Targeted Delivery of Diphtheria Toxin into VEGFR1/VEGFR2 Overexpressing Cells Induces Anti-angiogenesis Activity.

BACKGROUND: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated. METHODS: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays. RESULTS: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells. CONCLUSION: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells.

Differential toxic and antiepileptic features of Vigabatrin raceme and its enantiomers.

BACKGROUND: The study aimed to investigate the potential pharmacological and toxicological differences between Vigabatrin (VGB) and its enantiomers S-VGB and R-VGB. The researchers focused on the toxic effects and antiepileptic activity of these compounds in a rat model. METHODS: The epileptic rat model was established by intraperitoneal injection of kainic acid, and the antiepileptic activity of VGB, S-VGB, and VGB was observed, focusing on the improvements in seizure latency, seizure frequency and sensory, motor, learning and memory deficits in epileptic rats, as well as the hippocampal expression of key molecular associated with synaptic plasticity and the Wnt/ß-catenin/GSK 3ß signaling pathway. The acute toxic test was carried out and the LD50 was calculated, and tretinal damages in epileptic rats were also evaluated. RESULT: The results showed that S-VGB exhibited stronger antiepileptic and neuroprotective effects with lower toxicity compared to VGB raceme. These findings suggest that S-VGB and VGB may modulate neuronal damage, glial cell activation, and synaptic plasticity related to epilepsy through the Wnt/ß-catenin/GSK 3ß signaling pathway. The study provides valuable insights into the potential differential effects of VGB enantiomers, highlighting the potential of S-VGB as an antiepileptic drug with reduced side effects. CONCLUSION: S-VGB has the highest antiepileptic effect and lowest toxicity compared to VGB and R-VGB.

Polyhydroxybutyrate production by recombinant Escherichia coli based on genes related to synthesis pathway of PHB from Massilia sp. UMI-21.

BACKGROUND: Polyhydroxybutyrate (PHB) is currently the most common polymer produced by natural bacteria and alternative to conventional petrochemical-based plastics due to its similar material properties and biodegradability. Massilia sp. UMI-21, a newly found bacterium, could produce PHB from starch, maltotriose, or maltose, etc. and could serve as a candidate for seaweed-degrading bioplastic producers. However, the genes involved in PHB metabolism in Massilia sp. UMI-21 are still unclear. RESULTS: In the present study, we assembled and annotated the genome of Massilia sp. UMI-21, identified genes related to the metabolism of PHB, and successfully constructed recombinant Escherichia coli harboring PHB-related genes (phaA2, phaB1 and phaC1) of Massilia sp. UMI-21, which showed up to 139.41% more product. Also, the vgb gene (encoding Vitreoscilla hemoglobin) was introduced into the genetically engineered E. coli and gained up to 117.42% more cell dry weight, 213.30% more PHB-like production and 44.09% more product content. Fermentation products extracted from recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb were identified as PHB by Fourier Transform Infrared and Proton nuclear magnetic resonance spectroscopy analysis. Furthermore, the decomposition temperature at 10% weight loss of PHB extracted from Massilia sp. UMI-21, recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was 276.5, 278.7 and 286.3 °C, respectively, showing good thermal stability. CONCLUSIONS: Herein, we presented the whole genome information of PHB-producing Massilia sp. UMI-21 and constructed novel recombinant strains using key genes in PHB synthesis of strain UMI-21 and the vgb gene. This genetically engineered E. coli strain can serve as an effective novel candidate in E. coli cell factory for PHB production by the rapid cell growth and high PHB production.

Vigabatrin - new data on indications and safety in paediatric epilepsy.

INTRODUCTION: Vigabatrin (VGB), a second-generation antiepileptic drug, is effective for the treatment of infantile spasms and focal seizures, primarily in tuberous sclerosis complex (TSC) patients. However, reports of adverse events of VGB, including VGB-associated visual field loss and brain abnormalities in neuroimaging, have raised concerns about the broader use of VGB and thus significantly limited its application. AIM OF THE STUDY: The goal of this review was to summarise the recent therapeutic guidelines, the use of VGB in focal seizures and new VGB applications as a disease-modifying treatment in TSC patients. Moreover, we discuss the current opinions on potential VGB-associated toxicity and the safety of VGB.

Ocular evaluation and genetic test for an early Alström Syndrome diagnosis.

PURPOSE: We present 3 cases of Alström syndrome (ALMS) that highlight the importance of the ophthalmic exam, as well as the diagnostic challenges and management considerations of this ultra-rare disease. OBSERVATIONS: The first case is of a 2-year-old boy with history of spasmus nutans who presented with head bobbing and nystagmus. The second patient is a 5-year-old boy with history of infantile dilated cardiomyopathy status post heart transplant, Burkitt lymphoma status post chemotherapy, obesity, global developmental delay, and high hyperopia previously thought to have cortical visual impairment secondary to heart surgery/possible ischemic event. This patient presented with nystagmus, photophobia, and reduced vision. The third case involves a 8-year-old boy with history of obesity, bilateral optic nerve atrophy, hyperopic astigmatism, exotropia, and nystagmus. Upon presentation to the consulting pediatric ophthalmologist, none of the patients had yet been diagnosed with ALMS. All 3 cases were subsequently found to have an electroretinogram (ERG) that exhibited severe global depression and to carry ALMS1 pathogenic variants. CONCLUSIONS AND IMPORTANCE: ALMS is an autosomal recessive disease caused by ALMS1 variations, characterized by cone-rod dystrophy, obesity, progressive sensorineural hearing loss, cardiomyopathy, insulin resistance, and multiorgan dysfunction. Retinal dystrophy diagnosis is critical given clinical criteria and detection rates of genetic testing. Early diagnosis is extremely important because progression to flat ERG leads to the inability to differentiate between rod-cone or cone-rod involvement, either of which have their own differential diagnoses. In our series, the ophthalmic exam and abnormal ERG prompted further genetic testing and the subsequent diagnosis of ALMS. Multidisciplinary care ensures the best possible outcome with the ophthalmologist playing a key role.

Capsular Polysaccharide Biosynthesis from Recombinant E. coli and Chondroitin Sülfate Production.

Chondroitin sulfate (CS) is an important biomedical product. CS is the basic structural component of the mammalian extracellular matrix and is widely used in many applications in the fields of medicine, veterinary medicine, pharmaceuticals and cosmetics. For CS production, mainly animal sources are used. However, in today's conditions, due to various risks and artificial synthesis, there has been an increase in alternative sources of production methods for CS, instead of using animal resources. In this study as a powerful alternative microbial production of CS has been targeted. By using recombinant E. coli strains to integrate VHb /vgb+ and kfo+ systems, the aim was to obtain high purity CS from reliable biotechnological processes. Plasmid pUC8:15 bearing the vgb gene region, and plasmid pETM6-PACF carrying the kfoA, kfoC and kfoF genes responsible for chondroitin synthesis, were transferred to E. coli bacteria. Microbial CS was obtained by adding sulfate groups to chondroitin acquired after the treatments. The results were confirmed by HPLC and NMR analyses. The product, compared to its counterparts, was found to be an effective drug, potentially with a low molecular weight  value.

Enhancement of substrate supply and ido expression to improve 4-hydroxyisoleucine production in recombinant Corynebacterium glutamicum ssp. lactofermentum.

4-Hydroxyisoleucine (4-HIL) has potential value in treating diabetes. L-isoleucine dioxygenase (IDO) catalyzes the hydroxylation of L-isoleucine (Ile) to form 4-HIL with the concomitant oxidation of α-ketoglutarate (α-KG) and oxygen consumption. In our previous study, by expressing the ido gene in the Ile producer Corynebacterium glutamicum ssp. lactofermentum SN01, 4-HIL was de novo-synthesized from glucose without adding either Ile or α-KG. In this study, synergistically improving the substrates supply and IDO activity was applied to enhance the de novo biosynthesis of 4-HIL. Deletion of aceA and blocking of the glyoxylate pathway effectively enhanced α-KG supply and Ile synthesis and thus improved 4-HIL production to 69.47 ± 2.18 mM, 18.9% higher than in the original strain. Coexpression of mqo with ido further improved Ile synthesis but decreased 4-HIL production, partially due to the inadequate activity of IDO. Coexpression of another gene, ido3, with mqo and ido efficiently promoted IDO activity, thus improving 4-HIL production to 91.54 ± 8.29 mM. Further expression of vgb and promotion of the oxygen uptake rate did not change the 4-HIL titer significantly but increased the 4-HIL production rate in the first 72 h of fermentation. After fermentation in the optimized medium, 4-HIL production by the final strains increased to 112-117 mM, indicating these strains are promising candidates for producing 4-HIL. These results demonstrate that synergistically promoting substrate supply and improving IDO activity are efficient approaches to enhance 4-HIL production in C. glutamicum.

Enhancement of l-phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin.

l-Phenylalanine is an important amino acid that is widely used in the production of food flavors and pharmaceuticals. Generally, l-phenylalanine production by engineered Escherichia coli requires a high rate of oxygen supply. However, the coexpression of Vitreoscilla hemoglobin gene (vgb), driven bya tac promoter, with the genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase (aroF) and feedback-resistant chorismate mutase/prephenate dehydratase (pheAfbr ), led to increased productivity and decreased demand for aeration by E. coli CICC10245. Shake-flask studies showed that vgb-expressing strains displayed higher rates of oxygen uptake, and l-phenylalanine production under standard aeration conditions was increased. In the aerobic fermentation process, cell growth, l-phenylalanine production, and glucose consumption by the recombinant E. coli strain PAPV, which harbored aroF, pheAfbr , and tac-vgb genes, were increased compared to that in the strain harboring only aroF and pheAfbr (E. coli strain PAP), especially under oxygen-limited conditions. The vgb-expressing strain PAPV produced 21.9% more biomass and 16.6% more l-phenylalanine, while consuming only approximately 5% more glucose after 48 H of fermentation. This study demonstrates a method to enhance the l-phenylalanine production by E. coli using less intensive and thus more economical aeration conditions.

Increasing oxygen availability for improving poly(3-hydroxybutyrate) production by Halomonas.

Technologies enabling high-cell-density growth are required for economical industrial production of most biotechnological products. However, the key factor limiting cell density in bioreactors is the availability of oxygen during the late phases of fermentation. Although the expression of bacterial Vitreoscilla hemoglobin (VHb) is useful for enhanced oxygen availability, bacterial cell membrane makes efficient hemoglobin-oxygen contact a challenge. On the other hand, periplasmic spaces of Gram-negative microorganisms offer an excellent compartment for the intermittent storage of hemoglobin-bound oxygen. In this study, the cell growth was increased by a remarkable 100% using the twin-arginine translocase (Tat) pathway to export active VHb into the periplasm of Escherichia coli, Halomonas bluephagenesis TD01 and H. campaniensis LS21. Furthermore, eight low-oxygen-inducible vgb promoters were constructed in tandem to become a strong promoter cassette termed P8vgb, which better induces expression of both gene vgb encoding VHb and the PHB synthesis operon microaerobically. Both the P8vgb and VHb performed excellently in E. coli and two Halomonas spp., demonstrating their universal applicability for various organisms.

Drug-induced hypersensitivity syndrome caused by valproic acid as a monotherapy for epilepsy: First case report in Asian population.

Valproic acid (VPA) is a broad-spectrum antiseizure drug used for a variety of clinical conditions, such as epilepsy and mood disorders. Drug-induced hypersensitivity syndrome (DRESS) accompanied by hyponatremia, thrombocytopenia, hypoalbuminemia and elevated aminotransferase has never been reported as an adverse effect of VPA monotherapy during titration for epilepsy in Asian population. Hereby, we present the case of a 73-year-old Chinese male who suffered from DRESS and other complications two weeks after initiating VPA treatment for epilepsy. Understanding the risk associated with VPA-induced DRESS, and taking effective measures to avoid the severe side effects are necessary.

The Functional Anatomy of Nerves Innervating the Ventral Grooved Blubber of Fin Whales (Balaenoptera Physalus).

Nerves that supply the floor of the oral cavity in rorqual whales are extensible to accommodate the dramatic changes in tissue dimensions that occur during "lunge feeding" in this group. We report here that the large nerves innervating the muscle component of the ventral grooved blubber (VGB) in fin whales are branches of cranial nerve VII (facial nerve). Therefore, the muscles of the VGB are homologous to second branchial arch derived muscles, which in humans include the muscles of "facial expression." We speculate, based on the presence of numerous foramina on the dorsolateral surface of the mandibular bones, that general sensation from the VGB likely is carried by branches of the mandibular division (V3) of cranial nerve V (trigeminal nerve), and that these small branches travel in the lipid-rich layer directly underlying the skin. We show that intercostal and phrenic nerves, which are not extensible, have a different wall and nerve core morphology than the large VGB nerves that are branches of VII. Although these VGB nerves are known to have two levels of waviness, the intercostal and phrenic nerves have only one in which the nerve fascicles in the nerve core are moderately wavy. In addition, the VGB nerves have inner and outer parts to their walls with numerous large elastin fibers in the outer part, whereas intercostal and phrenic nerves have single walls formed predominantly of collagen. Our results illustrate that overall nerve morphology depends greatly on location and the forces to which the structures are exposed. Anat Rec, 300:1963-1972, 2017. © 2017 Wiley Periodicals, Inc.

A new approach for improving epothilone B yield in Sorangium cellulosum by the introduction of vgb epoF genes.

Epothilone B has drawn great attention due to its much stronger anticancer activity and weaker side effects compared with taxol. The relative low yield of epothilone B limited its application. In this study, we report the successful introduction of the vgb gene and the epoF gene into Sorangium cellulosum So ce M4 by electroporation for the first time, which was demonstrated by Southern blot analysis. Results of qRT-PCR, SDS-PAGE and western blot analysis confirmed the transcription and expression of the vgb and epoF genes. LC-MS results showed that the epothilones B, A yields were improved and epothilones D, C yields were decreased. The yields of epothilone B were improved by 57.9 ± 0.3, 62.7 ± 0.8 and 122.4 ± 0.7 % through the introduction of vgb gene, epoF gene and both genes into strain So ce M4, respectively. Our study provides a new approach for improving epothilone B yield in S. cellulosum.

Effect of sildenafil, a selective phosphodiesterase 5 inhibitor, on the anticonvulsant action of some antiepileptic drugs in the mouse 6-Hz psychomotor seizure model.

Sildenafil, a selective phosphodiesterase 5 inhibitor (PDE5), has been recently reported to have both pro- and anticonvulsant action in various experimental models of seizures and epilepsy. Furthermore, it affects anticonvulsant action of some antiepileptic drugs (AEDs) in mice seizure tests and both pharmacodynamic and pharmacokinetic interactions were noted. The present study was carried out to investigate influence of sildenafil on the threshold for 6 Hz-induced psychomotor seizures in mice. Effect of sildenafil on activity of some AEDs, i.e., phenobarbital (PB), clonazepam (CZP), ethosuximide (ETS), valproic acid (VPA), tiagabine (TGB), oxcarbazepine (OXC) and levetiracetam (LEV), in 6 Hz test was also examined. Moreover, combination of sildenafil with LEV was investigated in terms of influence on motor coordination (determined by the chimney test), muscular strength (evaluated in the grip-strength test) and long-term memory (assessed in the passive avoidance task) in mice. To determine the type of pharmacological interaction between sildenafil and LEV, free plasma and total brain concentrations of this AED were determined by LC-MS/MS method. Sildenafil at a dose ranging from 10 to 40 mg/kg statistically increased psychomotor seizure threshold in mice. Moreover, sildenafil enhanced the anticonvulsant action of all the studied AEDs in this test. Interactions between this PDE5 inhibitor and PB, CZP, ETS, TGB and OXC seem to be pharmacodynamic. Since sildenafil increased free plasma and total brain concentration of LEV, interactions between these drugs have pharmacokinetic nature. This kind of interaction was also noted between sildenafil and VPA. Neither LEV (2.32 mg/kg) nor its co-administration with sildenafil (40 mg/kg) produced any significant changes in motor coordination, muscular strength and long-term memory in mice.

Electroclinical features and long-term outcome of cryptogenic epilepsy in children with Down syndrome.

OBJECTIVE: To describe the electroclinical features and the long-term outcomes of epilepsy in a large cohort of males and females with Down syndrome who developed epilepsy in childhood. STUDY DESIGN: Subjects with Down syndrome and cryptogenic epilepsy with onset in childhood were identified retrospectively from the databases of 16 Italian epilepsy centers over a 40-year period. For each subject, age at onset of seizures, seizure semiology and frequency, electroencephalography characteristics, treatment with antiepileptic drugs, and long-term clinical and electroencephalography outcomes were analyzed. RESULTS: A total of 104 subjects (64 males [61.5%], 40 females [38.5%]) were identified. Seizure onset occurred within 1 year of birth in 54 subjects (51.9%), between 1 and 12 years in 42 subjects (40.4%), and after 12 years in 8 subjects (7.7%). Males had a younger age of seizure onset than females. Of the 104 subjects, 51 (49.0%) had infantile spasms (IS), 35 (33.7%) had partial seizures (PS), and 18 (17.3%) had generalized seizures (GS). Febrile seizures were recorded in 5 (4.8%) subjects. Intractable seizures were observed in 23 (22.1%) subjects, including 5 (9.8%) with IS, 8 (44.4%) with PS, and 10 (31.3%) with GS. CONCLUSION: Cryptogenic epilepsy in Down syndrome may develop during the first year of life in the form of IS or, successively, as PS or GS. Electroclinical features of IS resemble those of idiopathic West syndrome, with a favorable response to treatment with adrenocorticotropic hormone seen. Patients experiencing PS and GS may be resistant to therapy with antiepileptic drugs.

Epileptic spasms in tuberous sclerosis complex.

PURPOSE: To characterize epileptic spasms (ES) occurring after the age of two years in patients with tuberous sclerosis complex (TSC), particularly treatment response to vigabatrin (VGB), which is extremely effective for infantile spasms (IS) in TSC. METHODS: The authors retrospectively reviewed 19 patients with TSC and ES. Medical records were assessed for clinical and treatment data, neurocognitive, EEG, MRI data, and genetic analyses. RESULTS: Of 391 patients with TSC, 19 (4.8%) had ES. Of those with detailed clinical data, six had infantile spasms that persisted after 2 years old, six recurred after an initial remission of infantile spasms (range 2-24 years old), and four occurred de novo over the age of two (range 2-20 years old). All concurrently had other seizure types. One had hypsarrhythmia on EEG. All had brain MRI stigmata typical of TSC. Thirteen had a mutation in TSC2, and one in TSC1. Six patients became spasm-free with medication treatment, including four with VGB, one with VGB in combination with the low glycemic index dietary treatment, and one with felbamate. Five became spasm-free after epilepsy surgery. VGB was not effective for seven patients. The majority continued to have refractory epilepsy. CONCLUSIONS: ES are not uncommon in patients with TSC, especially those with TSC2 mutations. ES in TSC occur in the setting of other seizure types and refractory epilepsy. Hypsarrhythmia is rare. VGB can be effective, but the success of VGB for ES in TSC is not equivalent to that of IS in TSC.