Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in Streptomyces albus B4 chassis.

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. IMPORTANCE: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

Short-Term Efficacy of Cerebello-spinal tDCS and Body Weight-Supported Treadmill Training in the Hypertrophic Olivary Degeneration: a Rare Case Report.

The present case study reported a patient diagnosed with hypertrophic olivary degeneration, a rare condition characterized by a trans-neuronal degeneration and signal enhancement in T2-weighted images on magnetic resonance imaging, usually caused by cerebral hemorrhage, cerebral infarction, and trauma. Furthermore, the relevant literature review was performed. The existing pharmacological treatment has limited clinical benefits on the patient. Since spontaneous remission hardly occurs in the disease, there are no other effective treatments. In this case, the patient was a 55-year-old Chinese male who presented progressive gait difficulty for several months due to both-sided ataxia. Neurological examination revealed upper extremity and lower limb bilateral spasticity, ataxia, slurred speech, and dysmetria. Therefore, our study treated the patient through the inventive application of cerebello-spinal transcranial direct current stimulation and body weight-supported treadmill training. After a 4-week treatment, the patient could walk independently, without aid, speeding up by 7%, as well as the ataxia symptoms, and balance has improved significantly. It was demonstrated in this case report that the combination of cerebello-spinal tDCS and body weight-supported treadmill training can be an effective treatment for patients with Hypertrophic olivary degeneration.

Effects of Multimodal Exercise on Health-related Physical Fitness and Quality of Life in Patients with Nasopharyngeal Carcinoma during Radiotherapy.

BACKGROUND: The health-related physical fitness of patients with nasopharyngeal carcinoma can decrease significantly during radiotherapy, which can adversely affect their quality of life. AIM: This study was designed to evaluate the potential influence of a multimodal exercise program on the health-related physical fitness and quality of life of patients with nasopharyngeal carcinoma during radiotherapy. METHODS: Forty patients with nasopharyngeal carcinoma undergoing radiotherapy in the First Affiliated Hospital of Fujian Medical University from May to November 2019 were included. The participants in the control group (N=20) received routine nursing, while those in the intervention group (N=20) were also subjected to the multimodal exercise program during radiotherapy. RESULTS: The multimodal exercise program had a positive effect on participants. The step test index in the intervention group was significantly higher as compared to the control group (p < .05). The participants were subjected to 5 times slow speed (60°/s) and 10 times fast (180°/s) speed, and function of some extensor and flexor muscles of the elbow, shoulder, and knee joints in the intervention group was markedly improved (p < .05). In the intervention group, the grip strength of the right hand was observed to be significantly improved (p < .01). Furthermore, the upper limb scratch dorsal test of intervention group was significantly better than that of the control group (p < .05). The scores of physical, emotional, and social functions in the intervention group were found to be significantly higher as compared to the control group (p < .05). CONCLUSIONS: The multimodal exercise program significantly improved the health-related physical fitness and life quality of the patients with nasopharyngeal carcinoma during radiotherapy, though its long-term effects remain to be further analyzed.

A Magnaporthe Chitinase Interacts with a Rice Jacalin-Related Lectin to Promote Host Colonization.

The genome of rice blast fungus (Magnaporthe oryzae) encodes 15 glycoside hydrolase 18 family chitinases. In this study, we characterized the function of an M. oryzae extracellular chitinase, MoChi1, and its interaction with a host protein, OsMBL1, a jacalin-related Mannose-Binding Lectin (MBL) in rice (Oryza sativa). Deletion of MoChi1 resulted in reduced aerial hyphal formation and reduced virulence in rice by activating the expression of defense-responsive genes. We confirmed MoChi1 interaction with rice OsMBL1 in vitro and in vivo. OsMBL1 was induced by pathogen-associated molecular patterns and M. oryzae infection. Overexpression of OsMBL1 led to activation of rice defense-responsive genes and a chitin-induced reactive oxygen species burst, thereby enhancing resistance to M. oryzae Knockdown of OsMBL1 enhances susceptibility of rice plants to M. oryzae Furthermore, MoChi1 suppressed chitin-induced reactive oxygen species in rice cells and competed with OsMBL1 for chitin binding. Taken together, our study reveals a mechanism in which MoChi1 targets a host lectin to suppress rice immunity.

Cloning and functional analysis of squalene synthase gene from Dryopteris fragrans (L.) Schott.

Dryopteris fragrans (L.) Schott is a traditional herbal medicine containing medicinal sterols and triterpenoids. Squalene synthase (SQS) is the first crucial enzyme in the biosynthesis pathway of sterols and triterpenoids. The full-length cDNA named DfSQS1 was isolated by RACE. It was predicted that DfSQS1 contained an open reading frame (ORF) of 1239 bp coding 412 amino acid residues with molecular weight of 46.6 kDa. It had 18 potential phosphorylation sites, 1 potential N-glycosylation site and 2 transmembrane domains. In neighbor-joining (NJ) phylogenetic tree, DfSQS1 was away from branch of gymnosperms and angiosperms. One hydrophobic domain at the C-terminal of DfSQS1 was deleted to express soluble recombinant enzyme. The truncated DfSQS1 (tDfSQS1) was expressed in Escherichia coli BL21 (DE3). Then, tDfSQS1 was obtained and incubated with farnesyl diphosphate (FPP) to identify its enzymatic activity. The result demontrated that squalene, the product of enzyme catalyzed reaction, was detected by HPLC. Quantitative real-time PCR (qRT-PCR) analysis revealed that the transcription level of DfSQS1 in D. fragrans was the highest in roots, followed by leaves and rhizomes. This work is the first report on cloning, characteration and expression of SQS from D. fragrans. It will be helpful to understand the regulatory role of SQS on the biosynthesis of triterpenoids in the fern.

Heterologous activation and metabolites identification of the pks7 gene cluster from Saccharopolyspora erythraea.

The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.

Engineering Streptomyces albus B4 for Enhanced Production of (R)-Mellein: A High-Titer Heterologous Biosynthesis Approach.

The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in ß-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Case report: Beneficial effects of visual cortex tDCS stimulation combined with visual training in patients with visual field defects.

Background: Visual field defect (VFD) refers to the phenomenon that the eye is unable to see a certain area within the normal range of vision, which may be caused by eye diseases, neurological diseases and other reasons. Transcranial direct current stimulation (tDCS) is expected to be an effective treatment for the recovery or partial recovery of VFD. This paper describes the potential for tDCS in combination with visual retraining strategies to have a positive impact on vision recovery, and the potential for neuroplasticity to play a key role in vision recovery. Methods: This case report includes two patients. Patient 1 was diagnosed with a right occipital hemorrhage and homonymous hemianopia. Patient 2 had multiple facial fractures, a contusion of the right eye, and damage to the optic nerve of the right eye, which was diagnosed as a peripheral nerve injury (optic nerve injury). We administered a series of treatments to two patients, including transcranial direct current stimulation; visual field restoration rehabilitation: paracentric gaze training, upper and lower visual field training, VR rehabilitation, and perceptual training. One time per day, 5 days per week, total 6 weeks. Results: After 6 weeks of visual rehabilitation and tDCS treatment, Patient 1 Humphrey visual field examination showed a significant improvement compared to the initial visit, with a reduction in the extent of visual field defects, increased visual acuity, and improvement in most visual functions. Patient 2 had an expanded visual field, improved visual sensitivity, and substantial improvement in visual function. Conclusion: Our case reports support the feasibility and effectiveness of tDCS combined with visual rehabilitation training in the treatment of occipital stroke and optic nerve injury settings.

BCL2L12A localizes to the cell nucleus and induces growth inhibition through G2/M arrest in CHO cells.

BCL2L12, a newly identified member of Bcl-2 family, and its transcript variant BCL2L12A have been found to be associated with favorable prognosis in breast cancer patients while correlated with tumorigenesis of glioblastoma and colon cancer. However, the biological functions of BCL2L12 and especially those of BCL2L12A are largely unknown. Here, we report that, unlike other Bcl-2 family proteins, BCL2L12 and its transcript variant BCL2L12A are nuclear proteins. Interestingly, BCL2L12 forms speckle patterns in the nuclei and potently induces apoptosis in CHO cells. BCL2L12A had a diffuse distribution in the nuclei and inhibits cell growth by inducing cell cycle arrested at G2/M transition in CHO cells. More importantly, BCL2L12A-induced G2/M arrest was associated with a slight up-regulation of cyclin B1 and significant down-regulation of an active form of cyclin B1 phosphorylated at Ser147. Taken together, our study suggests that both BCL2L12 and BCL2L12A have negative effects on CHO cell growths, and that BCL2L12A is a potential cell cycle regulator that interferes with G2-M transition.

Knockdown of BCL2L12 leads to cisplatin resistance in MDA-MB-231 breast cancer cells.

BCL2L12, a newly identified member of Bcl-2 family, contains a BH2 domain and a putative BH3 domain. It was found to be highly expressed in normal breast tissues, and was associated with favorable prognosis in breast cancer patients. Here, we reported that the mRNA levels of BCL2L12 and its transcript variant BCL2L12A could be upregulated upon cisplatin treatment in MDA-MB-231 breast cancer cells. Knockdown of BCL2L12 and BCL2L12A dramatically inhibited cisplatin-induced apoptosis. In contrast, ectopic expressions of each of the proteins promoted cisplatin-induced apoptosis. These results indicated that decreased expressions or loss of BCL2L12 and BCL2L12A may contribute to the cisplatin resistance in breast cancer patients. Furthermore, we found that cisplatin-induced downregulation of beta-catenin was partially suppressed in BCL2L12- and BCL2L12A-knocked down MDA-MB-231 cells, which indicated that knockdown of these two proteins may stabilize beta-catenin in cisplatin-induced apoptosis. In short, we proposed that BCL2L12 and BCL2L12A may play an important role in cisplatin-induced apoptosis in MDA-MB-231 breast cancer cells.

MoIVD-Mediated Leucine Catabolism Is Required for Vegetative Growth, Conidiation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae.

Isovaleryl-CoA dehydrogenase (IVD), a member of the acyl-CoA dehydrogenase (ACAD) family, is a key enzyme catalyzing the conversion of isovaleryl-CoA to ß-methylcrotonyl-CoA in the third reaction of the leucine catabolism pathway and simultaneously transfers electrons to the electron-transferring flavoprotein (ETF) for ATP synthesis. We previously identified the ETF ortholog in rice blast fungus Magnaporthe oryzae (MoETF) and showed that MoETF was essential for fungal growth, conidiation and pathogenicity. To further investigate the biological function of electron-transferring proteins and clarify the role of leucine catabolism in growth and pathogenesis, we characterized MoIVD (M. oryzae isovaleryl-CoA dehydrogenase). MoIvd is highly conserved in fungi and its expression was highly induced by leucine. The Δmoivd mutants showed reduced growth, decreased conidiation and compromised pathogenicity, while the conidial germination and appressorial formation appeared normal. Consistent with a block in leucine degradation, the Δmoivd mutants accumulated isovaleric acid, grew more slowly, fully lacked pigmentation and completely failed to produce conidia on leucine-rich medium. These defects were largely rescued by raising the extracellular pH, suggesting that the accumulation of isovaleric acid contributes to the growth and conidiation defects. However, the reduced virulence of the mutants was probably due to their inability to overcome oxidative stress, since a large amount of ROS (reactive oxygen species) accumulated in infected host cell. In addition, MoIvd is localized to mitochondria and interacted with its electron receptor MoEtfb, the ß subunit of MoEtf. Taken together, our results suggest that MoIVD functions in leucine catabolism and is required for the vegetative growth, conidiation and full virulence of M. oryzae, providing the first evidence for IVD-mediated leucine catabolism in the development and pathogenesis of plant fungal pathogens.

HSP70 protects BCL2L12 and BCL2L12A from N-terminal ubiquitination-mediated proteasomal degradation.

BCL2L12 has been found to be associated with favorable prognosis in breast cancer patients while correlated with tumorigenesis of glioblastoma and colon cancer. Here, we report that BCL2L12 and its transcript variant BCL2L12A are degraded through ubiquitin-proteasome system (UPS). Interestingly, the ubiquitinations and degradations of BCL2L12 and BCL2L12A are independent of the internal lysine residues but the first N-terminal residues. In addition, HSP70 was identified to interact with BCL2L12 and BCL2L12A and protected them from ubiquitinations and degradations in mammalian cells. In summary, HSP70 protects BCL2L12 and BCL2L12A from N-terminal ubiquitination-mediated proteasomal degradation.