Carboxylesterases for the hydrolysis of acetoacetate esters and their applications in terpenoid production using Escherichia coli.

Pathway engineering is a useful technology for producing desired compounds on a large scale by modifying the biosynthetic pathways of host organisms using genetic engineering. We focused on acetoacetate esters as novel low-cost substrates and established an efficient terpenoid production system using pathway-engineered recombinant Escherichia coli. Functional analysis using recombinant E. coli proteins of 18 carboxylesterases identified from the microbial esterases and lipases database showed that the p-nitrobenzyl esterase (PnbA) from Bacillus subtilis specifically hydrolyzed two acetoacetate esters: methyl acetoacetate (MAA) and ethyl acetoacetate (EAA). We generated a plasmid (pAC-Mev/Scidi/Aacl/PnbA) co-expressing PnbA and six enzymes of the mevalonate pathway gene cluster from Streptomyces, isopentenyl diphosphate isomerase type I from Saccharomyces cerevisiae, and acetoacetyl-coenzyme A ligase from Rattus norvegicus. The plasmid pAC-Mev/Scidi/Aacl/PnbA was introduced into E. coli along with plasmid expressing carotenoid (lycopene) or sesquiterpene (ß-bisabolene) biosynthesis genes, and the terpenoid production was evaluated following the addition of acetoacetate esters as substrates. These recombinant E. coli strains used MAA and EAA as substrates for the biosynthesis of terpenoids and produced almost equivalent concentrations of target compounds compared with the previous production system that used mevalonolactone and lithium acetoacetate. The findings of this study will enable the production of useful terpenoids from low-cost substrates, which may facilitate their commercial production on an industrial scale in the future. KEY POINTS: • PnbA from Bacillus subtilis exhibits acetoacetate hydrolysis activity. • A plasmid enabling terpenoid synthesis from acetoacetate esters was constructed. • Acetoacetate esters as substrates enable a low-cost production of terpenoids.

Robinow syndrome in a newborn presenting with hydrocephalus and craniosynostosis.

INTRODUCTION: Robinow syndrome is a rare entity with a characteristic appearance, such as hypertelorism, short stature, mesomelic shortening of the limbs, hypoplastic genitalia, and rib as well as vertebral anomalies. We had treated a patient with Robinow syndrome who developed hydrocephalus and craniosynostosis which is not usually associated. CASE PRESENTATION: The ventricle enlargement was detected during pregnancy in a female infant. She did not develop hydrocephalus just after birth. Her facial appearance was fetus-like, so the pediatricians had suspected Robinow syndrome. During follow-up examinations, a rapidly enlarging head circumference was detected when she was 3 months old. Her conscious level was not disturbed, but she had a tight fontanel and sunset phenomenon was recognized. Hydrocephalus was diagnosed by radiographic imaging so that she underwent ventriculo-peritoneal shunting (VPS). Her irregular head enlargement seized. Six months after surgery, her parents noticed the brachycephalic shape of her head. A computed tomography (CT) and magnetic resonance (MR) scan were conducted and showed that her bilateral coronal, bilateral lambdoid, and the sagittal suture were fused in addition with a tonsillar herniation. Since the sutures were not remaining, we diagnosed that this was a primary pan synostosis rather than secondary craniosynostosis due to VPS. Posterior cranial vault distraction with foramen magnum decompression (FMD) was conducted. The distractor was extended by 1 mm per day up to 30 mm. After a consolidation period of 2 months, the distractors were removed. Through this intervention, a 15.4% increase (+196cc) of the intracranial space with an improvement of the chronic tonsillar herniation was achieved. CONCLUSION: To confirm the diagnosis of Robinow syndrome, a genetic test was conducted. The analysis showed ROR2 Exon3 (c233 c>t p. Thr 78 Met), which is found in the recessive type of Robinow syndrome. We report this patient as, to our best knowledge, the first case documented case of Robinow disease presenting with hydrocephalus and craniosynostosis. Posterior cranial vault distraction with FMD is a useful way to treat this condition.

Quality and Quantity-Cultured Human Mononuclear Cells Improve Human Fat Graft Vascularization and Survival in an In Vivo Murine Experimental Model.

BACKGROUND: Fat graft ischemia impedes us from having satisfying long-term results. The quality and quantity culture is a 1-week cell culture that increases the vasculogenic potential of peripheral blood mononuclear cells (PBMNC). This in vivo murine model investigates whether enrichment with quality and quantity-cultured human mononuclear cells (MNC-QQ) improves the vascularization in the human fat graft and whether this decreases the tissue loss. METHODS: Human adipose tissue, PBMNC, MNC-QQ, and stromal vascular fraction were prepared. First, PBMNC, MNC-QQ, and stromal vascular fraction were compared in vitro for vasculogenic potential by endothelial progenitor cell colony-forming and culture assays. Second, 0.25-g fat grafts were created with 1 × 106 PBMNC (n = 16), 1 × 106 MNC-QQ (n = 16), 1 × 106 stromal vascular fraction (n = 16), or phosphate-buffered saline as control (n = 16) before grafting in BALB/c nude mice. Grafts were analyzed for weight persistence, vessel formation by CD31 immunohistochemistry, and angiogenic markers by quantitative polymerase chain reaction. RESULTS: MNC-QQ develop more definitive endothelial progenitor cell colonies and more functional endothelial progenitor cells compared to PBMNC and stromal vascular fraction. Weight persistence after 7 weeks was significantly higher in grafts with MNC-QQ (89.8 ± 3.5 percent) or stromal vascular fraction (90.1 ± 4.2 percent) compared with control (70.4 ± 6.3 percent; p < 0.05). MNC-QQ-enriched grafts had the highest vessel density (96.6 ± 6.5 vessels/mm2; control, 70.4 ± 5.6 vessels/mm2; p < 0.05). MNC-QQ exerted a direct vasculogenic effect through vascular integration and a potential paracrine vascular endothelial growth factor-mediated effect. CONCLUSION: Quality and quantity-cultured human mononuclear cells containing endothelial progenitor cells stimulate fat graft vascularization and enhance graft survival in a rodent recipient.

Quantitative Analysis of Change in Intracranial Volume After Posterior Cranial Vault Distraction and Frontal Orbital Advancement/Remodeling.

INTRODUCTION: Patients with craniosynostosis with shortened occipitofrontal diameter are mainly treated with posterior cranial vault distraction osteogenesis (PVDO) in our institution. If further intracranial volume (ICV) expansion is needed, additional treatment with frontal orbital advancement (FOA) is done. On the contrary, frontal orbital remodeling (FOR) is done for better aesthetic results. In this study, post-treatment ICV changes in patients with craniosynostosis treated with these methods have been investigated. METHODS: Patients who underwent FOA or FOR in addition to PVDO at Juntendo University Hospital between 2011 and 2017 were reviewed for patient characteristics and pre/postoperative ICV measurements using 3-dimensional computed tomography scans. RESULTS: Nine patients aged from 5 months to 6 years 8 months at the time of PVDO were included. For PVDO, the ICV change was 113 to 328 mL, and the enlargement ratio of ICV was 109% to 152%. Two patients were further operated with FOA while the remaining 7 with FOR. With FOA, ICV change was 73 to 138 mL, while enlargement ratio of ICV was 107% to 114%. With FOR, ICV change was 3 to 45 mL (mean 20 mL), while enlargement ratio of ICV was 100% to 103%. CONCLUSION: The PVDO is our first line of treatment for ICV expansion and posterior cranial fossa decompression in patients with severe craniosynostosis. The FOA is performed if extra ICV increase is necessary. This approach seems to enable larger ICV expansions compared with other conventional methods. The FOR should be reserved for patients in whom adequate ICV levels are achieved with PVDO yet additional frontal reshaping is necessary.

The Rapid Development of Squamous Cell Carcinoma on the Nasal Dorsum of a Patient Receiving Immunosuppressive Therapy.

The risk of cancer is significantly increased in patients undergoing renal transplant surgery than in the general population. In particular, skin cancer is the most commonly occurring cancer in these patients.A 34-year-old man underwent living renal transplantation for focal segmental glomerulosclerosis. After 18 months, he developed a lesion on the nasal dorsum, approximately 1 cm in size, and the lesion rapidly expanded to cover the entire dorsum.Owing to its rapid expansion, the lesion was suspected to be a malignant tumor and wide excision was planned.We removed the lesion with a 6-mm margin. Squamous cell carcinoma was diagnosed through intraoperative rapid pathological examination. The nasal bone and septum were invaded by the tumor and, as a result, the entire external nose was removed. The patient's nose was subsequently reconstructed using a free forearm flap for lining, iliac bone graft for the nasal frame, and a scalping forehead flap for skin coverage.Selective target radiotherapy was administered at the closest margin around the lesion, and the dosage of immunosuppressants was reduced.At >2 years postoperatively, the patient showed good cosmetic results with no relapse or metastasis of the tumor.We report the unusual case of a young man who developed a rapidly progressing squamous cell carcinoma on his nasal dorsum after 18 months of immunosuppression. Squamous cell carcinoma in organ transplant recipients may be more aggressive and may progress differently than in regular patients. Therefore, special attention is required for patients who take immunosuppressive drugs after renal transplant surgery.

End-to-Side Neurorrhaphy as Schwann Cells Provider to Acellular Nerve Allograft and Its Suitable Application.

Axonal regeneration relies on support from proliferating host Schwann cells (SCs), and previous studies on acellular nerve allografts (ANGs) suggest that axons can regenerate into ANGs within a limited distance. Numerous studies have demonstrated that the supplementation of ANGs with exogenous factors, such as cultured SCs, stem cells, and growth factors, promote nerve regeneration in ANGs. However, there are several problems associated with their utilization. In this study, we investigated whether end-to-side (ETS) neurorrhaphy, which is an axonal provider, could be useful as an SC provider to support axonal elongation in ANGs. We found that ETS neurorrhaphy effectively promoted SC migration into ANGs when an epineurium window combined with partial neurectomy was performed, and the effectiveness increased when it was applied bilaterally. When we transplanted ANGs containing migrated SCs via ETS neurorrhaphy (hybrid ANGs) to the nerve gap, hybrid ANGs increased the number of regenerated axons and facilitated rapid axonal elongation, particularly when ETS neurorrhaphy was applied to both edges of the graft. This approach may represent a novel application of ETS neurorrhaphy and lead to the development of hybrid ANGs, making ANGs more practical in a clinical setting.

Levator lengthening technique using cartilage or fascia graft for paralytic lagophthalmos in facial paralysis.

INTRODUCTION: Lid loading using gold weights has been commonly used to treat paralytic lagophthalmos (PL); however, the procedure has a relatively high complication rate and the availability of these plates varies among social circumstances. We used a levator lengthening (LL) technique, which originally elongated the levator aponeurosis by inserting a fascia graft between the edge of the levator aponeurosis and the tarsal plate. However, because this procedure tends to result in a wide residual lagophthalmos, we changed the graft material from fascia to conchal cartilage. In this study, we describe in detail our experience with LL using the cartilage graft. METHODS: LL was performed in 18 patients with PL. Fascia grafts were used in seven patients and cartilage grafts in 11. Static reconstructions of the lower eyelid and eyebrow were also performed in most patients. Efficacy was evaluated from patient reports of ocular symptoms and by measuring the palpebral fissure width at opening and closing for both eyes. RESULTS: All patients experienced improved ophthalmological symptoms, which were more apparent in cartilage cases. The average palpebral fissure at eyelid closure was 1.8 mm in cartilage cases and 4.0 mm in fascia cases. In cases where an eyebrow lift was concurrently performed, the residual lagophthalmos became wider in fascia grafting but remained acceptable in cartilage grafting. DISCUSSION: LL is a simple and useful procedure for treating PL with higher efficacy when a cartilage graft is used. However, the level of the upper eyelid can be easily adjusted by changing the fixation position of the cartilage. Additional experience is required to obtain more consistent outcomes.

Effectiveness of platysma muscle flap in preventing Frey syndrome and depressive deformities after parotidectomy.

BACKGROUND: Frey syndrome (FS) or depressive deformity (DD) occurring after parotidectomy significantly reduces a patient's quality of life. However, there seems to be no effective treatment strategy against these complications. In this study, we report our experience of using platysma muscle flap (PMF) to prevent the development of FS and DD after parotidectomy, and evaluate its effect subjectively and objectively. METHODS: Superficial parotidectomy was performed for eight cases of parotid gland tumor, and a PMF was transferred to cover the site. The incidence of FS and DD were evaluated subjectively, using a questionnaire to the patients and board-certified reconstructive surgeons, and objectively, using Minor's starch-iodine test. RESULTS: In seven patients, the defect could be completely covered with PMF, and none of them developed FS or obvious DD. However, in one patient, the defect could be only partially covered, and the patient developed complications in the exact site that the flap did not cover. Overall scores from the questionnaire were high in relation to both cosmetic and functional perspectives from most of the patients and all the surgeons. No patients had major postoperative complications requiring revision. CONCLUSIONS: PMF can be useful to cover the defect and prevent complications after parotidectomy. PMF is relatively easy to perform with fewer complications; however, a complete coverage of the defect should be ensured to obtain optimal results.