A new Prdm1-Cre line is suitable for studying the second heart field development.

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

HuR promotes triglyceride synthesis and intestinal fat absorption.

Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the underlying mechanisms remain to be further studied. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3' UTR of Dgat2 mRNA and intron 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3' UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.

Clpf encodes pentatricopeptide repeat protein (PPR5) and regulates pink flesh color in watermelon (Citrullus lanatus L.).

KEY MESSAGE: The gene controlling pink flesh in watermelon was finely mapped to a 55.26-kb region on chromosome 6. The prime candidate gene, Cla97C06G122120 (ClPPR5), was identified through forward genetics. Carotenoids offer numerous health benefits; while, they cannot be synthesized by the human body. Watermelon stands out as one of the richest sources of carotenoids. In this study, genetic generations derived from parental lines W15-059 (red flesh) and JQ13-3 (pink flesh) revealed the presence of the recessive gene Clpf responsible for the pink flesh (pf) trait in watermelon. Comparative analysis of pigment components and microstructure indicated that the disparity in flesh color between the parental lines primarily stemmed from variations in lycopene content, as well as differences in chromoplast number and size. Subsequent bulk segregant analysis (BSA-seq) and genetic mapping successfully narrowed down the Clpf locus to a 55.26-kb region on chromosome 6, harboring two candidate genes. Through sequence comparison and gene expression analysis, Cla97C06G122120 (annotated as a pentatricopeptide repeat, PPR) was predicted as the prime candidate gene related to pink flesh trait. To further investigate the role of the PPR gene, its homologous gene in tomato was silenced using a virus-induced system. The resulting silenced fruit lines displayed diminished carotenoid accumulation compared with the wild-type, indicating the potential regulatory function of the PPR gene in pigment accumulation. This study significantly contributes to our understanding of the forward genetics underlying watermelon flesh traits, particularly in relation to carotenoid accumulation. The findings lay essential groundwork for elucidating mechanisms governing pigment synthesis and deposition in watermelon flesh, thereby providing valuable insights for future breeding strategies aimed at enhancing fruit quality and nutritional value.

Total Infectome Characterization of Respiratory Infections during the 2022-23 COVID-19 Outbreak in China Revealed Extensive Coinfections with Links to SARS-CoV-2 Status, Age, and Disease Severity.

Between 7 December 2022 and 28 February 2023, China experienced a new wave of COVID-19 that swept across the entire country and resulted in an increasing amount of respiratory infections and hospitalizations. The purpose of this study is to reveal the intensity and composition of coinfecting microbial agents. In total, 196 inpatients were recruited from The Third People's Hospital of Shenzhen, and 169 respiratory and 73 blood samples were collected for metagenomic next-generation sequencing. The total "Infectome" was characterized and compared across different groups defined by the SARS-CoV-2 detection status, age groups, and severity of disease. Our results revealed a total of 22 species of pathogenic microbes (4 viruses, 13 bacteria, and 5 fungi), and more were discovered in the respiratory tract than in blood. The diversity of the total infectome was highly distinguished between respiratory and blood samples, and it was generally higher in patients that were SARS-CoV-2-positive, older in age, and with more severe disease. At the individual pathogen level, HSV-1 seemed to be the major contributor to these differences observed in the overall comparisons. Collectively, this study reveals the highly complex respiratory infectome and high-intensity coinfection in patients admitted to the hospital during the period of the 2023 COVID-19 pandemic in China.

The clinical outcome of COVID-19 is strongly associated with microbiome dynamics in the upper respiratory tract.

OBJECTIVES: The respiratory tract is the portal of entry for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a variety of respiratory pathogens other than SARS-CoV-2 have been associated with severe cases of COVID-19 disease, the dynamics of the upper respiratory microbiota during disease the course of disease, and how they impact disease manifestation, remain uncertain. METHODS: We collected 349 longitudinal upper respiratory samples from a cohort of 65 COVID-19 patients (cohort 1), 28 samples from 28 recovered COVID-19 patients (cohort 2), and 59 samples from 59 healthy controls (cohort 3). All COVID-19 patients originated from the earliest stage of the epidemic in Wuhan. Based on a modified clinical scale, the disease course was divided into five clinical disease phases (pseudotimes): "Healthy" (pseudotime 0), "Incremental" (pseudotime 1), "Critical" (pseudotime 2), "Complicated" (pseudotime 3), "Convalescent" (pseudotime 4), and "Long-term follow-up" (pseudotime 5). Using meta-transcriptomics, we investigated the features and dynamics of transcriptionally active microbes in the upper respiratory tract (URT) over the course of COVID-19 disease, as well as its association with disease progression and clinical outcomes. RESULTS: Our results revealed that the URT microbiome exhibits substantial heterogeneity during disease course. Two clusters of microbial communities characterized by low alpha diversity and enrichment for multiple pathogens or potential pathobionts (including Acinetobacter and Candida) were associated with disease progression and a worse clinical outcome. We also identified a series of microbial indicators that classified disease progression into more severe stages. Longitudinal analysis revealed that although the microbiome exhibited complex and changing patterns during COVID-19, a restoration of URT microbiomes from early dysbiosis toward more diverse status in later disease stages was observed in most patients. In addition, a group of potential pathobionts were strongly associated with the concentration of inflammatory indicators and mortality. CONCLUSION: This study revealed strong links between URT microbiome dynamics and disease progression and clinical outcomes in COVID-19, implying that the treatment of severe disease should consider the full spectrum of microbial pathogens present.

Natural variation in the promoter of FLOWERING LOCUS T-LIKE 2 in pumpkin (Cucurbita moschata Duch.) is associated with flowering time under short-day conditions.

Late flowering is a serious bottleneck in pumpkin (Cucurbita moschata Duch.) agriculture production. Although key genes governing flowering time have been reported in many species, the regulatory network of flowering in pumpkin remains largely obscure, thereby impeding the resolution of industry-wide challenges associated with delayed fruit ripening in pumpkin cultivation. Here, we report an early flowering pumpkin germplasm accession (LXX-4). Using LXX-4 and a late flowering germplasm accession (HYM-9), we constructed an F2 segregation population. A significant difference in FLOWERING LOCUS T-LIKE 2 (FTL2) expression level was identified to be the causal factor of the flowering time trait discrepancy in LXX-4 and HYM-9. Moreover, we have shown that a 21 bp InDel in the FTL2 promoter was the key reason for the waxing and waning of its transcript level. The 21 bp deletion excluded a repressor-AGL19 and recruited activators-BBX7, WRKY40 and SVP to the FTL2 promoter in LXX-4. Together, our data add a useful element to our knowledge which could be used to simplify breeding efforts for early-maturing pumpkin.

HDAC3 aberration-incurred GPX4 suppression drives renal ferroptosis and AKI-CKD progression.

Acute kidney injury (AKI) progression to chronic kidney disease (CKD) represents a unique renal disease setting characterized by early renal cellular injury and regulated cell death, and later renal fibrosis, of which the critical role and nature of ferroptosis are only partially understood. Here, we report that renal tubular epithelial ferroptosis caused by HDAC3 (histone deacetylase 3) aberration and the resultant GPX4 suppression drives AKI-CKD progression. In mouse models of AKI-CKD transition induced by nephrotoxic aristolochic acid (AA) and folic acid (FA), renal tubular epithelial ferroptosis occurred early that coincided with preferential HDAC3 elevation and marked suppression of a core anti-ferroptosis enzyme GPX4 (glutathione peroxidase 4). Intriguingly, genetic Hdac3 knockout or administration of a HDAC3-selective inhibitor RGFP966 effectively mitigated the GPX4 suppression, ferroptosis and the fibrosis-associated renal functional loss. In cultured tubular epithelial cells, HDAC3 over-expression or inhibition inversely affected GPX4 abundances. Further analysis revealed that Gpx4 promoter contains a typical binding motif of transcription factor KLF5 (Kruppel-like factor 5). HDAC3 and KLF5 inducibly associated and bound to Gpx4 promoter upon AA treatment, leading to local histone hypoacetylation and GPX4 transactivation inhibition, which was blocked by RGFP966 and a KLF5 inhibitor ML264, respectively, suggesting that KLF5 co-regulated the HDAC3-incurred Gpx4 transcription inhibition. More importantly, in AKI-CKD mice receiving a GPX4 inactivator RSL3, the anti-ferroptosis and renoprotective effects of RGFP966 were largely abrogated, indicating that GPX4 is an essential downstream mediator of the HDAC3 aberration and renal ferroptosis during AKI-CKD transition. Together, our study identified a critical epigenetic pathway of ferroptosis during AKI-CKD transition and suggested that the strategies preserving GPX4 by HDAC3 inhibition are potentially effective to reduce renal ferroptosis and slow AKI-CKD progression.

SlbHLH152, a bHLH transcription factor positively regulates iron homeostasis in tomato.

The maintain of iron (Fe) homeostasis is essential for plant survival. In tomato, few transcription factors have been identified as regulators of Fe homeostasis, among which SlbHLH068 induced by iron deficiency, plays an important role. However, the upstream regulator(s) responsible for activating the expression of SlbHLH068 remain(s) unknown. In this study, the bHLH (basic helix-loop-helix) transcription factor SlbHLH152 was identified as an upstream regulator of SlbHLH068 using yeast one-hybrid screening. Deletion of SlbHLH152 led to a significant decline in Fe concentration, which was accompanied by reduced expression of Fe-deficiency-responsive genes. In contrast, SlbHLH152 overexpression plants displayed tolerance to iron deficiency, increased Fe accumulation, and elevated expression of Fe-deficiency-responsive genes. Further analysis indicated that SlbHLH152 directly activates the transcription of SlbHLH068. Taken together, our results suggest that SlbHLH152 may be involved in the regulation of iron homeostasis by directly activating the transcription of SlbHLH068 in tomato.

LonP1 Links Mitochondria-ER Interaction to Regulate Heart Function.

Interorganelle contacts and communications are increasingly recognized to play a vital role in cellular function and homeostasis. In particular, the mitochondria-endoplasmic reticulum (ER) membrane contact site (MAM) is known to regulate ion and lipid transfer, as well as signaling and organelle dynamics. However, the regulatory mechanisms of MAM formation and their function are still elusive. Here, we identify mitochondrial Lon protease (LonP1), a highly conserved mitochondrial matrix protease, as a new MAM tethering protein. The removal of LonP1 substantially reduces MAM formation and causes mitochondrial fragmentation. Furthermore, deletion of LonP1 in the cardiomyocytes of mouse heart impairs MAM integrity and mitochondrial fusion and activates the unfolded protein response within the ER (UPRER). Consequently, cardiac-specific LonP1 deficiency causes aberrant metabolic reprogramming and pathological heart remodeling. These findings demonstrate that LonP1 is a novel MAM-localized protein orchestrating MAM integrity, mitochondrial dynamics, and UPRER, offering exciting new insights into the potential therapeutic strategy for heart failure.

Deletion of PDK1 Caused Cardiac Malmorphogenesis and Heart Defects Due to Profound Protein Phosphorylation Changes Mediated by SHP2.

Phosphoinositide-dependent protein kinase-1 (PDK1), a master kinase and involved in multiple signaling transduction, participates in regulating embryonic cardiac development and postnatal cardiac remodeling. Germline PDK1 knockout mice displayed no heart development; in this article, we deleted PDK1 in heart tissue with different cre to characterize the temporospatial features and find the relevance with congenital heart disease(CHD), furthermore to investigate the underlying mechanism. Knocking out PDK1 with Nkx2.5-cre, the heart showed prominent pulmonic stenosis. Ablated PDK1 with Mef2cSHF-cre, the second heart field (SHF) exhibited severe hypoplasia. And deleted PDK1 with αMHC-cre, the mice displayed dilated heart disease, protein analysis indicated PI3K and ERK were activated; meanwhile, PDK1-AKT-GSK3, and S6K-S6 were disrupted; phosphorylation level of Akt473, S6k421/424, and Gsk3α21 enhanced; however, Akt308, S6k389, and Gsk3ß9 decreased. In mechanism investigation, we found SHP2 membrane localization and phosphorylation level of SHP2542 elevated, which suggested SHP2 likely mediated the disruption.

Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis.

T helper 17 (Th17) cells are a subset of CD4+ T helper cells involved in the inflammatory response in autoimmunity. Th17 cells secrete Th17 specific cytokines, such as IL-17A and IL17-F, which are governed by the master transcription factor RoRγt. However, the epigenetic mechanism regulating Th17 cell function is still not fully understood. Here, we reveal that deletion of RNA 5-methylcytosine (m5C) methyltransferase Nsun2 in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates Th17 cell-induced colitis pathogenesis. Mechanistically, RoRγt can recruit Nsun2 to chromatin regions of their targets, including Il17a and Il17f, leading to the transcription-coupled m5C formation and consequently enhanced mRNA stability. Our study demonstrates a m5C mediated cell intrinsic function in Th17 cells and suggests Nsun2 as a potential therapeutic target for autoimmune disease.

The second heart field: the first 20 years.

In 2001, three independent groups reported the identification of a novel cluster of progenitor cells that contribute to heart development in mouse and chicken embryos. This population of progenitor cells was designated as the second heart field (SHF), and a new research direction in heart development was launched. Twenty years have since passed and a comprehensive understanding of the SHF has been achieved. This review provides retrospective insights in to the contribution, the signaling regulatory networks and the epithelial properties of the SHF. It also includes the spatiotemporal characteristics of SHF development and interactions between the SHF and other types of cells during heart development. Although considerable efforts will be required to investigate the cellular heterogeneity of the SHF, together with its intricate regulatory networks and undefined mechanisms, it is expected that the burgeoning new technology of single-cell sequencing and precise lineage tracing will advance the comprehension of SHF function and its molecular signals. The advances in SHF research will translate to clinical applications and to the treatment of congenital heart diseases, especially conotruncal defects, as well as to regenerative medicine.

The lifestyle factors of physical activity and diet balance associated with HPV infection in China: The cross-sectional study.

Background: Human wellbeing has been linked with lifestyle factors such as physical activity, diet balance, sleep quality, depression, and anxiety. However, few studies illustrate the relationship between such lifestyle factors and HPV infection. In this study, we investigated the association between lifestyle factors, age, disease status and HPV infection. Participants and methods: Participants were recruited through a digital eHealth platform in Shenzhen, Mainland China. Both lifestyle factors and cervicovaginal mucus (CVM) samples to test for HPV outcomes were collected from each participant as a cross-sectional study. In addition, the eHealth platform recorded age and current or history diseases, which were adjusted to apply for both univariable and multivariable logistic regression. Furthermore, lifestyle factors were categorized as different levels to conduct stratification analysis. Results: We recruited 149 HPV positive and 346 HPV negative participants through HPV detection. Physical activity and diet balance were significantly associated with HPV infection in lifestyle factors (P values < 0.001) after adjusting for age and current or history diseases. However, stratified analysis showed three factors were insignificant for HPV infection - namely, sleep quality, depression, and anxiety. Most HPV infections involved a sole HPV serotype (83%), and diet balance was the most significant difference between sole and multiple HPV infections. Conclusions: Among lifestyle factors, physical inactivity or diet imbalance can significantly increase HPV infection risk. In particular, diet balance might be related to the number of HPV serotypes. Our results suggest that exercising and regulating diet may reduce the risk of HPV infection.

Vaginal microbiota and personal risk factors associated with HPV status conversion-A new approach to reduce the risk of cervical cancer?

Vaginal microbiota (VMB) is associated with changes in Human papilloma virus (HPV) status, which consequently influences the risk of cervical cancer. This association was often confounded by personal risk factors. This pilot research aimed to explore the relationship between vaginal microbiota, personal risk factors and their interactions with HPV status conversion to identify the vaginal microbiota that was associated with HPV clearance under heterogeneous personal risk factors. A total of 38 women participated by self-collecting a cervicovaginal mucus (CVM) sample that was sent for metagenomics sequencing. Most of the participants also filled in personal risk factors questionnaire through an eHealth platform and authorized the use of their previous HPV genotyping results stored in this eHealth platform. Based on the two HPV results, the participants were grouped into three cohorts, namely HPV negative, HPV persistent infection, and HPV status conversion. The relative abundance of VMB and personal factors were compared among these three cohorts. A correlation investigation was performed between VMB and the significant personal factors to characterize a robustness of the panel for HPV status change using R programming. At baseline, 12 participants were HPV-negative, and 22 were HPV-positive. Within one year, 18 women remained HPV-positive, 12 were HPV-negative and 4 participants showed HPV clearance. The factors in the eHealth questionnaire were systematically evaluated which identified several factors significantly associated with persistent HPV infection, including age, salary, history of reproductive tract infection, and the total number of sexual partners. Concurrent vaginal microbiome samples suggest that a candidate biomarker panel consisting of Lactobacillus gasseri, Streptococcus agalactiae, and Timona prevotella bacteria, which may be associated with HPV clearance. This pilot study indicates a stable HPV status-related vaginal microbe environment. To establish a robust biomarker panel for clinical use, larger cohorts will be recruited into follow-up studies.

To unwind the biological knots: The DNA/RNA G-quadruplex resolvase RHAU (DHX36) in development and disease.

The G-quadruplex (G4) sequences are short fragments of 4-interval triple guanine (G) with frequent and ubiquitous distribution in the genome and RNA transcripts. The G4 sequences are usually folded into secondary "knot" structure via Hoogsteen hydrogen bond to exert negative regulation on a variety of biological processes, including DNA replication and transcription, mRNA translation, and telomere maintenance. Recent structural biological and mouse genetics studies have demonstrated that RHAU (DHX36) can bind and unwind the G4 "knots" to modulate embryonic development and postnatal organ function. Deficiency of RHAU gives rise to embryonic lethality, impaired organogenesis, and organ dysfunction. These studies uncovered the pivotal G4 resolvase function of RHAU to release the G4 barrier, which plays fundamental roles in development and physiological homeostasis. This review discusses the latest advancements and findings in deciphering RHAU functions using animal models.

Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state.

Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos.

Targeted therapy of atherosclerosis by pH-sensitive hyaluronic acid nanoparticles co-delivering all-trans retinal and rapamycin.

Atherosclerosis, the leading cause of death in the elderly worldwide, is typically characterized by elevated reactive oxygen species (ROS) levels and a chronic inflammatory state at the arterial plaques. Herein, pH-sensitive nanoparticles (HRRAP NPs) co-delivering all-trans retinal (ATR), an antioxidant linked to hyaluronic acid (HA) through a pH-sensitive hydrazone bond, and rapamycin (RAP), an anti-atherosclerotic drug loaded into the nanoparticle core, are developed for targeted combination therapy of atherosclerosis. In this way, HRRAP NPs might simultaneously reduce ROS levels via ATR antioxidant activity and reduce inflammation via the anti-inflammatory effect of RAP. In response to mildly acidic conditions mimicking the lesional inflammation in vitro, HRRAP NPs dissociated and both ATR and RAP were effectively released. The developed HRRAP NPs effectively inhibited pro-inflammatory macrophage proliferation, and displayed dose- and time-dependent specific internalization by different cellular models of atherosclerosis. Also, HRRAP NP combination therapy showed an efficient synergetic anti-atherosclerotic effect in vitro by effectively inhibiting the inflammatory response and oxidative stress in inflammatory cells. More importantly, HR NPs specifically accumulated in the atherosclerotic plaques of apolipoprotein E-deficient (ApoE-/-) mice, by active interaction with HA receptors overexpressed by different cells of the plaque. The treatment with HRRAP NPs remarkably inhibited the progression of atherosclerosis in ApoE-/- mice which resulted in stable plaques with considerably smaller necrotic cores, lower matrix metalloproteinase-9, and decreased proliferation of macrophages and smooth muscle cells (SMCs). Furthermore, HRRAP NPs attenuated RAP adverse effects and exhibited a good safety profile after long-term treatment in mice. Consequently, the developed pH-sensitive HRRAP NP represent a promising nanoplatform for atherosclerosis combination therapy.

Med23 supports angiogenesis and maintains vascular integrity through negative regulation of angiopoietin2 expression.

The mammalian Mediator complex consists of over 30 subunits and functions as a transcriptional hub integrating signaling for tissue-specific gene expression. Although the role of the Mediator complex in transcription has been extensively investigated, the functions of distinct Mediator subunits in development are not well understood. Here, we dissected the role of the Mediator subunit Med23 in mouse cardiovascular development. Endothelial-specific Med23 deletion caused embryonic lethality before embryonic day 13.5 (E13.5). The mutant embryos exhibited intracranial hemorrhage and diminished angiogenesis with dilated blood vessels in the head region, where the expression of Med23 was abundant at E10.5. Med23 deficiency impaired vasculogenesis in the head region and impeded retinal angiogenesis. Knocking down Med23 in human umbilical vein endothelial cells (HUVECs) resulted in angiogenic defects, recapitulating the vascular defects in Med23-mutant mice in a cell-autonomous manner. RNA sequencing in HUVECs indicated that Med23 deficiency resulted in the interruption of angiogenesis and the upregulation of angiopoietin2 (Ang2), an inducing factor for vascular network instability. Inhibition of Ang2 partially rescued angiogenic sprouting and lumen dilation defects in tube formation assays. Collectively, our findings demonstrate that Med23 promotes angiogenesis and maintains vascular integrity, in part by suppressing Ang2 signaling.