Boosting the epoxidation of squalene to produce triterpenoids in Saccharomyces cerevisiae.

BACKGROUND: Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. RESULTS: In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and ß-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of ß-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). CONCLUSIONS: This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.

Eight weeks of dry dynamic breath-hold training results in larger spleen volume but does not increase haemoglobin concentration.

Purpose: It has previously been reported that repeated exposure to hypoxia increases spleen size and haemoglobin (HGB) level and recent reports on the effect apnoea has on spleen size and haematological parameters are contradictory. Therefore, this study aims to evaluate the effect apnoea training has on spleen size and haematological parameters. Methods: The breath-holding (BH) group was comprised of 12 local student-athletes with no BH exercise experience who performed BH jogging and BH jumping rope dynamic apnoea protocols, five times weekly for 8 weeks. The BH event duration was progressively increased as the apnoea tolerance of the athletes improved (20 to 35 s). The same training task was performed by the control group (n = 10) without BH. Spleen sizes were measured with an ultrasound system and a complete blood cell analysis was performed on the median cubital venous blood. Results: Spleen volume in the BH group increased from 109 ± 13 ml to 136 ± 13 ml (p < 0.001), and bulky platelets decreased from 70.50 ± 5.83 to 65.17 ± 5.87 (p = 0.034), but no changes were recorded for erythrocytes (p = 0.914), HGB (p = 0.637), PLTs (p = 0.346) and WBC (p = 0.532). No changes were recorded for the control group regarding spleen size or haematological parameters. Conclusion: Eight weeks of dry dynamic apnoea training increased spleen size and decreased the number of circulating bulky platelets in the athletes who were assessed in this study. However, the baseline RBC counts and HGB levels of the athletes were not altered by the training programme.

Engineering Saccharomyces cerevisiae for Hyperproduction of ß-Amyrin by Mitigating the Inhibition Effect of Squalene on ß-Amyrin Synthase.

The study aims to enhance ß-amyrin production in Saccharomyces cerevisiae by peroxisome compartmentalization. First, overaccumulated squalene was determined as a key limiting factor for the production of ß-amyrin since it could inhibit the activity of ß-amyrin synthase GgbAs1. Second, to mitigate the inhibition effect, the enhanced squalene synthesis pathway was compartmentalized into peroxisomes to insulate overaccumulated squalene from GgbAs1, and thus the specific titer of ß-amyrin reached 57.8 mg/g dry cell weight (DCW), which was 2.6-fold higher than that of the cytosol engineering strain. Third, by combining peroxisome compartmentalization with the "push-pull-restrain" strategy (ERG1 and GgbAs1 overexpression and ERG7 weakening), the production of ß-amyrin was further increased to 81.0 mg/g DCW (347.0 mg/L). Finally, through fed-batch fermentation in a 5 L fermenter, the titer of ß-amyrin reached 2.6 g/L, which is the highest reported to date. The study provides a new perspective to engineering yeasts as a platform for triterpene production.

Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction.

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.

Curcumin ameliorates hypertension via gut-brain communication in spontaneously hypertensive rat.

Gut dysbiosis and dysregulation of gut-brain communication have been identified in hypertensive patients and animal models. Previous studies have shown that probiotic or prebiotic treatments exert positive effects on the pathophysiology of hypertension. This study aimed to examine the hypothesis that the microbiota-gut-brain axis is involved in the antihypertensive effects of curcumin, a potential prebiotic obtained from Curcuma longa. Male 8- to 10-week-old spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats were divided into four groups: WKY rats and SHRs treated with vehicle and SHRs treated with curcumin in dosage of 100 or 300 mg/kg/day for 12 weeks. Our results show that the elevated blood pressure of SHRs was markedly decreased in both curcumin-treated groups. Curcumin treatment also altered the gut microbial composition and improved intestinal pathology and integrity. These factors were associated with reduced neuroinflammation and oxidative stress in the hypothalamus paraventricular nucleus (PVN). Moreover, curcumin treatment increased butyrate levels in the plasma, which may be the result of increased butyrate-producing gut microorganisms. In addition, curcumin treatment also activated G protein-coupled receptor 43 (GPR 43) in the PVN. These results indicate that curcumin reshapes the composition of the gut microbiota and ameliorates the dysregulation of the gut-brain communication to induce antihypertensive effects.

High-Level Production of Sesquiterpene Patchoulol in Saccharomyces cerevisiae.

Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.

Antihypertensive effects of exercise involve reshaping of gut microbiota and improvement of gut-brain axis in spontaneously hypertensive rat.

Exercise (Ex) has long been recognized to produce beneficial effects on hypertension (HTN). This coupled with evidence of gut dysbiosis and an impaired gut-brain axis led us to hypothesize that reshaping of gut microbiota and improvement in impaired gut-brain axis would, in part, be associated with beneficial influence of exercise. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise for 12 weeks, whereas, detrained groups underwent 8 weeks of moderate-intensity exercise followed by 4 weeks of detraining. Fecal microbiota, gut pathology, intestinal inflammation, and permeability, brain microglia and neuroinflammation were analyzed. We observed that exercise training resulted in a persistent decrease in systolic blood pressure in the SHR. This was associated with increase in microbial α diversity, altered ß diversity, and enrichment of beneficial bacterial genera. Furthermore, decrease in the number of activated microglia, neuroinflammation in the hypothalamic paraventricular nucleus, improved gut pathology, inflammation, and permeability were also observed in the SHR following exercise. Interestingly, short-term detraining did not abolish these exercise-mediated improvements. Finally, fecal microbiota transplantation from exercised SHR into sedentary SHR resulted in attenuated SBP and an improved gut-brain axis. These observations support our concept that an impaired gut-brain axis is linked to HTN and exercise ameliorates this impairment to induce antihypertensive effects.

Safety Precautions for the Corona Mortis using Minimally Invasive Ilioinguinal Approach in Treatment of Anterior Pelvic Ring Fracture.

OBJECTIVE: To explore the safety of the corona mortis of the minimally invasive plate insertion in treatment of the anterior pelvic ring fracture by studying the relationship between the vessel and the position of plate. METHOD: The corona mortis was dissected out of eight adult cadavers and were simulated for the insertion of the anterior ring minimally invasive plate, and the presence of the anastomotic branch (the corona mortis) in the suprapubic branch area was observed. After the Corona mortis stripped off, the data was measured, such as the length, vessel diameter, distance from the pubic tubercle, and the maximum vertical distance between the corona mortis and the pubis. The measured data and the previous literatures were analyzed to study the morphology of the corona mortis and the position relation between the corona mortis and the placement of subperiosteal tunnel through the minimally invasive ilioinguinal approach. RESULTS: Out of the 16 unilateral pelvises, the corona mortis were observed on 12 unilateral pelvises with an incidence rate of 75%. Amongst them, there were seven cases of vein anastomosis (incidence of 43.75%), three cases of arterial anastomosis (incidence of 18.75%), and two cases of both arterial anastomosis and vein anastomosis (incidence of 12.5%). The corona mortis length ranged between 24.5 and 37.5 mm (average of 30.7 ± 3.6 mm); the diameter ranged between 1.6 and 3.5 mm (average of 2.5 ± 0.5 mm) and the distance between the vessels and the pubic tubercle was between 53.9 and 65.2 mm (average of 59.0 ± 3.6 mm). Above the pubis, the corona mortis originated from the iliac or the inferior epigastric vessel. It crossed the pubic branch to the dorsal side of the pubis and proceeded downward to anastomize with the obturator vessels near the obturator. Toothless tweezers were used to peel and lift up the corona mortis from the pubic bone. The maximum vertical distance between the corona mortis and the pubis ranged between 8.8 and 18.3 mm (average of 12.6 ± 3.0 mm). CONCLUSION: The corona mortis have a high rate of incidence, with a large number of differences in the type and shape of blood vessels among patients. Following peeling, the movement between the corona mortis and pubic bone is limited. Nevertheless, the plate and bone exfoliator still passed safely. Therefore, when surgeons use the minimally invasive ilioinguinal approach to establish channels, the process of subperiosteal stripping must be performed to avoid any accidental injury.

Calcitriol ameliorated autonomic dysfunction and hypertension by down-regulating inflammation and oxidative stress in the paraventricular nucleus of SHR.

The hypothalamic paraventricular nucleus (PVN) plays crucial roles in central cardiovascular regulation. Increasing evidence in humans and rodents shows that vitamin D intake is important for achieving optimal cardiovascular function. The purpose of this study was to investigate whether calcitriol, an active form of vitamin D, improves autonomic and cardiovascular function in hypertensive rats and whether PVN oxidative stress and inflammation are involved in these beneficial effects. Male spontaneously hypertensive rats (SHR) and normotensive control Wistar Kyoto (WKY) rats were treated with either calcitriol (40 ng/day) or vehicle (0.11 µL/h) through chronic PVN infusion for 4 weeks. Blood pressure and heart rate were recorded continuously by radiotelemetry. PVN tissue, heart and plasma were collected for molecular and histological analysis. Compared to WKY rats, SHR exhibited increased systolic blood pressure, sympathetic drive, and cardiac hypertrophy and remodeling. These were associated with higher mRNA and protein expression levels of high mobility box 1 (HMGB1), receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), proinflammatory cytokines, NADPH oxidase subunit in the PVN. In addition, increased norepinephrine in plasma, elevated reactive oxygen species levels and activation of microglia in the PVN were also observed in SHR. Chronic calcitriol treatment ameliorated these changes but not in WKY rats. Our results demonstrate that chronic infusion of calcitriol in the PVN ameliorates hypertensive responses, sympathoexcitation and retains cardiovascular function in SHR. Reduced inflammation and oxidative stress within the PVN are involved in these calcitriol-induced effects.

External Fixator-assisted Ulnar Osteotomy: A Novel Technique to Treat Missed Monteggia Fracture in Children.

OBJECTIVE: The treatment of missed Monteggia fracture remains a challenge, despite the various surgical methods described. The purpose of this study was to explore a new surgical technique utilizing external fixator-assisted ulnar osteotomy and to assess the surgical results in a case series. METHODS: Thirteen patients with missed Monteggia fractures were treated at our institution using this new surgical technique from August 2012 to January 2016. Our series included 11 boys and 2 girls. The left elbow was involved in 6 patients and the right elbow was involved in 7 patients. According to the Bado classification, 10 fractures were classified as Bado type I with anterior radial head dislocation and 3 were classified as Bado type III with anterolateral dislocation. The average age at the time of surgery was 5 years 8 months (range, 2 years 2 months-10 years). The mean trauma-to-surgery interval was 12 months (range, 2-36 months). All patients underwent ulnar osteotomy with angulation and lengthening using a temporary external fixator, plate fixation of the osteotomy, and open reduction of the radial head dislocation without annular ligament reconstruction. RESULTS: The average follow-up was 27 months (range, 16-44 months). The average operation time was 175 min (range, 140-215 min). The average length of distraction was 0.7 cm (range, 0.5-1.2 cm) and the average angulation was 28° (range, 20°-30°) at the ulnar osteotomy site intraoperatively. The elbow performance score (Kim's) was excellent in 10 cases and good in 3 cases. No neurovascular complications, compartment syndrome or implant breakage occurred. No pain in the distal radioulnar joint or limited range of motion of the wrist occurred in any patient. The radial head remained reduced in all patients with no subluxation or redislocation. However, delayed ulnar union occurred in 3 cases, all of which were successfully treated with plaster cast immobilization within approximately 6 months postoperatively. One patient presented with cubitus valgus postoperatively with a carrying angle of 30°, which was 10° greater than the contralateral carrying angle. CONCLUSIONS: External fixator-assisted ulnar osteotomy offers substantial flexibility for achieving the optimal positioning of the transected ulna to reduce the radial head prior to the final ulnar osteotomy fixation with a plate, thereby facilitating an effective operative performance. Our procedure is a safe and effective method to treat missed pediatric Monteggia fractures.

[Relationship between clinical features and cognitive function in patients with childhood and adolescence-onset schizophrenia].

OBJECTIVE: To explore the factors influencing cognitive functions in patients with childhood and adolescence-onset schizophrenia. METHODS: The clinical data of 78 patients with childhood and adolescence-onset schizophrenia who met with the criteria of ICD-10 for schizophrenia were retrospectively reviewed. The cognitive functions were evaluated by the Chinese Wechsler Intelligence Scale for Children (C-WISC), the Wisconsin Card Sorting Test (WCST), digit span backward and P300. The clinical symptoms were evaluated by the Positive and Negative Syndrome Scale (PANSS). RESULTS: The patients with a lower education level or earlier onset of age had a longer P3 latency at the P300Fz area. The patients with a higher parental education level had higher scores of full intelligence quotient (FIQ), verbal intelligence quotient (VIQ), performance intelligence quotient (PIQ), conceptual level and completed categories of WCST and backward numeric order reciting. The patients with higher PANSS negative subscale scores had lower scores of FIQ, VIQ, PIQ, completed categories and conceptual level of WCST and backward numeric order reciting. The patients with a longer stabilization time had higher backward numeric order reciting scores. CONCLUSIONS: The severity of negative symptoms of the patients and the educational level of their parents are major factors influencing cognitive functions in patients with childhood and adolescence-onset schizophrenia.