Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect

Background: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol(Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis.Methods: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide(LPS). We screened for potential protein targets of Cel using activity-based protein profiling(ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays(CETSA) and surface plasmon resonance(SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown.Results: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS(P <0.05). Cel bound to Cys424 of pyruvate kinase M2(PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis(Warburg effect). Cel also bound to Cys106 in high mobility group box 1(HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin(IL)-1β. Cel bound to the Cys residues in lactate dehydrogenase A(LDHA).Conclusions: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA

Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.

Sparing lung tissue with virtual block method in VMAT planning for locally advanced non-small cell lung cancer

This study aimed to exploit a new virtual block method to spare normal lung tissue in VMAT planning for patients with locally advanced non-small cell lung cancer(LA-NSCLC).The previous method was used to manually restrict the angle of the beam passing through,which ignored the location and shape of large targets that varied between different slices and did not block the beamlets precisely.Unlike the previous method,this new virtual block method was used to block the beamlets when necessary by closing the multi-leaf collimator(MLC)at prerequisite angles.The algorithm for closing the MLC depended on the thickness of the beamlets passing through the lungs and avoided only the entrance radiation beamlet.Moreover,this block can be automatically contoured.A retrospective study was performed to compare the VMAT plans with and without the virtual block method for 17 LANSCLC patients,named the block plan(B-plan)/non-block plan(N-plan).All cases were selected in this study because of the large tumor size and unmet dose constraints of the lungs.In addition to the maximum dose constraint for the virtual block,B-plans adopted identical optimization parameters to N-plans for each patient.These two types of plans were compared in terms of dosimetric indices and plan scores.The results were statistically analyzed using the Wilcoxon nonparametric signed-rank test.B-plans have advantages in the following dosimetric metrics that have statistical significance(p<0.05):(1)lower V_(5)/V_(10)/D_(mean)/normal tissue complication probability(NTCP)of total lungs;(2)reductions in V_(5)/V_(10)for the contralateral lung;(3)decrease in Dmean/V_(40)of the heart;(4)decrease in esophagus V_(40);(5)reductions in Dmean,V_(5)/V_(10) of normal tissue.B-plans(82.51±7.07)achieved higher-quality scores than N-plans(80.74±7.22).The new virtual block spared the lungs as well as other normal structures in VMAT planning for LA-NSCLC.Thus,the block method may decrease the risk of radiation-related toxicity in patients.

Numerical simulation study on monoblock casting process of ultra-slender structural components and experimental validation

Substrate, a typical ultra-slender aluminum alloy structural components with a large aspect ratio and complex internal structure, was traditionally manufactured by re-assembly and sub-welding. In order to realize the monoblock casting of the substrate, the Pro/E software was utilized to carry out three-dimensional(3D) modeling of the substrate casting, and the filling and solidification processes were calculated, as well as the location and types of casting defects were predicted by the casting simulation software Anycasting. Results of the filling process simulation show that the metal liquid is distributed into each gap runner evenly and smoothly. There is no serious vortex phenomenon in the mold cavity, and the trajectory of the virtual particles is clear. Results of the solidification process simulation show that shrinkage cavities mainly appear at the junction of gap runners and the rail surface of the substrate. The average deformation is 0.6 mm in X direction, 3.8 mm in Y direction, and 8.2 mm in Z direction. Based on the simulation results, the casting process of the substrate was optimized, and qualified castings were successfully produced, which will provide a reference for the casting process design of other ultraslender aluminum alloy structural components.