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Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.

Acute myeloid leukaemia is a complex, highly aggressive hematopoietic disorder. Currently, in spite of great advances in radiotherapy and chemotherapy, the prognosis for AML patients with initial treatment failure is still poor. Therefore, the need for novel and efficient therapies to improve AML treatment outcome has become desperately urgent. In this study, we identified the expression of ZEB1 (a transcription factor) and focused on its possible role and mechanisms in the progression of AML. According to the data provided by the Gene Expression Profiling Interactive Analysis (GEPIA), high expression of ZEB1 closely correlates with poor prognosis in AML patients. Additionally, the overexpression of ZEB1 was observed in both AML patients and cell lines. Further functional experiments showed that ZEB1 depletion can induce AML differentiation and inhibit AML proliferation in vitro and in vivo. Moreover, ZEB1 expression was negatively correlated with tumour suppressor P53 expression and ZEB1 can directly bind to P53. Our results also revealed that ZEB1 can regulate PTEN/PI3K/AKT signalling pathway. The inhibitory effect of ZEB1 silencing on PTEN/PI3K/AKT signalling pathway could be significantly reversed by P53 small interfering RNA treatment. Overall, the present data indicated that ZEB1 may be a promising therapeutic target for AML treatment or a potential biomarker for diagnosis and prognosis.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating cancers without effective treatments. Amplified in breast cancer 1 (AIB1) is a member of the steroid receptor coactivator family that mediates the transcriptional activities of nuclear receptors. While AIB1 is associated with the initiation and progression of multiple cancers, the mechanism by which AIB1 contributes to PDAC progression remains unknown. In this study, we aimed to explore the role of AIB1 in the progression of PDAC and elucidate the underlying mechanisms. Methods: The clinical significance and mRNA level of AIB1 in PDAC were studied by database analysis. To demonstrate whether AIB1 mediates the malignant features of PDAC cells, namely, proliferation, migration, invasion, we performed real-time PCR and Western blot analysis, established xenograft models and used in vivo metastasis assay. With insights into the mechanism of AIB1, we performed RNA sequencing (Seq), ChIP-Seq, luciferase reporter assays and pull-down assays. Furthermore, we analyzed the relationship between AIB1 expression and its target expression in PDAC cells and patients and explored whether PDAC cells with high AIB1 levels are sensitive to inhibitors of its target. Results: We found that AIB1 was significantly upregulated in PDAC and associated with its malignancy. Silencing AIB1 impaired hedgehog (Hh) activation by reducing the expression of smoothened (SMO), leading to cell cycle arrest and the inhibition of PDAC cell proliferation. In addition, AIB1, via upregulation of integrin αv (ITGAV) expression, promoted extracellular matrix (ECM) signaling, which played an important role in PDAC progression. Further studies showed that AIB1 preferably bound to AP-1 related elements and served as a coactivator for enhancing the transcriptional activity of MafB, which promoted the expression of SMO and ITGAV. PDAC cells with high AIB1 levels were sensitive to Hh signaling inhibitors, suggesting that blocking Hh activation is an effective treatment against PDAC with high AIB1 expression. Conclusions: These findings reveal that AIB1 is a crucial oncogenic regulator associated with PDAC progression via Hh and ECM signaling and suggest potential therapeutic targets for PDAC treatment.

Cancer growth is usually accompanied by metastasis which kills most cancer patients. Here we aim to study the effect of cisplatin at different doses on breast cancer growth and metastasis. Methods: We used cisplatin to treat breast cancer cells, then detected the migration of cells and the changes of epithelial-mesenchymal transition (EMT) markers by migration assay, Western blot, and immunofluorescent staining. Next, we analyzed the changes of RNA expression of genes by RNA-seq and confirmed the binding of activating transcription factor 3 (ATF3) to cytoskeleton related genes by ChIP-seq. Thereafter, we combined cisplatin and paclitaxel in a neoadjuvant setting to treat xenograft mouse models. Furthermore, we analyzed the association of disease prognosis with cytoskeletal genes and ATF3 by clinical data analysis. Results: When administered at a higher dose (6 mg/kg), cisplatin inhibits both cancer growth and metastasis, yet with strong side effects, whereas a lower dose (2 mg/kg) cisplatin blocks cancer metastasis without obvious killing effects. Cisplatin inhibits cancer metastasis through blocking early steps of EMT. It antagonizes transforming growth factor beta (TGFβ) signaling through suppressing transcription of many genes involved in cytoskeleton reorganization and filopodia formation which occur early in EMT and are responsible for cancer metastasis. Mechanistically, TGFβ and fibronectin-1 (FN1) constitute a positive reciprocal regulation loop that is critical for activating TGFβ/SMAD3 signaling, which is repressed by cisplatin induced expression of ATF3. Furthermore, neoadjuvant administration of cisplatin at 2 mg/kg in conjunction with paclitaxel inhibits cancer growth and blocks metastasis without causing obvious side effects by inhibiting colonization of cancer cells in the target organs. Conclusion: Thus, cisplatin prevents breast cancer metastasis through blocking early EMT, and the combination of cisplatin and paclitaxel represents a promising therapy for killing breast cancer and blocking tumor metastasis.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.

MiR-183 is a tumor onco-miR and has been shown by our previous studies to be overexpressed in esophageal squamous cell carcinomas (ESCCs). In this study, we sought to determine the possible mechanisms of miR-183 in ESCC. In our study, cell migration and invasion, real-time PCR, Western blot, and chromatin immunoprecipitation (ChIP) assays were used to explore the mechanism of miR-183 in three ESCC cell lines. We found several potential transcription factors, including c-Jun, by bioinformatics methods. Using a ChIP assay, we identified that c-Jun binds to the promoter region of pre-miR-183 and that upregulated c-Jun expression is related to increased expression of miR-183. We found that downregulation of miR-183 significantly reduced the cell invasiveness and migration of ESCC cells, whereas upregulation of miR-183 via a mimic increased the cell migration and invasion of ESCC cells. We further discovered one direct miR-183 target gene, Smad4, which has been implicated in invasion and metastasis. Furthermore, miR-183 promoted epithelial-mesenchymal transition (EMT), which is involved in the invasion and migration of ESCC cells. Dysregulation of miR-183 has an important role in tumor growth and invasion because miR-183 targets Smad4. Therefore, suppression of miR-183 may provide a potential approach for treatment.

Poly(ionic liquid)s (PILs) such as polymeric quaternary ammonium compounds (PQACs) are among the most widely used disinfectant agents due to their broad-spectrum antimicrobial activity. However, the high cytotoxicity and difficult reclamation of PQACs remain as challenges for practical water treatment. Herein, we present the synthesis of sugar-incorporated PQACs, which could efficiently decrease the cytotoxicity toward mammalian cells (mouse fibroblast cells, L929). On the basis of the mussel-inspired surface chemistry, the functional glycopolymers could then be immobilized onto the surface of magnetic iron oxide nanoparticles, which are successfully used for the trapping and killing of bacteria. The sugar-containing PILs have combined the lectin-recognition behavior of glycopolymers and the antibacterial activity of PQACs. Antibacterial experiments demonstrate the high-performance of cationic hybrid nanocomposites in killing pathogenic

Auracyanin (Ac) is a blue copper protein that mediates the electron transfer between Alternative Complex III (ACIII) and downstream electron acceptors in both fort chains of filamentous anoxygenic phototrophs. Here, we extracted and purified the air-oxidized RfxAc from the photoheterotrophically grown Roseiflexus castenholzii, and we illustrated the structural basis underlying its electron transferring features. Spectroscopic and enzymatic analyses demonstrated the reduction of air-oxidized RfxAc by the ACIII upon oxidation of menaquinol-4 and menaquinol-7. Crystal structures of the air-oxidized and Na-dithionite-reduced RfxAc at 2.2 and 2.0 A resolutions, respectively, showed that the copper ions are coordinated by His77, His146, Cys141, and Met151 in minor different geometries. The Cu1–Sδ bond length increase of Met151, and the electron density Fourier differences at Cu1 and His77 demonstrated their essential roles in the dithionite-induced reduction. Structural comparisons further revealed that the RfxAc contains a Chloroflexus aurantiacus Ac-A-like copper binding pocket and a hydrophobic patch surrounding the exposed edge of His146 imidazole, as well as an Ac-B-like Ser- and Thr-rich polar patch located at a different site on the surface. These spectroscopic and structural features allow RfxAc to mediate electron transfers between the ACIII and redox partners different from those of Ac-A and Ac-B. These results provide a structural basis for further investigating the electron transfer and energy transformation mechanism of bacterial photosynthesis, and the diversity and evolution of electron transport chains.

Nondegradable PEGylated protein drugs are known to cause accumulation in the tissue and accelerated blood clearance effect, which inspire people to develop alternative polymers such as polyesters for bioconjugation. However, the hydrophobicity and slow degradation rate of polyesters remain as challenges for typical therapeutic applications. Here, we report the facile synthesis of biodegradable protein–poly(e-caprolactone) conjugates via enzymatic ring opening polymerization (eROP). Candida antarctica lipase B (CALB)–poly(N-hydroxyethyl acrylamide) conjugates with pendent hydroxyl groups were utilized as initiators and catalysts simultaneously for the eROP of e-caprolactone to form amphiphilic graft copolymers. The enzymatic degradation of ester-containing polymers in the conjugates could be significantly accelerated by lipase, leading to total degradation in several days.

Purpose To analyze the clinical characteristics of Pneumocystis pneumonia (PCP) in renal transplant recipients, identify early sensitivity indicators, and optimize clinical strategies. Patients and methods We retrospectively analyzed clinical data for 24 patients with confirmed PCP who underwent renal transplantation (RT) between 2010 and 2019, encompassing a mean follow-up of 29 (range, 11-49) d. Results A 71% incidence was observed for PCP during the first 6 months after RT. Progressive dyspnea (79%) was the most common symptom, followed by fever (75%) and dry cough (67%). In the initial phase of PCP, the most frequent computerized tomography (CT) finding was the presence of symmetric, apically distributed ground-glass opacities. Nine of 11 patients (82%) were diagnosed by induced sputum testing, 14 of 17 (82%) by bronchoalveolar lavage, and 1 of 24 (4%) by sputum smear. The 1,3-β-D-glucan level was elevated (mean, 259.16 ± 392.34 pg/mL) in 80% of patients, while 75% had elevated C-reactive protein levels (median, 37.85 mg/L). Two of 18 patients (11%) were positive for cytomegalovirus. All patients were treated with trimethoprim-sulfamethoxazole (3 doses of 1-6 g/kg) and third-generation cephalosporin or moxifloxacin monotherapy to prevent bacterial infection. The methylprednisolone dose (40-400 mg/d) varied according to illness. Most patients were treated using a nasal cannula or oxygen mask, and 2 by mechanical ventilation. CT showed improved lesions after treatment, and completely absorbed lesions or residual fibrosis at follow-up. The mean hospitalization cost was 14,644.73 ± 11,101.59 RMB. Conclusion Peak PCP incidence occurred during the first 6 months after surgery. Progressive dyspnea, fever, and dry cough are important indicators for PCP. Bilateral and diffuse ground-glass opacities involving both lung apexes are often the first indication for PCP diagnosis. Induced sputum testing may be the method-of-choice for pathogen detection. The cure rate can be improved through early antipathogen, glucocorticoid, and preventive anti-infection therapies, as well as respiratory support.

Fibroblast growth factor receptor 2 (FGFR2) is a membrane‐spanning tyrosine kinase that mediates FGF signaling. Various FGFR2 alterations are detected in breast cancer, yet it remains unclear if activation of FGFR2 signaling initiates tumor formation. In an attempt to answer this question, a mouse model berrying an activation mutation of FGFR2 (FGFR2‐S252W) in the mammary gland is generated. It is found that FGF/FGFR2 signaling drives the development of triple‐negative breast cancer accompanied by epithelial‐mesenchymal transition that is regulated by FGFR2‐STAT3 signaling. It is demonstrated that FGFR2 suppresses BRCA1 via the ERK‐YY1 axis and promotes tumor progression. BRCA1 knockout in the mammary gland of the FGFR2‐S252W mice significantly accelerated tumorigenesis. It is also shown that FGFR2 positively regulates PD‐L1 and that a combination of FGFR2 inhibition and immune checkpoint blockade kills cancer cells. These data suggest that the mouse models mimic human breast cancers and can be used to identify actionable therapeutic targets., In this study, it is demonstrated that activation of FGFR2 promotes triple‐negative breast cancer formation through regulating expression of BRCA1 negatively and PD‐L1 positively. Inhibition of FGFR2, which is widely expressed in human breast cancers, can be combined preclinically with immune checkpoint antibodies to enhance anticancer immunotherapy, warranting clinical evaluation of personalized targeted therapy.

Cable bacteria occur in many natural environments, and their electrogenic sulfide oxidation (e-SOx) may influence sediment biogeochemistry. The environmental factors determining the growth and diversity of cable bacteria are poorly known, especially in freshwater sediments. We conducted a laboratory incubation experiment, using freshwater sediments with different sulfide supply levels, to study how sulfide availability in sediment affects the metabolic activity and population dynamics of cable bacteria. A moderate increase in the sulfide availability in sediment significantly promoted metabolic activity and the proliferation of the cable bacteria population, as revealed by enhanced e-SOx intensity and increased bacteria abundance. In high-sulfide treatments there was a more significant increase in the population of cable bacteria in the deeper sediment layers, indicating that increased sulfide availability may expand the vertical scale impact of cable bacteria activities on sediment biogeochemistry. The relative proportions of co-existing species in the cable bacteria population also changed with sulfide supply levels, indicating that sulfide availability can be involved in determining the interspecies relationships of cable bacteria. Our findings provide new insight into the relationship between sediment sulfide availability and the growth, depth distribution, and species composition of cable bacteria, implying the consideration of regulating environmental sulfide availability as a potential management practice for the development of cable bacteria–based environmental biotechnologies.

Sirtuin-1 (SIRT1) is involved in various metabolic pathways, including fatty acid synthesis and gluconeogenesis in the liver. However, its role in initiation and progression of liver cancer remains unclear. Studying Sirt1 liver-specific knockout (LKO) mice in combination with diethylnitrosamine (DEN) treatment, we demonstrated that loss of Sirt1 rendered mice resistant to DEN-induced hepatocellular carcinoma (HCC) development. RNA-seq revealed that livers from LKO mice exhibited an enrichment in glutathione metabolism eight months after DEN challenge. Sirt1 deficiency elevated the expression of glutathione-s-transferase family genes by increasing the level of Nrf2, a key regulator of glutathione metabolism. Hence, LKO livers displayed a reductive environment with an increased ratio of GSH to GSSG and an elevated GSH level. Furthermore, using CRISPR knockout techniques, we confirmed that the impairment of HCC formation in LKO mice is mainly dependent on NRF2 signaling. Meanwhile, HCC induced by DEN could be blocked by the administration of N-acetyl cysteine (NAC) when administered one month after DEN challenge. However, NAC treatment starting five months after DEN injection was not able to prevent tumor development. In conclusion, our findings indicate that a reductive environment orchestrated by glutathione metabolism at an early stage can prevent the initiation of HCC.

Background: BRCA1 plays critical roles in mammary gland development and mammary tumorigenesis. And loss of BRCA1 induces mammary tumors in a stochastic manner. These tumors present great heterogeneity at both intertumor and intratumor levels. Methods: To comprehensively elucidate the heterogeneity of BRCA1 deficient mammary tumors and the underlying mechanisms for tumor initiation and progression, we conducted bulk and single cell RNA sequencing (scRNA-seq) on both mammary gland cells and mammary tumor cells isolated from Brca1 knockout mice. Results: We found the BRCA1 deficient tumors could be classified into four subtypes with distinct molecular features and different sensitivities to anti-cancer drugs at the intertumor level. Whereas within the tumors, heterogeneous subgroups were classified mainly due to the different activities of cell proliferation, DNA damage response/repair and epithelial-to-mesenchymal transition (EMT). Besides, we reconstructed the BRCA1 related mammary tumorigenesis to uncover the transcriptomes alterations during this process via pseudo-temporal analysis of the scRNA-seq data. Furthermore, from candidate markers for BRCA1 mutant tumors, we discovered and validated one oncogene Mrc2, whose loss could reduce mammary tumor growth in vitro and in vivo. Conclusion: Our study provides a useful resource for better understanding of mammary tumorigenesis induced by BRCA1 deficiency.

Cartilage-hair hypoplasia (CHH) is an autosomal recessive metaphyseal chondrodysplasia characterized by bone dysplasia and many other highly variable features. The gene responsible for CHH is the RNA component of the mitochondrial RNA-processing endoribonuclease (RMRP) gene. Currently, the pathogenesis of osteochondrodysplasia and extraskeletal manifestations in CHH patients remains incompletely understood; in addition, there are no viable animal models for CHH. We generated an rmrp KO zebrafish model to study the developmental mechanisms of CHH. We found that rmrp is required for the patterning and shaping of pharyngeal arches. Rmrp mutation inhibits the intramembranous ossification of skull bones and promotes vertebrae ossification. The abnormalities of endochondral bone ossification are variable, depending on the degree of dysregulated chondrogenesis. Moreover, rmrp mutation inhibits cell proliferation and promotes apoptosis through dysregulating the expressions of cell-cycle- and apoptosis-related genes. We also demonstrate that rmrp mutation upregulates canonical Wnt/β-catenin signaling; the pharmacological inhibition of Wnt/β-catenin could partially alleviate the chondrodysplasia and increased vertebrae mineralization in rmrp mutants. Our study, by establishing a novel zebrafish model for CHH, partially reveals the underlying mechanism of CHH, hence deepening our understanding of the role of rmrp in skeleton development.

Introduction Acute pulmonary embolism (APE) is a cardiovascular disease with high morbidity and mortality. Although the anatomical obstruction of the pulmonary vascular bed initiates APE, recent studies have suggested that vasoconstrictors in the renin-angiotensin system (RAS) play a role in the severity of APE. Materials and methods We performed a 5-year retrospective clinical study to analyze the key RAS components in APE patients, including angiotensin converting enzyme (ACE), ACE2, angiotensin II (Ang II) and angiotensin 1-7(Ang(1-7)). The role of RhoA-Rho associated kinase (ROCK) signaling in regulating RAS vasoconstrictors was detected in rat pulmonary artery endothelial cells and in an APE rat model. Results In clinical study, we found that the levels of RAS vasoconstrictors were correlated with the clinical classification of APE patients, ACE and Ang II were unregulated, whereas ACE2 and Ang(1-7) were downregulated in the high-risk group compared to the healthy volunteers. In animal study, we found that activated RhoA-ROCK signaling was responsible for the imbalance in RAS vasoconstrictors both in vitro and in vivo, and further evidence indicated that ROCK inhibitors (Y27632 or HA1077) and an ACE2 activator (Resorcinol naphthalein) restored the dysregulated RAS vasoconstrictors significantly and had a protective role in an APE rat model. Conclusions Our study revealed that RhoA-ROCK signaling leads to RAS imbalance in APE patients, and ACE2 activation might be a novel therapeutic target in APE treatment.

The mammary gland undergoes fast cell proliferation during early pregnancy, yet the mechanism to ensure genome integrity during this highly proliferative stage is largely unknown. We show that pregnancy triggers replicative stresses leading to genetic instability in mice carrying a mammary specific disruption of breast cancer associated gene-1 (BRCA1). The fast cell proliferation was correlated with enhanced expression of most genes encoding replisomes, which are positively regulated by estrogen/ERα signaling but negatively regulated by BRCA1. Our further analysis revealed two parallel signaling pathways, which are mediated by ATR-CHK1 and WEE1-MCM2 and are responsible for regulating DNA replication checkpoint. Upon DNA damage, BRCA1 deficiency markedly enhances DNA replication initiation and preferably impairs DNA replication checkpoint mediated by ATR and CHK1. Meanwhile, DNA damage also activates WEE1-MCM2 signaling, which inhibits DNA replication initiation and enables BRCA1-deficient cells to avoid further genomic instability. Finally, we demonstrated that overriding this defense by WEE1 inhibition in combination with cisplatin, which causes DNA damage, serves as a promising therapeutic approach for killing BRCA1-deficient cancer cells.

Background Metagenomic next-generation sequencing (mNGS) was suggested to potentially replace traditional microbiological methodology because of its comprehensiveness. However, clinical experience with application of the test is relatively limited. Methods From April 2017 to December 2017, 511 specimens were collected, and their retrospective diagnoses were classified into infectious disease (347 [67.9%]), noninfectious disease (119 [23.3%]), and unknown cases (45 [8.8%]). The diagnostic performance of pathogens was compared between mNGS and culture. The effect of antibiotic exposure on detection rate was also assessed. Results The sensitivity and specificity of mNGS for diagnosing infectious disease were 50.7% and 85.7%, respectively, and these values outperformed those of culture, especially for Mycobacterium tuberculosis (odds ratio [OR], 4 [95% confidence interval {CI}, 1.7-10.8]; P < .01), viruses (mNGS only; P < .01), anaerobes (OR, ∞ [95% CI, 1.71-∞]; P < .01) and fungi (OR, 4.0 [95% CI, 1.6-10.3]; P < .01). Importantly, for mNGS-positive cases where the conventional method was inconclusive, 43 (61%) cases led to diagnosis modification, and 41 (58%) cases were not covered by empirical antibiotics. For cases where viruses were identified, broad-spectrum antibiotics were commonly administered (14/27), and 10 of 27 of these cases were suspected to be inappropriate. Interestingly, the sensitivity of mNGS was superior to that of culture (52.5% vs 34.2%; P < .01) in cases with, but not without, antibiotic exposure. Conclusions mNGS could yield a higher sensitivity for pathogen identification and is less affected by prior antibiotic exposure, thereby emerging as a promising technology for detecting infectious diseases.

Pulmonary clear cell sarcoma is a rare malignant tumor that has rarely been reported and is challenging to diagnose, especially when differentiating from malignant melanoma. Currently, EWSR1-ATF1 is the key marker for distinguishing clear cell sarcoma from melanoma, but IHC has diagnostic limitations. We report a patient diagnosed with pulmonary clear cell sarcoma, in which an NGS was used to help with the pathological diagnosis. The exposure to the immune microenvironment in pulmonary clear cell sarcoma suggests that TIGIT-related drugs may be a new and effective treatment for this rare disease. Immune microenvironment-related markers, including PD-L1, CD8, TIM3, LAG3, and CD163, were negatively expressed in pulmonary clear cell sarcoma.

As an active glycoprotein with high nutritional value, lactoferrin is widely used in food and medical treatment. Therefore, it is very important to establish an accurate and efficient detection method for lactoferrin. At present, the detection of lactoferrin in milk faces many challenges, such as low separation degree and poor parallelism. To address this issue, we developed an aptamer affinity column (AAC) for purification and enrichment of lactoferrin in milk. The column was prepared by covalent conjugation of an amino-modified aptamer with NHS-activated Sepharose. The washing buffer type (0.01 mol/L phosphate buffer) and volume (10 mL) and the sodium chloride concentration (1 mol/L) in the elution buffer were optimized for the AAC method. The performance of the AAC was then evaluated in terms of the column capacity, specificity, stability, and reusability. The column capacity was 500 ± 13.7 μg and the column could be reused up to ten times with a large loss in performance. The AAC method combined with high-performance liquid chromatography gave excellent linearity over a wide range, good sensitivity with a limit of detection of 3 μg/mL, and acceptable recoveries for different concentrations of lactoferrin spiked in real raw milk samples from cattle. Finally, the AAC was successfully applied to analyze lactoferrin in milk. This method could be applied to routine analysis of samples for lactoferrin in testing laboratories and dairy factories.

Lung adenocarcinoma (LUAD) is one of the malignant lung tumors. However, its pathology has not been fully understood. The purpose of this study is to identify the hub genes associated with LUAD by bioinformatics methods. Three gene expression datasets including GSE116959, GSE74706, and GSE85841 downloaded from the Gene Expression Omnibus (GEO) database were used in this study. The differentially expressed genes (DEGs) related to LUAD were screened by using the limma package. Gene Ontology (GO) and KEGG analysis of DEGs were carried out through the DAVID website. The protein-protein interaction (PPI) of differentially expressed genes was drawn by the STRING website, and the results were imported into Cytoscape for visualization. Then, the PPI network was analyzed by using MCODE, and the modules with a score greater than 5 were found by using cytoHubba. Finally, the GEPIA database and UALCAN database were used to verify and analyze the survival of hub genes. We identified 67 upregulated genes and 277 downregulated genes from three LUAD datasets. The results of GO analysis showed that the downregulated genes were significantly enriched in matrix adhesion and angiogenesis and upregulated differential genes were significantly enriched in cell adhesion and vascular development. KEGG pathway analysis showed that the differential genes of LUAD were significantly enriched in viral carcinogenesis and adhesion spots. The PPI network of differentially expressed genes consists of 269 nodes and 625 interactions. In addition, three modules with scores greater than 5 and seven hub genes, namely, MCM4, BIRC5, CDC20, CDC25C, FOXM1, GTSE1, and RFC4, playing an important role in the PPI network were screened out. In this study, we obtained the hub genes and pathways related to LUAD, revealing the molecular mechanism and pathogenesis of LUAD, which is helpful for the early detection of LUAD and provides a new idea for the treatment of LUAD.

Various nanostructured surfaces have been developed recently to physically inactivate bacteria, for reducing the rapidly spreading threat of pathogenic bacteria. However, it generally takes several hours for these surfaces to inactivate most of the bacteria, which greatly limits their application in the fields favoring rapid bactericidal performance. Besides, the accumulated bacteria debris left on these surfaces is rarely discussed in the previous reports. Herein we report the nanotip-engineered ZnO nanoarrays (NAs) with ultrafast physical bactericidal rate and the ability to photocatalytically remove the bacteria debris. Neither chemical (Zn2+ or reactive oxygen species) nor photocatalytic effect leads to the ultrafast bactericidal rate, where 97.5% of E. coli and 94.9% of S. aureus are inactivated within only 1 min. The simulation analysis further supported our proposed mechanism attributing the ultrafast bactericidal activity to the great stress enabled by the uneven topography. Moreover, the re-exposure of the ZnO NAs nanotips can be achieved in only 10 min under a mild UV light source. This study not only presents an ultrafast physical bactericidal activity, but also demonstrates the potential of the recyclable and photocatalytic self-cleaning functions of theses surfaces for applications that desire rapid and sustainable bactericidal performance., Graphical abstract Unlabelled Image, Highlights • The ZnO nanoarrays exhibit an ultra-fast physico-bactericidal activity within 1 min. • The ultra-fast bactericidal mechanism is attributed to the greater stress enabled by the sharp tips and uneven topography. • The ZnO nanoarray tips can be re-exposed under a mild UV light source, and the surface has sustainable bactericidal properties.

Single-cell whole-exome sequencing (scWES) is a powerful approach for deciphering intratumor heterogeneity and identifying cancer drivers. So far, however, simultaneous analysis of single nucleotide variants (SNVs) and copy number variations (CNVs) of a single cell has been challenging. By analyzing SNVs and CNVs simultaneously in bulk and single cells of premalignant tissues and tumors from mouse and human BRCA1-associated breast cancers, we discover an evolution process through which the tumors initiate from cells with SNVs affecting driver genes in the premalignant stage and malignantly progress later via CNVs acquired in chromosome regions with cancer driver genes. These events occur randomly and hit many putative cancer drivers besides p53 to generate unique genetic and pathological features for each tumor. Upon this, we finally identify a tumor metastasis suppressor Plekha5, whose deficiency promotes cancer metastasis to the liver and/or lung., Single-cell whole-exome sequencing is a powerful method for deciphering intratumor heterogeneity and identifying cancer drivers. Here, the authors analyze SNVs and CNVs simultaneously in bulk and single cells of BRCA1-associated breast tissues and cancers and show that Plekha5 deficiency promotes metastasis.

Cable bacteria are filamentous sulfur-oxidizing microorganisms that couple the reduction of oxygen or nitrate in surface sediments with the oxidation of free sulfide in deeper sediments by transferring electrons across centimeter scale distances. The distribution and activities of cable bacteria in freshwater sediments are still poorly understood, especially the impact of cable bacteria on sulfur cycling. The goal of this study was to investigate electrogenic sulfide oxidation associated with cable bacteria in laboratory microcosm incubations of freshwater sediments using microsensor technology, 16S full-length rRNA sequencing, and fluorescence in situ hybridization (FISH) microscopy. Their activity was characterized by a pH maximum of 8.56 in the oxic zone and the formation of a 13.7 ± 0.6 mm wide suboxic zone after 25 days of incubation. Full-length 16S rRNA gene sequences related to cable bacteria were recovered from the sediments and exhibited 93.3%–99.4% nucleotide (nt) similarities with those from other reported freshwater cable bacteria, indicating that new species of cable bacteria were present in the sediments. FISH analysis indicated that cable bacteria density increased with time, reaching a maximum of 95.48 m cm−2 on day 50. The cells grew downwards to 40 mm but were mainly concentrated on the top 0–20 mm of sediment. The cable bacteria continuously consumed H2S in deeper layers and oxidized sulfide into sulfate in the 0–20 mm surface layers, thereby affecting the sulfur cycling within sediments. These findings provide new evidence for the existence of higher diversity of cable bacteria in freshwater sediments than previously known.

Purpose: This systematic review and meta-analysis was designed to evaluate the effects of acupuncture intervention on alleviating delayed onset of muscle soreness (DOMS) after intense exercise.Method: Randomized controlled trials (RCTs) were searched from online databases including Medline (PubMed), Cochrane Library, Web of Science, Embase, PsycINFO, China Knowledge Resource Integrated Database (CNKI), and Wanfang (Chinese) up to April 2019. Data points were extracted from the eligible RCTs at the time points of 24, 48, and 72 h post strenuous exercise-induced DOMS. The outcomes of muscle soreness rating (MSR), creatine kinase (CK), and maximal isometric force (MIF) were pooled into the meta-analysis to assess the acupuncture intervention on DOMS.Results: Six eligible RCTs were included in the meta-analysis, and the results showed that acupuncture intervention significantly decreased MSR [standardized mean difference (SMD) −0.49, 95%CI −0.73 to −0.24, P < 0.001, I2 = 34%] and the serum level of CK (SMD −0.91, 95%CI −1.27 to −0.56, P < 0.001, I2 = 30%), accompanied with the improvement of the muscle strength (MIF) (SMD 0.54, 95%CI 0.16 to 0.93, P = 0.006, I2 = 51%) after intense exercise. At the same time, the findings also revealed that acupuncture intervention had a long-lasting effect and tended to accumulate the effect size and that it had the most efficacy on alleviating DOMS at the time point of 72 h post exercise.Conclusion: The current evidence indicates that acupuncture intervention after intense exercise could be effective for alleviating DOMS and improving muscle recovery. The long-lasting effect of acupuncture intervention on DOMS started from 24 h and would reach a peak on the time point of 72 h post exercise.

For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several in vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of "cleared" recipients and mammary cells for implantation, the surgery process and how to assess the experimental results. Combined with manipulation of mammary cells via gene editing and /or drug treatment, this protocol could be very useful in the researches of mammary stem cells and mammary development.

Significance In patients with coronavirus disease 2019, a large number of T lymphocytes and mononuclear macrophages are activated, producing cytokines such as interleukin-6 (IL-6), which bind to the IL-6 receptor on the target cells, causing the cytokine storm and severe inflammatory responses in lungs and other tissues and organs. Tocilizumab, as a recombinant humanized anti-human IL-6 receptor monoclonal antibody, can bind to the IL-6 receptor with high affinity, thus preventing IL-6 itself from binding to its receptor, rendering it incapable of immune damage to target cells, and alleviating the inflammatory responses., After analyzing the immune characteristics of patients with severe coronavirus disease 2019 (COVID-19), we have identified that pathogenic T cells and inflammatory monocytes with large amount of interleukin 6 secreting may incite the inflammatory storm, which may potentially be curbed through monoclonal antibody that targets the IL-6 pathways. Here, we aimed to assess the efficacy of tocilizumab in severe patients with COVID-19 and seek a therapeutic strategy. The patients diagnosed as severe or critical COVID-19 in The First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) and Anhui Fuyang Second People’s Hospital were given tocilizumab in addition to routine therapy between 5 and 14 February 2020. The changes of clinical manifestations, computerized tomography (CT) scan image, and laboratory examinations were retrospectively analyzed. Fever returned to normal on the first day, and other symptoms improved remarkably within a few days. Within 5 d after tocilizumab, 15 of the 20 patients (75.0%) had lowered their oxygen intake, and 1 patient needed no oxygen therapy. CT scans manifested that the lung lesion opacity absorbed in 19 patients (90.5%). The percentage of lymphocytes in peripheral blood, which decreased in 85.0% of patients (17/20) before treatment (mean, 15.52 ± 8.89%), returned to normal in 52.6% of patients (10/19) on the fifth day after treatment. Abnormally elevated C-reactive protein decreased significantly in 84.2% of patients (16/19). No obvious adverse reactions were observed. All patients have been discharged on average 15.1 d after giving tocilizumab. Preliminary data show that tocilizumab, which improved the clinical outcome immediately in severe and critical COVID-19 patients, is an effective treatment to reduce mortality.

A severe pneumonia-associated respiratory syndrome caused by a new coronavirus was identified in December 2019 (COVID-19), spread rapidly and has become a world-wide public health challenge. About 25% of COVID-19 patients experienced severe complications including acute respiratory distress syndrome (ARDS), and even progressed into an intensive care unit (ICU) admission and died. The exploration for the mortality causes and advancing novel therapeutic development of severe COVID-19 is crucial at the moment. The biopsy samples analysis at autopsy suggested that increased alveolar exudate caused by aberrant host immune response and inflammatory cytokine storm probably impedes alveolar gas exchange and contributes to the high mortality of severe COVID-19 patients. Our research has identified that pathogenic T cells and inflammatory monocytes incite inflammatory storm with large amount of interleukin 6, therefore monoclonal antibody that targets the IL-6 pathways may potentially curb inflammatory storm. Moreover, Tocilizumab treatment that blocking IL-6 receptors showed inspiring clinical results including temperature returned to normal quickly and respiratory function improved. Therefore, we suggest that Tocilizumab is an effective treatment in severe patients of COVID-19 to calm the inflammatory storm and reduce mortality.

Abundant evidence has illustrated that long non-coding RNA (lncRNA) plays a vital role in the regulation of tumor development and progression. Most lncRNAs have been proven to have biological and clinical significance in acute myeloid leukemia (AML), but further investigation remains necessary. In this study, we investigated lncRNA NR-104098 in AML and its specific mechanism. The microarray analysis was performed on NB4 cells. Based on the related analysis results, we identified that lncRNA NR-104098 is a suppressor gene that is significantly upregulated in AML cells. LncRNA NR-104098 could inhibit proliferation and induce differentiation in AML cells in vitro and also play main role in the mouse xenografts. Mechanically, it was confirmed that lncRNA NR-104098 may effectively inhibit EZH2 transcription by directly binding to E2F1 and recruiting E2F1 to the EZH2 promoter. In addition, ATPR can significantly increase the expression of lncRNA NR-104098, whereas knocking down NR104098 can inhibit the inhibitory effect of ATPR on the proliferation and induction differentiation of AML cells. Taken together, these results lead to deeper insight into the mechanism of ATPR-induced AML differentiation and prevent proliferation by inhibiting EZH2 on the transcriptional level.

Photosynthetic prokaryotes evolved diverse light-harvesting (LH) antennas to absorb sunlight and transfer energy to reaction centers (RC). The filamentous anoxygenic phototrophs (FAPs) are important early branching photosynthetic bacteria in understanding the origin and evolution of photosynthesis. How their photosynthetic machinery assembles for efficient energy transfer is yet to be elucidated. Here, we report the 4.1 Å structure of photosynthetic core complex from Roseiflexus castenholzii by cryo-electron microscopy. The RC–LH complex has a tetra-heme cytochrome c bound RC encompassed by an elliptical LH ring that is assembled from 15 LHαβ subunits. An N-terminal transmembrane helix of cytochrome c inserts into the LH ring, not only yielding a tightly bound cytochrome c for rapid electron transfer, but also opening a slit in the LH ring, which is further flanked by a transmembrane helix from a newly discovered subunit X. These structural features suggest an unusual quinone exchange model of prokaryotic photosynthetic machinery., Filamentous anoxygenic phototrophs (FAPs) are phylogenetically distant from other anoxygenic photosynthetic bacteria. Here the authors present the 4.1 Å cryo-EM structure of the photosynthetic core complex from the FAP Roseiflexus castenholzii and propose a model for energy and electron transfer.

Breast cancer associated gene 1 (BRCA1) function has been shown to be regulated by phosphorylation but the role of acetylation has not been determined. Therefore, we tested whether BRCA1 can be acetylated by the acetyltransferases P300/CBP-associated factor (pCAF), GCN5, and p300. p300 exhibited the highest level of BRCA1 acetylation; however, there was also a decrease in the total level of BRCA1. Therefore, we focused on pCAF and GCN5 because they both acetylated BRCA1 without affecting BRCA1 expression. Further analysis indicated that the acetylated form of BRCA1 is deacetylated by wild-type (WT) SIRT1, but not deacetylase mutant SIRT1, suggesting that SIRT1 is a specific deacetylase of BRCA1. We demonstrated that lysine 830 of BRCA1 is a preferential acetylation site by pCAF and tested its function in embryonic stem (ES) cells by changing lysine 830 to arginine using a transcription activator-like effector nuclease (TALEN) system. After exposure to DNA damage-inducing UV radiation, the viability of BRCA1 K830R mutant cells is greater than the WT ES cells. Further analysis using additional cell lines indicated that the BRCA1 K830R mutation impairs the intra-S checkpoint. Also, checkpoint kinase 1 (CHK1) phosphorylation was less in K830R cells as compared with WT cells after UV exposure. These data suggest that acetylation of BRCA1 on lysine 830 activates BRCA1 function at the intra-S checkpoint after DNA damage.

Checkpoint inhibitors show promising efficacy in advanced lung cancer, especially in non-small-cell lung cancer (NSCLC). This meta-analysis was conducted to explore the therapeutic efficacy and safety of anti-PD-1/PD-L1 antibodies combined with chemotherapy or CTLA4 antibody as first-line treatments for patients with advanced lung cancer. A systematic search was performed in databases for this system review and quantitative meta-analysis. Twelve trials were finally enrolled in the meta-analysis. Our analyses revealed that the combined overall response rate (ORR) and disease control rate (DCR) for immune checkpoint inhibitors combined with chemotherapy for the treatment of NSCLC were 47.0% (95% CI: 34.2%-60.2%) and 80.9% (95% CI: 69.4%-88.7%), respectively. The combined ORR and DCR for CTLA4 antibody combined with chemotherapy for the treatment of small-cell lung cancer (SCLC) were 65.4% (61.1%-69.5%) and 87.6% (84.5%-90.2%), respectively. The combined six-month progression-free survival rates (PFSRs6m ) for NSCLC and SCLC were 50.2% (95% CI: 21.9%-78.4%) and 30.7% (21.2%-40.3%), respectively, and the OSRs1y were 56.4% (39.1%-73.7%) and 36.9% (33.3%-40.5%), respectively. In addition, the combined ORR and DCR for the checkpoint inhibitors plus CTLA4 antibody treatment group in NSCLC were 29.6% (95% CI: 11.4%-57.8%) and 48.7% (16.8%-81.7%), respectively. In subgroup analyses, a significant improvement in PFS was observed in NSCLC and SCLC, with a combined hazard ratio and 95% confidence interval of 0.841 (0.737-0.961) and 0.856 (0.756-0.968), respectively. In summary, synergistic activity and an acceptable safety profile were observed with checkpoint inhibitor plus chemotherapy combination treatment in lung cancer.

It is challenging to regenerate enzyme carriers when covalently immobilized enzymes suffered from inactivation during continuous operations. Hence, it is urgent to develop a facile strategy to immobilize enzymes reversibly. Herein, the non-covalent interaction between protein and carbohydrate was used to adsorb and desorb enzymes reversibly. Laccase was immobilized onto glycopolymer microspheres via protein-carbohydrate interaction using lectins as the intermediates. The enzyme loading and immobilization yield were up to 49 mg/g and 77.1% with highly expressed activity of 107.9 U/mg. The immobilized laccase exhibited enhanced pH stability and high activity in catalyzing the biodegradation of paracetamol. During ten successive recoveries, the immobilized laccases could be recycled while maintaining relatively high enzyme activity. The glycopolymer microspheres could be efficiently regenerated by elution with an aqueous solution of mannose or acid for further enzyme immobilization. This glycopolymer microspheres has excellent potential to act as reusable carriers for the non-covalent immobilization of different enzymes.

Tryptophanyl-tRNA synthetase (TrpRS) in vertebrates contains a N-terminal extension in front of the catalytic core. Proteolytic removal of the N-terminal 93 amino acids gives rise to T2-TrpRS, which has potent anti-angiogenic activity mediated through its extracellular interaction with VE-cadherin. Zinc has been shown to have anti-angiogenic effects and can bind to human TrpRS. However, the connection between zinc and the anti-angiogenic function of TrpRS has not been explored. Here we report that zinc binding can induce structural relaxation in human TrpRS to facilitate the proteolytic generation of a T2-TrpRS-like fragment. The zinc-binding site is likely to be contained within T2-TrpRS, and the zinc-bound conformation of T2-TrpRS is mimicked by mutation H130R. We determined the crystal structure of H130R T2-TrpRS at 2.8 Å resolution, which reveals drastically different conformation from that of wild-type (WT) T2-TrpRS. The conformational change creates larger binding surfaces for VE-cadherin as suggested by molecular dynamic simulations. Surface plasmon resonance analysis indicates more than 50-fold increase in binding affinity of H130R T2-TrpRS for VE-cadherin, compared to WT T2-TrpRS. The enhanced interaction is also confirmed by a cell-based binding analysis. These results suggest that zinc plays an important role in activating TrpRS for angiogenesis regulation.

Breast cancers display striking genetic and phenotypic diversities. To date, several hypotheses are raised to explain and understand the heterogeneity, including theories for cancer stem cell (CSC) and clonal evolution. According to the CSC theory, the most tumorigenic cells, while maintaining themselves through symmetric division, divide asymmetrically to generate non-CSCs with less tumorigenic and metastatic potential, although they can also dedifferentiate back to CSCs. Clonal evolution theory recapitulates that a tumor initially arises from a single cell, which then undergoes clonal expansion to a population of cancer cells. During tumorigenesis and evolution process, cancer cells undergo different degrees of genetic instability and consequently obtain varied genetic aberrations. Yet the heterogeneity in breast cancers is very complex, poorly understood and subjected to further investigation. In recent years, single cell sequencing (SCS) technology developed rapidly, providing a powerful new way to better understand the heterogeneity, which may lay foundations to some new strategies for breast cancer therapies. In this review, we will summarize development of SCS technologies and recent advances of SCS in breast cancer.

Summary Preadipocytes initiate differentiation into adipocytes through a cascade of events. Mitotic clonal expansion, as one of the earliest events, is essential for adipogenesis. However, the underlying mechanisms that regulate mitotic clonal expansion remain elusive. SIRT6 is a member of the evolutionarily conserved sirtuin family of nicotinamide adenine dinucleotide (NAD)+-dependent protein deacetylases. Here, we show that SIRT6 deficiency in preadipocytes blocks their adipogenesis. Analysis of gene expression during adipogenesis reveals that KIF5C, which belongs to the kinesin family, is negatively regulated by SIRT6. Furthermore, we show that KIF5C is a negative factor for adipogenesis through interacting with CK2α′, a catalytic subunit of CK2. This interaction blocks CK2α′ nuclear translocation and CK2 kinase activity and inhibits mitotic clonal expansion during adipogenesis. These findings reveal a crucial role of SIRT6 in adipogenesis and provide potential therapeutic targets for obesity.

Malyl-coenzyme A lyase (MCL) is a carbon–carbon bond lyase that catalyzes the reversible cleavage of coenzyme A (CoA) thioesters in multiple carbon metabolic pathways. This enzyme contains a CitE-like TIM barrel and an additional C-terminal domain that undergoes conformational changes upon substrate binding. However, the structural basis underlying these conformational changes is elusive. Here, we report the crystal structure of MCL from the thermophilic photosynthetic bacterium Roseiflexus castenholzii (RfxMCL) in the apo- and oxalate-bound forms at resolutions of 2.50 and 2.65 A, respectively. Molecular dynamics simulations and structural comparisons with MCLs from other species reveal the deflection of the C-terminal domain to close the adjacent active site pocket in the trimer and contribute active site residues for CoA coordination. The deflection angles of the C-terminal domain are not only related to the occupation but also the type of bound substrates in the adjacent active site pocket. Our work illustrates that a conformational switch of the C-terminal domain accompanies the substrate-binding of MCLs. The results provide a framework for further investigating the reaction mechanism and multifunctionality of MCLs in different carbon metabolic pathways.

Tolrestat and epalrestat have been characterized as noncompetitive inhibitors of aldo-ketone reductase 1B1 (AKR1B1), a leading drug target for the treatment of type 2 diabetes complications. However, clinical applications are limited for most AKR1B1 inhibitors due to adverse effects of cross-inhibition with other AKRs. Here, we report an atypical competitive binding and inhibitory effect of tolrestat on the thermostable AKR Tm1743 from Thermotoga maritima. Analysis of the Tm1743 crystal structure in complex with tolrestat alone and epalrestat-NADP+ shows that tolrestat, but not epalrestat, binding triggers dramatic conformational changes in the anionic site and cofactor binding pocket that prevents accommodation of NADP+ . Enzymatic and molecular dynamics simulation analyses further confirm tolrestat as a competitive inhibitor of Tm1743.

CRISPR/Cas9 is a powerful technology widely used for genome editing, with the potential to be used for correcting a wide variety of deleterious disease-causing mutations. However, the technique tends to generate more indels (insertions and deletions) than precise modifications at the target sites, which might not resolve the mutation and could instead exacerbate the initial genetic disruption. We sought to develop an improved protocol for CRISPR/Cas9 that would correct mutations without unintended consequences. As a case study, we focused on achondroplasia, a common genetic form of dwarfism defined by missense mutation in the Fgfr3 gene that results in glycine to arginine substitution at position 374 in mice in fibroblast growth factor receptor 3 (Fgfr3-G374R), which corresponds to G380R in humans. First, we designed a GFP reporter system that can evaluate the cutting efficiency and specificity of single guide RNAs (sgRNAs). Using the sgRNA selected based on our GFP reporter system, we conducted targeted therapy of achondroplasia in mice. We found that we achieved higher frequency of precise correction of the Fgfr3-G374R mutation using Cas9 protein rather than Cas9 mRNA. We further demonstrated that targeting oligos of 100 and 200 nucleotides precisely corrected the mutation at equal efficiency. We showed that our strategy completely suppressed phenotypes of achondroplasia and whole genome sequencing detected no off-target effects. These data indicate that improved protocols can enable the precise CRISPR/Cas9-mediated correction of individual mutations with high fidelity.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis., Author summary Adaptation to the host niche is an essential attribute of pathogens. Here we found that the Zn2Cys6-type transcription factor LeuB cross-regulates branched-chain amino acid biosynthesis, nitrogen metabolism, iron acquisition via siderophores, and proteasome activity in the mold Aspergillus fumigatus. Lack of this regulatory circuit impaired virulence in an insect infection model. Mammals do neither express Zn2Cys6-type transcription factors nor have the capacity to produce branched-chain amino acids or siderophores. Consequently, this regulatory circuit is a paradigm for fungal pathogen-specific adaptation to the host niche.

Angiogenesis is recognized as an important hallmark of cancer. Although telomerase is thought to be involved in tumor angiogenesis, the evidence and underlying mechanism remain elusive. Here, we demonstrate that human telomerase reverse transcriptase (hTERT) activates vascular epithelial growth factor (VEGF) gene expression through interactions with the VEGF promoter and the transcription factor Sp1. hTERT binds to Sp1 in vitro and in vivo and stimulates angiogenesis in a manner dependent on Sp1. Deletion of the mTert gene in the first generation of Tert null mice compromised tumor growth, with reduced VEGF expression. In addition, we show that hTERT expression levels are positively correlated with those of VEGF in human gastric tumor samples. Together, our results demonstrate that hTERT facilitates tumor angiogenesis by up-regulating VEGF expression through direct interactions with the VEGF gene and the Sp1 transcription factor. These results provide novel insights into hTERT function in tumor progression in addition to its role in telomere maintenance.

SIRT1 is the homologue of SIR2 that links to longevity in lower organisms and has a direct or indirect role that positively regulates the metabolic pathway via insulin signaling. RSV, a naturally occurring small polyphenol that activates SIRT1, has been reported to ameliorate the early development of metabolic syndrome of high-fat-diet-fed mice at a low dose (0.005 w/w) in 10 weeks through inhibition of fatty acid synthase and other relevant enzymes. However, what effect of a higher dose of dietary RSV on the long-term high-fat-diet-fed C57BL/6J mice has not been reported in view of regulating SIRT1 and related genes. This study investigated the effect of a higher dose of RSV (0.1 % w/w) on glucose and lipid metabolism in 13-week high-fat-diet-fed C57BL/6J mice as well as on mRNA and protein expression regulations with respect to relevant genes. The data showed that a higher dose of RSV supplementation significantly lowered the concentration of glucose, plasma total cholesterol, total triglyceride concentrations, and hepatic cholesterol in high-fat-diet-fed mice. Relative mRNA expressions of SIRT1, FOXO1, PPARγ, and PGC-1α were significantly up-regulated, and corresponding protein expressions were also enhanced.

Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus , resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (M pro s) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV M pro in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. IMPORTANCE Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (M pro ) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. M pro , conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV M pro . We also solved the structure of FIPV M pro complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV M pro through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible peptidomimetic inhibitor.

The breast cancer susceptibility gene 1 (BRCA1) is a major tumor suppressor gene and is most frequently mutated in hereditary breast cancer. BRCA1 plays a critical role in many biological processes, especially maintaining genomic stability in the nucleus, yet its role in the cytoplasm remains elusive. Here, it is revealed that BRCA1 maintains a healthy mitochondrial network through regulating mitochondrial dynamics, including fission and fusion. BRCA1 deficiency causes dysfunctional mitochondrial dynamics through increased expression of mitofusin1/2. With mitochondrial stress, BRCA1 is recruited to the mitochondrial outer membrane, where it plays an essential role in maintaining a healthy mitochondrial network. Consequently, BRCA1 deficiency impairs stress‐induced mitophagy through blocking ataxia‐telangiectasia mutated (ATM)‐AMP‐activated protein kinase (AMPK)‐Dynamin‐related protein 1 (DRP1)‐mediated mitochondrial fission and triggers NLRP3 inflammasome activation, which creates a tumor‐associated microenvironment, thereby facilitating tumor proliferation and metastasis. It is further shown that inflammasome inhibition can prevent tumor recurrence and metastasis. This study uncovers an important role of BRCA1 in regulating mitophagy and suggests a therapeutic approach for fighting this deadly disease., Breast cancer susceptibility gene 1 (BRCA1) not only negatively regulates mitochondrial fusion but also facilitates stress‐induced fission and mitophagy. BRCA1 deficiency causes oxidative stress and inflammasome activation, which creates a tumor‐associated microenvironment, thereby promoting breast cancer development. This study uncovers the function of BRCA1 in regulating mitophagy and provides a rational approach for treating BRCA1 mutant breast cancer with inflammasome inhibitors.

Lysine 2-hydroxyisobutyrylation is a newly discovered protein acylation and was reported to share acyltransferases and deacylases with the widely studied lysine acetylation. The well-known acetyltransferase Tip60 and histone deacetylases HDAC 2 and HDAC 3 were discovered to be "writer" and "eraser" of this new PTM on histones. However, the acyltransferases and deacylases for nonhistone proteins are still unclear. In this work, lysine 2-hydroxyisobutyrylome on both histones and nonhistone proteins upon SAHA treatment were intensively studied and 8765 lysine 2-hydroxyisobutyrylation sites on 2484 proteins were identified in A549 cells. This is the largest data set of lysine 2-hydroxyisobutyrylome in mammalian cells to date. It was found that lysine 2-hydroxyisobutyrylation participates in varieties of biological functions and processes including ribosome, glycolysis/gluconeogenesis, and transcription. More importantly, it was found that most quantified sites on core histones were up-regulated upon SAHA treatment for all 2-hydroxyisobutyrylation, crotonylation, and acetylation and the fold changes upon SAHA of 2-hydroxyisobutyrylation and crotonylation on nonhistone proteins were highly correlated, while their fold changes have little correlations with acetylation on nonhistone proteins.

Sediment is the one of most important storage of heavy metal. Microbiotas in sediment can be used as the effective indicators of heavy metals. The goal of this study was to understand the bacterial communities responding to heavy metal enrichment in sediments and prioritize some factors that affected significantly to bacterial community. Sediments were sampled from five river-lake systems in the Poyang Lake in dry season, and the bacterial community was analyzed using Illumina high-throughput sequencing. Relationships between sediment environment and the diversity and structure of bacterial communities were determined by correlation analysis and redundancy analysis (RDA). The result indicated that Cd and Sb were identified as the heavy metals of the great risk in sediments. Sediments from five river-lake systems shared 31.83% core operational taxonomic units (OTUs) of bacterial communities. Proteobacteria (33.54% of total sequences) and Actinobacteria (15.04%) were the dominant phyla across all sites. High enrichment of heavy metals (MRI and mCd) resulted in low diversity of bacterial communities (Simpson index). The RDA revealed pH, OC, mCd, and Efs of As, Pb, Cd were major factors related to bacterial community structure changes. The dominant phylum Actinobacteria was regarded as tolerant bacteria, while the dominant phylum Proteobacteria was named as resistant bacteria in sediment with high anthropogenic Cd enrichment.

The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer, arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5(+) cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5(+) cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors.

Arsenic is one of the most widespread global environmental toxicants associated with endemic poisoning. ATP-binding cassette (ABC) proteins are transmembrane channels that transport and dispose of lipids and metabolic products across the plasma membrane. The majority of ABC family members (including ABCB1 and ABCC1) are reported to play a role in the development of arsenic and drug resistance in mammals. Previously, we established a human arsenic-resistant ECV-304 (AsRE) cell line and identified ABCA1 as a novel arsenic resistance gene. In the current study, we further investigated the potential contribution of ABCA1, ABCB1, and ABCC1 to arsenic resistance through measurement of survival rates and arsenic accumulation in AsRE cells with RNA interference. The arsenic resistance capacity of ABCC1 was the strongest among the three genes, while those of ABCA1 and ABCB1 were similar. Double or triple gene knockdown of ABCA1, ABCB1, and ABCC1 via RNA interference led to a decrease significant in arsenic resistance when ABCA1/ABCB1 or ABCB1/ABCC1 were simultaneously silenced. Interestingly, no differences were evident between cells with ABCA1/ABCC1 and ABCC1 only knockdown. Our findings suggest that ABCA1 and ABCB1 proteins display similar arsenic resistance capabilities and possibly coordinate to promote arsenic resistance in AsRE cells.

Summary Objective We previously reported that genetic ablation of (Fibroblast Growth Factors Receptors) FGFR1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice, which suggests that FGFR1 is a potential targeting molecule for osteoarthritis (OA). Here, we identified R1-P1, an inhibitory peptide for FGFR1 and investigated its effect on the pathogenesis of OA in mice induced by destabilization of medial meniscus (DMM). Design Binding ability between R1-P1 and FGFR1 protein was evaluated by enzyme-linked immuno sorbent assay (ELISA) and molecular docking. Alterations in cartilage were evaluated histologically. The expression levels of molecules associated with articular cartilage homeostasis and FGFR1 signaling were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry (IHC). The chondrocyte apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. Results R1-P1 had highly binding affinities to human FGFR1 protein, and efficiently inhibited extracellular signal-regulated kinase (ERK)1/2 pathway in mouse primary chondrocytes. In addition, R1-P1 attenuated the IL-1β induced significant loss of proteoglycan in full-thickness cartilage tissue from human femur head. Moreover, this peptide can significantly restore the IL-1β mediated loss of proteoglycan and type II collagen (Col II) and attenuate the expression of matrix metalloproteinase-13 (MMP13) in mouse primary chondrocytes. Finally, intra-articular injection of R1-P1 remarkably attenuated the loss of proteoglycan and the destruction of articular cartilage and decreased the expressions of extracellular matrix (ECM) degrading enzymes and apoptosis in articular chondrocytes of mice underwent DMM surgery. Conclusions R1-P1, a novel inhibitory peptide for FGFR1, attenuates the degeneration of articular cartilage in adult mice, which is a potential leading molecule for the treatment of OA.

Hydroxy acid dehydrogenases, including l - and d -lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD + as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d -lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn 174 was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.