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Engineering a system with a high mass fraction of active ingredients, especially water-soluble proteins, is still an ongoing challenge. In this work, we developed a versatile surface camouflage strategy that can engineer systems with an ultrahigh mass fraction of proteins. By formulating protein molecules into nanoparticles, the demand of molecular modification was transformed into a surface camouflage of protein nanoparticles. Thanks to electrostatic attractions and van der Waals interactions, we camouflaged the surface of protein nanoparticles through the adsorption of carrier materials. The adsorption of carrier materials successfully inhibited the phase transfer of insulin, albumin, β-lactoglobulin, and ovalbumin nanoparticles. As a result, the obtained microcomposites featured with a record of protein encapsulation efficiencies near 100% and a record of protein mass fraction of 77%. After the encapsulation in microcomposites, the insulin revealed a hypoglycemic effect for at least 14 d with one single injection, while that of insulin solution was only ∼4 h.

Photoaffinity labeling is a powerful technique to interrogate drug-protein interactions in native cellular environments. Photo-cross-linkers are instrumental for this technique. However, the introduction of unnatural photo-cross-linkers may significantly reduce the bioactivity of the drug, thus impairing the chemoproteomic outcomes. Herein, we developed a common pharmacophore, isoxazole, into a natively embedded photo-cross-linker for chemoproteomics, which minimally perturbs the drug structure. The photo-cross-linking reactions of the isoxazole were thoroughly investigated for the first time. Functionalized isoxazoles were then designed and applied to protein labeling, demonstrating the superior photo-cross-linking efficiency. Subsequently, two isoxazole-based drugs, Danazol and Luminespib, were employed in chemoproteomic studies, revealing their potential cellular targets. These results provide valuable strategies for future chemoproteomic study and drug development.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from

The UFM1 conjugation system is a Ubiquitin (Ub) -like modification system that is essential for animal development and normal physiology of multiple tissues and organs. It consists of UFM1, a Ub-like modifier, and the UFM1-specific enzymes (namely E1 enzyme UBA5, E2 enzyme UFC1 E2, and E3 ligases) that catalyze conjugation of UFM1 to its specific protein targets. Clinical studies have identified rare genetic variants in human UFM1, UBA5 and UFC1 genes that were linked to early-onset encephalopathy and defective brain development, strongly suggesting the critical role of the UFM1 system in the nervous system. Yet the phsyiologica function of this system in adult brain remains not defined. In this study, we investigated the role of UFM1 E3 ligase in adult mouse and found that both UFL1 and UFBP1 proteins, two components of UFM1 E3 ligase, are essential for survival of mature neurons in adult mouse. Neuron-specific deletion of either UFL1 or UFBP1 led to significant neuronal loss and elevation of inflammatory response. Interestingly, loss of one allele of UFBP1 genes caused occurance of seizure-like events. Our study has provided genetic evidence for the indispensable role of UFM1 E3 ligase in mature neurons and further demonstrated the importance of the UFM1 system in the nervous system.

Isothermal exponential amplification technology has rarely been fabricated as a universal sensing platform for the detection of proteins. To broaden their application, we have developed a strategy, named protein-recognition-initiated exponential amplification reaction (PRIEAR) using protein recognition to induce DNA assembly, which converts protein recognition events into ssDNA amplicons and combines two-stage amplification to achieve exponential amplification. Taking advantage of this principle, diverse biomarkers can be quantified at sub-picomolar concentrations in a homogenous manner, making PRIEAR suitable for clinical settings. Therefore, this strategy can expand the powerful isothermal exponential amplification technology to protein targets and thus provide a new toolbox in clinical and biomedical applications.

Stem cell transplantation is a candidate method for the treatment of Leydig cell dysfunction‐related diseases. However, there are still many problems that limit its clinical application. Here, we report the establishment of CXCR4‐SF1 bifunctional adipose‐derived stem cells (CXCR4‐SF1‐ADSCs) and their reparative effect on Leydig cell dysfunction. CD29+ CD44+ CD34− CD45− ADSCs were isolated from adipose tissue and purified by fluorescence‐activated cell sorting (FACS). Infection with lentiviruses carrying the CXCR4 and SF1 genes was applied to construct CXCR4‐SF1‐ADSCs. The CXCR4‐SF1‐ADSCs exhibited enhanced migration and had the ability to differentiate into Leydig‐like cells in vitro. Furthermore, the bifunctional ADSCs were injected into BPA‐mediated Leydig cell damage model mice via the tail vein. We found that the CXCR4‐SF1‐ADSCs were capable of homing to the injured testes, differentiating into Leydig‐like cells and repairing the deficiency in reproductive function caused by Leydig cell dysfunction. Moreover, we investigated the mechanism underlying SF1‐mediated differentiation and testosterone synthesis in Leydig cells, and the B‐box and SPRY Domain Containing Protein (BSPRY) gene was proposed to be involved in this process. This study provides insight into the treatment of Leydig cell dysfunction‐related diseases.

Motivation Exploring drug–protein interactions (DPIs) provides a rapid and precise approach to assist in laboratory experiments for discovering new drugs. Network-based methods usually utilize a drug–protein association network and predict DPIs by the information of its associated proteins or drugs, called ‘guilt-by-association’ principle. However, the ‘guilt-by-association’ principle is not always true because sometimes similar proteins cannot interact with similar drugs. Recently, learning-based methods learn molecule properties underlying DPIs by utilizing existing databases of characterized interactions but neglect the network-level information. Results We propose a novel method, namely BridgeDPI. We devise a class of virtual nodes to bridge the gap between drugs and proteins and construct a learnable drug–protein association network. The network is optimized based on the supervised signals from the downstream task—the DPI prediction. Through information passing on this drug–protein association network, a Graph Neural Network can capture the network-level information among diverse drugs and proteins. By combining the network-level information and the learning-based method, BridgeDPI achieves significant improvement in three real-world DPI datasets. Moreover, the case study further verifies the effectiveness and reliability of BridgeDPI. Availability and implementation The source code of BridgeDPI can be accessed at https://github.com/SenseTime-Knowledge-Mining/BridgeDPI. The source data used in this study is available on the https://github.com/IBM/InterpretableDTIP (for the BindingDB dataset), https://github.com/masashitsubaki/CPI_prediction (for the C.ELEGANS and HUMAN) datasets, http://dude.docking.org/ (for the DUD-E dataset), repectively.

Lysozyme hydrolysis can accelerate waste-activated sludge (WAS) solubilisation, which can significantly shorten the process and promote the efficiency of anaerobic digestion. This study investigated the impact of divalent cations on lysozyme-induced solubilisation of WAS. The performance of lysozyme pretreatment was dramatically inhibited by Mg

Bioproduction of renewable chemicals is considered as an urgent solution for fossil energy crisis. However, despite tremendous efforts, it is still challenging to generate microbial strains that can produce target biochemical to high levels. Here, we report an example of biosynthesis of high-value and easy-recoverable derivatives built upon natural microbial pathways, leading to improvement in bioproduction efficiency. By leveraging pathways in solventogenic clostridia for co-producing acyl-CoAs, acids and alcohols as precursors, through rational screening for host strains and enzymes, systematic metabolic engineering-including elimination of putative prophages, we develop strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl butyrate. Techno-economic analysis results suggest the economic competitiveness of our developed bioprocess. Our principles of selecting the most appropriate host for specific bioproduction and engineering microbial chassis to produce high-value and easy-separable end products may be applicable to other bioprocesses., Esters can be used as fuels and specialty chemicals for food flavoring, cosmetic and pharmaceutical industries. Here, the authors systematically engineer clostridia, including discovery and deletion of prophages to increase strain stability, for the production of butyl acetate and butyl butyrate from corn stover at low cost.

Lysozyme can efficiently accelerate solubilization and hydrolysis of waste activated sludge (WAS) for anerobic digestion. However, the effect of lysozyme was easily to be inhibited by metal ions in WAS. The impact of magnesium ions (Mg

Proteases are widely found in organisms participating in the decomposition of proteins to maintain the organisms' normal life activities. Protease inhibitors regulate the activities of target proteases by binding to their active sites, thereby affecting protein metabolism. The key amino acid mutations in proteases and protease inhibitors can affect their physiological functions, stability, catalytic activity, and inhibition specificity. More active, stable, specific, environmentally friendly and cheap proteases and protease inhibitors might be obtained by excavating various natural mutants of proteases and protease inhibitors, analyzing their key active sites by using protein engineering methods. Here, we review the studies on proteases' key active sites and protease inhibitors to deepen the understanding of the active mechanism of proteases and their inhibitors.蛋白酶广泛存在于生物体中，参与分解蛋白质，维持生物体正常的生命活动。蛋白酶抑制剂通过与蛋白酶活性位点结合调控靶蛋白酶活性，从而影响蛋白质代谢。蛋白酶及其抑制剂关键氨基酸的突变，可以影响其生理功能、稳定性、催化活性、抑制特异性等。通过挖掘自然界蛋白酶及其抑制剂的各种突变体，分析它们的关键活性位点，并运用蛋白质工程手段改造和设计活性更强、稳定性更高、特异性更好、环境更友好、成本更低的蛋白酶及其抑制剂，已成为当前的热点研究之一。文中对近年来蛋白酶及其抑制剂关键活性位点研究进行了简要综述，以期深化人们对蛋白酶及其抑制剂活性作用机制的认识，并为蛋白酶及其抑制剂的生物学活性改造研究提供理论参考。.

Graphical abstract, Highlights • Ultrasonication was synergistically combined with microfiltration for milk processing. • Ultrasonication further extended the shelf life of microfiltered skim milk. • Ultrasonication above 1296 J/mL changed the proteome of milk serum. • Microfiltration combined with ultrasonication retains ˃80% bioactive proteins., To extend the shelf life and retain bioactive proteins in milk, this study utilized microfiltration (MF) combined with ultrasonication to treat skim milk and investigated its efficiency in removing bacteria and retaining bioactive proteins compared with HTST pasteurization and microfiltration alone. Results showed that microfiltration combined with ultrasonication at 1296 J/mL could completely remove the bacteria in skim milk. Ultrasonication further extended the shelf life (4 °C) of microfiltered skim milk, which could reach at least 40 days when MF was combined with ˃1296 J/mL ultrasonication. In addition, ELISA showed that HTST pasteurization significantly decreased the levels of IgG by ~30%, IgA by ~ 50%, IgM by ~60%, and lactoferrin by ~40%, whereas the activity of the enzymes lactoperoxidase and xanthine oxidase were also decreased by ~ 20%. Compared with HTST, MF alone or combined with ultrasonication retained these bioactive proteins to a larger degree. On the other hand, proteomics indicated both damage to casein micelle and fat globule structures in milk when ultrasonication at >1296 J/mL was applied, as shown by increases in caseins and milk fat globular proteins. Simultaneously, this ultrasound intensity also decreased levels of bioactive proteins, such as complement factors. Taken together, this study provided new insights that may help to implement this novel combination of non-thermal technologies for the dairy industry aimed at improving milk quality and functionality.

Most studies on ischemic preconditioning (IPC) use one or two ischemic stimuli before examining cardioprotection. To better simulate the clinical situation, we examined, in pigs, the effects of six episodes of 10 min coronary artery occlusion (CAO) 12 h apart, followed by 60 min CAO. We named this model the fourth window of IPC. To determine the novel mechanisms mediating cardioprotection in the fourth window, gene analysis was examined in fourth window IPC cardiac tissue 60 min after the last episode of 10 min CAO. Secreted frizzled-related protein 3 (sFRP3) was the most significantly upregulated gene that was unique to the fourth window, that is, not found in the first, second, or third window IPC. To study the effects of sFRP3 on cardioprotection, sFRP3 was injected in the hearts of wild-type (WT) mice. In the [CAO/coronary artery reperfusion (CAR)] model (30 min CAO followed by 24 h CAR), infarct size was less, P < 0.01, after sFRP3 injection (14% ± 1.7%) compared with vehicle injection (48% ± 1.6%). sFRP3 injection also protected the development of heart failure following permanent CAO for 2 wk. Left ventricular ejection fraction was significantly improved, P < 0.05, at 2 wk after CAO with sFRP3 (53% ± 5%) compared with vehicle (36% ± 2%) and was accompanied by significant, P < 0.01, reductions in myocardial fibrosis (53% ± 4%), myocyte size (17% ± 3%), apoptosis (100%), and mortality (56%). Thus, sFRP3, unique to the clinically relevant fourth window IPC model, is a novel mechanism mediating ischemic cardioprotection. NEW & NOTEWORTHY 1) This investigation identifies the novel fourth window of ischemic preconditioning. 2) sFRP3 was identified as the most significantly upregulated gene in the fourth window and was shown to induce cardioprotection when administered to the hearts of wild-type mice.

Cereal cyst nematodes cause serious yield losses of wheat in Hunaghuai winter wheat growing region in China. Beauveria bassiana 08F04 isolated from the surface of cysts is a promising biological control agent for cereal cyst nematodes. As the colonization capacity is a crucial criteria to assess biocontrol effectiveness for a microbial agent candidate, we aimed to label B. bassiana 08F04 for efficient monitoring of colonization in the soil. The binary pCAM-gfp plasmid containing sgfp and hph was integrated into B. bassiana 08F04 using the Agrobacterium tumefaciens-mediated transformation. The transformation caused a significant change in mycelial and conidial yields, and in extracellular chitinase activity in some transformants. The cultural filtrates of some transformants also decreased acetylcholinesterase activity and the survival of Heterodera filipjevi second-stage juveniles relative to the wild-type strain. One transformant (G10) had a growth rate and biocontrol efficacy similar to the wild-type strain, so it was used for a pilot study of B. bassiana colonization conducted over 13 weeks. Real-time PCR results and CFU counts revealed that the population of G10 increased quickly over the first 3 weeks, then decreased slowly over the following 4 weeks before stabilizing. In addition, the application of wild-type B. bassiana 08F04 and transformant G10 significantly reduced the number of H. filipjevi females in roots by 64.4% and 60.2%, respectively. The results of this study have practical applications for ecological, biological and functional studies of B. bassiana 08F04 and for bionematicide registration.

Benefiting from more precise imaging and radiotherapy, patients with locoregionally nasopharyngeal carcinoma (NPC) have a significantly higher survival rate. Nonetheless, distant metastasis is still the predominant mode of failure. Advances in cancer research have highlighted that pathological angiogenesis is necessary for tumor metastasis by offering oxygen, nutrients, or cell metastatic conduits. MicroRNAs (miRNAs), a class of small noncoding RNAs, are increasingly implicated in modulation of angiogenesis in physiological and pathological conditions. Currently, we detected that miR-23a was highly enriched in NPC tissues at the metastatic or premetastatic stage, and its levels in NPC were associated with microvessel density. Subsequently, we proved that alteration of miR-23a expression modulated the growth, migration, and tube formation of HUVECs in vitro and affected the blood vessel outgrowth in the zebrafish model. Considering the possibility that extracellular miR-23a was horizontally transferred from CNE2 cells to HUVECs, we analyzed miR-23a encapsulated in exosomes, showing that overexpression of exosomal miR-23a in NPC promoted angiogenesis both in vitro and in vivo. Moreover, we provided evidences that miR-23a regulated angiogenesis by directly targeting testis-specific gene antigen (TSGA10). Taken together, our findings revealed that metastasis-associated miR-23a from NPC-derived exosomes plays an important role in mediating angiogenesis by targeting TSGA10.

A new bench-stable reagent with double diazonium sites was designed and synthesized for protein crosslinking. Based on the highly efficient diazonium-Tyr coupling reaction, a direct mixture of the reagent and tobacco mosaic virus led to the formation of a new hydrogel, which could be degraded by chemicals and could be used to encapsulate small molecules for sustained release. Because plant viruses exhibit many chemical characteristics like protein labelling and nucleic acid packaging, the virus-based hydrogel will have large chemical space for further functionalization. Besides, this dual-diazonium reagent should be a generally useful crosslinker for chemical biology and biomaterials.

Recently, a magnetic protein was discovered, and a multimeric magnetosensing complex was validated, which may form the basis of magnetoreception. In this study, the magnetic protein was firstly used in biotechnology application, and a novel convenient one-step purification and immobilization method was established. A universal vector and three linker patterns were developed for fusion expression of magnetic protein and target protein. The magnetic protein was absorbed by iron beads, followed by target protein aggregation, purification, and immobilization. GFP, employed as a reporter protein, was successfully purified from cell lysate. Subsequently, three enzymes (lipase, α-L-arabinofuranosidase, pullulanase) with different molecular sizes testified the versatility of this magnetic-based approach. The specific activities of the purified enzymes were distinctly higher than those of the traditionally purified enzymes using affinity chromatography. The lipase immobilized on iron beads presented improved thermostability and enhanced pH tolerance compared to the free enzyme. The immobilized lipase could be easily recovered and reused for maximum utilization. After 20 cycles of reutilization, the magnetically immobilized lipase retained 71% of its initial activity. This investigation may help introduce magnetic protein into biotechnology applications, and the one-step purification and immobilization method may serve to illustrate an economically viable process for industry.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals(1,2). However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(ii) and the integrity of its binding motif His-XAsp, which is conserved in Fe-dependent dioxygenases(3). However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate(4), CMD1 uses l-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.

© 2019 The Author(s). Background: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has previously been demonstrated to play a pro-inflammatory role in allergic airways disease and COPD through the upregulation of the E3 ubiquitin ligase MID1 and the subsequent deactivation of protein phosphatase 2A (PP2A). Methods: Biopsies were taken from eight IPF patients presenting to the Second Affiliated Hospital of Jilin University, China between January 2013 and February 2014 with control samples obtained from resected lung cancers. Serum TRAIL, MID1 protein and PP2A activity in biopsies, and patients' lung function were measured. Wild type and TRAIL deficient Tnfsf10 -/- BALB/c mice were administered bleomycin to induce fibrosis and some groups were treated with the FTY720 analogue AAL(s) to activate PP2A. Mouse fibroblasts were treated with recombinant TRAIL and fibrotic responses were assessed. Results: TRAIL in serum and MID1 protein levels in biopsies from IPF patients were increased compared to controls. MID1 levels were inversely associated while PP2A activity levels correlated with DLco. Tnfsf10 -/- and mice treated with the PP2A activator AAL(s) were largely protected against bleomycin-induced reductions in lung function and fibrotic changes. Addition of recombinant TRAIL to mouse fibroblasts in-vitro increased collagen production which was reversed by PP2A activation with AAL(s). Conclusion: TRAIL signalling through MID1 deactivates PP2A and promotes fibrosis with corresponding lung function decline. This may provide novel therapeutic targets for IPF.

Natural products (NPs) are a promising source of anti-inflammatory molecules for the development of drugs. Despite there being an abundance of reports of large numbers of NPs having bioactivity in preliminary cell-based assays of anti-inflammatory potential, their further optimization and exploration are limited by the lack of a comprehensive understanding of their effective scaffold structure or biological targets. To facilitate target-based studies of anti-inflammatory NPs, the details of 665 NPs reported to have anti-inflammatory activity were extracted from the literature and compiled into a data set we termed InflamNat. The physicochemical properties of the NPs were analyzed, and the distribution of their structures and scaffolds is presented. A compound-target network was constructed from data in the PubChem Bioassay database. The results demonstrated that, compared to natural anticancer compounds in the NPACT database, compounds from the InflamNat data set contained a comparable distribution of compound types but with a higher proportion satisfying Lipinski's rule. The all-atom structures and scaffold of the compounds were diverse and barely convergent, with flavonoids and triterpenoids being the groups with the greatest abundance. The biological targets of the InflamNat compounds were identified as belonging to a variety of protein families that had varied function. Seventy-two percent of InflamNat compounds involved in the network were identified as having more than one biological target, highlighting the potential for multitarget anti-inflammatory drug development. In conclusion, anti-inflammatory NPs provide a good library for the screening of target-based leads or fragment-based drug design. Thus, elucidation of their biological targets is fundamental for either a specific single-target or multitarget drug development strategy. Meanwhile, a large proportion of the chemical space of anti-inflammatory NPs is still unexplored, with novel active scaffolds remaining to be discovered.

The startup and long term operation of enhanced biological phosphorus removal (EBPR) in a continuous-flow reactor (CFR) with granules were investigated in this study. Through reducing the settling time from 9min to 3min gradually, the startup of EBPR in a CFR with granules was successfully realized in 16days. Under continuous-flow operation, the granules with good phosphorus and COD removal performance were stably operated for more than 6months. And the granules were characterized with particle size of around 960μm, loose structure and good settling ability. During the startup phase, polysaccharides (PS) was secreted excessively by microorganisms to resist the influence from the variation of operational mode. Results of relative quantitative PCR indicated that granules dominated by polyphosphate-accumulating organisms (PAOs) were easier accumulated in the CFR because more excellent settling ability was needed in the system.

Neuronal inclusions of poly(GA), a protein unconventionally translated from G(4)C(2) repeat expansions in C9ORF72, are abundant in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) caused by this mutation. To investigate poly(GA) toxicity, we generated mice that exhibit poly(GA) pathology, neurodegeneration and behavioral abnormalities reminiscent of FTD and ALS. These phenotypes occurred in the absence of TDP-43 pathology and required poly(GA) aggregation. HR23 proteins involved in proteasomal degradation and proteins involved in nucleocytoplasmic transport were sequestered by poly(GA) in these mice. HR23A and HR23B similarly colocalized to poly(GA) inclusions in C9ORF72 expansion carriers. Sequestration was accompanied by an accumulation of ubiquitinated proteins and decreased xeroderma pigmentosum C (XPC) levels in mice, indicative of HR23A and HR23B dysfunction. Restoring HR23B levels attenuated poly(GA) aggregation and rescued poly(GA)-induced toxicity in neuronal cultures. These data demonstrate that sequestration and impairment of nuclear HR23 and nucleocytoplasmic transport proteins is an outcome of, and a contributor to, poly(GA) pathology.

Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin β, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import., Author summary Influenza viral RNA is encapsidated by three polymerase proteins and the NP protein to form the vRNP complex, which is transported to the nucleus of infected cells for viral transcription and replication. The active nuclear import of the vRNP complex is mediated by the interaction between NP and importin α through the nuclear import pathway. Because the interactions between NP and the components of the nuclear import pathway are indispensable in mediating the nuclear import of the vRNP complex, the host has evolved mechanisms to antagonize influenza virus infection that target this crucial step. In this study, we identified PLSCR1 as an interacting partner of the influenza NP protein. We found that PLSCR1 negatively regulates influenza virus replication by inhibiting the nuclear import of the NP/vRNP complex. Importantly, we found that PLSCR1 did not disrupt the interaction between NP and importin α. Instead, NP, PLSCR1, and importin α formed a stable complex that blocked the interaction between importin α and importin β, thereby inhibiting the import of NP/vRNP complex through the nuclear import pathway. Our findings provide an example of a host restriction factor binding simultaneously to a nuclear import adaptor and to a cargo protein to inhibit the import of that cargo into the nucleus.

The epithelial-to-mesenchymal transition (EMT) and the reverse process (the mesenchymal-to-epithelial transition [MET]) have been shown to be associated with tumor cell invasion and metastasis in different carcinomas. The EMT and MET have recently been shown to play a key role in the pathogenic processes of sarcomas, which are completely different from those of carcinomas. However, the definitive roles of the EMT in the tumorigenesis of synovial sarcomas remain unknown. Here, we explored whether transforming growth factor (TGF)-β signaling, an important oncogenic event in synovial sarcoma, modulates tumor cell characteristics related to the EMT, such as cell adhesion, migration, invasion, and proliferation. Interestingly, we found that TGF-β1 induced tumor cell activation, resulting in a tendency to aggregate and biphasic-like features. TGF-β1 also caused downregulation of E-cadherin and subsequent upregulation of N-cadherin, Snail, and Slug, which are responsible for EMT-like phenomena and increased cell motility and invasion. To further investigate the roles of TGF-β1 in the EMT, we established a SW982 cell line with stable TGF-β1 inhibition viaSB431542.These cells exhibited significantly decreased motility, migration, and proliferation (P = 0.001). Taken together, our data demonstrated that alterations in the TGF-β1/Smad signaling pathway could regulate the expression of EMT-related factors and the EMT process, resulting in changes in tumor cell invasion, migration, and proliferation in synovial sarcoma cells. These results may provide a important insights into therapeutic interventions and contribute to the present understanding of tumor progression in patients.

Summary MicroRNAs (miRNAs) are small non-coding RNAs, and they bind to complementary sequences in the three prime untranslated regions (3′ UTRs) of target mRNA transcripts, thereby inhibiting mRNA translation or promoting mRNA degradation. Excessive reactive oxygen species (ROS) can cause cell-damaging effects through oxidative modification of macromolecules leading to their inappropriate functions. Such oxidative modification is related to cancers, aging, and neurodegenerative and cardiovascular diseases. Here we report that miRNAs can be oxidatively modified by ROS. We identified that miR-184 upon oxidative modification associates with the 3′ UTRs of Bcl-xL and Bcl-w that are not its native targets. The mismatch of oxidized miR-184 with Bcl-xL and Bcl-w is involved in the initiation of apoptosis in the study with rat heart cell line H9c2 and mouse models. Our results reveal a model of ROS in regulating cellular events by oxidatively modifying miRNA.

Abnormally elevated bone morphogenetic protein 4 (BMP4) expression and mediated signaling play a critical role in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH). In this study, we investigated the expression level and functional significance of four reported naturally occurring BMP4 antagonists, noggin, follistatin, gremlin1, and matrix gla protein (MGP), in the lung and distal pulmonary arterial smooth muscle cell (PASMC). A 21-day chronic hypoxic (10% O2) exposure rat model was utilized, which has been previously shown to successfully establish experimental CHPH. Among the four antagonists, noggin, but not the other three, was selectively downregulated by hypoxic exposure in both the lung tissue and PASMC, in correlation with markedly elevated BMP4 expression, suggesting that the loss of noggin might account for the hypoxia-triggered BMP4 signaling transduction. Then, by using treatment of extrogenous recombinant noggin protein, we further found that noggin significantly normalized 1) BMP4-induced phosphorylation of cellular p38 and ERK1/2; 2) BMP4-induced phosphorylation of cellular JAK2 and STAT3; 3) hypoxia-induced PASMC proliferation; 4) hypoxia-induced store-operated calcium entry (SOCE), and 5) hypoxia-increased expression of transient receptor potential cation channels (TRPC1 and TRPC6) in PASMC. In combination, these data strongly indicated that the hypoxia-suppressed noggin accounts, at least partially, for hypoxia-induced excessive PASMC proliferation, while restoration of noggin may be an effective way to inhibit cell proliferation by suppressing SOCE and TRPC expression.

The fresh waste activated sludge (WAS) from a lab-scale sequencing batch reactor was used to determine the performance of the lysozyme for promoting its biodegradability. The results showed that a strict linear relationship presented between the degree of disintegration (DDM) of WAS and the lysozyme incubation time from 0 to 240min (R(2) was 0.992, 0.995 and 0.999 in accordance with the corresponding lysozyme/TS, respectively). Ratio of net SCOD increase augmented significantly by lysozyme digestion for evaluating the sludge biodegradability changes. Moreover, the protein dominated both in the EPS and SMP. In addition, the logarithm of SMP contents in supernatant presented an increasing trend similar with the ascending logarithmic relation with the lysozyme incubation time from 0 to 240min (R(2) was 0.960, 0.959 and 0.947, respectively). The SMP, especially the soluble protein, had an important contribution to the improvement of WAS biodegradability.

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the “c9RAN proteins” thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1336-5) contains supplementary material, which is available to authorized users.

Water is widely assumed to be essential for protein dynamics and function. In particular, the well-documented ``dynamical'' transition at $\ensuremath{\sim}200\text{ }\text{ }\mathrm{K}$, at which the protein changes from a rigid, nonfunctional form to a flexible, functional state, as detected in hydrogenated protein by incoherent neutron scattering, requires hydration. Here, we report on coherent neutron scattering experiments on perdeuterated proteins and reveal that a transition occurs in dry proteins at the same temperature resulting primarily from the collective heavy-atom motions. The dynamical transition discovered is intrinsic to the energy landscape of dry proteins.

Yersinia pestis (Y. pestis) has caused an alarming number of deaths throughout recorded human history, and novel prophylactics and therapeutics are necessary given its potential as a bioweapon. Only one monoclonal antibody has been identified to date that provides complete protection against Y. pestis. Here, we describe a second novel murine monoclonal antibody (F2H5) that provided complete protection against Y. pestis 141 infection when administered prophylactically to Balb/c mice (100 μg intravenously). We humanized F2H5, characterized its ability to bind to the Y. pestis F1 protein and further characterized the neutralizing epitope using computational and experimental approaches. While Western blot results suggested a linear epitope, peptide mapping using ELISA failed to identify an epitope, suggesting a conformational epitope instead. We adopted a computational approach based on Residue Contact Frequency to predict the site of antigen-antibody interaction and defined the F2H5/F1 binding site computationally. Based on computational approach, we determined that residues G104E105N106 in F1 were critical to F2H5 binding and that CDRH2 and CDRH3 of F2H5 interacted with F1. Our results show that combining computational approach and experimental approach can effectively identify epitopes.

Allergic asthma is a complex disease characterized by acute inflammation of the airways that over time leads to the development of significant structural changes termed remodeling. TNF-related apoptosis-inducing ligand (TRAIL) has an important regulatory role in acute allergic airways inflammation through up-regulation of the E3 ubiquitin ligase Midline-1 (MID-1), which limits protein phosphatase 2A (PP2A) activity and downstream dephosphorylation of proinflammatory signaling molecules. The relevance of TRAIL in the development of airways remodeling has yet to be determined. In this study, the lungs of wild-type (WT) BALB/c and Tnfsf10 knockout (TRAIL-/-) mice were chronically exposed to ovalbumin (OVA) for 12 weeks to induce hallmark features of chronic allergic airways disease, including airways hyperreactivity (AHR), subepithelial collagen deposition, goblet cell hyperplasia, and smooth muscle hypertrophy. TRAIL-/- mice were largely protected from the development of AHR and peribronchial eosinophilia and had reduced levels of mast cells in the airways. This correlated with lower levels of cytokines, including IL-4, -5, -10, and -13, and with lower levels of proinflammatory chemokines from cultured cells isolated from the draining lymph nodes. TRAIL-/- mice were also protected from the characteristic features of airways remodeling, including peribronchial fibrosis, smooth muscle hypertrophy, and mucus hypersecretion, which correlated with reduced TGF-β1 levels in the lungs. MID-1 expression was reduced in TRAIL-/- mice and up-regulated in allergic WT mice. Raising PP2A activity using 2-amino-4-(4-heptyloyphenol)-2-methylbutan-1-ol in allergic WT mice reduced eosinophilia, TGF-β1, and peribronchial fibrosis. This study shows that TRAIL promotes airways remodeling in an OVA-induced model of chronic allergic airways disease. Targeting TRAIL and its downstream proinflammatory signaling pathway involving PP2A may be of therapeutic benefit in reducing the hallmark features of airways remodeling observed in chronic allergic airways inflammation.

Ubiquitin-fold modifier 1 (Ufm1) is a new member of the ubiquitin-like protein family, and its biological function remains largely unknown, particularly in macrophages. In this study, we demonstrate that Ufm1 expression is increased in diabetic mouse resident peritoneal macrophages (RPMs) and in the mouse macrophage cell line, Raw264.7, subjected to endoplasmic reticulum (ER) stress. Following treatment of the cells with the ER stress inducers, thapsigargin (TG) or tunicamycin (TM), the lentiviral short hairpin RNA (shRNA)-mediated knockdown of Ufm1 increased the apoptosis of Raw264.7 cells. Furthermore, these cells had higher expression levels of immunoglobulin heavy chain-binding protein (BiP) and C/EBP homologous protein (CHOP), which are markers of the ER stress response. The overexpression of Ufm1 induced by lentiviral infection in the Raw264.7 cells treated with the ER stress inducers, TG or TM, resulted in the opposite effect. Taken together, our results suggest that Ufm1 is upregulated in diabetic mouse RPMs and in Raw264.7 cells in response to ER stress and that Ufm1 protects macrophages against apoptosis. Thus, Ufm1 may be a novel gene that protects against ER stress-induced apoptosis in macrophages.

A hexanucleotide repeat expansion within a non-coding region of the C9ORF72 gene is the most common mutation causative of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Elucidating how this bidirectionally transcribed G4C2·C4G2 expanded repeat causes "C9FTLD/ALS" has since become an important goal of the field. Likely pathogenic mechanisms include toxicity induced by repeat-containing RNAs, and loss of C9orf72 function due to epigenetic changes resulting in decreased C9ORF72 mRNA expression. With regards to the former, sense and antisense transcripts of the expanded repeat aberrantly interact with various RNA-binding proteins and form discrete nuclear structures, termed RNA foci. These foci have the capacity to sequester select RNA-binding proteins, thereby impairing their function. (G4C2)exp and (C4G2)exp transcripts also succumb to an alternative fate: repeat-associated non-ATG (RAN) translation. This unconventional mode of translation, which occurs in the absence of an initiating codon, results in the abnormal production of poly(GA), poly(GP), poly(GR), poly(PR) and poly(PA) peptides, collectively referred to as C9RAN proteins. C9RAN proteins form neuronal inclusions throughout the central nervous system of C9FTLD/ALS patients and may contribute to disease pathogenesis. This review aims to summarize the important findings from studies examining mechanisms of disease in C9FTLD/ALS, and will also highlight some of the many questions in need of further investigation.

Chitin synthase and chitinase play crucial roles in chitin biosynthesis and degradation during insect molting. Silencing of Dicer-1 results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. In this study, we found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) were regulated by miR-71 and miR-263 during nymph molting. The coding sequence of CHS1 and the 3’-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 displayed cellular co-localization with their target genes in epidermal cells and directly interacted with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppressed the expression of CHS1 and CHT10, which consequently altered chitin production of new and old cuticles and resulted in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increased CHS1 and CHT10 mRNA expression and led to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts., Author Summary Molting is a crucial process in the growth and development in insects. Disturbing the molting process represents an attractive strategy for developing safe and effective insecticides. The migratory locust is a hemimetabolous pest that undergoes five molting stages in its life cycle. Similar molting defects can be observed in expression silencing of the key genes both in miRNA processing and in chitin metabolism. However, any link between a specific miRNA to chitin metabolism has not yet been established. In this study, we elucidated a mechanism by which two miRNAs regulate chitin metabolism related to the molting process. We found that miR-71 and miR-263 directly repress two genes, chitin synthase1 (CHS1) and chitinase10 (CHT10), which are required for chitin biosynthesis and degradation in chitin metabolism. Manipulation of miR-71 and miR-263 expression blocked molting and resulted in abnormal molting by negatively regulating the expression of LmCHS1 and LmCHT10. Furthermore, both up-regulation and down-regulation of LmCHS1 and LmCHT10 by miRNA manipulation altered the chitin content of the new cuticle and old cuticles, leading to a similar aberrant molting phenotype. Our results demonstrate a balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation that controlled the precise molting process in locusts.

The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequences and functional annotation. It integrates, interprets and standardizes data from literature and numerous resources to achieve the most comprehensive catalog possible of protein information. The central activities are the biocuration of the UniProt Knowledgebase and the dissemination of these data through our Web site and web services. UniProt is produced by the UniProt Consortium, which consists of groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads.

Accumulating evidence indicates that miRNAs can be promising diagnostic and/or prognostic markers for various cancers. In this study, we identified a novel miRNA, miR-3607-3p, and its targets in non-small cell lung cancer (NSCLC). The expression of miR-3607-3p was measured and its correlation with patient prognosis was determined. Ectopic expression in NSCLC cells, xenografts, and metastasis models was used to evaluate the effects of miR-3607-3p on proliferation and migration of NSCLC. Luciferase assay and western blotting were performed to validate the potential targets of miR-3607-3p after preliminary screening by microarray analysis and computer-aided algorithms. We demonstrated that miR-3607-3p was downregulated in NSCLC tissues and that miR-3607-3p might act as an independent predictor for overall survival in NSCLC. Moreover, serum miR-3607-3p may be a novel and stable marker for NSCLC. We found that overexpression of miR-3607-3p inhibited cell proliferation, colony formation, migration and invasion, and hampered the cell cycle of NSCLC cell lines in vitro. Our results suggested that miR-3607-3p directly targets TGFBR1 and CCNE2. In accordance with in vitro studies, we confirmed that miR-3607-3p functions as a potent suppressor miRNA of NSCLC. We showed that miR-3607-3p agomir could reduce tumor growth and inhibit TGFBR1 and CCNE2 protein expression. Taken together, our findings indicate that miR-3607-3p can inhibit NSCLC cell growth and metastasis by targeting TGFBR1 and CCNE2 protein expression, and provide new evidence of miR-3607-3p as a potential non-invasive biomarker and therapeutic target for NSCLC., Author summary We first showed downregulation of miR-3607-3p in NSCLC tissues and demonstrated that miR-3607-3p may act as an independent predictor of overall survival. Serum miR-3607-3p may be a novel marker of patients with NSCLC. We further found that miR-3607-3p possesses the potency to suppress NSCLC growth and metastasis and induce cell cycle arrest by regulating TGFBR1 and CCNE2. Importantly, agomiR-3607-3p could reduce tumor growth and lung and brain metastasis and inhibit TGFBR1 and CCNE2 protein expression. Our findings suggest that miR-3607-3p is a tumor suppressor in NSCLC and holds promise as a prognostic biomarker and potential therapeutic target for NSCLC.

An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson–Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG–IEF and non-IPG–IEF (opposite to IPG–IEF) were developed to unveil stability mechanism of IPG–IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG–IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG–IEF system, (ii) there existed high diffusional current in non-IPG–IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG–IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG–IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG–IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique.

This paper presents a theoretical study of electrochemical affinity biosensors for the detection of DNA/protein that utilize nanoparticle labels for signal amplification. This study analyzes the effects of binding and mass transport of the analytes on biosensor performance by using numerical simulations. Four cases were considered: 1) nanoparticles used to increase the loading of an electroactive species, or used as catalysts under pseudo-first-order conditions; 2) nanoparticles used as ultramicroelectrode arrays for the electrolysis of large concentrations of substrate; 3) nanoparticles used as seeds to deposit electrochemically detectable species; and 4) nanoparticles used to mediate the deposition of electrocatalysts. By using nanoparticle labels, high sensitivity is possible under all conditions considered. However, theoretical findings suggested that nonspecific adsorption could be more problematic in cases 2-4 due to the mismatch between the chemistry of surface binding and the principle of signal amplification that originates from the effect of mass transport. Under these conditions, any given signal would plateau at a much lower analyte concentration, well before the analyte binding had actually reached a plateau. Views on possible solutions to the above limitations are also presented.

IL-27, as a pleiotropic cytokine, promotes the differentiation of naïve T cells to Th1, while suppressing Th2 and Th17 differentiation in the periphery. However, the role of IL-27 in the thymocyte development remains unknown. Here we showed that IL-27 was highly expressed in thymic plasmacytoid dendritic cells (pDCs) while its receptor expression was mainly detected in CD4+ single-positive (SP) thymocytes. Deletion of the p28 subunit in DCs resulted in a reduction of the most mature Qa-2+ subsets of CD4+ SP T cells. This defect was rescued by intrathymic administration of exogenous IL-27. In vitro differentiation assay further demonstrated that IL-27 alone was able to drive the maturation of the newly generated 6C10+CD69+CD4+ SP cells into Qa-2+ cells. Collectively, this study has revealed an important role of thymic DCs-derived IL-27 in the regulation of the phenotypic maturation of CD4+ SP thymocytes.

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues., Author Summary Hedgehog signaling regulates many important biological processes and has been linked to developmental disorders, wound healing, and cancer. Although the major components in the pathway have been well studied in Drosophila and vertebrates, how the signaling is regulated by different modulators is not well understood. Here, we take advantage of a fly forward genetic screen to isolate Ubr3. We show that it is a novel modulator that regulates Hh signaling. Loss of ubr3 leads to Hh signaling defects in developing eyes of Drosophila, and affects eye, and somite and sensory neuron development in zebrafish. However, Hh signaling is not affected in all cells known to be dependent on Hh signaling as loss of ubr3 in the fly wing and zebrafish inner ear are not affected. This suggests that Ubr3 is a modulator that is only required in some Hh dependent organs/cells. We have shown that Ubr3 down-regulates the levels of Cos2 and its mammalian homolog Kif7, key negative regulators of Hh signaling, through poly-ubiquitination. The poly-ubiquitination of Cos2 by Ubr3 is enhanced by Hh activation, suggesting that it functions in a positive feedback that modulates Hh activation.

In this study, a continuous-flow system with enhanced biological phosphorus removal (EBPR) granules was operated at different COD concentrations (200, 300 and 400mgL(-)(1)) to investigate the effect of COD loading on this system. The results showed that when the COD concentration in influent was increased to 400mgL(-)(1), the anaerobic COD removal efficiency and total phosphorus removal efficiency reduced obviously and the settling ability of granules deteriorated due to the proliferation of filamentous bacteria. Moreover, high COD loading inhibited the EPS secretion and destroyed the stability of granules. Results of high-through pyrosequencing indicated that filamentous bacteria had a competitive advantage over polyphosphate-accumulating organisms (PAOs) at high COD loading. The performance of system, settling ability of granules and proportion of PAOs gradually recovered to the initial level after the COD concentration was reduced to 200mgL(-)(1) on day 81.

MicroRNA (miRNA) may function as an oncogene or a tumor suppressor in tumorigenesis. However, the mechanism of miRNAs in adenoid cystic carcinoma (ACC) is unclear. Here, we provide evidence that miR-24-3p was downreglated and functions as a tumor suppressor in human lacrimal adenoid cystic carcinoma by suppressing proliferation and migration/invasion while promoting apoptosis. miR-24-3p down-regulated protein kinase C eta (PRKCH) by binding to its untranslated region (3’UTR). PRKCH increased the of the cell growth and migration/invasion in ACC cells and suppressed the expression of p53 and p21 in both mRNA and protein level. The overexpression of miR-24-3p decreased its malignant phenotype. Ectopic expression of PRKCH counteracted the suppression of malignancy induced by miR-24-3p, as well as ectopic expression of miR-24-3p rescued the suppression of PRKCH in the p53/p21 pathway. These results suggest that miR-24-3p promotes the p53/p21 pathway by down-regulating PRKCH expression in lacrimal adenoid cystic carcinoma cells.

Background Iron deficiency (ID) is common in toddlers in developing countries. Iron fortified or meat-based complementary foods may be effective to prevent ID. Objective Our objective was to compare iron status at 18 months and growth from 6 to 18 months in rural poor toddlers fed 3 different complementary foods. Methods The study was nested within a larger trial in which 6-month-old infants were randomized to receive 50g/d meat (MG), an equi-caloric fortified cereal supplement (FG) or local cereal supplement (LG) for 1 year. Hb, sTfR, HsCRP, ferritin and AGP were measured in 410 blood samples collected by a random sampling (MG, 137; FG, 140; LG, 133); calprotectin was measured in feces. Body iron = -[log (sTfR ×1000/ferritin)-2.8229] /0.1207. ID = ferritin

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Targeting three defects with one strategy The neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia are most commonly caused by a mutation in the C9orf72 gene. The mutation is an expanded hexanucleotide repeat in a noncoding region. The expanded repeat produces sense and antisense RNA transcripts, which accumulate in patient cells and appear to sequester RNA-binding proteins. The sense and antisense transcripts are also translated into dipeptide repeat proteins, which are aggregation-prone and accumulate in the brain and spinal cord. Last, loss of function from reduced expression of C9orf72 in neurons and glia could contribute to the disease. Kramer et al. targeted both sense and antisense repeats by blocking a single gene called SPT4 , which mitigated degeneration in human cells by reducing all three types of pathologies. Science , this issue p. 708

Motivation: The folding free energy is an important characteristic of proteins stability and is directly related to protein's wild-type function. The changes of protein's stability due to naturally occurring mutations, missense mutations, are typically causing diseases. Single point mutations made in vitro are frequently used to assess the contribution of given amino acid to the stability of the protein. In both cases, it is desirable to predict the change of the folding free energy upon single point mutations in order to either provide insights of the molecular mechanism of the change or to design new experimental studies. Results: We report an approach that predicts the free energy change upon single point mutation by utilizing the 3D structure of the wild-type protein. It is based on variation of the molecular mechanics Generalized Born (MMGB) method, scaled with optimized parameters (sMMGB) and utilizing specific model of unfolded state. The corresponding mutations are built in silico and the predictions are tested against large dataset of 1109 mutations with experimentally measured changes of the folding free energy. Benchmarking resulted in root mean square deviation = 1.78 kcal/mol and slope of the linear regression fit between the experimental data and the calculations was 1.04. The sMMGB is compared with other leading methods of predicting folding free energy changes upon single mutations and results discussed with respect to various parameters. Availability: All the pdb files we used in this article can be downloaded from http://compbio.clemson.edu/downloadDir/mentaldisorders/sMMGB_pdb.rar Contact: ealexov@clemson.edu Supplementary information: Supplementary data are available at Bioinformatics online.