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The crosstalk between clathrin-mediated endocytosis (CME) and the autophagy pathway has been reported in mammals; however, the interconnection of CME with autophagy has not been established in plants. Here, we report that the Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopies Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants in both autoimmunity and nutrient sensitivity. Accordingly, the autophagy pathway is significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, multiple assays demonstrate that CLC2 directly interacts with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP are subjected to autophagic degradation, and degradation of GFP-ATG8h is significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneous knockout of ATG8h and ATG8i by CRISPR-Cas9 results in enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results reveal a link between the function of CLCs and the autophagy pathway in Arabidopsis.

This article presents a study on the larviposition preference of Sarcophaga dux, a type of flesh fly, specifically focusing on their attraction to soy sauce. The researchers used gas chromatography-mass spectrometry to identify key odorants in soy sauce that are attractive to female S. dux. They developed a model that can estimate the attractiveness of unknown soy sauce samples based on the proportions of these key odorants. The model was validated through blind tests and showed consistent results. The study has implications for the food industry and highlights the need for further research on the effects of odorants on S. dux and food safety. [Extracted from the article]

Osteoporosis is a systemic skeletal disease that is characterized by low bone density and microarchitectural deterioration of bone tissue. The increasing prevalence of osteoporosis has attracted much attention. In this study, MC3T3-E1 pre-osteoblasts were treated with the natural compound, baicalein (0.1 μmol/L, 1 μmol/L, 10 μmol/L), to stimulate differentiation over a 14-day period. In addition, a canonical ovariectomized (OVX) mouse model was used to investigate the effect of 3-month baicalein treatment (10 mg/kg per day) in preventing postmenopausal osteoporosis. In vitro, we found that baicalein induced activation of alkaline phosphatase, stimulated the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, and induced expression of osteoblast differentiation markers, ie, osteocalcin, osterix, collagen Iα1, and runt-related transcription factor 2 (RUNX2), in osteoblasts. In vivo, several bone parameters, including trabecular thickness, trabecular bone mineral density, and trabecular number, in the distal femoral metaphysis were significantly increased in OVX mice treated intragastrically with baicalein for 3 months compared with OVX mice that were not treated with baicalein. We also found that expression of osteocalcin and RUNX2 was decreased in primary ossified tissue from the OVX group, and baicalein increased the levels of osteocalcin and RUNX2 in OVX mice. These data suggest that baicalein can stimulate MC3T3-E1 cells to differentiate into osteoblasts via activation of the mTORC1 signaling pathway, which includes protein kinases and transcription factors such as P-4E/BP1 and P-S6K1.

Objective: To identify the protein from host macrophages which interacted with GRA7 dense granule protein of Toxoplasma gondii, and reveal the relationship between protein interaction and infection process., Methods: The recombinant GRA7 protein with N-terminal GST tag were used as a bait in in vitro GST Pull-down experiment, the proteins of THP-1 monocytic macrophage cell line were captured and identified by LC-MS/MS proteomics method. The in vivo protein interaction was verified by Co-IP experiment The overexpression of the target host protein by pcDNA3.1 (+) vector in THP-1 macrophage was further used to analyze the relationship between protein interaction and infection process., Results: The captured THP-1 cell protein was about Mr 29000, which was identified as human carbonic anhydrase 1 (hCA1). The significant in vivo protein-protein interaction between GRA7 and hCA1 was verified by Co-IP assay. The overexpression of hCA1 gene in THP-1 macrophage induced a higher propagation speed of Tgondii and the formation of the parasitophorous vacuole, but did nmt influence the number of the parasite., Conclusion: There is a significant protein interaction between Toxoplasma GRA7 dense granule protein and hCA1 enzyme from host macrophages, which is positively related with the propagation speed of T. gondii.

Aim: To investigate the effect of endogenous n-3 polyunsaturated fatty acids (PUFAs) on bone marrow adipogenesis under osteoporosis conditions., Methods: A mouse osteoporosis model overexpressing the FAT1 gene from Caenorhabditis elegans and converting n-6 PUFAs to n-3 PUFAs endogenously was used., Results: The mice presented significantly lower bone marrow adiposity (adipocyte volume/tissue volume, mean adipocyte number) but increased the bone parameters (bone mineral density, bone mineral content, bone volume/total volume) in the distal femoral metaphysis., Conclusion: Endogenous n-3 PUFAs protect bone marrow adipogenesis, which provides a novel drug target.

Objective: To separate and identify the surface proteins and secreted proteins of Toxoplasma gondii tachyzoites of RH strain., Methods: T. gondii tachyzoites were cultured in Vero cells, and purified by filtration and Percoll cell separation solution. The biotin-labeled tachyzoites were lysed, and the surface and secreted proteins were separated by NeutrAvidin agarose beads. After condensation and SDS-PAGE, the protein were collected, digested and identified by LC/MS-MS., Results: A total of 785 T. gondii proteins were identified, 81 (10.3%) of which were originally annotated as the surface or secreted proteins. Among the highly-expressed (PSM>10) 65 proteins, 43 (66%) were originally annotated as surface or secreted proteins, while the others were predicted unknown proteins., Conclusion: The surface and secreted proteins of T. gondii are separated by biotin labeling and avidin chromatography, among which some potential new surface or secreted proteins of T. gondii are identified.

Objective: To analyze the epidemiologic features of Pneumocystis pneumonia (PCP) among non-HIV infected patients in China., Methods: Sputum or bronchoalveolar lavage fluid (BALF) specimens obtained from 851 pneumonia patients without HIV infection from Jan. 2006 to Oct. 2008 were detected, using PCR and Gomori' s methenamine silver (UMS) stain for Pneumocystis jirovecii., Results: Of the 615 sputum specimens, P. jirovecii positive rates of PCR and GMS stain were 20.3% and 10.2% respectively (P < 0.05). Of 236 BALF specimens, P. jirovecii positive rates of PCR and GMS stain were 32.6% and 25.5% respectively (P > 0.05). Of the total 851 pneumonia cases, 123 (14.5%) were GMS positive for P. jirovecii cyst and 202 cases (23.7%) were PCR positive for P. jirovecii DNA. In those immuno-suppressed patient group including patients with connective tissue diseases, organ transplant recipients, nephrotic, hematologic diseases and malignant tumor, P. jirovecii positive rate appeared the highest, 28.2% for GMS stain and 39.4% for PCR. There were also PCP patients in the immuno-competent pneumonia patient groups including senile patients with chronic diseases and patients without clear predisposing immuno-deficiencies. The positive rates of P. jirovecii GMS were 8.7% and 10.9%, respectively and 17.5% and 19.6% for P. jirovecii under PCR., Conclusion: PCR assay seemed sensitive for the detection of P. jirovecii in the sputum specimens and could be used for screening PCP patients without HIV infection. Our data showed that there was high risk of PCP in non-HIV infected patients in China.

Objective: To establish axenic cultivation of Pneumocystis carinii (P.c)., Methods: The organisms of P.c were isolated from the bronchoalveolar lavage fluid (BALF) of the rats with Pneumocystis carinii pneumonia (PCP) and cultured in a medium which was based on IMDM(GIBCO) supplemented with S-adenosyl-L-methionine, putrescine, N-acetyl glucosamine, putrescine, L-cysteine and L-glutamine, and newborn calf serum. The organisms cultured in the system were identified by observing the morphology of cysts in smears stained with Gomori's methenamine silver nitrate stain (GMS). Ultrastructure of the cysts/trophozoites was examined by transmission electron microscopy. The sequences of mitochondrial large ribosomal DNA subunit of the cultured organisms were compared with the Pneumocystis carinii f.sp. ratti variant isolate (GenBank No U20173) and Pneumocystis carinii f.sp.hominis (GenBank No M58605)., Results: Five isolates of P. carinii received from BALF of 8 rats with PCP were cultured axenically and continuously in the system. The cultured organisms could be stored in frozen condition and used to reinitiate culture, and were amplified by 19-22 times within 72 h. The morphology, ultrastructure and gene sequencing of the cultured organisms confirmed that the isolated organisms were P. carinii., Conclusion: Five continuously and axenicly cultured isolates of P. carinii have been received.