RNA editing increases the nucleotide diversity of SARS-CoV-2 in human host cells.

SARS-CoV-2 is a positive-sense, single-stranded RNA virus responsible for the COVID-19 pandemic. It remains unclear whether and to what extent the virus in human host cells undergoes RNA editing, a major RNA modification mechanism. Here we perform a robust bioinformatic analysis of metatranscriptomic data from multiple bronchoalveolar lavage fluid samples of COVID-19 patients, revealing an appreciable number of A-to-I RNA editing candidate sites in SARS-CoV-2. We confirm the enrichment of A-to-I RNA editing signals at these candidate sites through evaluating four characteristics specific to RNA editing: the inferred RNA editing sites exhibit (i) stronger ADAR1 binding affinity predicted by a deep-learning model built from ADAR1 CLIP-seq data, (ii) decreased editing levels in ADAR1-inhibited human lung cells, (iii) local clustering patterns, and (iv) higher RNA secondary structure propensity. Our results have critical implications in understanding the evolution of SARS-CoV-2 as well as in COVID-19 research, such as phylogenetic analysis and vaccine development.

Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome.

Surface biotinylation has been widely adapted in profiling the cellular proteome associated with the plasma membrane. However, the workflow is subject to interference from the cytoplasmic biotin-associated proteins that compete for streptavidin-binding during purification. Here we established a bioorthogonal conjugation-assisted purification (BCAP) workflow that utilizes the Staudinger chemoselective ligation to label and isolate surface-associated proteins while minimizing the binding of endogenous biotin-associated proteins. Label-free quantitative proteomics demonstrated that BCAP is efficient in isolating cell surface proteins with excellent reproducibility. Subsequently, we applied BCAP to compare the surface proteome of proliferating and senescent mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified and validated as a novel protein that is enhanced at the cell surface of senescent MEFs. We expect that BCAP will have broad applications in profiling cell surface proteomes in the future.

Novel multimodal analgesia regimen improves post-TACE pain in patients with hepatocellular carcinoma.

BACKGROUD: Transarterial chemoembolization (TACE) is the primary palliative treatment for patients with unresectable hepatocellular carcinoma (HCC). However, it is often accompanied by postoperative pain which hinder patient recovery. This study was to examine whether preemptive parecoxib and sufentanil-based patient controlled analgesia (PCA) could improve the pain management in patients receiving TACE for inoperable HCC. METHODS: From June to December 2016, 84 HCC patients undergoing TACE procedure were enrolled. Because of the willingness of the individuals, it is difficult to randomize the patients to different groups. We matched the patients' age, gender and pain scores, and divided the patients into the multimodal group (n = 42) and control group (n = 42). Patients in the multimodal group received 40 mg of parecoxib, 30 min before TACE, followed by 48 h of sufentanil-based PCA. Patients in the control group received a routine analgesic regimen, i.e., 5 mg of dezocine during operation, and 100 mg of tramadol or equivalent intravenous opioid according to patient's complaints and pain intensity. Postoperative pain intensity, percentage of patients as per the pain category, adverse reaction, duration of hospital stay, cost-effectiveness, and patient's satisfaction were all taken into consideration when evaluated. RESULTS: Compared to the control group, the visual analogue scale scores for pain intensity was significantly lower at 2, 4, 6, and 12 h (all P < 0.05) in the multimodal group and a noticeably lower prevalence of post-operative nausea and vomiting in the multimodal group (31.0% vs. 59.5%). Patient's satisfaction in the multimodal group was also significantly higher than that in the control group (95.2% vs. 69.0%). No significant difference was observed in the duration of hospital stay between the two groups. CONCLUSION: Preemptive parecoxib and sufentanil-based multimodal analgesia regime is a safe, efficient and cost-effective regimen for postoperative pain control in HCC patients undergoing TACE.

Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors.

Epigenetic reader proteins interpret histone epigenetic marks to regulate gene expression. Given their vital roles and the link between their dysfunction and various diseases, these proteins present compelling targets for therapeutic interventions. Nevertheless, designing selective inhibitors for these proteins poses significant challenges, primarily due to their unique properties such as shallow binding sites and similarities with homologous proteins. To overcome these challenges, we propose an innovative strategy that uses phage display with a genetically encoded noncanonical amino acid (ncAA) containing an epigenetic mark. This ncAA guides binding to the reader protein's active site, allowing the identification of peptide inhibitors with enhanced affinity and selectivity. In this study, we demonstrate this novel approach's effectiveness by identifying potent inhibitors for the ENL YEATS domain that plays a critical role in leukemogenesis. Our strategy involved genetically incorporating Nε-butyryl-l-lysine (BuK), known for its binding to ENL YEATS, into a phage display library for enriching the pool of potent inhibitors. One resultant hit was further optimized by substituting BuK with other pharmacophores to exploit a unique π-π-π stacking interaction with ENL YEATS. This led to the creation of selective ENL YEATS inhibitors with a KD value of 2.0 nM and a selectivity 28 times higher for ENL YEATS than its close homologue AF9 YEATS. One such inhibitor, tENL-S1f, demonstrated robust cellular target engagement and on-target effects to inhibit leukemia cell growth and suppress the expression of ENL target genes. As a pioneering study, this work opens up extensive avenues for the development of potent and selective peptidyl inhibitors for a broad spectrum of epigenetic reader proteins.

Dynamic Immune Landscape and VZV-Specific T Cell Responses in Patients With Herpes Zoster and Postherpetic Neuralgia.

Objectives: Varicella-zoster virus (VZV) can induce herpes zoster (HZ) and postherpetic neuralgia (PHN). Immune cells play an important role in regulating HZ and PHN pathogenesis, but the dynamic immune profiles and molecular mechanisms remain unclear. This study aimed to screen dynamic immune signatures during HZ progression and elucidate the mechanism of VZV-specific T cells in PHN. Methods: We used cytometry by time-of-flight (CyTOF) to analyze peripheral blood mononuclear cells (PBMC) samples from 45 patients with HZ and eight age-sex-matched healthy controls, eight PHN samples and seven non-PHN samples. Correlations between the immune subsets and clinical pain-related scores were performed. Further, the characteristics of VZV-specific T cells between PHN and non-PHN patients were evaluated by VZV peptide pools stimulation. The expression level of cytokines, including granzyme B, interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α was performed via cytometric bead array. Finally, we analyzed the alteration of Ca2+ signals in dorsal root ganglion (DRG)-derived cells after TNF-α stimulation. Results: We investigated the dynamic characteristics of the immune landscape of peripheral blood samples of patients with HZ and PHN, and depicted two major dynamic signatures in NK, CD4+ and CD8+ T subsets in patients with HZ, which closely correlated with clinical pain-related scores. The frequency of PD-1+CD4+ T cells, VZV-specific PD-1+CD4+ T cells, and the amount of TNF-α produced by VZV-specific T cells were higher in patients with PHN than without PHN. Furthermore, we showed that TNF-α could induce calcium influx in DRG-derived cells in a dose-dependent manner. Conclusions: Our results profiled the dynamic signatures of immune cells in patients with HZ and highlighted the important role of VZV-specific T cells in the pathogenesis of PHN.

Development of a Model for Predicting the Effectiveness of Pulsed Radiofrequency on Zoster-Associated Pain.

INTRODUCTION: Zoster-associated pain (ZAP), which may cause anxiety, depression, and sleep disorders and reduce quality of life, is often refractory to current standard treatments. Studies have shown that pulsed radiofrequency (PRF) can alleviate ZAP and reduce the incidence of postherpetic neuralgia (PHN). This study aimed to explore the clinical characteristics associated with PRF responsiveness, develop a model for identifying risk factors of inadequate PRF management, and help clinicians make better decisions. METHODS: Patients who underwent PRF for ZAP between January 2017 and October 2020 in our hospital were included in this study. Patients were evaluated using the numerical rating scale (NRS), Insomnia Severity Index, Patient Health Questionnaire-9, and 36-Item Short Form Health Survey (SF-36) before and 3 months after the procedure. Patient demographic data and blood test results were also collected. We defined the effectiveness of PRF for ZAP as relief of > 50% in NRS scores compared to pre-PRF. Least absolute shrinkage and selection operator (LASSO) regression analyses were subsequently performed to identify factors related to the therapeutic effect of PRF in patients with ZAP. The performance of the prediction model was assessed by the area under the receiver operating characteristic curve (AUC). RESULTS: The effectiveness of PRF in patients with ZAP was 69.6% (total 313 patients) after 3 months. LASSO regression analysis extracted the seven most powerful features in the developed prediction model: sex, stage of herpes zoster (HZ), pregabalin dose, bodily pain indicators of SF-36, lymphocyte count, and low-density lipoprotein cholesterol (LDLC) and complement C4 in peripheral blood. Model = 1.586 + 0.148 × lymphocyte + (-0.001) × bodily pain indicators of SF-36 + (-0.001) × pregabalin dose + 0.028 × LDLC + 0.001 × C4 + (-0.508) × sex + (-0.128) × stage of HZ. We generated the ROC curve for the prediction model, and the final AUC was 0.701. The sensitivity, specificity, and overall accuracy of the model were 90%, 33%, and 73%, respectively. CONCLUSIONS: Seven factors were significantly associated with poor PRF outcome: male sex, advanced stage of HZ, higher pregabalin dose, higher bodily pain indicators of SF-36, and lower lymphocyte count, LDLC, and complement C4 in the peripheral blood. PRF should be applied to patients with ZAP as early as possible to achieve satisfactory outcomes.

Encapsulating an acid-activatable phthalocyanine-doxorubicin conjugate and the hypoxia-sensitive tirapazamine in polymeric micelles for multimodal cancer therapy.

A zinc(ii) phthalocyanine (ZnPc) was conjugated to doxorubicin (Dox) via an acid-labile hydrazone linker. The resulting ZnPc-Dox conjugate was then encapsulated into polymeric micelles formed through self-assembly of a block copolymer of poly(ethylene glycol) and poly(d,l-lactide) both in the absence and presence of the hypoxia-activated prodrug tirapazamine (TPZ) to give ZnPc-Dox@micelles and ZnPc-Dox/TPZ@micelles respectively. These polymeric micelles exhibited an excellent stability in aqueous media, but underwent disassembly in an acidic environment. Upon internalisation into HT29 human colorectal carcinoma cells, fluorescence due to ZnPc and Dox could be observed in the cytoplasm and nucleus respectively for both nanosystems. This observation suggested the disassembly of the polymeric micelles and the cleavage of the hydrazone linker in ZnPc-Dox in the acidic intracellular compartments. These micelles were slightly cytotoxic against HT29 cells in the dark due to the chemotherapeutic effect of Dox and/or TPZ. Upon light irradiation, ZnPc-Dox@micelles showed higher cytotoxicity. The IC50 value under a normoxic condition (0.35 µM based on ZnPc-Dox) was significantly lower than that under hypoxia (>1 µM). With an additional therapeutic component, ZnPc-Dox/TPZ@micelles exhibited higher photocytotoxicity with IC50 values of 0.20 µM and 0.78 µM under normoxia and hypoxia respectively. It is believed that the photodynamic action of this nanosystem consumed the intracellular oxygen and hence triggered the hypoxia-mediated chemotherapeutic action of TPZ. The multimodal antitumor effects of these polymeric micelles were also validated on HT29 tumour-bearing nude mice.

Reliability and validity of a modified 8-item Morisky Medication Adherence Scale in patients with chronic pain.

BACKGROUND: The 8-item Morisky Medication Adherence Scale (MMAS-8) is a simple, economic and easy tool to evaluate the medication compliance of chronic disease. The reliability and validity of the MMAS-8 in patients with chronic pain were unclear. Therefore, we aimed to validate the MMAS-8 for detecting nonadherent patients with chronic pain. METHODS: A modified MMAS-8 was used to assess the medication compliance of patients with chronic pain who were treated at our hospital from July 2018 to October 2018. Cronbach's α was used to evaluate the internal consistency, and a factor analysis was used to examine the construct validity. Convergent validity was assessed by comparing the MMAS-8 and a medication adherence visual analog score (MA-VAS) through Pearson's correlation coefficient. RESULTS: A total of 113 patients were evaluated. The (t-test) results revealed that there was a significant difference in average scores between the low-score group (who scored less than 5 points) and the high-score group (who scored 8 points or above), indicating that the scale displayed a good degree of discrimination. Except for Items 4 and 5, all the other items exhibited a good correlation with the total score (correlation coefficient >0.5; P<0.05). The Cronbach's α coefficient was 0.625, indicating that the scale's internal consistency was relatively satisfactory. Two common factors, which explained 62.978% of the total variance, were extracted by factor analysis to examine the construct validity of the MMAS-8, and the load of the 6 items was greater than 0.4. The Pearson correlation coefficient was 0.845 (P<0.001); thus, convergent validity was high. CONCLUSIONS: The modified MMAS-8 exhibited acceptable reliability and validity in evaluating medication compliance in patients with chronic pain; thus, it can be applied to detect nonadherent patients with chronic pain.

Analysis of the genomic landscape of yolk sac tumors reveals mechanisms of evolution and chemoresistance.

Yolk sac tumors (YSTs) are a major histological subtype of malignant ovarian germ cell tumors with a relatively poor prognosis. The molecular basis of this disease has not been thoroughly characterized at the genomic level. Here we perform whole-exome and RNA sequencing on 41 clinical tumor samples from 30 YST patients, with distinct responses to cisplatin-based chemotherapy. We show that microsatellite instability status and mutational signatures are informative of chemoresistance. We identify somatic driver candidates, including significantly mutated genes KRAS and KIT and copy-number alteration drivers, including deleted ARID1A and PARK2, and amplified ZNF217, CDKN1B, and KRAS. YSTs have very infrequent TP53 mutations, whereas the tumors from patients with abnormal gonadal development contain both KRAS and TP53 mutations. We further reveal a role of OVOL2 overexpression in YST resistance to cisplatin. This study lays a critical foundation for understanding key molecular aberrations in YSTs and developing related therapeutic strategies.

Efficacy and safety of treating chronic nonspecific low back pain with radial extracorporeal shock wave therapy (rESWT), rESWT combined with celecoxib and eperisone (C + E) or C + E alone: a prospective, randomized trial.

BACKGROUND: To investigate whether respectively radial extracoporeal shock wave therapy (rESWT) or a combination of rESWT, celecoxib and eperisone (rESWT + C + E) are superior in reducing pain in patients with chronic nonspecific low back pain (cnsLBP) compared to C + E alone (a standard treatment of this condition in China). METHODS: 140 patients with cnsLBP were randomly allocated to rESWT (n = 47), rESWT + C + E (n = 45) or C + E alone (n = 48) for four weeks between November 2017 and March 2019. Outcome was evaluated using the Pain Self-Efficacy Questionnaire (PSEQ), Numerical Rating Scale (NRS), Oswestry Low Back Pain Disability Questionnaire and Patient Health Questionnaire 9, collected at baseline as well as one week (W1), W2, W3, W4 and W12 after baseline. RESULTS: All scores showed a statistically significant improvement over time. The PSEQ and NRS scores showed a significant Time × Treatment effect. Patients treated with rESWT had significantly lower mean NRS values than patients treated with rESWT + C + E at W1 and W3, as well as than patients treated with C + E alone at W3 and W4. No severe adverse events were observed. CONCLUSIONS: rESWT may not be inferior to respectively rESWT + C + E or C + E alone in reducing pain in patients with cnsLBP. LEVEL OF EVIDENCE: Level I, prospective, randomized, active-controlled trial. TRIAL REGISTRATION: Clinicaltrials.gov Identifier NCT03337607. Registered November 09, 2017, https://www.clinicaltrials.gov/ct2/show/NCT03337607 . LEVEL OF EVIDENCE: Level I; prospective, randomized, controlled trial.

Genomic characterization of co-existing neoplasia and carcinoma lesions reveals distinct evolutionary paths of gallbladder cancer.

Gallbladder carcinoma is the most common cancer of the biliary tract with dismal survival largely due to delayed diagnosis. Biliary tract intraepithelial neoplasia (BilIN) is the common benign tumor that is suspected to be precancerous lesions. However, the genetic and evolutionary relationships between BilIN and carcinoma remain unclear. Here we perform whole-exome sequencing of coexisting low-grade BilIN (adenoma), high-grade BilIN, and carcinoma lesions, and normal tissues from the same patients. We identify aging as a major factor contributing to accumulated mutations and a critical role of CTNNB1 mutations in these tumors. We reveal two distinct carcinoma evolutionary paths: carcinoma can either diverge earlier and evolve more independently or form through the classic adenoma/dysplasia-carcinoma sequence model. Our analysis suggests that extensive loss-of-heterozygosity and mutation events in the initial stage tend to result in a cancerous niche, leading to the subsequent BilIN-independent path. These results reframes our understanding of tumor transformation and the evolutionary trajectory of carcinogenesis in the gallbladder, laying a foundation for the early diagnosis and effective treatment of gallbladder cancer.

[Study on terahertz spectroscopy of Hotan jade].

Terahertz time-domain spectroscopy (THz-TDS) technique has a wide range of applications including illicit drugs and explosive detection, and organic molecules recognition. In the present paper, the spectral features of three kinds of Hotan jade were studied experimentally by THz-TDS technique and the characteristic absorption spectra and refractive index were obtained in the range of 0.2 to 2.6 THz. The experimental results show that different samples have different absorption characters, and the refractive index is 2.4-2.7 in the range of 0.2-2.6 THz. The results indicate that it is feasible to apply THz-TDS technique to identification of Hotan jade, which provides a new approach to the nondestructive examination of Hotan jade.

Genetic Polymorphisms of Cytokines Might Affect Postoperative Sufentanil Dosage for Analgesia in Patients.

OBJECTIVE: To explore the effect of genetic polymorphisms of cytokines on the dosage of sufentanil for patient-controlled intravenous analgesia (PCIA) after radical lung cancer surgery. METHODS: A total of 100 patients, aged 18 years and above, with ASA grade Ⅰ-Ⅱ and body mass index (BMI) 18.5 to 30, and who were scheduled for radical lung cancer surgery under total intravenous anaesthesia with PCIA of sufentanil from September 2015 to March 2016, were selected. DNA was collected from peripheral blood samples before surgery, and the iMLDRTM multiple single-nucleotide polymorphism typing kit was used to detect 16 related single-nucleotide polymorphism (SNP) sites of interleukin-1A (IL-1A), interleukin-1ß (IL-1ß), interleukin-1RN (IL-1RN), interleukin-6 (IL-6), C-X-C motif chemokine ligand 8 (CXCL8), interleukin-10 (IL-10), tumour necrosis factor (TNF), nuclear factor kappa-B1 (NFκB1), REL (REL proto-oncogene, NF-kB subunit), and nuclear factor kappa-B inhibitor alpha (NFκBIA). The general characteristics of patients, surgery and anaesthesia data, postoperative resting VAS pain scores, postoperative opioid dosages of sufentanil for PCIA and opioid-related adverse events were recorded. The effects of the examined genetic polymorphisms of the cytokines on the dosage of sufentanil were analysed. RESULTS: Eight of 100 patients withdrew for various reasons, and, eventually, 92 patients were included. The patients' resting visual analogue scale (VAS) scores at 24 h, 48 h, and 72 h after surgery were 2.3 ± 1.2, 2.0 ± 0.9, and 1.9 ± 1.0, respectively. The total amounts of sufentanil used were 34.7 ± 10.5 µg, 65.2 ± 13.7 µg, and 94.7 ± 11.6 µg, respectively. We found that the TT genotype of NFκBIA rs696 had higher PCIA sufentanil dosages than the CC genotype and the CT genotype at 48-72 h postoperation (p=0.023, p=0.025, respectively). CONCLUSION: The genetic polymorphisms of the cytokine NFκBIA rs696 might affect the dosage of sufentanil for PCIA after radical lung cancer surgery. The specific mechanism needs further study.

VGLUT2/Cdk5/p25 Signaling Pathway Contributed to Inflammatory Pain by Complete Freund's Adjuvant.

Vesicular glutamate transporter type 2 (VGLUT2) is known to play an important role in mediating heat hyperalgesia induced by inflammation. However, the underlying mechanism for this activity is poorly understood. Cyclin-dependent kinase 5 (Cdk5), serving as a key regulator in modulating release of glutamate, acted a key player in the formation of heat hyperalgesia of inflammatory pain. However, it remains unknown whether there is a bridge between Cdk5 and VGLUT2 for mediating inflammatory pain. Therefore, we designed the experiment to determine whether VGLUT2 signaling pathway is involved in inflammatory pain mediated by Cdk5 in the inflammatory pain model induced by complete Freund's adjuvant (CFA). Our results showed that the coexpression of Cdk5/VGLUT2 in small- and medium-sized neuronal cells of the dorsal root ganglion (DRG) and spinal cord between days 1 and 3 following subcutaneous injection of CFA was significantly increased. Moreover, our study revealed that the expression of VGLUT2 protein in the DRG and spinal cord was remarkably increased between days 1 and 3 following CFA injection and was significantly reduced by roscovitine, a selective antagonist of Cdk5. Additionally, p25 but not p35, an activator of Cdk5, protein was significantly increased by CFA and reduced by roscovitine. Our findings suggested that VGLUT2/Cdk5 signaling pathway contributes to inflammatory pain mediated by Cdk5/p25.

Effects of ghrelin on pGSK-3ß and ß-catenin expression when protects against neuropathic pain behavior in rats challenged with chronic constriction injury.

Ghrelin has been shown to alleviate neuropathic pain by inhibiting the release of proinflammatory cytokines. The purpose of this study was to investigate the role of GSK-3ß/ß-catenin signaling in mediating the effect of ghrelin on neuropathic pain and to understand the associated mechanisms. Chronic constriction injury (CCI) of the sciatic nerve was used to establish a rat model of neuropathic pain. Hyperalgesia and allodynia were evaluated by observing the mechanical withdrawal threshold and the thermal withdrawal latency. Wnt3a and ß-catenin protein expression and GSK-3ß phosphorylation were detected by western blotting analysis. The levels of tumor necrosis factor-α and IL-1ß were determined using an enzyme-linked immunosorbent assay. In addition, we used immunohistochemical analysis to determine the levels of GSK-3ß phosphorylation in the dorsal horn of the spinal cord. Intrathecal delivery of ghrelin effectively ameliorated CCI-induced mechanical allodynia and thermal hyperalgesia at 7 and 14 days and reduced the levels of tumor necrosis factor-α. Ghrelin inhibited CCI-induced GSK-3ß activation and ß-catenin overexpression in the spinal dorsal horn. Moreover, intrathecal injection of ghrelin suppressed the activation of GSK-3ß in the spinal dorsal horn of CCI rats, as assessed by immunohistochemical analysis. Our data indicated that ghrelin could markedly alleviate neuropathic pain by inhibiting the expression of ß-catenin, via the suppression of GSK-3ß activation, in the spinal cord of CCI rats.

A novel distyryl boron dipyrromethene with two functional tags for site-specific bioorthogonal photosensitisation towards targeted photodynamic therapy.

A distyryl boron dipyrromethene based photosensitiser substituted with 1,2,4,5-tetrazine and alkyne moieties was prepared. Through site-specific bioorthogonal reactions with the complementary functional tags anchored on the membrane of A431 human epidermoid carcinoma cells, this versatile photosensitiser exhibited enhanced cellular uptake and photocytotoxicity. The bioorthogonal ligation was also demonstrated in tumour-bearing nude mice.

Intrathecal morphine combined with ropivacaine induces spinal myoclonus in cancer patients with an implanted intrathecal drug delivery system: Three case reports.

RATIONALE: Although intrathecal opioid infusion has been used for decades for the treatment of severe pain, myoclonus as one of the complications of this therapeutic modality is now beginning to be recognized more. PATIENTS CONCERNS: Here, we report three patients who developed myoclonus after dose adjustment in intrathecal drug delivery system for the treatment of refractory cancer pain. DIAGNOSIS: Spinal myoclonus is a sudden, brief, shock-like muscle contractions originating from the central nervous system. In our cases, it occurred after opioid administration via intrathecal delivery system with no abnormality found in laboratory or imaging examinations. INTERVENTIONS: Spinal myoclonus can be treated effectively by reducing the dose or infusion rate as described in case 1, or changing from an intrathecal to systemic administration in case 2, or correcting infusion and bolus parameters mistakes in case 3. OUTCOMES: All patients recovered quickly after stopping or decreasing the intrathecal drug infusion. LESSONS: Prevention is more important than treatment as for spinal myoclonus. Pain management teams should be aware of this distressing complication. Dose of intrathecal drugs should not exceed the recommended maximal daily doses by guidelines and patient education is important for successful intrathecal analgesic therapy.

Constructing Yeast Chimeric Pathways To Boost Lipophilic Terpene Synthesis.

Synthetic chimeric biological system offers opportunities to illuminate principles of designing life, and a primary step is constructing synthetic chimeric pathways. Here, we constructed yeast chimeric pathways by transferring the genes from  Saccharomyces cerevisiae pathways into another budding yeast Yarrowia lipolytica for in vivo assembly. We efficiently diversified gene option, combination, localization order, and copy number as expected. Convergence of two yeast pathways, especially mevalonic acid (MVA) pathways, remarkably enhanced synthesis of a lipophilic terpene, lycopene. In the selected champion strain with 50-fold of enhanced lycopene production, the chimeric MVA pathway gathered three S. cerevisiae genes with particular copies and locations. Amazingly, therein we discovered distinct transcriptional up-regulation of three significant pathways correlated with acetyl-CoA supply and tuning of cellular lipid amounts and composition. Modulating these pathways further improved lycopene production to 150-fold, a final 259 mg/L (approximately 80 mg/g DCW). We primarily showed the capacity of boosting the synthesis of lipophilic products with yeast chimeric pathways.

Correction: RNA-seq transcriptome analysis of the immature seeds of two Brassica napus lines with extremely different thousand-seed weight to identify the candidate genes related to seed weight.

[This corrects the article DOI: 10.1371/journal.pone.0191297.].