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Abstract Background To identify the relationship between BMI or lipid metabolism and diabetic neuropathy using a Mendelian randomization (MR) study. Methods Body constitution-related phenotypes, namely BMI (kg/m2), total cholesterol (TC), and triglyceride (TG), were investigated in this study. Despite the disparate origins of these data, all were accessible through the IEU OPEN GWAS database ( https://gwas.mrcieu.ac.uk/ ). Instrumental variables and F-statistics for each exposure-outcome pair were determined in weighted mode, weighted median, MR-Egger and Inverse-Variance Weighted (IVW) MR analyses. The p-value threshold was consistently set at 5.00E−08, following established methodology. The preliminary analysis utilized the IVW method to explore potential causal relationships between body constitution-related phenotypes and diabetic neuropathy. Inverse variance weighting, a technique amalgamating random variables, assigns weights inversely proportional to each variable's variance, commonly used for merging findings from independent studies. The weighted median method provides a causal estimate even when up to 50% of the instruments are invalid, enhancing robustness. The weighted mode method identifies the most common causal effect, reducing bias when some instruments exhibit horizontal pleiotropy. The Wald ratio method was utilized to calculate exposure-outcome effects, employing a range of methodologies to ensure result accuracy across different scenarios. This study addresses the critical gap in understanding the causal relationship between BMI, lipid metabolism, and diabetic neuropathy (DN). Employing a MR approach, it highlights BMI as a predictive factor for DN progression, providing insights into potential risk management strategies. Results IVW analysis showed that BMI (P = 0.033, OR = 2.53, 95% CI 1.08–5.96) and triglycerides level (P = 0.593, OR = 1.11, 95% CI 0.77–1.60) were positively associated with the initiation of DN, indicating that the values of BMI and triglycerides are potentially the risk factors in DN development. Additionally, TC was negatively associated with the DN (P = 0.069, OR = 0.72, 95% CI = 0.50–1.03).The forest plot of advanced analysis between BMI and DN relationship indicated a positive correlation between increasing BMI and the risk of DN. In addition, it is evident that with the increase in BMI, the risk of diabetic polyneuropathy also rises. This research demonstrates a positive association between BMI and DN risk (P = 0.033, OR = 2.53, 95% CI = 1.08–5.96). However, no significant correlation was observed between triglycerides (P = 0.593) or total cholesterol (P = 0.069) and DN development, underscoring the complex interplay between lipid metabolism and DN. Conclusion This research demonstrates a positive association between the risk of DN and BMI, while no significant correlation exists between TG or TC and the development of DN. These results imply that BMI may serve as a predictive factor for the progression of DN.

Abstract Background Gastric cancer is a significant global malignancy with poor prognosis. Although the emergence of immune checkpoint inhibitors (ICIs) prolonged the duration of survival, resistance and progression are inevitable. We aim to evaluate the effectiveness of programmed death-1 (PD-1) inhibitors in immunotherapy beyond progression (IBP). Method We divided the advanced gastric cancer patients who received two lines immunotherapy into same regimen group (with same PD-1 inhibitor regime after IBP) and different regimen group (with different PD-1 inhibitor regime after IBP). Statistical analysis conducted to compare patient characteristics and evaluate survival differences between groups. Result The clinical outcome analysis showed that the same PD-1 inhibitor regime seemed to exhibit a higher disease control rate (DCR) (51.8% vs. 29.2%, P = 0.062), significantly prolonged progression-free survival 2 (PFS2) (162 vs. 75 days, P = 0.001) and overall survival (OS) (312 vs. 166 days, P = 0.022) when compared with those of cross line. In the multivariate analysis, when using different regimen group as reference, the same regimen group was found to be independently associated with improved PFS2 [hazard ratio (HR) = 0.467, 95% confidence interval (CI): 0.267–0.816, P = 0.008] and OS (HR = 0.508, 95%CI: 0.278–0.927, P = 0.027). Conclusion Continuation of the same type of PD-1 inhibitor regime in IBP shows clinical benefits and represents a promising therapeutic approach.

Abstract Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive thyroid cancer with poor prognosis. Killing cancer cells by inducing DNA damage or blockage of DNA repair is a promising strategy for chemotherapy. It is reported that aldehyde-reactive alkoxyamines can capture the AP sites, one of the most common DNA lesions, and inhibit apurinic/apyrimidinic endonuclease 1(APE1)-mediated base excision repair (BER), leading to cell death. Whether this strategy can be employed for ATC treatment is rarely investigated. The aim of this study is to exploit GSH-responsive AP site capture reagent (AP probe-net), which responses to the elevated glutathione (GSH) levels in the tumor micro-environment (TME), releasing reactive alkoxyamine to trap AP sites and block the APE1-mediated BER for targeted anti-tumor activity against ATC. In vitro experiments, including MTT andγ-H2AX assays, demonstrate their selective cytotoxicity towards ATC cells over normal thyroid cells. Flow cytometry analysis suggests that AP probe-net arrests the cell cycle in the G2/M phase and induces apoptosis. Western blotting (WB) results show that the expression of apoptotic protein increased with the increased concentration of AP probe-net. Further in vivo experiments reveal that the AP probe-net has a good therapeutic effect on subcutaneous tumors of the ATC cells. In conclusion, taking advantage of the elevated GSH in TME, our study affords a new strategy for targeted chemotherapy of ATC with high selectivity and reduced adverse effects.

Abstract Chemoresistance remains a significant challenge for effective breast cancer treatment which leads to cancer recurrence. CRISPR-directed gene editing becomes a powerful tool to reduce chemoresistance by reprogramming the tumor microenvironment. Previous research has revealed that Chinese herbal extracts have significant potential to overcome tumor chemoresistance. However, the therapeutic efficacy is often limited due to their poor tumor targeting and in vivo durability. Here we have developed a tumor microenvironment responsive nanoplatform (H-MnO2(ISL + DOX)-PTPN2@HA, M(I + D)PH) for nano-herb and CRISPR codelivery to reduce chemoresistance. Synergistic tumor inhibitory effects were achieved by the treatment of isoliquiritigenin (ISL) with doxorubicin (DOX), which were enhanced by CRISPR-based gene editing to target protein tyrosine phosphatase non-receptor type 2 (PTPN2) to initiate long-term immunotherapy. Efficient PTPN2 depletion was observed after treatment with M(I + D)PH nanoparticles, which resulted in the recruitment of intratumoral infiltrating lymphocytes and an increase of proinflammatory cytokines in the tumor tissue. Overall, our nanoparticle platform provides a diverse technique for accomplishing synergistic chemotherapy and immunotherapy, which offers an effective treatment alternative for malignant neoplasms. Graphical Abstract

Abstract Ferroptosis is a type of regulated cell death characterized by iron accumulation and uncontrolled lipid peroxidation, leading to plasma membrane rupture and intracellular content release. Originally investigated as a targeted therapy for cancer cells carrying oncogenic RAS mutations, ferroptosis induction now exhibits potential to complement chemotherapy, immunotherapy, and radiotherapy in various cancer types. However, it can lead to side effects, including immune cell death, bone marrow impairment, liver and kidney damage, cachexia (severe weight loss and muscle wasting), and secondary tumorigenesis. In this review, we discuss the advantages and offer an overview of the diverse range of documented side effects. Furthermore, we examine the underlying mechanisms and explore potential strategies for side effect mitigation.

Abstract Background The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism. Methods A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments. Results DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP. Conclusions miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.

Abstract Background Pure uterine serous carcinoma (p-USC) and mixed tumors with serous component (m-USC) are aggressive subtypes of endometrial cancer associated with high mortality rates. This retrospective study aimed to compare clinicopathologic features and outcomes of p-USC and m-USC in a single center. Methods This study retrospectively reviewed patients diagnosed with USC at Peking University People’s Hospital between 2008 and 2022. T-tests and chi-square tests were used to compare clinicopathological characteristics between p-USC and m-USC. Kaplan-Meier survival curve and Cox regression analysis were used to analyze the impact of clinical and pathological variables on OS and PFS. Results Among the 91 patients who underwent surgery, 65.9% (n = 60) were p-USC, and 34.1% (n = 31) were m-USC. Patients with p-USC had earlier menopause (P = 0.0217), a lower rate of progesterone receptor(PR) expression (P

Abstract Background The production of human chorionic gonadotropin (hCG) by the placental trophoblast cells is essential for maintaining a normal pregnancy. Aberrant hCG levels are associated with reproductive disorders. The protein of hCG is a dimer consisting of an α subunit and a β subunit. The β subunit is encoded by the CGB gene and is unique to hCG. Growth differentiation factor-11 (GDF-11), a member of the transforming growth factor-β (TGF-β) superfamily, is expressed in the human placenta and can stimulate trophoblast cell invasion. However, whether the expression of CGB and the production of hCG are regulated by GDF-11 remains undetermined. Methods Two human choriocarcinoma cell lines, BeWo and JEG-3, and primary cultures of human cytotrophoblast (CTB) cells were used as experimental models. The effects of GDF-11 on CGB expression and hCG production, as well as the underlying mechanisms, were explored by a series of in vitro experiments. Results Our results show that treatment of GDF-11 downregulates the expression of CGB and the production of hCG in both BeWo and JEG-3 cells as well as in primary CTB cells. Using a pharmacological inhibitor and siRNA-mediated approach, we reveal that both ALK4 and ALK5 are required for the GDF-11-induced downregulation of CGB expression. In addition, treatment of GDF-11 activates SMAD2/3 but not SMAD1/5/8 signaling pathways. Moreover, both SMAD2 and SMAD3 are involved in the GDF-11-downregulated CGB expression. ELISA results show that the GDF-11-suppressed hCG production requires the ALK4/5-mediated activation of SMAD2/3 signaling pathways. Conclusions This study not only discovers the biological function of GDF-11 in the human placenta but also provides important insights into the regulation of the expression of hCG. Video Abstract

Abstract Background Cystinuria and xanthinuria are both rare genetic diseases involving urinary calculi. However, cases combining these two disorders have not yet been reported. Case Presentation In this study, we report a case of cystinuria with xanthine stones and hyperuricemia. The 23-year-old male patient was diagnosed with kidney and ureteral stones, solitary functioning kidney and hyperuricemia after admission to the hospital. The stones were removed by surgery and found to be composed of xanthine. Conclusion Genetic testing by next-generation sequencing technology showed that the patient carried the homozygous nonsense mutation c.1113 C> A (p.Tyr371*) in the SLC3A1 gene, which was judged to be a functionally pathogenic variant. Sanger sequencing revealed that the patient’s parents carried this heterozygous mutation, which is a pathogenic variant that can cause cystinuria. The 24-h urine metabolism analysis showed that the cystine content was 644 mg (

Abstract Background Hepatocellular carcinoma (HCC) is associated with a high occurrence, mortality, and poor prognosis. MLX interacting protein like (MLXIPL) is an important regulator of glucolipid metabolism and is involved in tumor progression. We aimed to clarify the role of MLXIPL in HCC and its underlying mechanisms. Methods The level of MLXIPL was predicted using bioinformatic analysis and verified using quantitative real-time PCR (qPCR), immunohistochemical analysis, and western blot. We assessed the effects of MLXIPL on biological behaviors using the cell counting kit-8, colony formation, and Transwell assay. Glycolysis was evaluated using the Seahorse method. The interaction between MLXIPL and mechanistic target of rapamycin kinase (mTOR) was confirmed using RNA immunoprecipitation and co-immunoprecipitation. mTOR expression was detected in HCC cells using qPCR, immunofluorescence analysis, and western blot. Results The results showed that MLXIPL levels were elevated in both HCC tissues and HCC cell lines. Knockdown of MLXIPL impeded HCC cell growth, invasion, migration, and glycolysis. Moreover, MLXIPL combined with mTOR to induce phosphorylation of mTOR. Activated mTOR abrogated the effects on cellular processes induced by MLXIPL. Conclusion MLXIPL promoted the malignant progression of HCC by activating phosphorylation of mTOR, suggesting an important role of the combination of MLXIPL and mTOR in HCC.

Abstract Background Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown. Methods A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments. Results Western blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway. Conclusion This study increases the understanding of the physiological role of HB-EGF in human luteal functions. Video Abstract

Abstract Human endometrial epithelia undergo injury repair and regeneration with the menstrual cycle; however, mechanisms underpinning the roles of endometrial epithelial cells in endometrial lesions and regeneration remain incompletely understood, mainly owing to the difficulty in the isolation and expansion of primary endometrial epithelial cells and the lack of reliable models for in vitro and in vivo studies. In this report, we sought to improve methods for the isolation and expansion of human endometrial epithelial cells with a Rho-associated protein kinase (ROCK) inhibitor–modified medium and subsequently characterize endometrial epithelium generated with primary cells cultured in an air–liquid interface (ALI) state. Immunocytochemistry staining revealed the expression of epithelial cellular adhesion molecule (EpCam) and stage-specific embryonic antigen-1 (SSEA-1) but a lack of CD13 in endometrial epithelial cells. Meanwhile, a large number of proliferative Ki67+ cells were observed in isolated epithelial cells. Importantly, the EpCam+/CD13− cells were capable of forming spheroids, a characteristic of epithelial stem/progenitor cells. Interestingly, these cells also exhibited a capacity to reconstitute epithelial layers in an ALI state. Morphological analysis revealed mucosal secretion of differentiated epithelial cells with cilia and microvilli in ALI epithelial cells as determined by electronic microscopy. Immunoblotting assay further demonstrated the expression of endometrial epithelial cell markers keratin 17/19 and EpCam and stem cell marker OCT3/4 but not stromal cell marker Vimentin protein and CD13 in cell expansions. Furthermore, molecular analysis also showed that the exposure of cells to estrogen elevated the expression of estrogen receptor and progesterone receptors in ALI cultures. Our results shed light on the possibility of expanding sufficient numbers of endometrial epithelial cells for stem cell biology studies, and they provide a feasible and alternative model that can recapitulate the characteristics and physiology of endometrial epithelium in vivo.

Abstract Background Total en bloc spondylectomy (TES) is a recognized surgical approach for managing spinal tumors. With advancements in three-dimensional (3D) printing technology, the use of 3D-printed prosthetics for vertebral reconstruction post-tumor resection has gained traction. However, research on the clinical implications of these prosthetics remains limited. Methods This retrospective study evaluated patients who underwent TES for primary and metastatic thoracolumbar tumors at the Department of Spinal Surgery, Tianjin Hospital, between October 2017 and September 2020. These patients received anterior reconstruction with 3D-printed artificial vertebral bodies. Results 14 patients completed the surgery, with intraoperative blood loss ranging from 1,400 to 4,200 ml (mean 2,767 ± 790 ml) and operative duration between 240 and 520 min (mean 382 ± 75.9 min). The follow-up period extended from 7 to 43 months, with an average of 19.9 ± 9.5 months. Standardized prefabricated prosthetics were utilized in nine patients, while five received customized prosthetics. Throughout the follow-up, there were no reports of posterior connecting rod, 3D-printed prosthetic, or pedicle screw failures. Notably, one patient presented with significant prosthetic subsidence resulting in screw loosening, and three cases of prosthetic subsidence were observed. Conclusion The incorporation of 3D-printed prosthetics in TES procedures yielded favorable clinical outcomes. Further research is warranted to optimize these prosthetics for enhanced postoperative stability and patient-specific applications.

Abstract Background N6-Methyladenosine (m6A) methylation, a common form of RNA modification, play an important role in the pathogenesis of various diseases and in the ontogeny of organisms. Nevertheless, the precise function of m6A methylation in photoaging remains unknown. Objectives This study aims to investigate the biological role and underlying mechanism of m6A methylation in photoaging. Methods m6A dot blot, Real-time quantitative PCR (RT-qPCR), western blot and immunohistochemical (IHC) assays were employed to detect the m6A level and specific m6A methylase in ultraviolet ray (UVR)-induced photoaging tissue. The profile of m6A-tagged mRNA was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis. Finally, we investigated the regulatory mechanism of KIAA1429 by MeRIP-qPCR, RNA knockdown and immunofluorescence assay. Results m6A levels were increased in photoaging and were closely associated with the upregulation of KIAA1429 expression. 1331 differentially m6A methylated genes were identified in the UVR group compared with the control group, of which 1192 (90%) were hypermethylated. Gene ontology analysis showed that genes with m6A hypermethylation and mRNA downregulation were mainly involved in extracellular matrix metabolism and collagen metabolism-related processes. Furthermore, KIAA1429 knockdown abolished the downregulation of TGF-bRII and upregulation of MMP1 in UVR-irradiated human dermal fibroblasts (HDFs). Mechanically, we identified MFAP4 as a target of KIAA1429-mediated m6A modification and KIAA1429 might suppress collagen synthesis through an m6A-MFAP4-mediated process. Conclusions The increased expression of KIAA1429 hinders collagen synthesis during UVR-induced photoaging, suggesting that KIAA1429 represents a potential candidate for targeted therapy to mitigate UVR-driven photoaging.

Abstract Background Owing to a lack of data, this study aimed to explore the effect of cardiac preload on myocardial strain in patients with sepsis. Methods A total of 70 patients with sepsis in intensive care unit (ICU) of a tertiary teaching hospital in China from January 2018 to July 2019 and underwent transthoracic echocardiography were enrolled. Echocardiographic data were recorded at ICU admission and 24 h later. Patients were assigned to low left ventricular end-diastolic volume index (LVEDVI) and normal LVEDVI groups. We assessed the impact of preload on myocardial strain between the groups and analyzed the correlation of echocardiographic parameters under different preload conditions. Results Thirty-seven patients (53%) had a low LVEDVI and 33 (47%) a normal LVEDVI. Those in the low LVEDVI group had a faster heart rate (121.7 vs. 95.3, p

Abstract Background Diabetic kidney disease (DKD) is a primary microvascular complication of diabetes with limited therapeutic effects. Delving into the pathogenic mechanisms of DKD and identifying new therapeutic targets is crucial. Emerging studies reveal the implication of ferroptosis and immune dysregulation in the pathogenesis of DKD, however, the precise relationship between them remains not fully elucidated. Investigating their interplay is pivotal to unraveling the pathogenesis of diabetic kidney disease, offering insights crucial for targeted interventions and improved patient outcomes. Methods Integrated analysis, Consensus clustering, Machine learning including Generalized Linear Models (GLM), RandomForest (RF), Support Vector Machine (SVM) and Extreme Gradient Boosting (xGB), Artificial neural network (ANN) methods of DKD glomerular mRNA sequencing were performed to screen DKD-related ferroptosis genes.CIBERSORT, ESTIMATE and ssGSEA algorithm were used to assess the infiltration of immune cells between DKD and control groups and in two distinct ferroptosis phenotypes. The ferroptosis hub genes were verified in patients with DKD and in the db/db spontaneous type 2 diabetes mouse model via immunohistochemical and Western blotting analyses in mouse podocyte MPC5 and mesangial SV40-MES-13 cells under high-glucose (HG) conditions. Results We obtained 16 differentially expressed ferroptosis related genes and patients with DKD were clustered into two subgroups by consensus clustering. Five ferroptosis genes (DUSP1,ZFP36,PDK4,CD44 and RGS4) were identified to construct a diagnostic model with a good diagnosis performance in external validation. Analysis of immune infiltration revealed immune heterogeneity between DKD patients and controls.Moreover, a notable differentiation in immune landscape, comprised of Immune cells, ESTIMATE Score, Immune Score and Stromal Score was observed between two FRG clusters. GSVA analysis indicated that autophagy, apoptosis and complement activation can participate in the regulation of ferroptosis phenotypes. Experiment results showed that ZFP36 was significantly overexpressed in both tissue and cells while CD44 was on the contrary.Meanwhile,spearman analysis showed both ZFP36 and CD44 has a strong correlation with different immune cells,especially macrophage. Conclusion The regulation of the immune landscape in DKD is significantly influenced by the focal point on ferroptosis. Newly identified ferroptosis markers, CD44 and ZFP36, are poised to play essential roles through their interactions with macrophages, adding substantial value to this regulatory landscape.

Abstract Background The pattern recognition receptor Dectin-1 was initially discovered to play a pivotal role in mediating pulmonary antifungal immunity and promoting neutrophil-driven inflammation. Recent studies have revealed that Dectin-1 is overexpressed in asthma, but the specific mechanism remains elusive. Additionally, Dectin-1 has been implicated in promoting pyroptosis, a hallmark of severe asthma airway inflammation. Nevertheless, the involvement of the non-classical pyroptosis signal caspase-11/4 and its upstream regulatory mechanisms in asthma has not been completely explored. Methods House dust mite (HDM)-induced mice was treated with Dectin-1 agonist Curdlan, Dectin-1 inhibitor Laminarin, and caspase-11 inhibitor wedelolactone separately. Subsequently, inflammatory cells in bronchoalveolar lavage fluid (BALF) were analyzed. Western blotting was performed to measure the protein expression of caspase-11 and gasdermin D (GSDMD). Cell pyroptosis and the expression of chemokine were detected in vitro. The correlation between Dectin-1 expression, pyroptosis factors and neutrophils in the induced sputum of asthma patients was analyzed. Results Curdlan appeared to exacerbate neutrophil airway inflammation in asthmatic mice, whereas wedelolactone effectively alleviated airway inflammation aggravated by Curdlan. Moreover, Curdlan enhanced the release of caspase-11 activation fragments and N-terminal fragments of gasdermin D (GSDMD-N) stimulated by HDM both in vivo or in vitro. In mouse alveolar macrophages (MH-S cells), Curdlan/HDM stimulation resulted in vacuolar degeneration and elevated lactate dehydrogenase (LDH) release. In addition, there was an upregulation of neutrophil chemokines CXCL1, CXCL3, CXCL5 and their receptor CXCR2, which was suppressed by wedelolactone. In asthma patients, a positive correlation was observed between the expression of Dectin-1 on macrophages and caspase-4 (the human homology of caspase-11), and the proportion of neutrophils in induced sputum. Conclusion Dectin-1 activation in asthma induced caspase-11/4 mediated macrophage pyroptosis, which subsequently stimulated the secretion of chemokines, leading to the exacerbation of airway neutrophil inflammation. Graphical Abstract

Abstract Background Schizothorax o’connori is an endemic fish distributed in the upper and lower reaches of the Yarlung Zangbo River in China. It has experienced a fourth round of whole gene replication events and is a good model for exploring the genetic differentiation and environmental adaptability of fish in the Qinghai-Tibet Plateau. The uplift of the Qinghai-Tibet Plateau has led to changes in the river system, thereby affecting gene exchange and population differentiation between fish populations. With the release of fish whole genome data, whole genome resequencing has been widely used in genetic evolutionary analysis and screening of selected genes in fish, which can better elucidate the genetic basis and molecular environmental adaptation mechanisms of fish. Therefore, our purpose of this study was to understand the population structure and adaptive characteristics of S. o’connori using the whole-genome resequencing method. Results The results showed that 23,602,746 SNPs were identified from seven populations, mostly distributed on chromosomes 2 and 23. There was no significant genetic differentiation between the populations, and the genetic diversity was relatively low. However, the Zangga population could be separated from the Bomi, Linzhi, and Milin populations in the cluster analysis. Based on historical dynamics analysis of the population, the size of the ancestral population of S. o’connori was affected by the late accelerated uplift of the Qinghai Tibet Plateau and the Fourth Glacial Age. The selected sites were mostly enriched in pathways related to DNA repair and energy metabolism. Conclusion Overall, the whole-genome resequencing analysis provides valuable insights into the population structure and adaptive characteristics of S. o’connori. There was no obvious genetic differentiation at the genome level between the S. o’connori populations upstream and downstream of the Yarlung Zangbo River. The current distribution pattern and genetic diversity are influenced by the late accelerated uplift of the Qinghai Tibet Plateau and the Fourth Ice Age. The selected sites of S. o’connori are enriched in the energy metabolism and DNA repair pathways to adapt to the low temperature and strong ultraviolet radiation environment at high altitude.

Abstract Background Understanding the ecological and environmental functions of phototrophic biofilms in the biological crust is crucial for improving metal(loid) (e.g. Cd, As) bioremediation in mining ecosystems. In this study, in combination with metal(loid) monitoring and metagenomic analysis, we systematically evaluated the effect of biofilm in a novel biological aqua crust (biogenic aqua crust—BAC) on in situ metal(loid) bioremediation of a representative Pb/Zn tailing pond. Results We observed strong accumulation of potentially bioavailable metal(loid)s and visible phototrophic biofilms in the BAC. Furthermore, dominating taxa Leptolyngbyaceae (10.2–10.4%, Cyanobacteria) and Cytophagales (12.3–22.1%, Bacteroidota) were enriched in biofilm. Along with predominant heterotrophs (e.g. Cytophagales sp.) as well as diazotrophs (e.g. Hyphomonadaceae sp.), autotrophs/diazotrophs (e.g. Leptolyngbyaceae sp.) in phototrophic biofilm enriched the genes encoding extracellular peptidase (e.g. family S9, S1), CAZymes (e.g. CBM50, GT2) and biofilm formation (e.g. OmpR, CRP and LuxS), thus enhancing the capacity of nutrient accumulation and metal(loid) bioremediation in BAC system. Conclusions Our study demonstrated that a phototrophic/diazotrophic biofilm constitutes the structured communities containing specific autotrophs (e.g. Leptolyngbyaceae sp.) and heterotrophs (e.g. Cytophagales sp.), which effectively control metal(loid) and nutrient input using solar energy in aquatic environments. Elucidation of the mechanisms of biofilm formation coupled with metal(loid) immobilization in BAC expands the fundamental understanding of the geochemical fate of metal(loid)s, which may be harnessed to enhance in situ metal(loid) bioremediation in the aquatic ecosystem of the mining area. Video Abstract

Abstract Background The use of creatinine-based glomerular filtration rate (GFR)-estimating equations to evaluate kidney function in elderly individuals does not appear to offer any performance advantages. We therefore aimed to develop an accurate GFR-estimating tool for this age group. Methods Adults aged ≥ 65 years who underwent GFR measurement by technetium-99 m-diethylene triamine pentaacetic acid (99mTc-DTPA) renal dynamic imaging were included. Data were randomly split into a training set containing 80% of the participants and a test set containing the remaining 20% of the subjects. The Back propagation neural network (BPNN) approach was used to derive a novel GFR estimation tool; then we compared the performance of the BPNN tool with six creatinine-based equations (Chronic Kidney Disease-Epidemiology Collaboration [CKD-EPI], European Kidney Function Consortium [EKFC], Berlin Initiative Study-1 [BIS1], Lund-Malmö Revised [LMR], Asian modified CKD-EPI, and Modification of Diet in Renal Disease [MDRD]) in the test cohort. Three equation performance criteria were considered: bias (difference between measured GFR and estimated GFR), precision (interquartile range [IQR] of the median difference), and accuracy P30 (percentage of GFR estimates that are within 30% of measured GFR). Results The study included 1,222 older adults. The mean age of both the training cohort (n = 978) and the test cohort (n = 244) was 72 ± 6 years, with 544 (55.6%) and 129 (52.9%) males, respectively. The median bias of BPNN was 2.06 ml/min/1.73 m2, which was smaller than that of LMR (4.59 ml/min/1.73 m2; p = 0.03), and higher than that of the Asian modified CKD-EPI (-1.43 ml/min/1.73 m2; p = 0.02). The median bias between BPNN and each of CKD-EPI (2.19 ml/min/1.73 m2; p = 0.31), EKFC (-1.41 ml/min/1.73 m2; p = 0.26), BIS1 (0.64 ml/min/1.73 m2; p = 0.99), and MDRD (1.11 ml/min/1.73 m2; p = 0.45) was not significant. However, the BPNN had the highest precision IQR (14.31 ml/min/1.73 m2) and the greatest accuracy P30 among all equations (78.28%). At measured GFR

Abstract Background Anxiety disorder is a common non-motor symptom among individuals with Parkinson’s disease (PD). At present, there are no specific tools in China for assessing the anxiety level of patients with PD. This study aimed to test the reliability and validity of the Chinese version of the Parkinson Anxiety Scale (C-PAS) in Chinese patients with PD. Methods A total of 158 patients with PD at one hospital in Nanjing were recruited through convenience sampling. The C-PAS was translated into Chinese using a classic ‘forward-backward’ translation method. Reliability tests included internal consistency and test-reliability. And in addition to content, structure and criterion-related validity were performed for the validity tests. Criterion-related validity was evaluated with the Hospital Anxiety and Depression Scale-Anxiety Subscale (HADS-A). Results Results confirmed the three-factor structure of the original C-PAS with 12 items, including persistent anxiety (5 items), episodic anxiety (4 items) and avoidance behavior (3 items). Significant and positive correlations were obtained between C-PAS and HADS-A (r = 0.82, P

Abstract Objective To analyse and compare the biomechanical differences between 3D-printed prostheses, titanium mesh cages and poorly matched titanium mesh cages in total en bloc spondylectomy (TES). Methods The finite element model of T10-L2 for healthy adults was modified to make three models after T12 total spondylectomy. These models were a 3D-printed prosthesis, titanium mesh cage and prosthesis-endplate mismatched titanium mesh cage for reconstruction. The range of motion (ROM), stress distribution of the endplate and internal fixation system of three models in flexion and extension, lateral bending and axial rotation were simulated and analysed by ABAQUS. Result In flexion, due to the support of the anterior prosthesis, the fixation system showed the maximum fixation strength. The fixation strength of the 3D-printed prosthesis model was 26.73 N·m /°, that of the TMC support model was 27.20 N·m /°, and that of the poorly matched TMC model was 24.16 N·m /°. In flexion, the L1 upper endplate stress of the poorly matched TMC model was 35.5% and 49.6% higher than that of the TMC and 3D-printed prosthesis, respectively. It was 17% and 28.1% higher in extension, 39.3% and 42.5% higher in lateral bending, and 82.9% and 91.2% higher in axial rotation, respectively. The lower endplate of T11 showed a similar trend, but the magnitude of the stress change was reduced. In the stress analysis of the 3D-printed prosthesis and TMC, it was found that the maximum stress was in flexion and axial rotation, followed by left and right bending, and the least stress was in extension. However, the mismatched TMC withstood the maximum von Mises stress of 418.7 MPa (almost twice as much as the buckling state) in rotation, 3 times and 5.83 times in extension, and 1.29 and 2.85 times in lateral bending, respectively. Conclusion Different prostheses with good endplate matching after total spondylectomy can obtain effective postoperative stable support, and the reduction in contact area caused by mismatch will affect the biomechanical properties and increase the probability of internal fixation failure.

Abstract Aims To investigate the proportion and risk factors of diabetic retinopathy (DR) by stages in less-developed rural areas in Hunan Province of China. Background DR is common among people with diabetes but not well recognized in less-developed rural areas. There is insufficient evidence on the risk factors of DR by stages, making it challenging to develop targeted prevention and intervention programs for DR in primary care settings. Methods A multi-site cross-sectional survey was conducted among people with type 2 diabetes mellitus (T2DM) from four less-developed counties in Hunan Province of China. All participants underwent the screening of DR via digital fundus photography and completed self-reported questionnaires on their socio-demographic and clinical characteristics, diabetes self-efficacy, diabetes self-care behaviors, social support, family function, and health service accessibility. The multinomial logistic regression models were employed to explore the risk factors of DR by stage, which were selected based on the socio-ecological model, literature, and clinical experience. Results A total of 196 participants were included in this study with an average age of 57.43 ± 10.26. 59.6% (117/196) of the participants were identified as DR, including 37.2% (73/196) non-proliferative DR and 22.4% (44/196) proliferative DR. Compared to the non-DR group, the risk factors of non-proliferative DR and proliferative DR were diabetes duration (OR: 1.10, 95 CI%: 1.04–1.17; OR: 1.14, 95 CI% 1.06–1.22) and self-monitoring of blood glucose (OR: 1.09, 95 CI% 1.01–1.17; OR: 1.11, 95 CI%: 1.02–1.20); the protective factors of non-proliferative DR was accessible complication prevention and management education (OR: 0.37, 95 CI% 0.14–0.94) while the protective factors of proliferative DR were physical activities (OR: 0.89, 95 CI%: 0.80–0.98). Compared to the non-proliferative DR group, the protective factors of proliferative DR were physical activities (OR: 0.89, 95 CI% 0.02–0.89) and family function (OR: 0.84, 95 CI%: 0.04–0.84). Conclusion DR was prevalent among people with T2DM in less-developed rural areas, indicating the need of strengthening DR screening. Risk factors of DR varied by stage while sharing some common factors. Future DR prevention and intervention programs may benefit from improving these factors to reduce the risk of DR by stage.

Abstract Background Chemoresistance is one of the major obstacles that lead to poor prognosis in cervical cancer. linc00958 was reported to be an oncogene in cervical cancer. However, its role in mediating chemoresistance remains to be revealed. Purpose To explore the regulatory mechanisms of linc00958 in cisplatin-resistant cervical cancer cells and further validate in xenograft mice. Methods Online bioinformatic tools were used to conduct the pre-investigation of linc00958/miR-185-5p/RSF-1 and predict the associations between RSF-1 and AKT1/GSK3β/VEGFA in cervical cancer. RT-qPCR measured the RNA expression levels of linc00958/miR-185-5p/RSF-1 in SiHa and SiHa/DDP. Cell survival rates were evaluated by CCK8 methods after cells were exposed to differential concentrations of DDP. Dual-luciferase reporter methods were used to measure luciferase activity. Western blot measured RSF-1 protein and phosphorylated changes of AKT1/GSK3β. Immunofluorescence was employed to observe VEGFA secretion in vitro. Tube formation was applied to evaluate the in-vitro changes of angiogenesis. The SiHa/DDP cells stably transfected with pLKO-sh-NC or pLKO-sh-linc00958 plasmids, were injected into mice, establishing xenograft models. The changes in mice weight and tumor volumes were recorded. H&E staining and Immunohistochemistry (IHC) method was further performed. Results linc00958 expression was higher in SiHa/DDP cells. High linc00958 expression was associated with low overall survival. In SiHa/DDP cells linc00958/miR-185-5p/RSF-1 axis inhibited the cellular resistance to cisplatin and suppressed VEGFA and the tube formation through AKT1/GSK3β/VEGFA pathway. The knockdown of linc00958 inhibited RSF-1 and Ki67, curbing tumor growth; it also inhibited VEGFA and CD34, decreasing angiogenesis in mice. Conclusion linc00958/miR-185-5p/RSF-1 modulates cisplatin resistance and angiogenesis through AKT1/GSK3β/VEGFA pathway in cervical cancer.

Abstract Background An increasing number of long noncoding RNAs (lncRNAs) have been found to play important roles in sex differentiation and gonad development by regulating gene expression at the epigenetic, transcriptional and posttranscriptional levels. The ricefield eel, Monopterus albus, is a protogynous hermaphroditic fish that undergoes a sequential sex change from female to male. However, the roles of lncRNA in the sex change is unclear. Results Herein, we performed RNA sequencing to analyse lncRNA expression patterns in five different stages of M. albus development to investigate the roles of lncRNAs in the sex change process. A total of 12,746 lncRNAs (1503 known lncRNAs and 11,243 new lncRNAs) and 2901 differentially expressed lncRNAs (DE-lncRNAs) were identified in the gonads. The target genes of the DE-lncRNAs included foxo1, foxm1, smad3, foxr1, camk4, ar and tgfb3, which were mainly enriched in signalling pathways related to gonadal development, such as the insulin signalling pathway, MAPK signalling pathway, and calcium signalling pathway. We selected 5 highly expressed DE-lncRNAs (LOC109952131, LOC109953466, LOC109954337, LOC109954360 and LOC109958454) for full length amplification and expression pattern verification. They were all expressed at higher levels in ovaries and intersex gonads than in testes, and exhibited specific time-dependent expression in ovarian tissue incubated with follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The results of quantitative real-time PCR (qRT-PCR) analysis and a dual-luciferase assay showed that znf207, as the gene targeted by LOC109958454, was expressed in multiple tissues and gonadal developmental stages of M. albus, and its expression was also inhibited by the hormones FSH and hCG. Conclusions These results provide new insights into the role of lncRNAs in gonad development, especially regarding natural sex changes in fish, which will be useful for enhancing our understanding of sequential hermaphroditism and sex changes in the ricefield eel (M. albus) and other teleosts.

Abstract Background Lymphangioleiomyomatosis (LAM) is a rare multisystem disease characterized by cystic lung disease and extrapulmonary manifestations, including lymphatic system disorder. The objective of this study was to investigate the findings of 68Ga-NOTA-Evans Blue (NEB) PET/CT in LAM and compare it with that of 99mTc-ASC lymphoscintigraphy. Methods Ten patients diagnosed with LAM according to the American Thoracic Society/Japanese Respiratory Society guidelines for LAM were recruited in this study. PET/CT acquisition was performed at 20 to 40 min after subcutaneous injection of 68Ga-NEB into the first interdigital spaces of both feet (0.3 ml, 37 MBq/foot). All subjects also underwent 99mTc-antimony sulfide colloid (ASC) lymphoscintigraphy within a week for comparison. Results 68Ga-NEB PET/CT displayed various lymphatic system abnormalities in 10 (100%) of 10 patients. These included pulmonary lymphatic abnormalities in 10 (100%) of 10 patients, enlarged lymph nodes in 5 (50%), lymphangioleiomyomas in 2 (20%), dilation of the lumbar trunk and/or iliac lymph vessels in 5 (50%), thoracic duct dilation in 2 (20%), chylous effusion in 1 (10%). For pulmonary lymphatic abnormalities, the positive rates of 68Ga-NEB PET/CT and 99mTc-ASC lymphoscintigraphy were 100% (10/10) and 10% (1/10), respectively (P

Abstract Background This study compared the biomechanics of artificial pedicle fixation in spine reconstruction with a 3-dimensional (3D)-printed prosthesis after total en bloc spondylectomy (TES) by finite element analysis. Methods A thoracolumbar (T10–L2) finite element model was developed and validated. Two models of T12 TES were established in combination with different fixation methods: Model A consisted of long-segment posterior fixation (T10/11, L1/2) + 3D-printed prosthesis; and Model B consisted of Model A + two artificial pedicle fixation screws. The models were evaluated with an applied of 7.5 N·m and axial force of 200 N. We recorded and analyzed the following: (1) stiffness of the two fixation systems, (2) hardware stress in the two fixation systems, and (3) stress on the endplate adjacent to the 3D-printed prosthesis. Results The fixation strength of Model B was enhanced by the screws in the artificial pedicle, which was mainly manifested as an improvement in rotational stability. The stress transmission of the artificial pedicle fixation screws reduced the stress on the posterior rods and endplate adjacent to the 3D-printed prosthesis in all directions of motion, especially in rotation. Conclusions After TES, the posterior long-segment fixation combined with the anterior 3D printed prosthesis could maintain postoperative spinal stability, but adding artificial pedicle fixation increased the stability of the fixation system and reduced the risk of prosthesis subsidence and instrumentation failure.

Abstract Purpose Papillary renal cell carcinoma (RCC) is the second most common subtype of RCC, after clear cell RCC. This study aimed to investigate the usefulness of FDG PET/CT in primary and recurrent papillary RCC, and the role of staging FDG PET/CT in predicting survival. Methods A total of 66 patients with histopathologically confirmed papillary RCC who underwent either staging or restaging FDG PET/CT scans (30 had staging scans only, 28 had restaging scans only, 8 had both) were retrospectively included in this study. The sensitivity and specificity of restaging FDG PET/CT for detecting recurrence were assessed by histopathology and/or clinical follow-up as standard reference. Results Staging FDG PET/CT scans were performed in 38 patients, of which 31 (81.5%) showed FDG-positive primary renal lesions. The SUVmax of high-grade (WHO grade 3 and 4) papillary RCCs were significantly higher than that of low-grade (WHO grade 1 and 2) tumors (9.44 ± 6.18 vs 4.83 ± 3.19, P = 0.008). The SUVmax was not significantly different between type 1 and type 2 papillary RCCs (5.71 ± 2.88 vs. 6.99 ± 5.57, P = 0.563). Of the 38 patients, 12 developed disease progression during the follow-up period. Patients with primary tumor SUVmax> 5.85 were associated with significantly shorter progression-free survival (PFS) than those with tumor SUVmax≤5.85 (P = 0.005). Restaging FDG PET/CT scans were performed in 36 patients with suspected recurrent papillary RCCs. FDG PET/CT showed a sensitivity and specificity of 100 and 72.7% for detecting recurrent disease. Comparison of PET/CT scans with CT/MRI imaging was available in 23 patients. FDG PET/CT revealed additional findings in 11 patients, mainly including lymph node and bone metastases. FDG PET/CT findings led to change in management in 5.3% (2/38) of patients in the staging setting and 16.7 (6/36) of patients in the restaging setting. Conclusions FDG PET/CT had a sensitivity of 81.5% for detecting primary papillary RCC, and tumor SUVmax derived from staging FDG PET/CT was a predictor of PFS. In the restaging process of papillary RCC, FDG PET/CT was very effective for detecting recurrent disease.

Abstract Background Circular RNA (circRNA) is increasingly attracting attention in gastric cancer (GC). Hsa_circ_0032821 (circ_0032821) has been declared to be upregulated in human GC tissues. However, the biological role of circ_0032821 remains undisclosed in GC cells. Methods Expression of circ_0032821 was measured by real-time quantitative PCR. Cell proliferation, autophagy, Epithelial-mesenchymal transition (EMT), migration, and invasion were evaluated by Cell counting kit-8 assay, western blotting or transwell assays. Expression of proliferating cell nuclear antigen (PCNA), Matrix metalloproteinase 2 (MMP2), MMP9, Light chain 3 (LC3), p62, total and phosphorylated Extracellular signal-regulated kinase 1/2 (ERK1/2) and Mitogen-activated protein kinase’s kinase 1 (MEK1) was evaluated by western blotting. Xenograft tumor model was established to measure tumor growth in vivo. Results Circ_0032821 was significantly upregulated in human GC tumors and cells. Moreover, circ_0032821 might be a biomarker for the advanced Tumor node metastasis (TNM) stage, lymphoid node metastasis and poor prognosis in gastric cancer. Knockdown of circ_0032821 by transfection induced decrease of cell proliferation, EMT, migration and invasion, but increase of autophagy of AGS and HGC-27 cells in vitro, as well as induced tumor growth inhibition in vivo. Besides, overexpression of circ_0032821 by transfection functioned the opposite effects in human GC cells. Mechanically, the MEK1/ERK1/2 signaling pathway was activated when circ_0032821 upregulation, whereas inhibited when circ_0032821 silencing. Conclusion Circ_0032821 expression induced cell proliferation, EMT, migration, invasion, and autophagy inhibition in human GC cells in vitro and in vivo through activating MEK1/ERK1/2 signaling pathway, suggesting circ_0032821 as an oncogenic role in GC.

Abstract Background Biological α-olefins can be used as both biofuels and high value-added chemical precursors to lubricants, polymers, and detergents. The prototypic CYP152 peroxygenase family member OleTJE from Jeotgalicoccus sp. ATCC 8456 catalyzes a single-step decarboxylation of free fatty acids (FFAs) to form α-olefins using H2O2 as a cofactor, thus attracting much attention since its discovery. To improve the productivity of α-olefins, significant efforts on protein engineering, electron donor engineering, and metabolic engineering of OleTJE have been made. However, little success has been achieved in obtaining α-olefin high-producer microorganisms due to multiple reasons such as the tight regulation of FFA biosynthesis, the difficulty of manipulating multi-enzyme metabolic network, and the poor catalytic performance of OleTJE. Results In this study, a novel enzyme cascade was developed for one-pot production of α-olefins from low-cost triacylglycerols (TAGs) and natural oils without exogenous H2O2 addition. This artificial biocatalytic route consists of a lipase (CRL, AOL or Lip2) for TAG hydrolysis to produce glycerol and free fatty acids (FFAs), an alditol oxidase (AldO) for H2O2 generation upon glycerol oxidation, and the P450 fatty acid decarboxylase OleTJE for FFA decarboxylation using H2O2 generated in situ. The multi-enzyme system was systematically optimized leading to the production of α-olefins with the conversion rates ranging from 37.2 to 68.5%. Furthermore, a reaction using lyophilized CRL/OleTJE/AldO enzymes at an optimized ratio (5 U/6 μM/30 μM) gave a promising α-olefin yield of 0.53 g/L from 1500 μM (~1 g/L) coconut oil. Conclusions The one-pot enzyme cascade was successfully established and applied to prepare high value-added α-olefins from low-cost and renewable TAGs/natural oils. This system is independent of exogenous addition of H2O2, thus not only circumventing the detrimental effect of H2O2 on the stability and activity of involved enzymes, but also lower the overall costs on the TAG-to-olefin transformation. It is anticipated that this biotransformation system will become industrially relevant in the future upon more engineering efforts based on this proof-of-concept work.

Abstract Background Elytrigia lolioides (Kar. et Kir.) Nevski, which is a perennial, cross-pollinating wheatgrass that is distributed in Russia and Kazakhstan, is classified into Elytrigia, Elymus, and Lophopyrum genera by taxonomists on the basis of different taxonomic classification systems. However, the genomic constitution of E. lolioides is still unknown. To identify the genome constitution and evolution of E. lolioides, we used single-copy nuclear genes acetyl-CoA carboxylase (Acc1) and elongation factor G (EF-G), multi-copy nuclear gene internal transcribed space (ITS), chloroplast gene trnL-F together with fluorescence and genomic in situ hybridization. Results Despite the widespread homogenization of ITS sequences, two distinct lineages (genera Pseudoroegneria and Hordeum) were identified. Acc1 and EF-G sequences suggested that in addition to Pseudoroegneria and Hordeum, unknown genome was the third potential donor of E. lolioides. Data from chloroplast DNA showed that Pseudoroegneria is the maternal donor of E. lolioides. Data from specific FISH marker for St genome indicated that E. lolioides has two sets of St genomes. Both genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) results confirmed the presence of Hordeum genome in this species. When E genome was used as the probe, no signal was found in 42 chromosomes. The E-like copy of Acc1 sequences was detected in E. lolioides possibly due to the introgression from E genome species. One of the H chromosomes in the accession W6–26586 from Kazakhstan did not hybridize H genome signals but had St genome signals on the pericentromeric regions in the two-color GISH. Conclusions Phylogenetic and in situ hybridization indicated the presence of two sets of Pseudoroegneria and one set of Hordeum genome in E. lolioides. The genome formula of E. lolioides was designed as StStStStHH. E. lolioides may have originated through the hybridization between tetraploid Elymus (StH) and diploid Pseudoroegneria species. E and unknown genomes may participate in the speciation of E. lolioides through introgression. According to the genome classification system, E. lolioides should be transferred into Elymus L. and renamed as Elymus lolioidus (Kar. er Kir.) Meld.

Abstract Background The CYP152 family member OleTJE from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. To gain more insights into the decarboxylation mechanism and reaction bifurcation (decarboxylation versus hydroxylation), we turned to an alternative approach to explore the natural CYP152 resources for a better variety of enzyme candidates. Results We biochemically characterized three new P450 fatty acid decarboxylases including OleTJH, OleTSQ and OleTSA, with respect to their substrate specificity, steady-state kinetics, and salt effects. These enzymes all act as an OleTJE-like fatty acid decarboxylase being able to decarboxylate a range of straight-chain saturated fatty acids (C8–C20) to various degrees. Site-directed mutagenesis analysis to the lower activity P450 enzyme OleTSA revealed a number of key amino acid residues within the substrate-binding pocket (T47F, I177L, V319A and L405I) that are important for delicate substrate positioning of different chain-length fatty acids and thus the decarboxylation versus hydroxylation chemoselectivity, in particular for the mid-chain fatty acids (C8–C12). In addition, the three new decarboxylases exhibited optimal catalytic activity and stability at a salt concentration of 0.5 M, and were thus classified as moderate halophilic enzymes. Conclusion The P450 fatty acid decarboxylases OleTJE, OleTJH, OleTSQ and OleTSA belong to a novel group of moderate halophilic P450 enzymes. OleTJH from Jeotgalicoccus halophilus shows the decarboxylation activity, kinetic parameters, as well as salt tolerance and stability that are comparable to OleTJE. Site-directed mutagenesis of several key amino acid residues near substrate-binding pocket provides important guidance for further engineering of these P450 fatty acid decarboxylases that hold promising application potential for production of α-olefin biohydrocarbons.

Abstract Background Loquat (Eriobotrya japonica) is a subtropical tree bearing fruit that ripens during late spring and early summer, which is the off-season for fruit production. The specific flowering habit of loquat, which starts in fall and ends in winter, has attracted an increasing number of researchers who believe that it may represent an ideal model for studying flowering shift adaptations to climate change in Rosaceae. These studies require an understanding of gene expression patterns within the fruit and other tissues of this plant. Although ACTINs (ACTs) have previously been used as reference genes (RGs) for gene expression studies in loquats, a comprehensive analysis of whether these RGs are optimal for normalizing RT-qPCR data has not been performed. Results In this study, 11 candidate RGs (RIBOSOMAL-LIKE PROTEIN4 (RPL4), RIBOSOMAL-LIKE PROTEIN18 (RPL18), Histone H3.3 (HIS3), Alpha-tubulin-3 (TUA3), S-Adenosyl Methionine Decarboxylase (SAMDC), TIP41-like Family Protein (TIP41), (UDP)-glucose Pyrophosphorylase (UGPase), 18S ribosomal RNA (18S), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Plasma Intrinsic Protein 2 (PIP2) and ACTIN(ACT)) were assessed to determine their expression stability in 23 samples from different tissues or organs of loquat. Integrated expression stability evaluations using five computational statistical methods (GeNorm, NormFinder, ΔCt, BestKeeper, and RefFinder) suggested that a RG set, including RPL4, RPL18, HIS3 and TUA3, was the most stable one across all of the tested loquat samples. The expression pattern of EjCDKB1;2 in the tested loquat tissues normalized to the selected RG set demonstrated its reliability. Conclusions This study reveals the reliable RGs for accurate normalization of gene expression in loquat. In addition, our findings demonstrate an efficient system for identifying the most effective RGs for different organs, which may be applied to related rosaceous crops.

Abstract Background Anaerobic, mesophilic, and cellulolytic Clostridium papyrosolvens produces an efficient cellulolytic extracellular complex named cellulosome that hydrolyzes plant cell wall polysaccharides into simple sugars. Its genome harbors two long cellulosomal clusters: cip-cel operon encoding major cellulosome components (including scaffolding) and xyl-doc gene cluster encoding hemicellulases. Compared with works on cip-cel operon, there are much fewer studies on xyl-doc mainly due to its rare location in cellulolytic clostridia. Sequence analysis of xyl-doc revealed that it harbors a 5′ untranslated region (5′-UTR) which potentially plays a role in the regulation of downstream gene expression. Here, we analyzed the function of 5′-UTR of xyl-doc cluster in C. papyrosolvens in vivo via transformation technology developed in this study. Results In this study, we firstly developed an electrotransformation method for C. papyrosolvens DSM 2782 before the analysis of 5′-UTR of xyl-doc cluster. In the optimized condition, a field with an intensity of 7.5–9.0 kV/cm was applied to a cuvette (0.2 cm gap) containing a mixture of plasmid and late cell suspended in exponential phase to form a 5 ms pulse in a sucrose-containing buffer. Afterwards, the putative promoter and the 5′-UTR of xyl-doc cluster were determined by sequence alignment. It is indicated that xyl-doc possesses a long conservative 5′-UTR with a complex secondary structure encompassing at least two perfect stem-loops which are potential candidates for controlling the transcriptional termination. In the last step, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) as a quantitative reporter to analyze promoter activity and 5′-UTR function in vivo. It revealed that 5′-UTR significantly blocked transcription of downstream genes, but corn stover can relieve its suppression. Conclusions In the present study, our results demonstrated that 5′-UTR of the cellulosomal xyl-doc cluster blocks the transcriptional activity of promoter. However, some substrates, such as corn stover, can relieve the effect of depression of 5′-UTR. Thus, it is speculated that 5′-UTR of xyl-doc was a putative riboswitch to regulate the expression of downstream cellulosomal genes, which is helpful to understand the complex regulation of cellulosome.