Discovery of metal-binding proteins by thermal proteome profiling.

Metal-binding proteins (MBPs) have various and important biological roles in all living species and many human diseases are intricately linked to dysfunctional MBPs. Here, we report a chemoproteomic method named 'metal extraction-triggered agitation logged by thermal proteome profiling' (METAL-TPP) to globally profile MBPs in proteomes. The method involves the extraction of metals from MBPs using chelators and monitoring the resulting protein stability changes through thermal proteome profiling. Applying METAL-TPP to the human proteome with a broad-spectrum chelator, EDTA, revealed a group of proteins with reduced thermal stability that contained both previously known MBPs and currently unannotated MBP candidates. Biochemical characterization of one potential target, glutamine-fructose-6-phosphate transaminase 2 (GFPT2), showed that zinc bound the protein, inhibited its enzymatic activity and modulated the hexosamine biosynthesis pathway. METAL-TPP profiling with another chelator, TPEN, uncovered additional MBPs in proteomes. Collectively, this study developed a robust tool for proteomic discovery of MBPs and provides a rich resource for functional studies of metals in cell biology.

TREM2 acts as a receptor for IL-34 to suppress acute myeloid leukemia in mice.

The bone marrow microenvironment supports leukocyte mobilization and differentiation and controls the development of leukemias, including acute myeloid leukemia (AML). Here, we found that the development of AML xenotransplants was suppressed in mice with osteoclasts tuberous sclerosis 1 (Tsc1) deletion. Tsc1-deficient osteoclasts released a high level of interleukin-34 (IL-34), which efficiently induced AML cell differentiation and prevented AML progression in various preclinical models. Conversely, AML development was accelerated in mice deficient in IL-34. Interestingly, IL-34 inhibited AML independent of its known receptors but bound directly to triggering receptor expressed on myeloid cells 2 (TREM2), a key hub of immune signals. TREM2-deficient AML cells and normal myeloid cells were resistant to IL-34 treatment. Mechanistically, IL-34-TREM2 binding rapidly phosphorylated Ras protein activator like 3 and inactivated extracellular signal-regulated protein kinase 1/2 signaling to prevent AML cell proliferation and stimulate differentiation. Furthermore, TREM2 was downregulated in patients with AML and associated with a poor prognosis. This study identified TREM2 as a novel receptor for IL-34, indicating a promising strategy for overcoming AML differentiation blockade in patients with AML.

Sigma-1 Receptor Activation Improves Oligodendrogenesis and Promotes White-Matter Integrity after Stroke in Mice with Diabetic Mellitus.

Diabetes mellitus (DM) is a major risk factor for stroke and exacerbates white-matter damage in focal cerebral ischemia. Our previous study showed that the sigma-1 receptor agonist PRE084 ameliorates bilateral common-carotid-artery occlusion-induced brain damage in mice. However, whether this protective effect can extend to white matter remains unclear. In this study, C57BL/6 mice were treated with high-fat diets (HFDs) combined with streptozotocin (STZ) injection to mimic type 2 diabetes mellitus (T2DM). Focal cerebral ischemia in T2DM mice was established via injection of the vasoconstrictor peptide endothelin-1 (ET-1) into the hippocampus. Three different treatment plans were used in this study. In one plan, 1 mg/kg of PRE084 (intraperitoneally) was administered for 7 d before ET-1 injection; the mice were sacrificed 24 h after ET-1 injection. In another plan, PRE084 treatment was initiated 24 h after ET-1 injection and lasted for 7 d. In the third plan, PRE084 treatment was initiated 24 h after ET-1 injection and lasted for 21 d. The Y-maze, novel object recognition, and passive avoidance tests were used to assess neurobehavioral outcomes. We found no cognitive dysfunction or white-matter damage 24 h after ET-1 injection. However, 7 and 21 d after ET-1 injection, the mice showed significant cognitive impairment and white-matter damage. Only PRE084 treatment for 21 d could improve this white-matter injury; increase axon and myelin density; decrease demyelination; and increase the expressions of myelin regulator 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNpase) and myelin oligodendrocyte protein (MOG) (which was expressed by mature oligodendrocytes), the number of nerve/glial-antigen 2 (NG2)-positive cells, and the expression of platelet-derived growth factor receptor-alpha (PDGFRα), all of which were expressed by oligodendrocyte progenitor cells in mice with diabetes and focal cerebral ischemia. These results indicate that maybe there was more severe white-matter damage in the focal cerebral ischemia of the diabetic mice than in the mice with normal blood glucose levels. Long-term sigma-1 receptor activation may promote oligodendrogenesis and white-matter functional recovery in patients with stroke and with diabetes.

Room-temperature polariton lasing in GaN microrods with large Rabi splitting.

Room-temperature polariton lasing is achieved in GaN microrods grown by metal-organic vapor phase epitaxy. We demonstrate a large Rabi splitting (Ω = 2g0) up to 162 meV, exceeding the results from both the state-of-the-art nitride-based planar microcavities and previously reported GaN microrods. An ultra-low threshold of 1.8 kW/cm2 is observed by power-dependent photoluminescence spectra, with the linewidth down to 1.31 meV and the blue shift up to 17.8 meV. This large Rabi splitting distinguishes our coherent light emission from a conventional photon lasing, which strongly supports the preparation of coherent light sources in integrated optical circuits and the study of exciting phenomena in macroscopic quantum states.

Sample attrition analysis in a prospective cohort study of medical graduates in China.

BACKGROUND: A major challenge of prospective cohort studies is attrition in follow-up surveys. This study investigated attrition in a prospective cohort comprised of medical graduates in China. We described status of attrition, identified participants with higher possibility of attrition, and examined if attrition affect the estimation of the key outcome measures. METHODS: The cohort study recruited 3,620 new medical graduates from four medical universities in central and western China between 2015 and 2019. Online follow-up surveys were conducted on an annual basis. Follow-up status was defined as complete (meaning that the participant completed all the follow-up surveys) and incomplete, while incomplete follow-up was further divided into 'always-out', 'rejoin' and 'other'. Multivariable logistic and linear regressions were used to examine factors predicting attrition and the influence on the outcome measures of career development. RESULTS: 2364 (65.3%) participants completed all follow-up surveys. For those with incomplete follow-up, 520 (14.4%) were 'always-out', 276 (7.6%) rejoined in the 2020 survey. Willingness to participate in residency training (OR=0.80, 95%CI[0.66 - 0.98]) and willingness to provide sensitive information in the baseline survey predicted a lower rate of attrition (providing scores for university entrance exam OR=0.82, 95%CI[0.69 - 0.97]]; providing contact information (OR=0.46, 95%CI[0.32 - 0.66]); providing household income (OR=0.60, 95%CI[0.43 - 0.84]). Participants with compulsory rural service (OR=1.52, 95%CI[1.05 - 2.19]) and those providing university entrance scores (OR=1.64, 95%CI[1.15-2.33)) were more likely to rejoin in the follow-up survey. These factors associated with follow-up status did not have significant impact on key outcome measures of career development. CONCLUSIONS: Graduates who were unwilling to participate in residency training or not providing sensitive information should be targeted early in the cohort study to reduce attrition. More information about the study should be provided to those graduates early to facilitate their understanding of the meaning in participation. On the contrary, medical graduates with compulsory rural service and those who provided university entrance scores were more likely to rejoin in the cohort. The research team should invest more effort in contacting those graduates and returned them to the cohort.

Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

Transcription Factor RrANT1 of Rosa rugosa Positively Regulates Flower Organ Size in Petunia hybrida.

The flower is the main organ that produces essential oils in many plants. The yield of raw flowers and the number of secretory epidermal cells are the main factors for essential oil production. The cultivated rose species "Pingyin 1" in China was used to study the effect of RrANT1 on floral organ development. Eighteen AP2 transcription factors with dual AP2 domains were identified from Rosa rugosa genome. RrANT1 belonged to euANT. The subcellular localization results showed that RrANT1 protein is localized in the nucleus. The relative expression level of RrANT1 in the receptacle is higher than that in petals in the developmental stages, and both decreased from the initial phase to senescence. Compared with the RrANT1 expression level in petals in the blooming stage, RrANT1 expression level was significant in petals (~48.8) and highest in the receptacle (~102.5) in the large bud stage. It was only highly expressed in the receptacle (~39.4) in the blooming period. RrANT1 overexpression significantly increased petunia flower and leaf sizes (~1.2), as well as flower fresh weight (~30%). The total number of epidermis cells in the petals of overexpressing plants significantly increased (>40%). This study concluded that RrANT1 overexpression can increase the size and weight of flowers by promoting cell proliferation, providing a basis for creating new rose germplasm to increase rose and essential oil yield.

Activation of aryl hydrocarbon receptor by 6-formylindolo[3,2-b]carbazole alleviated acute kidney injury by repressing inflammation and apoptosis.

Acute kidney injury (AKI) is a multifactorial disease of various aetiologies. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that responds to ligands to induce or repress gene expressions, thereby regulating a diverse spectrum of biological or pathophysiologic effects. However, the effect of AhR on AKI remains unknown. A single intraperitoneal injection of 50% glycerol was performed to induce rhabdomyolysis in C57BL/6J mice. The bilateral renal pedicles were occluded for 30 minutes and then removed to stimulate renal I/R injury. 6-formylindolo[3,2-b]carbazole (FICZ), a photo-oxidation product of tryptophan with a high affinity for AhR, was used. The in vitro study was performed on HK-2 cells. Ferrous myoglobin and FICZ was dissolved in the medium in different cell groups. Treatment with AhR agonist FICZ significantly alleviated the elevation of serum creatinine and urea in AKI. AKI modelling-induced renal damage was attenuated by FICZ. AhR mainly expressed in proximal tubular cells and could be activated by FICZ administration. Meanwhile, AKI triggered the production of pro-inflammatory cytokines in injured kidneys, while FICZ inhibited their expressions. Furthermore, FICZ effectively reversed cell apoptosis in AKI models. Mechanistically, AKI stimulated the activation of NF-κB and JNK pathways in the kidneys, while FICZ significantly suppressed these corresponding protein expressions. For the in vitro study, FICZ also inhibited inflammation and apoptosis in myoglobin or H/R-stimulated HK-2 cells. In summary, agonism of AhR by FICZ alleviated rhabdomyolysis and I/R-induced AKI. FICZ inhibited inflammation and apoptosis via suppressing NF-κB and JNK pathways in proximal tubular cells.

Complement C5 activation promotes type 2 diabetic kidney disease via activating STAT3 pathway and disrupting the gut-kidney axis.

Diabetic kidney disease (DKD) is a severe DM complication. While complement C5 up-regulation and gut dysbiosis are found in T2DM, their roles in DKD are unclear. Here, we investigated the effect of C5 on the gut microbiota during DKD development. Renal C5a/C5a receptor (C5aR) expression changes were measured in T2DM patients and db/db mice. Db/db mice were treated with a C5aR antagonist (C5aRA), and renal function, gut microbiota and renal genome changes were analysed. The effects of C5a and short-chain fatty acids (SCFAs) on the signal transducer and activator of transcription 3 (STAT3) pathway were examined in vitro. C5a was up-regulated in glomerular endothelial cells (GECs) of T2DM patients and db/db mice. Although glucose and lipid metabolism were unchanged, C5aR blockade alleviated renal dysfunction, ECM deposition, macrophage infiltration and proinflammatory factor expression in db/db mice. C5aRA partly reversed the declines in gut microbiota diversity and abundance and gut SCFA levels in db/db mice. C5aRA down-regulated the expression of many immune response-related genes, such as STAT3, in db/db mouse kidneys. C5aRA and SCFAs suppressed C5a-induced STAT3 activation in human renal glomerular endothelial cells (HRGECs). Based on our results, C5 hyperactivation promotes DKD by activating STAT3 in GECs and impairing the gut-kidney axis, suggesting that this hyperactivation is a potential target for the treatment of DKD.

Legionella effector SetA as a general O-glucosyltransferase for eukaryotic proteins.

The identification of host protein substrates is key to understanding effector glycosyltransferases secreted by pathogenic bacteria and to using them for glycoprotein engineering. Here we report a chemical method for tagging, enrichment, and site-specific proteomic profiling of effector-modified proteins in host cells. Using this method, we discover that Legionella effector SetA α-O-glucosylates various eukaryotic proteins by recognizing a S/T-X-L-P/G sequence motif, which can be exploited to site-specifically introduce O-glucose on recombinant proteins.