Is the microbiome the cause of irritable bowel syndrome and inflammatory bowel disease? Lessons to consider from odontology.

BACKGROUND: A substantial amount of research is pointing to the disrupted microbiome and dysfunctional host-microbiome interaction as potential causes of Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). The true cause of the diseases is still not fully elucidated, and the various treatments used are not truly effective in the long run, especially for IBD, since a true cure is not known to exist. Treatment failure and surgery are common for IBD, many times leading to a perceived lower quality of life, not to mention the enormous cost for society for treatment up until that point and after. Although it is clear that the microbiome has a major role in the disease, it seems the majority of the research and treatments are still focused on treating and understanding the inflammation and not the primary cause of the inflammation in the first place. This was also the case for many decades in the search for the cause of periodontitis (PD) and gingivitis (GV), a destructive and non-destructive inflammatory disorder, respectively, the first resulting in loss of tissue supporting the teeth. There was much uncertainty and confusion until it was fully established that the microbiome was the cause. PD treatments primarily nowadays reflect the cause, i.e. the removal of microbes. There is no doubt, however, that the inflammatory pathways are important in both diseases and the purpose of this text is not to dispute this in respect to gastrointestinal disorders too. However, a different view on inflammation and associated disorders is explored to explain the nature of extraintestinal manifestations. PURPOSE: The aim of this report is not to systematically fully review the literature to try to strengthen causality, as there are many reviews that explore the microbial aspects of IBS and IBD. Instead, the objective is to above all reflect on what has been learned in the field of odontology/stomatology and discuss relevant gastrointestinal research in order to propose tentative hypotheses and questions regarding IBS and IBD aetiology. Perhaps it could help soften the confusion regarding the microbial aetiology and dysbiosis concept, while guiding future research and treatments, primarily regarding microbial transplants, antibiotics, and diet.

Fed-batch production assessment of a tetravalent bispecific antibody: A case study on piggyBac stably transfected HEK293 cells.

The transition from preclinical biological drug development into clinical trials requires an efficient upscaling process. In this context, bispecific antibody drugs are particularly challenging due to their propensity to form aggregates and generally produce low titers. Here, the upscaling process for a tetravalent bispecific antibody expressed by a piggyBac transposon-mediated stable HEK293 cell pool has been evaluated. The project was performed as a case study at Testa Center, a non-GMP facility for scale-up testing of biologics in Sweden, and encompassed media adaptation strategies, fed-batch optimization and a novel antibody purification technology. The cell pool was adapted to different culture media for evaluation in terms of cell viability and titers compared to its original Expi293 Expression Medium. These parameters were assessed in both sequential stepwise adaption and direct media exchanges. By this, a more affordable medium was identified that did not require stepwise adaptation and with similar titers and viability as in the Expi293 Expression Medium. Fed-batch optimizations resulted in culture densities reaching up to 20 × 106 viable cells/mL with over 90 % viability 12 days post-inoculum, and antibody titers three times higher than corresponding batch cultures. By implementing a novel high-speed protein A fiber technology (Fibro PrismA) with a capture residence time of only 7.5 s, 8 L of supernatant could be purified in 4.5 h without compromising the purity, structural integrity and function of the bispecific antibody. Results from this study related to medium adaptation and design of fed-batch protocols will be highly beneficial during the forthcoming scale-up of this therapeutic antibody.

Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.

The congenital disorder of glycosylation in PGM1 (PGM1-CDG) can cause severe cardiomyopathy and unexpected sudden cardiac death in childhood.

INTRODUCTION: Sudden cardiac death (SCD) in the young is rare and should always lead to suspicion of a genetic cardiac disorder. We describe a family, in which the proband was a girl deceased by sudden cardiac death in the playground at thirteen years of age. The index-patient had short stature, cleft palate but no previous cardiac symptoms. We found an uncommon cause of cardiomyopathy, due to a congenital disorder of glycosylation (CDG), previously described to cause a variable range of usually mild symptoms, and not previously found to cause SCD as the first symptom of the condition. METHODS: The index patient underwent postmortem genetic testing/molecular autopsy for genes known to cause SCD, without a detection of causative agent, why two siblings of similar phenotype as the deceased sister underwent clinical-exome genetic sequencing (next generation sequencing). All first-degree relatives underwent clinical examination including cardiac ultrasound, Holter-ECG, exercise stress test and biochemistry panel. RESULTS: A genetic variant in the gene for phosphoglucomutase 1 (PGM1) was identified in the index patient and her two brothers, all were found to be homozygous for the genetic variant (G230E) NM_002633.2:c.689 G > A in PGM1. This variant has been linked to a congenital disorder of glycosylation (PGM1-CDG), explaining the clinical picture of short stature, cleft palate, liver engagement and cardiomyopathy. During follow-up one of the brothers died unexpectedly after physical exertion during daily life at the age of twelve years. The other brother fainted during similar circumstances at the age of thirteen years. Both parents and three other siblings were found to be heterozygous gene carriers without risk for the disease. CONCLUSION: Our findings suggest that there is a need of multidisciplinary discussion and genetic testing after unexpected cardiac death in the young. We have to be more flexible in our evaluation of diseases and to consider even uncommon diseases including rare recessive inherited disorders. Our findings also suggest that the autosomal recessive PGM1-CDG might be highly associated with life-threatening cardiomyopathy with arrhythmia or sudden cardiac death as the first symptom presenting from childhood and adolescence.

Quality of pediatric anesthesia: A cross-sectional study of a university hospital in a low-income country.

OBJECTIVE: To evaluate the quality of pediatric anesthesia in a university hospital in Dar es Salaam, Tanzania. METHOD: A cross-sectional study conducted using a new tool that was developed from the literature and WHO recommendations including 28 parameters as standards for pediatric anesthesia. These 28 parameters consisted of 17 structure parameters of the equipment and medicines that should be present in theatre before any surgery starts, and 11 process parameters of actions taken by staff. Adverse events occurring during the anesthesia were recorded. RESULTS: 30 patients were included, aged between 1.5 months to 5 years with a mean of 2.4 years. 26 of the patients underwent elective surgery and 4 patients emergency surgery. Nine parameters were always present and one parameter (bag and mask) was not available for any of the patients. The structure index ranged from 71% to 94% with a mean of 84%. The process index had a mean score of 71% with a range from 50% to 90%: lower than the structure index (p<0.001). With the structure and process index combined the average score was 79% with a low of 67% and high of 89%. 70 adverse events were observed with a range from 0 to 7 adverse events per patient. The most common adverse event was hypoxia at extubation in 20 (69%) patients. Nine patients had an episode of severe hypoxia at extubation. CONCLUSION: Pediatric anesthesia in low resource settings suffers from deficiencies in the structures and processes of providing good quality care. Improvement efforts may be best focused on improving the consistency and quality of the process of care and a reduction in adverse events rather than the structures available. Use of the assessment tool developed for this research could be useful for systematic quality-improvement efforts and to assess the needs in different settings.

A 290 mV Sub-V(T) ASIC for Real-Time Atrial Fibrillation Detection.

A real-time detector for episodes of atrial fibrillation is fabricated as an application specific integrated circuit (ASIC). The basis for detection is a set of three parameters for characterizing the RR interval series, i.e., turning point ratio, root mean square of successive differences, and Shannon entropy. The developed hardware architecture targets ultra-low voltage operation, suitable for implantable loop recorders with ultra-low energy requirements. Algorithmic and architectural optimizations are performed to minimize area and energy dissipation, with a total area footprint reduction of 44%. The design is fabricated in 65-nm CMOS low-leakage high-threshold technology. Measurements with aggressively scaled supply voltage (VDD) in the subthreshold (sub-VT) region show energy savings of up to 41 X when operating at 1 kHz with a VDD of 300 mV compared to a nominal VDD of 1.2 V.

Production of recombinant antibodies in Drosophila melanogaster S2 cells.

One of the major bottlenecks in antibody discovery for research and therapeutic applications is the need for large quantities of protein in a short amount of time. Here we describe an alternative method using the Drosophila melanogaster S2 expression system to produce high levels of antibodies (both IgG and Fab) with equivalent binding properties to antibodies produced in mammalian cell expression systems. Using the Drosophila S2 expression system for antibody production has many advantages over current mammalian systems making antibody expression, purification, and evaluation a much less time-consuming process.

Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays.

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.