Nanobody in a Double "Y"-Shaped Assembly: A Promising Candidate for Lateral Flow Immunoassays.

Derived from camelid heavy-chain antibodies, nanobodies (Nbs) are the smallest natural antibodies and are an ideal tool in biological studies because of their simple structure, high yield, and low cost. Nbs possess significant potential for developing highly specific and user-friendly diagnostic assays. Despite offering considerable advantages in detection applications, knowledge is limited regarding the exclusive use of Nbs in lateral flow immunoassay (LFIA) detection. Herein, we present a novel double "Y" architecture, achieved by using the SpyTag/SpyCatcher and Im7/CL7 systems. The double "Y" assemblies exhibited a significantly higher affinity for their epitopes, as particularly evident in the reduced dissociation rate. An LFIA employing double "Y" assemblies was effectively used to detect the severe acute respiratory syndrome coronavirus-2 N protein, with a detection limit of at least 500 pg/mL. This study helps broaden the array of tools available for the development of Nb-based diagnostic techniques.

Lily LlHSFC2 coordinates with HSFAs to balance heat stress response and improve thermotolerance.

Heat stress transcription factors (HSFs) are core regulators of plant heat stress response. Much research has focused on class A and B HSFs, leaving those of class C relatively understudied. Here, we reported a lily (Lilium longiflorum) heat-inducible HSFC2 homology involved in thermotolerance. LlHSFC2 was located in the nucleus and cytoplasm and exhibited a repression ability by binding heat stress element. Overexpression of LlHSFC2 in Arabidopsis, tobacco (Nicotiana benthamiana), and lily, all increased the thermotolerance. Conversely, silencing of LlHSFC2 in lily reduced its thermotolerance. LlHSFC2 could interact with itself, or interact with LlHSFA1, LlHSFA2, LlHSFA3A, and LlHSFA3B of lily, AtHSFA1e and AtHSFA2 of Arabidopsis, and NbHSFA2 of tobacco. LlHSFC2 interacted with HSFAs to accelerate their transactivation ability and act as a transcriptional coactivator. Notably, compared with the separate LlHSFA3A overexpression, co-overexpression of LlHSFC2/LlHSFA3A further enhanced thermotolerance of transgenic plants. In addition, after suffering HS, the homologous interaction of LlHSFC2 was repressed, but its heterologous interaction with the heat-inducible HSFAs was promoted, enabling it to exert its co-activation effect for thermotolerance establishment and maintenance. Taken together, we identified that LlHSFC2 plays an active role in the general balance and maintenance of heat stress response by cooperating with HSFAs, and provided an important candidate for the enhanced thermotolerance breeding of crops and horticulture plants.

An AP2/ERF member LlERF012 confers thermotolerance via activation of HSF pathway in lily.

Heat stress transcription factors (HSFs) are core factors of plants in response to heat stress (HS), but their regulatory network is complicated and remains elusive in a large part, especially HSFBs. In this study, we reported that the LlERF012-LlHSFA1 module participates in heat stress response (HSR) by directly regulating HSF pathway in lily (Lilium longiflorum). LlHSFB1 was confirmed as a positive regulator in lily thermotolerance and a heat-inducible AP2/ERF member LlERF012 (Ethylene Response Factor 012) was further identified to be a direct trans-activator of LlHSFB1. Overexpression of LlERF012 elevated the thermotolerance of transgenic Arabidopsis and lily, but silencing LlERF012 reduced thermotolerance in lily. Further analysis showed LlERF012 interacted with LlHSFA1, which led to enhanced transactivation activity and DNA-binding capability of LlERF012. In addition, LlERF012 also directly activated the expression of LlHSFA1 by binding its promoter. As expected, we found that LlERF012 bound the promoters of LlHSFA2, LlHSFA3A, and LlHSFA3B to stimulate their expression, and LlERF012-LlHSFA1 interaction enhanced these activation effects. Overall, our data suggested that LlERF012 was a key factor for lily thermotolerance and the LlERF012-LlHSFA1 interaction synergistically regulated the activity of the HSF pathway including the class A and B members, which might be of great significance for coordinating the functions of different HSFs.

Establishment and pathophysiological evaluation of a novel model of acute compartment syndrome in rats.

BACKGROUND: Researches have used intra-compartmental infusion and ballon tourniquest to create high intra-compartmental pressure in animal models of Acute Compartment Syndrome (ACS). However, due to the large differences in the modeling methods and the evaluation criteria of ACS, further researches of its pathophysiology and pathogenesis are hindered. Currently, there is no ideal animal model for ACS and this study aimed to establish a reproducible, clinically relevant animal model. METHODS: Blunt trauma and fracture were caused by the free falling of weights (0.5 kg, 1 kg, 2 kg) from a height of 40 cm onto the lower legs of rats, and the application of pressures of 100 mmHg, 200 mmHg, 300 mmHg and 400 mmHg to the lower limbs of rats using a modified pressurizing device for 6 h. The intra-compartmental pressure (ICP) and the pressure change (ΔP) of rats with single and combined injury were continuously recorded, and the pathophysiology of the rats was assessed based on serum biochemistry, histological and hemodynamic changes. RESULTS: The ΔP caused by single injury method of different weights falling onto the lower leg did not meet the diagnosis criteria for ACS (< 30 mmHg). On the other hand, a combined injury method of a falling weight of 1.0 kg and the use of a pressurizing device with pressure of 300 mmHg or 400 mmHg for 6 h resulted in the desired ACS diagnosis criteria with a ΔP value of less than 30 mmHg. The serum analytes, histological damage score, and fibrosis level of the combined injury group were significantly increased compared with control group, while the blood flow was significantly decreased compared with control group. CONCLUSION: We successfully established a new preclinical ACS-like rat model, by the compression of the lower leg of rats with 300 mmHg pressure for 6 h and blunt trauma by 1.0 kg weight falling.

Analysis of the Secreted Peptidome from Omental Adipose Tissue in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is a preferential omental metastasis malignancy. Since omental adipose tissue is an endocrine organ, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to compare the peptides secreted from omental adipose tissues of HGSOC and benign serous ovarian cysts (BSOC). Among the differentially secreted peptides, we detected 58 upregulated peptides, 197 downregulated peptides, 24 peptides that were only in the HGSOC group and 20 peptides that were only in the BSOC group (absolute fold change ≥ 2 and P < 0.05). Then, the basic characteristics of the differential peptides were analyzed, such as lengths, molecular weights, isoelectric points, and cleavage sites. Furthermore, we summarized the possible functions according to the precursor protein functions of the differentially expressed peptides by Gene Ontology (GO) analysis with the Annotation, Visualization, and Integrated Discovery (DAVID) database and canonical pathway analysis with IPA. For the GO analysis, the differentially secreted peptides were mainly associated with binding in molecular function and cellular processes in biology process. For the canonical pathways, the differentially secreted peptides were related to calcium signaling, protein kinase A signaling, and integrin-linked kinase (ILK) signaling. We also identified 67 differentially secreted peptides that located in the functional domains of the precursor proteins. These functional domains were mainly related to energy metabolism and immunoregulation. Our study might provide drugs that could potentially treat HGSOC or omental metastases of HGSOC cells.

Low LdMYB12 expression contributes to petal spot deficiency in Lilium davidii var. unicolor.

Petal spots are widespread in plants, they are important for attracting pollinators and as economic traits in crop breeding. However, the genetic and developmental control of petal spots has seldom been investigated. To further clarify the development of petal spots formation, we performed comparative transcriptome analysis of Lilium davidii var. unicolor and Lilium davidii petals at the full-bloom stage. In comparison with the parental species L. davidii, petals of the lily variety L. davidii var. unicolor do not have the distinct anthocyanin spots. We show that among 7846 differentially expressed genes detected, LdMYB12 was identified as a candidate gene contributing to spot formation in lily petals. The expression level of LdMYB12 in the petals of L. davidii was higher than that in L. davidii var. unicolor petals. Moreover, overexpression of LdMYB12 led to the appearance of spots on the petals of L. davidii var. unicolor, accompanied by increased expression of anthocyanin synthesis-related genes. Taken together, these results indicate that abnormal expression of LdMYB12 contributes to petal spot deficiency in L. davidii var. unicolor.

Association between SCN5A R225Q variant and dilated cardiomyopathy: potential role of intracellular pH and WNT/ß-catenin pathway.

BACKGROUND: The SCN5A variant is a common cause of familial dilated cardiomyopathy (DCM). We previously reported a SCN5A variant (c.674G>A), located in the high-risk S4 segment of domain I (DI-S4) region in patients with idiopathic DCM and R225Q knockin (p.R225Q) mice carrying the c.674G>A variant exhibited prolonged baseline PR intervals without DCM phenotypes. In this study, we explored the association and mechanism between R225Q variant and DCM phenotype. METHODS: Prevalence of DI-S4 variant was compared between patients with idiopathic DCM and the control participants. R225Q knockin and wild-type (WT) mice were subjected to doxorubicin (DOX), D-galactose (D-gal) or D-gal combined with DOX. RESULTS: Clinical data suggested that the prevalence of DI-S4 variant was higher in DCM group than in the control group (4/90 (4.4%) vs 3/1339 (0.2%), p<0.001). Cardiomyocytes from R225Q knockin mice treated with D-gal and DOX exhibited more significant hypertrophic phenotype and weaker contraction/dilation function and an increased level of apoptosis as compared with WT mice. Mechanistically, we found that R225Q variant could increase intracellular pH and further induce the activation of the WNT/ß-catenin pathway as well as the overexpression of pro-hypertrophic and pro-apoptotic targets. WNT-C59 inhibitor improved cardiac function in the R225Q knockin mice treated with D-gal and DOX. CONCLUSION: Our results suggest that R225Q variant is associated with increased susceptibility to DCM. Ageing could enhance this process via activating WNT/ß-catenin signaling in response to increased intracellular pH. Antagonising the WNT/ß-catenin pathway might be a potential therapeutic strategy for mitigating R225Q variant-related DCM pathogenesis.

Phosphoproteome Profiling of uEVs Reveals p-AQP2 and p-GSK3ß as Potential Markers for Diabetic Nephropathy.

Diabetic nephropathy (DN) contributes to increased morbidity and mortality among patients with diabetes and presents a considerable global health challenge. However, reliable biomarkers of DN have not yet been established. Phosphorylated proteins are crucial for disease progression. However, their diagnostic potential remains unexplored. In this study, we used ultra-high-sensitivity quantitative phosphoproteomics to identify phosphoproteins in urinary extracellular vesicles (uEVs) as potential biomarkers of DN. We detected 233 phosphopeptides within the uEVs, with 47 phosphoproteins exhibiting significant alterations in patients with DN compared to those in patients with diabetes. From these phosphoproteins, we selected phosphorylated aquaporin-2 (p-AQP2[S256]) and phosphorylated glycogen synthase kinase-3ß (p-GSK3ß[Y216]) for validation, as they were significantly overrepresented in pathway analyses and previously implicated in DN pathogenesis. Both phosphoproteins were successfully confirmed through Phos-tag western blotting in uEVs and immunohistochemistry staining in kidney sections, suggesting that phosphoprotein alterations in uEVs reflect corresponding changes within the kidney and their potential as candidate biomarkers for DN. Our research proposes the utilization of phosphoproteins in uEVs as a liquid biopsy, presenting a highly feasible diagnostic tool for kidney disease.

Chemokine CCL2 from proximal tubular epithelial cells contributes to sepsis-induced acute kidney injury.

Damage-associated molecular patterns secreted from activated kidney cells initiate the inflammatory response, a critical step in the development of sepsis-induced acute kidney injury (AKI). However, the underlying mechanism remains to be clarified. Here, we established a mouse model of sepsis-induced AKI through intraperitoneal injection of lipopolysaccharide (LPS) and demonstrated that LPS induced dramatical upregulation of C-C motif chemokine ligand 2 (CCL2) at both the mRNA and protein levels in the kidney, which was mainly expressed by tubular epithelial cells (TECs), especially by proximal TECs. Proximal tubule-specific ablation of CCL2 reduced LPS-induced macrophage infiltration, proinflammatory cytokine expression, and attenuated AKI. In vitro, using a Transwell migration assay, we found that deficiency of CCL2 in TECs decreased macrophage migration ability. However, myeloid-specific depletion of CCL2 could not protect the kidneys from the aforementioned effects. Mechanistically, LPS activated Toll-like receptor (TLR)2 signaling in TECs, which induced activation of its downstream effector NF-κB. Blockade of TLR2 signaling or inhibition of NF-κB activation in TECs significantly suppressed LPS-induced CCL2 expression. Furthermore, chromatin immunoprecipitation analyses confirmed a direct binding of NF-κB p65 in the CCL2 promoter region, and LPS increased the binding of NF-κB p65 to the CCL2 promoter, suggesting that TLR2/NF-κB p65 regulates CCL2 expression in TECs. Together, these results demonstrate that endogenous CCL2 released from proximal TECs, not from myeloid cells, was responsible for sepsis-induced kidney inflammation and AKI. Specifically targeting tubular TLR2/NF-κB/CCL2 signaling may be a potential therapeutic strategy for the prevention or attenuation of septic AKI.NEW & NOTEWORTHY This study provides a mechanistic insight into how C-C motif chemokine ligand 2 (CCL2) is upregulated in renal tubular epithelial cells (TECs) and contributes to kidney dysfunction during sepsis. The data reveal that lipopolysaccharide induces CCL2 expression through the Toll-like receptor 2/NF-κB signaling pathway in TECs. Endogenous CCL2 released from TECs, not from myeloid cells, is responsible for sepsis-induced kidney inflammation and acute kidney injury.

Nuclear farnesoid X receptor attenuates acute kidney injury through fatty acid oxidation.

Acute kidney injury (AKI) is a life-threatening condition that is one of most common side effects of cisplatin therapy. Fatty acid oxidation (FAO) is the main source of energy production in kidney proximal tubular epithelial cells (PTECs) but it is inhibited in AKI. Recent work demonstrated that activation of the farnesoid X receptor (FXR) protects against AKI, but the underlying mechanism remains elusive. Using a model of cisplatin-induced AKI, we found that FXR and FAO-related genes were remarkably downregulated while kidney lipid accumulated. Proximal tubule-specific or whole body FXR knockout worsened, while pharmacological activation attenuated these effects. Conversely, FXR knockout in non-proximal tubules did not. RNA-sequencing of PTECs demonstrated increased transcripts involved in metabolic pathways in cells overexpressing FXR versus control after cisplatin treatment, specifically transcripts associated with FAO and peroxisome proliferator-activated receptor-γ (PPARγ) signaling. Furthermore, FXR overexpression or activation improved FAO and inhibited intracellular lipid accumulation in cisplatin-treated cells. In vivo studies have shown that pharmacological activation of PPARγ can prevent cisplatin-induced lipid accumulation, kidney tubule injury and kidney function decline. However, inhibition of PPARγ eliminated the protective effects of FXR compared to control mice during the cisplatin treatment phase and after ischemia-reperfusion injury. Consistent with findings in vivo, FXR/PPARγ reduced lipid accumulation by improving FAO in cisplatin-treated cells. Furthermore, the inhibition of carnitine palmitoyltransferase 1α abolished the protective effect of FXR in cisplatin-treated mice. Thus, FXR improves FAO and reduced lipid accumulation via PPARγ in PTECs of the kidney. Hence, reconstruction of the FXR/PPARγ/FAO axis may be a novel therapeutic strategy for preventing or treating AKI.

Chrysanthemum embryo development is negatively affected by a novel ERF transcription factor, CmERF12.

Embryo abortion often occurs during distant hybridization events. Apetala 2/ethylene-responsive factor (AP2/ERF) proteins are key transcription factor (TF) regulators of plant development and stress resistance, but their roles in hybrid embryo development are poorly understood. In this study, we isolated a novel AP2/ERF TF, CmERF12, from chrysanthemum and show that it adversely affects embryo development during distant hybridization. Transcriptome and real-time quantitative PCR demonstrate that CmERF12 is expressed at significantly higher levels in aborted ovaries compared with normal ones. CmERF12 localizes to the cell nucleus and contains a conserved EAR motif that mediates its transcription repressor function in yeast and plant cells. We generated artificial microRNA (amiR) CmERF12 transgenic lines of Chrysanthemum morifolium var. 'Yuhualuoying' and conducted distant hybridization with the wild-type tetraploid, Chrysanthemum nankingense, and found that CmERF12-knock down significantly promoted embryo development and increased the seed-setting rates during hybridization. The expression of various genes related to embryo development was up-regulated in developing ovaries from the cross between female amiR-CmERF12 C. morifolium var. 'Yuhualuoying'× male C. nankingense. Furthermore, CmERF12 directly interacted with CmSUF4, which is known to affect flower development and embryogenesis, and significantly reduced its ability to activate its target gene CmEC1 (EGG CELL1). Our study provides a novel method to overcome barriers to distant hybridization in plants and reveals the mechanism by which CmERF12 negatively affects chrysanthemum embryo development.

AIM2 inflammasome contributes to aldosterone-induced renal injury via endoplasmic reticulum stress.

Inflammatory response and renal fibrosis are the hallmarks of chronic kidney disease (CKD). However, the specific mechanism of aldosterone-induced renal injury in the progress of CKD requires elucidation. Emerging evidence has demonstrated that absent in melanoma 2 (AIM2)-mediated inflammasome activation and endoplasmic reticulum stress (ERS) play a pivotal role in the renal fibrosis. Here, we investigated whether overexpression or deficiency of AIM2 affects ERS and fibrosis in aldosterone-infused renal injury. Interestingly, we found that AIM2 was markedly expressed in the diseased proximal tubules from human and experimental CKD. Mechanically, overactivation of AIM2 aggravated aldosterone-induced ERS and fibrotic changes in vitro while knockdown of AIM2 blunted these effects in vivo and in vitro. By contrast, AIM2 deficiency ameliorated renal structure and function deterioration, decreased proteinuria levels and lowered systolic blood pressure in vivo; silencing of AIM2 blocked inflammasome-mediated signaling pathway, relieved ERS and fibrotic changes in vivo. Furthermore, mineralocorticoid receptor (MR) antagonist eplerenone and ERS inhibitor tauroursodeoxycholic acid (TUDCA) had nephroprotective effects on the basis of AIM2 overactivation in vitro, while they failed to produce a more remarkable renoprotective effect on the treatment of AIM2 silence in vitro. Notably, the combination of TUDCA with AIM2 knockdown significantly reduced proteinuria levels in vivo. Additionally, immunofluorescence assay identified that apoptosis-associated speck-like protein (ASC) recruitment and Gasdermin-D (GSDMD) cleavage respectively occurred in the glomeruli and tubules in vivo. These findings establish a crucial role for AIM2 inflammasome in aldosterone-induced renal injury, which may provide a novel therapeutic target for the pathogenesis of CKD.

YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of Fur in a SlyD-dependent manner.

Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.

Diagnostic value of total testosterone and free androgen index measured by LC-MS/MS for PCOS and insulin resistance.

BACKGROUND: The objective of the study was to explore the clinical significance of steroid hormones in the diagnosis of PCOS and PCOS-related insulin resistance through liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and chemiluminescent immunoassay (CLIA). METHODS: The study included 114 patients with PCOS and 100 controls. Steroid hormone levels in serum were measured using LC-MS/MS and CLIA. The Bland-Altman method was used to check the consistency between the two methods. The diagnostic value of the LC-MS/MS method for female hyperandrogenemia and PCOS was evaluated. RESULTS: Women with PCOS were younger than controls on average (p < 0.001). PCOS patients had higher luteal hormone (LH, p < 0.001), insulin (p = 0.002), estradiol (E2, p < 0.001), total testosterone (TT, p < 0.001), free androgen index (FAI, p = 0.021), dehydroepiandrosterone sulfate (DHEA, p = 0.021), insulin resistance index (HOMA-IR) (p = 0.034), and fasting glucose (p = 0.017) levels than controls as measured by CLIA. The diagnostic value of TT was the best, and the area under the AUC curve was 0.766. Women with PCOS had higher androstenedione (A2, p < 0.001), FAI (p < 0.001), TT (p < 0.001), and 17-hydroxyprogesterone (17-OHP, p < 0.001) levels than controls as measured by LC-MS/MS. The ROC curve showed that the diagnostic efficacy of A2, TT, and 17-OHP was 0.830, 0.851, and 0.714, respectively. The consistency of TT detected by LC-MS/MS and CLIA was poor according to the Bland-Altman method. Detected TT by LC-MS/MS had the highest diagnostic efficiency for PCOS. The diagnostic power of the LC-MS/MS results for PCOS-related insulin resistance was analyzed. The results showed that the FAI had the highest diagnostic power, with an ROC curve of 0.798. CONCLUSION: LC-MS/MS is more sensitive and accurate than CLIA in the determination of serum TT and FAI. TT is more effective for the diagnosis of PCOS, whereas FAI is more valuable in the diagnosis of insulin resistance.

Identification of microRNA expression profiles of CD44+ ovarian cancer stem cells.

PURPOSE: The aim of our study was to investigate microRNA (miRNA) expression profiles in CD44+ ovarian cancer stem cells (ovarian CSCs). METHODS: In this study, we enriched CD44+ ovarian CSCs using magnetic activated cell sorting (MACS). A combination of real-time quantitative PCR (qRT-PCR), western blot and sphere formation assays was used to demonstrate stem cell-like properties. RNA sequencing was used to detect the miRNA expression profiles in CD44+ ovarian CSCs. Transient transfection, qRT-PCR, western blot and sphere formation assays were further used to test the function of miR-181a-2-3p. RESULTS: We found that CD44+ ovarian CSCs showed enhanced sphere formation and expression of stemness-associated genes (NANOG, OCT4, SOX2) compared to ovarian cancer cells. The RNA sequencing results showed that the miRNA expression profiles of CD44+ ovarian CSCs were different from those of ovarian cancer cells. GO and KEGG pathway analyses indicated that these miRNAs regulate stem cell-like properties in CD44+ ovarian CSCs. In addition, miR-181a-2-3p negatively regulates the stem cell-like properties of CD44+ ovarian CSCs by targeting EGR1. CONCLUSION: Our data suggest that miRNAs play important roles in regulating the stem cell-like properties of CD44+ ovarian CSCs.

Disulfiram ameliorates ischemia/reperfusion-induced acute kidney injury by suppressing the caspase-11-GSDMD pathway.

Acute kidney injury (AKI) is a serious condition with high mortality. The most common cause is kidney ischemia/reperfusion (IR) injury, which is thought to be closely related to pyroptosis. Disulfiram is a well-known alcohol abuse drug, and recent studies have shown its ability to mitigate pyroptosis in mouse macrophages. This study investigated whether disulfiram could improve IR-induced AKI and elucidated the possible molecular mechanism. We generated an IR model in mouse kidneys and a hypoxia/reoxygenation (HR) injury model with murine tubular epithelial cells (MTECs). The results showed that IR caused renal dysfunction in mice and triggered pyroptosis in renal tubular epithelial cells, and disulfiram improved renal impairment after IR. The expression of proteins associated with the classical pyroptosis pathway (Nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3), apoptosis-related specific protein (ASC), caspase-1, N-GSDMD) and nonclassical pyroptosis pathway (caspase-11, N-GSDMD) were upregulated after IR. Disulfiram blocked the upregulation of nonclassical but not all classical pyroptosis pathway proteins (NLRP3 and ASC), suggesting that disulfiram might reduce pyroptosis by inhibiting the caspase-11-GSDMD pathway. In vitro, HR increased intracellular ROS levels, the positive rate of PI staining and LDH levels in MTECs, all of which were reversed by disulfiram pretreatment. Furthermore, we performed a computer simulation of the TIR domain of TLR4 using homology modeling and identified a small molecular binding energy between disulfiram and the TIR domain. We concluded that disulfiram might inhibit pyroptosis by antagonizing TLR4 and inhibiting the caspase-11-GSDMD pathway.

Starch Degradation and Sucrose Accumulation of Lily Bulbs after Cold Storage.

Functional lilies are a group of edible lily cultivars with great potential for landscape application. Low-temperature storage can significantly improve their taste, but the knowledge of this process is largely unknown. In this study, we used the functional lilies 'Fly Shaohua' and 'Fly Tiancheng' as materials. Through physiological observation and transcriptome analysis during the bulbs' cold storage, it was found that the starch degradation and sucrose accumulation in bulbs contributed to taste improvement. After 60 d of cold storage, the sucrose accumulation was highest and the starch content was lower in the bulbs, suggesting this time-point was optimal for consumption. Accompanying the fluctuation of sucrose content during cold storage, the enzyme activities of sucrose phosphate synthase and sucrose synthase for sucrose synthesis were increased. Transcriptome analysis showed that many differentially expressed genes (DEGs) were involved in the starch and sucrose metabolism pathway, which might promote the conversion of starch to sucrose in bulbs. In addition, the DEGs involved in dormancy and stress response were also determined during cold storage, which might explain the decreased sucrose accumulation with extended storage time over 60 d due to the energy consumption for dormancy release. Taken together, our results indicated sucrose accumulation was a main factor in the taste improvement of lily bulbs after cold storage, which is attributable to the different gene expression of starch and sucrose metabolism pathways in this process.

A Novel Lateral Organ Boundary-domain Factor CmLBD2 Positively Regulates Pollen Development by Activating CmACOS5 in Chrysanthemum morifolium.

Male sterility, as a common reproductive characteristic in plants, plays an important role in breeding, in which pollen abortion is a key factor leading to male sterility. Here, based on a low expression level gene CmACOS5 in transcriptome of pollen abortive chrysanthemum, a new transcription factor CmLBD2 of the Lateral Organ Boundaries Domain family, which could bind the promoter of CmACOS5 by yeast one-hybrid library was screened. This study revealed the origin and expression pattern of CmLBD2 in chrysanthemum and verified the functions of two genes in pollen development by transgenic means. Inhibiting the expression of CmACOS5 or CmLBD2 can lead to a large reduction in pollen and even abortion in chrysanthemum. Using yeast one-/two-hybrid, electrophoretic mobility shift assays, and luciferase reporter assays, it was verified that CmLBD2 directly binds to the promoter of CmACOS5. These results suggest that LBD2 is a novel, key transcription factor regulating pollen development. This result will provide a new research background for enriching the function of LBD family proteins and also lay a new foundation for the breeding of male sterile lines and the mechanism of pollen development.

Depletion of miR-21 in dendritic cells aggravates renal ischemia-reperfusion injury.

Dendritic cells (DCs) play an important role in the pathophysiology of renal ischemia-reperfusion injury (IRI). The mechanisms underlying DCs phenotypic modulation and their function are not fully understood. In this study, we examined the effect of miR-21 on the phenotypic modulation of DCs in vitro and in vivo, and further investigated the impact of miR-21-overexpression DC or miR-21-deficient DC on renal IRI. We found that treatment with hypoxia/reoxygenation (H/R) suppressed miR-21 expression in bone marrow-derived dendritic cells (BMDCs), and significantly increased the percentage of mature DCs (CD11c+ /MHC-II+ /CD80+ ). Using a selection of microRNA mimics, we successfully induced the upregulation of miR-21 in BMDCs, which induced immature DC phenotype and an anti-inflammatory DC response. However, deletion of miR-21 in BMDCs promoted maturation of DCs under H/R. Adoptive transfer of miR-21-overexpression BMDCs could alleviate renal IR-induced pro-inflammatory cytokines production and acute kidney injury (AKI). Mice with miR-21 deficiency in DCs subjected to renal IR showed more severe renal dysfunction and inflammatory response compared with wild-type mice. In addition, chemokine C receptor 7 (CCR7), a surface marker of mature DC, was a target gene of miR-21, and silencing of CCR7 in BMDCs led to reduced mature DCs under H/R. In conclusion, our findings highlight miR-21 as a key regulator of DCs subset phenotype and a potential therapeutic target in renal IRI.

Hemodilution is associated with underestimation of serum creatinine in cardiac surgery patients: a retrospective analysis.

BACKGROUND: Fluid overload is related to the development and prognosis of cardiac surgery-associated acute kidney injury (CSA-AKI). The study is to investigate the influence of serum creatinine (SCr) corrected by fluid balance on the prognosis of patients with cardiac surgery. METHODS: A retrospective study was conducted in 1334 patients who underwent elective cardiac surgery from January 1 to December 31, 2015. Kidney Disease: Improving Global Outcomes (KDIGO) criteria for AKI were applied to identify CSA-AKI. SCr was measured every 24 h during ICU period and was accordingly adjusted for cumulative fluid balance. Changes in SCr, defined as ∆Crea, were determined by difference between before and after adjustment for cumulative fluid balance. All patients were then divided into three groups: underestimation group (∆Crea ≥ P75), normal group (P25 < ∆Crea < P75) and overestimation group (∆Crea ≤ P25). RESULTS: The incidence of AKI increased from 29.5% to 31.8% after adjustment for fluid balance. Patients in underestimation group showed prolonged length of ICU stay compared with normal group and overestimation group (3.2 [1.0-4.0] vs 2.1 [1.0-3.0] d, P < 0.001; 3.2  [1.0-4.0] vs 2.3 [1.0-3.0] d, P < 0.001). Length of hospital stay and mechanical ventilation dependent days in underestimation group were significantly longer than normal group (P < 0.001). Multivariate analysis showed age, baseline SCr and left ventricular ejection fraction were independently associated with underestimation of creatinine. CONCLUSIONS: Cumulative fluid balance after cardiac surgery disturbs accurate measurement of serum creatinine. Patients with underestimation of SCr were associated with poor prognosis.