Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E.coli.

CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes.

Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in Caldicellulosiruptor saccharolyticus.

BACKGROUND: Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified ß-1,4-linked oligosaccharides, including ß-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization. RESULTS: Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used. CONCLUSION: These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

US-guided core-needle biopsy versus US-guided fine-needle aspiration of suspicious cervical lymph nodes for staging workup of non-head and neck malignancies: A propensity score matching study.

BACKGROUND AND OBJECTIVES: To compare US-guided core-needle biopsy (USCNB) and US-guided fine-needle aspiration (USFNA) of suspicious cervical lymph nodes for staging workup of malignancies in locations other than the head and neck using propensity score matching (PSM). METHODS: This retrospective cohort study included 108 patients who underwent USFNA and 1058 who underwent USCNB for suspicious cervical lymph nodes detected by imaging modalities during the staging workup for malignancies located in regions other than the head and neck. The primary outcome was defined as the inconclusive results. To evaluate the procedures according to the outcome, we performed univariate and multivariate logistic regression analyses and a 1:1 PSM of USCNB and USFNA. RESULTS: There was no complication for both USFNA and USCNB. The inconclusive results were 6.5% (7/108) for USFNA and 1.6% (17/1058) for USCNB. A significantly lower rate of the inconclusive results in USCNB than in USFNA before matching was maintained after a 1:1 PSM of 103 patients from each group (before PSM, odds ratio (OR) = 4.489, 95% confidence interval (CI) [1.803-11.177], P = 0.001; after PSM, OR = 1.060, 95%CI [1.013-1.109], P = 0.012). CONCLUSIONS: Because USCNB can reduce inconclusive results compared to USFNA, it could be more helpful for staging workup of malignancies in locations other than the head and neck in patients with suspicious cervical lymph nodes.

Diffusion-weighted imaging with reverse phase-encoding polarity: the added value to the conventional diffusion-weighted imaging in differentiating acute infarctions from hyperintense brainstem artifacts.

OBJECTIVES: The aim of our study was to assess the value of diffusion-weighted imaging with reverse phase-encoding polarity (R-DWI) in addition to conventional DWI using forward phase-encoding polarity (F-DWI) in differentiating acute brainstem infarctions from hyperintense artefacts. METHODS: Seventy-six patients with 38 hyperintense brainstem artefacts and 38 acute brainstem infarctions that had undergone F-DWI and R-DWI were retrospectively selected based on the clinicoradiological diagnosis. Four radiologists independently rated their confidence in diagnosing acute infarctions and ruling out brainstem artefacts in a blind manner, and then compared the diagnostic performance and confidence between F-DWI alone and F-DWI with R-DWI. RESULTS: The areas under the curve determined for F-DWI with R-DWI in diagnosing infarctions were significantly higher than F-DWI alone for all readers (resident 1, 0.908 vs 0.776; resident 2, 0.908 vs 0.789; neuroradiologist, 0.961 vs 0.868; emergency radiologist, 0.934 vs 0.855, all p < 0.05). All readers were more confident using F-DWI with R-DWI than F-DWI alone (all p < 0.05) for diagnosing acute brainstem infarction, and three readers (readers except the neuroradiologist) were more confident using F-DWI with R-DWI for ruling out brainstem artefacts (p ≤ 0.001). CONCLUSION: The addition of R-DWI to F-DWI is a valuable method for differentiating acute brainstem infarctions from hyperintense artefacts. KEY POINTS: • Hyperintense brainstem artefacts can be confused with acute infarctions on DWI. • Additional R-DWI to F-DWI reduces inter-reader variability in diagnosing brainstem infarctions. • Additional R-DWI improves performance and confidence for discriminating infarctions from artefacts.

Neurogenic muscle hypertrophy: a case report.

Muscular hypertrophy is caused mainly due to myopathic disorder. But, it is also rarely produced by neurogenic disorder. A 74-year-old woman complained of right calf pain with hypertrophy for several years. Recent lumbar spine magnetic resonance imaging (MRI) showed central and lateral canal narrowing at the L4-L5 intervertebral space. Lower extremity MRI revealed fatty change of right medial head of the gastrocnemius and soleus, causing right calf hypertrophy. Electrodiagnostic examinations including electromyography and nerve conduction velocity testing demonstrated 5th lumbar and 1st sacral polyradiculopathy. Integrating all the results, the diagnosis was neurogenic muscle hypertrophy. Neurogenic muscle hypertrophy is very rare, but we recommend that clinicians consider this problem when a patient complains of lower limb hypertrophy and pain.

Superior Cervical Sympathetic Ganglion: Normal Imaging Appearance on 3T-MRI.

OBJECTIVE: To identify superior cervical sympathetic ganglion (SCSG) and describe their characteristic MR appearance using 3T-MRI. MATERIALS AND METHODS: In this prospective study, we recruited 53 consecutive patients without history of head and neck irradiation. Using anatomic location based on literature review, both sides of the neck were evaluated to identify SCSGs in consensus. SCSGs were divided into definite (medial to internal carotid artery [ICA] and lateral to longus capitis muscle [LCM]) and probable SCSGs based on relative location to ICA and LCM. Two readers evaluated signal characteristics including intraganglionic hypointensity of all SCSGs and relative location of probable SCSGs. Interrater and intrarater agreements were quantified using unweighted kappa. RESULTS: Ninety-one neck sites in 53 patients were evaluated after exclusion of 15 neck sites with pathology. Definite SCSGs were identified at 66 (73%) sites, and probable SCSGs were found in 25 (27%). Probable SCSGs were located anterior to LCM in 16 (18%), lateral to ICA in 6 (7%), and posterior to ICA in 3 (3%). Intraganglionic hypointensity was identified in 82 (90%) on contrast-enhanced fat-suppressed T1-weighted images. There was no statistical difference in the relative location between definite and probable SCSGs of the right and left sides with intragnalionic hypointensity on difference pulse sequences. Interrater and intrarater agreements on the location and intraganglionic hypointensity were excellent (κ-value, 0.749-1.000). CONCLUSION: 3T-MRI identified definite SCSGs at 73% of neck sites and varied location of the remaining SCSGs. Intraganglionic hypointensity was a characteristic feature of SCSGs.

Intravoxel Incoherent Motion MR Imaging in the Head and Neck: Correlation with Dynamic Contrast-Enhanced MR Imaging and Diffusion-Weighted Imaging.

OBJECTIVE: To investigate the correlation between perfusion- and diffusion-related parameters from intravoxel incoherent motion (IVIM) and those from dynamic contrast-enhanced MR imaging (DCE-MRI) and diffusion-weighted imaging in tumors and normal muscles of the head and neck. MATERIALS AND METHODS: We retrospectively enrolled 20 consecutive patients with head and neck tumors with MR imaging performed using a 3T MR scanner. Tissue diffusivity (D), pseudo-diffusion coefficient (D(*)), and perfusion fraction (f) were derived from bi-exponential fitting of IVIM data obtained with 14 different b-values in three orthogonal directions. We investigated the correlation between D, f, and D(*) and model-free parameters from the DCE-MRI (wash-in, Tmax, Emax, initial AUC60, whole AUC) and the apparent diffusion coefficient (ADC) value in the tumor and normal masseter muscle using a whole volume-of-interest approach. Pearson's correlation test was used for statistical analysis. RESULTS: No correlation was found between f or D(*) and any of the parameters from the DCE-MRI in all patients or in patients with squamous cell carcinoma (p > 0.05). The ADC was significantly correlated with D values in the tumors (p < 0.001, r = 0.980) and muscles (p = 0.013, r = 0.542), despite its significantly higher value than D. The difference between ADC and D showed significant correlation with f values in the tumors (p = 0.017, r = 0.528) and muscles (p = 0.003, r = 0.630), but no correlation with D(*) (p > 0.05, respectively). CONCLUSION: Intravoxel incoherent motion shows no significant correlation with model-free perfusion parameters derived from the DCE-MRI but is feasible for the analysis of diffusivity in both tumors and normal muscles of the head and neck.

Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity.

A directed evolution and random mutagenesis were carried out with thermotolerant yeast Kluyveromyces marxianus ATCC 36907 for efficient xylitol production. The final selected strain, K. marxianus 36907-FMEL1, exhibited 120 and 39 % improvements of xylitol concentration and xylitol yield, respectively, as compared to the parental strain, K. marxianus ATCC 36907. According to enzymatic assays for xylose reductase (XR) activities, XR activity from K. marxianus 36907-FMEL1 was around twofold higher than that from the parental strain. Interestingly, the ratios of NADH-linked and NADPH-linked XR activities were highly changed from 1.92 to 1.30 when K. marxianus ATCC 36907 and K. marxianus 36907-FMEL1 were compared. As results of KmXYL1 genes sequencing, it was found that cysteine was substituted to tyrosine at position 36 after strain development which might cause enhanced XR activity from K. marxianus 36907-FMEL1.

How to manage the patients with unsatisfactory results after ethanol ablation for thyroid nodules: role of radiofrequency ablation.

PURPOSE: Although ethanol ablation (EA) is effective in the treatment of cystic thyroid nodules, it is less effective in nodules with solid component. Therefore refractory cases with solid component require another treatment modality such as radiofrequency ablation (RFA), which is effective in both solid and cystic thyroid nodules. We prospectively evaluated the efficacy of additional RFA and factors related to volume reduction in patients showing unsatisfactory results after a single session of EA. MATERIALS AND METHODS: Of 94 patients with predominantly cystic thyroid nodules who underwent EA, 20 patients underwent additional RFA because of incompletely resolved clinical problems (symptomatic score reduction <50%) and presence of residual solid component at 1-month follow-up on ultrasonography. Improvement of clinical symptoms and nodule volume reduction were evaluated 6 month later. We evaluated factors related to nodule volume reduction after EA and RFA. RESULTS: RFA after a single session of EA was effective in reducing mean symptom score from 4.8 to 1.1 (p<0.001), mean cosmetic score from 3.5 to 1.4 (p<0.001) and mean nodule volume from 11.3 to 0.9 mL (p<0.001). The only independent factor related to volume reduction after EA was the presence of a solid component (p<0.001), and EA was less effective in nodules when solid component >20% (p=0.001). We identified no factors related to volume reduction after RFA. CONCLUSION: RFA is effective in treatment of benign predominantly cystic thyroid nodules in patients whose clinical problems were incompletely resolved after EA.

Bilateral inferior petrosal sinus sampling by unilateral femoral venous approach.

PURPOSE: Bilateral inferior petrosal sinus sampling (IPSS) is a direct method of distinguishing between pituitary and ectopic ACTH secretion. We present unilateral femoral route technique avoiding bilateral femoral venous puncture using two 4F catheters into both sides of IPSS in these obese patients. MATERIALS AND METHODS: Unilateral femoral puncture using 9F guiding catheter allowed two 4F catheters which can be introduced in each side of inferior petrosal sinus. To reduce bleeding in the gap between 2 catheters at the diaphragm of the 9F femoral sheath, we introduced a short guidewire provided along with femoral sheath. After removing the 9F sheath after procedure, we applied manual compression as usual. We evaluated any technical difficulty and other complications including the presence of hematoma at the puncture site 1 day and 30 days later. RESULTS: Bilateral IPSS by using two 4F catheters in both inferior petrosal sinuses was possible via unilateral femoral route via 9F sheath. There was no technical difficulty introducing 4F catheters into each IPS of both sides. After removing 9F femoral sheath, there was no other complication and no hematoma at the puncture site at 1 day and 30 days later. CONCLUSION: Unilateral femoral venous approach with a 9-French sheath can be used in IPSS. This technique allowed to pass two 4F catheters for IPSS at both sides and could avoid unnecessary bilateral femoral puncture in these obese patients without any hematoma formation after the procedure.

MR spectroscopy and perfusion MR imaging findings of intracranial foreign body granuloma: a case report.

We report a case of intracranial foreign body granuloma that showed features of a high grade tumor on magnetic resonance (MR) imaging. However, the relative cerebral blood volume was not increased in the enhancing mass on perfusion MRI and the choline/creatine ratio only slightly increased on MR spectroscopy. The results suggest that the lesion is benign in nature. Perfusion MRI and MR spectroscopy may be helpful to differentiate a foreign body granuloma from a neoplastic condition.