The clinical utility and diagnostic implementation of human subject cell transdifferentiation followed by RNA sequencing.

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.

Physico-chemical properties of Moringa oleifera seed oil enzymatically interesterified with palm stearin and palm kernel oil and its potential application in food.

BACKGROUND: High oleic acid Moringa oleifera seed oil (MoO) has been rarely applied in food products due to the low melting point and lack of plasticity. Enzymatic interesterification (EIE) of MoO with palm stearin (PS) and palm kernel oil (PKO) could yield harder fat stocks that may impart desirable nutritional and physical properties. RESULTS: Blends of MoO and PS or PKO were examined for triacylglycerol (TAG) composition, thermal properties and solid fat content (SFC). EIE caused rearrangement of TAGs, reduction of U3 and increase of U2 S in MoO/PS blends while reduction of U3 and S3 following increase of S2 U and U2 S in MoO/PKO blends (U, unsaturated and S, saturated fatty acids). SFC measurements revealed a wide range of plasticity, enhancements of spreadability, mouthfeel and cooling effect for interesterified MoO/PS, indicating the possible application of these blends in margarines. However, interesterified MoO/PKO was not suitable in margarine application, while ice-cream may be formulated from these blends. A soft margarine formulated from MoO/PS 70:30 revealed high oxidative stability during 8 weeks storage with no significant changes in peroxide and p-anisidine values. CONCLUSION: EIE of fats with MoO allowed nutritional and oxidative stable plastic fats to be obtained, suitable for possible use in industrial food applications. © 2015 Society of Chemical Industry.

Physicochemical properties and potential food applications of Moringa oleifera seed oil blended with other vegetable oils.

Blends (30:70, 50:50 and 70:30 w/w) of Moringa oleifera seed oil (MoO) with palm olein (PO), palm stearin (PS), palm kernel oil (PKO) and virgin coconut oil (VCO) were prepared. To determine the physicochemical properties of the blends, the iodine value (IV), saponication value (SV), fatty acid (FA) composition, triacylglycerol (TAG) composition, thermal behaviour (DSC) and solid fat content (SFC) tests were analysed. The incorporation of high oleic acid (81.73%) MoO into the blends resulted in the reduction of palmitic acid content of PO and PS from 36.38% to 17.17% and 54.66% to 14.39% and lauric acid content of PKO and VCO from 50.63% to 17.70% and 51.26% to 26.05% respectively while oleic acid and degree of unsaturation were increased in all blends. Changes in the FA composition and TAG profile have significantly affected the thermal behavior and solid fat content of the oil blends. In MoO/PO blends the melting temperature of MoO decreased while, in MoO/PS, MoO/PKO and MoO/VCO blends, it increased indicating produce of zero-trans harder oil blends without use of partial hydrogenation. The spreadability of PS, PKO and VCO in low temperatures was also increased due to incorporation of MoO. The melting point of PS significantly decreased in MoO/PS blends which proved to be suitable for high oleic bakery shortening and confectionary shortening formulation. The finding appears that blending of MoO with other vegetable oils would enable the initial properties of the oils to be modified or altered and provide functional and nutritional attributes for usage in various food applications, increasing the possibilities for the commercial use of these oils.

Mycelium-bound lipase from a locally isolated strain of Geotrichum candidum.

Mycelium-bound lipase (MBL), from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL), harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.

Hypocholesterolemic activity of monascus fermented product in the absence of monacolins with partial purification for functional food applications.

Hypercholesterolemia is one of the most common chronic diseases in human. Along with chemical therapy traditional medication is used as hypocholesterolemic remedy, however, with unfavorable side effects. Recently, Monascus fermented product (MFP) has become a popular hypocholesterolemic natural supplement. In the present study, the hypocholesterolemic activity of Monascus purpureus FTC5391 fermented product ethanolic extract (MFPe) was investigated in hypercholesterolemic rats. Results showed that MFPe not only reduced the serum total cholesterol (TC), LDL-C, TG concentration, and TC/HDL-C ratio but also increased the HDL-C. Further, solid phase extraction (SPE) was carried out to obtain the hypocholesterolemic bioactive fraction. The high polar fraction of SPE increased the HDL-C (42%) and decreased the TC (53.3%), LDL-C (47%), and TG (50.7%) levels as well as TC/HDL-C ratio (69.1%) in serum. The GC-MS results of the active fraction revealed two main compounds, isosorbide and erythritol, which act as coronary vasodilator compounds.

Improvement of medium chain fatty acid content and antimicrobial activity of coconut oil via solid-state fermentation using a Malaysian Geotrichum candidum.

Coconut oil is a rich source of beneficial medium chain fatty acids (MCFAs) particularly lauric acid. In this study, the oil was modified into a value-added product using direct modification of substrate through fermentation (DIMOSFER) method. A coconut-based and coconut-oil-added solid-state cultivation using a Malaysian lipolytic Geotrichum candidum was used to convert the coconut oil into MCFAs-rich oil. Chemical characteristics of the modified coconut oils (MCOs) considering total medium chain glyceride esters were compared to those of the normal coconut oil using ELSD-RP-HPLC. Optimum amount of coconut oil hydrolysis was achieved at 29% moisture content and 10.14% oil content after 9 days of incubation, where the quantitative amounts of the modified coconut oil and MCFA were 0.330 mL/g of solid media (76.5% bioconversion) and 0.175 mL/g of solid media (53% of the MCO), respectively. MCOs demonstrated improved antibacterial activity mostly due to the presence of free lauric acid. The highest MCFAs-rich coconut oil revealed as much as 90% and 80% antibacterial activities against Staphylococcus aureus and Escherichia coli, respectively. The results of the study showed that DIMOSFER by a local lipolytic G. candidum can be used to produce MCFAs as natural, effective, and safe antimicrobial agent. The produced MCOs and MCFAs could be further applied in food and pharmaceutical industries.

Production of a solvent, detergent, and thermotolerant lipase by a newly isolated Acinetobacter sp. in submerged and solid-state fermentations.

The lipase production ability of a newly isolated Acinetobacter sp. in submerged (SmF) and solid-state (SSF) fermentations was evaluated. The results demonstrated this strain as one of the rare bacterium, which is able to grow and produce lipase in SSF even more than SmF. Coconut oil cake as a cheap agroindustrial residue was employed as the solid substrate. The lipase production was optimized in both media using artificial neural network. Multilayer normal and full feed forward backpropagation networks were selected to build predictive models to optimize the culture parameters for lipase production in SmF and SSF systems, respectively. The produced models for both systems showed high predictive accuracy where the obtained conditions were close together. The produced enzyme was characterized as a thermotolerant lipase, although the organism was mesophile. The optimum temperature for the enzyme activity was 45°C where 63% of its activity remained at 70°C after 2 h. This lipase remained active after 24 h in a broad range of pH (6-11). The lipase demonstrated strong solvent and detergent tolerance potentials. Therefore, this inexpensive lipase production for such a potent and industrially valuable lipase is promising and of considerable commercial interest for biotechnological applications.

Enhancement of viability of a probiotic Lactobacillus strain for poultry during freeze-drying and storage using the response surface methodology.

A rotatable central composite design (CCD) was used to study the effect of cryoprotectants (skim milk, sucrose and lactose) on the survival rate of a probiotic Lactobacillus strain, L. reuteri C10, for poultry, during freeze-drying and storage. Using response surface methodology, a quadratic polynomial equation was obtained for response value by multiple regression analyses: Y = 8.59546-0.01038 X(1)-0.09382 X(2)-0.07771 X(3)-0.054861 X(1)(2)-0.04603 X(3)(2)-0.10938 X(1)X(2). Based on the model predicted, sucrose exerted the strongest effect on the survival rate. At various combinations of cryoprotectants, the viability loss of the cells after freeze-drying was reduced from 1.65 log colony forming units (CFU)/mL to 0.26-0.66 log CFU/mL. The estimated optimum combination for enhancing the survival rate of L. reuteri C10 was 19.5% skim milk, 1% sucrose and 9% lactose. Verification experiments confirmed the validity of the predicted model. The storage life of freeze-dried L. reuteri C10 was markedly improved when cryoprotectants were used. At optimum combination of the cryoprotectants, the survival rates of freeze-dried L. reuteri C10 stored at 4°C and 30°C for 6 months were 96.4% and 73.8%, respectively. Total viability loss of cells which were not protected by cryoprotectants occurred after 12 and 8 weeks of storage at 4°C and 30°C, respectively.

Thermostable lipase from a newly isolated staphylococcus xylosus strain; process optimization and characterization using RSM and ANN

Normal feed forward back-propagation artificial neural network (ANN) and cubic backward elimination response surface methodology (RSM) were used to build a predictive model of the combined effects and optimization of culture parameters for the lipase production of a newly isolated staphylococcus xylosus. The results demonstrated a high predictive accuracy of artificial neural network compared to response surface methodology. The optimum operating condition obtained from the ANN model was found to be at 30ºC incubation temperature, pH 7.5, 60 hrs incubation period, 1.8 percent inoculum size and 60 rpm agitation. The lipase production increased 3.5 fold for optimal medium. The produced enzyme was characterized biochemically and this is the first report about a mesophilic staphylococci bacterium with a high thermostable lipase which is able to retain 50 percent of its activity at 70ºC after 90 min and at 60ºC after 120 min. This lipase is also acidic and alkaline resistant which remains active after 24 hrs in a broad range of pH (4-11).