De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories.

BACKGROUND: Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis. RESULTS: We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L. CONCLUSIONS: We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.

Development of Integrated Vectors with Strong Constitutive Promoters for High-Yield Antibiotic Production in Mangrove-Derived Streptomyces.

It is important to improve the production of bioactive secondary products for drug development. The Escherichia coli-Streptomyces shuttle vector pSET152 and its derived vector pIB139 containing a strong constitutive promoter ermEp* are commonly used as integrative vectors in actinomycetes. Four new integrative vectors carrying the strong constitutive promoter kasOp*, hrdBp, SCO5768p, and SP44, respectively, were constructed and proven to be functional in different mangrove-derived Streptomyces host strains by using kanamycin resistance gene neo as a reporter. Some biosynthetic genes of elaiophylins, azalomycin Fs, and armeniaspirols were selected and inserted into these vectors to overexpress in their producers including Streptomyces sp. 219807, Streptomyces sp. 211726, and S. armeniacus DSM 43125, resulting in an approximately 1.1-1.4-fold enhancement of the antibiotic yields.

Characterization of benzylisoquinoline alkaloid methyltransferases in Liriodendron chinense provides insights into the phylogenic basis of angiosperm alkaloid diversity.

Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with great pharmacological value. Their biosynthetic pathways have been extensively elucidated in the species from the Ranunculales order, such as poppy and Coptis japonica, in which methylation events play central roles and are directly responsible for BIA chemodiversity. Here, we combined BIA quantitative profiling and transcriptomic analyses to identify novel BIA methyltransferases (MTs) from Liriodendron chinense, a basal angiosperm plant. We identified an N-methyltransferase (LcNMT1) and two O-methyltransferases (LcOMT1 and LcOMT3), and characterized their biochemical functions in vitro. LcNMT1 methylates (S)-coclaurine to produce mono- and dimethylated products. Mutagenesis experiments revealed that a single-residue alteration is sufficient to change its substrate selectivity. LcOMT1 methylates (S)-norcoclaurine at the C6 site and LcOMT3 methylates (S)-coclaurine at the C7 site, respectively. Two key residues of LcOMT3, A115 and T301, are identified as important contributors to its catalytic activity. Compared with Ranunculales-derived NMTs, Magnoliales-derived NMTs were less abundant and had narrower substrate specificity, indicating that NMT expansion has contributed substantially to BIA chemodiversity in angiosperms, particularly in Ranunculales species. In summary, we not only characterized three novel enzymes that could be useful in the biosynthetic production of valuable BIAs but also shed light on the molecular origin of BIAs during angiosperm evolution.

Association of joint hypermobility with range of cervical motion and its impact on the motor unit loss in patients with Hirayama disease.

INTRODUCTION/AIMS: Some patients with Hirayama disease (HD) may have generalized joint hypermobility (GJH), which may excessively increase cervical range of motion (ROM) and then worsen the HD. The purpose of this study was to identify the frequency of GJH in HD patients and to analyze the effect of GJH on cervical ROM and the severity of HD. METHODS: The Beighton scoring system (≥4) was used to diagnose GJH in 84 HD patients. All patients underwent assessments of cervical-flexion/extension ROM; motor unit number estimation in bilateral abductor pollicis brevis (APB) muscles; handgrip strength; and the disabilities of the arm, shoulder, and hand assessments. RESULTS: Concomitant GJH was identified in 20 (23.8%) HD patients. The HD patients with GJH exhibited greater cervical-flexion (P < .001) and cervical-extension (P = .033) ROM than those without GJH. Both greater single motor unit potential amplitudes (symptomatic side: P = .005; less-symptomatic side: P = .011) and lower motor unit numbers (symptomatic side: P = .008; less-symptomatic side: P = .013) in bilateral APB, along with lower compound muscle action potential amplitudes on the symptomatic-side APB (P = .039), were observed in patients with GJH than those without GJH. There was a mild negative correlation between motor unit number and cervical-flexion ROM in HD patients (symptomatic side: r = -0.239, P = .028; less-symptomatic side: r = -0.242, P = .027). DISCUSSION: The frequency of GJH in HD patients may be higher than in the general population. Importantly, GJH may exacerbate excessive cervical-flexion ROM, thereby worsening motor unit loss in HD patients. A cautious approach should be taken when treating HD due to possible comorbid GJH.

microRNA profiling in the aqueous humor of highly myopic eyes using next generation sequencing.

PURPOSE: To characterize microRNAs (miRNAs) and their possible roles in high myopia by using next generation sequencing. METHODS: Aqueous humor samples were obtained from 25 highly myopic eyes and 25 cataract eyes at the onset of surgery. miRNA next generation sequencing and bioinformatics analyses were performed using RNA extracted from 30 samples. The remaining 20 samples were used for quantitative polymerase chain reaction validation of sequencing results. RESULTS: A total of 341 microRNAs were detected in the aqueous humor samples of highly myopic eyes; 201 miRNAs were detected in the aqueous humor samples of cataractous control eyes. A total of 249 mature miRNAs and 17 novel miRNAs were differentially expressed during myopia. Possible pathways regulated by these aberrantly expressed miRNAs included the TNF, MAPK, PI3K-Akt, and HIF-1 signaling pathways. The relative expression patterns of hsa-let-7i-5p, hsa-miR-127-3p, and hsa-miR-98-5p were confirmed by quantitative polymerase chain reaction. CONCLUSIONS: The current study provided an overall view of miRNA profiling in the aqueous humor of highly myopic eyes. These profiles may be associated with myopia pathogenesis, and are potential biomarkers.

AUTOLOGOUS BLOOD CLOT COVERING INSTEAD OF GAS TAMPONADE FOR MACULAR HOLES.

PURPOSE: This study evaluates the efficacy and usefulness of vitrectomy with internal limiting membrane peeling and autologous blood clot covering without gas tamponade in the treatment of macular holes (MHs). METHODS: All patients with a full-thickness MH with a minimum diameter of <600 µm and a base diameter of <1,200 µm underwent pars plana vitrectomy and internal limiting membrane peeling with autologous blood covering the MH at the end of the surgery. No fluid-air exchange or gas tamponade was performed. Postoperatively, all patients were instructed to adopt supine position overnight and thereafter any comfortable posture. RESULTS: A total of 18 eyes of 18 consecutive patients were included. The mean age of the patients (12 women and 6 men) was 59.06 ± 14.31 years (range, 21-81 years). The MHs composed of 13 idiopathic MHs, 2 MHs with high myopia (axial length > 26.5 mm), 2 traumatic MHs, and 1 MH associated with diabetic macular edema. Among them were five large MHs (minimum diameter > 400 µm). Complete MH closure was achieved in all eyes at the end of the follow-up period (range, 3-14 months). Visual acuity was significantly improved from preoperative 0.89 ± 0.41 logarithm of the minimum angle of resolution (20/155 Snellen) to 0.42 ± 0.33 logarithm of the minimum angle of resolution (20/53 Snellen) at the final visit (P < 0.001). CONCLUSION: The novel surgical protocol using vitrectomy, internal limiting membrane peeling, and autologous blood clot covering at the end of the MH surgery with limited diameters achieved highly effective closure and visual improvement and eliminated the gas tamponade and thus the associated adverse effects and the need for postoperative face-down positioning.

Modified deep anterior lamellar dissection for corneal opacity during vitrectomy: case reports.

BACKGROUND: To introduce a modified deep anterior lamellar dissection technique to improve visibility during surgery for vitreoretinal diseases with coexisting corneal opacity. CASE PRESENTATION: Two patients with retinal detachment and coexisting corneal blood staining or corneal decompensation underwent modified deep anterior lamellar dissections followed by vitrectomy. The modified deep anterior lamellar dissection techniques, unlike the dissection and removal of corneal lamellar in a typical deep anterior lamellar keratoplasty, included the creation and preservation of a deep lamellar corneal flap, the retroillumination to visualize and easily remove the remaining opaque stroma on the Descemet membrane, and the big air bubble technique in the eye with endothelial decompensation. The patient's own cornea flap was sutured back after vitrectomy was done. The modified dissection techniques provided adequate fundus view during vitrectomy while removing as less corneal tissue as possible and decreasing the surgical complications and the requirement of a fresh cornea. Postoperatively, in case 1, the corneal blood staining was gradually absorbed and the vision improved from light perception to counting fingers. In case 2, even though the cornea remained cloudy and the vision was poor, the cornea endothelial decompensation was stable and asymptomatic. Both retinas were attached after silicone oil removal at 6-month follow-up. CONCLUSIONS: This modified and limited deep anterior lamellar corneal dissection procedure appears to be a useful alternative to penetrating keratoplasty, ophthalmic endoscope and temporary keratoprosthesis during the vitrectomy with coexisting corneal opacity.

Characterization of the Biosynthetic Gene Cluster for the Antibiotic Armeniaspirols in Streptomyces armeniacus.

Armeniaspirols (1-3) are potent antibiotics against Gram-positive pathogens. Through a biosynthetic investigation, we identified four enzymes involved in the structural modification of 1-3. Manipulation of their activity led to the generation of 4-6 and nine novel analogues, 7-15. Bioactivity assessments revealed that the pyrrole chloro group and the methyl group are important for the antimicrobial activities of armeniaspirols, which lays the foundation for future structure optimization and mechanism of action studies of armeniaspirols.

Treatment of Myopic Foveoschisis with Air Versus Perfluoropropane: A Retrospective Study.

BACKGROUND The aim of this study was to compare the efficacy of air and perfluoropropane (C3F8) combined with vitrectomy to treat myopic foveoschisis (MF). MATERIAL AND METHODS A retrospective comparison of a consecutive series of surgical patients was performed. Ninety-seven eyes of 91 patients with MF were assigned to undergo 23G vitrectomy. After internal limiting membrane (ILM) peeling, the vitreous cavity was filled with air in 48 eyes of 45 patients (Air Group). Fluid-air exchange was performed in 49 eyes of 46 patients (C3F8 Group) followed by an injection of 14% C3F8. Patients were evaluated using best-corrected visual acuity (BCVA) and optical coherence tomography. RESULTS Preoperatively, there was no significant difference in clinical features between the groups. After surgery, BCVA was markedly improved and the foveoschisis height was reduced in both groups compared with baseline (P<0.01), but the difference between the groups was not significant (P>0.05). No significant differences were noted in BCVA improvement and retinal restoration (P=0.33 and 0.39, respectively) in the mild and moderate subgroups (foveoschisis height ≤400 µm) between the tamponades. However, in the severe group (foveoschisis height >400 µm), C3F8 had a more favorable cure rate and foveoschisis height reduction improvement compared with air (P=0.04 and 0.04, respectively) at the last visit. CONCLUSIONS Vitrectomy combined with ILM peeling is effective in the treatment of myopic foveoschisis, and the choice of tamponade depends on the severity of foveoschisis. Air can be used for patients with a foveoschisis height ≤400 µm, but C3F8 is more effective for patients with a foveoschisis height >400 µm.

Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer-Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone.

In the biosynthesis of pentalenolactone (1), PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (4) to the lactone pentalenolactone D (5), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 4 to neopentalenolactone D (6), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer-Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 4 to 6 as the major product. The recombinant PntE mutant harboring R484L exhibited reduced regiospecificity, generating a mixture of lactones containing more than 17% of 6. These in vitro results were corroborated by analysis of the complementation of the S. avermitilis ΔptlED double deletion mutant with pntE mutants, such that pntE mutants harboring L185S produced 6 as the major product, whereas complemention of the ΔptlED deletion mutant with pntE mutants carrying the R484L mutation gave 6 as more than 33% of the total lactone product mixture.

Optimization of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines as tubulin polymerization inhibitors.

Thirteen new N-aryl 1,2,3,4-tetrahydroquinoline compounds (4a-f, 6a-c, and 8a-d) were synthesized and evaluated for antitumor activity and drug-like properties. Compound 4a exhibited high inhibitory potency with low nanomolar GI50 values of 16-20 nM in cellular assays, including excellent activity against the P-glycoprotein overexpressing cell line KBvin. Compound 4a inhibited colchicine binding to tubulin and tubulin assembly with an IC50 value of 0.85 µM, superior to the reference compound CA4 (1.2 µM) in the same assay. In addition, 4a also exhibited highly improved water solubility (75 µg/mL) and a suitable logP value (3.43) at pH 7.4. With a good balance between antitumor potency and drug-like properties, compound 4a could be a new potential drug candidate for further development. Current results on SAR studies and molecular modeling provided more insight about this class of compounds as tubulin polymerization inhibitors targeting the colchicine site.

Using large clinical corpora for query expansion in text-based cohort identification.

In light of the heightened problems of polysemy, synonymy, and hyponymy in clinical text, we hypothesize that patient cohort identification can be improved by using a large, in-domain clinical corpus for query expansion. We evaluate the utility of four auxiliary collections for the Text REtrieval Conference task of IR-based cohort retrieval, considering the effects of collection size, the inherent difficulty of a query, and the interaction between the collections. Each collection was applied to aid in cohort retrieval from the Pittsburgh NLP Repository by using a mixture of relevance models. Measured by mean average precision, performance using any auxiliary resource (MAP=0.386 and above) is shown to improve over the baseline query likelihood model (MAP=0.373). Considering subsets of the Mayo Clinic collection, we found that after including 2.5 billion term instances, retrieval is not improved by adding more instances. However, adding the Mayo Clinic collection did improve performance significantly over any existing setup, with a system using all four auxiliary collections obtaining the best results (MAP=0.4223). Because optimal results in the mixture of relevance models would require selective sampling of the collections, the common sense approach of "use all available data" is inappropriate. However, we found that it was still beneficial to add the Mayo corpus to any mixture of relevance models. On the task of IR-based cohort identification, query expansion with the Mayo Clinic corpus resulted in consistent and significant improvements. As such, any IR query expansion with access to a large clinical corpus could benefit from the additional resource. Additionally, we have shown that more data is not necessarily better, implying that there is value in collection curation.

Tear Fluid Progranulin as a Noninvasive Biomarker for the Monitoring of Corneal Innervation Changes in Patients With Type 2 Diabetes Mellitus.

Purpose: This study aimed to investigate the expression levels of progranulin (PGRN) in the tears of patients with diabetic retinopathy (DR) versus healthy controls. Additionally, we sought to explore the correlation between PGRN levels and the severity of ocular surface complications in patients with diabetes. Methods: In this prospective, single-visit, cross-sectional study, patients with DR (n = 48) and age-matched healthy controls (n = 22) were included and underwent dry eye examinations. Tear fluid was collected, and its components were analyzed using the Luminex assay. The subbasal nerve plexus of all participants was evaluated by in vivo confocal microscopy. Results: Patients with DR exhibited more severe dry eye symptoms, along with a reduction in nerve fiber density, length, and branch density within the subbasal nerve plexus, accompanied by an increase in the number of dendritic cells. Tear PGRN levels were also significantly lower in patients with diabetes than in normal controls, and the levels of some inflammatory factors (TNF-α, IL-6, and MMP-9) were higher in patients with DR. Remarkably, the PGRN level significantly correlated with nerve fiber density (R = 0.48, P < 0.001), nerve fiber length (R = 0.65, P < 0.001), and nerve branch density (R = 0.69, P < 0.001). Conclusions: Tear PGRN levels might reflect morphological changes in the corneal nerve plexus under diabetic conditions, suggesting that PGRN itself is a reliable indicator for predicting the advancement of neurotrophic keratopathy in patients with diabetes. Translational Relevance: PGRN insufficiency on the ocular surface under diabetic conditions was found to be closely associated with nerve impairment, providing a novel perspective to discover the pathogenesis of diabetic complications, which could help in developing innovative therapeutic strategies.

Silicone oil as a corneal lubricant to reduce corneal edema and improve visualization during vitrectomy.

AIM: To evaluate the efficacy and safety of silicone oil (SO) as a corneal lubricant to improve visualization during vitrectomy. METHODS: Patients who underwent vitreoretinal surgery were divided into two groups. Group 1 was operated on with initial SO (Oxane 5700) as a corneal lubricant. Group 2 was operated on with initial lactated ringer's solution (LRS) and then replaced with SO as required. Fundus clarity was scored during the surgery. Fluorescein staining was performed to determine the damage to corneal epithelium. RESULTS: Totally 114 eyes of 114 patients were included. Single SO use maintained a clear cornea and provided excellent visualization of surgical image. In group 1, the fundus clarity was grade 3 in 41/45 eyes and grade 2 in 4/45 eyes. In group 2, corneal edema frequently occurred after initial LRS use. The fundus clarity was grade 3 in 19/69 eyes, 2 in 37/69 eyes and 1 in 13/69 eyes (P<0.05). SO was applied in 29 eyes of initial LRS use with subsequent corneal edema, which eliminated the corneal edema in 26 eyes. Corneal fluorescein staining score in group 1 was 0 in 28 eyes, 1 in 11 eyes and 2 in 6 eyes, and 40, 20 and 9, respectively, in group 2 (all P>0.05). CONCLUSION: The use of SO as a corneal lubricant is effective and safe for preserving and improving corneal clarity and providing clear surgical field during vitrectomy.

Clinical efficacy of EUS-guided celiac plexus neurolysis versus EUS-guided celiac ganglion irradiation with iodine-125 seeds for pain relief in advanced pancreatic cancer: A long-term retrospective study.

Background and Objective: To compare the efficacy of EUS-guided celiac plexus neurolysis (CPN) and celiac plexus irradiation with iodine-125 (125I) seeds with absolute ethanol for relieving pain in patients with advanced pancreatic cancer. Methods: We retrospectively analyzed data of 81 patients with advanced pancreatic cancer who underwent EUS-CPN or EUS-125I implantation between January 2017 and December 2020. Postoperative pain was assessed using visual analog scale (VAS) scores; self-assessments of quality of life and the median survival time were compared between the 2 groups. Results: EUS-CPN and 125I implantation were performed in 43 and 38 patients, respectively. Postoperative VAS scores were significantly lower than the preoperative levels in both groups. One week after the operation, 26 patients (60.5%) in the EUS-CPN group achieved partial pain relief, whereas no patients in the EUS-125I seed group experienced pain relief. However, after 4 weeks postoperatively, VAS scores had decreased, and the rate of partial pain relief was higher for EUS-125I seeds than for EUS-CPN. Self-assessments of quality of life were similar in both groups during the first 1 month after the procedure. Conclusions: Both EUS-CPN and EUS-125I seeds can safely and effectively relieve pain in patients with advanced pancreatic cancer. Although EUS-125I seeds take additional time to show effects, the extent and duration of pain relief are better compared with CPN, and interestingly, the median survival time was different.

Product-mediated regulation of pentalenolactone biosynthesis in Streptomyces species by the MarR/SlyA family activators PenR and PntR.

The orthologous penR and pntR genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and S. arenae TÜ469, respectively, were predicted to encode MarR/SlyA family transcriptional regulators, responsible for regulation of the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. The intrinsic target DNA sequences and small molecule ligands of purified recombinant PenR and PntR were identified by electrophoretic mobility shift assays. PenR bound to DNA from both the penR-gapN and penM-penH intergenic regions, while PntR bound only the corresponding pntR-gapR intergenic region. The targets of PenR and PntR were shown to be limited to conserved 37-bp DNA segments. Pentalenolactone and two late-stage biosynthetic intermediates, pentalenolactones D and F, act as ligands of both PenR and PntR, resulting in release of these proteins from their target DNA. The production of pentalenolactones was significantly decreased in the penR deletion mutant S. exfoliatus ΔpenR ZD27 but could be restored by complementation with either penR or pntR. Reverse transcription-PCR established that transcription of pentalenolactone biosynthetic and resistance genes decreased, while that of the penR gene itself increased in the penR deletion mutant S. exfoliatus ZD27 compared to the wild-type strain. The PenR protein thus serves as a positive regulator of pentalenolactone biosynthesis and self-resistance while acting as an autorepressor of penR.

Proximally evoked soleus H-reflex to S1 nerve root stimulation in sensory neuronopathies (ganglionopathies).

INTRODUCTION: Sensory neuronopathy (SNN) mimics distal sensory axonopathy. The conventional H-reflex elicited by tibial nerve stimulation (tibial H-reflex) is usually abnormal in both conditions. We evaluated the proximally evoked soleus H-reflex in response to S1 nerve root stimulation (S1 foramen H-reflex) in SNN. METHODS: Eleven patients with SNN and 6 with distal sensory axonopathy were studied. Tibial and S1 foramen H-reflexes were performed bilaterally in each patient. RESULTS: Tibial and S1 foramen H-reflexes were absent bilaterally in all patients with SNN. In the patients with distal sensory axonopathy, tibial H-reflexes were absent in 4 and demonstrated prolonged latencies in 2, but S1 foramen H-reflexes were normal. CONCLUSIONS: Characteristic absence of the H-reflex after both proximal and distal stimulation reflects primary loss of dorsal root ganglion (DRG) neurons and the distinct non-length-dependent impairment of sensory nerve fibers in SNN.