Search results for: thermophilic proteins

Authors: Atitallah Sofien, Bouyahia Olfa, Attar Souleima, Missaoui Nada, Ben Rabeh Rania, Yahyaoui Salem, Mazigh Sonia, Boukthir Samir

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Introduction: Ciliopathies are a spectrum of diseases that have in common a defect in the synthesis of ciliary proteins. It is a rare cause of neonatal cholestasis. Clinical presentation varies extremely, and the main affected organs are the kidneys, liver, and pancreas. Methodology: This is a descriptive case report of a newborn who was admitted for exploration of neonatal cholestasis in the Paediatric Department C at the Children’s Hospital of Tunis, where the investigations concluded with a rare genetic mutation. Results: This is the case of a newborn male with no family history of hepatic and renal diseases, born to consanguineous parents, and from a well-monitored uneventful pregnancy. He developed jaundice on the second day of life, for which he received conventional phototherapy in the neonatal intensive care unit. He was admitted at 15 days for mild bronchiolitis. On clinical examination, intense jaundice was noted with normal stool and urine colour. Initial blood work showed an elevation in conjugated bilirubin and a high gamma-glutamyl transferase level. Transaminases and prothrombin time were normal. Abdominal sonography revealed hepatomegaly, splenomegaly, and undifferentiated renal cortex with bilateral medullar micro-cysts. Kidney function tests were normal. The infant received ursodeoxycholic acid and vitamin therapy. Genetic testing showed a homozygous mutation in the DCDC2 gene that hadn’t been documented before confirming the diagnosis of renal-hepatic ciliopathy. The patient has regular follow-ups, and his conjugated bilirubin and gamma-glutamyl transferase levels have been decreasing. Conclusion: Genetic testing has revolutionized the approach to etiological diagnosis in pediatric cholestasis. It enables personalised treatment strategies to better enhance the quality of life of patients and prevent potential complications following adequate long-term monitoring.

Keywords: cholestasis, newborn, ciliopathy, DCDC2, genetic

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Authors: S. Fröhlich, M. Herold, M. Allmer

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Early stage quantitative analysis of host cell protein (HCP) variations is challenging yet necessary for comprehensive bioprocess development. High resolution mass spectrometry (HRMS) provides a high-end technology for accurate identification alongside with quantitative information. Hereby we describe a flexible HRMS assay platform to quantify HCPs relevant in microbial expression systems such as E. Coli in both up and downstream development by means of MVDA tools. Cell pellets were lysed and proteins extracted, purified samples not further treated before applying the SMART tryptic digest kit. Peptides separation was optimized using an RP-UHPLC separation platform. HRMS-MSMS analysis was conducted on an Orbitrap Velos Elite applying CID. Quantification was performed label-free taking into account ionization properties and physicochemical peptide similarities. Results were analyzed using SIEVE 2.0 (Thermo Fisher Scientific) and SIMCA (Umetrics AG). The developed HRMS platform was applied to an E. Coli expression set with varying productivity and the corresponding downstream process. Selected HCPs were successfully quantified within the fmol range. Analysing HCP networks based on pattern analysis facilitated low level quantification and enhanced validity. This approach is of high relevance for high-throughput screening experiments during upstream development, e.g. for titer determination, dynamic HCP network analysis or product characterization. Considering the downstream purification process, physicochemical clustering of identified HCPs is of relevance to adjust buffer conditions accordingly. However, the technology provides an innovative approach for label-free MS based quantification relying on statistical pattern analysis and comparison. Absolute quantification based on physicochemical properties and peptide similarity score provides a technological approach without the need of sophisticated sample preparation strategies and is therefore proven to be straightforward, sensitive and highly reproducible in terms of product characterization.

Keywords: process analytical technology, mass spectrometry, process characterization, MVDA, pattern recognition

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Authors: Don Anushka Sandaruwan Elvitigala, P. D. S. U. Wickramasinghe, Jehee Lee

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Cystatins are a large superfamily of proteins which act as reversible inhibitors of cysteine proteases. Papain proteases and cysteine cathepsins are predominant substrates of cystatins. Cystatin superfamily can be further clustered into three groups as Stefins, Cystatins, and Kininogens. Among them, stefines are also known as family 1 cystatins which harbors cystatin Bs and cystatin As. In this study, a homologue of family one cystatins more close to cystatin Bs was identified from Korean black rockfish (Sebastes schlegeli) using a prior constructed cDNA (complementary deoxyribonucleic acid) database and designated as RfCyt1. The full-length cDNA of RfCyt1 consisted of 573 bp, with a coding region of 294 bp. It comprised a 5´-untranslated region (UTR) of 55 bp, and 3´-UTR of 263 bp. The coding sequence encodes a polypeptide consisting of 97 amino acids with a predicted molecular weight of 11kDa and theoretical isoelectric point of 6.3. The RfCyt1 shared homology with other teleosts and vertebrate species and consisted conserved features of cystatin family signature including single cystatin-like domain, cysteine protease inhibitory signature of pentapeptide (QXVXG) consensus sequence and N-terminal two conserved neighboring glycine (⁸GG⁹) residues. As expected, phylogenetic reconstruction developed using the neighbor-joining method showed that RfCyt1 is clustered with the cystatin family 1 members, in which more closely with its teleostan orthologues. An SYBR Green qPCR (quantitative polymerase chain reaction) assay was performed to quantify the RfCytB transcripts in different tissues in healthy and immune stimulated fish. RfCyt1 was ubiquitously expressed in all tissue types of healthy animals with gill and spleen being the highest. Temporal expression of RfCyt1 displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCyt1 showed concentration-dependent papain inhibitory activity. Collectively these findings evidence for detectable protease inhibitory and immunity relevant roles of RfCyt1 in Sebastes schlegeli.

Keywords: Sebastes schlegeli, family 1 cystatin, immune stimulation, expressional modulation

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Authors: Sinethemba Yakobi, Lindiwe Zuma, Ofentse Pooe

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Gonococcal infections present a notable public health issue, and the major approach for treatment involves using β-lactam antibiotics that specifically target penicillin-binding protein 2 (PBP2) in Neisseria gonorrhoeae. This study examines the influence of flavonoids, namely rutin, on the structural changes of PBP2 in both penicillin-resistant (FA6140) and penicillin-susceptible (FA19) strains. The research clarifies the structural effects of particular mutations, such as inserting an aspartate residue at position 345 (Asp-345a) in the PBP2 protein. The strain FA6140, which is resistant to penicillin, shows specific changes that lead to a decrease in penicillin binding. These mutations, namely P551S and F504L, significantly impact the pace at which acylation occurs and the stability of the strain under high temperatures. Molecular docking analyses investigate the antibacterial activities of rutin and other phytocompounds, emphasizing its exceptional binding affinity and potential as an inhibitor of PBP2. Quercetin and protocatechuic acid have encouraging antibacterial effectiveness, with quercetin displaying characteristics similar to those of drugs. Molecular dynamics simulations offer a detailed comprehension of the interactions between flavonoids and PBP2, highlighting rutin's exceptional antioxidant effects and strong affinity for the substrate binding site. The study's wider ramifications pertain to the pressing requirement for antiviral treatments in the context of the ongoing COVID-19 epidemic. Flavonoids have a strong affinity for binding to PBP2, indicating their potential as inhibitors to impair cell wall formation in N. gonorrhoeae. Ultimately, this study provides extensive knowledge on the interactions between proteins and ligands, the dynamics of the structure, and the ability of flavonoids to combat penicillin-resistant N. gonorrhoeae bacteria. The verified simulation outcomes establish a basis for creating potent inhibitors and medicinal therapies to combat infectious illnesses.

Keywords: phytochemicals, penicillin-binding protein 2, gonococcal infection, ligand-protein interaction, binding energy, neisseria gonorrhoeae FA19, neisseria gonorrhoeae FA6140, flavonoids

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Authors: Ana Beltran Sanahuja, A. Valdés García, Saray Lopez De Pablo Gallego, Maria Soledad Prats Moya

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Tomato consumption has greatly increased worldwide in the last few years, mostly due to a growing demand for products like sofrito. In this sense, regular consumption of tomato-based products has been consistently associated with a reduction in the incidence of chronic degenerative diseases. The sofrito is a homemade tomato sauce typical of the Mediterranean area, which contains as main ingredients: tomato, onion, garlic and olive oil. There are also sofrito’s variations by adding other spices which bring at the same time not only color, flavor, smell and or aroma; they also provide medicinal properties, due to their antioxidant power. This protective effect has mainly been attributed to the predominant bioactive compounds present in sofrito, such as lycopene and other carotenoids as well as more than 40 different polyphenols. Regarding the cooking process, it is known that it can modify the properties and the availability of nutrients in sofrito; however, there is not enough information regarding this issue. For this reason, the aim of the present work is to evaluate the cooking effect on the antioxidant capacity of different variants of tomato-based sofrito combined with other spices, through the analysis of total phenols content (TPC) and to evaluate the antioxidant capacity by using the method of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Based on the results obtained, it can be confirmed that the basic sofrito composed of tomato, onion, garlic and olive oil and the sofrito with 1 g of rosemary added, are the ones with the highest content of phenols presenting greater antioxidant power than other industrial sofrito, and that of other variables of sofrito with added thyme or higher amounts of garlic. Moreover, it has been observed that in the elaboration of the tomato-based sofrito, it is possible to cook until 60 minutes, since the cooking process increases the bioavailability of the carotenoids when breaking the cell walls, which weakens the binding forces between the carotenoids and increases the levels of antioxidants present, confirmed both with the TPC and DPPH methods. It can be concluded that the cooking process of different variants of tomato-based sofrito, including spices, can improve the antioxidant capacity. The synergistic effects of different antioxidants may have a greater protective effect; increasing, also, the digestibility of proteins. In addition, the antioxidants help to deactivate the free radicals of diseases such as atherosclerosis, aging, immune suppression, cancer, and diabetes.

Keywords: antioxidants, cooking process, phenols sofrito

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Authors: Abtehal Y. Anaas, Mohd. Nazmi Bin Abd. Manap

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Meat Tenderness is one of the most important factors affecting consumers' assessment of meat quality. Variation in meat tenderness is genetically controlled and varies among breeds, and it is also influenced by environmental factors that can affect its creation during rigor mortis and postmortem. The final postmortem meat tenderization relies on the extent of proteolysis of myofibrillar proteins caused by the endogenous activity of the proteolytic calpain system. This calpain system includes different calcium-dependent cysteine proteases, and an inhibitor, calpastatin. It is widely accepted that in farm animals including chickens, the μ-calpain gene (CAPN1) is a physiological candidate gene for meat tenderness. This study aimed to identify the association of single nucleotide polymorphism (SNP) markers in the CAPN1 gene with the tenderness of chicken breast meat from two Malaysian native and commercial broiler breed crosses. Ten, five months old native chickens and ten, 42 days commercial broilers were collected from the local market and breast muscles were removed two hours after slaughter, packed separately in plastic bags and kept at -20ºC for 24 h. The tenderness phenotype for all chickens’ breast meats was determined by Warner-Bratzler Shear Force (WBSF). Thawing and cooking losses were also measured in the same breast samples before using in WBSF determination. Polymerase chain reaction (PCR) was used to identify the previously reported C7198A and G9950A SNPs in the CAPN1 gene and assess their associations with meat tenderness in the two breeds. The broiler breast meat showed lower shear force values and lower thawing loss rates than the native chickens (p<0.05), whereas there were similar in the rates of cooking loss. The study confirms some previous results that the markers CAPN1 C7198A and G9950A were not significantly associated with the variation in meat tenderness in chickens. Therefore, further study is needed to confirm the functional molecular mechanism of these SNPs and evaluate their associations in different chicken populations.

Keywords: CAPNl, chicken, meat tenderness, meat quality, SNPs

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Authors: Amlan Kumar Das, Avinash Marwal, Vikram Pareek

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Liposome plays an important role in medical and pharmaceutical science as e.g. nano scale drug carriers. Liposomes are vesicles of varying size consisting of a spherical lipid bilayer and an aqueous inner compartment. Magnet-driven liposome used for the targeted delivery of drugs to organs and tissues1. These liposome preparations contain encapsulated drug components and finely dispersed magnetic particles. Liposomes are vesicles of varying size consisting of a spherical lipid bilayer and an aqueous inner compartment that are generated in vitro. These are useful in terms of biocompatibility, biodegradability, and low toxicity, and can control biodistribution by changing the size, lipid composition, and physical characteristics2. Furthermore, liposomes can entrap both hydrophobic and hydrophilic drugs and are able to continuously release the entrapped substrate, thus being useful drug carriers. Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magneticor paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI)3. The biological synthesis of nanoparticles using plant extracts plays an important role in the field of nanotechnology4. Green-synthesized magnetite nanoparticles-protein hybrid has been produced by treating Iron (III)/Iron(II) chloride with the leaf extract of Dhatura Inoxia. The phytochemicals present in the leaf extracts act as a reducing as well stabilizing agents preventing agglomeration, which include flavonoids, phenolic compounds, cardiac glycosides, proteins and sugars. The magnetite nanoparticles-protein hybrid has been trapped inside the aqueous core of the liposome prepared by reversed phase evaporation (REV) method using oleic and linoleic acid which has been shown to be driven under magnetic field confirming the formation magnetic liposome (ML). Chemical characterization of stealth magnetic liposome has been performed by breaking the liposome and release of magnetic nanoparticles. The presence iron has been confirmed by colour complex formation with KSCN and UV-Vis study using spectrophotometer Cary 60, Agilent. This magnet driven liposome using nanoparticles-protein hybrid can be a smart vesicles for the targeted drug delivery.

Keywords: nanoparticles-protein hybrid, magnetic liposome, medical, pharmaceutical science

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Authors: Ayesha Waqar Khan, Mariam Altaf, Jamil Ahmad, Shaheen Shahzad

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Kidney fibrosis is an anticipated outcome of possibly all types of progressive chronic kidney disease (CKD). Epithelial-mesenchymal transition (EMT) signaling pathway is responsible for production of matrix-producing fibroblasts and myofibroblasts in diseased kidney. In this study, a discrete model of TGF-beta (transforming growth factor) and CTGF (connective tissue growth factor) was constructed using Rene Thomas formalism to investigate renal fibrosis turn over. The kinetic logic proposed by Rene Thomas is a renowned approach for modeling of Biological Regulatory Networks (BRNs). This modeling approach uses a set of constraints which represents the dynamics of the BRN thus analyzing the pathway and predicting critical trajectories that lead to a normal or diseased state. The molecular connection between TGF-beta, Smad 2/3 (transcription factor) phosphorylation and CTGF is modeled using GenoTech. The order of BRN is CTGF, TGF-B, and SMAD3 respectively. The predicted cycle depicts activation of TGF-B (TGF-β) via cleavage of its own pro-domain (0,1,0) and presentation to TGFR-II receptor phosphorylating SMAD3 (Smad2/3) in the state (0,1,1). Later TGF-B is turned off (0,0,1) thereby activating SMAD3 that further stimulates the expression of CTGF in the state (1,0,1) and itself turns off in (1,0,0). Elevated CTGF expression reactivates TGF-B (1,1,0) and the cycle continues. The predicted model has generated one cycle and two steady states. Cyclic behavior in this study represents the diseased state in which all three proteins contribute to renal fibrosis. The proposed model is in accordance with the experimental findings of the existing diseased state. Extended cycle results in enhanced CTGF expression through Smad2/3 and Smad4 translocation in the nucleus. The results suggest that the system converges towards organ fibrogenesis if CTGF remains constructively active along with Smad2/3 and Smad 4 that plays an important role in kidney fibrosis. Therefore, modeling regulatory pathways of kidney fibrosis will escort to the progress of therapeutic tools and real-world useful applications such as predictive and preventive medicine.

Keywords: CTGF, renal fibrosis signaling pathway, system biology, qualitative modeling

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Authors: Zlatina Genisheva, Pedro Ferreira-Santos, Margarida Soares, Cândida Vilarinho, Joana Carvalho

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The wine industry produces significant quantities of waste, much of which remains underutilized as a potential raw material. Typically, this waste is either discarded in the fields or incinerated, leading to environmental concerns. By-products of wine production, like lees and grape pomace, are readily available at relatively low costs and hold promise as raw materials for biochemical conversion into valuable products. Reusing these waste materials is crucial, not only for reducing environmental impact but also for enhancing profitability. The Vinhos Verdes demarcated region, the largest wine-producing area in Portugal, has remained relatively stagnant over time. This project aims to offer an alternative income source for producers in the region while also expanding the limited existing research on this area. The main objective of this project is the study of the sustainable valorization of grape pomace and lees from the production of DOC Vinho Verde. Extraction tests were performed to obtain high-value compounds, targeting phenolic compounds from grape pomace and protein-rich extracts from lees. An environmentally friendly technique, microwave extraction, was used for this process. This method is not only efficient but also aligns with the principles of green chemistry, reducing the use of harmful solvents and minimizing energy consumption. The findings from this study have the potential to open new revenue streams for the region’s wine producers while promoting environmental sustainability. The optimal conditions for extracting proteins from lees involve the use of NaOH at 150ºC. Regardless of the solvent employed, the ideal temperature for obtaining extracts rich in polyphenol compounds and exhibiting strong antioxidant activity is also 150ºC. For grape pomace, extracts with a high concentration of polyphenols and significant antioxidant properties were obtained at 210ºC. However, the highest total tannin concentrations were achieved at 150ºC, while the maximum total flavonoid content was obtained at 170ºC.

Keywords: antioxidants, circular economy, polyphenol compounds, waste valorization

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Authors: Evgeniya Y. Yuzbasheva, Tigran V. Yuzbashev, Natalia I. Perkovskaya, Elizaveta B. Mostova

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Cell-surface display of lipase is of great interest as it has many applications in the field of biotechnology owing to its unique advantages: simplified product purification, and cost-effective downstream processing. One promising area of application for whole-cell biocatalysts with surface displayed lipase is biodiesel synthesis. Biodiesel is biodegradable, renewable, and nontoxic alternative fuel for diesel engines. Although the alkaline catalysis method has been widely used for biodiesel production, it has a number of limitations, such as rigorous feedstock specifications, complicated downstream processes, including removal of inorganic salts from the product, recovery of the salt-containing by-product glycerol, and treatment of alkaline wastewater. Enzymatic synthesis of biodiesel can overcome these drawbacks. In this study, Lip2p lipase was displayed on Yarrowia lipolytica cells via C- and N-terminal fusion variant. The active site of lipase is located near the C-terminus, therefore to prevent the activity loosing the insertion of glycine-serine linker between Lip2p and C-domains was performed. The hydrolytic activity of the displayed lipase reached 12,000–18,000 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. In case of C-terminal fusion variant, the leakage was occurred due to the proteolytic cleavage within the linker peptide. In case of N-terminal fusion variant, the leaking enzyme was presented as three proteins, one of which corresponded to the whole hybrid protein. The calculated number of recombinant enzyme displayed on the cell surface is approximately 6–9 × 105 molecules per cell, which is close to the theoretical maximum (2 × 106 molecules/cell). Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, cell-bound lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. It retained 74% of original activity at 60°C for 5 min of incubation, and 83% of original activity after incubation at 50°C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1–% and 71.0–% methyl esters after 33–h and 45–h reactions, respectively.

Keywords: biodiesel, cell-surface display, lipase, whole-cell biocatalyst

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Authors: Rabah Iratni, Husain El Hasasna, Khawlah Athamneh, Halima Al Sameri, Nehla Benhalilou, Asma Al Rashedi

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Background: Cancer therapies have witnessed great advances in the recent past, however, cancer continues to be a leading cause of death, with colorectal cancer being the fourth cause of cancer-related deaths. Colorectal cancer affects both sexes equally with poor survival rate once it metastasizes. Phytochemicals, which are plant derived compounds, have been on a steady rise as anti-cancer drugs due to the accumulation of evidences that support their potential. Here, we investigated the anticancer effect of Rhus coriaria on colon cancer cells. Material and Method: Human colon cancer HT-29 cell line was used. Protein expression and protein phosphorylation were examined using Western blotting. Transcription activity was measure using Quantitative RT-PCR. Human tumoral clonogenic assay was used to assess cell survival. Senescence was assessed by the senescence-associated beta-galactosidase assay. Results: Rhus coriaria extract (RCE) was found to significantly inhibit the viability and colony growth of human HT-29 colon cancer cells. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, p16, downregulation of cyclin D1, p27, c-myc and expression of Senescence-associated-β-Galactosidase activity. Moreover, RCE induced non-canonical beclin-1independent autophagy and subsequent apoptotic cell death through activation of activation caspase 8 and caspase 7. The blocking of autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death. Further, RCE induced DNA damage, reduced mutant p53 protein level and downregulated phospho-AKT and phospho-mTOR, events that preceded autophagy. Mechanistically, we found that RCE inhibited the AKT and mTOR pathway, a regulator of autophagy, by promoting the proteasome-dependent degradation of both AKT and mTOR proteins. Conclusion: Our findings provide strong evidence that Rhus coriaria possesses strong anti-colon cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against colon cancer.

Keywords: autophagy, proteasome degradation, senescence, mTOR, apoptosis, Beclin-1

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

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Authors: Kitti Szoke, Laszlo Szoke, Attila Czompa, Arpad Tosaki, Istvan Lekli

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Autophagy plays an important role in cardiac hypertrophy, which is one of the most common causes of heart failure in the world. This self-degradative catabolic process, responsible for protein quality control, balancing sources of energy at critical times, and elimination of damaged organelles. The autophagic activity can be triggered by starvation, oxidative stress, or pharmacological agents, like rapamycin. This induced autophagy can promote cell survival during starvation or pathological stress. In this study, it is investigated the effect of the induced autophagic process on angiotensin induced hypertrophic H9c2 cells. In our study, it is used H9c2 cells as an in vitro model. To induce hypertrophy, cells were treated with 10000 nM angiotensin-II, and to activate autophagy, 100 nM rapamycin treatment was used. The following groups were formed: 1: control, 2: 10000 nM AT-II, 3: 100 nM rapamycin, 4: 100 nM rapamycin pretreatment then 10000 nM AT-II. The cell viability was examined via MTT (cell proliferation assay) assay. The cells were stained with rhodamine-conjugated phalloidin and DAPI to visualize F-actin filaments and cell nuclei then the cell size alteration was examined in a fluorescence microscope. Furthermore, the expression levels of autophagic and apoptotic proteins such as Beclin-1, p62, LC3B-II, Cleaved Caspase-3 were evaluated by Western blot. MTT assay result suggests that the used pharmaceutical agents in the tested concentrations did not have a toxic effect; however, at group 3, a slight decrement was detected in cell viability. In response to AT-II treatment, a significant increase was detected in the cell size; cells became hypertrophic. However, rapamycin pretreatment slightly reduced the cell size compared to group 2. Western blot results showed that AT-II treatment-induced autophagy, because the increased expression of Beclin-1, p62, LC3B-II were observed. However, due to the incomplete autophagy, the apoptotic Cleaved Caspase-3 expression also increased. Rapamycin pretreatment up-regulated Beclin-1 and LC3B-II, down-regulated p62 and Cleaved Caspase-3, indicating that rapamycin-induced autophagy can restore the normal autophagic flux. Taken together, our results suggest that rapamycin activated autophagy reduces angiotensin-II induced hypertrophy.

Keywords: angiotensin-II, autophagy, H9c2 cell line, hypertrophy, rapamycin

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Authors: Ane Escobar, Paula Angelomé, Mihaela Delcea, Marek Grzelczak, Sergio Enrique Moya

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The antibacterial capacity of bone-anchoring implants can be improved by the use of antibiotics that can be delivered to the media after the surgery. Mesoporous films have shown great potential in drug delivery for orthopedic applications, since pore size and thickness can be tuned to produce different surface area and free volume inside the material. This work shows the synthesis of mesoporous titania films (MTF) by sol-gel chemistry and evaporation-induced self-assembly (EISA) on top of glass substrates. Pores with a diameter of 12nm were observed by Transmission Electron Microscopy (TEM). A film thickness of 100 nm was measured by Scanning Electron Microscopy (SEM). Gentamicin was used to study the antibiotic delivery from the film by means of High-performance liquid chromatography (HPLC). The Staphilococcus aureus strand was used to evaluate the effectiveness of the penicillin loaded films toward inhibiting bacterial colonization. MC3T3-E1 pre-osteoblast cell proliferation experiments proved that MTFs have a good biocompatibility and are a suitable surface for MC3T3-E1 cell proliferation. Moreover, images taken by Confocal Fluorescence Microscopy using labeled vinculin, showed good adhesion of the MC3T3-E1 cells to the MTFs, as well as complex actin filaments arrangement. In order to improve cell proliferation Bone Morphogenetic Protein-2 (BMP-2) was adsorbed on top of the mesoporous film. The deposition of the protein was proved by measurements in the contact angle, showing an increment in the hydrophobicity while the protein concentration is higher. By measuring the dehydrogenase activity in MC3T3-E1 cells cultured in dually functionalized mesoporous titatina films with gentamicin and BMP-2 is possible to find an improvement in cell proliferation. For this purpose, the absorption of a yellow-color formazan dye, product of a water-soluble salt (WST-8) reduction by the dehydrogenases, is measured. In summary, this study proves that by means of the surface modification of MTFs with proteins and loading of gentamicin is possible to achieve an antibacterial effect and a cell growth improvement.

Keywords: antibacterial, biocompatibility, bone morphogenetic protein-2, cell proliferation, gentamicin, implants, mesoporous titania films, osteoblasts

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Authors: Alan Luo, Hunter N. B. Moseley

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Many macromolecular structure entries in the Protein Data Bank (PDB) have a range of regional (localized) quality issues, be it derived from x-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, or other experimental approaches. However, most PDB entries are judged by global quality metrics like R-factor, R-free, and resolution for x-ray crystallography or backbone phi-psi distribution statistics and average restraint violations for NMR. Regional quality is often ignored when PDB entries are re-used for a variety of structurally based analyses. The binding of ligands, especially ligands involved in energy metabolism, is of particular interest in many structurally focused protein studies. Using a regional quality metric that provides chemically interpretable information from electron density maps, a significant number of outliers in regional structural quality was detected across x-ray crystallographic PDB entries for proteins bound to biochemically critical ligands. In this study, a series of analyses was performed to evaluate both specific and general potential factors that could promote these outliers. In particular, these potential factors were the minimum distance to a metal ion, the minimum distance to a crystal contact, and the isotropic atomic b-factor. To evaluate these potential factors, Fisher’s exact tests were performed, using regional quality criteria of outlier (top 1%, 2.5%, 5%, or 10%) versus non-outlier compared to a potential factor metric above versus below a certain outlier cutoff. The results revealed a consistent general effect from region-specific normalized b-factors but no specific effect from metal ion contact distances and only a very weak effect from crystal contact distance as compared to the b-factor results. These findings indicate that no single specific potential factor explains a majority of the outlier ligand-bound regions, implying that human error is likely as important as these other factors. Thus, all factors, including human error, should be considered when regions of low structural quality are detected. Also, the downstream re-use of protein structures for studying ligand-bound conformations should screen the regional quality of the binding sites. Doing so prevents misinterpretation due to the presence of structural uncertainty or flaws in regions of interest.

Keywords: biomacromolecular structure, coenzyme, electron density discrepancy analysis, x-ray crystallography

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Authors: Shilpi, Indu Gaur, Neha Bhadauria, Susmita Goswami, Prabir K. Paul

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Cultivation and consumption of organically grown fruits and vegetables have increased by several folds. However, the presence of Human Enteric Pathogens on the surface of organically grown vegetables causing Gastro-intestinal diseases, are most likely due to contaminated water and fecal matter of farm animals. Human Enteric Pathogens are adapted to colonize the human gut, and also colonize plant surface. Microbes on plant surface communicate with each other to establish quorum sensing. The cross talk study is important because the enteric pathogens on phylloplane have been reported to mask the beneficial resident bacteria of plant. In the present study, HEPs and bacterial colonizers were identified using 16s rRNA sequencing. Microbial colonization patterns after interaction between Human Enteric Pathogens and natural bacterial residents on tomato phylloplane was studied. Tomato plants raised under aseptic conditions were inoculated with a mixture of Serratia fonticola and Klebsiella pneumoniae. The molecules involved in cross-talk between Human Enteric Pathogens and regular bacterial colonizers were isolated and identified using molecular techniques and HPLC. The colonization pattern was studied by leaf imprint method after 48 hours of incubation. The associated protein-protein interaction in the host cytoplasm was studied by use of crosslinkers. From treated leaves the crosstalk molecules and interaction proteins were separated on 1D SDS-PAGE and analyzed by MALDI-TOF-TOF analysis. The study is critical in understanding the molecular aspects of HEP’s adaption to phylloplane. The study revealed human enteric pathogens aggressively interact among themselves and resident bacteria. HEPs induced establishment of a signaling cascade through protein-protein interaction in the host cytoplasm. The study revealed that the adaptation of Human Enteric Pathogens on phylloplane of Solanum lycopersicum involves the establishment of complex molecular interaction between the microbe and the host including microbe-microbe interaction leading to an establishment of quorum sensing. The outcome will help in minimizing the HEP load on fresh farm produce, thereby curtailing incidences of food-borne diseases.

Keywords: crosslinkers, human enteric pathogens (HEPs), phylloplane, quorum sensing

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Authors: Anna Tokajuk, Agnieszka Zakrzeska, Ewa Chabielska, Halina Car

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Whey is a by-product generated mainly in the production of cheese and casein. Powder forms of whey are used widely in the food industry as well as a high-protein food for infants, for convalescents, by athletes and especially by bodybuilders to increase muscle mass during exercise. Whey protein concentrate-80 (WPC-80) is a source of bioactive peptides with beneficial effects on the cardiovascular system. It is known that whey proteins health beneficial properties include antidiabetic, blood pressure lowering, improving cardiovascular system function, antibacterial, antiviral and other effects. To study its influence on the development of thrombosis, venous thrombosis model was performed according to the protocol featured by Reyers with modification by Chabielska and Gromotowicz. Male Wistar-Crl: WI (Han) rats from researched groups were supplemented with two doses of WPC-80 (0.3 or 0.5 g/kg) for 7, 14 or 21 days and after these periods, one-hour venous thrombosis model was performed. Control group received 0.9 % NaCl solution and was sham operated. The statistical significance of results was computed by Mann – Whitney’s test. We observed that thrombus weight was decreased in animals obtaining WPC-8080 and that was statistically significant in 14 and 21-day supplemented groups. Blood count parameters did not differ significantly in rats with and without thrombosis induction whether they were fed with WPC-80 or not. Moreover, the number of platelets (PLT) was within the normal range in each group. The examined coagulation parameters in rats of the control groups were within normal limits. After WPC-80 supplementation there was the tendency to prolonged activated partial thromboplastin time (aPTT), but in comparison, the results did not turn out significant. In animals that received WPC-80 0.3 g·kg-1 for 21 days with and without induced thrombosis, prothrombin time (PT) and an international normalized ratio (INR) was somewhat decreased, remaining within the normal range, but the nature and significance of this observation are beyond the framework of the current study. Additionally, fibrinogen and thrombin time (TT) did not differ significantly between groups. Therefore the exact effect of WPC-80 on coagulation system is still elusive and requires further thorough research including mechanisms of action. Determining the potential clinical application of WPC-80 requires the selection of the optimal dose and duration of supplementation.

Keywords: antithrombotic, rats, venous thrombosis, WPC-80

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Authors: Varsha Salian, Shama Rao, N. Narendra, B. Mohana Kumar

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Evolving evidence proposes the existence of a highly tumorigenic subpopulation of undifferentiated, self-renewing cancer stem cells, responsible for exhibiting resistance to conventional anti-cancer therapy, recurrence, metastasis and heterogeneous tumor formation. Importantly, the mechanisms exploited by cancer stem cells to resist chemotherapy are very less understood. Oral squamous cell carcinoma (OSCC) is one of the most regularly diagnosed cancer types in India and is associated commonly with alcohol and tobacco use. Therefore, the isolation and in vitro characterization of cancer stem-like cells from patients with OSCC is a critical step to advance the understanding of the chemoresistance processes and for designing therapeutic strategies. With this, the present study aimed to establish and characterize cancer stem-like cells in vitro from OSCC. The primary cultures of cancer stem-like cell lines were established from the tissue biopsies of patients with clinical evidence of an ulceroproliferative lesion and histopathological confirmation of OSCC. The viability of cells assessed by trypan blue exclusion assay showed more than 95% at passage 1 (P1), P2 and P3. Replication rate was performed by plating cells in 12-well plate and counting them at various time points of culture. Cells had a more marked proliferative activity and the average doubling time was less than 20 hrs. After being cultured for 10 to 14 days, cancer stem-like cells gradually aggregated and formed sphere-like bodies. More spheroid bodies were observed when cultured in DMEM/F-12 under low serum conditions. Interestingly, cells with higher proliferative activity had a tendency to form more sphere-like bodies. Expression of specific markers, including membrane proteins or cell enzymes, such as CD24, CD29, CD44, CD133, and aldehyde dehydrogenase 1 (ALDH1) is being explored for further characterization of cancer stem-like cells. To summarize the findings, the establishment of OSCC derived cancer stem-like cells may provide scope for better understanding the cause for recurrence and metastasis in oral epithelial malignancies. Particularly, identification and characterization studies on cancer stem-like cells in Indian population seem to be lacking thus provoking the need for such studies in a population where alcohol consumption and tobacco chewing are major risk habits.

Keywords: cancer stem-like cells, characterization, in vitro, oral squamous cell carcinoma

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae bacteria is a global threat to human health due to an increase in multi-drug resistance among strains. The hypervirulent strains of Klebsiella pneumoniae is a major trouble due to their association with life-threatening infections in a healthy population. One of the major virulence factors of hyper virulent strains of Klebsiella pneumoniae is the T6SS (Type six secretary system) which is majorly involved in microbial antagonism and causes interaction with the host eukaryotic cells during infections. T6SS mediates some of the crucial factors for establishing infection by the bacteria, such as cell adherence, invasion, and subsequent in vivo colonisation. The antibacterial activity and the cell invasion property of the T6SS system is a major requirement for the establishment of K. pneumoniae infections within the gut. The T6SS can be an appropriate target for developing therapeutics. The T6SS consists of an inner tube comprising hexamers of Hcp (Haemolysin -regulated protein) protein, and at the top of this tube sits VgrG (Valine glycine repeat protein G); the tip of the machinery consists of PAAR domain containing proteins which act as a delivery system for bacterial effectors. For this study, immune response to recombinant VgrG protein was generated to establish this protein as a potential immunogen for the development of therapeutic leads. The immunogenicity of the selected protein was determined by predicting the B cell epitopes by the BCEP analysis tool. The gene sequence for multiple domains of VgrG protein (phage_base_V, T6SS_Vgr, DUF2345) was selected and cloned in pMAL vector in E. coli. The construct was subcloned and expressed as a fusion protein of 203 residue protein with mannose binding protein tag (MBP) to enhance solubility and purification of this protein. The purified recombinant VgrG fusion protein was used for mice immunisation. The antiserum showed reactivity with the recombinant VgrG in ELISA and western blot. The immunised mice were challenged with K. pneumoniae bacteria and showed bacterial clearance in immunised mice. The recombinant VgrG protein can further be used for studying downstream signalling of VgrG protein in mice during infection and for therapeutic MAb development to eradicate K. pneumoniae infections.

Keywords: immune response, Klebsiella pneumoniae, multi-drug resistance, recombinant protein expression, T6SS, VgrG

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Authors: M. Y. Maila, H. P. Makhubele

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Roasting is one of the oldest preservation method used in foods such as nuts and seeds. It is a process by which heat is applied to dry foodstuffs without the use of oil or water as a carrier. Groundnut seeds, also known as peanuts when sun dried or roasted, are among the oldest oil crops that are mostly consumed as a snack, after roasting in many parts of South Africa. However, roasting can denature proteins, destroy amino acids, decrease nutritive value and induce undesirable chemical changes in the final product. The aim of this study, therefore, was to evaluate the effect of various roasting times on the food value of the runner groundnut seeds. A constant temperature of 160 °C and various time-intervals (20, 30, 40, 50 and 60 min) were used for roasting groundnut seeds in an oven. Roasted groundnut seeds were then cooled and milled to flour. The milled sundried, raw groundnuts served as reference. The proximate analysis (moisture, energy and crude fats) was performed and the results were determined using standard methods. The antioxidant content was determined using HPLC. Mineral (cobalt, chromium, silicon and iron) contents were determined by first digesting the ash of sundried and roasted seed samples in 3M Hydrochloric acid and then determined by Atomic Absorption Spectrometry. All results were subjected to ANOVA through SAS software. Relative to the reference, roasting time significantly (p ≤ 0.05) reduced moisture (71%–88%), energy (74%) and crude fat (5%–64%) of the runner groundnut seeds, whereas the antioxidant content was significantly (p ≤ 0.05) increased (35%–72%) with increasing roasting time. Similarly, the tested mineral contents of the roasted runner groundnut seeds were also significantly (p ≤ 0.05) reduced at all roasting times: cobalt (21%–83%), chromium (48%–106%) and silicon (58%–77%). However, the iron content was significantly (p ≤ 0.05) unaffected. Generally, the tested runner groundnut seeds had higher food value in the raw state than in the roasted state, except for the antioxidant content. Moisture is a critical factor affecting the shelf life, texture and flavor of the final product. Loss of moisture ensures prolonged shelf life, which contribute to the stability of the roasted peanuts. Also, increased antioxidant content in roasted groundnuts is essential in other health-promoting compounds. In conclusion, the overall reduction in the proximate and mineral contents of the runner groundnuts seeds due to roasting is sufficient to suggest influences of roasting time on the food value of the final product and shelf life.

Keywords: dry roasting, legume, oil source, peanuts

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Authors: Mark Gavriel, Ariel J. Jaffa, Dan Grisaru, David Elad

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The human endometrium-myometrium interface (EMI) is the uterine inner barrier without a separatig layer. It is composed of endometrial epithelial cells (EEC) and endometrial stromal cells (ESC) in the endometrium and myometrial smooth muscle cells (MSMC) in the myometrium. The EMI undergoes structural remodeling during the menstruation cycle which are essential for human reproduction. Recently, we co-cultured a layer-by-layer in vitro model of EEC, ESC and MSMC on a synthetic membrane for mechanobiology experiments. We also treated the model with progesterone and β-estradiol in order to mimic the in vivo receptive uterus In the present study we analyzed the cytokines profile in a single layer of EEC the hormonal treated in vitro model of the EMI. The methodologies of this research include simple tissue-engineering . First, we cultured commercial EEC (RL95-2, ATCC® CRL-1671™) in 24-wellplate. Then, we applied an hormonal stimuli protocol with 17-β-estradiol and progesterone in time dependent concentration according to the human physiology that mimics the menstrual cycle. We collected cell supernatant samples of control, pre-ovulation, ovulation and post-ovulaton periods for analysis of the secreted proteins and cytokines. The cytokine profiling was performed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Inc., USA) that can detect105 human soluble cytokines. The relative quantification of all the cytokines will be analyzed using xMAP – LUMINEX. We conducted a fishing expedition with the 4 membranes Proteome Profiler. We processed the images, quantified the spots intensity and normalized these values by the negative control and reference spots at the membrane. Analyses of the relative quantities that reflected change higher than 5% of the control points of the kit revealed the The results clearly showed that there are significant changes in the cytokine level for inflammation and angiogenesis pathways. Analysis of tissue-engineered models of the uterine wall will enable deeper investigation of molecular and biomechanical aspects of early reproductive stages (e.g. the window of implantation) or developments of pathologies.

Keywords: tissue-engineering, hormonal stimuli, reproduction, multi-layer uterine model, progesterone, β-estradiol, receptive uterine model, fertility

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Authors: Surabhi Gautam, Uma Kumar, Rima Dada

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A sustained acute-phase response in Rheumatoid Arthritis (RA) is associated with increased joint damage and inflammation leading to progressive disability. It is induced continuously by consecutive stimuli of proinflammatory cytokines, following a wide range of pathophysiological reactions, leading to increased synthesis of acute phase proteins like C - reactive protein (CRP) and dysregulation in levels of immunomodulatory soluble Human Leukocyte Antigen-G (HLA-G) molecule. This study was designed to explore the effect of yoga and meditation based lifestyle intervention (YMLI) on inflammatory markers in RA patients. Blood samples of 50 patients were collected at baseline (day 0) and after 30 days of YMLI. Patients underwent a pretested YMLI under the supervision of a certified yoga instructor for 30 days including different Asanas (physical postures), Pranayama (breathing exercises), and Dhayna (meditation). Levels of CRP, IL-6, IL-17A, soluble HLA-G and erythrocyte sedimentation rate (ESR) were measured at day 0 and 30 interval. Parameters of disease activity, disability quotient, pain acuity and quality of life were also assessed by disease activity score (DAS28), health assessment questionnaire (HAQ), visual analogue scale (VAS), and World Health Organization Quality of Life (WHOQOL-BREF) respectively. There was reduction in mean levels of CRP (p < 0.05), IL-6 (interleukin-6) (p < 0.05), IL-17A (interleukin-17A) (p < 0.05) and ESR (p < 0.05) and elevation in soluble HLA-G (p < 0.05) at 30 days compared to baseline level (day 0). There was reduction seen in DAS28-ESR (p < 0.05), VAS (p < 0.05) and HAQ (p < 0.05) after 30 days with respect to the base line levels (day 0) and significant increase in WHOQOL-BREF scale (p < 0.05) in all 4 domains of physical health, psychological health, social relationships, and environmental health. The present study has demonstrated that yoga practices are associated with regression of inflammatory processes by reducing inflammatory parameters and regulating the levels of soluble HLA-G significantly in active RA patients. Short term YMLI has significantly improved pain perception, disability quotient, disease activity and quality of life. Thus this simple life style intervention can reduce disease severity and dose of drugs used in the treatment of RA.

Keywords: inflammation, quality of life, rheumatoid arthritis, yoga and meditation

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Authors: Himani Sharma, Amit Agrawal

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Stability of the Cassie and Wenzel wetting states depends on intrinsic contact angle and geometric features on a surface that was exploited in capturing biofluids in microwells. However, the mechanism of imbibition of biofluids in the microwells is not well implied in terms of wettability of a substrate. In this work, we experimentally demonstrated filling dynamics in hydrophilic and hydrophobic microwells by protein solutions. Towards this, we utilized lotus leaf as a mold to fabricate microwells on a Polydimethylsiloxane (PDMS) surface. Lotus leaf containing micrometer-sized blunt-conical shaped pillars with a height of 8-15 µm and diameter of 3-8 µm were transferred on to PDMS. Furthermore, PDMS surface was treated with oxygen plasma to render the hydrophilic nature. A 10µL droplets containing fluorescein isothiocyanate (FITC) - labelled bovine serum albumin (BSA) were rested on both hydrophobic (θa = 108o, where θa is the apparent contact angle) and hydrophilic (θa = 60o) PDMS surfaces. A time-dependent fluorescence microscopy was conducted on these modified PDMS surfaces by recording the fluorescent intensity over a 5 minute period. It was observed that, initially (at t=1 min) FITC-BSA was accumulated on the periphery of both hydrophilic and hydrophobic microwells due to incomplete penetration of liquid-gas meniscus. This deposition of FITC-BSA on periphery of microwell was not changed with time for hydrophobic surfaces, whereas, a complete filling was occurred in hydrophilic microwells (at t=5 mins). This attributes to a gradual movement of three-phase contact line along the vertical surface of the hydrophilic microwells as compared to stable pinning in the hydrophobic microwells as confirmed by Surface Evolver simulations. In addition, if the cavities are presented on hydrophobic surfaces, air bubbles will be trapped inside the cavities once the aqueous solution is placed over these surfaces, resulting in the Cassie-Baxter wetting state. This condition hinders trapping of proteins inside the microwells. Thus, it is necessary to impart hydrophilicity to the microwell surfaces so as to induce the Wenzel state, such that, an entire solution will be fully in contact with the walls of microwells. Imbibition of microwells by protein solutions was analyzed in terms fluorescent intensity versus time. The present work underlines the importance of geometry of microwells and surface wettability of substrate in wetting and effective capturing of solid sub-phases in biofluids.

Keywords: BSA, microwells, surface evolver, wettability

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Authors: A. Díaz-Roa, P. I. Silva Junior, F. J. Bello

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Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Dipterous maggots release diverse proteins and peptides contained in larval excretion and secretion (ES) products playing a key role in digestion. The most important mechanism for combating infection using larval therapy depends on larval ES. These larvae are protected against infection by a diverse spectrum of antimicrobial peptides (AMPs), one already known like lucifensin. Special interest in these peptides has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue and kill different bacteria during larval therapy. The action of larvae on wounds occurs through 3 mechanisms of action: removal of necrotic tissue, stimulation of granulation tissue, and antibacterial action of larval ES. Some components of the ES include calcium, urea, allantoin ammonium bicarbonate and reducing the viability of Gram positive and Gram negative bacteria. The Lucilia sericata fly larvae have been the most used, however, we need to evaluate new species that could potentially be similar or more effective than fly above. This study was thus aimed at identifying and characterizing S. magellanica AMPs contained in ES products for the first time and compared them with the common fly used L. sericata. These products were obtained from third-instar larvae taken from a previously established colony. For the first analysis, ES fractions were separate by Sep-Pak C18 disposable columns (first step). The material obtained was fractionated by RP-HPLC by using Júpiter C18 semi-preparative column. The products were then lyophilized and their antimicrobial activity was characterized by incubation with different bacterial strains. The first chromatographic analysis of ES from L. sericata gives 6 fractions with antimicrobial activity against Gram-positive bacteria Micrococus luteus, and 3 fractions with activity against Gram-negative bacteria Pseudomonae aeruginosa while the one from S. magellanica gaves 1 fraction against M. luteus and 4 against P. aeruginosa. Maybe one of these fractions could correspond to the peptide already known from L. sericata. These results show the first work for supporting further experiments aimed at validating S. magellanica use in larval therapy. We still need to search if we find some new molecules, by making mass spectrometry and ‘de novo sequencing’. Further studies are necessary to identify and characterize them to better understand their functioning.

Keywords: antimicrobial peptides, larval therapy, Lucilia sericata, Sarconesiopsis magellanica

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Authors: Giorgio Bertolazzi, Panayiotis Benos, Michele Tumminello, Claudia Coronnello

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MicroRNAs are small non-coding RNAs that post-transcriptionally regulate the expression levels of messenger RNAs. MicroRNA regulation activity depends on the recognition of binding sites located on mRNA molecules. ComiR (Combinatorial miRNA targeting) is a user friendly web tool realized to predict the targets of a set of microRNAs, starting from their expression profile. ComiR incorporates miRNA expression in a thermodynamic binding model, and it associates each gene with the probability of being a target of a set of miRNAs. ComiR algorithms were trained with the information regarding binding sites in the 3’UTR region, by using a reliable dataset containing the targets of endogenously expressed microRNA in D. melanogaster S2 cells. This dataset was obtained by comparing the results from two different experimental approaches, i.e., inhibition, and immunoprecipitation of the AGO1 protein; this protein is a component of the microRNA induced silencing complex. In this work, we tested whether including coding region binding sites in the ComiR algorithm improves the performance of the tool in predicting microRNA targets. We focused the analysis on the D. melanogaster species and updated the ComiR underlying database with the currently available releases of mRNA and microRNA sequences. As a result, we find that the ComiR algorithm trained with the information related to the coding regions is more efficient in predicting the microRNA targets, with respect to the algorithm trained with 3’utr information. On the other hand, we show that 3’utr based predictions can be seen as complementary to the coding region based predictions, which suggests that both predictions, from 3'UTR and coding regions, should be considered in a comprehensive analysis. Furthermore, we observed that the lists of targets obtained by analyzing data from one experimental approach only, that is, inhibition or immunoprecipitation of AGO1, are not reliable enough to test the performance of our microRNA target prediction algorithm. Further analysis will be conducted to investigate the effectiveness of the tool with data from other species, provided that validated datasets, as obtained from the comparison of RISC proteins inhibition and immunoprecipitation experiments, will be available for the same samples. Finally, we propose to upgrade the existing ComiR web-tool by including the coding region based trained model, available together with the 3’UTR based one.

Keywords: AGO1, coding region, Drosophila melanogaster, microRNA target prediction

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Authors: Mihriban Korukluoglu, Goksen Arik, Cagla Erdogan, Selen Kocakoglu

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Kefir is a traditional fermented refreshing beverage which is known for its valuable and beneficial properties for human health. Mainly yeast species, lactic acid bacteria (LAB) strains and fewer acetic acid bacteria strains live together in a natural matrix named “kefir grain”, which is formed from various proteins and polysaccharides. Different microbial species live together in slimy kefir grain and it has been thought that synergetic effect could take place between microorganisms, which belong to different genera and species. In this research, yeast and LAB were isolated from kefir samples obtained from Uludag University Food Engineering Department. The cell morphology of isolates was screened by microscopic examination. Gram reactions of bacteria isolates were determined by Gram staining method, and as well catalase activity was examined. After observing the microscopic/morphological and physical, enzymatic properties of all isolates, they were divided into the groups as LAB and/or yeast according to their physicochemical responses to the applied examinations. As part of this research, the antagonistic/synergistic efficacy of the identified five LAB and five yeast strains to each other were determined individually by disk diffusion method. The antagonistic or synergistic effect is one of the most important properties in a co-culture system that different microorganisms are living together. The synergistic effect should be promoted, whereas the antagonistic effect is prevented to provide effective culture for fermentation of kefir. The aim of this study was to determine microbial interactions between identified yeast and LAB strains, and whether their effect is antagonistic or synergistic. Thus, if there is a strain which inhibits or retards the growth of other strains found in Kefir microflora, this circumstance shows the presence of antagonistic effect in the medium. Such negative influence should be prevented, whereas the microorganisms which have synergistic effect on each other should be promoted by combining them in kefir grain. Standardisation is the most desired property for industrial production. Each microorganism found in the microbial flora of a kefir grain should be identified individually. The members of the microbial community found in the glue-like kefir grain may be redesigned as a starter culture regarding efficacy of each microorganism to another in kefir processing. The main aim of this research was to shed light on more effective production of kefir grain and to contribute a standardisation of kefir processing in the food industry.

Keywords: antagonistic effect, kefir, lactic acid bacteria (LAB), synergistic, yeast

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Authors: Qiaoyue Kuang, Yang Li, Mizuki Endo, Takeaki Ozawa

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G protein-coupled receptors (GPCR) are the largest family of receptor proteins that detect molecules outside the cell and activate cellular responses. Of the GPCRs, dopamine receptors, which recognize extracellular dopamine, are essential to mammals due to their roles in numerous physiological events, including autonomic movement, hormonal regulation, emotions, and the reward system in the brain. To precisely understand the physiological roles of dopamine receptors, it is important to spatiotemporally control the signaling mediated by dopamine receptors, which is strongly dependent on their surface expression. Conventionally, chemical-induced interactions were applied to trigger the endocytosis of cell surface receptors. However, these methods were subjected to diffusion and therefore lacked temporal and special precision. To further understand the receptor-mediated signaling and to control the plasma membrane expression of receptors, an optogenetic tool called E-fragment was developed. The C-terminus of a light-sensitive photosensory protein cyptochrome2 (CRY2) was attached to β-Arrestin, and the E-fragment was generated by fusing the C-terminal peptide of vasopressin receptor (V2R) to CRY2’s binding partner protein CIB. The CRY2-CIB heterodimerization triggered by blue light stimulation brings β-Arrestin to the vicinity of membrane receptors and results in receptor endocytosis. In this study, the E-fragment system was applied to dopamine receptors 1 and 2 (DRD1 and DRD2) to control dopamine signaling. First, confocal fluorescence microscope observation qualitatively confirmed the light-induced endocytosis of E-fragment fused receptors. Second, NanoBiT bioluminescence assay verified quantitatively that the surface amount of E-fragment labeled receptors decreased after light treatment. Finally, GloSensor bioluminescence assay results suggested that the E-fragment-dependent receptor light-induced endocytosis decreased cAMP production in DRD1 signaling and attenuated the inhibition effect of DRD2 on cAMP production. The developed optogenetic tool was able to induce receptor endocytosis by external light, providing opportunities to further understand numerous physiological activities by controlling receptor-mediated signaling spatiotemporally.

Keywords: dopamine receptors, endocytosis, G protein-coupled receptors, optogenetics

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Authors: Kevin Kellner, Nga Lao, Orla Coleman, Paula Meleady, Niall Barron

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Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. To improve yields and find beneficial bioprocess phenotypes genetic engineering plays an essential role in recent research. The miR-23 cluster, specifically miR-24 and miR-27, was first identified as differentially expressed during hypothermic conditions suggesting a role in proliferation and productivity in CHO cells. In this study, we used sponge decoy technology to stably deplete the miRNA expression of the cluster. Furthermore, we implemented the CRISPR/Cas9 system to knockdown miRNA expression. Sponge constructs were designed for an imperfect binding of the miRNA target, protecting from RISC mediated cleavage. GuideRNAs for the CRISPR/Cas9 system were designed to target the seed region of the miRNA. The expression of mature miRNA and precursor were confirmed using RT-qPCR. For both approaches stable expressing mixed populations were generated and characterised in batch cultures. It was shown, that CRISPR/Cas9 can be implemented in CHO cells with achieving high knockdown efficacy of every single member of the cluster. Targeting of one miRNA member showed that its genomic paralog is successfully targeted as well. The stable depletion of miR-24 using CRISPR/Cas9 showed increased growth and specific productivity in a CHO-K1 mAb expressing cell line. This phenotype was further characterized using quantitative label-free LC-MS/MS showing 186 proteins differently expressed with 19 involved in proliferation and 26 involved in protein folding/translation. Targeting miR-27 in the same cell line showed increased viability in late stages of the culture compared to the control. To evaluate the phenotype in an industry relevant cell line; the miR-23 cluster, miR-24 and miR-27 were stably depleted in a Fc fusion CHO-S cell line which showed increased batch titers up to 1.5-fold. In this work, we highlighted that the stable depletion of the miR-23 cluster and its members can improve the bioprocess phenotype concerning growth and productivity in two different cell lines. Furthermore, we showed that using CRISPR/Cas9 is comparable to the traditional sponge decoy technology.

Keywords: Chinese Hamster ovary cells, CRISPR/Cas9, microRNAs, sponge decoy technology

Procedia PDF Downloads 195

Authors: Daniela Istrati, Camelia Vizireanu, Camelia Grozavu, Rodica Mihaela Dinica

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In last years, eating habits have changed, and snacking has become more common. Snacking habits, including eating whole fruit, vegetables and crackers, were found to contribute to better overall diet quality, while consuming snacks such as cookies, pastries, sweets, milk desserts and soft drinks was associated with poorer diet quality. The nutritional quality of the snack is very important and choosing nutritious foods as snacks can be beneficial for our health. For this reason, the development of functional snacks bars represents a necessity for this niche market. The aim of this work was to develop some formulations of energizing snack bars with high dietary fibers and antioxidant activity. Snack bars contain both fruits with antioxidant activity and components (cereals and seeds) rich in carbohydrates and polyunsaturated fats that provide energy during sports activities, physical and mental stress. Three types of samples were prepared and stored in refrigerated conditions at 40°C for 30 days. The first sample (S1) contains wheat germs, raw pumpkin seeds, toasted oat flakes, flaxseeds flour, cinnamon honey, raw sunflower seeds, sea buckthorn, amaranth flour, cinnamon and olive oil. The second sample (S2) has the same composition as the first, less flour and cinnamon flour and the honey used was ginger, honey. The third sample (S3) is like the first less amaranth flour and the honey used was buckthorn sea honey. The physicochemical, antioxidant activity, polyphenolic and flavonoid content and sensorial characteristics of the samples were investigated. Results showed that snacks bars had important level of extracted phenolics, flavonoids, fibers, proteins, carbohydrates and fats. Therefore, snack bars may be a convenient functional food, offering an important source of flavonoids and polyphenols, a healthy alternative to a poor diet quality, with balanced nutritional and sensory characteristics that recommend it in the diet of all consumers concerned with maintaining health. Acknowledgment: This study has been founded by the Francophone University Agency, Project Réseau régional dans le domaine de la santé, la nutrition et la sécurité alimentaire (SaIN), No. at Dunarea de Jos University of Galati 21899/ 06.09.2017

Keywords: antioxidant activity, functional food, sea buckthorn, snack bars

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Authors: Javed Mohammed

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Right now, bio-medical infrastructure lags well behind the curve. Our healthcare system is dispersed and disjointed; medical records are a bit of a mess; and we do not yet have the capacity to store and process the crazy amounts of data coming our way from widespread whole-genome sequencing. And then there are privacy issues. Despite these infrastructure challenges, some researchers are plunging into bio medical Big Data now, in hopes of extracting new and actionable knowledge. They are doing delving into molecular-level data to discover bio markers that help classify patients based on their response to existing treatments; and pushing their results out to physicians in novel and creative ways. Computer scientists and bio medical researchers are able to transform data into models and simulations that will enable scientists for the first time to gain a profound under-standing of the deepest biological functions. Solving biological problems may require High-Performance Computing HPC due either to the massive parallel computation required to solve a particular problem or to algorithmic complexity that may range from difficult to intractable. Many problems involve seemingly well-behaved polynomial time algorithms (such as all-to-all comparisons) but have massive computational requirements due to the large data sets that must be analyzed. High-throughput techniques for DNA sequencing and analysis of gene expression have led to exponential growth in the amount of publicly available genomic data. With the increased availability of genomic data traditional database approaches are no longer sufficient for rapidly performing life science queries involving the fusion of data types. Computing systems are now so powerful it is possible for researchers to consider modeling the folding of a protein or even the simulation of an entire human body. This research paper emphasizes the computational biology's growing need for high-performance computing and Big Data. It illustrates this article’s indispensability in meeting the scientific and engineering challenges of the twenty-first century, and how Protein Folding (the structure and function of proteins) and Phylogeny Reconstruction (evolutionary history of a group of genes) can use HPC that provides sufficient capability for evaluating or solving more limited but meaningful instances. This article also indicates solutions to optimization problems, and benefits Big Data and Computational Biology. The article illustrates the Current State-of-the-Art and Future-Generation Biology of HPC Computing with Big Data.

Keywords: high performance, big data, parallel computation, molecular data, computational biology

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