ez-ADiCon: A novel glyco-remodeling based strategy that enables preparation of homogenous antibody-drug conjugates via one-step enzymatic transglycosylation with payload-preloaded bi-antennary glycan complexes.

The conserved N-linked glycan at the Fc domain of recombinant monoclonal antibodies is an attractive target for site-specific payload conjugation for preparation of homogenous antibody-drug conjugates (ADCs). Here, we report a novel ADC constructing strategy, named "ez-ADiCon", that is achieved by one-step enzymatic transglycosylation of a payload-preloaded bi-antennary glycan oxazoline onto a deglycosylated antibody. In this method, a mixture of different glycoforms of the Fc-glycan is replaced with a pre-defined payload-linked glycan. Since two payloads are linked on each donor glycan substrate, efficient conjugation results in a highly homogenous ADC with mostly-four drug molecules per antibody, facilitating hydrophobic interaction chromatography analysis and purification. We validated this conjugation strategy using Monomethyl auristatin E (MMAE) and an anti-Human epidermal growth factor receptor 2 (anti-Her2) antibody as the model ADC components and demonstrated its target-specific in vitro cytotoxicity. Our novel conjugation strategy, ez-ADiCon, provides a new approach for the preparation of next generation ADCs.

Clinical predictors of surgical outcomes of severe carpal tunnel syndrome patients: utility of palmar stimulation in a nerve conduction study.

BACKGROUND: Carpal tunnel syndrome is a common peripheral nerve compression disorder. However, there is no established opinion regarding the predictors of symptom improvement after surgery. This study aimed to identify the predictors of surgical outcomes of severe carpal tunnel syndrome patients. METHODS: In the patients who underwent a carpal tunnel syndrome surgery, we selected the patients who had a preoperative Bland's classification of grade 5 or 6, and assessed for the changes in Bland's classification grade before and after surgery. Those who showed improvement from preoperative grades 5-6 to postoperative grades 1-4 comprised the improvement group. In contrast, those who did not show improvement and had postoperative grades 5 or 6 comprised the non-improvement group. In a nerve conduction study, amplitudes of the compound muscle action potential and sensory nerve action potential of the palms were assessed between the improvement and non-improvement groups. RESULTS: Among the 60 hands of 46 patients who had a preoperative Bland's classification of grade 5 or 6, 49 hands of 37 patients comprised the improvement group, and 11 hands of 9 patients comprised the non-improvement group. The amplitudes of the compound muscle action potential and sensory nerve action potential of the palms before surgery were significantly higher in the improvement group. The degree of improvement in Bland's classification grade was correlated with the degree of clinical symptom improvement. CONCLUSIONS: Amplitudes of compound muscle action potential and sensory nerve action potential before surgery induced by palmar stimulation can predict improvements in nerve conduction study scores and clinical findings after surgical treatment.

Profiling Aglycon-Recognizing Sites of UDP-glucose:glycoprotein Glucosyltransferase by Means of Squarate-Mediated Labeling.

Because of its ability to selectively glucosylate misfolded glycoproteins, UDP-glucose:glycoprotein glucosyltransferase (UGGT) functions as a folding sensor in the glycoprotein quality control system in the endoplasmic reticulum (ER). The unique property of UGGT derives from its ability to transfer a glucose residue to N-glycan moieties of incompletely folded glycoproteins. We have previously discovered nonproteinic synthetic substrates of this enzyme, allowing us to conduct its high-sensitivity assay in a quantitative manner. In this study, we aimed to conduct site-selective affinity labeling of UGGT using a functionalized oligosaccharide probe to identify domain(s) responsible for recognition of the aglycon moiety of substrates. To this end, a probe 1 was designed to selectively label nucleophilic amino acid residues in the proximity of the canonical aglycon-recognizing site of human UGGT1 (HUGT1) via squaramide formation. As expected, probe 1 was able to label HUGT1 in the presence of UDP. Analysis by nano-LC-ESI/MS(n) identified a unique lysine residue (K1424) that was modified by 1. Kyte-Doolittle analysis as well as homology modeling revealed a cluster of hydrophobic amino acids that may be functional in the folding sensing mechanism of HUGT1.

Synthesis and bioactivity of ß-(1→4)-linked oligomannoses and partially acetylated derivatives.

The synthesis of ß-(1→4)-linked hexa- to octamannoses and their partially acetylated derivatives was efficiently carried out by assembly of appropriate oligomeric fragments using ß-selective glucosylation followed by gluco to manno epimerization at a late stage of the synthetic pathway. In the course of this study, we also observed that 2-O-acetylated oligomannoses coexisted in equilibrium with the 3-O-acetylated isomers due to intramolecular migration of the acetyl group. Bioactivity of the synthetic oligomannoses and partially acetylated derivatives was investigated in order to identify the possible smallest oligomer for induction of cytokines as that shown in the polysaccharides extracted from Dendrobium huoshanense.

An innovative strategy for sulfopeptides analysis using MALDI-TOF MS reflectron positive ion mode.

Sulfation of tyrosine residues is a key posttranslational modification in the regulation of various cellular processes. As such, the detection and localization of tyrosine sulfation is an essential step toward the elucidation of the physiological and pathological roles of this process. Despite substantial advances, intact sulfated peptides are still difficult to detect by MALDI-MS due to the extreme lability of the sulfo-moiety. The present report demonstrates for the first time how intact sulfated peptides can be directly and specifically detected by MALDI-MS in positive reflectron mode by using pyrenemethylguanidine (pmg) as a noncovalent derivatizing agent and an ionization enhancer. This new method allows the determination of the degree of sulfation of sulfopeptides pure or in mixtures. Moreover, the observation of specific peaks in the mass spectra enables a rapid and unambiguous discrimination between phospho- and sulfopeptides.

A case of cardiac arrest with ST elevation induced by contrast medium.

A 62-year-old man with local recurrence of pancreatic cancer underwent his 17th infusion of contrast medium. He had no history of allergy and had not experienced any side effects from the contrast medium during any of the previous examinations. During infusion, he complained of nausea, followed by a loss of consciousness. He was injected intramuscularly with 0.3 mg adrenalin; however, he temporally went into cardiopulmonary arrest. He was therefore injected with 100 mg hydrocortisone and the continuous infusion of dopamine for shock. His electrocardiogram revealed ST elevation. An urgent cardiac echo evaluation revealed hyperkinetic wall motion. As his blood pressure increased after the initiation of the treatment, the ST elevation started to normalize. After transportation to an intensive care unit, the patient did not show chest pain, ST elevation on cardiograms, or any increase in the levels of cardiac markers. Based on his clinical course, the cause of the patient's ST elevation was considered to be coronary vasospasm. Kounis syndrome is the concurrence of acute coronary syndromes with conditions associated with mast cell activation, including allergic or hypersensitivity and anaphylactic or anaphylactoid insults. In cases of coronary vasospasm with shock due to contrast medium, supportive therapy using catecholamine, which has coronary vasodilator activity, and a steroid might be effective to treat the coronary vasospasm. Attention should therefore be paid to the patient's complaints, the findings of real-time cardiosonography, electrocardiograms, and the levels of cardiac markers to ensure a correct diagnosis and to achieve a good treatment outcome.

A Case of Severe Common Carotid Artery Stenosis Who Developed Cerebellar Infarction after Cervical Irradiation.

Objective: We report a case of cerebellar infarction caused by radiation-induced common carotid artery stenosis. Case Presentation: The patient was a 72-year-old man who underwent irradiation for hypopharyngeal carcinoma 13 years ago. He was referred for asymptomatic left common carotid artery stenosis, but was brought to our hospital by ambulance with transient dysarthria and right facial dysesthesia 2 days after referral. Magnetic resonance imaging (MRI) revealed acute infarction in the left cerebellar hemisphere, and digital subtraction angiography (DSA) demonstrated that the blood flow in the left internal carotid artery perfused the left posterior inferior cerebellar artery (PICA) retrogradely through the left posterior communicating artery. The patient underwent carotid artery stenting (CAS) for left common carotid artery stenosis and blood flow in the left PICA improved; however, in-stent restenosis was revealed during follow-up. Percutaneous transluminal angioplasty (PTA) for in-stent restenosis was performed 9 months after the surgery. Conclusion: We reported a rare case of ischemia in the PICA area caused by radiation-induced common carotid artery stenosis. Although CAS is recommended for the treatment of radiation-induced carotid artery stenosis, careful treatment and follow-up are needed to prevent perioperative complications and detect in-stent restenosis after CAS.

Spontaneous cerebrospinal fluid otorrhea and pneumocephalus on the contralateral side of the previous cranial surgery.

BACKGROUND: Cerebrospinal fluid (CSF) leaks and pneumocephalus commonly occur due to head trauma or surgical procedures. Spontaneous CSF (sCSF) leaks, however, occur without any clear etiology and are relatively uncommon. CASE DESCRIPTION: An 84-year-old woman presented with the right-sided otorrhea. The patient had a history of a ventriculoperitoneal shunt placement following a subarachnoid hemorrhage treated by clip ligation of a left-sided ruptured cerebral aneurysm 7 years before presentation, with shunt catheter ligation after evidence of intraventricular pneumocephalus 6 years before presentation. At admission, computed tomography (CT) imaging of the head showed enlargement of the lateral ventricles, a right mastoid fluid collection, and a defect of the superior wall of the right petrous bone. We performed a right temporal craniotomy for the repair of the CSF leak. Intraoperatively, it was noted that temporal lobe parenchyma herniated into the mastoid air cells through lacerated dura and a partially defective tegmen mastoideum. The leak point was successfully obliterated with a pericranial graft and reinforced by a collagen sheet and fibrin glue. There was no recurrence of otorrhea postoperatively. CONCLUSION: This report presents a very unique case of a patient with a CSF leak and pneumocephalus occurring on the contralateral side of a previous cranial surgery. We accurately identified the defect site with CT imaging and repaired the CSF leak by temporal craniotomy. Awareness of the mechanisms by which sCSF leaks can be caused by aberrant arachnoid granulations is imperative for neurosurgeons.

Matrix-assisted laser desorption/ionization mass spectrometric analysis of polysulfated-derived oligosaccharides using pyrenemethylguanidine.

A better understanding of the biological roles of carbohydrates requires the use of tools able to provide efficient and rapid structural information. Unfortunately, highly acidic oligomers-such as polysulfated oligosaccharides-are very challenging to characterize because of their high polarity, structural diversity, and sulfate lability. These features pose special problems for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis because polysulfated carbohydrates exhibit poor ionization efficiency and usually do not produce any signal. The present report demonstrates how MALDI-MS can be used to derive structural and compositional information from pure and mixed fractions of polysulfated oligosaccharides. Indeed, pyrenemethylguanidine (pmg, a derivatizing agent and ionization efficiency enhancer) was used for the analysis of di- to decasaccharides, carrying from two to nine sulfate groups. The method is applied to various highly sulfated chondroitin and carrageenan oligosaccharides as well as to the analysis of mixtures of compounds. In the mass spectra, the observation of a unique pmg-complexed ladder of peaks in both ionization modes allows an easy and rapid determination of both the number of sulfate groups carried by the analyte and its molecular weight. Moreover, we have developed a software tool for the rapid and automatic structural elucidation of carrageenans based on the mass spectra obtained.

Amine-guanidine switch: a promising approach to improve DNA binding and antiproliferative activities.

A series of polyaromatic guanidino derivatives was synthesized and evaluated for growth inhibitory properties in several human carcinoma cell lines. The properties of these guanidino compounds were compared to those of their corresponding synthetic amino precursors. The size of the polyaromatic ring system as well as the length of the tether attached to the ring had a direct impact on the observed antiproliferative profiles, compound 14 having the broadest spectrum of activity. As both series intercalate DNA, guanidine derivatives showed a remarkable affinity for DNA and the guanidinium group appeared to be essential, yet not sufficient for caspase-3/7 activation. Compound 14 also showed significant in vivo activity against breast cancer cell xenografts in NOG/SCID mice. These results suggest that the electronic nature of chain tethering an intercalator not only influences the DNA-binding process but also controls the antitumoral activity of the whole compound.

Characterization of specific noncovalent complexes between guanidinium derivatives and single-stranded DNA by MALDI.

Noncovalently bound complexes between highly basic sites of 12 guanidinium compounds and single-stranded DNA were studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. 6-Aza-2-thiothymine (ATT) was used as the matrix in the presence of ammonium citrate, and spectra were recorded in the positive ion mode. Detailed control experiments confirmed unambiguously the high selectivity and specificity of the guanidinium moiety for phosphate groups of DNA. The results verify the binding stoichiometry and show preferential binding of hydrophobic binders (pyrene and anthracene guanidinium derivatives) to all sequences examined. In addition, we demonstrate that electrostatic noncovalent interactions are strengthened with phosphorothioate analogs of DNA. These results clearly highlight the structure-directing role of the self-assembling organic species and strongly emphasize the significance of concentration, hydrophobicity, hydrogen-bonding, and pi-pi interactions of the artificial receptor in the formation of these noncovalent complexes. Because of the ability of DNA-binding compounds to influence gene expression, and therefore cell proliferation and differentiation, the interactions described above could be important in providing a better understanding of the mechanism of action of these noncovalent genetic regulators.

Oxidative stress and heart rate variability in patients with vertigo.

Aims: Peripheral vertigo has been reported to result from oxidative stress or autonomic nervous dysfunction. Recently, heart rate variability has been used to evaluate autonomic nervous activity. Parasympathetic nervous dysfunction is associated with peripheral vertigo; however, the relationships between vertigo, oxidative stress, and autonomic nervous activity have not been investigated. The aim of this study was to elucidate the changes in oxidative stress and autonomic nervous activity in vertigo patients compared with healthy volunteers. Methods: Oxidative stress was assessed by evaluating biological antioxidant potential and reactive oxygen metabolites, and heart rate variability was measured to evaluate autonomic nervous activity. Thirty-four patients who complained of peripheral vertigo and were treated in our emergency department between January and August 2011 were enrolled in study 1. Oxidative stress and heart rate variability were measured and compared with those of healthy volunteers (n = 23). In study 2, oxidative stress in 18 vertigo patients and heart rate variability in 41 vertigo patients were measured between January and August 2012 before and after conventional treatment of vertigo to evaluate the effect of the treatment on oxidative stress and autonomic nervous activity. Results: Reactive oxygen metabolites were higher in vertigo patients than in healthy volunteers. Parasympathetic nervous activity was lower and the sympathetic/parasympathetic nervous activity ratio (autonomic nervous activity ratio) was higher in vertigo patients than in healthy volunteers. After treatment of vertigo, reactive oxygen metabolites decreased significantly and the autonomic nervous activity ratio became similar to that observed in healthy volunteers. Conclusions: Bedside monitoring of oxidative stress and heart rate variability may be useful for the diagnosis of vertigo and evaluation of the effect of treatment.

A schizophrenic patient with cerebral infarctions after hemorrhagic shock.

We herein report the fourth case of cerebral infarction, concomitant with hemorrhagic shock, in English literature. A 33-year-old male, who had been diagnosed with schizophrenia and given a prescription for Olanzapine, was discovered with multiple self-inflicted bleeding cuts on his wrist. On arrival, he was in hemorrhagic shock without verbal responsiveness, but his vital signs were normalized following infusion of Lactate Ringer's solution. The neuroradiological studies revealed multiple cerebral ischemic lesions without any vascular abnormality. He was diagnosed with speech apraxia, motor aphasia, and dysgraphia, due to multiple cerebral infarctions. As there was no obvious causative factor with regard to the occurrence of cerebral infarction in the patient, the hypoperfusion due to hemorrhagic shock, and the thromboembolic tendency due to Olanzapine, might have acted together to lead to the patient's cerebral ischemia.

Synthetic studies on oligosaccharides composed of 5-thioglucopyranose units.

Glycosylation reactions of 5-thioglucopyranosyl trichloroacetimidates bearing ethereal protective groups at the 2-O-position 14-15, and 37 proceed smoothly to give alpha-glycosides stereoselectively by using a catalytic amount of silyl triflate. This methodology allowed us to achieve syntheses of sulfur-substituted isomaltotetraoside 2 and maltotetraoside 3. These studies also revealed that benzoyl-protected 5-thioglucopyranosyl trichloroacetimidate 12 underwent beta-selective glycosylation with C6-OH glucopyranosyl acceptors upon activation by BF3OEt2. This was applied for preparation of sulfur-substituted gentiobiosides 1 and 46.

alpha-Selective glycosylation with 5-thioglucopyranosyl donors; synthesis of an isomaltotetraoside mimic composed of 5-thioglucopyranose units.

A general method for alpha-selective glycosylation with 5-thioglucopyranosyl donors followed by efficient deprotection of the resulting products was developed. This methodology was utilized in the synthesis of an isomaltotetraoside analogue.