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BackgroundMuch of what we know about insulin resistance is based on studies from metabolically active tissues such as liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance, however, the underlying mechanisms remain incompletely understood. ADP ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in endothelial cell (EC) function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance.MethodsWe used mouse models of constitutive EC-specific Arf6 deletion (Arf6f/-Tie2Cre) and tamoxifen inducible Arf6 knockout (Arf6f/fCdh5Cre). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose- and insulin-tolerance tests and hyperinsulinemic-euglycemic clamps. A fluorescence microsphere-based technique was used to measure tissue blood flow. Intravital microscopy was used to assess skeletal muscle capillary density.ResultsEndothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue (WAT) and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide (NO) bioavailability but independent of altered acetylcholine- or sodium nitroprusside-mediated vasodilation. In vitro Arf6 inhibition resulted in suppressed insulin stimulated phosphorylation of Akt and endothelial NO synthase. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow fed mice and glucose intolerance in high fat diet fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability.ConclusionResults from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

Both glucose tolerance and adaptive immune function exhibit significant age-related alterations. The influence of the immune system on obesity-associated glucose intolerance is well characterized; however, whether the immune system contributes to age-related glucose intolerance is not as well understood. Here, we report that advancing age results in an increase in T cell infiltration in the epididymal white adipose tissue (eWAT), liver, and skeletal muscle. Subtype analyses show that both CD4+, CD8+ T cells are greater with advancing age in each of these tissues and that aging results in a blunted CD4 to CD8 ratio. Anti-CD3 F(ab')2 fragments depleted CD4+ and CD8+ cells in eWAT, CD4+ cells only in the liver, and did not deplete quadriceps T cells. In old mice, T cells producing both interferon-γ and tumor necrosis factor-α are accumulated in the eWAT and liver, and a greater proportion of skeletal muscle T cells produced interferon-γ. Aging resulted in increased proportion and numbers of T regulatory cells in eWAT, but not in the liver or muscle. Aging also resulted in greater numbers of eWAT and quadriceps CD206- macrophages and eWAT, liver and quadriceps B cells; neither cell type was altered by anti-CD3 treatment. Anti-CD3 treatment improved glucose tolerance in old mice and was accompanied by improved signaling related to liver and skeletal muscle insulin utilization and decreased gluconeogenesis-related gene expression in the liver. Our findings indicate a critical role of the adaptive immune system in the age-related metabolic dysfunction.

Background Inhibitors of the EGFR-RAS-RAF-MEK (MAPK) pathway have been successfully developed to treat cancers. However, many patients do not benefit from these inhibitors due to primary or acquired resistance. In colorectal cancer (CRC), resistance to inhibitors of the MAPK pathway is especially prominent, reflected by low response and short survival. Reactivation of MAPK signaling is a common mechanism of acquired resistance in CRC. WNT pathway is hyperactivated in the majority of CRC patients due to genetic alterations of several genes involved in WNT signaling, e.g., APC, CTNNB1, RSPO and RNF43. Cooperation between RAS-RAF and WNT signaling drives the carcinogenesis of CRC. Concomitant blockade of WNT signaling has been shown to impede the emergence of BRAF inhibitor-resistant clones, thus offering the possibility to overcome acquired resistance. Methods To prove the concept that simultaneous inhibition of the WNT pathway and the MAPK pathway reduces primary or acquired resistance to CRC treatment, we have tested combinations of various inhibitors of both pathways in CRC models, including BRAFV600E ;RSPO3 PDX models, KRASG12C ;RSPO3 cell lines and APC-mutated cell lines with or without KRAS mutations, including KRASG12C ;APC CRC cell lines. We tested MAPK pathway inhibitors, such as cetuximab, selumetinib and cobimetinib (MEK1/2 inhibitors), encorafenib (BRAFV600E inhibitor), and AMG510/sotorasib (KRASG12C inhibitor). Inhibitors of the WNT pathway included a porcupine inhibitor, CGX1321, that blocks the secretion of WNT ligands, currently in clinical development, and a tankyrase inhibitor, CGX11071, that stabilizes AXIN-1/2 and degrades β-catenin. Synergistic effects between inhibitors of the MAPK pathway and the WNT pathway were observed in CRC models in vitro and in vivo. Notably, when CGX1321 was added to encorafenib and cetuximab in CRC PDX models with BRAFV600E and RSPO fusions, a decrease in tumor size (>50%) occurred in two weeks, compared with tumor progression with encorafenib and cetuximab or CGX1321 treatment alone. Notable is the synergy between tankyrase inhibitor CGX11071 and KRASG12C inhibitor AMG510/sotorasib in CRC cell lines with KRASG12C and RSPO fusion (or APC mutations). We launched a phase 1b clinical trial to study the efficacy of CGX1321 in combination with encorafenib and cetuximab in CRC patients with RSPO fusions and BRAFV600E mutations. (NCT02675946). Primary endpoint included safety and RP2D. Secondary endpoints included PK, overall response rate, CR and PR rate, and duration of response by RECIST 1.1 and irRECIST 1.1. The clinical protocol and available data of CGX1321 in combination with encorafenib and cetuximab in CRC patients with RSPO fusions and BRAFV600E mutations will be discussed. Citation Format: Jinjin Cai, Andrew Dorr, Laurie Rosenstein, Jim Labdon, Xiaojuan Li, Hang Zhou, Yanyuan Zhou, Wangjun He, Qiuping He, Xiaoyan Huang, Aparna R. Parikh, Marios Giannakis, Michael An. Synergism between inhibitors of the EGFR-RAS-RAF-MEK pathway and the WNT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT117.

Cardiovascular diseases (CVDs) remain the leading causes of death in the United States, and advancing age is a primary risk factor. Impaired endothelium-dependent dilation and increased stiffening of the arteries with aging are independent predictors of CVD. Increased tissue and systemic oxidative stress and inflammation underlie this age-associated arterial dysfunction. Calorie restriction (CR) is the most powerful intervention known to increase life span and improve age-related phenotypes, including arterial dysfunction. However, the translatability of long-term CR to clinical populations is limited, stimulating interest in the pursuit of pharmacological CR mimetics to reproduce the beneficial effects of CR. The energy-sensing pathways, mammalian target of rapamycin, AMPK, and sirtuin-1 have all been implicated in the beneficial effects of CR on longevity and/or physiological function and, as such, have emerged as potential targets for therapeutic intervention as CR mimetics. Although manipulation of each of these pathways has CR-like benefits on arterial function, the magnitude and/or mechanisms can be disparate from that of CR. Nevertheless, targeting these pathways in older individuals may provide some benefits against arterial dysfunction and CVD. The goal of this review is to provide a brief discussion of the mechanisms and pathways underlying age-associated dysfunction in large arteries, explain how these are impacted by CR, and to present the available evidence, suggesting that targets for energy-sensing pathways may act as vascular CR mimetics.

Mortality is an unfortunately common outcome of extremely and very preterm birth. Existing clinical prediction models capture mortality risk at birth but fail to account for the remainder of the hospital course. Infants born

In this paper, based on the fact that is still a small peasant economy in China and there are many small plots, this article studies small and medium sprayers. In this sprayer, the subdivision precision spraying control system, designed for precision agriculture applications, was simulated by the LabVIEW software, while an experimental setup was able to measure and record during laboratory experiments. The main pipeline of sprayer chose the A and B. And the two different pipelines were set in the different target spray volume. When the theoretical spray value was kept being unchanged, the flow rate was verified with the field sprayer speed which were set by the pulse generator. With that the pressure stability test was completed. Based on the analysis and experimental results, the flow control precision is 97.03%, and the pressure stability precision is 97.88%, the relative average of the pulse generator is 0.05. Finally, the subdivision system could control the flow of the two branches and was better than the traditional spray method in China, while it could achieve more precise control of spray.

Both glucose tolerance and adaptive immune function exhibit significant age-related alterations. The influence of the immune system on obesity-associated glucose intolerance is well characterized; however, whether the immune system contributes to age-related glucose intolerance is not as well understood. Here, we report that advancing age results in an increase in T cell infiltration in the epididymal white adipose tissue (eWAT), liver, and skeletal muscle. Subtype analyses show that both CD4+, CD8+ T cells are greater with advancing age in each of these tissues and that aging results in a blunted CD4 to CD8 ratio. Anti-CD3 F(ab')2 fragments depleted CD4+ and CD8+ cells in eWAT, CD4+ cells only in the liver, and did not deplete quadriceps T cells. In old mice, T cells producing both interferon-γ and tumor necrosis factor-α are accumulated in the eWAT and liver, and a greater proportion of skeletal muscle T cells produced interferon-γ. Aging resulted in increased proportion and numbers of T regulatory cells in eWAT, but not in the liver or muscle. Aging also resulted in greater numbers of eWAT and quadriceps CD206- macrophages and eWAT, liver and quadriceps B cells; neither cell type was altered by anti-CD3 treatment. Anti-CD3 treatment improved glucose tolerance in old mice and was accompanied by improved signaling related to liver and skeletal muscle insulin utilization and decreased gluconeogenesis-related gene expression in the liver. Our findings indicate a critical role of the adaptive immune system in the age-related metabolic dysfunction.

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes β-cell death and inhibits β-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional β-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive β-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive β-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, β-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a β-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in β-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of β-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

The Internet of Multimedia Things (IoMT) orchestration enables the integration of systems, software, cloud, and smart sensors into a single platform. The IoMT deals with scalar as well as multimedia data. In these networks, sensor-embedded devices and their data face numerous challenges when it comes to security. In this paper, a comprehensive review of the existing literature for IoMT is presented in the context of security and blockchain. The latest literature on all three aspects of security, i.e., authentication, privacy, and trust is provided to explore the challenges experienced by multimedia data. The convergence of blockchain and IoMT along with multimedia-enabled blockchain platforms are discussed for emerging applications. To highlight the significance of this survey, large-scale commercial projects focused on security and blockchain for multimedia applications are reviewed. The shortcomings of these projects are explored and suggestions for further improvement are provided. Based on the aforementioned discussion, we present our own case study for healthcare industry: a theoretical framework having security and blockchain as key enablers. The case study reflects the importance of security and blockchain in multimedia applications of healthcare sector. Finally, we discuss the convergence of emerging technologies with security, blockchain and IoMT to visualize the future of tomorrow's applications.

Wheat lines with shortened Th. ponticum chromatin carrying Fhb7 and molecular markers linked to Fhb7 will accelerate the transfer of Fhb7 to breeding lines and provide an important resource for future map-based cloning of this gene. Fusarium head blight is a major wheat disease globally. A major FHB resistance gene, designated as Fhb7, derived from Thinopyrum ponticum, was earlier transferred to common wheat, but was not used in wheat breeding due to linkage drag. The aims of this study were to (1) saturate this FHB resistance gene region; (2) develop and characterize secondary translocation lines with shortened Thinopyrum segments carrying Fhb7 using ph1b; (3) pyramid Fhb7 and Fhb1 by marker-assisted selection. Fhb7 was mapped in a 1.7 cM interval that was flanked by molecular markers XsdauK66 and Xcfa2240 with SSR, diversity arrays technology, EST-derived and conserved markers. KS24-2 carrying Fhb7 was analyzed with molecular markers and genomic in situ hybridization, confirming it was a 7DS.7el2L Robertsonian translocation. To reduce the Thinopyrum chromatin segments carrying Fhb7, a BC1F2 population (Chinese Spring ph1bph1b*2/KS24-2) was developed and genotyped with the markers linked to Fhb7. Two new translocation lines (SDAU1881 and SDAU1886) carrying Fhb7 on shortened alien segments (approximately 16.1 and 17.3 % of the translocation chromosome, respectively) were developed. Furthermore, four wheat lines (SDAU1902, SDAU1903, SDAU1904, and SDAU1906) with the pyramided markers flanking Fhb1 and Fhb7 were developed and the FHB responses indicated lines with mean NDS ranging from 1.3 to 1.6 had successfully combined Fhb7 and Fhb1. Three new molecular markers associated with Fhb7 were identified and validated in 35 common wheat varieties. The translocation lines with shortened alien segments carrying Fhb7 (and Fhb1) and the markers closely linked to Fhb7 will be useful for improving wheat scab resistance.

Even though Big Data is a hot technology in the ICT industry with a large effect on economy and society, Big Data is a relatively indefinite concept, like a “laundry list”,compared with cloud computing and Internet of things. Fortunately, there is a consensus that Big Data standardization can promote the exchange and scoping of data, guarantee the data completeness and timeliness, ensure the quality of further analysis, eliminate information islands and improve the industry efficiency. However, the existed methods to Big Data standard system are all technology-oriented. This paper proposes an integrated method, called data-oriented method, which combines bottom–up, top–down, method of expertise with investigation together, to build Big Data standard system based on the data essence in the special field. This approach makes the system get rid of pure technique thinking and adapt more to practical condition. Meanwhile, this research also gives a case study on Chinese financial industry in practice. According to that this data-oriented method sets a close and accurate links between special data and standard system, it can be applied on the construction of Big Data standard system in other fields with improving the system model and laying a good foundation for Big Data applications and creations.

RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or LRKO mice). Adipose tissue RBP4 expression and secretion remained intact in LRKO mice and increased as expected in the setting of diet-induced insulin resistance. However, circulating RBP4 was undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin resistance. Adipocyte RBP4, therefore, may have a more important autocrine or paracrine function that is confined within the adipose tissue compartment.

Objective— Impaired endothelial cell (EC) autophagy compromises shear stress–induced nitric oxide (NO) generation. We determined the responsible mechanism. Approach and Results— On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOS S1177 ) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOS S1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2′-deoxy- N 6 -methyladenosine 3′,5′-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOS S1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδ T505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress–induced activating phosphorylation of PKCδ T505 is negated by inhibiting autophagy, (2) shear-induced p-eNOS S1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKCδ) activation of PKCδ T505 , and (3) pharmacological (eg, rottlerin) and genetic (eg, PKCδ small interfering RNA) PKCδ inhibition prevents shear-induced p-eNOS S1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs. Conclusions— Targeted reactivation of purinergic signaling and PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

Segregation distortion can be defined as deviations from normal Mendelian segregation (Sandler et al. 1958). It is a common phenomenon found in most genetic mapping studies in many species (Faris et al. 1998). Lophopyrum ponticum (also syn. Agropyron elongatum (Host) Beau.) has been one of the most important perennial Triticeae germplasm sources for wheat improvement (Oliver et al. 2006). Chromosome 7E of L. ponticum is crucial because it is the carrier of a number of elite agronomic traits (Zhang et al. 2011). However, there is still no report about molecular markers showing segregation distortion region of L. ponticum chromosome 7E. Segregation distortion caused by preferential transmission may be affected by genetic background. Kong et al. (2008) reported that a segment of the Thinopyrum intermedium chromosome 7E present in the translocation line P98134 was preferentially transmitted through male gametes under different genetic background conditions. However, the transmission frequency of T. intermedium 7E segment present in another wheat T. intermedium translocation line, P961341, varied with different genetic backgrounds. Similarly, studies on the translocations involving T. ponticum chromosome 7el1 indicated that Thinopyrum chromosome 7el1 was transmitted normally in Thatcher and China Spring wheat backgrounds (McIntosh et al. 1976). However, high levels of distortion were observed when chromosome 7el1 was transferred in other genetic backgrounds (McIntosh et al. 1995). Both 7el1 and 7el2, are gametocidal chromosomes (KibirigeSebunya and Knott 1983; Prins et al. 1996). Gametocidal chromosomes cause abortion of gametes lacking the gametocidal chromosome, which results in partial sterility and exclusive transmission of these chromosomes (Endo 1990).

Summary: Autophagy is a homeostatic cellular process involved in the degradation of long-lived or damaged cellular components. The role of autophagy in adipogenesis is well recognized, but its role in mature adipocyte function is largely unknown. We show that the autophagy proteins Atg3 and Atg16L1 are required for proper mitochondrial function in mature adipocytes. In contrast to previous studies, we found that post-developmental ablation of autophagy causes peripheral insulin resistance independently of diet or adiposity. Finally, lack of adipocyte autophagy reveals cross talk between fat and liver, mediated by lipid peroxide-induced Nrf2 signaling. Our data reveal a role for autophagy in preventing lipid peroxide formation and its transfer in insulin-sensitive peripheral tissues. : Cai et al. describe how lack of autophagy in mature adipocytes causes insulin resistance with no change in body weight. Adipocytes lacking Atg3 or Atg16L1 accumulate dysfunctional mitochondria and have increased lipid peroxidation and Nrf2 and keap1 activation. This study reveals a role for lipid peroxides and Nrf2 signaling in an adipose-liver crosstalk. Keywords: autophagy, adipocytes, insulin resistance, inflammation, mitochondria, lipid peroxide, adipose tissue, adiponectin

To identify resistance to Fusarium head blight (FHB), cereal yellow dwarf virus (CYDV), stem rust (Sr), and powdery mildew (Pm) in 24 common wheat (Triticum aestivum)-Leymus multicaulis addition/translocation lines that were developed cytogenetically and to verify the authenticity of these lines using microsatellite (SSR) DNA markers. Resistance to FHB was identified in the wheat-L. multicaulis addition lines, Line 9 and Line 26, which both contained L. multicaulis-specific fragments as shown by SSR markers. The translocation line, Trans 1, and the addition lines, Line 5 and Line 29, have resistance to stem rust (IT 0). Resistance to CYDV was evaluated based on virus titers measured by enzyme linked immunosorbent assay (ELISA). The addition line, Line 23, showed low virus titer (0.15), indicating resistance to CYDV. The segregation distribution of CYDV resistance in 98 F2 plants of Line 23/CS showed a significant deviation from 3:1. Inoculation with a set of 14 differential Blumeria graminis f. sp. tritici (Bgt) isolates did not detect powdery mildew resistance in translocation line Trans 1, addition line Line 9 and the amphiploid of wheat-L. multicaulis. However, Line 26 exhibited the resistance response pattern of Kavkaz, which carries Pm8, indicating that Line 26 most likely has the powdery mildew resistance gene Pm8 inherited from its parent lines Feng Kang 7 or Feng Kang 10. Twelve SSR markers, distributed on different homeologous chromosome groups of wheat, which distinguished L. multicaulis addition/translocation chromosomes, were used to verify the presence of L. multicaulis chromatin in the putative wheat-L. multicaulis addition/translocation lines. Of the 24 addition/translocation lines investigated using the 12 polymorphic SSR markers, 18 wheat-L. multicaulis derivatives showed the expected L. multicaulis-specific fragments, indicating that all of these 18 addition/translocation lines would most likely have the introgressed L. multicaulis chromosome(s). Chromosomal rearrangements also were detected in some of the wheat-L. multicaulis introgression lines.

In protein identification through tandem mass spectrometry, it is critical to accurately predict the theoretical spectrum for a peptide sequence. The widely used prediction models, such as SEQUEST and MASCOT, ignore the intensity of the ions with important neutral losses, including water loss and ammonia loss. However, ignoring these neutral losses results in a significant deviation between the predicted theoretical spectrum and its experimental counterpart. Here, based on the "one peak, multiple explanations" observation, we proposed an expectation-maximization (EM) method to automatically learn the probabilities of water loss and ammonia loss for each amino acid. Then we employed these probabilities to design an improved statistical model for theoretical spectrum prediction. We implemented these methods and tested them on practical data. On a training set containing 1803 spectra, the experimental results show a good agreement with some known knowledge about neutral losses, such as the tendency of water loss from Asp, Glu, Ser, and Thr. Furthermore, on a testing set containing 941 spectra, the improved similarity between the experimental and predicted spectra demonstrates that this method can generate more reasonable predictions relative to the model that ignores neutral losses. As an application of the derived probabilities, we implemented a database searching method adopting the improved theoretical spectrum model with neutral loss ions estimated. Experimental results on Keller's data set demonstrate that this method can identify peptides more accurately than SEQUEST. In another application to validate SEQUEST's results, the reported peptide-spectrum pairs are reranked with respect to the similarity between experimental and predicted spectra. Experimental results on both LTQ and QSTAR data sets suggest that this reranking strategy can effectively distinguish the false negative predictions reported by SEQUEST.

In protein identification by tandem mass spectrometry, it is critical to accurately predict the theoretical spectrum for a peptide sequence. To date, the widely-used database searching methods adopted simple statistical models for predicting. For some peptide, these models usually yield a theoretical spectrum with a significant deviation from the experimental one. In this paper, in order to derive an improved predicting model, we utilized a non-linear programming model to quantify the factors impacting peptide fragmentation. Then, an iterative algorithm was proposed to solve this optimization problem. Upon a training set of 1803 spectra, the experimental result showed a good agreement with some known principles about peptide fragmentation, such as the tendency to cleave at the middle of peptide, and Pro's preference of the N-terminal cleavage. Moreover, upon a testing set of 941 spectra, comparison of the predicted spectra against the experimental ones showed that this method can generate reasonable predictions. The results in this paper can offer help to both database searching and de novo methods.

Mitochondrial dysfunction is implicated in many human diseases and occurs in normal aging. Mitochondrial health is maintained through organelle biogenesis and repair or turnover of existing mitochondria. Mitochondrial turnover is principally mediated by mitophagy, the trafficking of damaged mitochondria to lysosomes via macroautophagy (autophagy). Mitophagy requires autophagy, but is itself a selective process that relies on specific autophagy-targeting mechanisms and thus can be dissociated from autophagy under certain circumstances. Therefore, it is important to assess autophagy and mitophagy together and separately. We sought to develop a robust, high-throughput, quantitative method for monitoring both processes in parallel. Here we report a flow cytometry-based assay capable of rapid parallel measurements of mitophagy and autophagy in mammalian cells using a single fluorescent protein biosensor. We demonstrate the ability of the assay to quantify Parkin-dependent selective mitophagy in CCCP-treated HeLa cells. In addition, we show the utility of the assay for measuring mitophagy in other cell lines, as well as for Parkin-independent mitophagy stimulated by deferiprone. The assay makes rapid measurements (10,000 cells per 6 seconds) and can be combined with other fluorescent indicators to monitor distinct cell populations, enabling design of high-throughput screening experiments to identify novel regulators of mitophagy in mammalian cells.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKdelta, we disrupted its gene in mice and found that DGKdelta deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKdelta-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGKdelta knockout cells, indicating that DGKdelta regulates EGFR by modulating PKC signaling.

A number of different approaches have been proposed to predict elemental component formulas (or molecular formulas) of molecular ions in low and medium resolution mass spectra. Most of them rely on isotope patterns, enumerate all possible formulas for an ion, and exclude certain formulas violating chemical constraints. However, these methods cannot be well generalized to the component prediction of fragment ions in tandem mass spectra. In this paper, a new method, FFP (Fragment ion Formula Prediction), is presented to predict elemental component formulas of fragment ions. In the FFP method, the prediction of the best formulas is converted into the minimization of the distance between theoretical and observed isotope patterns. And, then, a novel local search model is proposed to generate a set of candidate formulas efficiently. After the search, FFP applies a new multiconstraint filtering to exclude as many invalid and improbable formulas as possible. FFP is experimentally compared with the previous enumeration methods, and shown to outperform them significantly. The results of this paper can help to improve the reliability of de novo in the identification of peptide sequences.

Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues.

The leaf rust resistance gene Lr19 and Fusarium head blight (FHB) resistance quantitative trait loci (QTL) derived from the wild wheatgrass Lophopyrum ponticum have been located on chromosome 7E. The main objectives of the present study were to develop a genetic map of chromosome 7E and map the two resistance loci using a population of 237 F(7:8) recombinant inbred lines (RILs) derived from a cross between two Thatcher-L. ponticum substitution lines, K11463 (7el(1)(7D)) and K2620 (7el(2)(7D)). 532 G-SSR, E-SSR and STS markers from wheat chromosome group 7 were screened in the parent lines. Of these, 118 markers were polymorphic, with a polymorphism frequency of 22.2%. A genetic map of L. ponticum chromosome 7E was constructed with 64 markers, covering 95.76 cM, with an average genetic distance of 1.47 cM between markers. The major FHB resistance locus, temporarily assigned as FhbLoP, was mapped to the very distal region of the long arm of chromosome 7E within a 3.71 cM interval flanked by Xcfa2240 and Xswes19, which accounts for 30.46% of the phenotypic variance. Lr19 was bracketed by Xwmc273 and XBE404744, with a map distance of 1.54 and 1.43 cM from either side, respectively. The closely linked markers identified in this study will be helpful for marker-assisted introgression of the L. ponticum-derived FhbLoP and Lr19 genes into elite cultivars of wheat, and the development of a genetic map will accelerate the map-based cloning of these two genes.

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

There are ten mammalian diacylglycerol kinases (DGKs) whose primary role is to terminate diacylglycerol (DAG) signaling. However, it is becoming increasingly apparent that DGKs also influence signaling events through their product, phosphatidic acid (PA). They do so in some cases by associating with proteins and then modifying their activity by generating PA. In other cases, DGKs broadly regulate signaling events by virtue of their ability to provide PA for the synthesis of phosphatidylinositols (PtdIns).

In protein identification through MS/MS spectrum, it is critical to accurately predict theoretical spectrum from a peptide sequence, which heavily depends on a quantitative understanding of the fragmentation process. To date, widely used database searching methods adopted a simple statistical model to predict theoretical spectrum, yielding a spectrum deviating significantly from the practical spectrum for some peptides and therefore preventing automated positive identification. Here, in order to derive an improved predicting model, we proposed a novel method to automatically learn the factors influencing fragmentation from a training set of MS/MS spectra. In this method, the determining of factors is converted into an optimization problem to minimize an objective function that measures the distance between experimental spectrum and theoretical one. Then, an iterative algorithm was proposed to minimize the non-linear objective function. We implemented the methods and tested them on experimental data. The examination of 1451 spectra is in good agreement with some known knowledge about peptide fragmentation, such as the tendency of cleavage towards the middle of peptide, and Pro's preference of N-terminal cleavage. Moreover, on a testing set containing 1425 spectra, comparison between predicted and practical spectra generates a median correlation of 0.759, showing this method's ability to predict a "realistic" spectrum. The results in this paper help to an accurate identification of protein through both database searching and de novo methods.