Treatment-responsive primary autoimmune cerebellar ataxia in a patient with IgG and IgM anticerebellar antibodies.

BACKGROUND AND PURPOSE: Primary autoimmune cerebellar ataxia (PACA) in the absence of another triggering disease represents an emerging category of neurological illness. We report such a case whose ataxia was markedly responsive to plasma exchange. We analyzed patient serum for the presence of IgM or IgG anticerebellar neuronal antibodies. METHODS: Case presentation: rat cerebellar slice cultures incubated with patient sera were studied for IgG and IgM antibody uptake, intracellular binding, and neuronal death. Patient serum was evaluated for anti-myelin associated glycoprotein (anti-MAG) and associated anti-glycolipid antibodies. RESULTS: Antibodies were taken up by viable cerebellar neurons and bound to intracellular antigens. Uptake and predominantly nuclear binding of IgG were seen in granule cells whereas cytoplasmic binding of IgM was observed predominantly in Purkinje cells. Intracellular antibody accumulation was not accompanied by neuronal death, consistent with the patient's excellent clinical response to plasma exchange. Anti-MAG or other associated anti-glycolipid antibodies were not detected. CONCLUSIONS: PACA may be associated with both IgG and IgM antibodies reactive with cerebellar neuronal antigens. Our patient's response to plasma exchange supports a role for antineuronal antibodies in disease pathogenesis and emphasizes the need for rapid diagnosis and treatment.

Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli.

Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an O-antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars (ptsA) from the media combined with conservation of carbon through the glyoxylate shunt (icl) improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b. Unexpectedly, overexpression of a gene involved in the production of DXP from pyruvate (dxs), which was previously seen to have a positive impact on glycosylation, was detrimental to process efficiency and the possible reasons for this are discussed.

Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome.

Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.

Generation of Recombinant N-Linked Glycoproteins in E. coli.

The production of N-linked recombinant glycoproteins is possible in a variety of biotechnology host cells, and more recently in the bacterial workhorse, Escherichia coli. This methods chapter will outline the components and procedures needed to produce N-linked glycoproteins in E. coli, utilizing Campylobacter jejuni glycosylation machinery, although other related genes can be used with minimal tweaks to this methodology. To ensure a successful outcome, various methods will be highlighted that can confirm glycoprotein production to a high degree of confidence, including the gold standard of mass spectrometry analysis.

An iTRAQ characterisation of the role of TolC during electron transfer from Shewanella oneidensis MR-1.

Anodophilic bacteria have the ability to generate electricity in microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigated the anode-specific responses of Shewanella oneidensis MR-1, an exoelectroactive Gammaproteobacterium, using for the first time iTRAQ and 2D-LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture. The regulated dataset produced was enriched in membrane proteins. Proteins shown to be more abundant in anaerobic electroactive anodic cells included efflux pump TolC and an uncharacterised tetratricopeptide repeat (TPR) protein, whilst the TonB2 system and associated uncharacterised proteins such as TtpC2 and DUF3450 were more abundant in microaerobic planktonic cells. In order to validate the iTRAQ data, the functional role for TolC was examined using a δTolC knockout mutant of S. oneidensis. Possible roles for the uncharacterised proteins were identified using comparative bioinformatics. We demonstrate that employing an insoluble extracellular electron acceptor requires multiple proteins involved in cell surface properties. All MS and processed data are available via ProteomeXchange with identifier PXD004090.

Inverse Metabolic Engineering for Enhanced Glycoprotein Production in Escherichia coli.

Inverse metabolic engineering (IME) provides a strategy to rapidly identify the genetic elements responsible for the desired phenotype of a chosen target organism. This methodology has been successfully applied towards enhancing the N-linked glycosylation efficiency of Escherichia coli. Here, we describe the generation of differentially sized libraries from the E. coli W3110 genome followed by high-throughput semiquantitative glycan specific screening. DNA sequenced targets demonstrating increased levels of glycan production were selected for forward engineering, protein overexpression, and absolute quantification of glycoproteins.

Escherichia coli as a glycoprotein production host: recent developments and challenges.

Chinese Hamster Ovary cells are the most popular host expression system for the large-scale production of human therapeutic glycoproteins, but, the race to engineer Escherichia coli to perform glycosylation is gathering pace. The successful functional transfer of an N-glycosylation pathway from Campylobacter jejuni to Escherichia coli in 2002 can be considered as the crucial first engineering step. Here, we discuss the recent advancements in the field of N-glycosylation of recombinant therapeutic proteins in E. coli cells, from the manipulation of glycan composition, to the improvement in glycosylation efficiency, along with the challenges that remain before E. coli can be available as an industry host cell for economically viable glycoprotein production.

Inverse metabolic engineering to improve Escherichia coli as an N-glycosylation host.

An inverse metabolic engineering strategy was used to select for Escherichia coli cells with an increased capability to N-glycosylate a specific target protein. We developed a screen for E. coli cells containing extra-chromosomal DNA fragments for improved ability to add precise sugar groups onto the AcrA protein using the glycosylation system from Campylobacter jejuni. Four different sized (1, 2, 4, and 8 kb) genomic DNA libraries were screened, and the sequences that conferred a yield advantage were determined. These advantageous genomic fragments were mapped onto the E. coli W3110 chromosome. Five candidate genes (identified across two or more libraries) were subsequently selected for forward engineering verification in E. coli CLM24 cells, utilizing a combination of internal standards for absolute quantitation and pseudo-selective reaction monitoring (pSRM) and Western blotting validation. An increase in glycosylated protein was quantified in cells overexpressing 4-α-glucantransferase and a phosphoenolpyruvate-dependent sugar phosphotransferase system, amounting to a 3.8-fold (engineered cells total = 5.3 mg L(-1) ) and 6.7-fold (engineered cells total = 9.4 mg L(-1) ) improvement compared to control cells, respectively. Furthermore, increased glycosylation efficiency was observed in cells overexpressing enzymes involved with glycosylation precursor synthesis, enzymes 1-deoxyxylulose-5-phosphate synthase (1.3-fold) and UDP-N-acetylglucosamine pyrophosphorylase (1.6-fold). To evaluate the wider implications of the engineering, we tested a modified Fc fragment of an IgG antibody as the target glycoprotein with two of our engineered cells, and achieved a ca. 75% improved glycosylation efficiency.

Opportunities for protein interaction network-guided cellular engineering.

As we move further into the postgenomics age where the mountain of systems biology-generated data keeps growing, as does the number of genomes that have been sequenced, we have the exciting opportunity to understand more deeply the biology of important systems, those that are amenable to genetic manipulation and metabolic engineering. This is, of course, if we can make 'head or tail' of what we have measured and use this for robust predictions. The use of modern mass spectrometry tools has greatly facilitated our understanding of which proteins are present in a particular phenotype, their relative and absolute abundances and their state of modifications. Coupled with modern bioinformatics and systems biology modelling tools, this has the opportunity of not just providing information and understanding but also to provide targets for engineering and suggest new genetic/metabolic designs. Cellular engineering, whether it be via metabolic engineering, synthetic biology or a combination of both approaches, offers exciting potential for biotechnological exploitation in fields as diverse as medicine and energy as well as fine and bulk chemicals production. At the heart of such effective designs, proteins' interactions with other proteins or with DNA will become increasingly important. In this work, we examine the work done until now in protein-protein interactions and how this network knowledge can be used to inform ambitious cellular engineering strategies. Some examples demonstrating small molecules/biofuels and biopharmaceuticals applications are presented.

Neurologic presentations of hepatic disease.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that develops in the context of portosystemic venous shunting, in the presence or absence of intrinsic hepatic disease. HE is clinically characterized by altered sensorium and a spectrum of neuropsychiatric abnormalities. Several hypotheses have been proposed to explain the underlying pathogenic mechanisms of altered brain function associated with advanced hepatic disease and portosystemic shunting. HE may lead to profound coma and death; however, in many cases it is reversible. This article discusses the most recent developments in understanding the pathophysiology of HE and its diagnosis and management.

Neurologic presentations of systemic vasculitides.

Vasculitis or angiitis refers to a group of inflammatory disorders of the blood vessels that cause structural damage to the affected vessel, including thickening and weakening of the vessel wall, narrowing of its lumen, and, usually, vascular necrosis. Systemic vasculitis is classified according to the vessel size and histopathologic and clinical features. Vasculitides with small vessel involvement typically include Henoch-Schönlein purpura and cryoglobulinemia. Polyarteritis nodosa and Wegener granulomatosis are small- and medium-sized vessel vasculitides, whereas temporal arteritis and Takayasu arteritis involve large vessels. In this article, the authors provide a review of the neurologic presentations of the major systemic vasculitides.

Cavernous angioma: a clinical study of 35 cases with review of the literature.

BACKGROUND: Cavernous angioma is a vascular malformation which can be found in any region within the central nervous system. OBJECTIVES: There are few clinical and demographic cavernous angioma studies with large sample sizes. Therefore, the present study was designed to provide further information on the clinical and demographic characteristics of cavernous angioma using a relatively large sample of Persian patients. METHODS: Patients with cavernous angioma were recruited from the outpatient neurology clinics in Isfahan, Iran, from October 2003 to October 2006. RESULTS: In all cases, the diagnosis of cavernous angioma was based on brain magnetic resonance imaging. There were 35 patients (female/male: 17 : 18) identified with cavernous angioma. The mean age at presentation was 28.8 years. Initial manifestations included seizures in 16, headache in 11 and intracranial hemorrhage in eight patients. During follow-up, all patients experienced seizures and 19 developed headaches. Depression, vertigo, nausea, vomiting, disequilibrium, loss of consciousness and sensorimotor symptoms were also observed. CONCLUSION: Some of the findings of the present study were in accordance with previous studies. However, more of our patients with positive family history had solitary rather than multiple lesions, and more of our patients had generalized tonic-clonic seizures rather than partial seizures. Moreover, our data demonstrated that if there is a history of cavernous angioma with intracranial hemorrhage in family members, the presenting cavernous angioma patient is more prone to intracranial hemorrhage.

Clinically presenting acute/subacute ischemic stroke: differential diagnosis of the non-enhanced CT hypodensity by advanced neuroimaging.

OBJECTIVES: Patients presenting to the emergency room with an acute or subacute onset of focal neurological deficits are evaluated initially by non-contrast computed tomogram (CT) of the brain. This is primarily carried out to differentiate an ischemic from hemorrhagic stroke. However, other neurological conditions may have a similar clinical presentation as well as only hypodensities on CT scan, thus mimicking ischemic stroke. This review focuses on the advanced neuroimaging modalities that help differentiate these other conditions from a cerebral infarction. METHODS: The literature was reviewed in order to ascertain what conditions would clinically and by CT mimic an acute/subacute ischemic infarction, and what advanced neuroimaging techniques would be most useful in differentiating these conditions. RESULTS: Several infectious, inflammatory, metabolic and vascular diseases were found with clinical presentations identical to subacute/acute ischemic cerebral infarction, which also could demonstrate only hypodensities on a non-enhanced CT scan. However, advanced neuroimaging techniques could readily differentiate these conditions from ischemic infarction. CONCLUSIONS: As presented in this review, although several diseases initially present a diagnostic dilemma upon presentation because of their clinical and non-enhanced CT similarities to cerebral infarction, advanced diagnostic neuroimaging readily establishes their unique pathologies.

The molecular and cellular pathogenesis of dementia of the Alzheimer's type an overview.

The pathogenesis of dementia of the Alzheimer's type (DAT) remains elusive. The neurodegeneration occurring in this disease has been traditionally believed to be the result of toxicity caused by the accumulation of insoluble amyloid-beta 42 (AB) aggregates, however recent research questions this thesis and has suggested other more convincing cellular and molecular mechanisms. Dysfunction of amyloid precursor protein metabolism, AB generation/aggregation and/or degredation/clearance, tau metabolism, protein trafficking, signal transduction, heavy metal homeostasis, acetylcholine and cholesterol metabolism, have all been implicated etiologically especially as to production of neurotoxic by-products occurring as a result of a specific process derangement. In this paper, these and other research directions are discussed as well as their implications for future therapies. The relationship of the proposed abnormal molecular and cellular processes to underlying genetic mutations is also scrutinized, all in an attempt to stimulate further insight into the pathogenesis of, and thus therapeutics for this increasingly prevalent disease.

Early onset dementia.

Dementia is characterized by a decline in cognitive faculties and occurrence of behavioral abnormalities which interfere with an individual's activities of daily living. Dementing disorders usually affect elderly individuals but may occur in individuals younger than 65 years (early-onset dementia or EOD). EOD is often misdiagnosed or its diagnosis is delayed due to the fact that it has a more varied differential diagnosis than late-onset dementia. EOD affects individuals at the height of their career and productivity and produces devastating consequences and financial loss for the patient's family as well as society. EOD is not uncommon and is diagnosed in up to a third of patients presenting with dementia. Most importantly, some of the causes of EOD are curable which makes the need for a specific and timely diagnosis crucial. The present chapter presents a systematic approach to the differential diagnosis of EOD and provides readers with the clinical and neuroimaging features of these disorders as well as important considerations for their diagnostic evaluation. Specifically, the nuances of assessing the history and examination are discussed with careful attention to the various methods of cognitive and behavioral evaluation. A step-wise approach to diagnostic testing is followed by a discussion of anatomical localization, which often aids in identifying specific etiologies. Finally, in order to organize the subject for the reader, the various etiologies are grouped under the general categories of vascular, infectious, toxic-metabolic, immune-mediated, neoplastic/metastatic, and neurodegenerative.

Kinesigenic reflex epilepsy associated with a glioma in the lateral peri-rolandic region.

A patient with kinesigenic focal motor seizures induced by tongue-jaw movement had a grade III astrocytoma clearly co-localizing with the epileptic network in the appropriate peri-rolandic, motor-sensory, lingual-jaw cortical area. The clinical seizure phenomena were time-locked with the EEG epileptic activity. [Published with video sequences].

Cryptococcal meningitis presenting as pseudosubarachnoid hemorrhage.

A 50-year-old man presented with progressive visual loss, headache, and two days of confusion. A computed tomography of his head suggested subarachnoid hemorrhage with accompanying right parietal ischemic infarction. The magnetic resonance image was consistent with right parietal perisulcal pial and superficial cortical inflammation; a subjacent vasogenic edema with a 1 cm diameter abscess was also present. Funduscopy revealed bilateral multifocal choroidal lesions and retinal perivascular sheathing. He was diagnosed with pseudosubarachnoid hemorrhage secondary to cryptococcal meningitis and choroidal microabscesses with retinal inflammation after a cerebrospinal fluid (CSF) examination revealed cryptococcal yeast forms, as well as high titers of CSF cryptococcal antigen, but no CSF red blood cells.

Measurement of total acid number (TAN) and TAN boiling point distribution in petroleum products by electrospray ionization mass spectrometry.

We report a new method for rapid measurement of total acid number (TAN) and TAN boiling point (BP) distribution for petroleum crude and products. The technology is based on negative ion electrospray ionization mass spectrometry (ESI-MS) for selective ionization of petroleum acid and quantification of acid structures and molecular weight distributions. A chip-based nanoelectrospray system enables microscale (<200 mg) and higher throughput (20 samples/h) measurement. Naphthenic acid structures were assigned based on nominal masses of a set of predefined acid structures. Stearic acid is used as an internal standard to calibrate ESI-MS response factors for quantification purposes. With the use of structure-property correlations, boiling point distributions of TAN values can be calculated from the composition. The rapid measurement of TAN BP distributions by ESI is demonstrated for a series of high-TAN crudes and distillation cuts. TAN values determined by the technique agree well with those by the titration method. The distributed properties compare favorably with those measured by distillation and measurement of TAN of corresponding cuts.