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.

Radicular pain caused by Schmorl's node: a case report / Dor radicular causada por nódulo de Schmorl: relato de caso

Abstract Schmorl's node a focal herniation of intervertebral disc through the end plate into the vertebral body. Most of the established Schmorl's nodes are quiescent. However, disc herniation into the vertebral marrow can cause low back pain by irritating a nociceptive system. Schmorl's node induced radicular pain is a very rare condition. Some cases of Schmorl's node which generated low back pain or radicular pain were treated by surgical methods. In this article, authors reported a rare case of a patient with radicular pain cause by Schmorl's node located at the inferior surface of the 5th lumbar spine. The radicular pain was alleviated by serial 5th lumbar transforaminal epidural blocks. Transforaminal epidural block is suggested as first conservative option to treat radicular pain due to herniation of intervertebral disc. Therefore, non-surgical treatment such as transforaminal epidural block can be considered a first treatment option for radicular pain caused by Schmorl's node.

Resumo O nódulo de Schmörl (NS) é a herniação focal do disco intervertebral através da placa terminal para dentro do corpo vertebral. A maioria dos nódulos de Schmörl já estabelecidos é quiescente. Porém, a hérnia de disco na medula vertebral pode causar dor lombar quando afeta um sistema nociceptivo. A dor radicular induzida por NS é uma condição muito rara. Alguns casos de NS que causaram dor lombar ou radicular foram tratados com procedimentos cirúrgicos. Neste artigo, relatamos o caso raro de um paciente com dor radicular causada por NS localizado na superfície inferior da quinta vértebra lombar (L5). A dor radicular foi atenuada mediante uma série de bloqueios peridurais transforaminais no nível L5. O bloqueio epidural transforaminal (BET) foi sugerido como primeira opção conservadora para tratar a dor radicular devido à herniação do disco intervertebral. Portanto, um tratamento não cirúrgico como o BET pode ser considerado como uma primeira opção de tratamento da dor radicular causada por NS.

[Radicular pain caused by Schmörl's node: a case report]. / Dor radicular causada por nódulo de Schmorl: relato de caso.

Schmörl's node is focal herniation of intervertebral disc through the end plate into the vertebral body. Most of the established Schmörl's nodes are quiescent. However, disc herniation into the vertebral marrow can cause low back pain by irritating a nociceptive system. Schmörl's node induced radicular pain is very rare condition. Some cases of Schmörl's node which generated low back pain or radicular pain were treated by surgical methods. In this article, authors reported a rare case of a patient with radicular pain cause by Schmörl's node located inferior surface of the 5th lumbar spine. The radicular pain was alleviated by serial 5th lumbar transforamnial epidural blocks. Transforamnial epidural block is suggested as first conservative option to treat radicular pain due to herniation of intervertebral disc. Therefore, non-surgical treatment such as transforamnial epidural block can be considered first treatment option of radicular pain caused by Schmörl's node.

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.

Individualized Acupuncture for Symptom Relief in Functional Dyspepsia: A Randomized Controlled Trial.

OBJECTIVES: This study was implemented to evaluate the effect of individualized acupuncture treatment (AT) on functional dyspepsia (FD). METHODS: A randomized, waitlist-controlled, two-center trial was performed. Seventy-six patients with FD were enrolled in the trial with partially individualized AT in a more realistic clinical setting performed twice a week for 15 minutes a session over 4 weeks. The participants were randomly allocated to a group receiving 8 sessions of AT for 4 weeks or a waitlist control group. After 4 consecutive weeks, the AT group was followed up without AT and the control group received the identical AT. The proportion of responders with adequate symptom relief, Nepean Dyspepsia Index (NDI), FD-related quality of life, Beck Depression Inventory, State-Trait Anxiety Inventory, Acupuncture Belief Scale, and acupuncture credibility test were assessed. RESULTS: After the first 4 weeks, the proportion of responders significantly improved (59% in AT group [n = 37] versus 3% in control group [n = 39]; p < 0.001). The difference was no longer significant at 8 weeks, at which point the waitlist control group showed similar improvement after receiving AT (68% in the AT group versus 79% in the control group). Total NDI scores were significantly reduced in the AT group compared with the waitlist group (p = 0.03). Among NDI items, discomfort (p = 0.01), burning (p = 0.02), fullness after eating (p = 0.02), and burping (p = 0.02) were significantly improved in the AT group compared with the control group. No significant differences were observed between groups in other secondary variables. CONCLUSION: Individualized AT adequately relieves symptoms in patients with FD, and this effect may persist up to 8 weeks.

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.

Availability of tongue diagnosis system for assessing tongue coating thickness in patients with functional dyspepsia.

Tongue diagnosis is an important procedure in traditional Korean medicine (TKM). In particular, tongue coating thickness (TCT) is deemed to show the progression of the disease. However, conventional tongue diagnosis has limitations because of various external factors. Therefore, it is necessary to investigate the availability of tongue diagnosis system (TDS) in the assessment of TCT. This study has been designed as a prospective clinical trial involving 60 patients with functional dyspepsia. Tongue images will be obtained by TDS twice with a 30 min interval. The system will measure the percentage of TCT and classify it as either no coating, thin coating, or thick coating according to the existing diagnostic criteria. After finishing the collection of 60 patients' tongue images, TCT on the images will be simultaneously evaluated by the conventional method to establish the gold standard for assessing TCT by 5 well-trained clinicians. The evaluation will be repeated by the same clinicians after 2 weeks, but the order of the images will be changed. This trial is expected to provide clinical evidence for the availability of TDS as a diagnostic tool and to contribute to the standardization of the diagnosis system used in TKM. This trial is registered with ClinicalTrials.gov NCT01864837.