The high brightness, excellent photostability, and nontoxic nature of the nanomaterial have enabled continuous imaging of the whole digestive system and tracking of the cellular and developmental processes of the living organism for several days.

Background Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins. Methodology/Principal Findings Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts - have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP. Conclusions/Significance We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.