Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping.

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration ≤ 50 µM is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipe's MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

Actin related protein 2/3 complex subunit 1 up-regulation in the hypothalamus prevents high-fat diet induced obesity.

Obesity is a major health crisis in the modern society. Studies have shown that the consumption of a high-fat diet (HFD) induces hypothalamic inflammation and leptin resistance, which consequently favours body mass gain. Actin related protein 2/3 complex subunit 1 (ARPC1B), an actin-binding protein, is highly expressed in immune cells. Recent studies have shown that ARPC1B has a certain anti-inflammatory effect. While ARPC1B expression is decreased in the hypothalamus of mice fed a HFD, the role of ARPC1B in HFD-induced obesity remains unclear. Thus, we investigated whether ARPC1B up-regulation in the hypothalamic arcuate nucleus (ARC) could inhibit the development of obesity. Herein, ARPC1B overexpression lentiviral particles were stereotaxically injected into the ARC of male C57BL/6J mice (7 weeks old) fed with HFD. Overexpression of ARPC1B in the hypothalamic ARC attenuated HFD-induced ARC inflammation, reduced body-weight gain and feed efficiency. Furthermore, up-regulation of ARC ARPC1B improved the glucose tolerance and reduced subcutaneous/epididymal fat mass accumulation, which decreased the serum total cholesterol, serum triglyceride and leptin levels. In addition, upon ARPC1B overexpression in the hypothalamic ARC, intraperitoneal injection of leptin increased the phosphorylation level of signal transducer and activator of transcription 3 (STAT3), an important transcription factor for leptin's action, in the ARC of obese mice. Accordingly, we suggest that up-regulation of ARPC1B in the hypothalamic ARC may improve the HFD-induced hypothalamic inflammation and leptin resistance. Our findings demonstrate that ARPC1B is a promising target for the treatment of diet-induced obesity.

[Clinical efficacy on xerosis conjunctivitis of liver and kidney yin deficiency treated with SHI's acupuncture manipulation].

OBJECTIVE: To compare the efficacy of the patients of xerosis conjunctivitis with liver and kidney yin deficiency among the combined therapy of acupuncture and Shi's manipulation, common acupuncture and artificial tears therapy. METHODS: One hundred and eight patients were randomized into an acupuncture group, a SHI's manipulation group and an artificial tears group, 36 cases in each group. A total of 15 cases dropped out before the end of the study, including 4 cases in the acupuncture group, 6 cases in the SHI's manipulation group, and 5 cases in the artificial tears group. In the acupuncture group, acupuncture was applied to Jingming (BL 1) and Qiuhou (EX-HN 7) on the affected side, and the bilateral Sanyinjiao (SP 6) and Taixi (KI 3). The needles were retained for 20 min. In the SHI's manipulation group, on the basis of the treatment as the acupuncture group, Shuigou (GV26) was added and stimulated with SHI's acupuncture manipulation. In these two groups, acupuncture was given 3 times a week totally for 3 weeks. In the artificial tears group, sodium hyaluronate eye drops were used, 5 times a day, for 3 weeks totally. Separately, before treatment, at the moment after the 1st treatment and 3 weeks after treatment, the subjective symptom score, Schirmer I test, breakup time (BUT) of tear film were observed in each group. RESULTS: (1) Subjective symptom score: at the moment after the 1st treatment and 3 weeks after treatment, the scores in each group were all reduced significantly as compared with those before treatment (all P < 0.05). At the moment after the 1st treatment, the score in the SHI's manipulation group and the artificial tears group was reduced apparently as compared with that in the acupuncture group (both P < 0.05). In 3 weeks of treatment, the score in the SHI's manipulation group was reduced apparently as compared with the acupuncture group and the artificial tears group (both P < 0.05). (2) For Schirmer I test, at the moment of the 1st treatment, the result in the SHI's manipulation group and the artificial tears group was improved significantly as compared with that before treatment (both P < 0.05). In 3 weeks of treatment, the result in the acupuncture group and the SHI's manipulation group group was improved significantly as compared with that before treatment (both P < 0.05). At the moment of the 1st treatment, the result in the artificial tears group was improved significantly as compared with the acupuncture group and the SHI's manipulation group (both P < 0.05). In 3 weeks of treatment, the result in the acupuncture group and the SHI's manipulation group was better than that in the artifi-cial tears group separately (both P < 0.05). (3) For BUT, the result in the acupuncture group and the SHI's manipulation group was prolonged significantly as compared with that before treatment and was prolonged apparently as compared with that in the artificial tears group (both P < 0.05) in 3 weeks of treatment. CONCLUSION: The intervention of SHI's acupuncture manipulation relieves the subjective symptoms of xerosis conjunctivitis of liver and kidney yin deficiency and achieves the same efficacy as the common acupuncture and artificial tears treatment. It does not present the apparent advantages as the common acupuncture in the short term for promoting the tear secretion and tears film repair.

7,8-dihydroxyflavone, a small-molecule tropomyosin-related kinase B (TrkB) agonist, attenuates cerebral ischemia and reperfusion injury in rats.

7,8-dihydroxyflavone (7,8-DHF) is a recently identified potent agonist of tropomyosin-related kinase B that can cross the blood-brain barrier after oral or intraperitoneal administration. The aim of the present study was to determine whether 7,8-DHF has neuroprotective effects against cerebral ischemia and reperfusion (I/R) injury and, if so, to investigate the possible underlying mechanisms. Cerebral I/R injury rats were induced by middle cerebral artery occlusion for 90 min followed by reperfusion for 24 h. 7,8-DHF was administered intraperitoneally at a dose of 5 mg/kg immediately after ischemia. Our results showed that 7,8-DHF significantly reduced neurological deficit scores, infarct volumes, and neuronal apoptosis in brains of I/R rats. Meanwhile, 7,8-DHF also increased Bcl-2 expression, decreased expression of cleaved caspase-3, Bax and inducible nitric oxide synthase, and inhibited nuclear factor-κB activation in ischemic cortex. Finally, malondialdehyde and nitric oxide contents were reduced, but activities of glutathione, glutathione peroxidase and superoxide dismutase were restored in ischemic cortex treated with 7,8-DHF. Taken together, our findings demonstrated that 7,8-DHF is able to protect against cerebral I/R injury, which may be, at least in part, attributable to its anti-apoptotic, anti-oxidative and anti-inflammatory actions.

Auxin flow in anther filaments is critical for pollen grain development through regulating pollen mitosis.

It was well known that auxin is critical for anther/pollen grain development, however, the clear distribution and detailed effects of auxin during floral development are still unclear. We have shown here that, through analyzing GUS activities of Arabidopsis lines harboring auxin response elements DR5-GUS, auxin was mainly accumulated in the anther during flower stages 10-12. Further studies employing the indoleacetic acid-lysine synthetase (iaaL) coding gene from Pseudomonas syringae subsp. savastanoi under control of the promoter region of Arabidopsis phosphatidylinositol monophosphate 5-kinase 1 gene, which conducts the anther filament-specific expression, showed that block of auxin flow of filaments resulted in shortened filaments and significantly defective pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct, confirming the effects of auxin flow in filaments on anther development. Detailed studies further revealed that the meiosis process of pollen grain was normal while the mitosis at later stage was significantly defected, indicating the effects of auxin flow in filaments on pollen grain mitosis process. Analysis employing [(14)C]IAA, as well as the observation on the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in anther filaments and pollen grains.