Hybrid blended polymer of bacterial surface protein (fimbriae = pili = BNT (bionanotube) as an important vaccinating and drug delivery tool: a biomedical design.
The functional properties of bacterial metabolites including bioenergetics, enzyme kinetics and fermentations have been recently reviewed. BNT (baionanotube) = Fimbriae= pili as elongated appendages of outer most surface of E.coli have been identified under SEM (scanning electron microscopy. These BNT=fimbriae=pili are basic properties of bacterial pathogenic activities including MRHU (D-mannose resistant haemagglutination of human erythrocytes.), and flocculation of volatile organic and inorganic substrates in wastewater. BNT= fimbriae= pili have been identified as elongated appendages of surface of bacteria. They are responsible for bacterial colonization, MRHA and flocculation. The elongations of BNT=fimbriae=pili are the part of protein, enhancing the process of flocculation and MRHA (D-mannose resistant haemagglutinations of mammalian erythrocytes) to the distant cells of same species, to increase adherence to the matrix and for colonization. Fimbriae=pili= BNT are supposed to be considered as elongated protein nano-channel to carry biological molecules, secondary metabolites. These BNT=fimbriae=pili have been found to be essential and important for the interest of medicine (drug delivery and monitoring of diseases) and for environmental micro unit flocculating activities, [1-7].
To understand the properties of bacterial adherence, colonization corresponding to fimbriae, MRHU (+) positive and negative (-) assays were essential to differentiate pathogenic, virulent strains from a-virulent, nonpathogenic bacterium (Escherichia coli). MRHU were furthermore correlated corresponding to plasmid profiles of the same strains in AGE, Fig.1 and Fig.2. MRHU may be caused by the transport of biological molecules (bacterial metabolites= haemagglutinine, flocculine) through BNT (Biological nano-tubes)=fimbriae= pili and were evaluated at different MRHA and Sero-typing, caused by viable cell and by the isolated fimbriae=pili=BNT. They were then put in logical and realistic views as a part of bacterial adherence enhanced by BNT=fimbriae=pili, [1-4]. Serological assays and MRHA (D-mannose resistant haemagglutination of mammalian erythrocytes) were investigated at different mammalian and avian erythrocytes, Brahma (1993) [5,6]. Plasmids profiles assays in AGE has been considered for establishing that expressions of fimbriae=pili=BNT and MRHU are basically controlled by plasmids and by their regulatatory properties of drug resistance antibiotics, Tn (teaspoons), flanked by IS (Insertion sequences.), Brahma et. al (1982) [7, 8]. The transport of biological molecules (bacterial metabolites= haemagglutinine, flocculate) through BNT (Biological nanotubes)= fimbriae= pili must be logically and realistically approved to define the cause of bacterial adherence made by BNT= fimbriae = pili, [1-4]. Serological assays and E.coli MRHA (D-mannose resistant haemagglutination of mammalian erythrocytes) were investigated at different mammalian and avian erythrocytes, Brahma (1993) . In vivo co conjugative donor MRHU (+) isolated plasmid was transformed in to E.coli K-12 and a hybrid, 5405 auxotrophic E.coli K-12, positive to MRHU (+) was isolated. Co-conjugations were made at multiple recipients and plasmid vectors conjugations. The 5405 hybrid E.coli K-12 C600 Yale strain was used to study various interesting, MRHU (+) and MRHU (-) expressions, to define bacterial adherence at auxotrophic E.coli K-12, and to realize the concept of using polymer matrix based drug delivery through BNT=fimbriae=pili as cost effective measures of vaccination and online monitoring of mammalian diseases, Brahma (2006)[1,3],
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Fimbriae=pili= BNT or biological nano-channels of E.coli and are basically recognized in medical science as for the cause of adherence, colonization. The same are used and conceptualized also for wastewater flocculation and other industrial matrix based adherence. The concept that fimbriae=pili=BNT are the elongated protein appendages, conditionally controlled by Plasmid DNA., causing MRHU and proved hereby that in absence of fimbriae=pili= BNT, MRHU can also be occurred but faster like fimbriae E.coli. Non-piliate and isolated fimbriae= pili= BNT, showed slow performance of MRHU. Mutants and hybrids of E.coli K-12, perform different MRHU similar to the donors.
From the above plasmid profiles, Fig.1, it becomes clear, that single plasmid carries only MRHU (+) positive expression and /or MRBO (+) positive expressions and prove subsequently that plasmid is responsible for MRHU (+) and that plasmid may be cloned and transfer into E.coli K-12 for designing GE-E.coli K-12 with MRHU (+) expression at auxorophic K-12, resistant to sm[R], tet[R]. The prediction and correlations for the cause of MRHU, adherence and flocculation have been considered as one step ahead with the transport of bacterial metabolites, adhesions and haemagglutine, pass through membrane to cell wall and are elongated as expanded BNT=fimbriae=pili in case of rapid transport of biological molecules (metabolites) and to adhere blood cells microbes and substrates to the distant regions of 2-5 microns ([mu]), during microbial pathogenic and activated sludge treatments. Either by the process of natural immobilization or by ionic and molecular (haemagglutinine, adhesions) transport through nano-channels of Fimbriae= BNT= pili, E.coli K-12 performed MRHU (+) and flocculation. This has been investigated as major interest for developing antiadherent antibodies in Balb/c mice to protect mice against the hemorrhagic diarrhea of 026:EPEC. Two important physical and biological properties were considered. 1st one was the preparations of mammalian and avian blood cells (erythrocytes) in presence of D-mannose and Na-citrate at 24[degrees]C room temperature after separations of blood cell by centrifugations. However the MRHU (+) was performed at 4[degrees]C on ice bath and at room temperatures. The 2nd one the studies of plasmid profiles of E.coli. The process was equally be interested for medical practitioners in case of rapid diagnosis and in case of engineers these may lead to the understanding for developing antiadherent antibacterial agents, which may protect bacteria to adhere to the solid matrix to protect corrosions and damaging industrial pipelines, , or to develop hybrid polymer matrix, which will attract microbes of waste water to flocculate, vis-a vis.
The studies of MRHU plasmid profile, Fig. 1, 2, were significant to understand the functional properties of BNT=fimbriae=pili, as elongated protein appendages at the lengths of 2-5 microns ([mu]), and at diameters of 150-200 nanometers. Fig.3,4. However the studies of BNT= fimbriae= pili in bacterial adherence was supported by the studies of plasmid genetic engineering and regulatory properties of gene and protein appendages expressions by the studies of transposable elements (Tn) and flanking insertion sequences (IS), [8-10]. To investigate bacterial plasmids, involved in MRHU and adherence, two different plasmid genetic engineering studies were performed. 1st one was the transformation of donor MRHU (+) plasmid in vitro by means of routine donor plasmid isolations and its transformations into E.coli K-12 auxotrophic K-12.[6,7]. The 2nd one was in vivo donor plasmid transformation by co-conjugations,. Parallel to the above experiments, in vitro the vector plasmid pBR322 DNA cloning was used to select and to get quicker auxotrophic E.coli K-12 hybrid transconjugant. All the above experiments were routine wise to isolate one GE 5405 E.coli K-12 to study the cause of expressions of donor MRHU (+) plasmids into E.coli K-12 auxotrophic phenogenotypes and thereby the changing surface antigenic properties of E.coli K-12, expressing BNT= fimbriae =pili on the outer most cell wall of E.coli K-12 at auxotrophic pheno-genotypes, [6,7]. GE hybrid isolation was essential to study the properties of MRHU (+) in presence of BNT = fimbriae = pili and without BNT = fimbriae = pili, suppressing thereby the prototrophic activities of donor E.coli. This was essential to evaluate the properties of BNT= fimbriae=pili, specific to adherence and MRHU in E.coli K-12. After the evaluation of MRHU (+) expression in an auxotorphic E.coli K-12, nal[R], sm [R] phenogenotypes, the process and expressions of MRHU (+) in presence and in absence of BNT = fimbriae = pili and without BNT have been studied for fundamental causes and for Balb/c mice experiments. MRHU (+) and adherence may be the fundamental cause to describe the transport of biological molecules (metabolites) through membrane to the cell wall and through BNT = fimbriae = pili to the distant cell and substrates. This phenomenon has been recently evaluated and was envisaged by the specific concepts of MRHU = adherence = flocculation, Brahma (1993, 2006) [1,8,13,16]. In this paper, the author has emphasized the study related to classical and molecular bacterio-genetics and physicochemical properties of transporting biological molecules through BNT, causing MRHU (+) and their uses as nano-channels for drug and anti adherent vaccine delivery in support hybrid surface protein associated polymer matrix.
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A. Large-Scale production of SA= fimbriae = pili =BNT of E.coli
1) Identify bacterial cells belonging to infectious and non-infectious group, i.e. ETEC (Enterotoxigenic Escherichia coli), EPEC (Enteropathogenic Escherichia coli) and UTIEC (Urinary tract infecting Escherichia coli) and classify them according to their MRHU positive and negative properties and by their MRHU(+/-) plasmid profiles to compare their in vitro haegglutinations of HU, BO, HO, CH and D-mannose sensitive of Guinea pig. (MSGP). (Hybrid or Wild types). Study DNA properties and plasmid in Agarose gel.
Grow the cells in selective medium, at sterile. Isolate the cell and dissolve the cells in Tris.HCL, pH 7.8 buffer.
2) Homogenize or shear the cells by specific stirrer and centrifuge the homogenized components along with fimbriae=pili=BNT protein at 10,000 rpm in cold at 4-10 [degrees]C.
3) Separate and decant the supernatant. Centrifuge the supernatant in 12-15,000 rpm. Decant the supernatant into a clean 100 ml. conical flask. Add ammonium sulphate [(NH4).sub.2]S[O.sub.4] sterile, till saturation at cold (4[degrees] to 10[degrees]C), and agitate the mixture overnight for (12 hours) by continuous magnet stirring in a 50-100 ml. small conical flask.
4) Centrifuge the precipitate at 20-25,000 rpm and decant the supernatant. Use Sucrose gradient for ultra-centrifugation and for making gradient to separate fimbriae = pili = BNT protein out of cell wall protein in 40, 000 rpm at 4 [degrees]C for 48 hours. 8K for one 026:EPEC donor, 13, 15K for 08:ETEC and 24-40K for hybrid E.coli K-12 were isolated, Fig., according to Salit et al 
5) Isolate pure fimbriae=pili=BNT protein bands and dialyze them in parchment membrane to separate all salt ions undesirable.
6) Measure the concentration of protein fimbriae bands, according to Warburg and Christian[ ] Dissolve the precipitate (ppt) in sterile by clean Tris.HCl buffer at pH 7.8 and run SDS-PAGE
7) Use both the isolated hybrid E.coli, the isolated SA (surface antigens) pili= fimbriae= nano-tubes, in animal tests for antiadhrent= antidiarheal immune response, according to Brahma (1999)[5-8]
8) Pure fimbriae protein was used both for the test of Balb/c mice immune responses and to make hybrid polymer for slow drug delivery for protecting domestic animal against diarrhea of E.coli and other bacterial species. Blended araldite polymer has been shown in Fig.
B. Characterizations and isolations of plasmids in AGE (agarose gelelctrophoresis).
1) Make Small culture in a test tube
2) Centifuge the cells
3) Alkaline treatment and cell Lyse Precipitate the plasmid DNA by addition of NaCl and SDS and keep them at 16[degrees]C overnight.
4) Precipitation of DNA Add Na-Acetate and isopropyle alcohol at the total volume of 75 ml (micro lit).
5) Freeze them instant either in Liquid Nitrogen at 180[degrees]C or in C[O.sub.2]+ Alcohol at 45 [degrees]C or keep them at 75[degrees]C in deep freeze.
6) Melt them at room temp of 22-24[degrees]C and centrifuge in microfuge at 800-1000 rpm for two minute.
7) For the removals all alcohol you keep all the Tubes inverted at air on Tissue paper.
8) Dissolve the DNA at the bottom again in Tris. Buffer Prepare Agarose gel at 0.7-1.0 % Put the dissolved DNA for separations.
9) Study Plasmids and chromosomes in Agaose gel and run the gel.
10) Add ethidium bromide to visualize the DNA and photograph them for documentation in Polaroid camera
C. Measurement of Pressure drop ([DELTA]P) Across the Blended hybrid Membrane of Araldite and Bacterial Surface Protein BNT (Bionanotube).
This experiment will deal the pressure drop measurements through nano-channels of bacterial surface protein (fimbriae = pili = BNT) in an araldite-blended polymer, Fig. 5 and 6 mixed with various concentration of surface protein. The pressure drop ([DELTA]P) was measured on prepared araldite membrane vertically cross flow to the blended hybrid membrane of araldite, mixed with surface antigenic proteins of bionantotube (BNT)= fimbriae protein of bacteria. BNT protein or similar isolated surface protein has been used to prepare the blended Araldite simple membrane in the laboratory at the thickness of 1-2 mm.. The surface properties of the pores compared to pure araldite (0 pores) have been measured under the microscope at the magnifications of X 400 and counted the diameter of pores would be within the range of 150-200 nm (0.1 to 02. micron= [mu]), as shown in Fig.5 and 6. The performance of membrane have been compared virtually to demonstrate that simple pure araldite membrane does not allow any water flow, whereas the hybrid araldite BNT membrane performs water flow at syringe pressure, as shown in Fig. 7.In addition to that, present paper will perform also the calculation for indirect measurement of pores that are being measured microscopically. This will provide an indirect impact on biotechnological activities of microbes. This will lead to understand how microbes adhere to the surface and how their adherence are to be understood in the context of shear stress and exploitation of microbial pathogenic activities, that secondary metabolites of microbes, like adhesions, haemagglutinine, and flocculine are supposed to be needed for bacterial adherence. Adhesions and haemagglutinine are proposed to be carried out by means of facilitated transport through the nanochannel of fimbriae= BNT (bionanotubes). This was essential in case of bacterial colonization and spreading of infections. A concept associated with antiadherent antibody development has been communicated to some other journals to prove that adherence of microbes are basically derived by two species, i.e. microbes-microbes, microbes and tissues or microbes and matrixes (animate and inanimate) properties, caused either by van der Waal's attraction or by protein mediators that are being discharged from bacterial surface and are elongated as fimbriae = BNT (bionanotube) for attracting distant molecules.. This concept has a tremendous impact on industrial wastewater treatments, pipeline and sculpture damages and spreading of inactions. Pressure drop ([DELTA]P) measurement will highlight both the physicochemical and biological properties of microbes to understand how microbes actually controlling micro fluidic activities in a very small pressure gradient across the nano-channel, and how their fluid transport can be considered corresponding to non-Newtonian and Newtonian fluids in the form of Re < 1 and mathematical derivations. Fig. 5&6.
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Results and Discussion
microbial assays and specially in case of Escherichia coli K-12 infections, according to Brahma [1-4, 8-10]. Agars-Peptone, -Loeb, -Simca, -Minimal, antibiotics (chloramphenicol, streptomycin, tetracycline, gentamycine, nalidixic acids, antibiotics were used and were purchased from local suppliers. Sigma, Boeringer, and Indian standard HCL analar chemicals were used. For large-scale DNA isolations, CsCl, density gradient were used. DNA were marked with Et.-Br.(Ethidium Bromide), TRIS, EDTA(Ethylene diamine tetra acetic acid) and RE (Restriction enzymes). DNA pBR322-vector plasmid, Radio isotopic [[[alpha].sup.32] p] d ATP (Adenosine triphosphate)-probes, Agarose gel, were purchased from Boeringer Manheim, Germany. The methods related to MRHU and serotyping were made according to Brahma , Plasmid profile analysis in AGE (Agarose gel electrophoresis) and RE digestions, radio labeling, auto radiography, SDS-PAGE were made according to Brahma [6,8]. Except the modification in CsCl isopicnique density gradient,[5,8], all rest experimental methods were involved in chromosome and plasmid isolations and were made according to Brahma et.al . Plasmid isolations were made according to Binboim and Dolly . Plasmid of MRHU (+) at the range of 100-120 kb = 60-70 MD, were carefully separated and were studied in AGE and for RE digestions,[5,6]. The adherence with host and bacterial cells in presence of adhesions and receptors were also studied. The haemagglutination patterns were characterized by different blood granules agglutinated in MRHU, and were characterized by mammalian and avian erythrocytes and with different E.coli, [5,6].
[FIGURE 7 OMITTED]
However the characteristics of fimbriae and nonfimbrial haema gglutinations are still under investigation. MRHU may be caused either by the transport of haemagglutine through BNT channel or by direct contacts of cell wall without BNT=fimbriae=pili. However the present investigation envisaged that the transport of biological molecules (metabolites) are being made by BNT, from cell membrane to the targeted cell via BNT=fimbriae=pili. Fig.3, and Fig. 4 (a-c). Table1 corresponding to Plasmid MRHU have been studied according to Brahma (1999a) It was found that a 8 micron ([mu]) large RBC cell can procure 8 E.coli cells and subsequently the chain of reactions that spontaneously supports blood cell RBC (Red Blood corpuscles) to agglutinate, in presence of more than 2 x [10.sup.10] cells per 10 [micro]l = 1 drop of blood cells, MRHU have been studied as shown in Fig.1 and were used to prove indirectly the pathogenic activities of E.coli with MRHU (+) and without negative MRHU(+), i.e. the loss and gain of adherence for pathogenic activity, [5-7].Fig.2, shows the fimbriae=pili=BNT properties of various E.coli bacterium, involved in different types of colonization and adherence. BNT = fimbriae =pili properties, signified the possible transport of biological molecules involved in MRHU. The present paper describes the logical phenomena of BNT and their possible uses in drug application and diagnosis of infections. A pressure device has been used to measure and set up the membrane, Fig.7 to create pressure across the membrane by syringe. It has been proposed that in future a compatible membrane would perhaps be designed to introduce the same polymer matrix into a living system, higher animal tests with Balb/c mice. The concept is to allow blood and serum through the membrane pores and to get the signal of immune responses from the pores of the matrix to activate the body immures system in a continuous mode, measured and controlled either by sensors or by electronics devices. Nanopores of the fimbriae = pili =BNT of bacterium surface protein (antigen) has been derived, starting since sex-pili of E.coli that allows DNA molecules of donors (F+) to transport into recipients (F-) during conjugations. As per the SEM studies and measurement it was established that the length and thickness of fimbriae=pili=BNT could be varied, starting since 10-200 nm of fimbriae diameter to the length of 2-10 micron. BNT of bacterium or the surface protein of bacterium may be used for drug and vaccine delivery as polymer hybrid blend matrix to study the change of viscous fluid flow across the matrix, imbedded by fimbriae and without fimbriae (BNT). However the future experiment would be emphasized to place fimbriae in an elongated manner to allow the flow without any hindrance, Fig.4 and that could be made by the studies of transport of biological molecules through hybrid polymer patches and measuring pressure drop across the membrane by using pressure drop measuring sensors or transducers.
BNT= fimbriae = pili were basically being isolated as appendage elongated protein in a scattered distributions, Fig.3 (a-c) [1-4]. With the above objectives, this study, attempted to perform the logical predictions of BNT = fimbriae = pili in adherence and biological molecular transport correlated by plasmid genetic engineering. So in the first step the various MRHU (+) properties in presence and in absence of BNT = fimbriae = pili would be essential. In the 2nd step the correlations of BNT expressions was searched on the basis of plasmid and the presence of IS1 sequences in plasmids of MRHU (+) of an individual E.coli and the changing patterns of MRHU cause by BNT= fimbriae= pili. After identifications of MRHU (+) plasmid in 08:ETEC and 026:EPEC, the methods of isolation of donor and hybrid palsmid and chopping of plasmids were carried out to study some plasmid genetic components and origin of MRHU (+) expressions, corresponding to BNT = fimbriae = pili. Parallel to this investigation, the DNA--recombination was used to transfer MRHU (+) plasmids of donos into E.coli K-12 auxotrophic recipients. The presence of IS1 was searched among the plasmids, according to Brahma . The hybridization effects of IS1 have been characterized with donor, mutant and hybrid 5405 plasmids, Fig. 1. Based on plasmids and MRHU studies of E.coli, industrially important microbes and other enterobacterial species along with hybrid 5405 bacteria, it may be concluded that BNT= fimbriae= pili could be useful for effectively drug delivery, monitoring and diagnosing of pathogenic diseases. Based on transport model it has been proposed that due to presence of BNT= Fimbriae = pili inside the polymer matrix the biological molecular transport would be enhanced compared to non-BNT polymer matrix, as estimated by nonporous araldite polymer. The isolated scattered elongated nanotubes were immobilized parallel to the axis of flow, Fig. 3, 4. This polymer matrix condition will support the facilitate transport of biological molecules specially in artificial system as nano-devices for monitoring and diagnosis of diseases. Pressure drop related molecular protein diffusion was conditionally observed both in case of matrix without BNT=fimbriae=pili and matrix with BNT=fimbriae=pili. The same mechanisms has been effectively used in case of living microbes, that they convert them in spore formations during stress situation and expands their fimbriate,= piliate and BNT surface protein, when they are in suitable environment and are being activated to infect host by the process of colonization and flocculation in case biodegradation of wastes, Brahma (2000).
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The paper has been dedicated to Prof. V.R. Kalvey, Hon'ble Dy-Director, IIT-Kharagpur-721302, Prof. S.Basu, Ex Hon'ry Editor Indian Chemical Engineers Journal and Emeritus Professor of Biomedical and Chemical Engineering of IIT-Bombay, JU, and my wife Tapati.
Born in Brahmanbaria / Bangladesh on 01-11-1946, as a youngest son of Advocate late Jatindra Mohan Brahma, mother late Mrs. Debabala Brahma, the author Dipl.-Ing. Nitosh Kumar Brahma continued his teaching and research activities since (1976) in Germany and in India (J.U and IIT-Kharagpur), since the author obtained distinction graduation (B.Sc.) in Human Physiology, from the Calcutta University (1966), at the age of 19/20, Chemical Technology Graduation degree (1972), and Post graduation professional Title degree (1975) on Chemical and biochemical Process Technology, at the age of 28/29 from the Technical University, Berlin, Germany. During his teaching and research activities (1975-1986), the author worked at various famous Institutes, like; Institute of Chemical Engineering and Technology (TU-Berlin); Institute of Process and Combustion Engineering and Medical College (TU-Vienna); Institute of Polymer Science, (Dutch State Mines, Holland); Institute of Hygiene and Institute of Biology-III (Genetic Engineering) and finally in Max-Plank-Institute in Germany and completed his (Aufbaustudium) studies on Dr.-Ing.// Dr.-rer-.nat., as Doctorand on Artificial kidney and the Genesis of Bacterial Adherence; and submitted a Dr.-thesis to ALU on" Fimbriae of pathogenic Escherichia coli" to complete his Viva (defense). The author completed several teaching, research and innovative work on Artificial Kidney; Membrane Separations; Ultra filtration (UF) and Reverse Osmosis (RO); Bubble Column; Microbial technology in waste managements; microbial infections, "Pathogenesis and Vaccinations" by developing a Genetically Engineered Escherichia coli K-12 C600, strains, commonly used in fermentation and Bioreactor designs. The author obtained Ciba Geigy participation Medal for paper, proposed for " Vaccination Model of Balb/c mice" (1984) from University Basel, Switzerland. And several best paprs awards from IIChE, AMI and other parts of world. A joint paper of author with Rahul Gupta M.Tech student has been accepted in recent IEEE International onferenceAISPC'07.ISBN 87-90834-99-2ISSN 0908-1224
Nitosh Kumar Brahma *
Department of Chemical Engineering, Indian Institute of technology, Kharagpur-721302, India. firstname.lastname@example.org, email@example.com,
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|Author:||Brahma, Nitosh Kumar|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jul 1, 2007|
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