High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma.

Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR = 0.74, 95% CI = 0.68-0.81, p = 3.08 × 10-11) and 17q12 (rs226241; OR = 1.42, 95% CI = 1.26-1.60, p = 1.62 × 10-8). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified PHF2 at locus 9q22.33 as a susceptibility gene. PHF2 encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene PHF2 by inhibiting the enhancer activity of its long-range (4.3 Mb) cis-regulatory element, which promoted proliferation of NPC cells. In addition, we identified CDKN2B-AS1 as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of CDKN2B-AS1 by increasing its enhancer activity. The overexpression of CDKN2B-AS1 facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.

Discovery of a selective TRF2 inhibitor FKB04 induced telomere shortening and senescence in liver cancer cells.

Telomere repeat binding factor 2 (TRF2), a critical element of the shelterin complex, plays a vital role in the maintenance of genome integrity. TRF2 overexpression is found in a wide range of malignant cancers, whereas its down-regulation could cause cell death. Despite its potential role, the selectively small-molecule inhibitors of TRF2 and its therapeutic effects on liver cancer remain largely unknown. Our clinical data combined with bioinformatic analysis demonstrated that TRF2 is overexpressed in liver cancer and that high expression is associated with poor prognosis. Flavokavain B derivative FKB04 potently inhibited TRF2 expression in liver cancer cells while having limited effects on the other five shelterin subunits. Moreover, FKB04 treatment induced telomere shortening and increased the amounts of telomere-free ends, leading to the destruction of T-loop structure. Consequently, FKB04 promoted liver cancer cell senescence without modulating apoptosis levels. In corroboration with these findings, FKB04 inhibited tumor cell growth by promoting telomeric TRF2 deficiency-induced telomere shortening in a mouse xenograft tumor model, with no obvious side effects. These results demonstrate that TRF2 is a potential therapeutic target for liver cancer and suggest that FKB04 may be a selective small-molecule inhibitor of TRF2, showing promise in the treatment of liver cancer.

Quantitative detection of Epstein-Barr virus DNA methylation in the diagnosis of nasopharyngeal carcinoma by blind brush sampling.

Detecting EBV DNA load in nasopharyngeal (NP) brushing samples for the diagnosis of nasopharyngeal carcinoma (NPC) has attracted widespread attentions. Currently, NP brush sampling mostly relies on endoscopic guidance, and there are few reports on diagnostic markers suitable for nonguided conditions (blind brush sampling), which is of great significance for extending its application. One hundred seventy nasopharyngeal brushing samples were taken from 98 NPC patients and 72 non-NPC controls under the guidance of endoscope, and 305 blind brushing samples were taken without endoscopic guidance from 164 NPC patients and 141 non-NPC controls (divided into discovery and validation sets). Among these, 38 cases of NPC underwent both endoscopy-guided NP brushing and blind brushing. EBV DNA load targeting BamHI-W region and EBV DNA methylation targeting 11029 bp CpG site located at Cp-promoter region were detected by quantitative polymerase chain reaction (q-PCR). EBV DNA load showed good classification accuracy for NPC in endoscopy-guided brushing samples (AUC = 0.984). However, in blind bushing samples, the diagnostic performance was greatly reduced (AUC = 0.865). Unlike EBV DNA load, the accuracy of EBV DNA methylation was less affected by brush sampling methods, whether in endoscopy-guided brushing (AUC = 0.923) or blind brushing (AUC = 0.928 in discovery set and AUC = 0.902 in validation set). Importantly, EBV DNA methylation achieved a better diagnostic accuracy than EBV DNA load in blind brushing samples. Overall, detection of EBV DNA methylation with blind brush sampling shows great potential in the diagnosis of NPC and may facilitate its use in nonclinical screening of NPC.

Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma.

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.

Clinical and genome-wide association analysis of chemoradiation-induced hearing loss in nasopharyngeal carcinoma.

Chemoradiation-induced hearing loss (CRIHL) is one of the most devasting side effects for nasopharyngeal carcinoma (NPC) patients, which seriously affects survivors' long-term quality of life. However, few studies have comprehensively characterized the risk factors for CRIHL. In this study, we found that age at diagnosis, tumor stage, and concurrent cisplatin dose were positively associated with chemoradiation-induced hearing loss. We performed a genome-wide association study (GWAS) in 777 NPC patients and identified rs1050851 (within the exon 2 of NFKBIA), a variant with a high deleteriousness score, to be significantly associated with hearing loss risk (HR = 5.46, 95% CI 2.93-10.18, P = 9.51 × 10-08). The risk genotype of rs1050851 was associated with higher NFKBIA expression, which was correlated with lower cellular tolerance to cisplatin. According to permutation-based enrichment analysis, the variants mapping to 149 hereditary deafness genes were significantly enriched among GWAS top signals, which indicated the genetic similarity between hereditary deafness and CRIHL. Pathway analysis suggested that synaptic signaling was involved in the development of CRIHL. Additionally, the risk score integrating genetic and clinical factors can predict the risk of hearing loss with a relatively good performance in the test set. Collectively, this study shed new light on the etiology of chemoradiation-induced hearing loss, which facilitates high-risk individuals' identification for personalized prevention and treatment.

Genomic Landscapes of Epstein-Barr Virus in Pulmonary Lymphoepithelioma-Like Carcinoma.

Epstein-Barr virus (EBV) infection is associated with multiple malignancies, including pulmonary lymphoepithelioma-like carcinoma (pLELC), a particular subtype of primary lung cancer. However, the genomic characteristics of EBV related to pLELC remain unclear. Here, we obtained the whole-genome data set of EBV isolated from 78 pLELC patients and 37 healthy controls using EBV-captured sequencing. Compared with the reference genome (NC_007605), a total of 3,995 variations were detected across pLELC-derived EBV sequences, with the mutational hot spots located in latent genes. Combined with 180 published EBV sequences derived from healthy people in Southern China, we performed a genome-wide association study and identified 32 variations significantly related to pLELC (P < 2.56 × 10-05, Bonferroni correction), with the top signal of single nucleotide polymorphism (SNP) coordinate T7327C (OR = 1.22, P = 2.39 × 10-15) locating in the origin of plasmid replication (OriP). The results of population structure analysis of EBV isolates in East Asian showed the EBV strains derived from pLELC were more similar to those from nasopharyngeal carcinoma (NPC) than other EBV-associated diseases. In addition, typical latency type-II infection were recognized for EBV of pLELC at both transcription and methylation levels. Taken together, we defined the global view of EBV genomic profiles in pLELC patients for the first time, providing new insights to deepening our understanding of this rare EBV-associated primary lung carcinoma. IMPORTANCE Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare, distinctive subtype of primary lung cancer closely associated with Epstein-Barr virus (EBV) infection. Here, we gave the first overview of pLELC-derived EBV at the level of genome, methylation and transcription. We obtained the EBV sequences data set from 78 primary pLELC patients, and revealed the sequences diversity across EBV genome and detected variability in known immune epitopes. Genome-wide association analysis combining 217 healthy controls identifies significant variations related to the risk of pLELC. Meanwhile, we characterized the integration landscapes of EBV at the genome-wide level. These results provided new insight for understanding EBV's role in pLELC tumorigenesis.

Peptidome-wide association analysis of Epstein-Barr virus identifies epitope repertoires associated with nasopharyngeal carcinoma.

Human leukocyte antigen (HLA) molecules are essential for presenting Epstein-Barr virus (EBV) antigens and are closely related to nasopharyngeal carcinoma (NPC). This study aims to systematically investigate the association between HLA-bound EBV peptides and NPC risk through in silico HLA-peptide binding prediction. A total of 455 NPC patients and 463 healthy individuals in NPC endemic areas were recruited, and HLA-target sequencing was performed. HLA-peptide binding prediction for EBV, followed by peptidome-wide logistic regression and motif analysis, was applied. Binding affinity changes for EBV peptides carrying high-risk mutations were analyzed. We found that NPC-associated EBV peptides were significantly enriched in immunogenic proteins and core linkage disequilibrium (LD) proteins related to evolution, especially those binding HLA-A alleles (p = 3.10 × 10-4 for immunogenic proteins and p = 8.10 × 10-5 for core LD proteins related to evolution). These peptides were clustered and showed binding motifs of HLA supertypes, among which supertype A02 presented an NPC-risk effect (padj = 3.77 × 10-4 ) and supertype A03 presented an NPC-protective effect (padj = 4.89 × 10-4 ). Moreover, a decreased binding affinity toward risk HLA supertype A02 was observed for the peptide carrying the NPC-risk mutation BNRF1 V1222I (p = 0.0078), and an increased binding affinity toward protective HLA supertype A03 was observed for the peptide carrying the NPC-risk mutation BALF2 I613V (p = 0.022). This study revealed the distinct preference of EBV peptides for binding HLA supertypes, which may contribute to shaping EBV population structure and be involved in NPC development.

The interaction and mediation effects between the host genetic factors and Epstein-Barr virus VCA-IgA in the risk of nasopharyngeal carcinoma.

Previous studies have demonstrated strong associations between host genetic factors and Epstein-Barr virus (EBV) VCA-IgA with the risk of nasopharyngeal carcinoma (NPC). However, the specific interplay between host genetics and EBV VCA-IgA on NPC risk is not well understood. In this two-stage case-control study (N = 4804), we utilized interaction and mediation analysis to investigate the interplay between host genetics (genome-wide association study-derived polygenic risk score [PRS]) and EBV VCA-IgA antibody level in the NPC risk. We employed a four-way decomposition analysis to assess the extent to which the genetic effect on NPC risk is mediated by or interacts with EBV VCA-IgA. We consistently found a significant interaction between the PRS and EBV VCA-IgA on NPC risk (discovery population: synergy index [SI] = 2.39, 95% confidence interval [CI] = 1.85-3.10; replication population: SI = 3.10, 95% CI = 2.17-4.44; all pinteraction < 0.001). Moreover, the genetic variants included in the PRS demonstrated similar interactions with EBV VCA-IgA antibody. We also observed an obvious dose-response relationship between the PRS and EBV VCA-IgA antibody on NPC risk (all ptrend < 0.001). Furthermore, our decomposition analysis revealed that a substantial proportion (approximately 90%) of the genetic effects on NPC risk could be attributed to host genetic-EBV interaction, while the risk effects mediated by EBV VCA-IgA antibody were weak and statistically insignificant. Our study provides compelling evidence for an interaction between host genetics and EBV VCA-IgA antibody in the development of NPC. These findings emphasize the importance of implementing measures to control EBV infection as a crucial strategy for effectively preventing NPC, particularly in individuals at high genetic risk.

A systematic review and meta-analysis on the efficacy outcomes of selective serotonin reuptake inhibitors in depression in Alzheimer's disease.

BACKGROUND: Depressive symptoms are the most common neuropsychiatric symptoms in patients with Alzheimer's disease (AD). However, despite being common, no definite consensus recommendations exist for the management of depression in AD. OBJECTIVE: To assess the effects of selective serotonin reuptake inhibitors (SSRIs) on the alleviation of depressive symptoms in patients with AD. MATERIAL AND METHODS: Medline, Scopus, Web of Science, Google Scholar, and PsychINFO were electronically searched from inception until October 2022. Response to therapy and mean depression scores between the treatment (or before) and placebo (or after) groups were the primary outcomes. For depression scores, the standard mean deviation and accompanying 95% confidence interval were determined. The risk of bias was determined using the funnel plot, trim and fill, Egger's and Begg's analyses. RESULTS: SSRIs attenuated depressive symptoms in patients with AD (0.905 SMD, 95%CI, 0.689 to 1.121, p < 0.000). At individual SSRI level, escitalopram, paroxetine, and sertraline significantly alleviated depressive symptoms in AD patients (0.813 SMD, 95%CI, 0.207 to 1.419, p = 0.009, 1.244 SMD, 95%CI, 0.939 to 1.548, p < 0.000, and 0.818 SMD, 95%CI, 0.274 to 1.362, p < 0.000). The funnel plot, trim and fill, Begg's test (p = 0.052), and Egger's test (p = 0.148), showed no significant risk of publication bias. CONCLUSION: Our meta-analysis supports the use of SSRIs for the alleviation of depression in patients with AD. However, we recommend larger randomized clinical trials that would compare the efficacy of different SSRIs in AD patients with depression.

Discovery of ß-cyclocitral-derived mono-carbonyl curcumin analogs as anti-hepatocellular carcinoma agents via suppression of MAPK signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high recurrence and mortality rate. In this study, a series of ß-cyclocitral-derived mono-carbonyl curcumin analogs were synthesized and their anticancer properties were evaluated. Among the series, A19 exhibited the strongest cytotoxic activity by inhibiting cell viability and colony formation, inducing cell cycle G2/M phase arrest and cell apoptosis of HCC HepG2 and Huh-7 cells, while having almost no cytotoxicity on normal liver MIHA cells. Mechanistically, our results demonstrated that A19 triggered intense DNA damage via suppression of the ERK/JNK/p38 MAPK signaling pathway. Additionally, a combination of A19 with sorafenib significantly induced synergistic cytotoxicity in HCC cells. Overall, our results indicate the potential of A19 as a novel chemotherapeutic drug administered either separately or in combined therapy for HCC treatment.

Research progress on metabolomics in the quality evaluation and clinical study of Panax ginseng.

Panax ginseng, an essential component of traditional medicine and often referred to as the king of herbs, has played a pivotal role in medicine globally for several millennia. Previously, traditional phytochemical methods were mainly used for quality evaluation and pharmacological mechanism studies of ginseng, resulting in the lack of systematicness and innovation and hindering the development and utilization of ginseng resources. Since the beginning of the new century, systems biology technology represented by metabolomics has shown unique advantages in the modernization and internationalization of herbal medicine, establishing a bridge for communication between traditional medicine and modern medicine. P. ginseng, a special herb used in medicine and food, is one of the main research objects for qualitative and quantitative analysis of metabolomics and has gradually become the focus of researchers globally. Here, we conducted a comprehensive summary and analysis of numerous studies published in ginseng metabolomics. This review aims to provide more novel ideas for the quality evaluation, development, and clinical application of ginseng in the future and offer more useful technical references for the modernization and internationalization of herbal medicine based on metabolomics.

Efficacy and Postoperative Complication Rates of Different Surgical Approaches for Calcaneal Fractures: A Network Meta-analysis.

Context: Calcaneal fractures (CFs) are the most common kind of tarsal fracture. The choice of surgical approach is a key element in the management of CFs, but the best method remains in dispute. Also, no single approach is appropriate for all kinds of CFs. Objective: The study intended to evaluate the relationship between six surgical approaches for clinical treatment of CFs and prevention of postoperative complications, to provide an evidence-based approach for treatment. Design: The research team performed a meta-analysis using the data from a previously published review and updating that data through a new narrative review. The team performed a systematic search in PubMed, Embase, the Cochrane Library, and the Chinese National Knowledge Internet (CNKI) from inception until January 2022, with no language restrictions. The search used the following keywords for the search: calcaneus, heel bone, surgical wounds, surgical incisions, prospective trials, prospective trials, and randomized controlled trials. Outcome Measures: The research team compared the complication rates, American Orthopedic Foot and Ankle Society (AOFAS) scores, and Bohler's angles for the six surgical approaches, which were: (1) the extensive lateral approach (ELA), (2) the sinus tarsi approach (STA), (3) the horizontal arc approach (HAA), (4) the longitudinal approach (LA), (5) the oblique lateral incision (OLI), and (6) the modified incision (MI)). The team summarized the results using a random effects model. Results: The research team analyzed the data from 19 RCTs with 1521 participants. They all were randomized controlled trials (RCTs). The complication rates were available for 18 studies, which included 1474 participants. The rates were significantly lower: (1) for HAA compared to ELA, [OR=-2.03; 95% CrI: [-3.63, -0.43)]; (2) for LA compared to ELA (OR=-1.83; 95% CrI: [-2.83, -0.84]); and (3) for STA compared to ELA (OR=- 1.22; 95% CrI: [-1.67, -0.78]). Of the 19 studies, 11 RCTs, with 942 participants, used the AOFAS scale. The probabilities for the surface under the cumulative ranking curve (SUCRA) indicated that OLI (0.694 ) >LA (0.596) >HAA (0.51) >STA (0.477) >ELA (0.224). In addition, ELA had the worst SUCRA (0.224). Of the 19 studies, 15 RCTs, with 1376 participants, used the Bohler angle as an outcome measure. The probability of SUCRA for the surgical approaches indicated that LA (0.723) >ELA (0.667) >STA (0.468) >HAA (0.373) >MI (0.27). Conclusions: The meta-analysis provides an evidence-based approach to the clinical treatment of CFs for six surgical approaches. HAA had the best outcomes, and ELA had the worst.

PHB2 Alleviates Neurotoxicity of Prion Peptide PrP106-126 via PINK1/Parkin-Dependent Mitophagy.

Prion diseases are a group of neurodegenerative diseases characterized by mitochondrial dysfunction and neuronal death. Mitophagy is a selective form of macroautophagy that clears injured mitochondria. Prohibitin 2 (PHB2) has been identified as a novel inner membrane mitophagy receptor that mediates mitophagy. However, the role of PHB2 in prion diseases remains unclear. In this study, we isolated primary cortical neurons from rats and used the neurotoxic prion peptide PrP106-126 as a cell model for prion diseases. We examined the role of PHB2 in PrP106-126-induced mitophagy using Western blotting and immunofluorescence microscopy and assessed the function of PHB2 in PrP106-126-induced neuronal death using the cell viability assay and the TUNEL assay. The results showed that PrP106-126 induced mitochondrial morphological abnormalities and mitophagy in primary cortical neurons. PHB2 was found to be indispensable for PrP106-126-induced mitophagy and was involved in the accumulation of PINK1 and recruitment of Parkin to mitochondria in primary neurons. Additionally, PHB2 depletion exacerbated neuronal cell death induced by PrP106-126, whereas the overexpression of PHB2 alleviated PrP106-126 neuronal toxicity. Taken together, this study demonstrated that PHB2 is indispensable for PINK1/Parkin-mediated mitophagy in PrP106-126-treated neurons and protects neurons against the neurotoxicity of the prion peptide.

C1632 suppresses the migration and proliferation of non-small-cell lung cancer cells involving LIN28 and FGFR1 pathway.

Chemoresistance and migration represent major obstacles in the therapy of non-small-cell lung cancer (NSCLC), which accounts for approximately 85% of lung cancer patients in clinic. In the present study, we report that the compound C1632 is preferentially distributed in the lung after oral administration in vivo with high bioavailability and limited inhibitory effects on CYP450 isoenzymes. We found that C1632 could simultaneously inhibit the expression of LIN28 and block FGFR1 signalling transduction in NSCLC A549 and A549R cells, resulting in significant decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metalloproteinase-9. Consequently, C1632 effectively inhibited the migration and invasion of A549 and A549R cells. Meanwhile, C1632 significantly suppressed the cell viability and the colony formation of A549 and A549R cells by inhibiting DNA replication and inducing G0/G1 cell cycle arrest. Interestingly, compared with A549 cells, C1632 possesses the same or even better anti-migration and anti-proliferation effects on A549R cells, regardless of drug resistance. In addition, C1632 also displayed the capacity to inhibit the growth of A549R xenograft tumours in mice. Altogether, these findings reveal the potential of C1632 as a promising anti-NSCLC agent, especially for chemotherapy-resistant NSCLC treatment.

The novel FGFR inhibitor F1-7 induces DNA damage and cell death in colon cells.

BACKGROUND: Fibroblast growth factor receptor (FGFR) signaling influenced tumour occurrence and development. Overexpression of FGFR had been observed in many types of cancers, including colon cancer. FGFR inhibitor is considered to be effective in treating colon cancer patients. METHODS: First, the kinase inhibition rate was determined. MTT, western blotting, colony formation, EdU and comet assays were performed to evaluate the anti-tumour effects of F1-7 in vitro. RNA-seq and bioinformatics analysis were used for further verification. Additionally, a xenograft model was generated to investigate the anti-tumour effect of F1-7. RESULTS: F1-7 can inhibit the proliferation of colon cancer cells in vitro. It could significantly inhibit FGFR phosphorylation and its downstream signaling pathway. Whole-genome RNA-seq analysis found that the changed genes were not only functionally focused on MAPK signaling pathway but also related to cell apoptosis and ferroptosis. Experimental evidence demonstrated that F1-7 can directly increase the level of cellular DNA damage. The occurrence of DNA damage led to cell cycle arrest and inhibition of cell metastasis and cell apoptosis. Mouse model experiments also confirmed that F1-7 could inhibit tumour growth by inhibiting the FGFR pathway. CONCLUSIONS: F1-7 exhibits anti-tumour activity by inhibiting the FGFR pathway. It could be a novel therapeutic agent for targeting colon cancer cells.

Genomic landscape of Epstein-Barr virus in familial nasopharyngeal carcinoma.

To better understand the genomic characteristics of Epstein-Barr virus (EBV) in familial nasopharyngeal carcinoma (NPC), we sequenced the EBV genomes by whole-genome capture in 38 unrelated patients with NPC family history in first-degree relatives and 47 healthy controls, including 13 with family history and 34 without. Compared with type 1 reference genome, mutation hotspots were observed in the latent gene regions of EBV in familial NPC cases. Population structure analysis showed that one cluster has a higher frequency in familial cases than in controls (OR=5.33, 95 % CI 2.50-11.33, P=1.42×10-5), and similar population structure composition was observed among familial and sporadic NPC cases in high-endemic areas. By genome-wide association analysis, four variants were found to be significantly associated with familial NPC. Consistent results were observed in the meta-analysis integrating two published case-control EBV sequencing studies in NPC high-endemic areas. High-risk haplotypes of EBV composed of 34 variants were associated with familial NPC risk (OR=13.85, 95 % CI 4.13-46.44, P=2.06×10-5), and higher frequency was observed in healthy blood-relative controls with NPC family history (9/13, 69.23 %) than those without family history (16/34, 47.06%). This study suggested the potential contribution of EBV high-risk subtypes to familial aggregation of NPC.

Ultra-Performance Liquid Chromatography-Q-Exactive Orbitrap-Mass Spectrometry Analysis for Metabolic Communication between Heart and Kidney in Adriamycin-Induced Nephropathy Rats.

BACKGROUND/AIMS: Although the adriamycin-induced nephropathy model is frequently employed in the study of nephrotic syndrome and focal segmental glomerulosclerosis, the accompanying myocardial damage has always been a cause for concern. Therefore, there is a great need to study cardiorenal communication in this model. METHODS: An adriamycin-induced nephropathy model was established via tail vein injection. The levels of the biochemical indicators serum albumin, serum globulin, serum total protein, serum cholesterol, serum creatinine (SCr), urinary protein, and urinary creatinine (UCr) were measured, and histopathological changes in the heart and kidneys were assessed using hematoxylin-eosin staining. Metabolomic changes in the heart, blood, and kidneys were analyzed using the metabolomics method based on ultra-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry. RESULTS: Compared with the control group, the model group showed significant decreases in serum protein and total protein levels, albumin/globulin ratio, and creatinine clearance rate as well as significant increases in serum cholesterol, SCr, urinary protein, and UCr levels. Significant pathological changes were observed in the renal pathology sections in the model group, including diffusely merged glomerular epithelial cells, inflammatory infiltration, and vacuolated glomerular cells. Additionally, thickened myocardial fibers, swollen nuclei, inflammatory infiltration, and partial myocardial necrosis could be seen in the cardiac pathology sections in the model group. Based on multivariate statistical analysis, a total of 20 differential metabolites associated with 15 metabolic pathways were identified in the heart, 7 differential metabolites with 7 metabolic pathways were identified in the blood, and 16 differential metabolites with 21 metabolic pathways were identified in the kidney. Moreover, 6 common metabolic pathways shared by the heart and kidney were identified: arginine and proline metabolism; arginine biosynthesis; glutathione metabolism; alanine, aspartate, and glutamate metabolism; beta-alanine metabolism; and histidine metabolism. Among these metabolic pathways, alanine, aspartate, and glutamate metabolism was shared by the heart, blood, and kidney. Succinic acid was found to be the key regulatory metabolite in cardiorenal metabolic communication. CONCLUSION: Six metabolic pathways were found to be involved in cardiorenal metabolic communication in an adriamycin-induced nephropathy model, in which alanine, aspartate, and glutamate metabolism may be the metabolic link between the heart and kidney in the development and maintenance of oxidative stress and inflammation. Succinic acid may serve as a key regulatory metabolic switch or marker of cardiac and renal co-injury, as shown in an adriamycin-induced nephropathy model.

Highly efficient biosynthesis of spermidine from L-homoserine and putrescine using an engineered Escherichia coli with NADPH self-sufficient system.

Spermidine is an important polyamine that can be used for the synthesis of various bioactive compounds in the food and pharmaceutical fields. In this study, a novel efficient whole-cell biocatalytic method with an NADPH self-sufficient cycle for spermidine biosynthesis was designed and constructed by co-expressing homoserine dehydrogenase (HSD), carboxyspermidine dehydrogenase (CASDH), and carboxyspermidine decarboxylase (CASDC). First, the enzyme-substrate coupled cofactor regeneration system from co-expression of NADP+-dependent ScHSD and NADPH-dependent AfCASDH exactly provides an efficient method for cofactor cycling. Second, we identified and characterized a putative CASDC with high decarboxylase activity from Butyrivibrio crossotus DSM 2876; it showed an optimum temperature of 35 °C and an optimum pH of 7.0, which make it better suited for the designed synthetic route. Subsequently, the protein expression level of each enzyme was optimized through the variation of the gene copy number, and a whole-cell catalyst with high catalytic efficiency was constructed successfully. Finally, a yield of 28.6 mM of spermidine was produced in a 1-L scale of E. coli whole-cell catalytic system with a 95.3% molar conversion rate after optimization of temperature, the ratio of catalyst-to-substrate, and the amount of NADP+, and a productivity of 0.17 g·L-1·h-1 was achieved. In summary, this novel pathway of constructing a whole-cell catalytic system from L-homoserine and putrescine could provide a green alternative method for the efficient synthesis of spermidine. KEY POINTS: • A novel pathway for spermidine biosynthesis was developed in Escherichia coli. • The enzyme-substrate coupled system provides an NADPH self-sufficient cycle. • Spermidine with 28.6 mM was obtained using an optimized whole-cell system.

Biosynthesis of phenylpyruvic acid from l-phenylalanine using chromosomally engineered Escherichia coli.

The efficiency of whole-cell biotransformation is often affected by the genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for the synthesis of phenylpyruvic acid (PPA) from l-phenylalanine. First, l-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into Escherichia coli BL21 (DE3) chromosome and the copy numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). Fifty-nine copies of pmLAAD were obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of isopropyl-ß-d-thiogalactopyranoside, the resulting strain, E. coli BL8△recA△lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37% increase in PPA production compared to E. coli BL8△recA. Finally, the engineered E. coli BL8△recA△lacI strain achieved 19.14 g/L PPA at 24 h in a 5-L bioreactor.

Converting the 3-quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4-chloroacetoacetate reductase by site-directed mutagenesis.

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat /Km ) for COBE was 51.36 s-1 ·mM-1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.