Integrated application of technologies into Chemical Phenomenology within Neurodegenerative sindromes throughout Bio Markers

Eduardo Terranova, VTMD.

Neurochemistry research and Technology transfer arrangement advisor within private activity; General Manager of Grupo Cuenca

[email protected]

www.grupocuenca.com.uy

3th. Latin American School & Symposium on Brain & Mind Evolution (B&ME).

IBRO - LARC and PEDECIBA School.

Novembre, 2021

Final essay about the conception of an Integrated Network to get better success using technology. Reflection based 25 year of specific kind of research work in neuro chemistry, functional structures and clinical manifestations of them within different diseases but showing common issues as they will be defined as Biomarkers.

Background:

Since 1987, under our professional veterinary practice, we were most attracted to seek some no answers to several questions about different evolution pathways during the onset to the final destiny of patients affected with Viral encephalitis caused by Distemper virus in canines. We also studied the main diseases (same Morbilivirus family) within other species like sea lions and the clinical manifestations in Human (Measles). The surveys and literature diving, drove us into a network of some different kinds of diseases related with also different etiology, like Prions (Bovine Spongiform Encephalitis, BSE) and Bovine Respiratory syncytial virus (BRSV).

All cases of study were designed as clinical approaches to understand the pathophysiology of such sindromes. Same kind of techniques were applied including Histopathology, Protein serum profiles associated with the Central Nervous System (CNS) damage, from the beginning in dogs (1), but they were applied to the rest of species mentioned.

From the observation of same patterns of several protein manifestations (abundance and/or variation of concentration), we continued to study the serum proteins in parallel with Cerebrospinal fluid protein patterns by the same technique, in chronological order: Cellulose acetate gel electrophoresis; Dodecyl Sodium Sulfate Gel stained with Coomassie blue (SDS Page); Immunoelectrofocussing, Western blotting using Monoclonal Antibodies anti Canine

Distemper virus Nucleocapsid and Hemagglutinin Proteins, supplied by Rhone Merieux (1995). Using the main antibodies, Western blot assays were performed and immunohistochemistry on histological necropsy blocks from Brain cuts of dead patients (They were named as patients, because they didn’t used as experimental reproductions, because the objective was the observation of therapeutic response in natural cases).

Doing parallel assays with molecular techniques as Nested PCR 1995 (the unique technique available that time) (2), we tried to develop a comparative study between protein profile analysis vs Viral detection in many tissues by PCR techniques. After those, PCR evolutioned to Real Time PCR which were introduced for that goal in Science Faculty UDELAR in 1999.

As a result of the common observation between histopathological patterns and protein profiles with correlations in both: Serum and CSF, we observed a 115 KDa of MW protein band which appeared (3).

In absence of budget to develop and keep laboratory platform, we needed to contact several different laboratories and researchers in different Faculties of UDELAR and IIBCE: Dr. Navarrete and Pizarrosa (Neuropathology professors from Medicine Faculty UDELAR, Dr. J. Siciliano Intracel signalization laboratory from Histology Department, Medicine Faculty, UDELAR), Dr. Carlos Cervenansky; Dr. Jose Sotello

ft. IIBCE. Dr. Carlos Oehninger, Neurology and Biochemistry Professor from Medicine Faculty UDELAR.

From the growed and huge profit of such relationship, several patterns were detected within different groups of specific patients throughout the species as well as different kind of therapeutic responses and clinical evolution were observed in natural affected animal rather than the Human patients with same diseases or under the same groups, but all together sharing a common issue, which was the presence of protein pattern in CSF and Serum by the mentioned techniques (Oehninger C. MD; Terranova E. VMD).

Since those times, we have worked together in search of the Biomarkers roles within the clinical aspects of CNS Diseases and such roles as an indicator of damage grade of that very prior to the clinical onset.

To get the best result on the main goal, we have worked together with the main technology companies toward obtaining valuable practical information from samples and patients, under a network of analytical pathways mutually integrated to get only one whole information about the pathophysiology pathway of the Diseases as we have been interested in.

As a result of this action, a great networking group has been created linking Genetic Analysis Technology (Sequencing -Sanger first and NGS now; Real Time PCR), Mass

Spectrometry for Proteomics / Metabolomics; Pulsed Gel Field Electrophoresis (PFGE) tasked to real time sorting and separation of target fragment for both: DNA or Proteins. Currently we are performing the New Single Molecule Immunoassay (SIMoA) tasked with detection of Molecular Biomarkers related with several CNS diseases.

A description is given about the conception of a network to get better performance enhancing results from different technologies all together linked to the same goal within predictive diagnosis and pathophysiology pathways of CNS Disease using Biomarkers but beyond them, using the technology integrated network as itself.

Introduction:

The knowledge of biomarkers related to CNS disease has been evolutioned throughout the last years under exponential mode.

Otherwise, the evolution of research within Neuroscience has been done in two parallel ways: one on the knowledge generation of structural architecture of Neural Systems and another way tasked to know further about functional characteristics of the main systems deeper as time goes by and the analytical technology advances.

Two prolific ways in which they do not necessarily get contact between them.

Within this work, we’ll try to highlight some cases to do

successful integration of different technologies toward one common result.

We viewed that the Biomarkers would be the best example to demonstrate that the networking of technology platforms would be the best way to get enhanced information as well as Big Data processing better, under an intelligent system.

Throughout the evolution, the changes on structures and functions in brain and knowledge development have been done under a systematic coordinate and complex modifications along the time, under two discussed modes (analogic developed structures, or Homologous). By the way, deep and strong surveys and investigations have been developed until now based on the demonstration of the changes upon the structure architecture after that they would be related with functions and behavior tasks of CNSs. (4,5,6, 8).

But not only is it based on structured organization. The functionality through the neurochemistry and functional and also structural molecules studied in situ and within peripheral fluids, like CSF, Blood, Serum and Urine, used to bring highly valued information about such origins, functions in situ and destiny of such final location. The follow and scope of those molecules commit to better understanding of some pathways of disease which they couldn’t explain under structure analysis.

In fact, a Kuhninan paradox has had place in discussion about tool development, which is most applicable to our current view within this paper (7).

Nowadays, there is a tendency to approximate both kinds of works, belonging to a morphofunctional conception from the transcriptomic tool utility which is bringing facilities to create networking atmospheres. (9).

It’s seems to be significant to highlight as example of the use of molecules related with function, the case of switching of neuron differentiation from non enolase neuron specific to enolase, using that molecule as a biomarker which has specific function but it is also a biomarker per se, when it’s associate with certain neuroglia regeneration in adults, under some inflammatory onsets from neurodegenerative diseases. (32, 34).

We shall present as the main issue to study, the Biomarkers as the huge representative of functional and structure relationships under dynamic mode.

The technologies applied to do studies of these molecules include Mass Spectrometry (Chemical Characterization of molecules): The biomarkers would be identified as such chemical structure and also they would be quantified whenever they are detected. Genetic Analysis (Sequencing / Real Time PCR) Once the Biomarkers have been identified,

they must be related with such genetic origins and regulation by transcriptomic, qPCR, Seq. surveys. Pulsed Gel Field Electrophoresis -PFGE- These techniques used to rescue the main manual techniques prior to the dawn of the current massive techniques applied to both: DNA and Proteins. These surveys complain to sort in real time the main molecules selected and customized studied with classical techniques as western blot, or RFLP, because of their low abundance and then, sorted to deliver to massive technologies.

Single Molecule ImmunoAssay -SIMoA-. It’s a wonderful technique to detect specific Biomarkers in peripheral fluids, easy to do and cheaper than anyone else. The application of that technology allows the researcher and clinicians to use the technology network, to get the best data as possible, as well as the dynamic monitoring at low cost and easy to do of the evolution of diseases and interpretation along the time of such pathophysiology.

As an example of Biomarkers and Diseases related, we mention some of these described in table 1 and 2. (10 - 31,33).

Table 1: Relationship between Biomarkers-Affected Cells & Diseases

Table 2: Relationship between Biomarkers-Affected Neurons specifficaly & Diseases

As it seems, through time and history, the Science research

and Technology development have gone in parallel ways, perhaps with asymptotic trayectories, but often less mutually crossed over.

Both universes correspond by themselves to the general scientific knowledge, but in fact they used to stand with their backs joined together in contact, but either looking to the opposite point.

The main goal from our experience, is to turnover each one and let them see to the same target, with complementary criteria to get the whole vision of phenomena involved in Neuroscience.

Methodology and Discussion:

A conception of Technology Network, as well as a Technology Transfer Arrangement methods would accomplish to get better Data and deeper knowledge from the integrated morphofunctional visualization of some research phenomena.

The different technologies have different kinds of data processing and interpretation, as well as different time, and analytic requirements to process samples. In fact, such results might be presented under different parameters which must be integrated under a common language toward an efficient interpretation tasked to diagnose, evaluate, monitor or discover.

The cryteria mentioned, is diagrammed in the figure 1,

Figure 1: Technology Networking sketch

In this sketch, we summarize the global vision of the eventual same phenomena with different input of different kinds of data and interpretations of their particular specificity.

We define Knowledge Generating Plants (KGP), all academic centers (Universities; Development / Research institutions and such research groups). The Executive Units (EU), might be the institutions which perform and put in

function, the Technology Core Facilities and techniques to be applied at field level after taking the scaled prototype belt at KGPs. This biunivoque dynamic balance, warrants the constant growing and rudgeness earning of the entire ecosystem of development.

The EUs keep watching to the raising needs from the field and may translate them to the KGPs, with the generation of positive feedback between all partners of the system.

There’s no software or machine involved in the integration pathway. In fact it means a Behavior.

The scientist users of each kind of technology might only look into their own universe (Genomic / Epigenomic application = Sequencing, qPCR, MicroArray Technology) (Proteomic / Metabolomic = Mass Spectrometry, Chromatography, Capillary Electrophoresis). (Imagenology = MNR, PET, CT, Histology). The complex and diversification of each universe requires their researchers full dedication and they used to be full busy working and generating new data from that. It’s unevitable to get some slant or bias to interpret their results under the analytical; molecular; or imagenology optics, depending on case. That it’s very important but is relatively often that they do not get communications and exchange data between the other kind of scientists. Under specific associations and communities, they used to publish and enhance their knowledge and deep performance of the

main technologies. This situation puts much more distance between them. Outside of this special polygon of knowledge sources, there are the Clinicians, entrepreneurs, field professionals, which used to be so far to all other universes mentioned, but they have the responsibility to take decisions over their own study or working objects as the destiny of the applied knowledge transformed on technology.

As we mentioned that the integration is a Behavior, we discuss the need to perform multidisciplinary teams who might interact with all vertices of the multisectorial poligon and link them in. They must interpret the needs that came from the field scope, input them to the Academic Generating Plants, take their scaled solutions, input the Executive Units and co-design the application mode back to field.

These packages of actions must perform what we define as TECHNOLOGY HUB, which is extending links between all actors and creating a network. The concept of HUB implies strong communication and exchaninging from / to all technology universes and targets at the field.

Acting as an integrative Neuron within neural nuclei, the HUB must be an intelligent Node which has the adaptability and constant enhance due to the dynamic balance of knowledge sources, communication between them and pragmatic application to the field in parallel to the basic science which their growth is warranted by the dynamic and

constant resources flowing under the HUB system.

All disciplines are useful; all professionals need to; all kinds of applications must be considered. That is the kind of relation into a HUB.

Biomarkers and Single Molecule ImmunoAssay Technology as examples of integration and being the vortex of a TECHNOLOGY HUB.

Figure 2: Technology Hub Sketch:

CNS: Nervous SystemBM: Biomarkers; IMCH: Immunochemistry; LC/MSMS: Liquid Chromatography Mass Spectrometry; Seq.: Sequencing; qPCR: Quantitative PCR, PGFE: Pulsed Gel Field Electrophoresis; MNR: Magnetic Nuclear Resonance; PET: Positron Emission Tomography; CT: Computed Tomography; SIMoA: Single Molecule ImmunoAssay

In this sketch, several great technologies are shown as part of a greatest system which involves them and it makes a bundle with one exit channel for the final report delivered to the application field.

The always growing amount and depth of knowledge about Biomarkers allow the scientists and technicians to improve and expand the meant network as a HUB.

The special biological characteristics of these biomarkers, define them not only as a marker per se but such main functions, original locations and later translocation in cases of disease onsets, used to bring a lot of huge information about, both, structures, function, dynamic of responses and plasticity, and much more other issues which would be discovered.

Otherwise, the Single Molecule Immunoassay technology by which they detected with high specificity and sensitivity (Subattomolar level of concentration) as well as the biological feature of Biomarkers to be found at low but detectable concentration and abundance by SIMoA techniques, in peripheral fluids like CSF, Blood, Serum and also Urine. Both issues: Biomarkers and SIMOA perform a huge couple of tools to start up and develop the rudged HUB as it could be created.

SIMoA used to settle within the intersection of different technology universes like Mass Spectrometry and Genetic

Analysis. SIMoA features commit to process and arrange the main information brought by each technology as a common information within a reciprocal mode (generate output to the field and input from that to there).

SIMoA allows monitoring and getting constant information after the main results of higher platforms at very low costs, but with high practicality.

Conclusions

These examples, as we have done under our professional practice, try to illustrate the need for deep knowledge of features and benefits of each technology, but meanwhile the attitude to compliance, bundle, closing and linking their universe into a greatest system which arrives necessary to a paradigm to grow.

The goal is to create the Best as possible Technology Transfer Arrangement.

Watching the world of science from a cloud, and from that, see the frontier to achieve.

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