Polarimetric Signature Imaging Of Anisotropic Bio-Medical Tissues

Summary of Protein Technologies

Discuss about the Polarimetric Signature Imaging of Anisotropic.

Proteins are polymers constituting of amino acid monomers. Of the known types of proteins, the current paper reviews enzymes. Enzymes are remarkably similar in structure to the other proteins but their distinct functionality is always fascinating. Various methods have proved vital in the study of different enzymes with respect to their composition, functionality and enzyme kinetics and enzyme assay. This review focuses on the different technologies or methods that have been used to study enzyme assay.

The study conducted by Spinazzi and his team noted that mitochondrial dysfunction significantly contributes to disorders in humans including mitochondrial abnormalities. Some of these conditions affect the mitochondrial itself while some affect nuclear DNA contributing to ailments such aging, neurodegenerative conditions (Parkinson’s disease and Alzheimer’s disease) and diabetes, to mention a few[1].

A clear understanding of mitochondrial activities is necessary to avert undesirable outcomes. Various technological approaches have been used to study mitochondrial respiratory chain (RC) during enzymatic activities. In the current study, the enzymatic activities of RC complexes I–IV were assayed using spectrophotometric techniques[2]. Like in many other studies, the results were standardized to the aggregate muscle protein composition and to the activity of citrate synthase to mirror mitochondrial matrix enzyme. However, the technique posed one glaring problem of the assays: at all times, the enzymatic reactions are assessed using in vitro environments[3]. These conditions are hardly physiological with respect to pH, substrate concentrations, osmolarity and cellular context and disallow the estimation of respiratory pairing. Despite that challenge, spectrophotometric technology offered pertinent quantitative data regarding the top catalytic reactions of the RC complexes. In addition the procedure are easy to duplicate and can be undertaken by use of iced up tissue and cellular samples.

An acceptable into mitochondrial enzymatic activities ought to encompass a blend of other assays including determination of polarographical oxygen utilizations in intact insulated mitochondria, ATP synthesis and permeabilized cells or tissues. Assessment of the mitochondrial membrane potential is also necessary to determine any dysfunction. Nonetheless, the aforementioned techniques have certain shortcomings in that investigators require fresh samples and are time consuming due to the complexity of the procedures involved. As such, spectrophotometric technology remains a first-line method both for research investigations on mitochondrial ailments and for diagnostics.

Studies from recent years indicate that most published protocols for spectrophotometric assays were dissatisfactory in that there were complex biochemical hindrances that led to enzymatic inhibition or to unsatisfactory linearity of the kinetics with regards to protein concentration. Such analytical shortcomings have been known to severely prejudice sensitivity and precision, bringing about analytical discrepancies and considerable disagreements of results from various laboratories. The protocols in the current study were designed to overpower or negate most of these challenges, in an attempt to improve the sensitivity, specificity and precision with little adjustments to the framework of the procedure. The spectrophotometric technology depicted herein specify the steps for mitochondrial RC enzyme reaction analysis in which small quantities of muscle tissue from mammals and cultured skin fibroblasts were used compared to previously established procedures. The success of this undertaking imply that these technology is applicable to other tissues for example the heart, liver and brain in which case if the tissue homogenates are used, the sample preparation phase will have to be optimized for every tissue.

Spectrophotometric Technology

Proper application of spectrophotometric technology in the current study required measurement of Mitochondrial RC enzymatic reactions in crude homogenates as well as secluded mitochondria. It is important to note that the technology can also be extended for experiments in other organisms including yeast, human and bovine muscles, in which case isolated mitochondria is prepared in a similar quantities and similar quantities. Spectrophotometric technology makes it possible for all the assays to be reliably performed on muscle homogenates with requiring mitochondrial isolation. For convenience purposes and membrane disruption to ease substrates access to the enzymes, freezing of the samples is normally recommended. This ensures maximization of enzyme activity. Nonetheless, a fractional loss of activity is likely to follow for complex II+III in frozen muscle[4]. As such, it is critical to subject all the samples to the same treatment before the investigation. Though, mitochondria from fresh muscle as well as from various other tissues are still usable after isolation. When assessing of RC enzymes using cultured cells, every assays with the exception of those for precipitates of I and I+III are doable on cell lysates. Nonetheless for the analysis of compound I reactions when using cultured cells, it is mandatory that the investigator utilizes supplemented mitochondrial portions to as not to overwhelm nonspecific rotenone-insensitive reactions.

Wu et al. emphasize that polarimetric technology is vital procedure molecular imaging of cell, protein and tissues. These materials are optically anisotropic and tend to scatter photons in manner which makes polarimetric procedures possible.

The anisotropic trait of proteins is determinable from the polarity attributes of the light dispersed and/or diffused from the material. Polarization is one of the remarkable properties of light. The rectilinear and non-rectilinear complete polarization photosensitive characteristics of an anisotropic material has been depicted by a 4 ´ 4 Mueller matrix[5]. This is a modeling theory newly postulated to ease scientific inquiries of the optical polarization attributes of proteins as well as other light bio-material. The Mueller matrix scanning protocols have been newly improved for investigations of the optical polarization characteristic of hydrolases. Analogous to other polarimetric inquiries, the visual structures and the imaging information results showed existence of relative few physical models for the discernment of the essential optical polarization characteristics of protein samples.

This theory supports the basis for the model analysis scrutiny of the polarization features of protein media and correlates well to the polarization optical signatures protein molecules together with its infinitesimal electronic constituents[6]. Founded on binary photon dispersal, the extents of direct -dichroic polarization disposal by protein molecules was arithmetically computed and corresponded to past experiments. The Mueller matrix data has revealed that the protein biomolecules are optically anisotropic and exceedingly scattering but show an imperfectly diffusion. As such, the Mueller Stokes scanning technology anchored upon the penta-principal Mueller matrix e fundamentals of m01, m11, m22, m23 and m33 is practical for researching the anisotropic[7], photon-dispersal and polarization/depolarization photosensitive properties proteins and all biomolecule media. To examine the probable biomedical use and to grasp the anisotropic optical characteristics of organic samples, the study utilized modest experimentation for Stokes vector scanning volume. The investigators chose to transmit the optical design via a microscope system[8]. Three investigatory steps were utilized to standardize the Mueller matrix signature of the protein molecules under study. The precise imageries of four autonomous polarization limits and the consequent distribution functions of the penta-principal Mueller matrix parameters were also taken into account.

Polarimetric Technology

The authors emphasize that manometric technology is an essential component in the analytical processes for solid media or solid environmental samples that require water or buffer solutions. Application of this technology requires dilution of the enzyme to fall below the detection threshold. This is followed by a time consuming and a time costly procedure of concentrating the sample. Some of the commonly used approaches used approaches in concentrating the sample include extracting the enzymes from solid media followed by addition of a buffer or water. The mixture is stirred and left to stand still for a certain period of time and the suspension is separated from the clear liquid using a filter paper.  The enzymatic activities are afterwards assessed in terms of the filtrates obtained. The technology makes use of relatively large samples during the extraction process. In addition, when screening for multiple properties of enzymes, many culture flasks, bottles are used. Scientists who desire to recovery of enzymes, they minimize the number of steps because easy step promote loss of enzyme activity. Reduction steps can further moderate preparation costs and analytical process, and even keep in check the quantity of replicate growth sub-units[9]. Any reduction, no matter how infinitesimal it may appear cuts the practical steps and cost involved. The most preferred tactic is to inflate the measurement sensitivity by making use of fluorogenic substrates and determination concentration with the suspensions before extracting. The authors described an approach where minute samples of agar media with or deprived of supplements of natural organic matter were experimented on and analyzed for enzyme activation form outside the cell[10].

Electrode technology has been extensively in use because electrodes are electrochemical sensors. Electrodes show elevated and potential-dependent background current in solutions that are aqueous in order to support the high current and intricate multi-step surface reduction-oxidation processes. This is somehow an unwanted occurrence because it renders it strenuous to realize substantial signal-to-background ratios describable as minute detection thresholds. In comparison to bulk Au, Au nanoparticles (NPs) indicate improved electrocatalytic reactions in most electroactive species[11].

In most cases, Indium tin oxide (ITO) electrodes show insignificant electrocatalytic reactions, nevertheless constructively little and flat background current. As such, ITO electrode adjustment with a small exterior exposure of Au NPs is likely to permit high electrocatalytic reactions as well as low background charge. In addition, utilization of marginal Au is likely to shrink the cost of electrochemical sensors during enzyme detection. Any variation of electrode coats with the use metal NPs may be realized through the electrodeposition of metal ions as well as the immobilization of presynthesized metal NPs[12].

Nevertheless, the fast rate of electrodeposition renders it strenuous to regulate the size and exterior coating of NPs, thus negligible surface coating of metal NPs is hardily realizable with both approaches. The arrangement of metal NPs with the use of electrostatic adsorption of metal ions trailed by a cutback has also be been accepted as one of the strategies. This can be exemplified by use of Pt NPs with amine-functionalized on Si substrates that have synthesized with the help of enzymes[13].

The increasing use of fluorescent proteins (FPs) shows that it is increasingly being adopted in microbiological and biomedical studies. In less than two decades, when the initial phase of the Aequorea victoria jellyfish wild-kind green FP was first use to highlight sensory proteins in nematode, more advances have been made. The race to produce improved FPs with a wider coverage and better photostability has been touch. The microbiologists have also been keen in desensitizing their FPs specimen with regards to pH as well as improving the maturation rates.

For smooth application of fluorescent, the wild type (wtGFP) has since been undergone numerous modifications to yield variants emitting in the blue (BFP), cyan (CFP) and yellow (YFP) localities. However, the orange and the red spectral localities have proving difficult to achieve with Aequorea GFP seeming astoundingly obscure until the unanticipated finding of the initial red FP from a nonbioluminescent reef coral in the late 1990s[14]. The development gave leeway to a second chapter of the innovation, the persistent expedition for the holygrail of fluorescent enzymatic proteins. The search is still on to the present times. Considering the investigative interest, it can be said that progress has largely been very inspiring, despite the huge gap in many FP spectral modules.

The recent FP development stratagems are funneled towards fine-tuning the photophysical characteristics of blue to yellow modifications originated from the Aequorea victoria jellyfish GFP as well as the progress of monomeric FPs particularly that of organisms that have been confirmed to perceive light in the yellow-orange to far-red spectra. Advancement towards this direction has been satisfactory, and almost-infrared emitting FPs may be released to the market. The most recent endeavors in jellyfish modifications have yielded in better monomeric BFP, and YFP alternative and the unrelenting hunt for a bright, monomeric and fast-maturing red FP has given a myriad of desirable contenders, despite nothing has produced optimal results for the known applications. In the meantime, photoactivatable FPs are blossoming as an influential category of inquiries for intracellular undercurrents and, unpredictably, as important tools for the advancement of terrific resolution microscopy applications.

Notwithstanding the current improvements in FP technology, a large group of microbiologists  still make use of an augmented version of wild-type GFP (EGFP), in conjunction with the initial cyan and yellow products (ECFP and EYFP), for a significant percentage of their imaging applications[15]. The disinclination of a huge number of microbiologists to switch to current FP variants is necessitated by the unpredictable accessibility of different FPs, compounded with (often supportable) reservations regarding narrated claims of increased brightness and utility in fusions. I a lot of circumstances, merely locating a source for a new FP is not necessarily time wasting and as such an obvious challenge. The deficiency of reliable market sources regularly necessitates scientists to rely on the charity of the prototype laboratory, which has been known to be overwhelmed with an avalanche of applications immediately following the reporting of new protein or fusion construct.

Prohibiting the enactment of a reasonable and competent system for the circulation of FP variants among scientists seems to be persistent and bars them adopting the technology fully. The paper attempted to iron out regular misunderstandings that are bound to happen when microbiologists are transitioning to newly developed variants with a focus on enzymes. The paper further discussed recent developments in enzyme engineering protocols in an attempt to unmask steps to improvements of the color palette as well as the new photoactivatable FPs. The authors further gave suggestions of the most desirable choices in single and multi-color scanning.

The wide array of FP genetic variants advanced during the past decades focus on fluorescence emission reports traversing almost the complete visible light spectrum. Numerous regulatory motifs have surfaced to support the essential origins and control of the emission color. Localized environmental parameters across the chromophore on top of the location of charged amino acid deposits coupled with hydrophobic associations in the protein matrix have been fund to yield blue or red spectral alterations with regards to absorption and emission maximaal to a tune of 40 nm[16]. Wider spectral alterations that characterize the overall FP spectral classes of CFP and GFP are largely ascribed to divergences in the covalent arrangement and the magnitude of –orbital conjugation of the chromophore. More investigations into the multiple characteristics of FP chromophores give inklings related to the structural composition and functional connection with the polypeptide shape, the undertaking of genetically engineering exceptionally tuned color variants and widening of the spectral scope of valuable enzymatic proteins becomes smooth.

References

Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nature protocols. 2012 Jun 1;7(6):1235.

Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, 287–295 (2007).

Palmer, J.W., Tandler, B. & Hoppel, C.L. Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations. Arch. Biochem. Biophys. 236, 691–702 (1985).

Jonckheere, A.I., Smeitink, J.A. & Rodenburg, R.J. Mitochondrial ATP synthase: architecture, function and pathology. J. Inherit. Metab Dis. (2011).

Barrientos, A., Fontanesi, F. & Diaz, F. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr. Protoc. Hum. Genet. 63, 19.3.1–1 (2009).

Lin, M.T. & Beal, M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 (2006).

Winklhofer, K.F. & Haass, C. Mitochondrial dysfunction in Parkinson’s disease. Biochim. Biophys. Acta 1802, 29–44 (2010).

Hauptmann, S. et al. Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice. Neurobiol. Aging 30, 1574–1586 (2009).

Reisch, A.S. & Elpeleg, O. Biochemical assays for mitochondrial activity: assays of TCA cycle enzymes and PDHc. Methods Cell Biol. 80, 199–222 (2007)

Wu SH, Nee SF, Yang DM, Chiou A, Nee TW. Polarimetric signature imaging of anisotropic bio-medical tissues. Biomed. Eng. Res.. 2013 Mar;2:20-9.

Heinonsalo, J., Kabiersch, G., Niemi, R. M., Simpanen, S., Ilvesniemi, H., Hofrichter, M., ... & Steffen, K. T. (2012). Filter centrifugation as a sampling method for miniaturization of extracellular fungal enzyme activity measurements in solid media.Fungal Ecology, 5(2), 261-269

Coakley WT, Brown RC, James CJ, Gould RK, 1973. The inactivation of enzymes by ultrasonic cavitation at 20 kHz. Archives of Biochemistry and Biophysics 159: 722e729.

Dinis MJ, Bezerra RMF, Nunes F, Dias AA, Guedes CV, Ferreira LMM, Cone JW, Marques GSM, Barros ARN, Rodrigues MAM, 2009. Modification of wheat straw lignin by solid state fermentation with white-rot fungi. Bioresource Technology 100: 4829e4835.

Aziz, M.A., Patra, S. and Yang, H., 2008. A facile method of achieving low surface coverage of Au nanoparticles on an indium tin oxide electrode and its application to protein detection.Chemical Communications, (38), pp.4607-4609.

Shaner NC, Patterson GH, Davidson MW. Advances in fluorescent protein technology. Journal of cell science. 2007 Dec 15;120(24):4247-60.

Brakemann T, Stiel AC, Weber G, Andresen M, Testa I, Grotjohann T, Leutenegger M, Plessmann U, Urlaub H, Eggeling C, Wahl MC. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nature biotechnology. 2011 Oct 1;29(10):942-7.