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SummaryProteins with destabilizing N-terminal residues are degraded via K48-linked ubiquitination-dependent N-degron pathway, during which the HECT-type E3 ligase Ufd4 can further augment polyubiquitination events on these proteins to accelerate their degradation. How Ufd4 increase polyubiquitination on ubiquitinated N-degrons remains unclear. Here, we biochemically determined that Ufd4 preferentially ubiquitinates the proximal ubiquitin in K48-linked ubiquitin chain in K29-linkage specific manner and revealed high-resolution cryo-EM structures of Ufd4 accepting (E2-to-E3 state, 3.52 Å) and transferring ubiquitin-thioester (E3-to-substrate state, 3.31 Å), respectively. The N-terminal ARM region and HECT domain C-lobe of Ufd4 were identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximal Ub to the active cysteine of Ufd4 for K29-linked branched ubiquitination. These structures not only provide mechanistic insights into the architecture of the Ufd4 complex but also show the full picture of the Ub transferring cascades of HECT-type E3 ligase.

Objective Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. Methods A miR122 target (122T) sequence was incorporated into the 3′ untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. Results The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. Conclusion HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

Protein ubiquitination regulates almost all eukaryotic cellular processes, and is of very high complexity due to the diversity of ubiquitin (Ub) modifications including mono-, multiply mono-, homotypic poly-, and even heterotypic poly-ubiquitination. To accurately elucidate the role of each specific Ub signal in different cells with spatiotemporal resolutions, a variety of chemical biology tools have been developed. In this review, we summarize some recently developed chemical biology tools for ubiquitination studies and their applications in molecular and cellular biology.

Protein ubiquitination regulates almost every process in eukaryotic cells. The study of the many enzymes involved in the ubiquitination system and the development of ubiquitination-associated therapeutics are important areas of current research. Synthetic tools such as ubiquitin-based chemical probes have been making an increasing contribution to deciphering various biochemical components involved in ubiquitin conjugation, recruitment, signaling, and deconjugation. In the present minireview, we summarize the progress of ubiquitin-based chemical probes with an emphasis on their various structures and chemical synthesis. We discuss the utility of the ubiquitin-based chemical probes for discovering and profiling ubiquitin-dependent signaling systems, as well as the monitoring and visualization of ubiquitin-related enzymatic machinery. We also show how the probes can serve to elucidate the molecular mechanism of recognition and catalysis. Collectively, the development and application of ubiquitin-based chemical probes emphasizes the importance and utility of chemical protein synthesis in modern chemical biology., This article reviews the design, synthesis, and application of different classes of Ub-based chemical probes.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

It is very important to seek a sustainable improvement in human well-being under a limited resource supply and to promote the scientific and coordinated development of urban economic development, ecological environment protection, and human well-being. This paper constructs a human well-being index that includes economic well-being, culture and education well-being, and social development well-being as factors, and it incorporates the human well-being index into the evaluation system for urban well-being energy eco-efficiency (WEE). It uses the super-slack-based measure (SBM) model, which considers undesirable output, to measure the WEE of 10 prefecture-level cities in Shaanxi Province, China, from 2005 to 2019. The social network analysis (SNA) is used to describe the characteristics of the spatial correlation network of WEE and its spatiotemporal evolutionary trend, and the quadratic assignment procedure (QAP) analysis method is used to identify the driving factors that affect the spatial correlation network. The results show that, first, the WEE in Shaanxi is relatively low as a whole and varies greatly among regions, with the highest level in northern Shaanxi, followed by Guanzhong; the lowest level is in southern Shaanxi. Second, in Shaanxi, WEE has transcended geographical proximity into a complex, multi-threaded spatial correlation network, and Yulin is at the center of the network. Third, the network shows four sectors: the net overflow, main benefit, two-way overflow, and broker. Members in each sector have not fully exploited their advantages, and the whole network can be improved. Fourth, the differences in the economic development level, openness, industrial structure, and population are the main driving factors influencing the formation of the spatial correlation network.

The leading cause of heart failure is cardiomyopathy and damage to the cardiomyocytes. Adult mammalian cardiomyocytes have the ability to regenerate, but this cannot wholly compensate for myocardial cell loss after myocardial injury. Studies have shown that exercise has a regulatory role in the activation and promotion of regeneration of healthy and injured adult cardiomyocytes. However, current research on the effects of aerobic exercise in myocardial regeneration is not comprehensive. This review discusses the relationships between aerobic exercise and the regeneration of cardiomyocytes with respect to complex molecular and cellular mechanisms, paracrine factors, transcriptional factors, signaling pathways, and microRNAs that induce cardiac regeneration. The topics discussed herein provide a knowledge base for physical activity-induced cardiomyocyte regeneration, in which exercise enhances overall heart function and improves the efficacy of cardiac rehabilitation.

Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.

ObjectiveTo determine whether gut microbiota, fatty metabolism and cytokines were associated with immune thrombocytopenia (ITP).MethodsIn total, 29 preliminarily diagnosed ITP patients and 33 healthy volunteers were enrolled. Fecal bacterial were analyzed based on 16S rRNA sequencing. Plasma cytokines and motabolites were analyzed using flow cytometry and liquid chromatography-mass spectrometry (LC-MS), respectively.ResultsBacteroides, Phascolarctobacterium, and Lactobacillus were enriched at the genus level in ITP patients, while Ruminococcaceae UCG-002, Eubacterium coprostanoligeues, Megamonas, and Lachnospiraceae NC2004 were depleted. At the phylum level, the relative abundance of Proteobacteria and Chloroflexi increased in ITP patients, while Firmicutes, Actinobacteria, and the Firmicutes/Bacteroidetes ratio decreased. Plasma levels of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-12-epi-leukotriene B4 (6t,12e-LTB4), and resolvin D2 (RvD2) were upregulated, and stachydrine, dowicide A, dodecanoylcarnitine were downregulated in ITP patients. Furthermore, RvD2 is positively correlated with order Bacteroidetes VC2.1 Bac22, 5-HETE is positively correlated with genus Azospirillum, and 6t,12e-LTB4 is positively correlated with genus Cupriavidus. In addition, stachydrine is positively correlated with family Planococcaceae, dowicide A is positively correlated with class MVP-15, and dodecanoylcarnitine is positively correlated with order WCHB1-41. Plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were upregulated in ITP patients.ConclusionOur study revealed a relationship between microbiota and fatty metabolism in ITP. Gut microbiota may participate in the pathogenesis of ITP through affecting cytokine secretion, interfering with fatty metabolism.

Mutations in genes encoding PINK1 (PTEN-induced kinase 1) and Parkin (E3 ubiquitin ligase) are identified in familial Parkinson’s disease. However, it remains unclear whether the phosphorylated Ub ...

Solitary, persistent wave packets called solitons hold potential to transfer information and energy across a wide range of spatial and temporal scales in physical, chemical, and biological systems. Mechanical solitons characteristically emerge either as a single wave packet or uncorrelated propagating topological entities through space and/or time, but these are notoriously difficult to control. Here, we report a theoretical framework for programming static periodic topological solitons into a metamaterial, and demonstrate its implementation in real metamaterials computationally and experimentally. The solitons are excited by deformation localizations under quasi-static compression, and arise from buckling-induced kink-antikink bands that provide domain separation barriers. The soliton number and wavelength demonstrate a previously unreported size-dependence, due to intrinsic length scales. We identify that these unanticipated solitons stem from displacive phase transitions with periodic topological excitations captured by the well-known \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\varphi }^{4}$$\end{document}φ4 theory. Results reveal pathways for robust regularizations of stochastic responses of metamaterials., Mechanical solitons are notoriously difficult to control. Here, the authors report a theoretical framework for programming static periodic topological solitons into a metamaterial, and demonstrate its implementation in real metamaterials computationally and experimentally.

New synthetic strategies that exploited the strengths of both chemoselective ligation and recombinant protein expression were developed to prepare K27 di-ubiquitins (diUb), which enabled mechanistic studies on the molecular recognition of K27-linked Ubs by single-molecule Förster resonance energy transfer (smFRET) and X-ray crystallography. The results revealed that free K27 diUb adopted a compact conformation, whereas upon binding to UCHL3, K27 diUb was remodeled to an open conformation. The K27 isopeptide bond remained rigidly buried inside the diUb moiety during binding, an interesting unique structural feature that may explain the distinctive biological function of K27 Ub chains.

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.

The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalysed by Ubr1.

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development., Graphical abstract, rAAV vector production requires large amounts of bacterial-derived plasmids, which may contain lipopolysaccharide contamination. In this manuscript, we used a genetically modified ClearColi K12 bacterial strain to generate rAAV vectors and showed that the vectors trigger lower endotoxic response in mammalian cells both in vitro and in vivo.

p97, also known as valosin-containing protein (VCP), processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Mutations in human p97 are associated with multisystem proteinopathy (MSP), a dominantly inherited degenerative disorder that can affect muscle, bone and the central nervous system. It is also a drug target for cancer therapy with various inhibitors developed over the past decade. Despite significant structural insights into the fungal homologue of p97, Cdc48, little is known about how human p97 processes its substrates and how the activity is allosterically affected by inhibitors. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Angstrom that captured power stroke-like motions of both the D1 and D2 ATPase rings of p97. The structures elucidated how the unfolded substrate is engaged in the pore at atomic level. Critical conformational changes of the inter-subunit signaling (ISS) motifs were revealed, providing molecular insights into substrate translocation. Furthermore, we also determined cryo-EM structures of human p97 in complex with NMS-873, the most potent p97 inhibitor, at a resolution of 2.4 Angstrom. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change, thus blocking substrate translocation allosterically. Finally, using NMS-873 at a substoichiometric concentration, we captured a series of intermediate states, suggesting how the cofactor Npl4 coordinates with the D1 ring of p97 to initiate the translocation.

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex., The human AAA+protein p97 plays an important role in cellular protein homeostasis. Here, the authors use cryo-EM to obtain further insights into how p97 interacts with its co-factor Npl4 and they observe three distinct conformational states of Npl4 in complex with human p97, which suggests that a seesaw motion is essential for the unfolding activity of the p97 complex.

Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.

Osteoporosis currently afflicts 8 million postmenopausal women in the US, increasing the risk of bone fractures and morbidity, and reducing overall quality of life. We sought to define moderate exercise protocols that can prevent postmenopausal osteoporosis. Our previous findings singled out higher walking speed and pre-exercise meals as necessary for suppression of bone resorption and increasing of markers of bone formation. Since both studies were amenable to alternate biomechanical, nutritional, and circadian interpretations, we sought to determine the relative importance of higher speed, momentum, speed-enhanced load, duration of impulse, and meal timing on osteogenic response. We hypothesized that: (1) 20 min of exercise one hour after eating is sufficient to suppress bone resorption as much as a 40-min impulse and that two 20 min exercise bouts separated by 7 h would double the anabolic effect, (2) early morning exercise performed after eating will be as effective as mid-day exercise for anabolic outcome, and (3) the 08:00 h 40-min. exercise uphill would be as osteogenic as the 40-min exercise downhill. Healthy postmenopausal women, 8 each, were assigned to a no-exercise condition (SED) or to 40- or 20-min exercise bouts, spaced 7 h apart, for walking uphill (40 Up and 20 Up) or downhill (40 Down and 20 Down) to produce differences in biomechanical variables. Exercise was initiated at 08:00 h one hour after eating in 40-min groups, and also 7 h later, two hours after the midday meal, in 20-min groups. Measurements were made of CICP (c-terminal peptide of type I collagen), osteocalcin (OC), and bone-specific alkaline phosphatase (BALP), markers of bone formation, and of the bone resorptive marker CTX (c-terminal telopeptide of type 1 collagen). The osteogenic ratios CICP/CTX, OC/CTX, and BALP/CTX were calculated. Only the 40-min downhill exercise of suprathreshold speed-enhanced momentum, increased the three osteogenic ratios, demonstrating the necessity of a 40-min, and inadequacy of a 20-min, exercise impulse. The failure of anabolic outcome in 40-min uphill exercise was attributed to a sustained elevation of PTH concentration, as its high morning elevation enhances the CTX circadian rhythm. We conclude that postmenopausal osteoporosis can be prevented or mitigated in sedentary women by 45 min of morning exercise of suprathreshold speed-enhanced increased momentum performed shortly after a meal while walking on level ground, or by 40-min downhill, but not 40-min uphill, exercise to avoid circadian PTH oversecretion. The principal stimulus for the anabolic effect is exercise, but the prerequisite for a pre-exercise meal demonstrates the requirement for nutrient facilitation.

Bicistronic transgene expression mediated by internal ribosome entry site (IRES) elements has been widely used. It co-expresses heterologous transgene products from a message RNA driven by a single promoter. Hematologic gene delivery is a promising treatment for both inherited and acquired diseases. A combined strategy was recently documented for potential genome editing in hematopoietic cells. A transduction efficiency exceeding ~90% can be achieved by capsid-optimized recombinant adeno-associated virus serotype 6 (rAAV6) vectors. In this study, to deliver an encephalomyocarditis virus (EMCV) IRES-containing rAAV6 genome into hematopoietic cells, we observed that EMCV IRES almost completely shut down the transgene expression during the process of mRNA&ndash, protein transition. In addition, position-dependent behavior was observed, in which only the EMCV IRES element located between a promoter and the transgenes had an inhibitory effect. Although further studies are warranted to evaluate the involvement of cellular translation machinery, our results propose the use of specific IRES elements or an alternative strategy, such as the 2A system, to achieve bicistronic transgene expression in hematopoietic cells.

Type 2 diabetes increases bone fracture risk in postmenopausal women. Usual treatment with anti-resorptive bisphosphonate drugs has some undesirable side effects, which justified our interest in the osteogenic potential of nutrition and exercise. Since meal eating reduces bone resorption, downhill locomotion increases mechanical stress, and brief osteogenic responsiveness to mechanical stress is followed by several hours of refractoriness, we designed a study where 40-min of mechanical stress was manipulated by treadmill walking uphill or downhill. Exercise preceded or followed two daily meals by one hour, and the meals and exercise bouts were 7 hours apart. Fifteen subjects each performed two of five trials: No exercise (SED), uphill exercise before (UBM) or after meals (UAM), and downhill exercise before (DBM) or after meals (DAM). Relative to SED trial, osteogenic response, defined as the ratio of osteogenic C-terminal propeptide of type I collagen (CICP) over bone-resorptive C-terminal telopeptide of type-I collagen (CTX) markers, increased in exercise-after-meal trials, but not in exercise-before-meal trials. CICP/CTX response rose significantly after the first exercise-after-meal bout in DAM, and after the second one in UAM, due to a greater CICP rise, and not a decline in CTX. Post-meal exercise, but not the pre-meal exercise, also significantly lowered serum insulin response and homeostatic model (HOMA-IR) assessment of insulin resistance.

Structural integrity of bones is supported by mechanical loading, energy intake, and sex hormones. It is unclear why postmenopausal (PM) diabetics experience more bone breaks despite higher body weight and positive energy balance than non-diabetics. We hypothesized that greater bone fragility in PM diabetics may result from peripheral insulin resistance and reduced nutrient access to the bone. We engaged 15 diabetic women, age 57.7y, BMI 27.2 kg/m2, in two of five day-long experiments : SED (no exercise), UBM and UAM (40 min of uphill treadmill exercise, respectively one h before, or one h after eating two daily isocaloric meals at 10 and 17 h and containing 50% carbohydrate, 15% protein, and 25% fat), and DBM and DAM (downhill exercise at -6o slope with meals and exercise timed the same way). Markers of bone formation osteocalcin (OCN) and CICP (c-terminal propeptide of type I collagen) and of resorption, CTX (c-terminal telopeptide of type I collagen), anabolic growth hormone and catabolic cortisol were measured hourly in the serum. Glucose and insulin were measured to assess insulin resistance by the homeostatic HOMA-IR method from 7-h postprandial (PP) insulin and glucose areas under the curve (AUCs).Peak ground-reaction forces (GRFs) were monitored with Novel Pedar mechanosensitive shoe insoles, and physical effort was assessed as percent of VO2max with a metabolic cart. During uphill trials, mean effort was 75.2% , and GRFs were 780.7 N. During downhill trials, the corresponding values were 47.9% of VO2max and 1104.8 N (both >0.05). A similar CTX decline in all 5 trials after the 10h meal was larger than after the 17 h meal (7h negative PP AUCs: 1.52 vs 1.37 ng/ml*h, respectively) and coincided with PP insulin AUCs. CICP rise in DAM trial was 44% greater than in DBM trial after 10h meal (AUCs, 685.9 vs 384.1 ng/ml*h, respectively) and produced a 40 percent greater osteogenic CICP/CTX ratio after 10h meal in the in the DAM than DBM trial. No group difference was seen in brief postprandial serum glucose rises, while PP insulin was highest in UBM and SED trials and low in the two downhill and UAM trials. HOMA-IR in the two downhill trials (355.6) was reduced to 47% of that in two uphill trials (759.1) and to 62% of the SED trial (571.4). Circulating OCN and hormones showed no clear relationship to either the loading or meal-exercise timing patterns. We conclude that In PM diabetics, downhill (as opposed to uphill) exercise after a meal conveys a clear benefit in stimulating a bone- forming CICP, more so after the 10h than the 17 h meal. Meal eating, independently of exercise, lowers the bone resorption marker CTX. In diabetic PM women, Increased GRFs of downhill exercise reduce HOMA-IR insulin resistance to high-carbohydrate meals more than uphill exercise or SED control. Postprandial downhill exercise has the highest osteogenic potential.

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.

Objective: To assess effect of comprehensive nursing intervention on emergency treatment of acute myocardial infarction patients. Methods: 80 patients were invested to join our study who were diagnosed as acute myocardial infarction. They went to the hospital for treatment from March 2017 to July 2019. The participants were randomly assigned to two groups, that included a control group (n = 40) and an intervention group (n = 40). The control group participants received conventional nursing in treatment process. The intervention group participants receive comprehensive nursing intervention. All participants provided the data after their condition stabilized, the data contains first aid time, average hospital stay, the situation of cardiovascular adverse events and patient satisfaction. Result: Base on recording result, first aid time and average hospital stay of intervention group are shorter than that of control group, and those are statistical significance. In cardiovascular adverse events research, intervention group has better performance in cardiovascular adverse, it contains post-infarction angina pectoris [5 (12.5%) vs 10 (25.0%)], heart failure [1 (2.5%) vs 5 (12.5%)], Myocardial infarction recur [1 (2.5%) vs 6 (15.0%)] and cardiac death [0 (0%) vs 1 (2.5%)]. In satisfaction research, intervention group has more very good assessments in the patient report (21 vs 12). Conclusion: the comprehensive nursing intervention has strongly improvement to acute myocardial infarction patients in emergency treatment. Its influence contains not only reducing first aid time and average hospital stay but also reducing the cases of cardiovascular adverse events. Additionally, it also can improve patient satisfaction in the treatment process.

A robust, microwave‐assisted, highly efficient, solid‐phase peptide synthesis method for preparing isopeptide‐linked 62‐mer and 76‐mer isoubiquitins and polyubiquitin is developed. The strategy avoids the use of costly resins and pseudoprolines, and the isopeptide‐linked building blocks can be assembled with high initial purity within 1 day. All seven diubiquitins are successfully synthesized on a multi‐milligram scale; a four‐segment, three‐ligation method is used to obtain a K33‐/K11‐linked mixed triubiquitin in excellent yield. Circular dichroism and crystallographic analyses are used to verify the structures of the well‐folded, synthetic polyubiquitin chains. The facile synthetic strategy is expected to be generally applicable for the rapid synthesis of isopeptide‐linked isoUbs and to pave the way for the study of longer polyubiquitin chains.

Graphical abstract: Hydrothermal-synthesized NiCo{sub 2}O{sub 4} mesowall films exhibit porous structure and high capacity as well as good cycling life for supercapacitor application. - Highlights: • Hierarchical porous NiCo{sub 2}O{sub 4} nanowall films are prepared by a hydrothermal method. • NiCo{sub 2}O{sub 4} nanowall films show excellent electrochemical performance. • Hierarchical porous film structure is favorable for fast ion/electron transfer. - Abstract: Hierarchical porous NiCo{sub 2}O{sub 4} films composed of nanowalls on nickel foam are synthesized via a facile hydrothermal method. Besides the mesoporous walls, the NiCo{sub 2}O{sub 4} nanowalls are interconnected with each other to form hierarchical porous structure. These unique porous structured films possess a high specific surface area. The supercapacitor performance of the hierarchical porous NiCo{sub 2}O{sub 4} film is fully characterized. A high capacity of 130 mA h g{sup −1} is achieved at 2 A g{sup −1} with 97% capacity maintained after 2,000 cycles. Importantly, 75.6% of capacity is retained when the current density changes from 3 A g{sup −1} to 36 A g{sup −1}. The superior electrochemical performance is mainly due to the unique hierarchical porous structure with large surface area as well as shorter diffusion length for ion and charge transport.

Self-supported NiCo 2 O 4 nanoflake arrays on nickel foam are prepared by a facile hydrothermal method. The obtained NiCo 2 O 4 nanoflakes with thicknesses of ∼25 nm grow vertically to the nickel foam substrate and form an interconnected porous network with pore diameters of 50–500 nm. As anode material of LIBs, the NiCo 2 O 4 nanoflake arrays show a high initial coulombic efficiency of 76%, as well as good cycling stability with a capacity of 880 mAh g −1 at 0.5 A g −1 , and 523 mAh g −1 at 1.5 A g −1 after 50 cycles. As the cathode of alkaline batteries, a high capacity of 95 mAh g −1 is achieved at 2 A g −1 and 94% retention is maintained after 10,000 cycles. The superior electrochemical performance is mainly due to the unique nanoflake arrays structure with large surface area and shorter diffusion length for mass and charge transport.

Lysine 2,3-aminomutase (KAM; EC 5.4.3.2) catalyzes the interconversion of l -lysine and l -β-lysine. The transcription and regulation of the kam locus, including lysine-2,3-aminomutase-encoding genes, in Bacillus thuringiensis were analyzed in this study. Reverse transcription-PCR (RT-PCR) analysis revealed that this locus forms two operons: yodT ( yodT-yodS-yodR-yodQ-yodP-kamR ) and kamA ( kamA-yokU-yozE ). The transcriptional start sites (TSSs) of the kamA gene were determined using 5′ rapid amplification of cDNA ends (RACE). A typical −12/−24 σ 54 binding site was identified in the promoter P kamA , which is located upstream of the kamA gene TSS. A β-galactosidase assay showed that P kamA , which directs the transcription of the kamA operon, is controlled by the σ 54 factor and is activated through the σ 54 -dependent transcriptional regulator KamR. The kamA operon is also controlled by σ K and regulated by the GerE protein in the late stage of sporulation. kamR and kamA mutants were prepared by homologous recombination to examine the role of the kam locus. The results showed that the sporulation rate in B. thuringiensis HD(Δ kamR ) was slightly decreased compared to that in HD73, whereas that in HD(Δ kamA ) was similar to that in HD73. This means that other genes regulated by KamR are important for sporulation.

Racemic or quasi-racemic crystallography recently emerges as a useful technology for solution of the crystal structures of biomacromolecules. It remains unclear to what extent the biomacromolecules of opposite handedness can differ from each other in racemic or quasi-racemic crystallography. Here we report a finding that monomeric d-ubiquitin (Ub) has propensity to cocrystallize with different dimers, trimers, and even a tetramer of l-Ub. In these cocrystals the unconnected monomeric d-Ubs can self-assemble to form pseudomirror images of different oligomers of l-Ub. This monomer/oligomer cocrystallization phenomenon expands the concept of racemic crystallography. Using the monomer/oligomer cocrystallization technology we obtained, for the first time the X-ray structures of linear M1-linked tri- and tetra-Ubs and a K11/K63-branched tri-Ub.

A novel star-[polystyrene-b-poly(ferrocenyloxy ethyl acryl ate)]6 with hexa- functional cyclotriphosphazene core (star-(PS-b-PFcEA)6) was synthesized by atom -transfer radical polymerization(ATRP) and its electrochemical behaviors and anion recognition was investigated through CV. The results indicated that increasing of molecular weight resulted in obvious decreasing of reduction peak potential Epc, and the oxidation peak potential Epa kept comparatively stable. Moreover, when titrating anion into polymer solution, Epc increased while Epa reduced evidently, which suggested that star-(PS-b-PFcEA)6 provided with high anion recognition capacity of .

In this paper, the recent development of branched and functional branched polyethylene prepared using Ni- and Pd-based catalysts are reviewed. The influence of ligand species, steric effect, electronic effect and polymerization condition on branching density of resultant polymer prepared using Ni- and Pd-based catalysts is discussed. In addition, the review is also focused on functional branched polyethylene produced by copolymerization of ethylene and polar monomer and copolymerization mechanism.

Molar heat capacities of 4-(aminomethyl)benzoic acid were precisely measured with a small sample precision automated adiabatic calorimeter over the temperature range from 80 to 400 K, and the heat capacity data were fitted to a polynomial equation: C(p,m) = 162.01+91.771X+6.4372X(2)+1.7517X(3)-4.991X(4)+8.909X(5)+1.585X(6), X = (T235)/155. The constant-volume energy of combustion of the compound was measured by means of a precision oxygen bomb combustion calorimeter. The standard enthalpy values of combustion and formation were determined to be: Delta(c)H(m)(circle minus) = -(4300 +/- 4) kJ.mol(-1) and Delta(f)H(m)(circle minus) = -(420 +/- 5) kJ.mol(-1).

In order to determine the effect of bkdR deletion on Cry protein production. We analyzed the transcriptional regulation of bkd gene cluster and the phenotype of bkdR mutant.Sequence of bkd gene cluster in Bacillus thuringiensis was analyzed by sequence alignment. RT-PCRwas used to reveal the transcriptional units of the bkd gene cluster. bkdR insertion mutant was constructed by homologous recombination. Transcriptional activity was analyzed by promoter fusions with lacZ gene. Comparison of the CrylAc protein production was determined by protein quantitation.The bkd gene cluster was composed of eight genes. The ptb-bkdB formed one transcriptional unit. The transcriptional activity of ptb sharply decreased in sigL and bkdR mutants. Deletion of bkdR decreased the motility of cells, but no effect on growth, sporulation efficiency and Cry protein production. The bkd gene cluster is controlled by Sigma 54 and activated by BkdR. Deletion of bkdR has no effect on Cry protein production, but decreased the motility of the cells.The bkd gene cluster is controlled by Sigma 54 and activated by BkdR. Deletion of bkdR has nb effect on Cry protein production, but decreased the motility of the cells. It suggested that deletion of bkdR do not affect the Cry protein production the same as sigL mutant. It means decreasing of Cry protein productioninsigL mutant was not caused by only one EBP mutation, but might be multiple roles.

To optimize the expression of cry genes in a Bacillus thuringiensis sigK mutant failing in crystal releasing, the transcriptional activity of the cry promoters cry1A, cry3A, cry4A, and cry8E was compared using lacZ gene fusions. A beta-galactosidase assay indicated that the cry8E promoter showed the highest transcriptional activity. A novel Escherichia coli-B. thuringiensis shuttle vector pHT315-8E21b was constructed for cry gene expression using the cry8E promoter and the multiple cloning sites from vector pET21b, based on vector pHT315. SDS-PAGE analysis showed that the expression of the cry1Ac gene directed by the cry8E promoter was increased by approximately 2.4-fold over the expression directed by the cry3A promoter. The cry1Ba gene was expressed in the sigK mutant with the constructed vector pHT315-8E21b. Normal bipyramidal crystals encapsulated in mother cell were observed by transmission electron microscopy (TEM). The encapsulated Cry1Ba protein expressed in the sigK mutant showed activity against Ostrinia furnacalis and Plutella xylostella similar to that of the released Cry1Ba protein expressed in the acrystalliferous strain HD73 and can be protected from inactivation by UV light. All these results suggest that the cry8E promoter can be an efficient transcriptional element for cry gene expression in sigK mutants and can be utilized for the construction of a genetically engineered strain.