Search results for: whey protein isolated

Authors: Anna Cameron, Chunxia Zhao, Haofei Wang, Yun Liu, Guang Ze Yang

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Since the first publication in 1775, cancer research has built a comprehensive understanding of how cellular components of the tumor niche promote disease development. However, only within the last decade has research begun to establish the impact of non-cellular components of the niche, particularly the extracellular matrix (ECM). The ECM, a three-dimensional scaffold that sustains the tumor microenvironment, plays a crucial role in disease progression. Cancer cells actively deregulate and remodel the ECM to establish a tumor-promoting environment. Recent work has highlighted the need to further our understanding of the complexity of this cancer-ECM relationship. In vitro models use hydrogels to mimic the ECM, as hydrogel matrices offer biological compatibility and stability needed for long term cell culture. However, natural hydrogels are being used in these models verbatim, without tuning their biophysical characteristics to achieve pathophysiological relevance, thus limiting their broad use within cancer research. The biophysical attributes of these gels dictate cancer cell proliferation, invasion, metastasis, and therapeutic response. Evaluating the three most widely used natural hydrogels, Matrigel, collagen, and agarose gel, the permeability, stiffness, and pore-size of each gel were measured and compared to the in vivo environment. The pore size of all three gels fell between 0.5-6 µm, which coincides with the 0.1-5 µm in vivo pore size found in the literature. However, the stiffness for hydrogels able to support cell culture ranged between 0.05 and 0.3 kPa, which falls outside the range of 0.3-20,000 kPa reported in the literature for an in vivo ECM. Permeability was ~100x greater than in vivo measurements, due in large part to the lack of cellular components which impede permeation. Though, these measurements prove important when assessing therapeutic particle delivery, as the ECM permeability decreased with increasing particle size, with 100 nm particles exhibiting a fifth of the permeability of 10 nm particles. This work explores ways of adjusting the biophysical characteristics of hydrogels by changing protein concentration and the trade-off, which occurs due to the interdependence of these factors. The global aim of this work is to produce a more pathophysiologically relevant model for each tumor type.

Keywords: cancer, extracellular matrix, hydrogel, microfluidic

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Authors: Lukas J. Evans, Danilo D. Fernando

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Shrub willows (Salix spp.) have many characteristics which make them suitable for a variety of applications such as riparian zone buffers, environmental contaminant sequestration, living snow fences, and biofuel production. In some cases, these functions are limited due to physical or financial obstacles associated with the number of individuals needed to reasonably satisfy that purpose. One way to increase the efficiency of willows is to bioengineer them with the genetic improvements suitable for the desired use. To accomplish this goal, an optimized in vitro transformation protocol via Agrobacterium tumefaciens is necessary to reliably express genes of interest. Therefore, the aim of this study is to observe the influence of tissue culture with different willow cultivars, hormones, and explants on the percentage of calli expressing reporter gene green florescent protein (GFP) to find ideal transformation conditions. Each callus was produced from 1 month old open-pollinated seedlings of three Salix miyabeana cultivars (‘SX61’, ‘WT1’, and ‘WT2’) from three different explants (lamina, petiole, and internodes). Explants were cultured for 1 month on an MS media with different concentrations of 6-Benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA) (No hormones, 1 mg⁻¹L BAP only, 3 mg⁻¹L NAA only, 1 mg⁻¹L BAP and 3 mg⁻¹L NAA, and 3 mg⁻¹L BAP and 1 mg⁻¹L NAA) to produce a callus. Samples were then treated with Agrobacterium tumefaciens at an OD600 of 0.6-0.8 to insert the transgene GFP for 30 minutes, co-cultivated for 72 hours, and selected on the same media type they were cultured on with added 7.5 mg⁻¹L of Hygromycin for 1 week before GFP visualization under a UV dissecting scope. Percentage of GFP expressing calli as well as the average number of fluorescing GFP units per callus were recorded and results were evaluated through an ANOVA test (α = 0.05). The WT1 internode-derived calli on media with 3 mg-1L NAA+1 mg⁻¹L BAP and mg⁻¹L BAP alone produced a significantly higher percentage of GFP expressing calli than each other group (19.1% and 19.4%, respectively). Additionally, The WT1 internode group cultured with 3 mg⁻¹L NAA+1 mg⁻¹L BAP produced an average of 2.89 GFP units per callus while the group cultivated with 1 mg⁻¹L BAP produced an average of 0.84 GFP units per callus. In conclusion, genotype, explant choice, and hormones all play a significant role in increasing successful transformation in willows. Future studies to produce whole callus GFP expression and subsequent plantlet regeneration are necessary for a complete willow transformation protocol.

Keywords: agrobacterium, callus, Salix, tissue culture

Procedia PDF Downloads 103

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: Obuzor Eze Obuzor, Ekpoke Okurube Sliver

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The study was carried out at Debee farms at Ahoada West Local Government area, Rivers State, Nigeria. To evaluate the impact of two different cost-effective available feed composition on growth performance of weaned piglets. Thirty weaned uncontrolled cross bred (Large white x pietrain) piglets of average initial weight of 3.04 Kg weaned at 30days were assigned to three dietary treatments, comprising three replicates of 10 weaned piglets each, piglets were kept at 7 °C in different pens with dimensions of 4.50 × 4.50 m. The design of the experiment was completely randomized design, data from the study were subjected to one-way analysis of variance (ANOVA) and significant means were separated using Duncan's Multiple Range Test using Statistical Analysis System (SAS) software for windows (2 0 0 3), statistical significance was assessed at P < 0.05 (95% confidence interval) while survival rate was calculated using simple percentage. A standard diet was prepared to meet the nutrient requirements of weaned piglets at (20.8% crude protein). The three diets were fed to the animals in concrete feeding trough, control diet (C) had soybean meal while first treatment had spent grain (T1) and the second treatment had wheat offal (T2) respectively. The experiment was partitioned into four weeks periods (days 1-7, 8-14, 15-21 and 22-28). Feed and water were given unrestrictedly throughout the period of the experiment. The feed intake and weights of the pigs were recorded on weekly basis. Feed conversion ratio and daily weight gain were calculated and the study lasted for four weeks. There was no significant (P>0.05) effect of diet on survival rate, final body weight, average daily weight gain, daily feed intake and feed conversion ratio. The overall performance showed that treatment one (T1) had survival rate (93%), improved daily weight gain (36.21 g), average daily feed intake (120.14 g) and had the best feed conversion ratio (0.29) similar high mean value with the control while treatment two (T2) had lowest and negative response to all parameters. It could be concluded that feed formulated with spent grain is cheaper than control (soybean meal) and also improved the growth performance of weaned piglets.

Keywords: piglets, weaning, feed conversions ratio, daily weight gain

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Authors: Nilgün Öztürk, Hakan Sabahtin Ali, Hülya Tuba Kıyan

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The genus Chenopodium belongs to Amaranthaceae, is represented by approximately 250 species in the world and 15 species and three subspecies in Turkey. Chenopodium species are traditionally used to treat chest and abdominal pain, shortness of breath, cough and neurological disorders. Chenopodium quinoa Willd. (Quinoa) is native to Andes region of South America (especially Peru and Bolivia) and cultivated in many countries include also Turkey in the world nowadays. The seeds of quinoa are rich in protein, and the phytochemical composition consists of antioxidant substances such as polyphenolic compounds, flavonoids, vitamins, and minerals; anticancer and neuroprotective compounds such as tocotrienols; anti-inflammatory compounds such as carotenoids and anthocyanins and also saponins and starch. Food products of quinoa such as quinoa cereal bar, pasta and cornflakes are used in the diet made during many disorders like obesity, cardiovascular disorder, hypertension and Celiac disease. Also quinoa seems to have antimicrobial, anti-inflammatory and cholesterol-lowering properties because of its bioactive compounds. In this present study, the aqueous ethanolic extracts of the seeds of three different coloured genotypes of quinoa were investigated for their antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelating effect, ferric-reducing antioxidant power, ABTS radical cation decolorization assays and total phenolic contents using Folin-Ciocalteu assay. Among the three genotypes of quinoa; the aqueous ethanolic extract of the red genotype had the highest total phenolic content (83.54 ± 2.12 mg gallic acid/100 g extract) whereas the extract of the white genotype had the lowest total phenolic content (70.66 ± 0.25 mg gallic acid/100 g). According to the antioxidant activity results; the extracts showed moderate reducing power effect whereas weak ABTS radical cation decolorization and ferrous ion-chelating effect and also too weak DPPH radical scavenging activity when compared to the positive standards.

Keywords: amaranthaceae, antioxidant activity, Chenopodium quinoa willd., total phenolic content

Procedia PDF Downloads 162

Authors: M. Chukhryaeva, R. Skhalyakho, J. Kagazegeva, E. Pocheshkhova, L. Yepiskopossyan, O. Balanovsky, E. Balanovska

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The Middle East is crossroad of different populations at different times. The Kurds are of particular interest in this region. Historical sources suggested that the origin of the Kurds is associated with Medes. Therefore, it was especially interesting to compare gene pool of Kurds with other supposed descendants of Medes-Tats. Yezidis are ethno confessional group of Kurds. Yezidism as a confessional teaching was formed in the XI-XIII centuries in Iraq. Yezidism has caused reproductively isolation of Yezidis from neighboring populations for centuries. Also, isolation helps to retain Yezidian caste system. It is unknown how the history of Yezidis affected its genу pool because it has never been the object of researching. We have examined the Y-chromosome variation in Yezidis and Kurdish males to understand their gene pool. We collected DNA samples from 90 Yezidi males and 24 Kurdish males together with their pedigrees. We performed Y-STR analysis of 17 loci in the samples collected (Yfiler system from Applied Biosystems) and analysis of 42 Y-SNPs by real-time PCR. We compared our data with published data from other Kurdish groups and from European, Caucasian, and West Asian populations. We found that gene pool of Yezidis contains haplogroups common in the Middle East (J-M172(xM67,M12)- 24%, E-M35(xM78)- 9%) and in South Western Asia (R-M124- 8%) and variant with wide distribution area - R-M198(xM458- 9%). The gene pool of Kurdish has higher genetic diversity than Yezidis. Their dominants haplogroups are R-M198- 20,3 %, E-M35- 9%, J-M172- 9%. Multidimensional scaling also shows that the Kurds and Yezidis are part of the same frontier Asian cluster, which, in addition, included Armenians, Iranians, Turks, and Greeks. At the same time, the peoples of the Caucasus and Europe form isolated clusters that do not overlap with the Asian clusters. It is noteworthy that Kurds from our study gravitate towards Tats, which indicates that most likely these two populations are descendants of ancient Medes population. Multidimensional scaling also reveals similarity between gene pool of Yezidis, Kurds with Armenians and Iranians. The analysis of Yezidis pedigrees and their STR variability did not reveal a reliable connection between genetic diversity and caste system. This indicates that the Yezidis caste system is a social division and not a biological one. Thus, we showed that, despite many years of isolation, the gene pool of Yezidis retained a common layer with the gene pool of Kurds, these populations have common spectrum of haplogroups, but Yezidis have lower genetic diversity than Kurds. This study received primary support from the RSF grant No. 16-36-00122 to MC and grant No. 16-06-00364 to EP.

Keywords: gene pool, haplogroup, Kurds, SNP and STR markers, Yezidis

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Authors: Keiko Tamura, Munenari Inoguchi, Michiyo Tsuji

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When disasters occur, in order to raise the speed of each decision making and response, it is common that delegation of authority is carried out. This tendency is particularly evident when the department or branch of the organization are separated by the physical distance from the main body; however, there are some issues to think about. If the department or branch is too dependent on the head office in the usual condition, they might feel lost in the disaster response operation when they are face to the situation. Avoiding this problem, an organization should decide how to delegate the authority and also who accept the responsibility for what before the disaster. This paper will discuss about the method which presents an approach for executing the delegation of authority process, implementing authorities, management by objectives, and preparedness plans and agreement. The paper will introduce the examples of efforts for the three research centers of Niigata University, Japan to arrange organizations capable of taking necessary actions for disaster response. Each center has a quality all its own. One is the center for carrying out the research in order to conserve the crested ibis (or Toki birds in Japanese), the endangered species. The another is the marine biological laboratory. The third one is very unique because of the old growth forests maintained as the experimental field. Those research centers are in the Sado Island, located off the coast of Niigata Prefecture, is Japan's second largest island after Okinawa and is known for possessing a rich history and culture. It takes 65 minutes jetfoil (high-speed ferry) ride to get to Sado Island from the mainland. The three centers are expected to be easily isolated at the time of a disaster. A sense of urgency encourages 3 centers in the process of organizational restructuring for enhancing resilience. The research team from the risk management headquarters offer those procedures; Step 1: Offer the hazard scenario based on the scientific evidence, Step 2: Design a risk management organization for disaster response function, Step 3: Conduct the participatory approach to make consensus about the overarching objectives, Step 4: Construct the unified operational action plan for 3 centers, Step 5: Simulate how to respond in each phase based on the understanding the various phases of the timeline of a disaster. Step 6: Document results to measure performance and facilitate corrective action. This paper shows the result of verifying the output and effects.

Keywords: delegation of authority, disaster response, risk management, unified command

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Authors: Fana Woldetsadik

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Ethiopia has a huge livestock population, including sheep, that has been contributing a considerable portion to the economy of the country and still promising to rally around the economic advancement of the country. However, feed shortage is a limiting factor in the production and productivity of sheep among Ethiopian smallholder farmers. Therefore, the aim of this study was to prove the role of the locally available brewery by-products called dried Atella as a supplement in feed intake, digestibility, live weight gain, carcass yield, and economic benefit in comparison with commercially purchased supplements known as niger seed cake (NSC). This on-station feeding experiment was conducted on the Zenzelma Campus of Bahir Dar University animal farm. The experimental design used for this research was a completely randomized design (CRD) with five replications. The crude protein (CP) content of dried Atella, wheat bran (WB), natural pasture hay (NPH) and NSC were about 25.07%, 16.57%, 4.48% and 38.04%, respectively, while the neutral detergent fibre (NDF),acid detergent fibre (ADF) and acid detergent lignin (ADL) content of dried Atella, WB, NPH and NSC were around 31.75%, 8.31%, 8.14%; 42.05%, 22.64%, 4.04%; 74.21%, 50.81%, 8.66%; 42.31%, 26.95% and 6.9%, respectively. The result depicted that a higher(P < 0.001) feed intake, nutrient intake, and digestibility for lambs supplemented with Atella than those supplemented with NSC. Furthermore, daily body weight gain and carcass characteristics were better (P < 0.05) for the sheep supplemented with dried Atella than NSC. On the other hand, in terms of profitability, although there was no substantial difference (P > 0.05) between T2 (animals fed NPH,NSC and WB) and T3 (animals fed NPH, Atella and WB), slightly better benefit was recorded in T3 groups. However, loss of money was recorded in T1 (animals fed NPH and WB). Hence, from the biological performance of lambs, it was concluded that Atella could be a potential supplementary feed for sheep fattening among smallholder farmers than NSC despite no profitability difference. Nevertheless, further investigation is recommended to examine the consequence of supplementation of NPH with NSC and NPH with Atella on fatty acid profile analysis, the physicochemical composition of meat, and meat composition.

Keywords: Attela, Bahir Dar university, Carcass yield, digestibility, natural pasture hay, Niger seed cake, smallholder farmers, weight gain, Ethiopia

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Authors: M. Naimi, M. B. Khaled

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The aim of the present work was to test in vitro inhibition of food pathogens and spoilage bacteria by crude bacteriocins from autochthonous lactic acid bacteria. Thirty autochthonous lactic acid bacteria isolated previously, belonging to the genera: Lactobacillus, Carnobacterium, Lactococcus, Vagococcus, Streptococcus, and Pediococcus, have been screened by an agar spot test and a well diffusion assay against Gram-positive and Gram-negative harmful bacteria: Bacillus cereus, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, and Pseudomonas aeruginosa under conditions means to reduce lactic acid and hydrogen peroxide effect to select bacteria with high bacteriocinogenic potential. Furthermore, crude bacteriocins semiquantification and heat sensitivity to different temperatures (80, 95, 110°C, and 121°C) were performed. Another exploratory test concerning the response of St. aureus ATCC 6538 to the presence of crude bacteriocins was realized. It has been observed by the agar spot test that fifteen candidates were active toward Gram-positive targets strains. The secondary screening demonstrated an antagonistic activity oriented only against St. aureus ATCC 6538, leading to the selection of five isolates: Lm14, Lm21, Lm23, Lm24, and Lm25 with a larger inhibition zone compared to the others. The ANOVA statistical analysis reveals a small variation of repeatability: Lm21: 0.56%, Lm23: 0%, Lm25: 1.67%, Lm14: 1.88%, Lm24: 2.14%. Conversely, slight variation was reported in terms of inhibition diameters: 9.58± 0.40, 9.83± 0.46, and 10.16± 0.24 8.5 ± 0.40 10 mm for, Lm21, Lm23, Lm25, Lm14and Lm24, indicating that the observed potential showed a heterogeneous distribution (BMS = 0.383, WMS = 0.117). The repeatability coefficient calculated displayed 7.35%. As for the bacteriocins semiquantification, the five samples exhibited production amounts about 4.16 for Lm21, Lm23, Lm25 and 2.08 AU/ml for Lm14, Lm24. Concerning the sensitivity the crude bacteriocins were fully insensitive to heat inactivation, until 121°C, they preserved the same inhibition diameter. As to, kinetic of growth , the µmax showed reductions in pathogens load for Lm21, Lm23, Lm25, Lm14, Lm24 of about 42.92%, 84.12%, 88.55%, 54.95%, 29.97% in the second trails. Inversely, this pathogen growth after five hours displayed differences of 79.45%, 12.64%, 11.82%, 87.88%, 85.66% in the second trails, compared to the control. This study showed potential inhibition to the growth of this food pathogen, suggesting the possibility to improve the hygienic food quality.

Keywords: exploratory test, lactic acid bacteria, crude bacteriocins, spoilage, pathogens

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Authors: Shveta Bathla, Preeti Rawat, Sudarshan Kumar, Rubina Baithalu, Jogender Singh Rana, Tushar Kumar Mohanty, Ashok Kumar Mohanty

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Pregnancy is a complex process which includes series of events such as fertilization, formation of blastocyst, implantation of embryo, placental formation and development of fetus. The success of these events depends on various interactions which are synchronized by endocrine interaction between a receptive dam and competent embryo. These interactions lead to change in expression of hormones and proteins. But till date no protein biomarker is available which can be used to detect successful completion of these events. We employed quantitative proteomics approach to develop putative serological biomarker which has diagnostic applicability for early detection of pregnancy in cows. For this study, sera were collected from control (non-pregnant, n=6) and pregnant animals on successive days of pregnancy (7, 19, 45, n=6). The sera were subjected to depletion for removal of albumin using Norgen depletion kit. The tryptic peptides were labeled with iTRAQ. The peptides were pooled and fractionated using bRPLC over 80 min gradient. Then 12 fractions were injected to nLC for identification and quantitation in DDA mode using ESI. Identification using Mascot search revealed 2056 proteins out of which 352 proteins were differentially expressed. Twenty proteins were upregulated and twelve proteins were down-regulated with fold change > 1.5 and < 0.6 respectively (p < 0.05). The gene ontology studies of DEPs using Panther software revealed that majority of proteins are actively involved in catalytic activities, binding and enzyme regulatory activities. The DEP'S such as NF2, MAPK, GRIPI, UGT1A1, PARP, CD68 were further subjected to pathway analysis using KEGG and Cytoscape plugin Cluego that showed involvement of proteins in successful implantation, maintenance of pluripotency, regulation of luteal function, differentiation of endometrial macrophages, protection from oxidative stress and developmental pathways such as Hippo. Further efforts are continuing for targeted proteomics, western blot to validate potential biomarkers and development of diagnostic kit for early pregnancy diagnosis in cows.

Keywords: bRPLC, Cluego, ESI, iTRAQ, KEGG, Panther

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Authors: Norhidayu Muhamad Zain

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Gelatin, a purified protein derived mostly from porcine and bovine sources, is used widely in food manufacturing, pharmaceutical, and cosmetic industries. However, the presence of any porcine-related products are strictly forbidden for Muslim and Jewish consumption. Therefore, analytical methods offering reliable results to differentiate the sources of gelatin are needed. The aim of this study was to differentiate the sources of gelatin (porcine and tilapia fish) using Fourier transform infrared- attenuated total reflection (FTIR-ATR) combined with two dimensional infrared (2DIR) correlation analysis. Porcine gelatin (PG) and tilapia fish gelatin (FG) samples were diluted in distilled water at concentrations ranged from 4-20% (w/v). The samples were then analysed using FTIR-ATR and 2DIR correlation software. The results showed a significant difference in the pattern map of synchronous spectra at the region of 1000 cm⁻¹ to 1100 cm⁻¹ between PG and FG samples. The auto peak at 1080 cm⁻¹ that attributed to C-O functional group was observed at high intensity in PG samples compared to FG samples. Meanwhile, two auto peaks (1080 cm⁻¹ and 1030 cm⁻¹) at lower intensity were identified in FG samples. In addition, using 2D correlation analysis, the original broad water OH bands in 1D IR spectra can be effectively differentiated into six auto peaks located at 3630, 3340, 3230, 3065, 2950 and 2885 cm⁻¹ for PG samples and five auto peaks at 3630, 3330, 3230, 3060 and 2940 cm⁻¹ for FG samples. Based on the rule proposed by Noda, the sequence of the spectral changes in PG samples is as following: NH₃⁺ amino acid > CH₂ and CH₃ aliphatic > OH stretch > carboxylic acid OH stretch > NH in secondary amide > NH in primary amide. In contrast, the sequence was totally in the opposite direction for FG samples and thus both samples provide different 2D correlation spectra ranged from 2800 cm-1 to 3700 cm⁻¹. This method may provide a rapid determination of gelatin source for application in food, pharmaceutical, and cosmetic products.

Keywords: 2 dimensional infrared (2DIR) correlation analysis, Fourier transform infrared- attenuated total reflection (FTIR-ATR), porcine gelatin, tilapia fish gelatin

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Authors: Fahad Al Yahyaey, Ihab Shaat, Russell Bush

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This study was conducted at the Livestock Research Centre, Ministry of Agriculture and Fisheries, Oman, on two local goat breeds (Jabbali and Sahrawi) due to their importance to Omani livestock production and food security. The Jabbali is characterized by increased growth rates and a higher twinning rate, while the Sahrawi has increased milk production. The aim of the study was to investigate the effect of Spirulina supplementation on live weight (BWT), average daily gain (ADG), and body conformation measurements; chest girth (CG), wither height (WH), body length (BL), and body condition score (BCS). Thirty-six males (approximately nine-months-old and 16.44 ± 0.33 kg average of initial body weight) were used across an eleven-week study from November–February 2019-2020. Each breed was divided into three groups (n = 6/group) and fed one of three rations: (1) concentrate mixture (Control) with crude protein 14% and energy 11.97% MJ/kg DM; (2) the same concentrate feed with the addition of 2 gm /capita daily Spirulina platensis (Treatment 1) and (3) the same concentrate feed with the addition of 4 gm /capita daily Spirulina platensis (Treatment 2). Analysis of weekly data collections for all traits indicated a significant effect of feeding Spirulina on all the studied traits except WH and BL. Analysis of variance for fixed effects in this study (damage and kid birth type i.e., single, twin or triple) were not significant for all studied traits. However, the breed effect was highly significant (P < 0.001) on BWT, ADG, BCS, and CG traits. On the other hand, when the analysis was done for the treatment effect within breeds for ADG, the Sahrawi breed had a significant effect (P < 0.05) at 56.52, 85.51, and 85.50 g/day for control, treatment 1 and treatment 2, respectively. This is a 51% difference between the control and treatment 1 (2 gm /capita). Whereas for the Jabbali breed, the treatment effect was not significant for ADG (P =0.55), and the actual ADG was 104.59, 118.84, and 114.25 g/day for control, treatment 1, and treatment 2, respectively, providing a 14% difference between the control group and the treated group (4 gm /capita). These findings indicate using Spirulina supplementation in Omani goat diets is recommended at 2 gm per capita as there was no benefit in feeding at 4 gm per capita for either breed. Farmers feeding Spirulina supplementation to kids after weaning at six-months could increase their herd performance and growth rate and facilitate buck selection at an earlier age.

Keywords: body conformation, goats, live weight, spirulina

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Authors: Man-Yun Liu, Emily Chia-Yu Su

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Alzheimer's disease (AD) is the public health crisis of the 21st century. AD is a degenerative brain disease and the most common cause of dementia, a costly disease on the healthcare system. Unfortunately, the cause of AD is poorly understood, furthermore; the treatments of AD so far can only alleviate symptoms rather cure or stop the progress of the disease. Currently, there are several ways to diagnose AD; medical imaging can be used to distinguish between AD, other dementias, and early onset AD, and cerebrospinal fluid (CSF). Compared with other diagnostic tools, blood (plasma) test has advantages as an approach to population-based disease screening because it is simpler, less invasive also cost effective. In our study, we used blood biomarkers dataset of The Alzheimer’s disease Neuroimaging Initiative (ADNI) which was funded by National Institutes of Health (NIH) to do data analysis and develop a prediction model. We used independent analysis of datasets to identify plasma protein biomarkers predicting early onset AD. Firstly, to compare the basic demographic statistics between the cohorts, we used SAS Enterprise Guide to do data preprocessing and statistical analysis. Secondly, we used logistic regression, neural network, decision tree to validate biomarkers by SAS Enterprise Miner. This study generated data from ADNI, contained 146 blood biomarkers from 566 participants. Participants include cognitive normal (healthy), mild cognitive impairment (MCI), and patient suffered Alzheimer’s disease (AD). Participants’ samples were separated into two groups, healthy and MCI, healthy and AD, respectively. We used the two groups to compare important biomarkers of AD and MCI. In preprocessing, we used a t-test to filter 41/47 features between the two groups (healthy and AD, healthy and MCI) before using machine learning algorithms. Then we have built model with 4 machine learning methods, the best AUC of two groups separately are 0.991/0.709. We want to stress the importance that the simple, less invasive, common blood (plasma) test may also early diagnose AD. As our opinion, the result will provide evidence that blood-based biomarkers might be an alternative diagnostics tool before further examination with CSF and medical imaging. A comprehensive study on the differences in blood-based biomarkers between AD patients and healthy subjects is warranted. Early detection of AD progression will allow physicians the opportunity for early intervention and treatment.

Keywords: Alzheimer's disease, blood-based biomarkers, diagnostics, early detection, machine learning

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Authors: Xiaojie Cheng, Ulrike Frank, Feng Zhao, Karin Pritsch

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Ragweed (Ambrosia artemisiifolia) is an invasive weed that has become an increasing global problem. In addition to affecting land use and crop yields, ragweed has a strong impact on human health as it produces highly allergenic pollen. Global warming will result in an earlier and longer pollen season enhanced pollen production and an increase in pollen allergenicity with a negative effect on atopic patients. The aims of this study were to investigate the effects of increasing temperature, the future climate scenario in the Munich area, southern Germany, predicted on the basis of RCP8.5 until the end of 2050s, or/and NO₂, a major air pollutant, 1) on the vegetative and reproductive characteristics of ragweed plants, 2) on the total allergenicity of ragweed pollen, 3) on the total pollen proteomic patterns. Ragweed plants were cultivated for the whole plant vegetation period under controlled conditions either under ambient climate conditions or 4°C higher temperatures with or without additional NO₂. Higher temperature resulted in bigger plant sizes, longer male inflorescences, and longer pollen seasons. The total allergenic potential of the pollen was accessed by dot blot using serum from ragweed pollen sensitized patients. The comparative immunoblot analysis revealed that the in vivo fumigation of ragweed plants with elevated NO₂-concentrations significantly increased the allergenic potential of the pollen, and in combination with increased temperature, the allergenic potential was even higher. On the other hand, label-free protein quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed. The results showed that more proteins were significantly up- and down-regulated under higher temperatures with/without elevated NO₂ conditions. Most of the highly expressed proteins were participating intensively in the metabolic process, the cellular process, and the stress defense process. These findings suggest that rising temperature and elevated NO₂ are important environmental factors for higher abiotic stress activities, catalytic activities, and thus higher allergenic potential observed in pollen proteins.

Keywords: climate change, NO₂, pollen proteome, ragweed, temperature

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Authors: Nazish Badar

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In early 2009, Pandemic type A (H1N1) Influenza virus emerged globally. Since then, it has continued circulation causing considerable morbidity and mortality. The purpose of this study was to evaluate the evolutionary changes in Influenza A (H1N1) pdm09 viruses from 2009-15 and their relevance with the current vaccine viruses. Methods: Respiratory specimens were collected with influenza-like illness and Severe Acute Respiratory Illness. Samples were processed according to CDC protocol. Sequencing and phylogenetic analysis of Haemagglutinin (HA) and neuraminidase (NA) genes was carried out comparing representative isolates from Pakistan viruses. Results: Between Jan2009 - Feb 2016, 1870 (13.2%) samples were positive for influenza A out of 14086. During the pandemic period (2009–10), Influenza A/ H1N1pdm 09 was the dominant strain with 366 (45%) of total influenza positives. In the post-pandemic period (2011–2016), a total of 1066 (59.6%) cases were positive Influenza A/ H1N1pdm 09 with co-circulation of different Influenza A subtypes. Overall, the Pakistan A(H1N1) pdm09 viruses grouped in two genetic clades. Influenza A(H1N1)pdm09 viruses only ascribed to Clade 7 during the pandemic period whereas viruses belong to clade 7 (2011) and clade 6B (2015) during the post-pandemic years. Amino acid analysis of the HA gene revealed mutations at positions S220T, I338V and P100S specially associated with outbreaks in all the analyzed strains. Sequence analyses of post-pandemic A(H1N1)pdm09 viruses showed additional substitutions at antigenic sites; S179N,K180Q (SA), D185N, D239G (CA), S202A (SB) and at receptor binding sites; A13T, S200P when compared with pandemic period. Substitution at Genetic markers; A273T (69%), S200P/T (15%) and D239G (7.6%) associated with severity and E391K (69%) associated with virulence was identified in viruses isolated during 2015. Analysis of NA gene revealed outbreak markers; V106I (23%) among pandemic and N248D (100%) during post-pandemic Pakistan viruses. Additional N-Glycosylation site; HA S179N (23%), NA I23T(7.6%) and N44S (77%) in place of N386K(77%) were only found in post-pandemic viruses. All isolates showed histidine (H) at position 275 in NA indicating sensitivity to neuraminidase inhibitors. Conclusion: This study shows that the Influenza A(H1N1)pdm09 viruses from Pakistan clustered into two genetic clades, with co-circulation of some variants. Certain key substitutions in the receptor binding site and few changes indicative of virulence were also detected in post-pandemic strains. Therefore, it is imperative to continue monitoring of the viruses for early identification of potential variants of high virulence or emergence of drug-resistant variants.

Keywords: Influenza A (H1N1) pdm09, evolutionary analysis, post pandemic period, Pakistan

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Authors: S. Mohd Afzal, R. O'Connell

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Kaposi's sarcoma (KS) is the most common HIV-related cancer, and ocular manifestations constitute at least 25% of all KS cases. However, ocular presentations often occur in the context of systemic KS, and isolated lesions are rare. We report a unique case of ocular KS masquerading as subconjunctival haemorrhage, and only developing systemic manifestations after initiation of HIV treatment. Case: A 49-year old man with previous hypertensive stroke and newly diagnosed HIV infection presented with an acutely red left eye following repeated bouts of coughing. Given the convincing history of poorly controlled hypertension and cough, a diagnosis of subconjunctival haemorrhage was made. Over the next week, his ocular lesion began to improve and he subsequently started anti-retroviral therapy. Prior to receiving anti-retroviral therapy, his CD4+ lymphocyte count was 194 cells/mm3 with HIV viral load greater than 1 million/ml. This rapidly improved to a viral load of 150 copies/ml within 2 weeks of starting treatment. However, a few days after starting HIV treatment, his ocular lesion recurred. Ophthalmic examination was otherwise normal. He also developed widespread lymphadenopathy and multiple dark lesions on his torso. Histology and virology confirmed KS, systemically triggered by Immune Reconstitution Syndrome (KS-IRIS). The patient has since undergone chemotherapy successfully. Discussion: Kaposi's sarcoma is an atypical tumour caused by human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV). In immunosuppressed patients, KSHV can also cause lymphoproliferative disorders such as primary effusion lymphoma and Castleman's disease (in our patient’s case, this was excluded through histological analysis of lymph nodes). KSHV is one of the seven currently known human oncoviruses, and its pathogenesis is poorly understood. Up to 13% of patients with HIV-related KS experience worsening of the disease after starting anti-retroviral treatment, due to a sudden increase in CD4 cell counts. Histology remains the diagnostic gold standard. Current British HIV Association (BHIVA) guidelines recommend treatment using anti-retroviral drugs, with either intralesional vinblastine for local disease or systemic chemotherapy for disseminated KS. Conclusion: This case is unique as ocular KS as initial presentation is rare and our patient's diagnosis was only made after systemic lesions were triggered by immune reconstitution. KS should be considered as an important differential diagnosis for red eyes in all patients at risk of acquiring HIV infection.

Keywords: human herpesvirus 8, human immunodeficiency virus, immune reconstitution syndrome, Kaposi’s sarcoma, Kaposi’s sarcoma-associated herpesvirus

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Authors: Sze Wing Ho, Nagendra P. Shah

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Lactic acid bacteria (LAB) have shown the beneficial effects on human gastrointestinal tract, such as protects diarrhea induced by lactose intolerance or enteric pathogens. Citrulline is a non-protein amino acid and also the precursors of arginine and nitric oxide, it has shown to enhance intestinal barrier function. Citrulline has shown to improve the growth of some strains of LAB, it is important for LAB to have a sufficient cell concentration to contribute the effects. Therefore, the aims of this study were to investigate the effect of combining citrulline with LAB on the anti-adhesion effect against pathogens and the effect on the cell integrity. The effect of citrulline on selected LAB was determined by incubating in 0%, 0.1% or 0.2% citrulline enriched MRS broth for 18 h. The adhesion ability of LAB and the anti-adhesion effect of LAB and citrulline against pathogens were performed on IPEC-J2 cell line. Transepithelial electrical resistance (TEER) assay was used to measure the tight junction (TJ) integrity. TJ proteins (claudin-1, occludin and zonula occluden-1 (ZO-1)) were determined by western blot analysis. It found that the growth of Lactobacillus helveticus ASCC 511 was significantly stimulated by 0.2% citrulline compared with control during 18 h fermentation. The adhesion of L. helveticus ASCC 511 and Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) ASCC 756 was increased when supplemented with citrulline. Citrulline has shown significant inhibitory effect on the adhesion of Escherichia coli PELI0480 (O157:H7), Shigella sonnei ATCC 25931, Staphyloccocus aureus CMCC26003 and Cronobacter sakazakii ATCC 29544. The anti-adhesion effect of L. helveticus ASCC 511, L. bulgaricus ASCC 756 and Lactobacillus paracasei ASCC 276 against Cronobacter sakazakii ATCC 29544 was significantly enhanced with citrulline supplementation. Treatments with citrulline and LAB were able to maintain the TEER of IPEC-J2 cell and shown the positive effect on the TJ proteins. In conclusion, citrulline had stimulating effect on some strains of LAB and determined to improve the adhesion of LAB on intestinal epithelial cell, to enhance the inhibitory effect on enteric pathogens adhesion as well as had beneficial effects on maintaining cell integrity. It implied LAB supplemented with citrulline might have advantageous effects on gastrointestinal tracts.

Keywords: citrulline, lactic acid bacteria, amino acid, anti-adhesion effect, cell integrity

Procedia PDF Downloads 221

Authors: Irina M. Kolesnikova, Andrey M. Gaponov, Sergey A. Roumiantsev, Tatiana V. Grigoryeva, Dilyara R. Khusnutdinova, Dilyara R. Kamaldinova, Alexander V. Shestopalov

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Background. Obese patients have unequal risks of metabolic disorders. It is accepted to distinguish between metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUHO). MUHO patients have a high risk of metabolic disorders, insulin resistance, and diabetes mellitus. Among the other things, the gut microbiota also contributes to the development of metabolic disorders in obesity. Obesity is accompanied by significant changes in the gut microbial community. In turn, bacterial translocation from the intestine is the basis for the blood microbiome formation. The aim was to study the features of the blood microbiome in patients with various metabolic types of obesity. Patients, materials, methods. The study included 116 healthy donors and 101 obese patients. Depending on the metabolic type of obesity, the obese patients were divided into subgroups with MHO (n=36) and MUHO (n=53). Quantitative and qualitative assessment of the blood microbiome was based on metagenomic analysis. Blood samples were used to isolate DNA and perform sequencing of the variable v3-v4 region of the 16S rRNA gene. Alpha diversity indices (Simpson index, Shannon index, Chao1 index, phylogenetic diversity, the number of observed operational taxonomic units) were calculated. Moreover, we compared taxa (phyla, classes, orders, and families) in terms of isolation frequency and the taxon share in the total bacterial DNA pool between different patient groups. Results. In patients with MHO, the characteristics of the alpha-diversity of the blood microbiome were like those of healthy donors. However, MUHO was associated with an increase in all diversity indices. The main phyla of the blood microbiome were Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Cyanobacteria, TM7, Thermi, Verrucomicrobia, Chloroflexi, Acidobacteria, Planctomycetes, Gemmatimonadetes, and Tenericutes were found to be less significant phyla of the blood microbiome. Phyla Acidobacteria, TM7, and Verrucomicrobia were more often isolated in blood samples of patients with MUHO compared with healthy donors. Obese patients had a decrease in some taxonomic ranks (Bacilli, Caulobacteraceae, Barnesiellaceae, Rikenellaceae, Williamsiaceae). These changes appear to be related to the increased diversity of the blood microbiome observed in obesity. An increase of Lachnospiraceae, Succinivibrionaceae, Prevotellaceae, and S24-7 was noted for MUHO patients, which, apparently, is explained by a magnification in intestinal permeability. Conclusion. Blood microbiome differs in obese patients and healthy donors at class, order, and family levels. Moreover, the nature of the changes is determined by the metabolic type of obesity. MUHO linked to increased diversity of the blood microbiome. This appears to be due to increased microbial translocation from the intestine and non-intestinal sources.

Keywords: blood microbiome, blood bacterial DNA, obesity, metabolically healthy obesity, metabolically unhealthy obesity

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Authors: Galuh Asri Bestari, Hajar Shofiyya

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Double burden of malnutrition (DBM) has been a global problem in these last decades occurs in both developed and developing countries. Overweight in adults and stunting among preschool children have dramatically increased and become the main problems of malnutrition that should be solved immediately since they are directly related with the health status and productivity. Reformulation of food product by using the local sea resources called terasi shrimp (Acetes indicus) has a potential possibility in facing the DBM. A study was carried out in Indonesia to determine the acceptability of terasi shrimp powder through sensory evaluation. Terasi shrimps were processed into powder form through sun drying and pounding methods. The powder form was directly added in food as alternative seasonings and tested among stunted and normal preschool children. Meanwhile, a further processing method is given to the shrimp powder tested in overweight and normal-weighed adults. The shrimp powder was mixed with sago flour and formed into balls, then steamed for 15-20 minutes, and finally served as alternative snacks. Based on the sensory evaluation, the shrimp powder has a good acceptance in taste (54%), shape (60%), and color properties (63%), while the shrimp balls has a good acceptance in size (65%), shape (50%), color (48%), taste (40%), and texture (36%). Terasi shrimp powder can be stored for a month in room temperature. In addition, carried out chemical analysis revealed that terasi shrimp (Acetes indicus) has higher percentage of protein, calcium, and iron than other animal sources, but conversely contains zero sodium and very low percentage of fat. Terasi shrimp’s shell also contains a substance called chitosan which acts by forming gels in the intestinal tract to entrap lipids, thus interfering with their absorption. After going through some processing methods, the shrimp powder and balls did not show any significant changes in their nutrient contents. So that, terasi shrimp powder is good to be consumed not only by overweight adults, but also by children to support their optimum growth. Intervention of terasi shrimp powder should be implemented step by step from national up to global governance program to face the DBM.

Keywords: Acetes indicus, alternative food, double burden of malnutrition, sensory evaluation

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Authors: P. Joris, E. Lombard, X. Cameleyre, G. Navarro, A. Paillet, N. Gorret, S. E. Guillouet

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Today, on the international space station, multiple supplies are needed per year to supply food and spare parts and to take out waste. But as it is planned to go longer and further into space these supplies will no longer be possible. The astronaut life support system must be able of continuously transform waste into valuable compounds. Two types of production were identified as critical and could be be supplemented by microorganisms. On the one hand, since microgravity causes rapid muscle loss, single cell proteins (SCPs) could be used as protein rich feed or food. On the other hand, having enough building materials to build an advanced habitat will not be possible only by transporting space goods from earth to mars for example. The bacterium Cupriavidus. necator is well known for its ability to produce a large amount of proteins or of polyhydroxyalkanoate biopolymers (PHAs) depending on its implementation. By coupling the life support system to a 3D-printer, astronauts could be supplied with an unlimited amount of building materials. Additionally, based on the design of the life support system, waste streams have been identified: urea from the crew urine and volatile fatty acids (VFAs) from a first stage of organic waste (excrement and food waste) treatment through anaerobic digestion. Thus, the objective of this, within the Spaceship.Fr project, was to demonstrate the feasibility of producing SCPs and PHAs from VFAs and urea in bioreactor. Because life support systems operate continuously as loops, continuous culture experiments were chosen and the effect of the bioreactor dilution rate on biomass composition was investigated. Total transformation of the carbon source into biomass with high SCP or PHA content was achieved in all cases. We will present the transformation performances of VFAs and urea by the bacteria in bioreactor in terms of titers, yields and productivities but also in terms of the quality of SCP and PHA produced, nucleic acid content. We will further discuss the envisioned integration of our process within life support systems.

Keywords: life support system, space travel, waste treatment, single cell proteins, polyhydroxyalkanoates, bioreactor

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Authors: Pedro Batista, André Vinha, Filipe Castelo, Bárbara Costa, Ricardo Sousa, Raquel Ricardo, André Pinto

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Background: Septic arthritis is a diagnosis that must be considered in any patient presenting with acute joint swelling and fever. Among the several risk factors for septic arthritis, such as age, rheumatoid arthritis, recent surgery, or skin infection, diabetes mellitus can sometimes be the main risk factor. Staphylococcus aureus is the most common pathogen isolated in septic arthritis; however, it is uncommon in monomicrobial necrotizing fasciitis. Objectives: A case report of concomitant septic arthritis and necrotizing fasciitis in a patient with undiagnosed diabetes based on clinical history. Study Design & Methods: We report a case of a 58-year-old Portuguese previously healthy man who presented to the emergency department with fever and left knee swelling and pain for two days. The blood work revealed ketonemia of 6.7 mmol/L and glycemia of 496 mg/dL. The vital signs were significant for a temperature of 38.5 ºC and 123 bpm of heart rate. The left knee had edema and inflammatory signs. Computed tomography of the left knee showed diffuse edema of the subcutaneous cellular tissue and soft tissue air bubbles. A diagnosis of septic arthritis and necrotising fasciitis was made. He was taken to the operating room for surgical debridement. The samples collected intraoperatively were sent for microbiological analysis, revealing infection by multi-sensitive Staphylococcus aureus. Given this result, the empiric flucloxacillin (500 mg IV) and clindamycin (1000 mg IV) were maintained for 3 weeks. On the seventh day of hospitalization, there was a significant improvement in subcutaneous and musculoskeletal tissues. After two weeks of hospitalization, there was no purulent content and partial closure of the wounds was possible. After 3 weeks, he was switched to oral antibiotics (flucloxacillin 500 mg). A week later, a urinary infection by Pseudomonas aeruginosa was diagnosed and ciprofloxacin 500 mg was administered for 7 days without complications. After 30 days of hospital admission, the patient was discharged home and recovered. Results: The final diagnosis of concomitant septic arthritis and necrotizing fasciitis was made based on the imaging findings, surgical exploration and microbiological tests results. Conclusions: Early antibiotic administration and surgical debridement are key in the management of septic arthritis and necrotizing fasciitis. Furthermore, risk factors control (euglycemic blood glucose levels) must always be taken into account given the crucial role in the patient's recovery.

Keywords: septic arthritis, Necrotizing fasciitis, diabetes, Staphylococcus Aureus

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Authors: Himalaya Patir, Bitupon Baruah, Sanjay Gayary, Subhajit Ray

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In recent age significant importance is given for the development of nutritious and health beneficial foods. Fruit juices collected from different fruits when blended that improves not only the physicochemical and nutritional properties but also enhance the sensorial or organoleptic properties. The study was carried out to determine the physico-chemical, nutritional, microbiological analysis and sensory evaluation of mixed fruit juice blend. Juice of orange (Citrus sinensis), apple (Malus domestica), mosambi (Citrus limetta) were blended in the ratio of sample-I (30% apple:30% orange:40% mosambi), sample-II ( 40% apple :30% orange :30% mosambi), sample-III (30% apple :40% orange :30% mosambi) , sample-IV (50% apple :30% orange :20% mosambi), sample-V (30% apple:20% orange:50% mosambi), sample-VI (20% apple :50% orange :30% mosambi) to evaluate all quality characteristics. Their colour characteristics in terms of hue angle, chroma and colour difference (∆E) were evaluated. The physico-chemical parameters analysis carried out were total soluble solids (TSS), total titratable acidity (TTA), pH, acidity (FA), volatile acidity (VA), pH, and vitamin C. There were significant differences (p˂0.05) in the TSS of the samples. However, sample-V (30% apple: 20% orange: 50% mosambi) provides the highest TSS of 9.02gm and significantly differed from other samples (p˂0.05). Sample-IV (50% apple: 30% orange: 20% mosambi) was shown the highest titratable acidity (.59%) in comparison to other samples. The highest value of pH was found as 5.01 for sample-IV (50% apple: 30% orange: 20% mosambi). Sample-VI (20% apple: 50% orange :30% mosambi) blend has the highest hue angle, chroma and colour changes of 72.14,25.29 and 54.48 and vitamin C, i.e. Ascorbic acid (.33g/l) content compared to other samples. The nutritional compositions study showed that, sample- VI (20% apple: 50% orange: 30% mosambi) has the significantly higher carbohydrate (51.67%), protein (.78%) and ash (1.24%) than other samples, while sample-V (30% apple: 20% orange: 50% mosambi) has higher dietary fibre (12.84%) and fat (2.82%) content. Microbiological analysis of all samples in terms of total plate count (TPC) ranges from 44-60 in 101 dilution and 4-5 in 107 dilutions and was found satisfactory. Moreover, other pathogenic bacterial count was found nil. The general acceptability of the mixed fruit juice blend samples were moderately liked by the panellists, and sensorial quality studies showed that sample-V (30% apple: 20% orange: 50% mosambi) contains highest overall acceptability of 8.37 over other samples and can be considered good for consumption.

Keywords: microbiological, nutritional, physico-chemical, sensory properties

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Authors: M. S. Raza, A. Kapil, Sonu Tyagi, H. Gautam, S. Mohapatra, R. Chaudhry, S. Sood, V. Goyal, R. Lodha, V. Sreenivas, B. K. Das

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Background: Acute bacterial meningitis remains the major cause of mortality and morbidity. More than half of the survivors develop the significant lifelong neurological abnormalities. Diagnosis of the hospital acquired acute bacterial meningitis (HAABM) is challenging as it appears either in the post operative patients or patients acquire the organisms from the hospital environment. In both the situations, pathogens are exposed to high dose of antibiotics. Chances of getting multidrug resistance organism are very high. We have performed this experiment to find out the etiological agents of HAABM and its antibiotics susceptibility pattern. Methodology: A perspective study was conducted at the Department of Microbiology, All India Institute of Medical Sciences, New Delhi. From March 2015 to April 2018 total 400 Cerebro spinal fluid samples were collected aseptically. Samples were processed for cell count, Gram staining, and culture. Culture plates were incubated at 37°C for 18-24 hours. Organism grown on blood and MacConkey agar were identified by MALDI-TOF Vitek MS (BioMerieux, France) and antibiotic susceptibility tests were performed by Kirby Bauer disc diffusion method as per CLSI 2015 guideline. Results: Of the 400 CSF samples processed, 43 (10.75%) were culture positive for different bacteria. Out of 43 isolates, the most prevalent Gram-positive organisms were S. aureus 4 (9.30%) followed by E. faecium 3 (6.97%) & CONS 2 (4.65%). Similarly, E. coli 13 (30.23%) was the commonest Gram-negative isolates followed by A. baumannii 12 (27.90%), K. pneumonia 5 (11.62%) and P. aeruginosa 4(9.30%). Most of the antibiotics tested against the Gram-negative isolates were resistance to them. Colistin was most effective followed by Meropenem and Imepenim for all Gram-negative HAABM isolates. Similarly, most of antibiotics tested were susceptible to S. aureus and CONS. However, E. faecium (100%) were only susceptible to vancomycin and teicoplanin. Conclusion: Hospital acquired acute bacterial meningitis (HAABM) is becoming the emerging challenge as most of isolates are showing resistance to commonly used antibiotics. Gram-negative organisms are emerging as the major player of HAABM. Great care needs to be taken especially in tertiary care hospitals. Similarly, antibiotic stewardship should be followed and antibiotic susceptibility test (AST) should be performed regularly to update the antibiotic patter and to prevent from the emergence of resistance. Updated information of the AST will be helpful for the better management of the meningitis patient.

Keywords: CSF, MALDI-TOF, hospital acquired acute bacterial meningitis, AST

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Authors: Lee Yujin, Han Jiye, Oh Jin-Woo

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The adverse effects of endocrine disrupting chemicals (EDCs) has attracted considerable public interests. The benzene-like EDCs structure mimics the mechanisms of hormones naturally occurring in vivo, and alters physiological function of the endocrine system. Although, some of the most representative EDCs such as polychlorinated biphenyls (PCBs) and phthalates compounds already have been prohibited to produce and use in many countries, however, PCBs and phthalates in plastic products as flame retardant and plasticizer are still circulated nowadays. EDCs can be released from products while using and discarding, and it causes serious environmental and health issues. Here, we developed virus-based structurally coloured nanostructure that can detect minute EDCs concentration sensitively and selectively. These structurally coloured nanostructure exhibits characteristic angel-independent colors due to the regular virus bundle structure formation through simple pulling technique. The designed number of different colour bands can be formed through controlling concentration of virus solution and pulling speed. The virus, M-13 bacteriophage, was genetically engineered to react with specific ECDs, typically PCBs and phthalates. M-13 bacteriophage surface (pVIII major coat protein) was decorated with benzene derivative binding peptides (WHW) through phage library method. In the initial assessment, virus-based color sensor was exposed to several organic chemicals including benzene, toluene, phenol, chlorobenzene, and phthalic anhydride. Along with the selectivity evaluation of virus-based colour sensor, it also been tested for sensitivity. 10 to 300 ppm of phthalic anhydride and chlorobenzene were detected by colour sensor, and showed the significant sensitivity with about 90 of dissociation constant. Noteworthy, all measurements were analyzed through principal component analysis (PCA) and linear discrimination analysis (LDA), and exhibited clear discrimination ability upon exposure to 2 categories of EDCs (PCBs and phthalates). Because of its easy fabrication, high sensitivity, and the superior selectivity, M-13 bacteriophage-based color sensor could be a simple and reliable portable sensing system for environmental monitoring, healthcare, social security, and so on.

Keywords: M-13 bacteriophage, colour sensor, genetic engineering, EDCs

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Authors: Zahra Shokrolahi, Mohammad Reza Atashzar

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The goals of treating patients with breast cancer are to cure the disease, prolong survival, and improve quality of life. Immune cells in the tumor microenvironment have an important role in regulating tumor progression. The term of cellular immunotherapy refers to the administration of living cells to a patient; this type of immunotherapy can be active, such as a dendritic cell (DC) vaccine, in that the cells can stimulate an anti-tumour response in the patient, or the therapy can be passive, whereby the cells have intrinsic anti-tumour activity; this is known as adoptive cell transfer (ACT) and includes the use of autologous or allogeneic lymphocytes that may, or may not, be modified. The most important breast cancer cellular immunotherapies involving the use of T cells and natural killer (NK) cells in adoptive cell transfer, as well as dendritic cells vaccines. T cell-based therapies including tumour-infiltrating lymphocytes (TILs), engineered TCR-T cells, chimeric antigen receptor (CAR T cell), Gamma-delta (γδ) T cells, natural killer T (NKT) cells. NK cell-based therapies including lymphokine-activated killers (LAK), cytokine-induced killer (CIK) cells, CAR-NK cells. Adoptive cell therapy has some advantages and disadvantages some. TILs cell strictly directed against tumor-specific antigens but are inactive against tumor changes due to immunoediting. CIK cell have MHC-independent cytotoxic effect and also need concurrent high dose IL-2 administration. CAR T cell are MHC-independent; overcome tumor MHC molecule downregulation; potent in recognizing any cell surface antigen (protein, carbohydrate or glycolipid); applicable to a broad range of patients and T cell populations; production of large numbers of tumor-specific cells in a moderately short period of time. Meanwhile CAR T cells capable of targeting only cell surface antigens; lethal toxicity due to cytokine storm reported. Here we present the most popular cancer cellular immunotherapy approaches and discuss their clinical relevance referring to data acquired from clinical trials .To date, clinical experience and efficacy suggest that combining more than one immunotherapy interventions, in conjunction with other treatment options like chemotherapy, radiotherapy and targeted or epigenetic therapy, should guide the way to cancer cure.

Keywords: breast cancer , cell therapy , CAR T cell , CIK cells

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Authors: R. A. Abdel Rahman, A. E. Abdel Wahab, E. A. Goghneimy, H. F. Mohamed, E. M. Salama

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Infectious diseases are a major cause of death worldwide. The treatment of infections continues to be problematic in modern time because of the severe side effects of some drugs and the growing resistance to antimicrobial agents. Hence, the search for newer, safer and more potent antimicrobials is a pressing need. Herbal medicines have received much attention as a source of new antibacterial drugs since they are considered time-tested and comparatively safe both for human use and the environment. In the present study, the antimicrobial activity of marjoram (Origanum majorana L.) essential oil on some gram positive and gram negative reference bacteria, as well as some hospital resistant microbes, was tested. Marjoram oil was extracted and the oil chemical constituents were identified using GC/MS analysis. Staphylococcus aureas ATCC 6923, Pseudomonus auregonosa ATCC 9027, Bacillus subtilis ATCC 6633, E. coli ATCC 8736 and two hospital resistant microbes isolates 16 and 21 were used. The two isolates were identified by biochemical tests and 16s rRNA as proteus spp. and Enterococcus facielus. The effect of different concentrations of essential oils on bacterial growth was tested using agar disk diffusion assay method to determine the minimum inhibitory concentrations and using micro dilution method to determine the minimum bactericidal concentrations. Marjoram oil was found to be effective against both reference and hospital resistance strains. Hospital strains were more resistant to marjoram oil than reference strains. P. auregonosa growth was completely inhibited at a low concentration of oil (4µl/ml). The other reference strains showed sensitivity to marjoram oil at concentrations ranged from 5 to 7µl/ml. The two hospital strains showed sensitivity at media containing 10 and 15µl/ml oil. The major components of oil were terpineol, cis-beta (23.5%), 1,6 – octadien –3-ol,3,7-dimethyl, 2 aminobenzoate (10.9%), alpha terpieol (8.6%) and linalool (6.3%). Scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis were used to determine the difference between treated and untreated hospital strains. SEM results showed that treated cells were smaller in size than control cells. TEM data showed that cell lysis has occurred to treated cells. Treated cells have ruptured cell wall and appeared empty of cytoplasm compared to control cells which shown to be intact with normal volume of cytoplasm. The results indicated that marjoram oil has a positive antimicrobial effect on hospital resistance microbes. Natural crude extracts can be perfect resources for new antimicrobial drugs.

Keywords: antimicrobial activity, essential oil, hospital resistance microbes, marjoram

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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: Ethel E. Phiri, Lionel Hartzenberg, Percy Chimwamuromba, Emmanuel Nepolo, Jens Kossmann, James R. Lloyd

Abstract:

Orphan crops are underresearched and underutilized food plant species that have not been categorized as major food crops, but have the potential to be economically and agronomically significant. They have been documented to have the ability to tolerate extreme environmental conditions. However, limited research has been conducted to uncover their potential as food crop species. The New Partnership for Africa’s Development (NEPAD) has classified Marama bean, Tylosema esculentum, as an orphan crop. The plant is one of the 101 African orphan crops that must have their genomes sequenced, assembled, and annotated in the foreseeable future. Marama bean is a perennial leguminous plant that primarily grows in poor, arid soils in southern Africa. The plants produce large tubers that can weigh as much as 200kg. While the foliage provides fodder, the tuber is carbohydrate rich and is a staple food source for rural communities in Namibia. Also, the edible seeds are protein- and oil-rich. Marama Bean plants respond rapidly to increased temperatures and severe water scarcity without extreme consequences. Advances in molecular biology and biotechnology have made it possible to effectively transfer technologies between model- and major crops to orphan crops. In this research, the aim was to assemble the first transcriptomic analysis of Marama Bean RNA-sequence data. Many model plant species have had their genomes sequenced and their transcriptomes assembled. Therefore the availability of transcriptome data for a non-model crop plant species will allow for gene identification and comparisons between various species. The data has been sequenced using the Ilumina Hiseq 2500 sequencing platform. Data analysis is underway. In essence, this research will eventually evaluate the potential use of Marama Bean as a crop species to improve its value in agronomy. data for a non-model crop plant species will allow for gene identification and comparisons between various species. The data has been sequenced using the Ilumina Hiseq 2500 sequencing platform. Data analysis is underway. In essence, this researc will eventually evaluate the potential use of Marama bean as a crop species to improve its value in agronomy.

Keywords: 101 African orphan crops, RNA-Seq, Tylosema esculentum, underutilised crop plants

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Authors: Camille Rich, Nadine Ferris France, Ann Nolan, Webster Mavhu, Vongai Munatsi

Abstract:

Background and Aim: Zimbabwe has one of the highest HIV rates in the world, with a 12.7% adult prevalence rate. Young adults are a key group affected by HIV, and one-third of all new infections in Zimbabwe are amongst people ages 18-24 years. Stigma remains one of the main barriers to managing and reducing the HIV crisis, especially for young adults. There are several types of stigma, including enacted stigma, the outward discrimination towards someone and self-stigma, the negative self-judgments one has towards themselves. Self-stigma can have severe consequences, including feelings of worthlessness, shame, suicidal thoughts, and avoidance of medical help. This can have detrimental effects on those living with HIV. However, the unique beliefs and impacts of self-stigma amongst key groups living with HIV have not yet been explored. Therefore, the focus of this study is on the beliefs and experiences of HIV-related self-stigma, as experienced by young adults living in Harare, Zimbabwe. Research Methods: A qualitative approach was taken for this study, using sixteen semi-structured interviews with young adults (18-24 years) who are living with HIV in Harare. Participants were conveniently and purposefully sampled as members of Africa, an organization dedicated to young people living with HIV. Interviews were conducted over Zoom due to the COVID-19 pandemic, recorded and then coded using the software NVivo. The data was analyzed using both inductive and deductive Thematic Analysis to find common themes. Results: All of the participants experienced HIV-related self-stigma, and both beliefs and experiences were explored. These negative self-perceptions included beliefs of worthlessness, hopelessness, and negative body image. The young adults described believing they were not good enough to be around HIV negative people or that they could never be loved due to their HIV status. Developing self-stigmatizing thoughts came from internalizing negative cultural values, stereotypes about people living with HIV, and adverse experiences. Three main themes of self-stigmatizing experiences emerged: disclosure difficulties, relationship complications, and being isolated. Fear of telling someone their status, rejection in a relationship, and being excluded by others due to their HIV status contributed to their self-stigma. These experiences caused feelings of loneliness, sadness, shame, fear, and low self-worth. Conclusions: This study explored the beliefs and experiences of HIV-related self-stigma of these young adults. The emergence of negative self-perceptions demonstrated deep-rooted beliefs of HIV-related self-stigma that adversely impact the participants. The negative self-perceptions and self-stigmatizing experiences caused the participants to feel worthless, hopeless, shameful, and alone-negatively impacting their physical and mental health, personal relationships, and sense of self-identity. These results can now be used to pursue interventions to target the specific beliefs and experiences of young adults living with HIV and reduce the adverse consequences of self-stigma.

Keywords: beliefs, HIV, self-stigma, stigma, Zimbabwe

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Authors: L. M. H. Mustonen, S. A. Heikkilä

Abstract:

The focus of the paper is to describe team teaching as a HAMK’s pedagogical method, and its impacts to the teachers work. Background: Traditionally it is thought that teaching is a job where one mostly works alone. More and more teachers feel that their work is getting more stressful. Solutions to these problems have been sought in Häme University of Applied sciences’ (From now on referred to as HAMK). HAMK has made a strategic change to move to the group oriented working of teachers. Instead of isolated study courses, there are now larger 15 credits study modules. Implementation: As examples of the method, two cases are presented: technical project module and summer studies module, which was integrated into the EU development project called Energy Efficiency with Precise Control. In autumn 2017, technical project will be implemented third time. There are at least three teachers involved in it and it is the first module of the new students. Main focus is to learn the basic skills of project working. From communicational viewpoint, they learn the basics of written and oral reporting and the basics of video reporting skills. According to our quality control system, the need for the development is evaluated in the end of the module. There are always some differences in each implementation but the basics are the same. The other case summer studies 2017 is new and part of a larger EU project. For the first time, we took a larger group of first to third year students from different study programmes to the summer studies. The students learned professional skills and also skills from different fields of study, international cooperation, and communication skills. Benefits and challenges: After three years, it is possible to consider what the changes mean in the everyday work of the teachers - and of course – what it means to students and the learning process. The perspective is HAMK’s electrical and automation study programme: At first, the change always means more work. The routines born after many years and the course material used for years may not be valid anymore. Teachers are teaching in modules simultaneously and often with some subjects overlapping. Finding the time to plan the modules together is often difficult. The essential benefit is that the learning outcomes have improved. This can be seen in the feedback given by both the teachers and the students. Conclusions: A new type of working environment is being born. A team of teachers designs a module that matches the objectives and ponders the answers to such questions as what are the knowledge-based targets of the module? Which pedagogical solutions will achieve the desired results? At what point do multiple teachers instruct the class together? How is the module evaluated? How can the module be developed further for the next execution? The team discusses openly and finds the solutions. Collegiate responsibility and support are always present. These are strengthening factors of the new communal university teaching culture. They are also strong sources of pleasure of work.

Keywords: pedagogical development, summer studies, team teaching, well-being at work

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