Metabolic engineering to produce palmitic acid or palmitoleic acid in an oleic acid-producing Corynebacterium glutamicum strain.

Focusing on the differences in the catalytic properties of two type I fatty acid synthases FasA and FasB, the fasA gene was disrupted in an oleic acid-producing Corynebacterium glutamicum strain. The resulting oleic acid-requiring strain whose fatty acid synthesis depends only on FasB exhibited almost exclusive production (217 mg/L) of palmitic acid (C16:0) from 1% glucose under the conditions supplemented with the minimum concentration of sodium oleate for growth. Plasmid-mediated amplification of fasB led to a 1.47-fold increase in palmitic acid production (320 mg/L), while fasB disruption resulted in no fatty acid production, with excretion of malonic acid (30 mg/L). Next, aiming at conversion of the palmitic acid producer to a producer of palmitoleic acid (POA, C16:1Δ9), we introduced the Pseudomonas nitroreducens Δ9-desaturase genes desBC into the palmitic acid producer. Although this resulted in failure, we noticed the emergence of suppressor mutants that exhibited the oleic acid-non-requiring phenotype. Production experiments revealed that one such mutant M-1 undoubtedly produced POA (17 mg/L) together with palmitic acid (173 mg/L). Whole genomic analysis and subsequent genetic analysis identified the suppressor mutation of strain M-1 as a loss-of-function mutation for the DtxR protein, a global regulator of iron metabolism. Considering that DesBC are both iron-containing enzymes, we investigated the conditions for increased iron availability to improve the DesBC-dependent conversion ratio of palmitic acid to POA. Eventually, supplementation of both hemin and the iron chelator protocatechuic acid in the engineered strain dramatically enhanced POA production to 161 mg/L with a conversion ratio of 80.1%. Cellular fatty acid analysis revealed that the POA-producing cells were really equipped with unnatural membrane lipids comprised predominantly of palmitic acid (85.1% of total cellular fatty acids), followed by non-native POA (12.4%).

The accD3 gene for mycolic acid biosynthesis as a target for improving fatty acid production by fatty acid-producing Corynebacterium glutamicum strains.

We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers. A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3A433T), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3A433T, increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.

Cognitive outcome of very-low-birthweight infants at 6 years of age.

BACKGROUND: The aim of this study was to determine the characteristics of the cognitive function in very-low-birth-weight infants (VLBWI) at 6 years of age and investigate significant factors during neonatal intensive care unit admission that affect cognitive outcomes. METHODS: One hundred and eighty-nine VLBWI (28.4 weeks, 1024 g), including 93 extremely low-birthweight (EL) infants whose birthweight was <1000 g (26.8 weeks, 759 g) and 96 very low-birthweight (VL) infants whose birthweight was 1000-1499 g (30.0 weeks, 1281 g), were enrolled. The cognitive function was measured using the Wechsler Intelligence Scale for Children version 3, three IQ tests, four factor indices and 13 subtest scores. Regression analyses were performed to analyze the cognitive indices and clinical variables during neonatal intensive care unit admission. RESULTS: The full-scale IQ (FIQ) in the EL infants was 85.3 ± 13.4, which was significantly lower than the 91.8 ± 9.7 observed in the VL infants. The verbal IQ and performance IQ in the EL infants were also lower than those observed in the VL infants. The rate of difference between verbal IQ and performance IQ >14 was 20% in the EL infants and 22% in the VL infants. A multiple linear regression analysis revealed a significant relation between FIQ and HC (P = 0.002) and FIQ and dexamethasone (P = 0.012). CONCLUSION: In comparison with that observed in the VL infants, the intelligence quotient of the EL infants was inferior and exhibited more inter-individual variation. Intra-individual imbalances of the cognitive function were highly observed irrespective of the EL or VL status. Restriction of intrauterine brain growth and greater doses of dexamethasone may be harmful for subsequent cognitive outcomes.

Facial nerve function insufficiency after radiosurgery versus microsurgery.

BACKGROUND: Due to the synergic role of the facial nerve and the nervus intermedius in the mechanical protection of the eye and taste, vestibular schwannomas and/or their treatment may prove to be dangerous for the visual function and taste. Our goal was to evaluate and compare the impact of the tumor itself and the impact of microsurgery (MS) or Gamma Knife radiosurgery (GKS). MATERIALS AND METHODS: A functional questionnaire evaluating, among other items, patient complaints related to the eye and taste has been given out to a series of 200 patients 3 years after the GKS of a unilateral vestibular schwannoma not previously resected. Their answers were compared with those of a group of 200 patients operated on microsurgically. A Schirmer test was additionally performed before radiosurgery (RS) and more than 2 years after RS in 66 patients. RESULTS: The risk of dry eye and burning eye is much higher in patients operated by MS compared to patients operated by GKS due to the high incidence of facial palsy (FP) in the former (57/99) and its absence in the later (0/80). In the population operated on microsurgically, the presence of a permanent FP (57 patients among 99 responding to the questionnaire) was, of course, associated with a high rate of complaint, with burning eye in 27 and crying eye in 39. In patients from the two arms with no FP, a dry eye was reported in 8/64 after GKS and 7/42 after MS (not significant) and a burning eye in 9/64 after GKS and 9/42 after MS (not significant). Thus, 14% of patients with no clinical signs of impairment of the VIIth motor nerve presented signs indicating the injury of the intermedius nerve, with the same probability whatever the kind of surgery. When no permanent FP was observed, a crocodile tear syndrome was more frequently observed after MS (4/42 versus 1/64; p = 0.07). This suggests an early lesion of the VIIth motor nerve and nervus intermedius and a subsequent abnormal regrowth. The only patient reporting a crocodile tear syndrome after GKS turned out to have a transiently presented mild deficit of the orbicular muscle signing a transient partial facial nerve injury. In the absence of FP, a 'crying eye' was reported more frequently after MS (16/42 vs. 9/64; p = 0.01) leading us to suspect a frequent subclinical injury of the VIIth nerve in those patients operated on using MS with no obvious FP. Patients tested with the Schirmer test before and more than 2 years later were improved in 27.3%, stable in 56.1% and worse in 16.7% of cases. The answers about taste showed that 8.1% of patients after GKS and 45.5% of patients after MS complained of taste. CONCLUSIONS: This study is the first demonstrating that RS can induce nervus intermedius injury in a small percentage of cases (14%). These patients have been treated 11 years ago with what we can consider as 'archeo-GKS technology' compared to today's radiosurgical instruments. Influence of modern GKS on the nervus intermedius is currently under evaluation in our group. However, symptoms related to the eye and taste either due to the injury of the nervus intermedius or the VIIth motor nerve or both are much more frequent after MS than after RS.

Influence of nerve radiation dose in the incidence of trigeminal dysfunction after trigeminal neuralgia radiosurgery.

OBJECTIVE: The authors conducted a comparative study to analyze dosimetry and results to understand the significant difference in the rate of trigeminal dysfunction after gamma knife radiosurgery for trigeminal neuralgia between two centers using the same target. METHODS: The data of 358 patients (109 patients from Brussels and 259 patients from Marseilles) were analyzed. Three different dosimetric strategies were found: treatment with less than 90 Gy and no selective beam channel blocking (Group 1; patients from Marseilles only), treatment with 90 Gy and no selective beam channel blocking (Group 2; patients from Brussels and Marseilles), or treatment with 90 Gy and use of selective beam channel blocking (Group 3; patients from Brussels only). RESULTS: The prescription dose and the use of selective beam channel blocking have been significantly associated with a higher energy received by the retrogasserian trigeminal nerve root. The different radiation dose delivered to the nerve root in these three groups of patients was significantly associated with the incidence of mild (15, 21, and 49% for Groups 1, 2, and 3, respectively) and bothersome (1.4, 2.4, and 10% for Groups 1, 2, and 3, respectively) trigeminal dysfunction. The good and excellent rates of pain relief were 81 and 66%, respectively, for Group 1, 85 and 77%, respectively, for Group 2, and 90 and 84%, respectively, for Group 3, and were also related to the amount of energy received by nerve root volume. CONCLUSION: Using a similar target, the incidence of trigeminal dysfunction and the pain relief rate can vary according to the radiation energy received by the retrogasserian part of the trigeminal nerve root. The prescription dose and the use of beam channel blocking modify the integrated dose delivered to the nerve and may contribute to the different rates of trigeminal numbness and pain outcome. The radiobiological effect of gamma knife radiosurgery may be related to the energy delivered to nerve root volume, rather than to the maximal dose delivered.

Effect of beam channel plugging on the outcome of gamma knife radiosurgery for trigeminal neuralgia.

PURPOSE: We studied the influence of using plugs for brainstem protection during gamma knife radiosurgery (GKR) of trigeminal neuralgia (TN), with special emphasis on irradiation doses delivered to the trigeminal nerve, pain outcomes, and incidence of trigeminal dysfunction. METHODS AND MATERIALS: A GKR procedure for TN using an anterior cisternal target and a maximum dose of 90 Gy was performed in 109 patients. For 49 patients, customized beam channel blocking (plugs) were used to reduce the dose delivered to the brainstem. We measured the mean and integrated radiation doses delivered to the trigeminal nerve and the clinical course of patients treated with and without plugs. RESULTS: We found that blocking increases the length of trigeminal nerve exposed to high-dose radiation, resulting in a significantly higher mean dose to the trigeminal nerve. Significantly more of the patients with blocking achieved excellent pain outcomes (84% vs. 62%), but with higher incidences of moderate and bothersome trigeminal nerve dysfunction (37% mild/10% bothersome with plugs vs. 30% mild/2% bothersome without). CONCLUSIONS: The use of plugs to protect the brainstem during GKR treatment for TN increases the dose of irradiation delivered to the intracisternal trigeminal nerve root and is associated with an important increase in the incidence of trigeminal nerve dysfunction. Therefore, beam channel blocking should be avoided for 90 Gy-GKR of TN.

Long-term complications after gamma knife surgery for arteriovenous malformations.

OBJECT: The authors analyzed of the long-term complications that occur 2 or more years after gamma knife surgery (GKS) for intracranial arteriovenous malformations (AVMs). METHODS: Patients with previously untreated intracranial AVMs that were managed by GKS and followed for at least 2 years after treatment were selected for analysis (237 cases). Complete AVM obliteration was attained in 130 cases (54.9%), and incomplete obliteration in 107 cases (45.1%). Long-term complications were observed in 22 patients (9.3%). These complications included hemorrhage (eight cases), delayed cyst formation (eight cases), increase of seizure frequency (four cases), and middle cerebral artery stenosis and increased white matter signal intensity on T2-weighted magnetic resonance imaging (one case of each). The long-term complications were associated with larger nidus volume (p < 0.001) and a lobar location of the AVM (p < 0.01). Delayed hemorrhage was associated only with incomplete obliteration of the nidus (p < 0.05). Partial obliteration conveyed no benefit. Delayed cyst formation was associated with a higher maximal GKS dose (p < 0.001), larger nidus volume (p < 0.001), complete nidus obliteration (p < 0.01), and a lobar location of the AVM (p < 0.05). CONCLUSIONS: Incomplete obliteration of the nidus is the most important factor associated with delayed hemorrhagic complications. Partial obliteration does not seem to reduce the risk of hemorrhage. Complete obliteration can be complicated by delayed cyst formation, especially if high maximal treatment doses have been administered.