Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

The chromosome-scale reference genome of mirid bugs (Adelphocoris suturalis) genome provides insights into omnivory, insecticide resistance, and survival adaptation.

BACKGROUND: Adelphocoris suturalis (Hemiptera: Miridae) is a notorious agricultural pest, which causes serious economic losses to a diverse range of agricultural crops around the world. The poor understanding of its genomic characteristics has seriously hindered the establishment of sustainable and environment-friendly agricultural pest management through biotechnology and biological insecticides. RESULTS: Here, we report a chromosome-level assembled genome of A. suturalis by integrating Illumina short reads, PacBio, 10x Chromium, and Hi-C mapping technologies. The resulting 1.29 Gb assembly contains twelve chromosomal pseudomolecules with an N50 of 1.4 and 120.6 Mb for the contigs and scaffolds, respectively, and carries 20,010 protein-coding genes. The considerable size of the A. suturalis genome is predominantly attributed to a high amount of retrotransposons, especially long interspersed nuclear elements (LINEs). Transcriptomic and phylogenetic analyses suggest that A. suturalis-specific candidate effectors, and expansion and expression of gene families associated with omnivory, insecticide resistance and reproductive characteristics, such as digestion, detoxification, chemosensory receptors and long-distance migration likely contribute to its strong environmental adaptability and ability to damage crops. Additionally, 19 highly credible effector candidates were identified and transiently overexpressed in Nicotiana benthamiana for functional assays and potential targeting for insect resistance genetic engineering. CONCLUSIONS: The high-quality genome of A. suturalis provides an important genomic landscape for further investigations into the mechanisms of omnivory, insecticide resistance and survival adaptation, and for the development of integrated management strategies.

Rubisco accumulation factor 1 (Raf1) plays essential roles in mediating Rubisco assembly and carboxysome biogenesis.

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.

Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes.

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.

Convenient Synthesis of a Ru Catalyst Containing Single Atoms and Nanoparticles on Nitrogen-Doped Carbon with Superior Hydrogen Evolution Reaction Activity in a Wide pH Range.

Ruthenium, which is relatively cheap in precious metals, has become a popular alternative for a hydrogen evolution reaction (HER) catalyst because of its corrosion resistance and appropriate metal-H bond strength. Convenient synthesis and active site regulation are conducive to stimulating the excellent catalytic performance of Ru as much as possible. Herein, using the mature mesoporous nitrogen-doped carbon material as the support, the catalytic materials containing both single atom Ru and Ru nanoparticles were synthesized by impregnation using the solid-phase reduction method. The effect of reduction temperature on the dispersion state and electronic structure of Ru species has been fully studied using electronic and spectroscopic characterizations. The sample reduced at 300 °C has excellent HER activity with overpotentials of 10.8 and 53.8 mV to deliver 10 mA/cm2 in alkaline and acidic media, respectively, which is among the best activities in the reported results. Electrochemical impedance analysis shows that the reduction temperature has a great influence on the number of active sites and charge transfer impedance of the catalyst.

Development of marker-free rice with stable and high resistance to rice black-streaked dwarf virus disease through RNA interference.

The rice black-streaked dwarf virus (RBSDV) disease causes severe rice yield losses in Asia. RNA interference (RNAi) has been widely applied to develop antiviral varieties in plants. So far, only a few studies reported the application of RNAi in rice against RBSDV and most of them are lack of enough data to support its breeding potential, which limited the progress on developing RBSDV-resistant variety. In this study, we generated three RNAi constructs to specifically target three RBSDV genes (S1, S2 and S6), respectively. We confirmed that RNAi targeting RBSDV S6 conferred rice with almost full immunity to RBSDV through phenotyping test in eight consecutive years in both artificial inoculation and field trials, while RNAi of S1 or S2 only leads to partially increased resistance. The S6RNAi was also found conferring strong resistance to southern rice black-streaked dwarf virus (SRBSDV), a novel species closely related to RBSDV that outbroke recently in Southern China. In particular, no adverse effects on agronomical and developmental traits were found in S6RNAi transgenic lines. The marker-free transgenic lines with S6RNAi, driven by either maize ubiquitin-1 promoter or rice rbcS green tissue expression promoter, in elite rice background should have great potential in breeding of resistant varieties to both RBSDV and SRBSDV and provide a basis for further safety evaluation and commercial application.

Targeting DNAJC19 overcomes tumor growth and lung metastasis in NSCLC by regulating PI3K/AKT signaling.

BACKGROUND: Some driver oncogenes are still unknown in non-small-cell lung cancer (NSCLC). DNAJC19, a major component of the translocation machinery of mitochondrial membranes, is a disease-associated protein. Herein, we report the role of DNAJC19 in NSCLC cell growth and metastasis. METHODS: Immunohistochemistry (IHC) was performed to investigate DNAJC19 expression in NSCLC clinical samples. For knockdown or overexpression assays in A549 or NCI-H1299 lung cancer cells, lentiviral vectors were used. After assessment of cell functions, DNAJC19-knockdown A549 cells were further applied to establish mouse xenograft and metastasis tumor models. Assessments based on the RNA-seq data, western blotting, PCR and IHC were performed for the mechanistic study. RESULTS: Expression of DNAJC19 was higher in tumors than in noncancerous adjacent tissues. Survival analysis indicated that low DNAJC19 levels were correlated with an increased progression-free survival rate. ShRNA-mediated knockdown of DNAJC19 markedly inhibited cell growth, viability, migration and invasion. Moreover, RNA-seq analysis revealed that the PI3K/AKT signaling pathway was involved in molecular events when A549 cells were treated with shDNAJC19. In addition, DNAJC19 knockdown decreased PI3Kp85a, AKT and p-AKT expression in A549 cells, and cellular functions were greatly rescued in DNAJC19-knockdown A549 cells by ectopic overexpression of AKT. Furthermore, tumor xenograft growth and lung metastasis were markedly repressed in the shDNAJC19 group compared to the control group. As expected, the expression levels of DNAJC19, PI3K and AKT in xenograft mouse samples were also lower in the shDNAJC19 group than in the shCtrl group. CONCLUSIONS: DNAJC19 greatly promotes NSCLC cell growth and lung metastasis by regulating PI3K/AKT signaling, providing a novel therapeutic target for treating NSCLC patients.

Clinical Application Value of the Prognostic Nutritional Index for Predicting Survival in Patients with Esophageal Squamous Cell Carcinoma Undergoing Chemoradiotherapy or Radiotherapy.

BACKGROUND: The prognostic nutrition index (PNI) has been shown to have prognostic value for several common cancers. The study aim was to explore the clinical application value of the PNI for prognosis of patients with esophageal squamous cell carcinoma (ESCC) treated with radical chemoradiotherapy (CRT) or radiotherapy (RT). METHODS: Overall, 193 patients with ESCC who received radiotherapy with or without chemotherapy at Sichuan Cancer Hospital from March 20, 2012 to December 25, 2017 were retrospectively analyzed. Based on serum measurements before treatment, the PNI at ESCC recurrence was calculated as albumin (g/L) + 5 × total lymphocyte count. The Kaplan-Meier method and Cox proportional regression model were used to analyze the relationship between PNI and overall survival (OS). RESULTS: The average PNI of 193 ESCC patients was 49.01 ± 4.68. The optimal cutoff value of PNI was 47.975, and the patients were divided into a low-PNI group (<47.975) and a high-PNI group (≥47.975). PNI was related to tumor length, T-stage and synchronous chemotherapy in ESCC patients (P < 0.05). The median OS for the entire group was 22.37 mo,. The median OS of patients in the high-PNI group and low-PNI group were 32.63 mo, and 15.4 mo, respectively, the 3-year survival rates were 47.5% and 32.2% and the 5-year survival rates were 37.7% and 16.8%, respectively, (all P = 0.001). Univariate analysis showed that PNI, tumor length, T-stage and synchronous chemotherapy were related to the prognosis of ESCC patients (P < 0.05). Multivariate analysis showed that tumor length (P = 0.019), synchronous chemotherapy (P = 0.009) and PNI (P = 0.003) were independent prognostic factors affecting the prognosis of patients in ESCC treated with RT or CRT. CONCLUSIONS: The calculation of PNI value is simple, reliable and repeatable and can improve the accuracy of a patient's prognosis. Confirmation of these results by a large-sample prospective study is desirable.

Clinical utility of the systemic immune-inflammation index for predicting survival in esophageal squamous cell carcinoma after radical radiotherapy.

Aim: To explore the clinical utility of the systemic immune-inflammation index (SII) for predicting the prognosis of esophageal squamous cell carcinoma (ESCC). Patients & methods: After calculating the SII in 180 patients with ESCC, the relationship between SII values and the pre-/post-radiotherapy SII ratio and overall survival was determined. Results: The median overall survival was 649 days for the entire group and 909 and 466 days for the high and low pre-/post-radiotherapy SII ratio groups, respectively. Multivariate analysis identified Karnofsky performance status (p = 0.045), lymphatic metastasis (p = 0.032), mid-radiotherapy SII (p < 0.001) and pre-/post-radiotherapy SII ratio (p = 0.003) as independent prognostic factors. Conclusion: The pre-/post-radiotherapy SII ratio and mid-radiotherapy SII are potentially effective markers for predicting ESCC prognosis.

Lay abstract The systemic immune-inflammation index (SII) is calculated from the counts of peripheral blood platelets (P), neutrophils (N) and lymphocytes (L) per liter according to the formula SII = P × N/L. The SII is associated with poor survival in certain cancer types. However, some reports have examined the prognostic value of the SII in patients with esophageal squamous cell carcinoma (ESCC) who were undergoing radiotherapy or radical chemoradiotherapy. As such, the current study sought to investigate the clinical prognostic value of the SII during radiotherapy and the ratio of the SII before and after radiotherapy in patients with ESCC who were undergoing chemoradiotherapy or radiotherapy. The study found that the pre-/post-radiotherapy SII ratio and mid-radiotherapy SII are potentially effective markers for predicting ESCC prognosis.

Landscape forest impacts the potential activity time of an invasive lizard and its possibilities for range expansion in Taiwan under climate warming.

Climate warming may have an impact on invasive species and their ecological consequences. Invasive reptiles, which have temperature-dependent physiology, are expected to be greatly impacted by climate warming, though data supporting this is limited. We investigated the potential impact of a warmer climate on an invasive lizard, Eutropis multifasciata, in Taiwan. A mechanistic model, NicheMapR, was used to simulate the maximum activity time available at three elevations, with varying forest densities, under the current climate and a warmer scenario. The results show that climate warming will provide this species more time for activity in the currently occupied lowland region but not in the mountain areas, which are covered with dense forests. However, if the landscape becomes more open in mountain areas, it will become more suitable for this species and may enable an expansion upslope. Our results show that climate warming has a positive impact on this species, and that landscape's characteristics profoundly modulate its impact and the possibilities for elevational expansion in the future.

Luteolin Inhibits Breast Cancer Stemness and Enhances Chemosensitivity through the Nrf2-Mediated Pathway.

Cancer stem cells (CSCs) are subpopulations of tumor masses with unique abilities in self-renewal, stemness maintenance, drug resistance, and the promotion of cancer recurrence. Recent studies have suggested that breast CSCs play essential roles in chemoresistance. Therefore, new agents that selectively target such cells are urgently required. Reactive oxygen species (ROS)-producing enzymes are the reason for an elevated tumor oxidant status. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor, which upon detecting cellular oxidative stress, binds to the promoter region of antioxidant genes. By triggering a cytoprotective response, Nrf2 maintains cellular redox status. Cripto-1 participates in the self-renewal of CSCs. Herein, luteolin, a flavonoid found in Taraxacum officinale extract, was determined to inhibit the expressions of stemness-related transcriptional factors, the ATP-binding cassette transporter G2 (ABCG2), CD44, aldehyde dehydrogenase 1 activity as well as the sphere formation properties of breast CSCs. Furthermore, luteolin suppressed the protein expressions of Nrf2, heme oxygenase 1 (HO-1), and Cripto-1 which have been determined to contribute critically to CSC features. The combination of luteolin and the chemotherapeutic drug, Taxol, resulted in enhanced cytotoxicity to breast cancer cells. These findings suggest that luteolin treatment significantly attenuated the hallmarks of breast cancer stemness by downregulating Nrf2-mediated expressions. Luteolin constitutes a potential agent for use in cancer stemness-targeted breast cancer treatments.

Low cold tolerance of the invasive lizard Eutropis multifasciata constrains its potential elevation distribution in Taiwan.

1. The invasive many-lined sun skink, Eutropis multifasciata, is established in much of southern Taiwan and is spreading northward. We investigated whether winter temperatures constrain further dispersion of this skink by comparing its cold tolerance to the spatial distribution of winter temperatures in Taiwan. 2. We measured the 28-day survival rate of this species at 4 constant temperatures (10-16 °C in 2 °C increments) and its critical thermal minimum (CTmin), i.e., the body temperature at which the righting reflex is lost during the cooling process. For comparison with the spatial distribution of temperatures over Taiwan, we used the biophysical model Niche Mapper™ in order to simulate the soil temperatures, where lizards are inactive in the winter, during the coldest month of the year, January, under three climatic scenarios (average temperature, average-3 °C, average+3 °C). 3. Our results indicate that this species has low tolerance to cold. Combining cold tolerance data with soil temperature data suggests that its upper elevation limit could range from 1000 m to 1500 m, above which the weather is lethal and precludes overwintering. The locations of sightings of E. multifasciata are consistent with this prediction, with no known locations above 500 m elevation. 4. This study highlights that the winter climate is a major factor in determining population establishment and hence in limiting this species' range. Future studies would benefit from accounting for low winter temperatures and their potential influence on range limits of invasive species.

Absence of OsßCA1 causes a CO2 deficit and affects leaf photosynthesis and the stomatal response to CO2 in rice.

Plants always adjust the opening of stomatal pores to adapt to the environment, for example CO2 concentration ([CO2 ]), humidity and temperature. Low [CO2 ] will trigger the opening of stomatal pores to absorb extra CO2 . However, little is known about how CO2 supply affects the carbon fixation and opening of stomatal pores in rice. Here, a chloroplast-located gene coding for ß-carbonic anhydrase (ßCA) was found to be involved in carbon assimilation and the CO2 -mediated stomatal pore response in rice. OsßCA1 was constitutively expressed in all tissues and its transcripts were induced by high [CO2 ] in leaves. Both T-DNA mutant and RNA interference lines showed phenotypes of lower biomass and CA activities. Knockout of OsßCA1 obviously decreased photosynthetic capacity, as demonstrated by the increased CO2 compensation point and decreased light saturation point in the mutant, while knockout increased the opening ratio of stomatal pores and the rate of water loss. Moreover, the mutant showed a delayed response to low [CO2 ], and stomatal pores could not be closed to the same degree as those of wild type even though the stomatal pores could rapidly respond to high [CO2 ]. Genome-wide gene expression analysis via RNA sequencing demonstrated that the transcript abundance of genes related to Rubisco, photosystem compounds and the opening of stomatal pores was globally upregulated in the mutant. Taken together, the inadequate CO2 supply caused by the absence of OsßCA1 reduces photosynthetic efficiency, triggers the opening of stomatal pores and finally decreases their sensitivity to CO2 fluctuation.

The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice.

Calcium-dependent protein kinases (CPKs) are serine/threonine protein kinases that function in plant stress responses. Although CPKs are recognized as key messengers in signal transduction, the specific roles of CPKs and the molecular mechanisms underlying their activity remain largely unknown. Here, we characterized the function of OsCPK24, a cytosol-localized calcium-dependent protein kinase in rice. OsCPK24 was universally and highly expressed in rice plants and was induced by cold treatment. Whereas OsCPK24 knockdown plants exhibited increased sensitivity to cold compared to wild type (WT), OsCPK24-overexpressing plants exhibited increased cold tolerance. Plants overexpressing OsCPK24 exhibited increased accumulation of proline (an osmoprotectant) and glutathione (an antioxidant) and maintained a higher GSH/GSSG (reduced glutathione to oxidized glutathione) ratio during cold stress compared to WT. In addition to these effects in response to cold stress, we observed the kinase activity of OsCPK24 varied under different calcium concentrations. Further, OsCPK24 phosphorylated OsGrx10, a glutathione-dependent thioltransferase, at rates modulated by changes in calcium concentration. Together, our results support the hypothesis that OsCPK24 functions as a positive regulator of cold stress tolerance in rice, a process mediated by calcium signaling and involving phosphorylation and the inhibition of OsGrx10 to sustain higher glutathione levels.

Loss-of-function mutation of rice SLAC7 decreases chloroplast stability and induces a photoprotection mechanism in rice.

Plants absorb sunlight to power the photochemical reactions of photosynthesis, which can potentially damage the photosynthetic machinery. However, the mechanism that protects chloroplasts from the damage remains unclear. In this work, we demonstrated that rice (Oryza sativa L.) SLAC7 is a generally expressed membrane protein. Loss-of-function of SLAC7 caused continuous damage to the chloroplasts of mutant leaves under normal light conditions. Ion leakage indicators related to leaf damage such as H2 O2 and abscisic acid levels were significantly higher in slac7-1 than in the wild type. Consistently, the photosynthesis efficiency and Fv/Fm ratio of slac7-1 were significantly decreased (similar to photoinhibition). In response to chloroplast damage, slac7-1 altered its leaf morphology (curled or fused leaf) by the synergy between plant hormones and transcriptional factors to decrease the absorption of light, suggesting that a photoprotection mechanism for chloroplast damage was activated in slac7-1. When grown in dark conditions, slac7-1 displayed a normal phenotype. SLAC7 under the control of the AtSLAC1 promoter could partially complement the phenotypes of Arabidopsis slac1 mutants, indicating a partial conservation of SLAC protein functions. These results suggest that SLAC7 is essential for maintaining the chloroplast stability in rice.

Major alterations in transcript profiles between C3-C4 and C4 photosynthesis of an amphibious species Eleocharis baldwinii.

Engineering C4 photosynthetic metabolism into C3 crops is regarded as a major strategy to increase crop productivity, and clarification of the evolutionary processes of C4 photosynthesis can help the better use of this strategy. Here, Eleocharis baldwinii, a species in which C4 photosynthesis can be induced from a C3-C4 state under either environmental or ABA treatments, was used to identify the major transcriptional modifications during the process from C3-C4 to C4. The transcriptomic comparison suggested that in addition to the major differences in C4 core pathway, the pathways of glycolysis, citrate acid metabolism and protein synthesis were dramatically modified during the inducement of C4 photosynthetic states. Transcripts of many transporters, including not only metabolite transporters but also ion transporters, were dramatically increased in C4 photosynthetic state. Many candidate regulatory genes with unidentified functions were differentially expressed in C3-C4 and C4 photosynthetic states. Finally, it was indicated that ABA, auxin signaling and DNA methylation play critical roles in the regulation of C4 photosynthesis. In summary, by studying the different photosynthetic states of the same species, this work provides the major transcriptional differences between C3-C4 and C4 photosynthesis, and many of the transcriptional differences are potentially related to C4 development and therefore are the potential targets for reverse genetics studies.

Total Flavonoids of Aurantii Fructus Immaturus Regulate miR-5100 to Improve Constipation by Targeting Fzd2 to Alleviate Calcium Balance and Autophagy in Interstitial Cells of Cajal.

Aurantii Fructus Immaturus total flavonoids (AFIF) is the main effective fraction extracted from AFI, which has a good effect on promoting gastrointestinal motility. This study aimed to investigate AFIF which regulates miR-5100 to improve constipation symptoms in mice by targeting Frizzled-2 (Fzd2) to alleviate interstitial cells of Cajal (ICCs) calcium ion balance and autophagy apoptosis. The constipated mouse model was induced by an antibiotic suspension, and then treated with AFIF. RNA-seq sequencing, luciferase assay, immunofluorescence staining, transmission electron microscopy, ELISA, flow cytometry, quantitative polymerase chain reaction (PCR), and Western blot were applied in this study. The results showed that AFIF improved constipation symptoms in antibiotic-induced constipated mice, and decreased the autophagy-related protein Beclin1 levels and the LC3-II/I ratio in ICCs. miR-5100 and its target gene Fzd2 were screened as key miRNAs and regulator associated with autophagy. Downregulation of miR-5100 caused increased expression of Fzd2, decreased proliferation activity of ICCs, increased apoptotic cells, and enhanced calcium ion release and autophagy signals. After AFIF treatment, miR-5100 expression was upregulated and Fzd2 was downregulated, while autophagy-related protein levels and calcium ion concentration decreased. Furthermore, AFIF increased the levels of SP, 5-HT, and VIP, and increased the expression of PGP9.5, Sy, and Cx43, which alleviated constipation by improving the integrity of the enteric nervous system network. In conclusion, AFIF could attenuate constipation symptoms by upregulating the expression of miR-5100 and targeting inhibition of Fzd2, alleviating calcium overload and autophagic death of ICCs, regulating the content of neurotransmitters, and enhancing the integrity of the enteric nervous system network.

MiR-26b-3p Promotes Intestinal Motility Disorder by Targeting FZD10 to Inhibit GSK3ß/ß-Catenin Signaling and Induce Enteric Glial Cell Apoptosis.

Enteric glial cells (EGCs) are the major component of the enteric nervous system and affect the pathophysiological process of intestinal motility dysfunction. MicroRNAs (miRNAs) play an important role in regulating gastrointestinal homeostasis. However, the mechanism of miRNA-mediated regulation of EGCs in intestinal dysmotility remains unclear. In this study, we investigated the effect of EGC apoptosis on intestinal dysmotility, and the effect of miR-26b-3p on EGC proliferation and apoptosis in vivo and in vitro. A loperamide hydrochloride (Lop)-induced constipated mouse model and an in vitro culture system of rat EGCs were established. The transcriptome was used to predict the differentially expressed gene miR-26b-3p and the target gene Frizzled 10 (FZD10), and their targeting binding relationship was verified by luciferase. EGCs were transfected with miR-26b-3p mimic or antagomir, and the FZD10 expression was down-regulated by siRNA. Immunofluorescence and flow cytometry were used to detect EGC apoptosis. MiR-26b-3p and FZD10 expressions were examined using quantitative real-time PCR (qRT-PCR). The CCK-8 assay was used to detect EGC proliferation. The protein levels were detected by Western blotting and enzyme-linked immunosorbent assay (ELISA). The results showed that miR-26b-3p was up-regulated in the Lop group, whereas FZD10 was down-regulated, and EGC apoptosis was increased in the colon of intestinal dysmotility mice. FZD10 down-regulation and miR-26b-3p mimic significantly increased glycogen synthase kinase-3ß phosphorylation (p-GSK3ß) levels, decreased ß-catenin expression, and promoted EGC apoptosis. MiR-26b-3p antagomir alleviated intestinal dysmotility, promoted EGC increased activity of EGCs, and reduced EGC apoptosis in vivo. In conclusion, this study indicated that miR-26b-3p promotes intestinal motility disorders by targeting FZD10 to block GSK3ß/ß-catenin signaling and induces apoptosis in EGCs. Our results provide a new research target for the treatment and intervention of intestinal dysmotility.

Uncovering the roles of the scaffolding protein CsoS2 in mediating the assembly and shape of the α-carboxysome shell.

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes. IMPORTANCE: Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications.