A Herniorrhaphy Lamination Technique for the Reconstruction of Midline Abdominal Wall Defects.

The primary goal of abdominal wall reconstruction is to prevent hernia recurrence through robust and durable repair. Synthetic mesh utilization can provide sound strength but is susceptible to extrusion, infection, and intestinal fistulization. The use of autologous fasciae latae to reinforce the primary fascial reapproximation has mostly been abandoned, presumably because synthetic patches are readily available. There is a specific demand for a sustainable, less-invasive, and ready-to-use repair method without mesh. The authors devised a herniorrhaphy lamination technique using local musculofascial flaps inspired by composite laminates. In this procedure, the primary fascial reapproximation is reinforced with 3 additional laminated musculofascial layers: (1) turnover hinge flaps of the anterior sheath of the rectus abdominis, (2) bilateral rectus abdominis, and (3) advancement flaps of newly generated edges of the fascia of the rectus sheath. Our technique's stability is essentially due to the mechanical superiority of the centralized pipe-like structure of musculofascia. Between February 2009 and November 2019, we used the lamination technique to repair midline incisional hernias in 10 patients. The operative procedure was successful in all patients, and there has been no evidence of recurrence. The follow-up period ranged from 12 to 69 months, with a mean follow-up of 35 months. The herniorrhaphy lamination technique to reinforce the primary repair can help prevent hernia recurrence. Although our technique is suitable for a small-sized defect, it is less invasive, and can be readily applied. Because it does not include any mesh, it is suitable for the contaminated abdominal wall reconstruction.

Atlantoaxial Rotatory Fixation after Microtia Reconstruction Surgery.

Nontraumatic atlantoaxial rotatory fixation after microtia reconstruction surgery is a rare complication. Intraoperative cervical hyperextension and/or excessive rotation and postoperative inflammation have been reported as causes of atlantoaxial rotatory fixation. We herein describe cases of atlantoaxial rotatory fixation after microtia reconstruction surgery. METHODS: This was a retrospective study of 80 patients (165 surgeries) who underwent microtia reconstruction surgery in Dokkyo Medical University Hospital between April 2006 and December 2012. The patient- and operation-related variables were obtained from medical charts. Neck radiographs and computed tomography scans of patients with atlantoaxial rotatory fixation were evaluated to check for cervical spine abnormalities. RESULTS: Five cases of atlantoaxial rotatory fixation after microtia reconstruction surgery were recorded. Three of these five cases were diagnosed with Klippel-Feil syndrome after the onset of atlantoaxial rotatory fixation. No significant difference was found in the operative duration and other variables between patients with atlantoaxial rotatory fixation and those without. All patients immediately underwent conservative treatment and showed complete recovery and no recurrences. CONCLUSIONS: Although atlantoaxial rotatory fixation is a rare complication, surgeons should consider it in patients with neck problems following microtia reconstruction surgery. A patient with microtia may have unrecognized Klippel-Feil syndrome. Patients with Klippel-Feil syndrome are more likely to develop atlantoaxial rotatory fixation, which may have severe consequences. Thus, it is crucial to preoperatively identify Klippel-Feil syndrome with neck radiography and to detect atlantoaxial rotatory fixation at the earliest.

Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome.

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1beta (IL-1beta), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1beta expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression.

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity.

Pseudomonas stutzeri L-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with L-rhamnose and D-allose which has different configurations of C4 and C5 from L-rhamnose, were determined at a resolution of 2.0 A, 1.97 A, and 1.97 A, respectively. P. stutzeri L-RhI has a large domain with a (beta/alpha)(8) barrel fold and an additional small domain composed of seven alpha-helices, forming a homo tetramer, as found in E. coli L-RhI and D-xylose isomerases (D-XIs) from various microorganisms. The beta1-alpha1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding beta1-alpha1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with L-rhamnose and D-allose show that both substrates are nicely fitted to the substrate-binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the beta1-alpha1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of L-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.

Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidusD-arabinose isomerase.

D-Arabinose isomerase catalyzes the isomerization of D-arabinose to D-ribulose. Bacillus pallidus D-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of D-arabinose, L-fucose, L-xylose, L-galactose and D-altrose. Recombinant B. pallidus D-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 A. Diffraction data were collected to 2.3 A resolution.

Increased numbers of macrophages in tracheal aspirates in premature infants with funisitis.

BACKGROUND: Funisitis is a manifestation of the fetal inflammatory response syndrome, and intrauterine inflammation is involved in the pathogenesis of lung injury in premature infants. The aim of the present paper was to examine the relationship between funisitis and lung injury in premature infants born at <28 weeks gestation. The present study focuses on the number of macrophages in tracheobronchial aspirate fluid (TAF). METHODS: The numbers of CD68-positive cells in cell cytopreps in TAF collected at <24 h of age were determined on immunocytochemistry. The funisitis (+) group (n > 8) was compared with the funisitis (-) group (n > 16). RESULTS: There were no significant differences in gestational age and birthweight between these groups. The duration of intermittent positive pressure ventilation was significantly longer in the funisitis (+) group compared with the funisitis (-) group (P < 0.05). Funisitis (+) infants had increases of CD68+ macrophages in their TAF. The appearance of Wilson-Mikity syndrome (WMS), characteristic of the severe variant of chronic lung disease (CLD), was associated with funisitis (+) infants with higher numbers of macrophages. CONCLUSION: The presence of macrophages at birth plays an important role in the neonatal lung with funisitis.

Purification, crystallization and preliminary X-ray diffraction studies of D-tagatose 3-epimerase from Pseudomonas cichorii.

D-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of D-psicose has not been reported with epimerases other than P. cichorii D-TE and D-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 A, beta = 102.82 degrees . Diffraction data were collected to 2.5 A resolution. The asymmetric unit is expected to contain four molecules.

Aseptic meningitis associated with cephalosporins in an infant with trisomy 21.

The authors report a case of aseptic meningitis associated with cephalosporins in an infant. A 1-year-old boy with trisomy 21 received several antimicrobials including cefotaxime and ceftriaxone for bacterial meningitis caused by Haemophilus influenzae b. High fever continued for more than a month, and discontinuation of cefotaxime broke the fever and improved the findings of cerebrospinal fluid. Because third-generation cephalosporins are the first choice against bacterial meningitis for infants, recognition and diagnosis of this rare occurrence of drug-induced aseptic meningitis is important. It is treatable by withdrawal of the drug, and recurrence can be prevented.

Costal cartilage graft with perichondrium, a possible anti-adhesive material.

BACKGROUND: Adhesion occurs as a part of the wound healing process, but it sometimes compromises patients' daily activities. The authors were looking for materials and methods that could prevent adhesion, and noticed that the costal cartilage has possibility. The anti-adhesive property of the costal cartilage was examined histologically. METHODS: Thirty-five patients with microtia who provided consent for participating in this study were enrolled between April 2008 and March 2015. In the first stage of microtia reconstruction surgery, the excess cartilage was used to create these three types of specimens: (A) a piece of cartilage retaining the perichondrium on one side, (B) a piece of only cartilage parenchyma sliced with a plane parallel to the long axis of costal cartilage, and (C) the costal cartilage in a plane perpendicular to the long axis sliced pieces. These specimens were implanted into the subcutaneous fat of the chest. After at least 6 months in the second stage of surgery (i.e. auricular elevation), these specimens, wearing a little around the adipose tissue, we removed and examined histologically. RESULT: A fibrosis formation of the perichondrium side of Specimen A was thicker significantly than that of the cartilage side. A fibrosis formation of Specimen B was thicker significantly than that of the cartilage side of Specimen A. CONCLUSION: It was suggested that, if there is perichondrium, the costal cartilage parenchyma surface makes less adhesion with surrounding tissues. Costal cartilage with unilateral perichondrium is likely to be an effective surgical material for adhesion prevention.

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation.

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.

Intraventricular ascorbic acid administration decreases hypoxic-ischemic brain injury in newborn rats.

Neuronal cell damage following hypoxic-ischemic (HI) brain injury is partly caused by production of free radicals and reactive oxygen species (ROS). Ascorbic acid (AA) is a potent antioxidant, which scavenges various types of ROS. Some studies have shown that it is neuroprotective, however, the issue is still controversial. In this study, we examined the effect of intraventricular AA administration on immature HI brain using the Rice-Vannucci model. After unilateral carotid artery ligation under isoflurane anesthesia, 7-day-old rat pups received varying concentrations of AA (0.04, 0.2, 1 and 5 mg/kg) by intraventricular injection and were exposed to 8% oxygen for 90 min. Vehicle controls received an equal volume of phosphate saline buffer. We assessed the neuroprotective effect of AA at 7 days post-HI. The percent brain damage measured by comparing the wet weight of the ligated side of hemisphere with that of contralateral one was reduced in both 1 and 5 mg/kg groups but not in either 0.04 or 0.2 mg/kg groups compared to vehicle controls (5 mg/kg 16.0 +/- 4.3%, 1 mg/kg 10.9 +/- 5.0%, vs. controls 36.7 +/- 3.6%, P < 0.05). Macroscopic evaluation of brain injury revealed the neuroprotective effect of AA in both 1 and 5 mg/kg groups (5 mg/kg 1.1 +/- 0.4, 1 mg/kg 0.4 +/- 0.3, vs. controls 2.9 +/- 0.3, P < 0.05). Western blots of fodrin on the ligated side also showed that AA significantly suppressed 150/145-kDa bands of fodrin breakdown products, which suggested that AA suppressed activation of calpain. Neuropathological quantitative analysis of cell death revealed that 1 mg/kg of AA injection significantly reduced the number of necrotic cells in cortex, caudate putamen, thalamus and hippocampus CA1, whereas that of apoptotic cells was only reduced in cortex. These findings show that intraventricular AA injection is neuroprotective after HI in immature rats.

Elastogenesis in cultured dermal fibroblasts from patients with lysosomal beta-galactosidase, protective protein/cathepsin A and neuraminidase-1 deficiencies.

The human GLB1 gene encodes a lysosomal beta-galactosidase (beta-Gal) and an elastin-binding protein (EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex among neuraminidase-1 (NEU1) and protective protein/cathepsin A (PPCA) is suggested responsible for impaired elastogenesis in autosomal recessive beta-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects of GLB1, PPCA and NEU1 gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H and W273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, the W273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.

Crystallization and preliminary X-ray diffraction studies of L-rhamnose isomerase from Pseudomonas stutzeri.

L-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeri L-RhI) catalyzes not only the reversible isomerization of L-rhamnose to L-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeri L-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 A, beta = 106.8 degrees . Diffraction data have been collected to 2.0 A resolution. The molecular weight of the purified P. stutzeri L-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI-TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules.

Novel substrate specificity of D-arabinose isomerase from Klebsiella pneumoniae and its application to production of D-altrose from D-psicose.

d-Arabinose isomerase from Klebsiella pneumoniae 40bXX was purified 12-fold with a 62.5% yield indicated by its electrophoretic homogeneity. The purified enzyme showed the highest activities toward d-arabinose and l-fucose as substrates at optimum conditions (50 mM glycine-NaOH, pH 9.0, 40 degrees C). The enzyme had a broad range of substrate specificities toward various d/l-aldoses, i.e., d-arabinose, l-fucose, d/l-xylose, d-mannose, d/l-lyxose, l-glucose, d-altrose and d/l-galactose. The equilibrium ratios between d-arabinose and d-ribulose, l-fucose and l-fuculose, d-altrose and d-psicose, and l-galactose and l-tagatose were 90:10, 90:10, 13:87 and 25:75, respectively. Using a combination of the immobilized d-tagatose 3-epimerase and d-arabinose isomerase, we achieved the production of d-altrose from d-fructose in a batch reactor. We successfully produced approximately 12 g of d-altrose from 200 g of d-fructose in a reaction series with an overall yield of 6%. The product obtained was confirmed to be d-altrose by HPLC and (13)C-NMR. To the best of our knowledge, this is the first report on the production of d-altrose from a cheap sugar, d-fructose, using an enzymatic method.

Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization.

Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.

Novel reactions of L-rhamnose isomerase from Pseudomonas stutzeri and its relation with D-xylose isomerase via substrate specificity.

Escherichia coli strain JM 109 harboring 6 x His-tag L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri allowed a 20-fold increase in the volumetric yield of soluble enzyme compared to the value for the intrinsic yield. Detailed studies on the substrate specificity of the purified His-tagged protein revealed that it catalyzed previously unknown common and rare aldo/ketotetrose, aldo/ketopentose, and aldo/ketohexose substrates in both D- and L-forms, for instance, erythrose, threose, xylose, lyxose, ribose, glucose, mannose, galactose, altrose, tagatose, sorbose, psicose, and fructose. Using a high enzyme-substrate ratio in extended reactions, the enzyme-catalyzed interconversion reactions from which two different products from one substrate were formed: L-lyxose, L-glucose, L-tagatose and D-allose were isomerized to L-xylulose and L-xylose, L-fructose and L-mannose, L-galactose and L-talose, and D-psicose and D-altrose, in that order. Kinetic studies, however, showed that L-rhamnose with Km and Vmax values of 11 mM and 240 U/mg, respectively, was the most preferred substrate, followed by L-mannose, L-lyxose, D-ribose, and D-allose. Based on the observed catalytic mode of action, these new findings reflected a hitherto undetected interrelation between L-RhI and D-xylose isomerase (D-XI).

Usefulness of exercise-induced changes in plasma levels of brain natriuretic peptide in predicting right ventricular contractile reserve after repair of tetralogy of Fallot.

Elevated levels of brain natriuretic peptide (BNP) have been associated with ventricular dysfunction, and exercise tests have been used for assessing cardiac contractile reserve. We examined the relation between BNP and right ventricular (RV) contractile reserve during exercise in patients after repair of tetralogy of Fallot (TOF). A total of 45 patients, 26 of whom underwent repair of TOF at 2 to 3 years of age and 19 age-matched healthy children, were studied. Plasma levels of BNP were measured at baseline and at maximal exercise. Echocardiography combined with tissue Doppler imaging (TDI) was performed at rest and during supine bicycle submaximal exercise. The peak value of the first derivation of RV pressure (peak dP/dt) was measured by the continuous-wave Doppler method. The severity of pulmonary regurgitation (PR) (mild, moderate, or severe) was based on color Doppler findings. Plasma BNP levels were significantly higher in patients with TOF than in controls (44 +/- 34 vs 6 +/- 4 pg/ml, p <0.01). Exercise was associated with increased plasma BNP levels in both groups. A larger increment in BNP was noted in patients with TOF than in normal subjects (15 +/- 12 vs 2 +/- 2 pg/ml, p <0.01). The peak systolic myocardial velocity (Sa) and peak dP/dt values increased significantly in both groups during exercise; however, the magnitude of increase in both of these values was significantly less in patients with TOF than in controls (36 +/- 19% vs 70 +/- 19% and 42 +/- 11% vs 81 +/- 12%, respectively; p <0.01). There were significant correlations between the increment in BNP and changes in Sa and peak dP/dt values (r = -0.67 and -0.53, p <0.01, respectively), and the severity of PR (r = 0.74, p <0.01). Thus, exercise increases plasma levels of BNP, and greater increases are associated with impaired RV contractile reserve in patients with TOF with various degrees of PR.

Calpain inhibitor MDL 28170 protects hypoxic-ischemic brain injury in neonatal rats by inhibition of both apoptosis and necrosis.

MDL 28170 is a CNS-penetrating calpain inhibitor, and we examined the effects of MDL 28170 on hypoxic-ischemic brain injury in immature brain using the Rice-Vannucci model. Immediately after hypoxic exposure, 24 mg/kg of MDL 28170 was injected intraperitoneally as an initial dose, followed by 12 mg/kg every 4 h for a total dose of 60 mg/kg over 12 h post-HI. A vehicle control group received peanut oil injection instead. Macroscopic evaluation of brain injury revealed the neuroprotective effect of MDL 28170 after 12 h post-HI. Neuropathological quantitative analysis of cell death showed that MDL 28170 significantly decreased the number of necrotic cells in all the examined regions except for cingular cortex, and the number of apoptotic cells in caudate putamen, parietal cortex, hippocampus CA1, and laterodorsal thalamus. Western blots showed that MDL 28170 suppressed 145/150 kDa subunits of alpha-spectrin breakdown products (SBDP) in cortex, hippocampus, thalamus, and striatum, and also 120-kDa subunit of SBDP in all regions except for striatum. This suggests that MDL 28170 inhibited activation of calpain and caspase-3, respectively. Our results indicate that post-hypoxic MDL 28170 injection is neuroprotective in HI newborn rat brain by decreasing both necrosis and apoptosis. SBDP expression also suggests that MDL 28170 injection inhibits both calpain and caspase-3 activation after HI insult.

Prolonged hypothermia protects neonatal rat brain against hypoxic-ischemia by reducing both apoptosis and necrosis.

Although hypothermia is an effective treatment for perinatal cerebral hypoxic-ischemic (HI) injury, it remains unclear how long and how deep we need to maintain hypothermia to obtain maximum neuroprotection. We examined effects of prolonged hypothermia on HI immature rat brain and its protective mechanisms using the Rice-Vannucci model. Immediately after the end of hypoxic exposure, the pups divided into a hypothermia group (30 degrees C) and a normothermia one (37 degrees C). Rectal temperature was maintained until they were sacrificed at each time point before 72h post HI. Prolonged hypothermia significantly reduced macroscopic brain injury compared with normothermia group. Quantitative analysis of cell death using H&E-stained sections revealed the number of both apoptotic and necrotic cells was significantly reduced by hypothermia after 24h post HI. Hypothermia seemed to decrease the number of TUNEL-positive cells. Immunohistochemistry and Western blot showed that prolonged hypothermia suppressed cytochrome c release from mitochondria to cytosol and activation of both caspase-3 and calpain in cortex, hippocampus, thalamus and striatum throughout the experiment. These results showed that prolonged hypothermia significantly reduced neonatal brain injury even when it was started after HI insult. Our results suggest that prolonged hypothermia protects neonatal brain after HI by reducing both apoptosis and necrosis.