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Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e7 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived exosomes, leading to the robust quantification of 1,020 cell and exosome surface proteins. We identified a newly-recognized subset of exosome-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer exosomes. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

In Southern China, the bark residues from Eucalyptus trees are collected and burned to generate electricity. They are prone to self-heating and even spontaneous combustion when stored in large-scale stockpiles. Pyrolysis plays an important role in these processes. In this work, the pyrolysis characteristics of Eucalyptus bark were investigated by using thermogravimetric analysis coupled with Fourier transform infrared spectrometry. The mass loss process of Eucalyptus bark pyrolysis consists of three stages: dehydration, decomposition of organic matters and decomposition of oxalate, lignin and other components. Except the decomposition of minerals, Eucalyptus bark pyrolysis was described by a four pseudo-component reaction scheme and the kinetic parameters were obtained by using the genetic algorithm. The major evolved gas species and their generation temperature ranges during Eucalyptus bark pyrolysis were identified by Fourier transform infrared spectrometry. It was found that the temperature ranges of the evolved gases were consistent with the decomposition temperature ranges of pseudo-cellulose and pseudo-hemicellulose, in particular with that of pseudo-cellulose.

The transition from smoldering to flaming is basically a homogeneous gas-phase ignition reaction that has a high potential to engulf the entire fuel bed in flames. However, the transition mechanism, especially under windy environment, is poorly understood due to lack of theoretical study. This work proposes a two-dimensional computational model with multiphase formulations to simulate the forward horizontal smoldering and spontaneous transition to flaming of cellulosic insulation. A chemical scheme of three-step solid decomposition reactions combined with one global gas-phase reaction is used in the model. The multi-physical coupling of heat, mass and momentum fields is implemented to reveal the interactions between the packed insulation bed and the surrounding free flow. This model shows that the hotter surface char layer acts as an ignition source for heating the gas mixture, and both the surface char oxidation and sample pyrolysis reactions behave like a gaseous fuel supplier for gas-phase reaction. With favorable conditions of high-temperature combustible fuel gas and sufficient oxygen supply under windy environment, the transition ultimately occurs at 2-3 cm above the leading edge of smolder front on the top surface, which is totally different from the transition within the porous fuel bed under windless condition. The predicted effects of wind velocity and bulk density of fuel bed on the smoldering velocity agree well with literature data. The modeling results for the first time give an interpretation of the spontaneous smoldering to flaming transition over the fuel bed surface.

The smoldering combustion of natural organic layers such as peatlands leads to the largest and most persistent wildland fires on the Earth. The atmospheric oxygen concentration (mass fraction of oxygen: Y O 2 ) significantly influences the smoldering characteristics of peatlands. This work investigates the effects of Y O 2 on horizontal peat smoldering behaviors by experimental and numerical means. The experimental results indicate that as Y O 2 increases, the upper surface of the dry peat undergoes a transition from weak charring (without combustion) to surface glowing combustion. The smoldering peak temperature increases with increasing Y O 2 , and when YO2 is beyond a certain value (35% in this work), the surface smoldering spread rate starts to deviate from the centerline smoldering spread rate. The decrease in the averaged volume ratio of X CO / X C O 2 and X C H 4 / ( X C H 4 + X CO + X C O 2 ) with increasing Y O 2 manifests the rise in CO2 production owing to intense surface oxidation reactions. For the first time, the model predicts the overhang structure of peat during horizontal smoldering spread. A theoretical criterion for the transition to surface glowing combustion is proposed as the onset of the second surface β-char oxidation, which needs sufficient oxygen and high enough surface temperature. Finally, two nonlinear transition thresholds varying with oxygen concentration and wind velocity are predicted.

The ash thickness formed above the combustion front of peat, which is affected by the inorganic content and the piled bed height of peat column, plays both positive and negative effects on peat smouldering fires. This work investigated the effects of the inorganic content and the piled bed height (5, 10 and 20 cm) on the natural downward smouldering mechanism of peat, both experimentally and numerically. It was observed that the char formation and char oxidation processes are stable and dominant during the downward smouldering. The spread rate of the pyrolysis front was found to decrease almost linearly with the inorganic content, while the spread rate of the char oxidation front was less dependent on the inorganic content and the piled bed height at higher inorganic content. The spread rate of the char oxidation front levelled off when the ash layer above the char layer was thick enough, which was also well-predicted by a 1-D numerical model. The model also predicted the nonlinearly increasing peak temperature and decreasing downward spread rate with depths in experiments. This work provides a better understanding of the multiple role of inorganic content in peat smouldering fires, especially for the fires in the in-depth peat layer with abundant inorganics in natural peatlands.

Inducing protein degradation by proteolysis targeting chimeras (PROTACs) has gained tremendous momentum for its promise to discover and develop new therapies. Based upon our previously reported PROTAC MDM2 degraders, we have designed and synthesized additional analogues. Surprisingly, we found that simple structural modifications of MD-222, a bona fide MDM2 PROTAC degrader, converts it into a "molecular glue", as exemplified by MG-277. MG-277 induces only moderate MDM2 degradation and fails to activate wild-type p53 but is highly potent in inhibition of tumor cell growth in a p53-independent manner. Our mechanistic investigation established that MG-277 is not a PROTAC MDM2 degrader but instead works as a molecular glue, inducing degradation of a translation termination factor, GSPT1 to achieve its potent anticancer activity. Our study provides the first example that simple structural modifications can convert a bona fide PROTAC degrader into a molecular glue compound, which has a completely different mechanism of action.

A functional HIV cure requires immune reconstitution for lasting viremia control. A major immune dysfunction persisting in HIV infection is the impairment of T helper cell migration and homing to lymphoid tissues such as GALTs (gut-associated lymphoid tissues). ART (antiretroviral therapy) does not fully restore T cell motility for tissue repopulation. The molecular mechanism dictating this persistent T cell dysfunction is not understood. Cofilin is an actin-depolymerizing factor that regulates actin dynamics for T cell migration. Here, we demonstrate that blood CD4 T cells from HIV-infected patients (n = 193), with or without ART, exhibit significantly lower levels of cofilin phosphorylation (hyperactivation) than those from healthy controls (n = 100; ratio, 1.1:2.3; P < 0.001); cofilin hyperactivation is also associated with poor CD4 T cell recovery following ART. These results suggest an HIV-mediated systemic dysregulation of T cell motility that cannot be repaired solely by ART. We further demonstrate that stimulating blood CD4 T cells with an anti-human α4β7 integrin antibody can trigger signal transduction and modulate the cofilin pathway, partially restoring T cell motility in vitro. However, we also observed that severe T cell motility defect caused by high degrees of cofilin hyperactivation was not repairable by the anti-integrin antibody, demonstrating a mechanistic hindrance to restore immune functions in vivo. Our study suggests that cofilin is a key molecule that may need to be therapeutically targeted early for T cell tissue repopulation, immune reconstitution, and immune control of viremia.

Smoldering combustion of natural organic layers like peatlands leads to the largest fires on Earth. The multidimensional smoldering structure of peatlands is observed in the field, but the mechanism is poorly understood. In this work, a 2-D cylindrical axi-symmetric model that considers volume shrinkage is developed for the natural downward smoldering of peat with different inorganic contents. Numerical results show that the model captures the two-dimensional multisubfront structure of the peat smoldering process. For the peat with lower inorganic content, the predicted bidimensionality of wall-attachment is more obvious, the mass loss rate and the spread rate are higher, and the smoldering front is thinner. For the peat with higher inorganic content, smoldering is more sensitive to lateral heat loss. Both the inorganic content and the lateral heat loss have negative effects on the volume shrinkage, peak temperature, and in-depth spread rate. Additionally, two groups of experiments using peat samples with...

An ignition model of reactive material (RM) by a hot particle, taking into account the interface thermal resistance (ITR) and reactant consumption is established. The heat transfer process between RM and the particle was described by a radiation boundary condition at the interface. Five regimes were identified from the ignition model through numerical simulations. The criticality of reaction heat on the ignition regime was determined by the sensitivity analysis technique. It was found that a larger ITR would result in a longer ignition delay time, a smaller burnout area and a higher critical reaction heat of RM, and a higher critical radius and initial temperature of the particle. The effects of particle size, initial particle temperature and particle shape on the ignition delay time were more significant at a larger ITR. This work shows that both ITR and reactant consumption have unusual and significant influences on the ignition process of RM by a hot particle, and may have an instructive significance for the hot-spot ignition theory and RM management.

Thermal characteristics and gas compositions during peat pyrolysis under helium atmosphere were investigated using the TG–FTIR-MS coupling technique. The majority of gas products were released during the decomposition temperature of 150–550 °C, resulting in about 60% mass loss of peat. The main gas species detected during 150–400 °C were CO 2 , CO, CH 4 , C 2 H 6 , HCOOH, CH 3 CH 2 COOH, C 4 H 8 and CH 2 CHCHO. From 400 °C to 550 °C, some aromatic hydrocarbons were identified, including benzaldehyde, phenol, benzoic acid and toluene. The release of CO 2 , CO, CH 4 and some aromatic hydrocarbons over 550 °C provided the evidence of char pyrolysis. A five-step pyrolysis scheme including water evaporation, hemicellulose, cellulose and lignin degradation, and char pyrolysis was used to simulate peat decomposition process in inert atmosphere. The well agreement between the calculated and experimental curves indicated that major aliphatic gases were derived from hemicellulose and cellulose pyrolysis, while aromatic hydrocarbons were mainly from lignin and char pyrolysis. This work provides a deep insight into the utilization of peat for gaseous biofuels.

Reduced-scale mock-ups had been developed to assess and regulate smoldering ignition resistance of residential upholstered furniture (RUF). However, there are limited data available on the effects of the mock-up test configuration and foam thickness on smoldering propensity and how they affect the degree of correlation with full-scale performance. In this work, the smoldering propensity for standard mock-ups (SMs, where the foam is in contact with a support frame) and modified mock-ups (MMs, where a 13 ± 1 mm air gap is introduced between the foam and the frame), were computationally simulated and compared to experimental results. The model results indicated that the buoyant airflow at the bottom of the mock-up was enhanced in the MM, giving rise to a higher foam oxidation rate, a higher peak smoldering temperature and higher mass loss rate as compared to the SM, and; that oxygen supply was dominated by diffusion-driven transport from the boundaries in proximity of the heating source in the SM. Additionally, the effects of foam thickness on smoldering propensity in the SM and MM were studied with foam thicknesses of 51 mm (2 in.) and 76 mm (3 in.). With an increase in the foam thickness, the smoldering propensity is weakened in the MM but enhanced in the SM. The model was able to predict the ranking of smoldering propensity quantified by the mass loss (ML) in experiments: ML(t)SM2

Human murine double minute 2 (MDM2) protein is a primary endogenous cellular inhibitor of the tumor suppressor p53 and has been pursued as an attractive cancer therapeutic target. Several potent, non-peptide small-molecule inhibitors of MDM2 are currently in clinical development. In this paper, we report our design, synthesis and evaluation of small-molecule MDM2 degraders based on the proteolysis targeting chimera (PROTAC) concept. The most promising compound (MD-224) effectively induces rapid degradation of MDM2 at concentrations

Proteins of the bromodomain and extra-terminal (BET) family are epigenetics “readers” and promising therapeutic targets for cancer and other human diseases. We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders. Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations. QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules. QCA570 is the most potent and efficacious BET degrader reported to date.

A functional HIV cure requires immune reconstitution for lasting viremia control. A major immune dysfunction persisting in HIV infection is the impairment of T helper cell migration and homing to lymphoid tissues such as GALTs (gut-associated lymphoid tissues). ART (antiretroviral therapy) does not fully restore T cell motility for tissue repopulation. The molecular mechanism dictating this persistent T cell dysfunction is not understood. Cofilin is an actin-depolymerizing factor that regulates actin dynamics for T cell migration. Here, we demonstrate that blood CD4 T cells from HIV-infected patients (

Smoldering combustion of peat receives considerable attention for the substantial destruction of carbon storage and potential health threats from high yields of noxious gaseous products. In this work, the combustion characteristics of peat were investigated by using thermogravimetric analysis coupled with Fourier transform infrared spectrometry (TG-FTIR) at five heating rates (5, 10, 15, 20 and 30 K min−1). The results showed that the combustion process included three stages, corresponding to the processes of dehydration (302–423 K), decomposition of organic matters of peat (423–643 K) and char oxidation (643–873 K). Tiny light organic gas products were generated in a lower temperature range (473–643 K), primarily ascribing to the decomposition of organic matters. Abundant CO2 and slight CO were released in a higher temperature range (643–873 K) during the combustion process, mainly due to char oxidation. A four-step kinetic scheme was developed through Genetic Algorithm and described the combustion kinetics very well. These results provide a better insight into the smoldering combustion characteristics and the gas emission behaviors of peat.

We report herein the design, synthesis, and evaluation of macrocyclic peptidomimetics that bind to WD repeat domain 5 (WDR5) and block the WDR5–mixed lineage leukemia (MLL) protein–protein interaction. Compound 18 (MM-589) binds to WDR5 with an IC50 value of 0.90 nM (Ki value 40 times better than the previously reported compound MM-401. Cocrystal structures of 16 and 18 complexed with WDR5 provide structural basis for their high affinity binding to WDR5. Additionally, we have developed and optimized a new AlphaLISA-based MLL HMT functional assay to facilitate the functional evaluation of these designed compounds. Compound 18 represents the most potent inhibitor of the WDR5–MLL interaction reported to date, and further optimization of 18 may yield a new therapy for acute leukemia.

This work studied the forced forward smoldering mechanism of foam by a transient one-dimensional numerical model. A three-step reaction scheme, including foam oxidation and pyrolysis, char oxidation was adopted. Based on the first principles of mass, momentum, and energy, the mass fractions of solid and gas species as well as temperature evolutions in self-propagation regime were examined. Foam oxidation and pyrolysis fronts were found to be ahead of char oxidation front where oxygen is not completely consumed. The critical kinetic parameters for self-sustained smoldering were determined. It was found that pyrolysis endothermic reaction, heat losses, and limited kinetics of char oxidation are favorable for smoldering extinction. Especially, smoldering extinction is more sensitive to kinetic parameters of char oxidation reaction than that of other two reactions. The halfway quenching of smoldering is mainly due to the weakening of char oxidation reaction, which cannot offset the pyrolysis endothermicity and heat loss to environment.

The E3 ubiquitin ligase MDM2 (Murine Double Minute 2) is the most important negative regulator of p53 tumor suppressor primarily by targeting it for ubiquitination and proteasomal degradation. Amplification or overexpression of the MDM2 gene occurs in many human cancers contributing to tumor development, progression and metastasis. Targeting MDM2 to restore p53 function has become an attractive therapeutic strategy for cancers harboring wild-type TP53. Currently, numerous drugs inhibiting the MDM2-p53 interaction, such as RG7112 (Hoffmann-La Roche), MI-773 (Sanofi) and DS-3032b (Daiichi Sankyo), have entered different stages of clinical trials. However, even in p53 wild-type tumors, MDM2 inhibitors can exert limited efficacy as monotherapy in some models, probably due to inadequate p53 induction and side effects. Therefore novel and improved strategies to effectively target the MDM2-p53 pathway are needed. Recently, we have designed a series of MDM2 degraders by conjugating a small molecule MDM2 inhibitor to phthalimide. The phthalimide moiety interacts with its target protein Cereblon (CRBN) and recruits the CUL4-DDB1-CRBN (also known as CRL4CRBN) E3 ubiquitin ligase complex to promote ubiquitination and proteasome degradation of MDM2. Our initial evaluation focused on acute leukemias that are mostly p53 wild-type and express high levels of MDM2. The MDM2 degraders exert significantly improved growth inhibitory activity compared to the inhibitor in human acute leukemia cell lines with IC50s < 5 nM. We performed Western Blots for MDM2 and p53 protein levels and qRT-PCR analysis for mRNA levels of p53 target genes to demonstrate MDM2 protein degradation and activation of p53 downstream pathways. Flow cytometry analysis confirmed the induction of apoptosis by MDM2 degraders in leukemia cells. In agreement with our design, the action of our MDM2 degraders depends on binding to Cereblon, as demonstrated by competition with free phthalimide, and on proteasome function, as demonstrated by proteasome inhibition. siRNA knockdown of p53 significantly abolished the activity of the degraders demonstrating the dependence upon p53 activation. Our studies demonstrate that the MDM2 degraders robustly activate wild-type p53 by inducing rapid degradation of MDM2 and activation of p53 pathways leading to strong apoptosis in leukemia cell lines in a CRBN-binding, proteasome and p53 dependent manner. More importantly, the MDM2 degraders showed great efficacy in inducing complete tumor regression or strong tumor growth inhibition in human leukemia xenograft models and significantly improved survival in disseminated human leukemia models in mice at well-tolerated dose-schedules. Our data provide strong preclinical rationale to further develop MDM2 degraders as a new class of therapy for the treatment of human acute leukemia and potentially other types of human cancer. Citation Format: Jiuling Yang, Yangbing Li, Angelo Aguilar, Donna McEachern, Sally Przybranowski, Ester Fernandez-Salas, Jianfeng Lu. Targeted MDM2 degradation as a novel and efficacious cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4870.

BACKGROUND: Lysosomal protein transmembrane 4 beta (LAPTM4B) is a novel cancer-related gene which has two alleles designated LAPTM4B*1 and LAPTM4B*2. In this study we investigated the correlation of LAPTM4B genotype with prognosis and clinicopathologic features in patients who had undergone curative resection for gallbladder carcinoma (GBC). METHODOLOGY/PRINCIPAL FINDINGS: PCR assay was performed to determine the LAPTM4B genotype in 85 patients. The correlation of LAPTM4B genotype with clinicopathologic parameters was assessed with the Chi-squared test. Differences in patient survival were determined by the Kaplan-Meier method. Multivariate analysis of prognostic factors was carried out with Cox regression analysis. Patients with LAPTM4B *2 had both significantly shorter overall survival (OS) and shorter disease-free survival (DFS) (both P