High-yield anaerobic succinate production by strategically regulating multiple metabolic pathways based on stoichiometric maximum in Escherichia coli.

BACKGROUND: Succinate has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals, which can be used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Escherichia coli are now one of the most important succinate producing candidates. However, the stoichiometric maximum succinate yield under anaerobic conditions through the reductive branch of the TCA cycle is restricted by NADH supply in E. coli. RESULTS: In the present work, we report a rational approach to increase succinate yield by regulating NADH supply via pentose phosphate (PP) pathway and enhancing flux towards succinate. The deregulated genes zwf243 (encoding glucose-6-phosphate dehydrogenase) and gnd361 (encoding 6-phosphogluconate dehydrogenase) involved in NADPH generation from Corynebacterium glutamicum were firstly introduced into E. coli for succinate production. Co-expression of beneficial mutated dehydrogenases, which removed feedback inhibition in the oxidative part of the PP pathway, increased succinate yield from 1.01 to 1.16 mol/mol glucose. Three critical genes, pgl (encoding 6-phosphogluconolactonase), tktA (encoding transketolase) and talB (encoding transaldolase) were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose. Furthermore, introducing Actinobacillus succinogenes pepck (encoding phosphoenolpyruvate carboxykinase) together with overexpressing sthA (encoding soluble transhydrogenase), further increased succinate yield to 1.31 mol/mol glucose. In addition, removing byproduct formation through inactivating acetate formation genes ackA-pta and heterogenously expressing pyc (encoding pyruvate carboxylase) from C. glutamicum led to improved succinate yield to 1.4 mol/mol glucose. Finally, synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield to 1.54 mol/mol glucose, representing 52 % increase relative to the parent strain and amounting to 90 % of the strain-specific stoichiometric maximum (1.714 mol/mol glucose). CONCLUSIONS: It's the first time to rationally regulate pentose phosphate pathway to improve NADH supply for succinate synthesis in E. coli. 90 % of stoichiometric maximum succinate yield was achieved by combining further flux increase towards succinate and engineering its export. Regulation of NADH supply via PP pathway is therefore recommended for the production of products that are NADH-demanding in E. coli.

Emerging strategies for prospective discovery of molecular glue degraders.

Molecular glue (MG) degraders are monovalent small molecule compounds that co-opt E3 ubiquitin ligases to target neo-substrates for proteasomal degradation. Here, we provide a concise review of recent advances in rational MG discovery, which are categorized into two major strategies, ligand modification and de novo discovery. We also highlight the structural mechanisms underlying the formation of MG-enabled ternary complexes and their thermodynamic properties. Finally, we summarize the broader category of proximity inducers including MGs, proteolysis-targeting chimeras (PROTACs), peptides, and viral proteins. MGs are specified as a unique class of proximity inducers with chemical simplicity and a requirement of pre-existing weak protein-protein interactions. We propose that leveraging the weak basal interaction provides a starting point to prospectively develop MGs to degrade high-value therapeutic targets.

DCAF15 control of cohesin dynamics sustains acute myeloid leukemia.

The CRL4-DCAF15 E3 ubiquitin ligase complex is targeted by the aryl-sulfonamide molecular glues, leading to neo-substrate recruitment, ubiquitination, and proteasomal degradation. However, the physiological function of DCAF15 remains unknown. Using a domain-focused genetic screening approach, we reveal DCAF15 as an acute myeloid leukemia (AML)-biased dependency. Loss of DCAF15 results in suppression of AML through compromised replication fork integrity and consequent accumulation of DNA damage. Accordingly, DCAF15 loss sensitizes AML to replication stress-inducing therapeutics. Mechanistically, we discover that DCAF15 directly interacts with the SMC1A protein of the cohesin complex and destabilizes the cohesin regulatory factors PDS5A and CDCA5. Loss of PDS5A and CDCA5 removal precludes cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication, and apoptosis. Collectively, our findings uncover an endogenous, cell autonomous function of DCAF15 in sustaining AML proliferation through post-translational control of cohesin dynamics.

Preoperative urine sediment chromosomal instability level predicts urothelial cancer prognosis.

PURPOSE: Urothelial carcinomas (UCs) are often characterized by frequent recurrences after surgery, making UC one of the costliest cancers. Chromosomal instability (CIN) has been proven to be a hallmark of UCs and is related to the prognosis of many cancer types. In this study, we evaluated CIN of urine sediments as a prognostic indicator for UCs. METHODS: Patients with UC were prospectively recruited. Preoperative urine samples were collected for whole genome sequencing and urine cytology tests. Patients underwent standard-of-care treatment and were followed up until disease relapse or study ended. Concordance and accuracy of CIN alone or in combination with cytology in predicting disease relapse were assessed. The value of CIN combined with European Organization for Research and Treatment of Cancer (EORTC) model were also analyzed. RESULTS: A total of 137 patients with UCs were included in this study. Median follow-up was 44.2 months and 41.61% patients suffered from cancer relapse. Patients with CIN-high indicated higher relapse rate, and this distinction was significant for patients underwent transurethral resection of bladder tumor (57.89% vs. 34.29%, P = 0.016). Combination of cytology and CIN result could further classified patients into subgroups with distinct relapse risks. Meanwhile, the combination of CIN and EORTC model significantly improved the prediction accuracy compared with EORTC alone (Harrel's C-index: 0.71 vs. 0.65). CONCLUSION: CIN level of preoperative urine exfoliated cells had robust prognostic value for bladder cancer patients underwent TURBT. The prognostic model by combining CIN and EORTC may help in stratifying patients to optimize follow-up regimen.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Multiproxy probing of anthropogenic influences on the different components of dissolved organic matter in coastal rainwater.

In an environment that is tightly linked to humankind, how anthropogenic activity affects the quality and quantity of dissolved organic matter (DOM) in atmospheric depositions is not well understood. In this study, dissolved organic carbon (DOC), UV-vis spectra combined with molecular markers, including formic acid (FA), acetic acid (AA) and dissolved black carbon (DBC), were applied to track the temporal variation and influential factors of rainwater DOM at a coastal site. The ranges of DOC, light absorption at 254 nm (a254), FA, AA and DBC were 23.2-471 µmol L-1, 0.16-10.6 m-1, 0.12-23.5 µmol L-1, 0.44-37.8 µmol L-1 and 0.02-4.8 µmol L-1, respectively. The negative correlations between DOC, a254, AA and precipitation amount revealed a dilution effect. The concentrations of all measured DOM components were statistically different among different seasons with the highest value in spring. Higher DOM concentrations also occurred in the rain with backward trajectories influenced by the land. Compared to the nearby riverine DOM, the DOC-specific UV absorbance (SUVA254) of rainwater was lower, suggesting lower aromaticity of rainwater DOM. Significant correlations among different DOM components suggest that they shared similar sources or were affected by the same processes, while the significant correlations with anions (SO42-, F- and NO3-) and the ratio of FA to AA all suggested that the direct emission and secondary production from anthropogenic emissions (fossil fuel burning, biomass and biofuel burning) played important roles in regulating the level of DOM concentration in rainwater. Correlations with environmental variables (PM2.5, CO and NO2) further confirmed the input from anthropogenic activities. Furthermore, the monthly wet atmospheric deposition fluxes of DOM components (except DBC) can be successfully simulated by monthly precipitation and monthly average values of PM2.5 and NO2. Future studies should examine how atmospheric deposition affects the biogeochemical cycles in coastal regions.

Opioids in cancer: The κ­opioid receptor (Review).

The κ­opioid receptor (KOR) is one of the primary receptors of opioids and serves a vital role in the regulation of pain, anesthesia, addiction and other pathological and physiological processes. KOR is associated with several types of cancer and may influence cancer progression. It has been proposed that KOR may represent a new tumor molecular marker and provide a novel basis for molecular targeted therapies for cancer. However, the association between KOR and cancer remains to be explored comprehensively. The present review introduces KOR and its association with different types of cancer. Improved understanding of KOR may facilitate development of novel antitumor therapies.

Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor.

c-Myc (hereafter, Myc) is a cancer driver whose abundance is regulated by the SCFFbw7 ubiquitin ligase and proteasomal degradation. Fbw7 binds to a phosphorylated Myc degron centered at threonine 58 (T58), and mutations of Fbw7 or T58 impair Myc degradation in cancers. Here, we identify a second Fbw7 phosphodegron at Myc T244 that is required for Myc ubiquitylation and acts in concert with T58 to engage Fbw7. While Ras-dependent Myc serine 62 phosphorylation (pS62) is thought to stabilize Myc by preventing Fbw7 binding, we find instead that pS62 greatly enhances Fbw7 binding and is an integral part of a high-affinity degron. Crystallographic studies revealed that both degrons bind Fbw7 in their diphosphorylated forms and that the T244 degron is recognized via a unique mode involving Fbw7 arginine 689 (R689), a mutational hotspot in cancers. These insights have important implications for Myc-associated tumorigenesis and therapeutic strategies targeting Myc stability.

Copy number variation of urine exfoliated cells by low-coverage whole genome sequencing for diagnosis of prostate adenocarcinoma: a prospective cohort study.

BACKGROUND: Non-invasive, especially the urine-based diagnosis of prostate cancer (PCa) remains challenging. Although prostate cancer antigen (PSA) is widely used in prostate cancer screening, the false positives may result in unnecessary invasive procedures. PSA elevated patients are triaged to further evaluation of free/total PSA ratio (f/t PSA), to find out potential clinically significant PCa before undergoing invasive procedures. Genomic instability, especially chromosomal copy number variations (CNVs) were proved much more tumor specific. Here we performed a prospective study to evaluate the diagnostic value of CNV via urine-exfoliated cell DNA analysis in PCa. METHODS: We enrolled 28 PSA elevated patients (≥ 4 ng/ml), including 16 PCa, 9 benign prostate hypertrophy (BPH) and 3 prostatic intraepithelial neoplasia (PIN). Fresh initial portion urine was collected after hospital admission. Urine exfoliated cell DNA was analyzed by low coverage Whole Genome Sequencing, followed by CNV genotyping by the prostate cancer chromosomal aneuploidy detector (ProCAD). CNVs were quantified in absolute z-score (|Z|). Serum free/total PSA ratio (f/t PSA) was reported altogether. RESULTS: In patients with PCa, the most frequent CNV events were chr3q gain (n = 2), chr8q gain (n = 2), chr2q loss (n = 4), and chr18q loss (n = 3). CNVs were found in 81.2% (95% Confidence Interval (CI) 53.7-95.0%) PCa. No CNV was identified in BPH patients. A diagnosis model was established by incorporating all CNVs. At the optimal cutoff of |Z|≥ 2.50, the model reached an AUC of 0.91 (95% CI 0.83-0.99), a sensitivity of 81.2% and a specificity of 100%. The CNV approach significantly outperformed f/t PSA (AUC = 0.62, P = 0.012). Further analyses showed that the CNV positive rate was significantly correlated with tumor grade. CNVs were found in 90.9% (95% CI 57.1-99.5%) high grade tumors and 60.0% (95% CI 17.0-92.7%) low grade tumors. No statistical significance was found for patient age, BMI, disease history and family history. CONCLUSIONS: Urine exfoliated cells harbor enriched CNV features in PCa patients. Urine detection of CNV might be a biomarker for PCa diagnosis, especially in terms of the clinically significant high-grade tumors.

Study on the Effect of Different Doses of Hydromorphone on the Time Response and Postoperative Analgesia of Ropivacaine in Ultrasound-Guided Suprailiac Fascia Inguinal Block.

OBJECTIVE: To investigate the effects of different doses of hydromorphone under the guidance of ultrasound on ropivacaine blocking the superior inguinal iliac fascia and postoperative analgesia. METHODS: From January 2020 to June 2021, 90 American Society of Anesthesiologists (ASA) I-II patients undergoing elective total hip arthroplasty (THA) were selected and randomly divided into 3 groups, 30 patients in each one. Ultrasound-guided superior inguinal iliac fascia block was performed in the patients of the 3 groups before operation. The L group: 0.3% ropivacaine 30 ml; the H1 group: 0.3% ropivacaine + 25 µg/kg hydromorphone 30 ml; the H2 group: 0.3% ropivacaine + 50 µg/kg hydromorphone 30 ml. The time until the occurrence of pain, pain intensity, sufentanil remedial dose, the number of PCIA presses, and effective times were compared among the 3 groups. The VAS and Ramsay scores of 3 groups were recorded at 12 h, 24 h, 36 h, and 48 h after operation. RESULTS: The time from the end of surgery to the appearance of pain in the H2 group was higher than that in the H1 group and the L group, and the time in the H1 group was higher than that in the L group (P < 0.05). The VAS score in the H2 group was lower than that in the H1 group and the L group, and the VAS score in the H1 group was lower than that in the L group (P < 0.05). The VAS scores of 12 h, 24 h, 36 h, and 48 h after operation in the H2 group were lower than those of the H1 group and the L group, and the H1 group was lower than the L group (P < 0.05). The Ramsay scores at 12 h, 24 h, 36 h, and 48 h after operation in the H2 group and the H1 group were higher than those in the L group (P < 0.05), and the difference was not statistically significant in the H2 group and the H1 group (P > 0.05). The remedial dosage of sufentanil, times of PCIA compression, and effective times in the H2 group were lower than those in the H1 group and the L group, and the level in the H1 group was lower than that in the L group (P < 0.05). The incidence rates of adverse reactions in the L group, the H1 group, and the H2 group were 13.33%, 23.33%, and 30.00%, respectively. There was no significant difference in the incidence rate of adverse reactions among the 3 groups (P > 0.05). CONCLUSION: 25 µg/kg and 50 µg/kg hydromorphone used in the ultrasound-guided superior inguinal iliac fascia block can enhance the time effect of ropivacaine and improve analgesic effects, with good safety. In addition, time effect and analgesic effect of 50 µg/kg hydromorphone in enhancing ropivacaine were more obvious.

Cascades between miRNAs, lncRNAs and the NF-κB signaling pathway in gastric cancer (Review).

Gastric cancer is a common digestive tract malignancy that is mainly treated with surgery combined with perioperative adjuvant chemoradiotherapy and biological targeted therapy. However, the diagnosis rate of early gastric cancer is low and both postoperative recurrence and distant metastasis are thorny problems. Therefore, it is essential to study the pathogenesis of gastric cancer and search for more effective means of treatment. The nuclear factor-κB (NF-κB) signaling pathway has an important role in the occurrence and development of gastric cancer and recent studies have revealed that microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are able to regulate this pathway through a variety of mechanisms. Understanding these interrelated molecular mechanisms is helpful in guiding improvements in gastric cancer treatment. In the present review, the functional associations between miRNAs, lncRNAs and the NF-κB signaling pathway in the occurrence, development and prognosis of gastric cancer were discussed. It was concluded that miRNAs and lncRNAs have complex relations with the NF-κB signaling pathway in gastric cancer. miRNAs/target genes/NF-κB/target proteins, signaling molecules/NF-κB/miRNAs/target genes, lncRNAs/miRNAs/NF-κB/genes or mRNAs, lncRNAs/target genes/NF-Κb/target proteins, and lncRNAs/NF-κB/target proteins cascades are all important factors in the occurrence and development of gastric cancer.

Influence of Dexmedetomidine on Diaphragm Function and Postoperative Outcomes in ICU Patients with Mechanical Ventilation.

OBJECTIVE: To probe into the influence of dexmedetomidine (DEX) on diaphragm function and postoperative outcomes of mechanically ventilated patients in the intensive care unit (ICU). METHODS: 84 patients with mechanical ventilation (MV) in the ICU of our hospital were selected as the research participants, including 38 patients in the control group (CG) sedated with midazolam (MZ) and 46 patients in the research group (RG) with DEX sedation. Ramsay sedation score, visual analogue scale (VAS), and restlessness score (RS) were used to evaluate their state before sedation (T0), as well as 2 h (T1), 6 h (T2), and 24 h (T3) after sedation, and the alterations of mean arterial pressure (MAP) and heart rate (HR) were recorded. Serum cortisol (Cor), adrenocorticotropic hormone (ACTH), superoxide dismutase (SOD), malondialdehyde (MDA), interleukin- (IL-) 1ß, IL-6, and tumor necrosis factor-α (TNF-α) were measured before and 24 h after sedation. The end-inspiratory diaphragm thickness (DTei) and end-expiratory diaphragm thickness (DTee) were measured within 2 h after the initiation of MV and 5 min after the spontaneous breathing test (SBT), and the diaphragm thickening fraction (DTF) was calculated. Finally, the ventilator weaning, MV time, and the incidence of adverse reactions (ADs) of the two groups were counted. RESULTS: T0 and T3 witnessed no distinct difference in Ramsay, VAS, and RS scores between the two arms (P > 0.05), but at T1 and T2, RG had better sedation state and lower VAS and RS scores than CG (P < 0.05), with more stable vital signs (P < 0.05). After sedation, the contents of oxidative stress and inflammatory factors in RG were lower, while DTee, DTei, and DTF were higher, versus CG (P < 0.05). Moreover, RG presented higher success rate of first ventilator weaning, less MV time, and lower incidence of ADs than CG (P < 0.05). CONCLUSIONS: DEX is effective in mechanically ventilated patients in the ICU, which can protect patients against diaphragm function damage, improve the success rate of ventilator weaning, and benefit the postoperative outcome, with excellent and rapid sedation effect and less stress damage to patients.

Interplay between cyclooxygenase­2 and microRNAs in cancer (Review).

Tumor­associated inflammation and aberrantly expressed biomarkers have been demonstrated to play crucial roles in the cancer microenvironment. Cyclooxygenase­2 (COX­2), a prominent inflammatory factor, is highly expressed in tumor cells and contributes to tumor growth, recurrence and metastasis. Overexpression of COX­2 may occur at both transcriptional and post­transcriptional levels. Thus, an improved understanding of the regulatory mechanisms of COX­2 can facilitate the development of novel antitumor therapies. MicroRNAs (miRNAs) are a group of small non­coding RNAs that act as translation repressors of target mRNAs, and play vital roles in regulating cancer development and progression. The present review discusses the association between miRNAs and COX­2 expression in different types of cancer. Understanding the regulatory role of miRNAs in COX­2 post­transcription can provide novel insight for suppressing COX­2 expression via gene silencing mechanisms, which offer new perspectives and future directions for the development of novel COX­2 selective inhibitors based on miRNAs.

MicroRNA and cyclooxygenase-2 in breast cancer.

Cancer remains a major public health problem worldwide and the latest statistics show that breast cancer (BC) is among the most frequent in women. MicroRNAs (miRNAs; miRs) and cyclooxygenase-2 (COX-2) are new diagnostic and therapeutic biomarkers for monitoring BC. COX-2 is a prominent tumor-associated inflammatory factor highly expressed in human tumor cells, including BC. Expression of COX-2 contributes to tumor growth, metastasis and recurrence. MiRs are a group of short (~22 nucleotides), noncoding regulatory RNAs that downregulate gene expression post-transcriptionally and play vital roles in regulating cancer development and progression. Interestingly, there are a group of miRNAs differentially expressed in breast tumor tissue. Understanding the pathway linking miRNAs to COX-2 can provide novel insight for suppressing COX-2 expression via gene silencing thereby leading to the development of selective miRNA inhibitors. Further research can also reveal key intermediate players and their potential as therapeutic targets. Given the association between different miRNAs and COX-2 expression in BC, this review presents a comprehensive overview of the current literature concerning how miRNAs and COX-2 signaling interact in BC progression.

Noninvasive Detection of Urothelial Carcinoma by Cost-effective Low-coverage Whole-genome Sequencing from Urine-Exfoliated Cell DNA.

PURPOSE: Urothelial carcinoma is a malignant cancer with frequent chromosomal aberrations. Here, we investigated the application of a cost-effective, low-coverage whole-genome sequencing technology in detecting all chromosomal aberrations. EXPERIMENTAL DESIGN: Patients with urothelial carcinomas and nontumor controls were prospectively recruited in clinical trial NCT03998371. Urine-exfoliated cell DNA was analyzed by Illumina HiSeq XTen, followed by genotyping with a customized bioinformatics workflow named Urine Exfoliated Cells Copy Number Aberration Detector (UroCAD). RESULTS: In the discovery phase, urine samples from 126 patients with urothelial carcinomas and 64 nontumor disease samples were analyzed. Frequent chromosome copy-number changes were found in patients with tumor as compared with nontumor controls. A novel diagnosis model, UroCAD, was built by incorporating all the autosomal chromosomal changes. The model reached performance of AUC = 0.92 (95% confidence interval, 89.4%-97.3%). At the optimal cutoff, |Z| ≥ 3.21, the sensitivity, specificity, and accuracy were 82.5%, 96.9%, and 89.0%, respectively. The prediction positivity was found correlated with tumor grade (P = 0.01). In the external validation cohort of 95 participants, the UroCAD assay identified urothelial carcinomas with an overall sensitivity of 80.4%, specificity of 94.9%, and AUC of 0.91. Meanwhile, UroCAD assay outperformed cytology tests with significantly improved sensitivity (80.4% vs. 33.9%; P < 0.001) and comparable specificity (94.9% vs. 100%; P = 0.49). CONCLUSIONS: UroCAD could be a robust urothelial carcinoma diagnostic method with improved sensitivity and similar specificity as compared with cytology tests. It may be used as a noninvasive approach for diagnosis and recurrence surveillance in urothelial carcinoma prior to the use of cystoscopy, which would largely reduce the burden on patients.

Comparison of the effect and clinical value in general anesthesia and combined spinal-epidural anesthesia in elderly patients undergoing hip arthroplasty.

Effect and clinical value in general anesthesia and combined spinal-epidural anesthesia in elderly patients undergoing hip arthroplasty were compared. One hundred and six patients with hip arthroplasty in the Affiliated Nanhua Hospital, University of South China from May 2013 to July 2015 were selected as the research subjects, including 50 patients in the study group who received combined spinal-epidural anesthesia by ondansetron hydrochloride tablets combined with spinal-epidural puncture kit, and 56 patients in the control group who received general anesthesia by fast-induced endotracheal intubation. Retrospective analysis was performed in terms of anesthesia effect, complete block time, anesthesia onset time, hemodynamic parameters at different time points before and after the surgery, and adverse reactions after the surgery. The study group had a statistically shorter onset time and a statistically shorter complete block time than the control group (P<0.05). No significant difference in the heart rate, systolic blood pressure or diastolic blood pressure before the surgery in the two groups was shown (P>0.05); the heart rate, systolic blood pressure, and diastolic blood pressure in the study group 20 min after the start of the operation and 15 min before the end of the operation were significantly higher those in the control group (P<0.05); the adverse reactions such as venous thrombosis, pulmonary infection, and nausea and vomiting in the study group were fewer than those in the control group (P<0.05). For elderly patients with fracture surgery, both the general anesthesia and the combined spinal-epidural anesthesia can maintain a good anesthesia effect, but the combined spinal-epidural anesthesia can shorten the onset time and has less impact on the patient's hemodynamic parameters and less incidence of complications, thus worthy of clinical promotion.

Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.

Indisulam and related sulfonamides recruit the splicing factor RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase, resulting in RBM39 ubiquitination and degradation. Here, we used a combination of domain mapping and random mutagenesis to identify domains or residues that are necessary for indisulam-dependent RBM39 ubiquitination. DCAF15 mutations at Q232 or D475 prevent RBM39 recruitment by indisulam. RBM39 is recruited to DCAF15 by its RRM2 (RNA recognition motif 2) and is ubiquitinated on its N terminus. RBM23, which is an RBM39 paralog, is also recruited to the CRL4-DCAF15 ligase through its RRM2 domain and undergoes sulfonamide-dependent degradation. Indisulam alters the expression of more than 3,000 genes and causes widespread intron retention and exon skipping. All of these changes can be attributed to RBM39, and none are the consequence of RBM23 degradation. Our findings demonstrate that indisulam selectively degrades RBM23 and RBM39, the latter of which is critically important for splicing and gene expression.

Aldehyde dehydrogenase 1A1 increases NADH levels and promotes tumor growth via glutathione/dihydrolipoic acid-dependent NAD+ reduction.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is a member of the aldehyde dehydrogenase superfamily that oxidizes aldehydes to their corresponding acids, reactions that are coupled to the reduction of NAD+ to NADH. We report here that ALDH1A1 can also use glutathione (GSH) and dihydrolipoic acid (DHLA) as electron donors to reduce NAD+ to NADH. The GSH/DHLA-dependent NAD+-reduction activity of ALDH1A1 is not affected by the aldehyde dehydrogenase inhibitor or by mutation of the residues in its aldehyde-binding pocket. It is thus a distinct biochemical reaction from the classic aldehyde-dehydrogenase activity catalyzed by ALDH1A1. We also found that the ectopic expression of ALDH1A1 decreased the intracellular NAD+/NADH ratio, while knockout of ALDH1A1 increased the NAD+/NADH ratio. Simultaneous knockout of ALDH1A1 and its isozyme ALDH3A1 in lung cancer cell line NCI-H460 inhibited tumor growth in a xenograft model. Moreover, the ALDH1A1 mutants that retained their GSH/DHLA-dependent NAD+ reduction activity but lost their aldehyde-dehydrogenase activity were able to decrease the NAD+/NADH ratio and to rescue the impaired growth of ALDH1A1/3A1 double knockout tumor cells. Collectively, these results suggest that this newly characterized GSH/DHLA-dependent NAD+-reduction activity of ALDH1A1 can decrease cellular NAD+/NADH ratio and promote tumor growth.

Metabolic engineering of Escherichia coli and in silico comparing of carboxylation pathways for high succinate productivity under aerobic conditions.

A novel aerobic succinate production system was strategically designed that allows Escherichia coli to produce and accumulate succinate with high specific productivity under aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhA, iclR) and byproduct formation pathways (poxB, ackA-pta, mgsA) of E. coli were created to construct the glyoxylate cycle and oxidative branch of the TCA cycle for aerobic succinate production. Strain ZJG13 (ΔsdhA, ΔackA-pta, ΔpoxB, ΔmgsA, ΔiclR) exhibited normal growth behavior and accumulated succinate with an average specific productivity of 0.50mmolg CDW(-1)h(-1) during the fermentation. The glyoxylate shunt operon aceKBA was overexpressed by introducing plasmid pT9aceKAB to ZJG13; the resulting strain had minor effect on productivity improvement. To fully understand the effect of the carboxylation reactions on succinate production, three reactions catalyzed by pyruvate carboxylase (PYC), malic enzyme (MAEA) and phosphoenolpyruvate carboxylase (PPC) were analyzed by a Computational Approach for Strain Optimization aiming at high Productivity (CASOP). Based on the CASOP analysis, carboxylation reaction catalyzed by PYC was the most suitable one to obtain high productivity. When pyc was overexpressed in ZJG13, the specific succinate productivity further increased to 0.76mmolg CDW(-1)h(-1). Fed-batch culture of the strain ZJG13/pT184pyc led to a titer of 36.1g/L succinate, with a specific productivity of 2.75mmolg CDW(-1)h(-1) which stands for the highest value among currently reported aerobic bacterial succinate producers. These results indicate that the CASOP strategy is useful as a guiding tool for the rational strain design with high productivity.