DESC:FIELD OF INVENTION
The invention relates to field of biotechnology particularly growth of biofilm on liquid nutrient medium and to obtain a composition comprising reverse micelle containing exRNA which enables reduction in the viral load in Dengue infection.
BACKGROUND OF THE INVENTION
Biofilm formation is considered a virulence determinant in micro-organisms and it strongly contributes to microbial resistance to conventional antimicrobial agents, host protective immune responses, hostile environment pressures/ stresses, predation and shear forces.
Biofilm culturing format is always unique to the study and can depend on several factors. Culturing formats designed to be accessible to microscopy are not always suited for other types of analysis, such as harvesting biofilm biomass for biochemical measurements.
A common feature of biofilm culturing methods is a mechanism for separating adherent bacteria from those growing planktonically. Abiotic attachment surfaces can be replaced with pathogenically relevant biotic surfaces.
Available methods for biofilm culturing are:
1. Microtiter plate assays
2. Flow cells
3. Tube biofilms
4. Colony biofilms
5. Biofilm growth on peg lipids
6. Rotating disk & concentric cylinder reactors
Although biofilm of any micro-organism is cause of various diseases, treatment of which are not available till date and may result in fatal disease, biofilm produced may also be used for production of drug used for the treatment of various deadly diseases. The biofilm-based drugs may also be used for treating various viral disease such as Dengue, Chikungunya, Hepatitis A, Hepatitis C etc.
Conventionally used medium for biofilm growth is solid phase medium, The state of the art does not report induction of biofilm on a broth media. Cultivation of biofilm using conventionally used medium is very costly, time taking and are scalable to large scale or be easily duplicated in a fermenter.
The invention herein discloses bacterial biofilm formation on liquid nutrient medium using easily available and cost-effective raw material., With the artificial complex medium including ingredients such as soya, water etc. can induce bacterial growth and biofilm formation. The purpose behind the present invention is produce bacterial biofilm at large scale and reduce the cost of media that is required for the growth of product.
The World Health Organization (WHO) classifies dengue fever as a neglected tropical disease Dengue fever is still one of the most serious mosquito-borne illnesses in the world, and spreads t by the bite of female mosquitos Aedes aegypti or Aedes albopictus. The pathogenic agent is a mosquito- borne, single-stranded, positive-sense RNA virus of the Flaviviridae family and belongs to the genus Flavivirus. DENV (Dengue virus) is 40-60 nm in size. DENV serotypes (DENV 1-4) are classified as having 65-70 percent sequence similarity. Dengue serotype 2 (DEN-2) has been the most common serotype in India during the last 50 years, however serotypes 3 and 4 have also been seen in several epidemics (3). Dengue fever's global prevalence has risen drastically, with over half of the world's population now at risk. Although an estimated 100-400 million infections occur each year, the majority are mild and asymptomatic. During 2021, India reported around 1.6 lakh dengue cases according to ‘The Economic Times’. National Dengue Day, observed annually on 16th may, an innovative initiative of the Ministry of Health and Family Welfare, India. Weak mosquito control regulations, afforestation, climate change, and global warming are only a few of the causes. Dengue/severe dengue has no particular therapy. Early diagnosis of disease development associated with severe dengue and availability to competent medical care reduces severe dengue mortality rates to less than 1%.

DENV genome encodes total 10 structural and non-structural proteins (Fig 1). The genome of DENV is 11 Kb in length. The DENV capsid (C) protein is a 12 kDa basic protein due to its 26 Arg or Lys residues. Protein envelope is found on the virus's surface and is crucial in viral attachment to host cells via cell receptors such as heparan sulphate, DC-SIGN, and others. It is the most critical protein for viral entrance into the cell. Domain I (structural domain), domain II (dimmers), and domain III comprise this protein (binding domain). The dimmer domain connects the structural and binding domains. Dengue virus enters a host cell when the viral envelop protein attaches to a receptor and responds to the decreased pH of an endosome via conformational rearrangement (6). Through phosphorylation, E protein can activate PKA and PKG, which inhibits platelet activation and causes thrombocytopenia. The DENV prM (Pre-membrane) protein is essential for viral particle production and maturation. The mature DENV virion's glycoprotein shell contains 180 copies of the E and M proteins (8). The immature virion originates with the E and prM proteins. This immature viral particle buds into the endoplasmic reticulum and subsequently proceeds to the Golgi apparatus through the secretory route. The virion is exposed to low pH when it moves through the trans- Golgi network. This acidic environment triggers a conformational change in the E protein, causing it to dissociate from the prM protein and create E homodimers, which lay flat against the viral surface, giving the mature virion a smooth look (9). Furin, the host serine endoprotease, cleaves peptide from M peptide during this maturation. The M protein then functions as a transmembrane protein beneath the mature virion's E-protein shell. Until the viral particle is discharged into the extracellular environment, the pr peptide remains attached to the E protein. The release of pr at neutral pH during exocytosis completes viral maturation into an infectious particle (10).
DENV NS1 is a 48-kDa glycoprotein produced on the surface of infected cells that might serve as an antibody target for dengue infection (11). NS1 can directly trigger the release of inflammatory immune molecules, resulting in vascular leak. Through TLR4 activation, the NS1 protein has been found to increase permeability in endothelial cells (12). Some studies suggested that DENV NS1-induced vascular leakage was independent of inflammatory cytokines such as TNF-a, IL-6, and IL-8, but was dependent on endothelial glycocalyx degradation through activation of cathepsin L and heparanase-1 (HPA-1) (Binding of NS1 to endothelial cells induces HPA-1 activation via cathepsin L, leading to endothelial glycocalyx degradation and vascular leakage) (13).
Non-structural protein 2A is a 22-kDa hydrophobic transmembrane protein that is known to connect with the endoplasmic reticulum membrane. NS2A is involved in virion assembly; a R84A mutation in NS2A prevents DENV-2 assembly. After flavivirus polyprotein translation, an unknown host protease cleaves the N terminus of NS2A from NS1, and the viral NS2B-NS3 protease-cofactor complex cleaves the C terminus from NS2B in the cytoplasm. Viruses stimulate the interferon (IFN) signaling pathway when infected, however dengue develops interferon resistance. To avoid the immunological response, NS2a, NS4a, and NS4b inhibit the interferon cascade. By preventing STAT-1 phosphorylation, NS4b suppresses the interferon cascade (Munoz). NS2B functions as a cofactor for NS3 (14).
NS3 is a multifunctional protein that acts as a protease, helicase, RTPase, and NTPase. NS3 also participates in RNA replication and participates in the regulation of polyprotein processing (15). NS2B-NS3 could specifically cleave human stimulator of interferon genes (STING, also known as MITA, MPYS and encoded by TMEM173), play a role to escape STING mediated antiviral pathway. TRIM69 significantly reduced amount of NS2B-NS3 protein, impaired the cleavage of STING. NS4B is a tiny, hydrophobic protein that is found in the endoplasmic reticulum. It may inhibit STAT 1 phosphorylation after stimulation by type I interferons alpha and beta. In fact, when Tyk2 kinase activity falls in conjunction with dengue virus, so does STAT 1 phosphorylation. Furthermore, the innate immune system's reaction to the virus is slowed because the 'NS4B' protein inhibits interferon-stimulating gene (ISG) production. STAT 1 suppression may potentially include the NS4B cofactors NS2A and NS4A (16).
At its C-terminus, the DENV NS5 protein has RNA-dependent RNA polymerase (RdRp) and methyltransferase activity. When this 105-kDa protein is produced alone, it inactivates STAT2. When a protease (NS2B3) cleaves NS5 with NS4B, it can destroy STAT2 (17). The NS5 serine-threonine phosphorylation is a flavivirus-specific characteristic. The lack of NS5 interaction with the NS3 is associated with NS5 phosphorylation (18).
The human body contains several receptors via which DENV may enter various cells. DC-SIGN, HSP70, HSP90, GRP78, CD14, Laminin receptor, Heparan sulphate, and other receptors are examples. The primary mechanism of DENV internalization has been identified as clathrin- mediated endocytosis (19). Clathrin is a protein complex made up of three heavy (190 kDa) and three light chains (25 kDa). Dynamin, encoded by the gene DNM1, is a GTPase that is essential for clathrin-dependent endocytosis. It functions as a pair of molecular scissors for freshly generated vesicles that emerge from the plasma membrane. In cells, dynamin assembles into the neck of the budding membrane and undergoes GTP-dependent conformational changes that result in membrane fission (Fig. 2) (21).

In endocytic trafficking, actin and microtubules play a dynamic function. Actin may be involved in the movement of endocytic vesicles into the cytosol once they are detached from the plasma membrane. ARPC1B, ARPC2 and ARPC3 (different subunit of actin related protein complex). Microtubules are necessary for effective transcytosis and early endosome trafficking to the late endosome. Rab 5 is critical for the formation, identification, and activity of early endosomes. Rab7 defines late endosomes. The involvement of V-ATPase (H+ ATPase) in the acidification of intracellular vesicles such as endosomes is well established. V-ATPase acidifies the surface of endosomes, modulating essential physiological activities such as receptor endocytosis and vesicular trafficking (22).
Clathrin-coated vesicles lose their clathrin covering and become acidified after budding into the cytoplasm. Most enveloped viruses that enter the host cell by endocytosis require the low- pH stage to cause viral envelope fusion with the endosome membrane and nucleocapsid release into the cytosol. The presence of Rab 5 protein in early endosomes, where pH is 6.5, suggests that the endosome is maturing. The mature endosome (late endosome) is identified by Rab 7 and a pH of 5.5. The acidic pH causes E-protein to alter shape. D I, D II, and D III are E protein domains that are attached to the viral membrane. Domain II of the E protein is bent outward from the viral surface, exposing the fusion loop and causing the E protein to reorganize. The fusion loop at the tip of Domain II interacts with the host endosomal membrane, causing the E protein to trimerize. Trimerization occurs from the tip to the base, causing conformational changes such as Domain III rotation, shifts, and trimer displacement, resulting in the fusing of the viral envelope with the host's endosomal membrane and the release of the viral DNA into the cytoplasm. This delivered positive sense viral RNA can function as both mRNA for protein synthesis and a template strand for viral genome replication (23).
The viral genome delivered to the endoplasmic reticulum (ER) performs two functions: viral protein synthesis and viral genome replication. The same positive-sense RNA strand can act as a template for both protein synthesis and the complementary negative strand. The positive sense RNA strand can be used to provide a template for the negatively complementary strand. The newly produced negative strand remains attached to the positive strand, resulting in the formation of dsRNA. This negative strand acts as a template for the format.

The interaction between positively charged capsid protein and negatively charged RNA results in viral genome encapsidation, also known as nucleocapsid formation. The newly synthesised positive sense RNA interacts to NS2 via a contact between the RNA 3' UTR and the NS2 R94, K95, and K99 residues (24). Along with NS2B-NS3, NS2 recruits translated C-prM-E protein. The pr domain of prM seals the fusion loop of the E protein, preventing the immature virion from fusing prematurely to the host membrane. During viral maturation, furin cleaves prM into pr and M in the TGN (trans golgi network). The mature virus is subsequently exocytosed from the infected cell and complete the infection cycle (Fig. 3) (23).
Signalase cleavage in the ER releases prM and E from polyprotein. The viral NS2B-NS3 protease cleaves the capsid protein. Non-structural proteins are mostly processed in the cytoplasm by the viral proteases NS2B-NS3, with the exception of NS1, which is removed from NS2A by an unidentified protease in the ER (25).
Langerhans cells (LCs), dendritic cells (DCs), and dermal cells are the first few cells, which DENV encounters around the site of the mosquito bite. After a mosquito bite injects DENV into the skin, Langerhans cells and dendritic cells (DCs) come into the picture (26). DCs attract NK cells to the location, whereas macrophages attract neutrophils. Infected macrophages provide the processed antigen to the T cell, which then interacts with the B cell to help in B cell growth. To elicit distinct cell-mediated immune responses against the virus, different kinds of T cells are activated. The "cytokine storm" occurs when a flood of cytokines is released. As a normal immunological response to any pathogen, humoral immunity, represented by B-cell-mediated antibody production from plasma cells, produces ADE in DENV infection (23).

To activate neutrophils and drive them to the site of infection, macrophages secrete IL-8, IFN-beta, and TNF-alpha. TNF-alpha and defensins are produced by neutrophils and have an antiviral impact. TLR3 recognises dengue dsRNA intermediates, which activates TRIF, which in turn activates IRFs (27).
According to certain research, EGF levels were shown to be considerably lower in DENV infected populations. It might be caused due to thrombocytopenia, as thrombocytes are a major source of EGF. B cells attach to DENV antigens to elicit a first IgM response, which is followed by a complex DENV-serotype-specific IgG response. The secondary infection is marked by a decreased IgM response but an increased IgG response. The antibodies target the DENV proteins NS1, E, and prM. The participation of B cells also suggests that T cells are involved in the DENV infection response. While CD4+ T cells primarily target the capsid as well as the NS2A/B, NS3, NS5, and E proteins, CD8+ T cells primarily target the capsid as well as the NS3, NS4A/B, NS5, and E proteins. These responses cause CD8+ T cytotoxic cells to release perforins and granzymes, while CD4+ T cells to engage in Th1 effector activities (secreting IFN-? and TNF-a). Furthermore, other T cell subsets, such as Treg cells, control inflammation via TGF-ß and IL-10, and Follicular T cells assist B cells in producing highly specific antibodies with appropriate affinity (28). Although the whole immune system works together to attack DENV, some of these responses may induce host harm through an occurrence known as the "cytokine storm." Humoral immunity is characterized by B-cell-mediated antibody generation from plasma cells, which results in antibody-dependent enhancement of DENV infection (23).
Dengue Hemorrhagic Fever (DHF) is distinguished by high vascular permeability, which results in vascular leakage. DHF can eventually evolve to Dengue Shock Syndrome (DSS), which is characterized by reduced peripheral perfusion, which causes tissue damage and organ failure (29). DHF is classified by the WHO into four classes, from least severe grade 1 to most severe grade 4. Grades 3 and 4 are often known as dengue shock syndrome (DSS) (30). During DHF/DSS, a "cytokine storm" occurs, which refers to increased cytokine production. IL-1, IL-2, IL-10, CXCL- 10 and 11, CCL-2, VEGF, TNF-, IFN-, and IFN-, IL-18, IL-9, IL-8, RANTES, CCL-21, and CCL-19 are seen to be upregulated in both DHF and DSS. VEGF controls vascular permeability. TNF- alpha and IL-1 beta levels are elevated, which raises VEGF levels and, as a result, vascular permeability, resulting in plasma leakage and, eventually, organ failure. Because of plasma leakage, it would be extremely difficult for our bodies to maintain osmotic equilibrium, resulting in shock. IL-12 has a significant influence on Th1 cell upregulation, whereas its absence switches the balance towards Th2 cytokines. IL-12 helps to avoid severe dengue sickness by sustaining a Th1 response. A high level of IL-8 is a sign of the severity of DHF. Overproduction of IL-10 inhibits IFN responses while also stimulating vascular leakage. IL-10 and IL-6 stimulate the production of the suppressor of cytokine signaling-3 (SOCS-3) gene, which inhibits the JAK- STAT pathway (31). Cyclooxygenase-2 (Cox-2) is activated as a result of DENV infection. Upon activation, it catalyses the conversion of arachidonic acid to prostaglandin-G2. Prostaglandin-G2 is converted to prostaglandin-H2 (PGH-2) by peroxidase. PGH-2 is a PGE-2 precursor. Prostaglandin E2 (PGE 2) is a key pro-inflammatory cytokine that promotes viral polymerase activity (DENV RdRp) and viral proliferation (32).
The phenomenon of ADE occurs when a virus binds to inadequate antibodies and improves its access into the host cell. When antibodies that attach to viral particles fail to effectively kill the virus, ADE occurs. This might be due to the antibodies that bind to viral epitopes other than those required for cell attachment and entrance or the existence of sub-neutralizing antibody concentrations (binding to viral epitopes below the neutralization threshold). In either case, these antibodies promote virus entrance into target cells, resulting in enhanced viral infection, known as antibody-dependent enhancement. The most commonly accepted explanation for explaining the elevated risk of DHF/DSS is ADE. IgG antibodies mostly mediate the ADE. The ADE-DENV complex binds to the Fc-receptor, activating SARM (Sterile-alpha and armadillo motif-containing protein) and TANK (Tumor necrosis factor receptor associated factor [TRAF] family member associated NF-Kb activator). SARM and TANK activation inhibited MYD88-dependent and independent pathways by blocking TLR-3, 4, and 7. SARM inhibits TRIF and TANK inhibits TRAF 6, both of which suppress type I IFN and NF-kB (33).
In vitro and in patients with severe secondary DENV infection, the RIG-I/MDA-5 antiviral signaling pathway is also downregulated during DENV-ADE (34). When immune complexes enter target cells, they activate the negative regulators dihydroxyacetone kinase (DAK) and autophagy-related 5-autophagy-related 12 (Atg5-Atg12), causing the RIG-I/MDA-5 (Retinoic acid-inducible gene I/ (melanoma differentiation-associated protein 5) signaling cascade to be disrupted. This suppresses Type I IFN production as well as IFN-mediated antiviral responses.
Binding of immune complexes to cellular activating FcRs is known to activate the ITAM- spleen tyrosine kinase-signal transducer and activator of transcription 1 (STAT1) signaling pathway, which leads to ISG induction and viral replication inhibition. Inhibiting FcR-dependent ISG transcription is thus critical for DENV replication and is thought to be caused by co-ligation of Leukocyte Immunoglobulin like Receptor B1 (LILR-B1) by FcR-bound antibody-opsonized DENV (35, 36). LILR-B1 is an inhibitory receptor that belongs to the ITIM family and is expressed on immune effector cells such as monocytes, macrophages, and dendritic cells. The DENV-antibody immunological complex interacts to two cellular receptors at sub-neutralizing antibody concentrations: FcR (through antibody) and LILR-B1 (through DENV E protein). By binding to LILR-B1, Src homology phosphatase-1 (SHP-1) is recruited, which dephosphorylates and inactivates Syk and suppresses ISG production (Kulkarni). SHP-1 signaling also inhibits DENV acidification and lysosomal degradation (37).
The type 1 IFN pathway is a key innate immune response that protects the host from pathogen invasion. When IFN-a and IFN-ß bind to JAK1 and Tyk2, they phosphorylate. JAK1 and Tyk2 phosphorylation activates STAT1 and STAT2 through tyrosine phosphorylation. Phosphorylated STAT1 and STAT2 heterodimer binds to interferon-regulated gene (IRG-9) and forms IFN- stimulated gene factor 3 transcriptional complex (ISGF-3). ISGF3 enters the nucleus and binds to the IFN-stimulated response element (ISRE) to activate the production of IFN-stimulated genes (ISGs) such as 2'-5'-oligoadenylate synthetase, PKR, IFITM, viperin, and others that inhibit various phases of viral replication (38).
Viperin, an ER-associated S-adenosyl-L-methionine domain-containing enzyme, has been shown to be a significant IFN-induced antiviral protein against DENV. ISG20 is a 3'-5' exonuclease that has been proven to decrease RNA virus infection (40).
Cyclic GMP-AMP synthetase (cGAS) is a cytosolic DNA sensor that identifies and triggers the host defensive response against microbial DNA. According to a recent study, cGAS plays a key role in preventing positive sense ssRNA viral infection, particularly since RNA is not known to activate cGAS. Virus entry into cells disrupts the mitochondria, resulting in the release of mitochondrial DNA. cGAS detects dsDNA and initiates the production of cGAMP. cGAMP activates STING. Activated STING further activates IKK and TBK1. IKK and TBK1 activate NF- kB and IRF3, respectively, resulting in cytokines and type 1 IFN responses (41). Mitochondrial DNA also triggers the TLR-9-mediated IFN response (42).
MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules of about 22 nucleotides. They can regulate the post-transcriptional expression of target genes by cleaving or repressing mRNAs, and they play a crucial role in organism's gene regulation. MiR-30e*, which targets I?ßa, boosts IFN production to prevent DENV replication (43). Other differentially regulated miRNAs that are typically implicated in EC permeability (miR-126, miR-155) and inflammation (miR-126, miR-221, etc.) and are enhanced post-DENV infection stand out as potential therapeutic agents against DENV-induced vascular leak syndrome (44, 45, 46, 47). Active dengue virus replication caused apoptosis in several cell lines by increasing p53 expression. Overexpression of miR-1204 resulted in cell death, which might be one of the mechanisms through which p53-induced apoptosis occurred (48). MiR-491-5p has also been proven a p53 regulator and an anti-apoptotic gene, BCL- XL. MiR-512-5p is abundant in acute dengue patients and targets the nucleotide binding oligomerization domain-2 (NOD-2). NOD2 suppression causes retardation of IFN, NF-kB, JNK, MAPK pathways (49).
Dengue fever is managed by rest and cold sponging to keep temperature below 39oC, usually antipyretics may be used to lower the body temperature. Aspirin/NSAID like Ibuprofen etc may cause gastritis, vomiting, acidosis and platelet disfunction and should be avoided. Oral fluid and electrolyte therapy are recommended for patients with excessive sweating or vomiting. If signs of complications are observed during the afebrile phase of DHF hospitalization is recommended. The haematocrit, platelet count and vital signs is carried out examined to assess the patient is condition and intravenous fluid therapy started. If haematocrit is decreasing, fresh whole blood transfusion 10ml/kg/dose is given.
Apart from management, novel ways of disease management are also being researched, US US9,186,397B2, published on 15th October, 2015 provides polynucleotides and polypeptides encoded therefrom having advantageous properties, including an ability to induce an immune response to flaviviruses. The polypeptides and polynucleotides of the invention are useful in methods of inducing immune response against flaviviruses, including dengue viruses. Compositions and methods for utilizing polynucleotides and polypeptides of the invention are also provided.
US 10117924B2, published on 13th October, 2017 provides compositions and methods of use comprising a chimeric dengue virus E glycoprotein comprising a dengue virus E glycoprotein backbone, which comprises amino acid substitutions that introduce a dengue virus E glycoprotein domain I and domain II hinge region from a dengue virus serotype that is different from the dengue virus serotype of the dengue virus E glycoprotein backbone.
US 11,059,883 B2 23rd May, 2019 discloses antibody molecules that specifically bind to dengue virus are disclosed. The antibody molecule binds to dengue virus serotypes DV - 1, DV - 2, DV - 3, and DV – 4. The antibody molecules can be used to treat, prevent, and / or diagnose dengue virus.
Although the management of dengue is being done for a long time, still there remains a need to find out better pharmaceutically active molecules/ compound /composition that can effectively treat dengue. The present study therefore aims to identity better options for the treatment of Dengue virus that can effectively manage the disease and gaps in the management of dengue.

OBJECT OF THE INVENTION
Accordingly, the main object of the present invention is to induce biofilm formation in the liquid media.
One of the object of the present invention is to develop a reverse micelle preparation for the inhibition of dengue virus.
Another object of the present invention is to develop an effective composition for the management of dengue infection.
Another object of the present invention is composition that have minimal side-effect in view of the immune response and attack on the thrombocytopenia in dengue patients.
Another object of the present invention is to the down-regulate of the inflammatory genes that are over-expressed in dengue infection.

SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that is further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.
The present invention discloses a composition of the invention discloses a liquid culture medium for culturing bacterial biofilm and a preparation method for the liquid culture medium. The liquid culture medium consists of 15-20g of soya flour, 0.2-0.7% of peptone, 0.5-0.9% of NaCl, 1-2% of tryptone and the balance of water. The invention further discloses the preparation method for the liquid culture medium for culturing bacterial biofilm. The liquid culture medium for culturing biofilm is extensive in raw material source and low in cost; the liquid culture medium is slightly in a brown sugar colour, and is transparent, clear and easy to observe; moreover, the bacterial biofilm proliferates easily on this medium and take less time to proliferate.
Therefore, the liquid culture medium for culturing bacterial biofilm is easy to make and is cost-effective. The product issued from Soya based liquid medium extensively used as therapeutic for treatment of Dengue & is able to normalize the cytokine & chemokine storm i.e., result of high anti-inflammatory activity.
The present invention discloses a method of down-regulating pro-inflammatory cytokines comprising; reverse micelle useful in the treatment of Dengue, wherein the down regulation of the pro-inflammatory cytokines is carried out by growth of biofilm on liquid nutrient medium composition comprising reverse micelle

BRIEF DESCRIPTION OF FIGURES:
The above-mentioned objectives and descriptions, features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:

Figure 1 depicts (A) Growth of bacteria and biofilm formation on liquid media (B) Structure of Reverse micelle showing Nano scale particles composed of an outward hydrophobic tail which consists of double stranded extracellular RNA attached with hydrogen bond and inward hydrophilic head.;

Figure 2 depicts fluorescence microscopy image of biofilm derived reverse micelle containing exRNA entering in the cytoplasm of A549 Cell;

Figure 3 depicts Zeta view image of biofilm derived reverse micelle containing exRNA. Propidium Iodide dyes bind to dsRNA present on the surface of nucleus shows red outline forming reverse micelle as positive charge hydrophilic head is towards inwards and negative charge ds RNA is towards outwards;

Figure 4 depicts Reverse micelle containing exRNA inhibits DENV1 serotype infection in Vero cells. A) MTT assay to check cytotoxicity of purified EPS composition in Vero cells. B) Western Blotting to check DENV 1 infection using anti-DENV NS3 antibody;

Figure 5 depicts Flowcytometry to show that the Reverse micelle containing exRNA inhibits DENV 1 infection in Vero cells in a dose response manner. A) FACS representative plots showing DENV infection in Vero cells. B) Histogram Plots showing DENV infection in Vero cells C) Bar graphs showing normalized infection in Vero cells;

Figure 6 depicts Flowcytometry to show that the Reverse micelle containing exRNA inhibits DENV 1 infection in Vero cells. A) FACS representative plots showing DENV infection in Vero cells. B) Histogram Plots showing DENV infection in Vero cells C) Bar graph showing normalized infection in Vero cells (Replicates).
Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of the aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar ‘’language throughout this specification may, but do not necessarily, all refer to the same embodiment The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.

Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Invention discloses a liquid culture medium for biofilm formation of gram-positive bacteria on it & preparation method for the liquid culture medium. The liquid culture medium consists of Soya water, NaCl, peptone and tryptone.
Liquid nutrient medium has wide material source, the cheap abundant useful element i.e. Soya as it is source of high quality of protein, carbohydrate, fat and also contains various active or metabolic proteins and enzymes such as trypsin inhibitors, hemagglutinins and cysteine proteases that contains the cultivation bacterial biofilm of the present invention make raw material, be applicable to the growth of biofilm, this liquid nutrient medium is brown sugar look slightly, transparent, limpid and easy to observe.
With the adoption of the liquid culture method for biofilm cultivation, the technical problems of long production cycle, high energy consumption, high potential risk and the like in the prior art can be solved; the liquid culture method for biofilm has the advantages that the production cycle is reduced, the contents of effective ingredients of biofilm product are increased, the growth conditions are easily controlled, and the industrial production can be carried out conveniently.
Below in conjunction with embodiment, the present invention is illustrated further.
The present invention discloses a composition of growth of biofilm on liquid nutrient medium comprising reverse micelle confirmed by Fluorescence microscopy.
In one embodiment the invention discloses a liquid culture medium for biofilm formation of gram-positive bacteria on it & preparation method for the liquid culture medium. The liquid culture medium consists of soya flour, NaCl, peptone and tryptone.
In one embodiment the liquid culture medium consists of 15-20g soya powder, 5-10g tryptone for 1 L.
In one embodiment, the Reverse micelle is encapsulated with exRNA.
In still another embodiment the reverse micelle may comprise an exRNA and the exRNA may be selected from a group comprising microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), PlWI-interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), transfer RNAs (tRNAs) and tRNA fragments, YRNAs, ribosomal RNAs (rRNAs), mitochondrial RNAs, protein-coding RNAs, mRNA, double stranded RNA (dsRNA) preferably dsRNA.
The composition that inhibits dengue virus in a dose- dependent manner with significant reduction in DENV infection with increase in dose.
In one aspect of the invention, a method of extraction of biofilm derived reverse micelle containing exRNA is disclosed comprising precipitation of suspended biofilm with ethanol.
Examples
The following examples are for illustration purposes and are not to be construed as limiting the invention disclosed in this document to only the embodiments disclosed in these examples.
Example 1
Soya flour of about 15-20g was added water to make up its volume. Then the medium is autoclaved for 30 minutes at 15 psi. This processed Soya water was further used with supplements required for promoting the growth of bacterial biofilm. Main component of this medium is Soya water supplemented with 0.2-0.7% of peptone, 0.5-0.9% of Nacl, 1-2% of tryptone after proper mixing of all the supplemented components the medium is autoclaved for 30 minutes at 15 psi, and is transferred to the utensils about 30 ml which will be used for inoculation in aseptic conditions and is left under UV for about 15 minutes for double sterilization. Proper sterilization of the medium results in very less chances of contamination for the bacterium growth and release biofilm in the provided medium. After proper cooling of the medium the medium is inoculated with the bacterium required for the cultivation of biofilm and was incubated at 37? for about 24 hours. In 24 hours, growth is observed on the topmost part of the medium which get separated for further processing of the required biofilm-based drug. The separation performed is done with proper handling otherwise it may get distorted.

After separation of the required biofilm the biofilm is processed with 5- 10% of ethanol and is mixed properly and is centrifuged at 3000 rpm for about 30 minutes and the process is repeated for the second time. After proper centrifugation and separation, the biofilm-based drug is filtered with the help of 0.22 µm of syringe filter for proper separation of bacterial spores and colonies from the end product. Further quality test was performed for its sRNA content check where 260/280 ration reading of the product was taken on spectrophotometer.

Cells and Viruses

Vero cells were cultured in DMEM media (Gibco USA) supplemented with 10% FBS (Gibco USA) and 1% antibiotic/antimycotic (Gibco USA) in incubator with 5% CO2 at 37 °C. C636 cells were used to propagate DENV. Monolayer of Vero cells were formed and infected with DENV and inoculum was removed and cells were supplemented with culture media with 2% FBS for 4-6 days (till cytopathic effect was observed). Culture supernatants containing viruses were harvested and kept at -80 °C until further use.

Cell viability assay
The viability of cells was determined by MTT (3-(4,5-dimethylthiazolyl-2)- 2,5diphenyltetrazolium bromide) assay. Vero cells seeded (2 × 104 cells per well) in a 96 well plate, were treated with different concentrations of purified EPS composition for 24 h and 48 h. Post incubation, 0.5 mg/ml MTT stock in 1× PBS along with incomplete cell culture media was added and the cells and incubated at 37 °C for 3 h. After incubation, the supernatant was carefully discarded and DMSO was added to solubilize the insoluble formazan crystals inside the cells. Lastly, absorbance was recorded at 570 nm.

DENV infection studies using Flowcytometry

Vero cells were seeded overnight at a density of 5 × 104 cells in a 12-well plate and infected with DENV for 90 min, following which viral inoculum was removed, infected cells were washed with 1× PBS and 400 µl of DMEM media (2% FBS) with or without purified EPS composition was added and incubated until 24 h. The culture supernatant after the incubation was collected and stored at - 80 °C for further use. The infected cells were then washed with 1× PBS, 4% paraformaldehyde was used for fixation of the cells for 15 min at RT and permeabilized with 1× Permwash buffer (Biolegend) and incubated with anti-Dengue 1, 2, 3 and 4 (GeneTex) primary antibodies diluted in 1× Permwash buffer overnight at 4 °C. After primary antibody incubation, the cells were incubated with anti-mouse Alexa-488-IgG (Invitrogen) for 1 h at 37 °C. After the incubation with the secondary antibody, the cells were washed and resuspended in FACS buffer and acquired on BD Accuri Plu s Flow cytometer. The analysis for identifying DENV positive cells was carried out through Flowjo software.

Western Blotting:

Uninfected or DENV infected cells were washed with cold 1x PBS, lysed using mammalian lysis buffer supplemented with 1X Protease inhibitor cocktail (Sigma Aldrich) as described earlier. Total proteins in the lysates were estimated using Bicinchoninic Acid (BCA) protein assay (G Biosciences) and equal concentration of proteins was loaded on Polyacrylamide SDS-PAGE (12%). Proteins were transferred on to a nitrocellulose membrane (HiMedia) through electroblotting. Further, membranes were blocked with 5% skimmed milk (HiMedia) 1X PBS and incubated with primary antibodies (Abs) at 4°C overnight. The primary antibodies used were rabbit Dengue Virus NS3 (Genetex), mouse anti-ß actin (Cell Signaling Technology). Following primary antibody incubation, membranes were washed with 1X TBST and incubated with appropriate secondary Abs for 1 h at room temperature. Proteins bands were visualized using femtoLUCENT™ chemiluminescent substrate (G Biosciences) and chemiluminiscence was captured by ChemiDoc Touch Imaging System (BioRad). Densitometry analysis of protein bands on blots was done using NIH software Image J.

Biofilm derived reverse micelle containing exRNA inhibits DENV infection in Vero cells.

In order to check anti-DENV effects of reverse micelle containing exRNA, the cytotoxicity of molecule in Vero cells by MTT assay was first determined. The reverse micelle containing exRNA did not induce death up to a concentration of 100 µl/ml (106 molecules/ml) (Fig. 1A). The non-toxic concentrations of reverse micelle were evaluated for anti-DENV effects. Vero cells were infected with DENV (1 MOI) followed by treatment of reverse micelle containing exRNA for 24h. It was observed that purified EPS significantly inhibits DENV infection demonstrated by reduction in dengue antiNS3 protein expression in reverse micelle containing exRNA treated cells quantified through immunoblotting (Fig. 1B). Anti-DENV effects of reverse micelle containing exRNA were further determined by using Flowcytometry. DENV infected Vero cells were treated with different concentration of reverse micelle containing exRNA and incubated with anti-DENV antibody followed by incubation with appropriate alexa-488 tagged secondary antibody. The stained cells were acquired in Flow cytometer and evaluated for DENV positive cells. It was observed that reverse micelle containing exRNA treated cells show less percentage of DENV positive cells compared to DENV infected cells and were comparable with uninfected cells. It was also observed that anti-DENV effect of reverse micelle containing exRNA therapeutics is dose dependent as with the increase in dose, DENV infection also significantly reduced. These results depict that reverse micelle containing exRNA inhibits DENV 1 (MOI 1) infection in Vero cells (Fig 2, 3 replicates). However, the mechanisms through which the RNA based drug inhibits the Dengue infections require in depth study.
While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein. The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.

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,CLAIMS:We Claim,

1. A media composition of growth of biofilm on liquid nutrient medium comprising reverse micelle comprising soya water supplemented with 0.2-0.7% of peptone, 0.5-0.9% of NaCl, 1-2% of tryptone, wherein soya water comprises 15-20% of soya suspended in distilled water.
2. The composition as claimed in claim 1, wherein the media promotes biofilm formation wherein the reverse micelle is encapsulated with exRNA.
3. The composition as claimed in claim 1, wherein the reverse micelles comprise the exRNA is selected from a group comprising microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), PlWI-interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), transfer RNAs (tRNAs) and tRNA fragments, YRNAs, ribosomal RNAs (rRNAs), mitochondrial RNAs, protein-coding RNAs, mRNA, double stranded RNA (dsRNA) preferably dsRNA.
4. The composition as claimed in claim 1, wherein the reverse micelle encapsulated with exRNA obtained from biofilm inhibits dengue virus in a dose-dependent manner with a significant reduction in dengue virus infection with an increase in dose