Spatiotemporal transcriptome atlas reveals the regional specification of the developing human brain.

Different functional regions of brain are fundamental for basic neurophysiological activities. However, the regional specification remains largely unexplored during human brain development. Here, by combining spatial transcriptomics (scStereo-seq) and scRNA-seq, we built a spatiotemporal developmental atlas of multiple human brain regions from 6-23 gestational weeks (GWs). We discovered that, around GW8, radial glia (RG) cells have displayed regional heterogeneity and specific spatial distribution. Interestingly, we found that the regional heterogeneity of RG subtypes contributed to the subsequent neuronal specification. Specifically, two diencephalon-specific subtypes gave rise to glutamatergic and GABAergic neurons, whereas subtypes in ventral midbrain were associated with the dopaminergic neurons. Similar GABAergic neuronal subtypes were shared between neocortex and diencephalon. Additionally, we revealed that cell-cell interactions between oligodendrocyte precursor cells and GABAergic neurons influenced and promoted neuronal development coupled with regional specification. Altogether, this study provides comprehensive insights into the regional specification in the developing human brain.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Mrhst4 gene, coding for NAD+-dependent deacetylase is involved in citrinin production of Monascus ruber.

AIMS: In this study, Mrhst4, encoding a member of NAD+-dependent histone deacetylase (HDAC), was deleted to evaluate its regulation on the production of Monascus azaphilone pigments (MonAzPs) and mycotoxin, as well as the developmental process in Monascusruber. METHODS AND RESULTS: Agrobacterium tumefaciens-mediated transformation was applied in this study to generate the Mrhst4 null strain. Mrhst4-deleted strain did not display obvious differences in the sexual and asexual reproduction, colonial morphology, and micro-morphology. UV-Vis scan and UPLC detection showed that disruption of Mrhst4 significantly increased the MonAzPs yields, and citrinin content was dramatically enhanced during the tested period. RT-qPCR results showed that the absence of Mrhst4 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. The Western blot assay suggested that deletion of Mrhst4 could significantly elevate the acetylation levels of H3K4, H3K9, H3K18, H3K56, and H4K12, but attenuated the lysine acetylation modification of H4Pan, H4K8, and H4K16. CONCLUSION: MrHst4 is an important regulator involved in secondary metabolism in Monascus ruber. In particular, MrHst4 plays a pivotal role in regulation of citrinin production.

Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification.

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

Identifying acute mesenteric ischemia via mesenteric fractional flow reserve in patients with spontaneous isolated superior mesenteric artery dissection: case report.

There is no definitive approach for assessing mesenteric ischemia and determining the optimal timing for endovascular intervention in the management of spontaneous isolated dissection of the superior mesenteric artery (SISMAD). A 56-year-old male with acute abdominal pain was diagnosed with SISMAD. After evaluating mesenteric ischemia through mesenteric fractional flow reserve (FFR), FFR was 0.72, and the patient was recommended conservative treatment for SISMAD, which involves fasting, total parenteral nutrition, and anticoagulation. The patient's syndrome was relieved after conservative treatment for 14 days without stent implantation. Over the next 5 years, no recurrence of abdominal pain or worsening of SISMAD was observed in the patient. Assessing the severity of mesenteric ischemia can be done through mesenteric FFR. Upon confirmation of the exclusion of risks related to dilatation or rupture of SISMAD aneurysm, an approach in favor of conservative management for SISMAD may indeed be considered pragmatic when the FFR exceeds 0.72.

Effective enhancement of the ability of Monascus pilosus to produce lipid-lowering compound Monacolin K via perturbation of metabolic flux and histone acetylation modification.

Monacolin K (MK), also known as lovastatin, is a polyketide compound with the ability to reduce plasma cholesterol levels and many other bio-activities. Red yeast rice (also named Hongqu) rich in MK derived from Monascus fermentation has attracted widespread attention due to its excellent performance in reducing blood lipids. However, industrial Monascus fermentation suffers from the limitations such as low yield of MK, long fermentation period, and susceptibility to contamination. In this study, we firstly blocked the competitive pathway of MK biosynthesis to create polyketide synthase gene pigA (the key gene responsible for the biosynthesis of Monascus azaphilone pigments) deficient strain A1. Then, based on the strategies to increase precursor supply for MK biosynthesis, acetyl-CoA carboxylase gene acc overexpression strains C1 and C2 were constructed with WT and A1 as the parent, respectively. Finally, histone deacetylase gene hos2 overexpression strain H1 was constructed by perturbation of histone acetylation modification. HPLC detection revealed all these four strains significantly increased their abilities to produce MK. After 14 days of solid-state fermentation, the MK yields of strains A1, C1, C2, and H1 reached 2.03 g/100 g, 1.81 g/100 g, 2.45 g/100 g and 2.52 g/100 g, which increased by 28.5 %, 14.7 %, 43.9 % and 36.1 % compared to WT, respectively. RT-qPCR results showed that overexpression of hos2 significantly increased the expression level of almost all genes responsible for MK biosynthesis after 5-day growth. Overall, the abilities of these strains to produce MK has been greatly improved, and MK production period has been shortened to 14 days from 20 days, providing new approaches for efficient production of Hongqu rich in MK.

New Horizons of Covalent Complex of Plant-Derived Recombinant Human Lactoferrin (OsrhLF) Combined with Different Polyphenols: Formation, Physicochemical Properties, and Gastrointestinal Fate.

Four typical dietary polyphenols ((-)-epigallocatechin gallate (EGCG), quinic acid (QA), caffeic acid (CA), and ferulic acid (FA)) were covalently prepared with rice recombinant human lactoferrin (OsrhLF) and bovine lactoferrin (bLF), and their structure and physicochemical properties were investigated, different lycopene emulsions were made by ultrasonic emulsification to analyze gastrointestinal fate. The results indicated that the covalent modification polyphenols changed the secondary/tertiary structure of LF, significantly improving the surface hydrophilicity, thermal stability, and antioxidant activity of LF. Compared with the bLF group, the OsrhLF group was more hydrophilic and the thermal denaturation temperature of the OsrhLF-CA reached 104.4 °C. LF-polyphenol emulsions significantly enhanced the photochemical stability and bioavailability of lycopene and achieved effective encapsulation and protection of lycopene compared to free lycopene, and the OsrhLF-EGCG reached 58.94% lycopene bioavailability. In short, OsrhLF does not differ much from bLF in terms of physicochemical properties and has a strong potential in the field of dietary supplements.

Comprehensive bioinformatics analysis revealed potential key genes and pathways underlying abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a permanent, asymptomatic segmental dilatation of the abdominal aorta, with a high mortality risk upon rupture. Identification of potential key genes and pathways may help to develop curative drugs for AAA. We conducted RNA-seq on abdominal aortic tissues from both AAA patients and normal individuals as a control group. Integrated bioinformatic analysis was subsequently performed to comprehensively reveal potential key genes and pathways. A total of 1148 differential expressed genes (DEGs) (631 up-regulated and 517 down-regulated) were identified in our study. Gene Ontology (GO) analysis revealed enrichment in terms related to extracellular matrix organization, while KEGG analysis indicated enrichment in hematopoietic cell lineage and ECM-receptor interaction. Protein-protein interaction (PPI) network analysis revealed several candidate key genes, and differential expression of 6 key genes (CXCL8, CCL2, PTGS2, SELL, CCR7, and CXCL1) was validated by Gene Expression Omnibus (GEO) datasets. Receiver operating characteristic curve (ROC) analysis demonstrated these genes' high discriminatory ability between AAA and normal tissues. Immunohistochemistry indicated that several key genes were highly expressed in AAA tissues. Single-cell RNA sequencing revealed differential distribution patterns of these identified key genes among various cell types. 26 potential drugs linked to our key genes were found through DGIdb. Overall, our study provides a comprehensive evaluation of potential key genes and pathways in AAA, which could pave the way for the development of curative pharmacological therapies.

Decoding the temporal and regional specification of microglia in the developing human brain.

Region-related heterogeneity and state transitions of microglia are important for brain development and neurological pathogenesis. However, regional specialization and state transition in microglia during early human CNS development remain unclear. Here, we profile single-cell transcriptomes of microglia from distinct regions of the developing human brain, and combined with experimental verification, we define and characterize early microglial fate determinations related to regional specification and state transition. We identified several subclasses of neuronal gene-enriched microglia with regional specification that dynamically and transiently appeared as early brain regions formed. In contrast, immune-related microglia were regionally specialized at later stages of CNS development. Surprisingly, we discovered that region-specialized immune-related microglia exit from a relative resting state and transition into distinct active states. In addition, we experimentally verified the microglial state transition. Finally, we showed that the state transition is conserved but that there are molecular differences in developing microglia in humans and mice.

CYR61, regulated by miR-22-3p and MALAT1, promotes autophagy in HK-2 cell inflammatory model.

BACKGROUND: Renal tubular epithelial cells play an important role in renal function and are a major site of injury from inflammation. Emerging evidence suggests that CYR61 is involved in the regulation of autophagy. However, there are few studies on CYR61 in nephropathy and associated inflammation. This study aimed to clarify how CYR61 regulates autophagy in human renal epithelial cells while in an inflammatory state and regulates the upstream pathway of CYR61 levels. METHODS: The human renal tubular epithelial cells (HK-2) cell line treated by lipopolysaccharide (LPS) was used as an inflammatory model of human epithelial cells. Short hairpin RNA (shRNA) was used to down-regulate CYR61, and the changes in the transcription and expression levels of related molecules, as well as the morphological changes of HK-2 cells, were detected by quantitative real time-PCR (qRT-PCR), western blot (WB), and transmission electron microscopy. Either CYR61 or MALAT1 were up-regulated by overexpression vectors, or MALAT1 was down-regulated by miR-22-3p mimics. Subsequently, the levels of CYR61, MALAT1, related inflammatory factors, and autophagy factors were measured by qPCR, WB, and enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was detected by flow cytometry and acridine-orange assay. RESULTS: We observed that down-regulation of CYR61 could down-regulate 1B-light chain 3 (LC3) level and inhibit autophagy in the LPS-induced inflammation model of HK-2 cells. The expression levels of CYR61, Beclin1, Atg5, LC3, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were significantly increased by upregulating CYR61 or MALAT1 by overexpression vector, while the expression level of p62 was significantly decreased, intracellular reactive oxygen species (ROS) content was increased, and the proportion of autophagy and apoptosis was increased. The use of miR-22-3p mimics significantly reversed the changes induced by up-regulation of CYR61 or MALAT1 at the molecular and cellular levels. CONCLUSIONS: Our data indicated that CYR61 positively regulates autophagy of HK-2 cells under an inflammatory state, and was negatively regulated by miR-22-3p, while miR-22-3p and MALAT1 were negatively regulated by each other.

A study revealing volatile aroma produced by Pediococcus pentosaceus in dough fermentation.

Pediococcus pentosaceus is important probiotics in Chinese Laomian. Its role in meat and fermented vegetable has been largely demonstrated, but few studies have investigated the role of P. pentosaceus in Chinese Laomian. For this purpose, we simulated Laomian fermentation using Saccharomyces cerevisiae and P. pentosaceus. Volatile aroma was detected by headspace solid-phase microextraction gas-chromatography-mass spectrometry. Real-time fluorescent quantitative polymerase chain reaction was used to determine dynamic growth of S. cerevisiae and P. pentosaceus in fermentation. Extracellular proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Folin-Ciocalteu method was used to detect extracellular protease activity in different pH values. Owing to addition of P. pentosaceus, the types and contents of esters increase, the relative contents of acetic acid hexyl ester, formic acid octyl ester, and heptanoic acid ethyl ester rise obliviously; especially, the relative content of hexanoic acid ethyl ester was highly correlated with P. pentosaceus by increasing 20.61%. As the gel electrophoresis results display, due to mixed fermentation of S. cerevisiae and P. pentosaceus, the 25k Da and 51k Da proteins expression quantity of P. pentosaceus clearly increased. Under neutral and alkaline culture conditions, the extracellular protease activity of P. pentosaceus is higher. This research benefits to gain insight into the fermentation actions of P. pentosaceus in Chinese Laomian.

[Study on the inclusion technology of volatile oil in Shiquandabu pills].

OBJECTIVE: To extract volatile oil from Shiquandabu pills and study technological process of its inclusion compound with beta-Cyclodextrin (beta-CD) to improve its stability. METHODS: The study was carried out with steam distillation and orthogonal design. The process conditions were studied by determining the utilization and ratio of oil from Shiquandabu pills volatile oil and extract ratio of in-clusion compound. The most important factor was the ratio of oil to beta-CD. and the inclusion compound was identified by FT-IR spectra and XRD. RESULTS: The optimum preparation conditions for inclusion were best-established as oil: beta-CD was 1:8, the inclusion time and temperature were for 1.0 hours. at 20 degrees C. CONCLUSION: Tne method can be used for increasing volatile oil's stability and its solubility. It is suitable for production of medicinal preparation.

Raloxifene inhibits bone loss and improves bone strength through an Opg-independent mechanism.

The osteoblast-derived paracrine factor osteoprotegerin (OPG) is considered to play a key role in inhibition of osteoclast formation and activity. Recently, raloxifene, a nonsteroidal benzothiophene, was found to exert anti-resorptive effects via modulating OPG expression in osteoblasts. To explore whether raloxifene regulates bone metabolism via an OPG-dependant pathway in vivo, we investigated the effects of raloxifene on bone loss in Opg-deficient mice. The results show that bone mineral density and bone strength are increased in mice deficient for Opg after treatment with raloxifene for 30 days. Histomorphometric analysis shows that raloxifene can increase bone trabecular area and decrease the number of osteoclasts in Opg (-/-) mice. Moreover, raloxifene reduces Rankl transcription and serum level of Rankl, which is dramatically increased in Opg knockout mice. These results suggest that raloxifene-induced inhibition of bone resorption may be independent of Opg pathway in mice.