Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

Development of a peptide nucleic acid-based multiprobe real-time PCR method targeting the hsp65 gene for differentiation among Mycobacterium abscessus strains.

Recently, the need to distinguish between members of the Mycobacterium abscessus group has gained increasing attention. Here, we introduced a novel peptide nucleic acid (PNA) real-time PCR method targeting the hsp65 gene in order to distinguish between four subspecies within the M. abscessus group (M. abscessus and 3 types of M. massiliense).

Separation of Mycobacterium abscessus into subspecies or genotype level by direct application of peptide nucleic acid multi-probe- real-time PCR method into sputa samples.

BACKGROUND: Recently, we introduced a novel peptide nucleic acid (PNA) multi-probe real time PCR method targeting the hsp65 gene (hsp65 PNA RT-PCR) to distinguish Mycobacterium abscessus groups. METHODS: Here, we evaluated the usefulness of the hsp65 PNA RT-PCR for the direct identification of the M. abscessus group at the subspecies and genotype levels from sputa samples. The method was applied to total sputa DNA from 60 different patients who were identified as having mycobacterial infections via rpoB PCR restriction analysis of the same cultures. RESULTS: The hsp65 PNA RT-PCR method had higher sensitivity than the multi-probe real-time PCR assay targeting hsp65 (HMPRT-PCR) for the detection of M. abscessus from sputum [96.7 % (29/30 samples) vs. 70 % (21/30 samples); 100 % specificity]. CONCLUSIONS: These results suggest that the PNA-based method is feasible for the detection of M. abscessus members not only from cultures but also directly from sputa.

Mycobacterium paragordonae sp. nov., a slowly growing, scotochromogenic species closely related to Mycobacterium gordonae.

A previously undescribed, slowly growing, scotochromogenic mycobacterial strain (49061(T)) was isolated from a patient with pulmonary infections during the hsp65-sequence-based identification of Korean clinical isolates. Its 16S rRNA gene sequence was unique and the phylogenetic analysis based on 16S rRNA gene sequence (1393 bp) placed the organism into the slow-growing Mycobacterium group close to Mycobacterium gordonae (99.0 % sequence similarity). Growth characteristics and acid-fastness also supported the placement of this species into the genus Mycobacterium. Phenotypically, this strain was generally similar to Mycobacterium gordonae; however, of particular interest, the optimal growth temperature of strain 49061(T) was 25-30 °C, and it was not able to grow at 37 °C on 7H10 agar slants. Unique MALDI-TOF MS profiles of lipids, phylogenetic analysis based on another two gene sequences (hsp65 and rpoB) and a low DNA-DNA relatedness (46.52±0.7) strongly supported the taxonomic status of this strain as a representative of a distinct species from M. gordonae. It was concluded that the strain represents a novel species for which the name Mycobacterium paragordonae is proposed with the type strain 49061(T) ( = JCM 18565(T) = KCTC 29126(T)).

Melatonin Improves Glucose Homeostasis and Insulin Sensitivity by Mitigating Inflammation and Activating AMPK Signaling in a Mouse Model of Sleep Fragmentation.

Sleep fragmentation (SF) can increase inflammation and production of reactive oxygen species (ROS), leading to metabolic dysfunction. SF is associated with inflammation of adipose tissue and insulin resistance. Several studies have suggested that melatonin may have beneficial metabolic effects due to activating AMP-activated protein kinase (AMPK). However, it is unclear whether melatonin affects the AMPK signaling pathway in SF-induced metabolic dysfunction. Therefore, we hypothesize that SF induces metabolic impairment and inflammation in white adipose tissue (WAT), as well as altered intracellular homeostasis. We further hypothesize that these conditions could be improved by melatonin treatment. We conducted an experiment using adult male C57BL/6 mice, which were divided into three groups: control, SF, and SF with melatonin treatment (SF+Mel). The SF mice were housed in SF chambers, while the SF+Mel mice received daily oral melatonin. After 12 weeks, glucose tolerance tests, insulin tolerance tests, adipose tissue inflammation tests, and AMPK assessments were performed. The SF mice showed increased weight gain, impaired glucose regulation, inflammation, and decreased AMPK in WAT compared to the controls. Melatonin significantly improved these outcomes by mitigating SF-induced metabolic dysfunction, inflammation, and AMPK downregulation in adipose tissue. The therapeutic efficacy of melatonin against cardiometabolic impairments in SF may be due to its ability to restore adipose tissue homeostatic pathways.

Mycobacterium parakoreense sp. nov., a slowly growing non-chromogenic species related to Mycobacterium koreense, isolated from a human clinical specimen.

A previously undescribed, slowly growing, non-chromogenic Mycobacterium strain (299(T)) was isolated from the sputum sample of a patient with a symptomatic pulmonary infection. Phenotypically, strain 299(T) was generally similar to Mycobacterium koreense DSM 45576(T) and Mycobacterium triviale ATCC 23292(T). The 16S rRNA gene sequence of strain 299(T) was similar to that of M. koreense DSM 45576(T) (GenBank accession no. AY734996, 99.5% similarity); however, it differed substantially from that of M. triviale ATCC 23292(T) (X88924, 98.2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 299(T) clustered together with M. koreense DSM 45576(T) and M. triviale ATCC 23292(T), supported by high bootstrapping values (99%). Unique mycolic acid profiles and phylogenetic analysis based on two different chronometer molecules, the hsp65 and rpoB genes, strongly supported the taxonomic status of this strain as representing a distinct species. These data support the conclusion that strain 299(T) represents a novel mycobacterial species, for which the name Mycobacterium parakoreense sp. nov. is proposed. The type strain is 299(T) (=DSM 45575(T)=KCTC 19818(T)).

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-36Y, belonging to the INT5 genotype.

Here we report the complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-36Y, previously grouped into the INT5 genotype among the 5 genotypes of M. intracellulare. This genome sequence will serve as a valuable reference for understanding the disparity in virulence and epidemiologic traits between M. intracellulare-related strains.

Physiologically based pharmacokinetics of zearalenone.

The objectives of this study were to (1) develop physiologically based pharmacokinetic (PBPK) models for zearalenone following intravenous (i.v.) and oral (p.o.) dosing in rats and (2) predict concentrations in humans via interspecies scaling. The model for i.v. dosing consisted of vein, artery, lung, liver, spleen, kidneys, heart, testes, brain, muscle, adipose tissue, stomach, and small intestine. To describe the secondary peak phenomenon observed after p.o. administration, the absorption model was constructed to reflect glucuronidation, biliary excretion, enterohepatic recirculation, and fast and slow absorption processes from the lumenal compartment. The developed models adequately described observed concentration-time data in rats after i.v. or p.o. administration. Upon model validation in rats, steady-state zearalenone concentrations in blood and tissues were simulated for rats after once daily p.o. exposures (0.1 mg/kg/d). The average steady-state blood zearalenone concentration predicted in rat was 0.014 ng/ml. Subsequently, a daily human p.o. dose needed to achieve the same steady-state blood concentration found in rats (0.014 ng/ml) was determined to be 0.0312 mg/kg/d or 2.18 mg/70 kg/d. The steady-state zearalenone concentration-time profiles in blood and tissues were also simulated for human after multiple p.o. administrations (dose 0.0312 mg/kg/d). The developed PBPK models adequately described the pharmacokinetics in rats and may be useful in predicting human blood and tissue concentrations for zearalenone under different p,o, exposure conditions.

Disposition, oral bioavailability, and tissue distribution of zearalenone in rats at various dose levels.

This study was conducted to characterize the disposition, oral bioavailability, and tissue distribution of zearalenone in rats. The pharmacokinetics and tissue distribution of zearalenone were studied after intravenous (i.v.) or oral (p.o.) administration at doses ranging from 1 to 8 mg/kg in intact and bile duct-cannulated rats. Serum, bile, and urine concentrations were determined by liquid chromatography and mass spectroscopy (LC/MS/MS) and tissue concentrations by high-performance liquid chromatography (HPLC)/fluorescence detection assays. Noncompartmental methods were used for pharmacokinetic analysis. Average Cl(s) (range 5.0-6.6 L/h/kg) and V(dss) (range 2-4.7 L/kg) remained unaltered over an i.v. dose range from 1 to 8 mg/kg, and area under the concentration-time curve (AUC) and initial peak concentrations increased linearly with dose. Minimal quantities of zearalenone were excreted unchanged in urine (f(e,urine) 0.5 +/- 0.2%) and bile (f(e,bile) 0.91 +/- 0.64%). After p.o. administration of 8 mg/kg, zearalenone was rapidly absorbed and serum concentration-time profiles showed a distinct second peak. The absolute oral bioavailability was low (2.7%). Comparing bile duct-cannulated to intact rats at a dose of 8 mg/kg, the impact of biliary excretion on overall pharmacokinetics was more pronounced after p.o. than after i.v. administration. Upon i.v. infusion to steady state, the highest zearalenone concentration was found in small intestine, followed by kidneys, liver, adipose tissue, and lung. Zearalenone concentrations in stomach, heart, brain, spleen, muscle, and testes were lower than those found in serum. The pharmacokinetics and tissue distribution data from this study may be useful to develop physiologically based pharmacokinetic (PBPK) models for zearalenone and subsequently to predict the pharmacokinetics and toxicity in humans.

Prediction of drug bioavailability in humans using immobilized artificial membrane phosphatidylcholine column chromatography and in vitro hepatic metabolic clearance.

This study reports a rapid screening method for the prediction of oral drug bioavailability in humans based on combined immobilized artificial membrane (IAM) chromatographic capacity factor (k(IAM)) and in vitro stability in hepatic microsomes. The fraction of drug absorbed (F(a)) in humans was predicted for a set of 15 structurally diverse commercial drugs based on k(IAM) values using a mobile phase consisting of acetonitrile: Dulbecco's phosphate-buffered saline. The hepatic intrinsic clearance (CL'(int)) was calculated from in vitro disappearance half-life, and the oral bioavailability was predicted using in vitro hepatic clearance (CL(h)) and F(a). Significant correlations were observed for the relationships between predicted hepatic extraction ratios (ER(h)) and actual presystemic metabolism (r = 0.854) and between predicted and observed oral bioavailabilities (r = 0.805; p < 0.01). The IAM capacity factor together with the hepatic microsomal disappearance half-life may be useful in identifying compounds with high oral absorption potential in early drug discovery processes.

Determination of zearalenone by liquid chromatography/tandem mass spectrometry and application to a pharmacokinetic study.

Zearalenone, a mycotoxin biosynthesized by various Fusarium fungi, is widely found as a contaminant in grains and animal feeds. This study describes a rapid and sensitive LC/MS/MS assay method for the quantification of zearalenone in rat serum. The assay was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy and precision. The multiple reaction monitoring was based on the transition of m/z 317.0 --> 130.9 for zearalenone and 319.0 --> 204.8 for zearalanone (internal standard). The assay utilized a single liquid-liquid extraction with t-butyl methyl ether and isocratic elution, and the LLOQ was 0.5 ng/mL using 0.1 mL rat serum. The assay was linear over a concentration range from 0.5 to 200 ng/mL, with correlation coefficients >0.9996. The mean intra- and inter-day assay accuracy was 101.2-112.9 and 96.3-108.0%, respectively. The mean intra- and inter-day precision was between 1.3-7.6 and 3.6-10.6%, respectively. The developed assay was applied to a pharmacokinetic study after a bolus intravenous injection of zearalenone in rats.

Effect of Lateral Ligament Augmentation Using Suture-Tape on Functional Ankle Instability.

BACKGROUND:: Although lateral ligament augmentation using suture-tape has been effective for restoration of mechanical ankle stability, few data are available regarding changes of peroneal strength, proprioception, and postural control. The aim of this study was to determine effects of suture-tape augmentation on functional ankle instability (FAI). METHODS:: Twenty-four patients who underwent suture-tape augmentation were eligible and were followed more than 2 years postoperatively. Functional outcomes were evaluated with the Cumberland Ankle Instability Tool (CAIT), Foot and Ankle Ability Measure (FAAM). Changes of peroneal strength, proprioception and postural control were analyzed with an isokinetic dynamometer and a modified Romberg test. RESULTS:: CAIT and FAAM (average of daily and sports activity scores) significantly improved to average 27.2 points and 86.7 points, respectively, at final follow-up. Peak torque for eversion in 60 degrees/s angular velocity significantly improved to 10.6 Nm at final follow-up. Deficit ratio of peak torque for eversion significantly improved from mean 39.5% to 20.9%, and significant side-to-side difference was revealed ( P < .001). There were no significant differences in joint position sense. A significant improvement in balance retention time was revealed at final follow-up, and the relative deficit ratio compared to the unaffected side was 30.9%. CONCLUSIONS:: Patient-reported functional outcomes significantly improved after lateral ligament augmentation using suture-tape. Although this procedure demonstrated significant effects on FAI based on improvement of isokinetic peroneal strength and postural control, recovery rates compared to the unaffected side were not significant at the intermediate-term follow-up. In addition, there was no positive effect on proprioception of the ankle. LEVEL OF EVIDENCE:: Level IV, prospective case series.

Oral bioavailability and enterohepatic recirculation of otilonium bromide in rats.

This study was conducted to examine the oral bioavailability and the possibility of enterohepatic recirculation of otilonium bromide in rats. A sensitive LC/MS/MS assay (LLOQ 0.5 ng/mL) was developed for the determination of otilonium and applied to i.v. and oral administration studies in bile duct cannulated (BDC) and non-BDC rats. After i.v. injection to BDC rats (1 mg/ kg as otilonium), average t(1/2), CL, Vz and AUC were 7.9 +/- 1.9 h, 8.7 +/- 3.1 mL/min/kg, 5.7 +/- 1.4 L/kg and 2,088 +/- 676 ng h/mL, respectively, and these values were comparable to those found in non-BDC rats. The percentages of i.v. dose excreted unchanged in bile and urine in BDC rats were 11.6 +/- 3.0 and 3.1 +/- 0.7%, respectively. Upon oral administration to non-BDC rats (20 mg/kg as otilonium), t(1/2), Cmax, Tmax and AUC were 6.4 +/- 1.3 h, 182.8 +/- 44.6 ng/mL, 1.9 +/- 1.6 h and 579 +/- 113 ng h/mL, respectively. The absolute oral bioavailability was low (1.1%), while the drug was preferentially distributed to gastrointestinal tissues. A secondary peak was observed in the serum concentration-time profiles in non-BDC rats following both i.v. and oral administration, indicating that otilonium bromide was subject to enterohepatic recirculation.

Supracondylar process syndrome: two cases of median nerve neuropathy due to compression by the ligament of Struthers.

The supracondylar process is a beak-shaped bony process on the anteromedial aspect of the distal humerus. The ligament of Struthers is a fibrous band extending from the tip of the process to the medial epicondyle. The median nerve and brachial artery pass under the ligament of Struthers and consequently can be compressed, causing supracondylar process syndrome. As a rare cause of proximal median nerve entrapment, supracondylar process syndrome is triggered when the median nerve is located in the superficial or deep layer of the ligament of Struthers as a result of anatomical variation. The supracondylar process can be easily detected on X-ray images obtained in oblique views but may not be identified in only anteroposterior or lateral views. In this article, we present 2 cases of supracondylar process syndrome and describe the process of diagnosis and treatment and results of a literature review.

Hypothermia Presenting in Wernicke Encephalopathy: A Case Report.

Wernicke encephalopathy (WE) is a neurologic disorder characterized by clinical symptoms, such as nystagmus, ataxia, and mental confusion. Hypothermia in patients with WE is a rare complication, and its pathogenic mechanism and therapy are yet to be ascertained. Herein, we presented a case of a 61-year-old man who was diagnosed with WE 3 months earlier. We investigated the cause of hypothermia (35.0℃) that occurred after an enema (bowel emptying). Brain magnetic resonance imaging revealed mammillary body and hypothalamus atrophy. In the autonomic function test, the sympathetic skin response (SSR) test did not evoke SSR latencies on both hands. In addition, abnormal orthostatic hypotension was observed. Laxative and stool softener medication were administered, and his diet was modified, which led to an improvement in constipation after 2 weeks. Moreover, there was no recurrence of hypothermic episode. This is the first reported case of late-onset hypothermia secondary to WE.

Heart Rate Variability for Quantification of Autonomic Dysfunction in Fibromyalgia.

OBJECTIVE: To quantify autonomic dysfunction in fibromyalgia patients compared to healthy controls using heart rate variability (HRV). METHODS: Sixteen patients with fibromyalgia and 16 healthy controls were recruited in this case control study. HRV was measured using the time-domain method incorporating the following parameters: total heartbeats, the mean of intervals between consecutive heartbeats (R-R intervals), the standard deviation of normal to normal R-R intervals (SDNN), the square root of the mean squared differences of successive R-R intervals (RMSSD), ratio of SDNN to RMSSD (SDNN/RMSSD), and difference between the longest and shortest R-R interval under different three conditions including normal quiet breathing, rate controlled breathing, and Valsalva maneuver. The severity of autonomic symptoms in the group of patients with fibromyalgia was measured by Composite Autonomic Symptom Scale 31 (COMPASS 31). Then we analyzed the difference between the fibromyalgia and control groups and the correlation between the COMPASS 31 and aforementioned HRV parameters in the study groups. RESULTS: Patients with fibromyalgia had significantly higher SDNN/RMSSD values under both normal quiet breathing and rate controlled breathing compared to controls. Differences between the longest and shortest R-R interval under Valsalva maneuver were also significantly lower in patients with fibromyalgia than in controls. COMPASS 31 score was negatively correlated with SDNN/RMSSD values under rate controlled breathing. CONCLUSION: SDNN/RMSSD is a valuable parameter for autonomic nervous system function and can be used to quantify subjective autonomic symptoms in patients with fibromyalgia.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation.

AIM: To develop a Fok-I nested polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) method for the detection of hepatitis B virus X region (HBx) V5M mutation. METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma (HCC) and 77 carrier patients]. To identify V5M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I (GGA TGN9↓) was done. For size comparison, the enzyme-treated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator. RESULTS: The assay enabled the identification of 69 patients (sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5M prevalence in HCC patients was significantly higher than in carrier patients (47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBxAg V5M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections. CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBxAg V5M mutation in chronic patients with genotype C2 infection.

Extracorporeal shock wave therapy for painful chronic neurogenic heterotopic ossification after traumatic brain injury: a case report.

Neurogenic heterotopic ossification (NHO) is a process of benign bone formation and growth in soft tissues surrounding major synovial joints and is associated with central nervous system (CNS) injuries. It is a common complication in major CNS injuries, such as traumatic brain injury, spinal cord injury, and stroke. Here, we report the case of a 72-year-old male, who experienced a traumatic brain injury and painful chronic NHO around the left hip joint. Three applications of extracorporeal shock wave therapy (ESWT) were administered to the area of NHO, which resulted in pain relief and an improvement in the loss of motion in the left hip joint. Improvements were also noted in walking performance and activities of daily living, although the size of NHO remained unchanged. Therapeutic effects of ESWT lasted for 12 weeks.

Molecular evidence of lateral gene transfer in rpoB gene of Mycobacterium yongonense strains via multilocus sequence analysis.

Recently, a novel species, Mycobacterium yongonense (DSM 45126(T)), was introduced and while it is phylogenetically related to Mycobacterium intracellulare, it has a distinct RNA polymerase ß-subunit gene (rpoB) sequence that is identical to that of Mycobacterium parascrofulaceum, which is a distantly related scotochromogen, which suggests the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. The aims of this study are to prove the presence of the LGT event in the rpoB gene of the M. yongonense strains via multilocus sequence analysis (MLSA). In order to determine the potential of an LGT event in the rpoB gene of the M. yongonense, the MLSA based on full rpoB sequences (3447 or 3450 bp) and on partial sequences of five other targets [16S rRNA (1383 or 1395 bp), hsp65 (603 bp), dnaJ (192 bp), recA (1053 bp), and sodA (501 bp)] were conducted. Incongruences between the phylogenetic analysis of the full rpoB and the five other genes in a total of three M. yongonense strains [two clinical strains (MOTT-12 and MOTT-27) and one type strain (DSM 45126(T))] were observed, suggesting that rpoB gene of three M. yongonense strains may have been acquired very recently via an LGT event from M. parascrofulaceum, which is a distantly related scotochromogen.

Whole-Genome Sequence of Mycobacterium intracellulare Clinical Strain MOTT-H4Y, Belonging to INT5 Genotype.

Here, we report the draft genome sequence of the Mycobacterium intracellulare clinical strain MOTT-H4Y, grouped previously into the INT5 genotype of the 5 genotypes of M. intracellulare.