Genome mining has revealed that Penicillium spp. possess numerous down-regulated or cryptic biosynthetic gene clusters (BGCs). This finding hinted that our investigation of fungal secondary metabolomes is limited. Herein, we report a genetically-modified activation strategy to characterize the spectrum of sesquiterpenoids produced by Penicillium brasilianum CGMCC 3.4402. The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene berA responsible for berkeleyacetals biosynthesis from Neosartorya glabra. Chemical analysis of the extracts from the mutant strain Pb-OE::berA enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C (1), one daucane-type arpenicarotane C (4), along with four known sesquiterpenoids including arpenibisabolane A (2), eupenicisirenins A (3), arpenicarotane B (5) and aspterric acid (6). The assignments of their structures were elucidated from detailed analyses of spectroscopic data, electronic circular dichroism calculation, and biogenetic considerations. The bioassay of isolated compounds (1-6) exhibited no cytotoxic activities against three tumor cells including MCF-7, HepG2, and A549. Arpenibisabolane C (1) and A (2) showed weak inhibition bioactivities on aquatic pathogens Vibrio owensii and Vibrio algivorus. Moreover, phylogenetic analysis and sequence alignments of crucial sesquiterpene synthases were performed. Based on the chemical structures and biogenetic investigations, a hypothetic pathway of new compounds (1, 4) was proposed.

Anti-Müllerian hormone (AMH) regulates key steps in folliculogenesis, yet unlike other TGF-β family members, no soluble AMH-binding proteins have been identified. We hypothesized that AMH may bind shuttling proteins in human follicular fluid (FF). Discarded pooled FF was collected from women undergoing oocyte retrieval at an academic fertility center. AMH-binding soluble proteins in FF were co-immunoprecipitated with AMH, identified by mass spectrometry, and their association was validated by western blot. We characterized the spatiotemporal expression of candidate binding protein glucose-regulated protein 78 (GRP78) in the mouse ovary across the cycle by immunohistochemistry. We used enzyme-linked immunoassay (ELISA) to measure AMH and GRP78 concentrations in patient FF. We used transient transfections in Chinese hamster ovary (CHO) cells to understand the interaction between AMH and GRP78. Co-immunoprecipitation of AMH in FF identified multiple soluble AMH-binding candidates in the heat-shock protein family, which was confirmed by reciprocal co-immunoprecipitation. We found that GRP78, the most abundant candidate, was enriched in antral follicles of mice at estrus and was present in higher concentrations in human FF than AMH. Finally, we found that AMH overexpression increased endoplasmic reticulum stress and induced GRP78 expression in CHO cells; further ectopic overexpression of GRP78 facilitated the secretion of AMH. This work describes AMH-binding protein candidates identified in human FF and suggests that GRP78 may chaperone AMH during secretion and remain bound in FF. Further study is warranted to understand how heat shock proteins may modulate other biological aspects of AMH and their effects on fertility.

Despite significant advancements in identifying novel therapeutic targets and compounds, cancer stem cells (CSCs) remain pivotal in driving therapeutic resistance and tumor progression in gastric cancer (GC). High-resolution knowledge of the transcriptional programs underlying the role of CSC niche in driving tumor stemness and progression is still lacking. Herein, spatial and single-cell RNA sequencing of 32 human gastric mucosa tissues at various stages of malignancy, illuminating the phenotypic plasticity of tumor epithelium and transcriptional trajectory from mature gastric chief cells to the CSC state, which is associated with activation of EGFR and WNT signaling pathways, is conducted. Moreover, the CSCs interact with not only the immunosuppressive CXCL13+ T cells and CCL18+ M2 macrophages to evade immune surveillance, but also the inflammatory cancer-associated fibroblasts (iCAFs) to promote tumorigenesis and maintain stemness, which construct the CSC niche leading to inferior prognosis. Notably, it is uncovered that amphiregulin (AREG) derived from iCAFs promotes tumor stemness by upregulating the expression of SOX9 in tumor cells, and contributes to drug resistance via the AREG-ERBB2 axis. This study provides valuable insight into the characteristics of CSC niche in driving tumor stemness and progression, offering novel perspective for designing effective strategies to overcome GC therapy resistance.

Introduction: Extracellular sulfatases (Sulf1, Sulf2) are enzymes that catalyze the removal of 6-O-sulfate group from heparan sulfate proteoglycans (HSPG) in the extracellular matrix. The resulting sulfation remodeling modifies the interactions with many proteins, which adjusts important cellular functions. Both enzymes have a highly conserved N-terminal region containing the consensus sulfatase motifs, and a unique active-site aldehyde residue, formylglycine (FGly). The recognition and binding of Sulf1 and Sulf2 to their substrates is mediated by several factors, including the overall structure and charge distribution of the HS chains, and the presence of specific sulfation patterns. Despite their biological significance, structure and function of the Sulf1 and Sulf2 enzymes remain insufficiently characterized largely due to the lack of the enzyme-sources and/or specific activity assays. Results: To understand their structure and function and to evaluate potential inhibitors, we have developed human expression systems for the production of recombinant Sulf1 and Sulf2 enzymes in high titers. We produce functional recombinant proteins in up to 3mg/L, as analyzed by SDS PAGE and Western blot. We characterize activity of the Sulf enzymes by an optimized arylsulfatase assay (4-MUS) and by a newly developed 6-O-endosulfatase assay. This assay is highly specific for Sulf1 and Sulf2 activity using a synthetic oligosaccharide (HS mimics) GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP as a substrate in an HPLC assay monitoring the para-nitrophenol (pNP) absorbance of de-sulfated products separated from the substrate by ion exchange chromatography. In our assays, Sulf2 recombinant protein displayed high enzymatic activity, while Sulf1 exhibited low enzymatic activity in both assays. To enhance the enzymatic activity of Sulf1, we co-transfected the Sulf1 HEK293T overexpression system with FGE (formylglycine generating enzyme, also known as SUMF1). This led to a significant recovery of Sulf1 enzymatic activity in both assays. Furthermore, the quantification of Sulf1 and Sulf2 enzymatic activities facilitated screening of potential inhibitors using chemical and/or biological compounds. We identified a novel marine fucosylated chondroitin sulfate inhibitor and demonstrated its ability to block the activity of Sulf1 and Sulf2 recombinant proteins in vitro. Conclusion: Our optimized expression systems and newly developed enzymatic assays provide us with the capability to comprehensively analyze Sulf1 and Sulf2 enzymatic activity. Furthermore, we can effectively evaluate inhibitors of the enzymes. It is noteworthy that the Sulf1 and Sulf2 activity have been associated with poor survival outcomes in various malignancies, which highlights their significance as essential diagnostic and therapeutic targets.

Sheep congenital microtia is characterized by underdeveloped ears and provides an ideal basis for studying human microtia. This study identified the causal mutation and regulatory mechanisms underlying this disorder. Whole-genome association analysis was conducted using 23 ear tissue samples from sheep with microtia and 28 samples from normal-eared sheep. A significant correlation was found between microtia and a 76-base pair duplication in the enhancer region of the HMX1 gene. Further analysis of offspring phenotypes confirmed an autosomal dominant inheritance pattern. Genotypic analysis showed that individuals that are homozygous for this duplication were earless, heterozygous individuals exhibited shortened ears, and wild-type individuals had normal ears. Moreover, luciferase assays confirmed that this duplication increased HMX1 gene expression, and duplication knock-in mice also exhibited shorter and narrower external ears compared to wild-type mice. Transcriptomic analysis further demonstrated that this duplication enhanced HMX1 gene expression in animal models. This study characterized the causal regulatory mutation underlying sheep microtia.

Hypertension associated with hyperhomocysteinemia (HHcy) accounts for 75% of hypertension in China. HHcy plays a synergistic role with hypertension in vascular injury and significantly increases the incidence of cardiovascular and cerebrovascular diseases. The present study aimed to explore the molecular mechanism of HHcy-induced arterial injury in hypertension. Spontaneously hypertensive rats (SHR) were injected intraperitoneally with DL-homocysteine (Hcy) to construct the model of hypertension associated with HHcy (HHcy + SHR). Biological network was employed to identify the material basis of arterial injury in hypertension associated with HHcy. The prediction molecules in oxidative stress and inflammation pathways were experimentally verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) analysis. The HHcy + SHR group significantly increased oxidative stress pathway molecules: nicotinamide adenine dinucleotide phosphate oxidase (Nox); inflammatory pathway molecules: vascular adhesion protein-1 (VAP-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a); as well as inflammatory pathway regulatory factors: nuclear factor-κ-gene binding (NF-κB) p65 and protein kinase B (Akt1). Among them, IL-6 was also significantly increased in the HHcy group. Both oxidative stress and inflammation contributed to the arterial injury of hypertension associated with HHcy, and inflammation mechanism might play a leading role in HHcy aggravating arterial injury, at least partially through the Akt1/NF-κB p65/IL-6 signaling pathway.

What is already known about this topic?:Numerous studies conducted in China have reported on the prevalence of birth defects (BDs). However, the limited surveillance periods in select studies curtail comprehensive trends and cause evaluation. Accordingly, the surveillance duration of BDs is extended, and a comprehensive analysis of their prevailing trends is conducted to provide a basis for government intervention and policy implementation.What is added by this report?:There is a distinct increase in the incidence of BDs observed in Jinan. In rural areas, a pronounced upward trend was observed, and the increase was more rapid than in urban areas. BD prevalence among mothers over 35 years old and under 20 years old was substantially higher than BD prevalence rates in other maternal age brackets. Specifically, the period from 2005 to 2022 saw the prevalence of congenital heart disease surge, the fastest average annual growth rate among all birth defects.What are the implications for public health practice?:It's essential to prioritize pregnant women in rural areas and those at both ends of the maternal age spectrum. Implementing comprehensive initiatives is crucial to address the high prevalence of congenital heart disease.

Supplementary Figure Legends 1-2 from Stromal Deletion of the APC Tumor Suppressor in Mice Triggers Development of Endometrial Cancer

Supplementary Figure 2 from Stromal Deletion of the APC Tumor Suppressor in Mice Triggers Development of Endometrial Cancer

Supplementary Figure 1 from Stromal Deletion of the APC Tumor Suppressor in Mice Triggers Development of Endometrial Cancer

The traditional homologous recombination (HR) gene-editing method faces problems such as low editing efficiency and absence of marker genes. CRISPR–Cas9-editing efficiency is high and has been widely used in bacteria and yeast. In comparison with CRISPR–Cas9, CRISPR–Cas12a has many outstanding advantages. Here, we report an Acidaminococcus sp. BV3L6 (As) Cas12a-based genome-editing method used for Starmerella bombicola . To demonstrate the high efficiency of the CCMGE system, we verified a counter-selectable marker in S. bombicola , orotidine 5’-phosphate decarboxylase (100% for URA3 ). We also tested the common gene UDP-glucosyltransferase (100% for UGTA ) using a 300 bp donor containing hygromycin expression cassette. This toolkit was further extended to simultaneously edit two genes (18% for UGTA and leu ) and three genes (13.8% for UGTA, leu and URA3 ). The system greatly reduces the screening time for such multi-site editing. Based on the CCMGE system, the PHA (polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase ( PHAC ). The PHA content and DCW reached 11.8% and 30.1 g/L, respectively. The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.

Qufeng Zhitong capsule (QZC), a Chinese patent medicine officially approved in China for the treatment of rheumatoid arthritis (RA) and other diseases, possesses the primary effects of dispelling wind, relieving pain, and promoting blood circulation, whose clinical applications have been confined owing to the incomplete elucidation of its chemical compositions and the underlying molecular mechanism for the treatment of RA. In this study, 61 compounds including 16 phenylpropanoids, 15 organic acids, 13 alkaloids, seven flavonoids, six iridoids, one saccharide, two aldehydes, and one saponin in QZC were simultaneously identified and traced to their herbal origins by ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC/Q-Orbitrap-MS), where 31 of them were unambiguously identified by reference compounds, and the other 30 were tentatively characterized. Besides, all these compounds were proven to have potential pharmacological activity in the treatment of RA based on network pharmacology analysis. In conclusion, this study first investigated the chemical composition and potential pharmacological effects of the main chemical compounds in QZC, which will contribute to the revelation of bioactive compounds in QZC and provide evidence for clinical application.

Background China was one of the few countries to achieve the Millennium Development Goals 5. China had taken many effective measures to reduce maternal mortality ratio (MMR) and has achieved encouraging progress. These measures were worth sharing for other countries to reduce the MMR, but the introduction of these measures from the national perspective was too grand, and the measures implemented in a city and the results achieved were more valuable. However, there were few studies on the prevalence and trends of prolonged maternal mortality in a city. In this study, we mainly introduced the prevalence of the MMR in Jinan,China from 1991 to 2020, analyzed the causes of trends and put forward some solutions to the difficulty existing in the process of reducing the MMR,hoping to serve as a model for some developing cities to reduce MMR. Methods We collected maternal mortality data from paper records, electronic files and network platforms. The time trend of MMR was tested by Cochran-Armitage Test (CAT). We divided the study period into three stages with 10 years as a stage and the Chi-square test or Fisher’s exact test was used to test the difference in MMR of different periods. Results From 1991 to 2020, We counted 1,804,162 live births and 323 maternal deaths, and the MMR was 17.93 per 100,000 live births. The MMR declined from 44.06 per 100,000 live births in 1991 to 5.94 per 100,000 live births in 2020, with a total decline of 86.52% and an annual decline of 2.89%. The MMR declined by 88.54% in rural areas, with an average annual decline 2.95%, faster than that in urban areas (82.06, 2.73%). From 1991 to 2020, the top five causes of maternal deaths were obstetric haemorrhage (4.55 per 100,000 live births), amniotic fluid embolism (3.27 per 100,000 live births), pregnancy-induced hypertension (2.61 per 100,000 live births), heart disease (2.33 per 100,000 live births) and other medical complications (2.05 per 100,000 live births). Postpartum hemorrhage, amniotic fluid embolism, pregnancy-induced hypertension showed a downward trend ( P < 0.05) and other medical complications showed an upward trend ( P < 0.05). Conclusions Subsidy for hospitalized delivery of rural women, free prenatal check-ups for pregnant women and rapid referral system between hospitals have contributed to reducing MMR in Jinan. However, it was still necessary to strengthen the treatment of obstetric hemorrhage by ensuring blood supply, reduce the MMR due to medical complications by improving the skills of obstetricians to deal with medical diseases, and reduce the MMR by strengthening the allocation of emergency equipment in county hospitals and the skills training of doctors.

Abstract Background Commercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol. Results The xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L− 1 xylitol in 120 h from 300 g L− 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L− 1 h− 1 and the conversion rate was 92.40 %. Conclusions In conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

Abstract Background Hypertension associated with hyperhomocysteinemia (HHcy) is correlated with a high risk of vascular diseases. However, the mechanisms of HHcy-associated hypertensive vascular damage and the efficacy of folic acid (FA) as a treatment have not been fully elucidated. The aim of the present study was to evaluate the role of immune/inflammatory molecules and oxidizing factors in HHcy-associated hypertensive vascular damage, and to observe the intervention effect of FA on the two vascular injury factors. Methods Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were administered DL-Hcy intraperitoneally to mimic HHcy and hypertension associated with HHcy for 12 weeks. WKYs and SHRs were randomized into WKY group, HHcy group, SHR group, SHR + HHcy group and SHR + HHcy + FA group. Mean tail artery blood pressure, plasma Hcy, serum SOD and MDA of rats in each group were compared. The thoracic aorta and bilateral carotid artery of rats were harvested for morphometric and immunostaining analyses. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of immune/inflammatory molecules such as TNF-α, IL-6, NF-κB p65/Rela and NF-κB2 and oxidative factors such as Nox2 and Nox4. Results We found that vascular inflammatory factors of vascular adhesion protein-1 (VAP-1), interleukin-6 (IL-6), and nuclear factor-κ-gene binding (NF-κB) p65/Rela in HHcy-associated hypertensive rats were significantly higher than those in SHRs (P༜0.05). While the oxidative stress indicators of Nox2 and Nox4 in HHcy-associated hypertensive rats were not significantly higher than those in SHRs (P༞0.05). Compared with SHRs, FA intervention in HHcy-associated hypertensive rats significantly increased serum superoxide dismutase (SOD) levels (P = 0.000) and significantly reduced vascular inflammatory factors of IL-6 and NF-κB p65/Rela (P༜0.05), but did not significantly change the oxidative stress indicators of Nox2 and Nox4 (P༞0.05). Conclusions HHcy-induced immune/inflammatory response plays a dominant role in vascular damage of HHcy-associated hypertensive rats. In addition to reducing the negative effects of HHcy, FA might involve unique antioxidant effects and inhibition of immune/inflammatory overreaction for HHcy-associated hypertensive rats.

Abstract Background Hypertensive disorders of pregnancy (HDP) is one of the leading causes of maternal and neonatal mortality, increasing the long-term incidence of cardiovascular diseases. Preeclampsia and gestational hypertension are the major components of HDP. The aim of our study is to establish a prediction model for pregnant women with new-onset hypertension during pregnancy (increased blood pressure after gestational age > 20 weeks), thus to guide the clinical prediction and treatment of de novo hypertension. Methods A total of 117 pregnant women with de novo hypertension who were admitted to our hospital’s obstetrics department were selected as the case group and 199 healthy pregnant women were selected as the control group from January 2017 to June 2018. Maternal clinical parameters such as age, family history and the biomarkers such as homocysteine, cystatin C, uric acid, total bile acid and glomerular filtration rate were collected at a mean gestational age in 16 to 20 weeks. The prediction model was established by logistic regression. Results Eleven indicators have statistically significant difference between two groups (P < 0.05). These 11 factors were substituted into the logistic regression equation and 7 independent predictors were obtained. The equation expressed including 7 factors. The calculated area under the curve was 0.884(95% confidence interval: 0.848–0.921), the sensitivity and specificity were 88.0 and 75.0%. A scoring system was established to classify pregnant women with scores ≤15.5 as low-risk pregnancy group and those with scores > 15.5 as high-risk pregnancy group. Conclusions Our regression equation provides a feasible and reliable means of predicting de novo hypertension after pregnancy. Risk stratification of new-onset hypertension was performed to early treatment interventions in high-risk populations.

Tyrosol is a pharmacologically active phenolic compound widely used in the pharmaceutical and chemical industries. Microbial fermentation has potential value as an environmentally friendly approach to tyrosol production, but suffers from low tyrosol yields and the need for expensive media additives. In this study, Escherichia coli MG1655 was modified by integrating an E. coli codon-optimized version of the Saccharomyces cerevisiae phenylpyruvate decarboxylase gene, named ARO10*, into the lacI locus. The resulting strain (YMGA*) produced 0.14 mM tyrosol from 2% glucose without the need for expensive media supplements. Subsequent deletion of E. coli genes designed to eliminate competing metabolic pathways (feaB, pheA, tyrB) or undesirable gene regulation (tyrR) produced a strain (YMGA*R) that produced 3.11 mM tyrosol. Tyrosol production was then increased to 10.92 mM by increasing the ARO10* copy number to five copies (strain YMG5A*R). Finally, tyrosol production was increased to 28 mM (ca. 3.9 g/L) by optimizing fermentation conditions in a 5 L fermenter. Engineering a productive E. coli strain with high tyrosol titer from glucose using a medium that does not require added amino acids, inducer, or antibiotic provides a solid basis to produce tyrosol through microbial fermentation.

Abstract Background: Epithelial ovarian cancers are the fifth leading cause of cancer-related deaths in American women, with a five-year overall survival rate for patients diagnosed at advanced stages of less than 30%. High-grade serous ovarian cancers (HGSOCs), the most common histologic subtype, often initially responds to platinum-based chemotherapy. However, over 80% of patients experience chemoresistant recurrence in advanced-stage disease. Methods: We have established over 40 patient-derived HGSOC cell lines from ascites of patients with recurrences and hypothesized that these refractory HGSOC cell lines may each possess genetic liabilities and be dependent on unique pathways for survival that can be identified using an unbiased screening approach for the development of personalized therapies. In one such example, we identified candidate genes that are uniquely essential to a single HGSOC patient (ptD), as proof of concept, using a genome-wide lentiviral sgRNA library, containing 187,536 sgRNAs targeting 18,543 protein-coding human genes and 1,504 nontargeting control sgRNAs in a Cas9-containing vector. Results: The unbiased CRISPR screen identified BRAF (p=2E-6) as a specific susceptibility of ptD, confirming the known oncogene addiction of this primary cell line carrying a V600E mutation, as well as a significant sensitivity to JUNB deletion (p=6E-6). Knockout of the JUNB gene, using lentiviruses expressing guide RNAs and Cas9 protein targeted to JUNB, repressed cell proliferation in vitro in ptD cells but not other primary cell lines. Moreover, in a xenograft mouse model, JUNB KO ptD cells displayed slower tumor growth than control JUNB WT cells. Conclusion: We have confirmed in a single patient cell line our ability to identify and validate unique genetic liabilities, suggesting the approach can be scaled to rapidly screen primary HGSOC samples for personalized therapy. Citation Format: Nobuhiro Takahashi, Heather Keys, Raghav Mohan, Lihua Zhang, Yi Li, Hatice D. Saatcioglu, Nicholas Nagykery, Patricia K. Donahoe, David Sabatini, David Pépin. Whole-genome CRISPR/CAS9 screen using patient samples reveals JunB as a unique genetic liability [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A63.

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time‐consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR‐associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR–Cas9 cassette or a transient CRISPR–Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis . Based on this system, we present a platform for the biosynthesis of β‐carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.