An immune cell map of human lung adenocarcinoma development reveals an anti-tumoral role of the Tfh-dependent tertiary lymphoid structure.

The immune responses during the initiation and invasion stages of human lung adenocarcinoma (LUAD) development are largely unknown. Here, we generated a single-cell RNA sequencing map to decipher the immune dynamics during human LUAD development. We found that T follicular helper (Tfh)-like cells, germinal center B cells, and dysfunctional CD8+ T cells increase during tumor initiation/invasion and form a tertiary lymphoid structure (TLS) inside the tumor. This TLS starts with an aggregation of CD4+ T cells and the generation of CXCL13-expressing Tfh-like cells, followed by an accumulation of B cells, and then forms a CD4+ T and B cell aggregate. TLS and its associated cells are correlated with better patient survival. Inhibiting TLS formation by Tfh or B cell depletion promotes tumor growth in mouse models. The anti-tumoral effect of the Tfh-dependent TLS is mediated through interleukin-21 (IL-21)-IL-21 receptor signaling. Our study establishes an anti-tumoral role of the Tfh-dependent TLS in the development of LUAD.

An immune cell atlas reveals the dynamics of human macrophage specification during prenatal development.

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.

Cryopreservation did not affect sperm DNA methylation levels of genes related to fertilization and embryonic development of cynomolgus macaque (Macaca fascicularis).

DNA methylation alters gene expression in numerous biological processes, including embryonic development. It is little known about the effect of cryopreservation on sperm DNA methylation. The present study has investigated whether cryopreservation causes abnormal DNA methylation in cynomolgus macaque sperm for five critical genes that includes the maternally imprinted gene (SNRPN), genes associated with male infertility (HSPA1L, MTHFR) and genes involved in embryonic development (TET3, LZTR1). Our results showed that sperm motility, the percentage of acrosomal integrity, DNA integrity and mitochondrial membrane potential were decreased after cryopreservation either being frozen with penetrating cryoprotectant, glycerol (Gly) or ethylene glycol (EG), compared to fresh sperm (p = 0.000), but the methylation patterns of the five target genes from cynomolgus macaque sperm samples were not affected after cryopreservation as evaluated by the Bisulfite Sequencing PCR (BSP) method. The data indicates that the current protocol for sperm cryopreservation of cynomolgus macaque is safe in terms of DNA methylation levels in these genes related to critical sperm functions.

Simultaneous and trace level quantification of two potential genotoxic impurities in valsartan drug substance using UPLC-MS/MS.

A sensitive and selective Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the identification and quantification of two potential genotoxic impurities (PGIs) - viz. methyl N-((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)-N-nitroso-L-valinate (PGI-1) and N-nitroso Valsartan (PGI-2) - in the angiotensin II receptor blocker valsartan. Among these impurities, PGI-1 is a distinctive compound which has never been reported. For this, chromatographic separation was performed using a Waters XBridge BEH C18 column (150 mm × 4.6 mm, 2.5 µm), with ammonium acetate aqueous solution (0.01 mol/L) as mobile phase A and acetonitrile as mobile phase B, in a gradient elution mode at a 0.5 mL/min flow rate. Mass spectrometric conditions were optimized using electrospray ionization (ESI) in positive mode. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying 2 PGIs at 0.016 ppm in samples at 50 mg/mL concentration. This validated approach presented good linearity over the concentration range of 0.016-0.06 ppm for 2 PGIs. The correlation coefficient of each impurity was observed greater than 0.999. The accuracy of this method was in the range of 83-113% for the aforementioned PGIs. In addition, expert knowledge rules (Derek-based) and statistical (Q) SAR evaluation system (Sarah-based) were used to evaluate and classify the genotoxicity of both valsartan-related PGIs as well as to define their standard limits. The predicted results were positive and classified into the third category, and the total nitrosamine limit was set to 0.03 ppm. As such, this approach represents a good quality control system for the simultaneous and precise quantitation of PGIs in valsartan.

Identifying of Anti-Thrombin Active Components From Curcumae Rhizoma by Affinity-Ultrafiltration Coupled With UPLC-Q-Exactive Orbitrap/MS.

Recent studies concerning products that originate from natural plants have sought to clarify active ingredients, which both explains the mechanisms of the function and aids in quality control during production. As a traditional functional plant, Curcumae Rhizoma (CR) has been proven to be effective in promoting blood circulation and removing blood stasis. However, the components that play a role in its huge compound library are still unclear. The present study aimed to develop a high-throughput screening method to identify thrombin inhibitors in CR and validate them by in vitro and in vivo experiments. The effect of CR on thrombin in HUVECs cells was determined by ELISA, then an affinity-ultrafiltration-UPLC-Q-Exactive Orbitrap/MS approach was applied. Agatroban and adenosine were used as positive and negative drugs respectively to verify the reliability of the established method. The in vitro activity of the compounds was determined by specific substrate S-2238. The in vivo effect of the active ingredients was determined using zebrafish. Molecular docking was used to understand the internal interactions between compounds and enzymes. ELISA results showed that CR had an inhibitory effect on thrombin. The screening method established in this paper is reliable, by which a total of 15 active compounds were successfully identified. This study is the first to report that C7, 8, and 11 have in vitro thrombin-inhibitory activity and significantly inhibit thrombosis in zebrafish models at a safe dose. Molecular docking studies were employed to analyze the possible active binding sites, with the results suggesting that compound 16 is likely a better thrombin inhibitor compared with the other compounds. Based on the affinity-ultrafiltration-UPLC-Q-Exactive Orbitrap/MS approach, a precisely targeted therapy method using bio-active compounds from CR might be successfully established, which also provides a valuable reference for targeted therapy, mechanism exploration, and the quality control of traditional herbal medicine.

[Analysis of volatile oil components of different species of Curcumae Rhizoma based on GC-MS and chemometrics].

The volatile oil of Curcumae Rhizoma has many active components,which are the key to the quality of Curcumae Rhizoma. Exploring the difference between volatile oil of different kinds of Curcumae Rhizoma facilitates the quality control and rational application of resources. In this study,GC-MS was applied to realize online qualitative and semi-quantitative analysis of the chemical composition spectrum of volatile oil from Curcuma wenyujin( CW),C. phaeocaulis( CP),and C. kwangsiensis( CK). Forty components were identified and their fingerprints were compared and evaluated. Hierarchical cluster analysis( HCA),principal component analysis( PCA),and orthogonal partial least squares discrimination analysis( OPLS-DA) were adopted to analyze the overall and outlier data. The results showed that the whole data could be divided into three kinds according to each analysis mode,and the volatile components of Curcumae Rhizoma vary greatly among species. PCA explored the difference between outliers and the mean value of the group and found that some volatile oils from CW may be greatly affected by the origin. By OPLS-DA,the samples from Zhejiang were able to gather,but those from Guizhou remained isolated,indicating the influence of growing environment on Curcumae Rhizoma metabolites. Based on VIP results combined with the heat map,characteristic volatile oil components of Curcumae Rhizoma from different varieties were screened out: curdione and linalool for CW; 2-undecanone for CP; humulene,γ-selinene,and zederone for CK. The GCMS method established in this study describes Curcumae Rhizoma samples comprehensively and accurately,and the characteristic components screened based on chemometrics can be used to distinguish Curcumae Rhizoma from different varieties and give them unique pharmacodynamic significance,which is fast,convenient,stable,and reliable and supports the rational application of Curcu-mae Rhizoma resources. It is found that the region of origin has great influence on CW,which is worthy of further study.

Downregulation of cyclooxygenase­1 stimulates mitochondrial apoptosis through the NF­κB signaling pathway in colorectal cancer cells.

The prognosis of patients with colorectal cancer (CRC) remains poor owing to diagnosis typically occurring at advanced stages of the disease. The understanding of the molecular regulatory signatures of CRC may lead to the identification of biomarkers for the early detection, prevention and clinical intervention of CRC. Epidemiological studies have indicated that cyclooxygenase­1 (COX­1) serves an active function in colon carcinogenesis. However, the molecular mechanism underlying COX­1 regulation in CRC remains unknown. In the present study, COX­1 was identified to be markedly upregulated in colorectal tissues of patients with CRC, and in the CRC cell lines HCT116 and HT29. To determine the function of COX­1 in cancer development, short hairpin RNA knockdown of COX­1 was employed in HCT116 and HT29 CRC cells in the present study. The results demonstrated that silencing of COX­1 depolarized the mitochondrial membrane potential, inhibited adenosine triphosphate production, induced the generation of intracellular reactive oxygen species and triggered caspase­dependent mitochondrial apoptosis. Furthermore, depletion of COX­1 suppressed anti­apoptotic B­cell lymphoma 2 and enhanced pro­apoptotic Bcl­2­associated X protein expression by inhibiting the p65 subunit phosphorylation of nuclear factor κB (NF­κB). Taken together, the results of the present study indicated that COX­1 inhibition significantly triggered cell death by destroying the mitochondrial function that is associated with deactivation of the NF­κB signaling pathway. These results suggest COX­1 as a potential anticancer target in CRC.

The Dual Role of MicroRNAs in Colorectal Cancer Progression.

Colorectal cancer (CRC) is responsible for one of the major cancer incidence and mortality worldwide. It is well known that MicroRNAs (miRNAs) play vital roles in maintaining the cell development and other physiological processes, as well as, the aberrant expression of numerous miRNAs involved in CRC progression. MiRNAs are a class of small, endogenous, non-coding, single-stranded RNAs that bind to the 3'-untranslated region (3'-UTR) complementary sequences of their target mRNA, resulting in mRNA degradation or inhibition of its translation as a post-transcriptional regulators. Moreover, miRNAs also can target the long non-coding RNA (lncRNA) to regulate the expression of its target genes involved in proliferation and metastasis of CRC. The functions of these dysregulated miRNAs appear to be context specific, with evidence of having a dual role in both oncogenes and tumor suppression depending on the cellular environment in which they are expressed. Therefore, the unique expression profiles of miRNAs relate to the diagnosis, prognosis, and therapeutic outcome in CRC. In this review, we focused on several oncogenic and tumor-suppressive miRNAs specific to CRC, and assess their functions to uncover the molecular mechanisms of tumor initiation and progression in CRC. These data promised that miRNAs can be used as early detection biomarkers and potential therapeutic target in CRC patients.

Bcl-2 delays cell cycle through mitochondrial ATP and ROS.

Bcl-2 inhibits cell proliferation by delaying G0/G1 to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G0/G1 phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G0/G1 arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1-3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.