Building a highly efficient Streptomyces super-chassis for secondary metabolite production by reprogramming naturally-evolved multifaceted shifts.

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae. We initially identified and characterized a set of multiplex targets (afsQ1, bldD, bldA, and miaA) that contribute to SM (jadomycin) production when subjected to ethanol shock. Subsequently, we developed an ethanol-induced orthogonal amplification system (EOAS), enabling dynamic and precise control over targets. Ultimately, we integrated these multiplex targets into functional units governed by the EOAS, generating a universal and plug-and-play Streptomyces super-chassis. In addition to achieving the unprecedented titer and yield of jadomycin B, we also evidenced the potential of this super-chassis for production of diverse heterologous SMs, including antibiotic oxytetracycline, anticancer drug doxorubicins, agricultural herbicide thaxtomin A, and plant growth regulator guvermectin, all with the yields of >10 mg/g glucose in a simple mineral medium. Given that the production of SMs all required complexed medium and the cognate yields were usually much lower, our achievement of using a universal super-chassis and engineering pipeline in a simple mineral medium is promising for convenient heterologous production of SMs.

Forensic age prediction and age classification for critical age thresholds via 3.0T magnetic resonance imaging of the knee in the Chinese Han population.

OBJECTIVES: To assess the performance of knee MRI for forensic age prediction and classification for 12-, 14-, 16-, and 18-year thresholds. METHODS: The ossification stages of distal femoral epiphyses and proximal tibial epiphyses were assessed using an integrated staging system by Schmeling et al. and Kellinghaus et al. for knee 3.0T MRI with T1-weighted turbo spin-echo (T1-TSE) in sagittal orientation among 852 Chinese Han individuals (483 males and 369 females) aged 7-30 years. Regression models for age prediction were constructed and their performances were evaluated based on mean absolute deviation (MAD) values. In addition, the performances of age classification were assessed using receiver operating characteristic (ROC) analyses. RESULTS: The intra- and inter-observer agreement levels were very good (κ > 0.80). The complete fusion of those two types of epiphyses took place before 18.0 years in our study participants. The minimum MAD values were 2.51 years (distal femur) and 2.69 years (proximal tibia) in males, and 2.75 years (distal femur) and 2.87 years (proximal tibia) in females. The specificity values of constructed prediction models were all above 90% for the 12-, 14-, and 16-year thresholds, compared to the 74.8-84.6% for the 18-year threshold. Better performances of age prediction and classification were observed in males by distal femoral epiphyses. CONCLUSIONS: Ossification stages via 3.0T MRI of the knee with T1-TSE sequence using an integrated staging system could be a reliable noninvasive method for age prediction or for age classification for 12-, 14-, and 16-year thresholds, especially in males by distal femoral epiphyses. However, assessments based on the full bony fusion of the distal femoral epiphysis and proximal tibial epiphysis seemed not reliable for age classification for the 18-year threshold in the Chinese Han population.

Micromonospora rubida sp. nov., a novel actinobacterium isolated from soil of Harbin.

A novel actinobacterium, designated strain NEAU-HG-1T, was isolated from soil collected from Harbin, Heilongjiang Province, Northeast China and characterised using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain NEAU-HG-1T belonged to the genus Micromonospora, and shared high sequence similarities with Micromonospora auratinigra DSM 44815T (98.9%) and Micromonospora coerulea DSM 43143T (98.7%). Morphological and chemotaxonomic characteristics of the strain also supported its assignment to the genus Micromonospora. Cell wall contained meso-diaminopimelic acid and the whole-cell sugars were arabinose and xylose. The polar lipid contained diphosphatidylglycerol, phosphatidylethanolamine, glycolipid and phosphatidylinositol. The predominant menaquinones were MK-10(H2), MK-10(H4) and MK-10(H6). The major fatty acids were C17:0 cycle, iso-C15:0, and iso-C16:0. Furthermore, strain NEAU-HG-1T displayed a DNA-DNA relatedness of 33.8 ± 2.2% with M. coerulea DSM 43143T. The level of digital DNA-DNA hybridization between strain NEAU-HG-1T and M. auratinigra DSM 44815T was 27.2% (24.8-29.7%). The value was well below the criteria for species delineation of 70% for dDDH. Whole-genome average nucleotide identity analyses result also indicated that the isolate should be assigned to a new species under the genus Micromonospora. Therefore, it is concluded that strain NEAU-HG-1T represents a novel species of the genus Micromonospora, for which the name Micromonospora rubida sp. nov. is proposed, with NEAU-HG-1T (= CGMCC 4.7479T = JCM 32386T) as the type strain.

Pseudonocardia tritici sp. nov., a novel actinomycete isolated from rhizosphere soil of wheat (Triticum aestivum L.).

An aerobic, non-motile, Gram-stain positive actinomycete, designated strain NEAU-YY211T, was isolated from the rhizosphere soil of wheat (Triticum aestivum L.) collected from Zhumadian, Henan Province, mid-eastern China, and characterised taxonomically using a polyphasic approach. Comparative analysis of the 16S rRNA gene sequence indicated that strain NEAU-YY211T belongs to the genus Pseudonocardia, showing high similarities with respect to Pseudonocardia ammonioxydans H9T (99.1%) and Pseudonocardia antitumoralis SCSIO 01299T (99.0%), respectively. The cell wall was found to contain meso-diaminopimelic acid and the whole cell sugars were identified as arabinose and galactose. The predominant menaquinone of strain NEAU-YY211T was identified as MK-8(H4) and the major fatty acids were identified as iso-C16:0, C17:1ω8c and iso-C16:1. The phospholipid profile was found to consist of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and an unidentified phospholipid. The G+C content of the genomic DNA was determined to be 72.6 mol%. Levels of DNA-DNA relatedness with P. ammonioxydans JCM 12462T and P. antitumoralis DSM 45322T were 54.5 ± 3.5% and 49.8 ± 2.5% (mean ± SD), respectively. Based on phylogenetic analysis, phenotypic and genotypic data, it is concluded that the isolate can be distinguished from closely related type strains and thus represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia tritici sp. nov. is proposed. The type strain is NEAU-YY211T (= DSM 106068T = CGMCC 4.7474T).

Probing the influence of phosphonate bonding modes to uranium(VI) on structural topology and stability: a complementary experimental and computational investigation.

Systematic control of the reactions between U(VI) and 1,4-phenylenebis(methylene))bis(phosphonic acid) (pmbH4) allows for alterations in the bonding between these constituents and affords three uranyl phosphonate compounds with chiral one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D) structures, namely, [TPA][UO2(pmbH3)(pmbH2)H2O]·2H2O (1), [NH4]2[UO2(pmb)] (2), UO2(pmbH2) (3), and the first uranyl mixed phosphite/phosphonate compound [TMA]2[(UO2)2(pmb)(HPO3)] (4) (TPA = NPr4+, TMA = NMe4+). These compounds crystallize in the space groups P212121, P1̅, P21/c, and Cmcm, respectively. Further investigation of the local uranyl coordination environment reveals that in 1 only oxygen atoms from P=O moieties ligate the uranium centers; whereas in 2 only P-O(-) oxygen atoms are involved in bonding and yield a layered topology. Compound 3 differs sharply from the first two in that conjugated P=O and P-O(-) oxygen atoms chelate the uranium centers resulting in a 3D framework. In compound 4, a phosphonate group bridges three uranyl centers further coordinated with a phosphite ligand HPO32­, which is a product of pmbH4 decomposing, forming a 2D layered structure. Compounds 3 and 4 also contain a different coordination environment for U(VI) than that found in 1 or 2. In this case, tetragonal bipyramidal UO6 units occur instead of the far more common UO7 pentagonal bipyramids found in 1 and 2. Interestingly, 1 converts to 3 at elevated reaction temperatures, indicating that the formation of 1 is likely under kinetic control. This is supported by thermal analysis, which reveals that 3 has higher thermal stability than 1 or 2. UV-vis-near-IR absorption and fluorescence spectroscopy show that the absorption and photoluminescence intensity increases from 1 to 4. Density functional theory electronic structure calculations provide insight into the nature of the interactions between U(VI) and the phosphonate ligands.

Anti-invasive effects of minoxidil on human breast cancer cells: combination with ranolazine.

A plethora of ion channels have been shown to be involved systemically in the pathophysiology of cancer and ion channel blockers can produce anti-metastatic effects. However, although ion channels are known to frequently function in concerted action, little is known about possible combined effects of ion channel modulators on metastatic cell behaviour. Here, we investigated functional consequences of pharmacologically modulating ATP-gated potassium (KATP) channel and voltage-gated sodium channel (VGSC) activities individually and in combination. Two triple-negative human breast cancer cell lines were used: MDA-MB-231 and MDA-MB-468, the latter mainly for comparison. Most experiments were carried out on hypoxic cells. Electrophysiological effects were studied by whole-cell patch clamp recording. Minoxidil (a KATP channel opener) and ranolazine (a blocker of the VGSC persistent current) had no effect on cell viability and proliferation, alone or in combination. In contrast, invasion was significantly reduced in a dose-dependent manner by clinical concentrations of minoxidil and ranolazine. Combining the two drugs produced significant additive effects at concentrations as low as 0.625 µM ranolazine and 2.5 µM minoxidil. Electrophysiologically, acute application of minoxidil shifted VGSC steady-state inactivation to more hyperpolarised potentials and slowed recovery from inactivation, consistent with inhibition of VGSC activation. We concluded (i) that clinically relevant doses of minoxidil and ranolazine individually could inhibit cellular invasiveness dose dependently and (ii) that their combination was additionally effective. Accordingly, ranolazine, minoxidil and their combination may be repurposed as novel anti-metastatic agents.

Polyketide pesticides from actinomycetes.

Natural product derived pesticides have increased in popularity worldwide because of their high efficacy, eco-friendly nature and favorable safety profile. The development of polyketide pesticides from actinomycetes reflects this increase in popularity in the past decades. These pesticides, which include avermectins, spinosyns, polynactins, tetramycin and their analogues, have been successfully applied in crop protection. Moreover, the advance of biotechnology has led to continuous improvement in the discovery and production processes. In this review, we summarize these polyketide pesticides, their activities and provide insight into their development. We also discuss engineering strategies and the current status of industrial production for these pesticides. Given that actinomycetes are known to produce a wide range of bioactive secondary metabolites, the description of pesticide development and high yield strain improvement presented herein will facilitate further development of these valuable polyketide pesticides from actinomycetes.

ERCC5 quantification for age estimation of peripheral blood in a Chinese Han population.

Age estimation is an important topic of human identification in forensic practice, especially coming to biological samples in crime scene, such as blood, saliva, semen. As rate-limiting enzyme in Nucleotide excision repair (NER) that was associated with aging, Excision repair cross-complementation group 5 (ERCC5) was considered to be a candidate biomarker for individual age estimation. The ERCC5 mRNA and protein expression levels association with age have been demonstrated in our previous study. However, very little is known about relationship DNA-based quantification of ERCC5 with age. In this study, we detected ERCC5 level in peripheral blood from a Chinese Han population by SYBR qPCR assay to gain better insight into the quantitative relationship with age. The results showed ERCC5 level declined with individual age with a negative correlation(r = -0.8, R2 = 0.63, P < 0.001). The data model for age estimation based on ERCC5 level was Y = -31.352X + 14.436 ± 10.28 (Y: age, year; X: CqTBP-CqERCC5; standard error: year). The accuracy about the data model for age estimation was about 73.33%. The mean absolute difference (MAD) values were 8.22, 8.09 and 8.38 in total, male and female, respectively. Furthermore, ERCC5 quantification for age estimation was also applicable for stored blood samples under low temperature up to 6 months. It was suggested that the ERCC5 quantification was expected to be a valuable additional method for individual age estimation, especially in cases where traditional morphologic method is absent or inefficient in forensic practice.

Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus.

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (ΔpyrF) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.

Underreporting characteristics of osteoporotic vertebral fracture in back pain clinic patients of a tertiary hospital in China.

AIM: Osteoporotic vertebral compressive fractures (VCFs) â€‹are known to be commonly missed in X-rays indicated for pulmonary or heart diseases. In this study, we investigated the underreporting status of VCF in back pain clinic patients when the spine was the focus of interest. MATERIALS AND METHODS: This is a retrospective analysis of 105 female cases (mean: 72 years, range: 55-93 years) from a tertiary hospital in China (facility A, FA). The patients with back and/or leg pain were referred for a spine X-ray. The images were retrieved and transferred to a central reading facility (facility B, FB), where images were double-read by two readers experienced in evaluating osteoporotic vertebral compressive deformity (VCD)/VCF. A qualitative VCD with <20%, 20-25%, 25-40%, and >40% vertebral body height loss was recorded as minimal, mild, moderate, and severe grades, respectively. A â€‹VCD coexisted with endplate/cortex fracture (ECF) was VCF. FB readings were considered as the reference. RESULTS: There were 34 true negative cases where FA and FB had a consensus. In 7 cases with minimal VCD, 3 cases with ECF, and 7 cases with minimal or mild VCFs, the FA readings were false negative. No standalone singular moderate or severe VCD/VCF in a patient was missed in FA's reports. In 25 FA reading positive cases with multiple VCFs, one VCF was missed in 8 cases, more than one VCF was missed in 15 cases, and one additional ECF was missed in 2 cases. In 14 cases, FA and FB had VCF number agreement, with the term 'vertebral fracture' was used appropriately in FA reports. In 15 cases, FA and FB had agreement in VCF number; however, the appropriate term 'vertebral fracture' was not used in FA reports; instead the terms of 'compressive change' or 'wedging change' were used. In most VCFs, severity grading was not given in FA. In 13 VCFs where grading was reported, all were marked as 'mild', including seven mild VCFs, five moderate VCFs, and even one severe VCF. CONCLUSION: Among the patients with VCD/VCF, the false negative rate among was 23.9% (17/71), but the missed cases were all minimal or mild grades. One or more VCFs were missed in 32.4% (23/71) of the cases with multiple VCFs. Appropriate severity grading was not reported for most cases. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The underreporting rate of osteoporotic vertebral compressive fracture in back pain clinic patients in a typical tertiary hospital setting in China compared favorably with literature reports. However, there is a general lack of awareness of vertebral endplate/cortex fracture sign and vertebral fracture severity grading, while minimal and mild VCD with endplate/cortex fracture may have clinical significance. Moreover, after one VCF is spotted in a patient, it is highly advisable to carefully check the whole spine so that multiple VCFs will not be missed.

Diffusion MRI Derived per Area Vessel Density as a Surrogate Biomarker for Detecting Viral Hepatitis B-Induced Liver Fibrosis: A Proof-of-Concept Study.

Liver vessel density can be evaluated by an imaging biomarker diffusion-derived vessel density (DDVD): DDVD/area(b0b2) = Sb0/ROIarea0 - Sb2/ROIarea2, where Sb0 and Sb2 refer to the liver signal when b is 0 or 2 (s/mm2); ROIarea0 and ROIarea2 refer to the region of interest (ROI) on b = 0 or 2 images; and Sb2 may be replaced by Sb15 (b = 15). This concept was validated in this study. Liver diffusion images were acquired at 1.5 T. For a scan-rescan repeatability study of six subjects, b values of 0 and 2 were used. The validation study was composed of 26 healthy volunteers and 19 consecutive suspected chronic viral hepatitis B patients, and diffusion images with b values of 0, 2, 15, 20, 30, 45, 50, 60, 80, 100, 200, 300, 600, and 800 were acquired. Four patients did not have liver fibrosis, and the rest were four stage 1, three stage 2, four stage 3, and one stage 4 patients, respectively. The intraclass correlation coefficient for repeatability was 0.994 for DDVD/area(Sb0Sb2) and 0.978 for DDVD/area(Sb0Sb15). In the validation study, DDVD/area(Sb0Sb2) and area(Sb0Sb15) were 14.80 ± 3.06 and 26.58 ± 3.97 for healthy volunteers, 10.51 ± 1.51 and 20.15 ± 2.21 for stage 1-2 fibrosis patients, and 9.42 ± 0.87 and 19.42 ± 1.89 for stage 3-4 fibrosis patients. For 16 patients where IVIM analysis was performed, a combination of DDVD/area, PF, and Dfast achieved the best differentiation for nonfibrotic livers and fibrotic livers. DDVD/area were weakly correlated with PF or Dfast. Both DDVD/area(Sb0Sb2) and area(Sb0Sb15) are useful imaging biomarkers to separate fibrotic and nonfibrotic livers, with fibrotic livers having lower measurements.

Recognizing osteoporotic vertebral deformity on frontal view radiograph: a cohort analysis and a pictorial review.

PURPOSE: Opportunities exist to detect osteoporotic vertebral deformities (VDs) on frontal radiograph (FR) indicated for lung or abdominal diseases, while literature have been mostly based on lateral radiograph (LR). This study analyzed the detectability of moderate and severe grades VD on FR. METHODS: There were 105 female cases (mean 72 years, range 55~93 year), who were referred for digital spine FR and LR with back and/or leg pain. The LR and FR were read, osteoporotic VDs with < 20%, 20-25%, 25-40%, and > 40% vertebral body height loss were recorded as minimal, mild, moderate, and severe grades, respectively. After a 10-month interval, only FRs were read again, and each vertebra was classified as (1) no notable VD, (2) with notable VD, and (3) ambiguous. The first reading was the reference, while the second reading was allowed to miss minimal/mild VCD and endplate/cortex fracture. RESULTS: Counting by subjects, for 98 cases, the two reading sessions had agreement, including 43 "true negative" cases and 55 true positive cases. There were two false positive cases, and five ambiguous cases. In total, 1286 vertebra were assessed, FR reading had 1126 vertebrae "true negative," 130 vertebrae true positive, one vertebra false negative, 3 vertebrae false positive, and 26 ambiguous vertebrae (65.4% being true negative and 34.6% being true positive). Most of the disagreements were associated with kyphosis or poor X-ray projection. Nineteen illustrative cases are presented graphically. CONCLUSION: Moderate and severe grades of VD are identifiable on FR as long as the involved vertebrae are clearly filmed.

Intravoxel incoherent motion derived liver perfusion/diffusion readouts can be reliable biomarker for the detection of viral hepatitis B induced liver fibrosis.

BACKGROUND: Recent two studies reported that intravoxel incoherent motion (IVIM) analysis can separate healthy livers and viral hepatitis B (VHB) induced liver fibrosis. However, in these two studies the starting b value for bi-exponential decay analysis was b =10 and 15 s/mm2 respectively. The current study has two primary aims. The first is to further confirm the diagnostic value of IVIM in detecting liver fibrosis. The second is to test whether by sampling very low b value densely, then b =0 s/mm2 image could be included to improve IVIM's diagnostic performance. METHODS: This was a prospective study with data acquired at the Third Xiangya Hospital of Central South University, Changsha, China. Healthy volunteers and patients suspected of VHB induced liver fibrosis with liver biopsy performed, as well as hepatocellular carcinoma patients scheduled for surgery, were recruited. All the hepatocellular carcinoma patients had liver fibrosis. After exclusions based on pre-defined criteria for image data quality, for IVIM analysis this study included 20 healthy volunteers; 4 chronic VHB patients with biopsy showing no liver fibrosis; 11 stage-1 liver fibrosis patients, 10 stage-2 liver fibrosis patients, 2 stage-3 liver fibrosis patients, and 5 stage-4 liver fibrosis patients. In the liver fibrosis patients, 1, 19, and 8 cases had inflammation grade-0, grade-1, and grade-2 respectively. The reference IVIM bi-exponential decay curve fitting analysis was segmented fitting performed with b =2 s/mm2 image as the starting point and a threshold-b of 60 s/mm2. This reference fitting method was compared with threshold-b of 40 s/mm2, full fitting, fitting starting from b =0, 5, and 10 s/mm2 respectively. The potential correlation between IVIM readouts and liver function was assessed for the liver fibrosis patients. RESULTS: Based on the smaller coefficient of variation (CoV) for the volunteer group and the smaller patient/volunteer ratios [= (mean measurement for patient groups)/(mean measurement for healthy volunteers)], the comparison of fitting methods favored the reference approach starting from b =2 s/mm2 with a threshold-b of 60 s/mm2. The IVIM measures of four patients without liver fibrosis resembled those of healthy subjects. PF offered the best diagnostic value for separating healthy livers and fibrotic livers, and a threshold of PF =0.1406 separated all fibrotic livers and healthy livers with an exception of one hepatocellular carcinoma patient (fibrosis grade-2/inflammation grade-2). The correlation between fibrosis grading and inflammation grading was weakly positive; while compared with fibrotic livers with inflammation grade-1, fibrotic livers with inflammation grade-2 showed a trend of higher Dfast. A weak correlation is shown with lower PF and lower Dfast associated with lower total protein, lower albumin; higher alanine transaminase, higher aspartate transaminase; higher total bilirubin, and higher direct bilirubin. CONCLUSIONS: Segmented-fitting with threshold-b =60 s/mm2 and starting from non-zero very low b value outperforms other methods. IVIM has high sensitivity in detecting liver fibrosis, and PF and Dfast have potential correlation with serum liver function biomarkers. IVIM measures and liver fibrosis grading are not in a linear relationship.