In studying the ecophysiology of fungal phytopathogens, several stages are involved (in vitro, greenhouse, in planta). Most in vitro studies extensively utilise the general growth media such as Potato Dextrose Agar and Malt Extract Agar. Although the crop components in these media serve as excellent carbon sources and yield luxuriant growth, they are not naturally contaminated with Aspergillus flavus and thus might result in under- or overestimation of its actual toxigenic potentials. Empirical data on the formulation of semi-synthetic growth medium mimicking the natural crop commonly contaminated by A. flavus for the ecophysiological studies in vitro are scarce. The present work was aimed at investigating the ecophysiology of A. flavus on commercial growth media (PDA, MEA); formulating maize- and peanut-based semi-synthetic growth media using two methods of raw material preparation (milling, hot water extraction) at different concentrations (1, 3, 5, 7, 9% w/v), and comparing the ecophysiological parameters between commercial and formulated growth media. Growth rates were obtained by computing the hyphal expansion data into y = mx + c equation. AFB1 was quantified using high performance liquid chromatography with fluorescence detector. Formulated media were found to yield significantly higher growth rates when compared to commercial media. However, commercial media yielded significantly higher AFB1 when compared to all formulated media. Between the two formulations, milling yielded significantly higher growth rates and AFB1 when compared to hot water extraction. Although in vitro data cannot directly extrapolate in planta performance, results obtained in the present work can be used to gauge the actual toxigenic potential of A. flavus in maize and peanut agro-ecosystems.
The present study focused on lipopeptide biosurfactant production by Streptomyces sp. PBD-410L in batch and fed-batch fermentation in a 3-L stirred-tank reactor (STR) using palm oil as a sole carbon source. In batch cultivation, the impact of bioprocessing parameters, namely aeration rate and agitation speed, was studied to improve biomass growth and lipopeptide biosurfactant production. The maximum oil spreading technique (OST) result (45 mm) which corresponds to 3.74 g/L of biosurfactant produced, was attained when the culture was agitated at 200 rpm and aeration rate of 0.5 vvm. The best aeration rate and agitation speed obtained from the batch cultivation was adopted in the fed-batch cultivation using DO-stat feeding strategy to further improve the lipopeptide biosurfactant production. The lipopeptide biosurfactant production was enhanced from 3.74 to 5.32 g/L via fed-batch fermentation mode at an initial feed rate of 0.6 mL/h compared to that in batch cultivation. This is the first report on the employment of fed-batch cultivation on the production of biosurfactant by genus Streptomyces.
Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits.
As pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) are the two major cell types that comprise the immunosuppressive tumor microenvironment of pancreatic cancer, we aimed to investigate the role of conditioned medium derived from PCCs and PSCs co-culture on the viability of lymphocytes. The conditioned medium (CM) collected from PCCs and/or PSCs was used to treat peripheral blood mononuclear cells (PBMCs) to determine CM ability in reducing lymphocytes population. A proteomic analysis has been done on the CM to investigate the differentially expressed protein (DEP) expressed by two PCC lines established from different stages of tumor. Subsequently, we investigated if the reduction of lymphocytes was directly caused by CM or indirectly via CM-induced MDSCs. This was achieved by isolating lymphocyte subtypes and treating them with CM and CM-induced MDSCs. Both PCCs and PSCs were important in suppressing lymphocytes, and the PCCs derived from a metastatic tumor appeared to have a stronger suppressive effect than the PCCs derived from a primary tumor. According to the proteomic profiles of CM, 416 secreted proteins were detected, and 13 DEPs were identified between PANC10.05 and SW1990. However, CM was found unable to reduce lymphocytes viability through a direct pathway. In contrast, CM that contains proteins secreted by PCC and/or PSC appear immunogenic as they increase the viability of lymphocytes subtypes. Lymphocyte subtype treated with CM-induced MDSCs showed reduced viability in T helper 1 (Th1), T helper 2 (Th2), and T regulatory (Treg) cells, but not in CD8+ T cells, and B cells. As a conclusion, the interplay between PCCs and PSCs is important as their co-culture displays a different trend in lymphocytes suppression, hence, their co-culture should be included in future studies to better mimic the tumor microenvironment.
Nowadays, many researches were conducted in minimizing tissue culture technology due to the overhead of cost needed. The purpose of this study was to investigate the effects of using five kinds of organic additives at four level concentrations responsive to the number of shoots produced for eight weeks in culture. Stem segment explants of Celosia sp. were cultured on MS medium that have been supplemented with different kinds of extract juice that serve as organic additives which are mature coconut, young coconut, papaya, banana and tomato at 20, 30, 50 and 70 ml L-1. The numbers of shoot on each explant were recorded and the mean of ten replicates explants were calculated. Among the media used, young coconut water at 70 ml L1- induced the highest shoot regeneration (14.21+/-8.26), followed by mature coconut water at 50 ml L-1 (13.14+/-10.33). Banana and tomato juice promote highest shoot regeneration of stem segments at 50 ml L-1 that produced 9.57+/-4.68 and 9.28+/-5.82 shoots per explants, respectively. While the lowest concentration which at 20 ml L-1 of papaya juice showed highest shoot regeneration (10.50+/-3.45) produced among the three other concentration tested. Statistical results showed that there were significant differences interactions effects (p<0.05) in terms of number of shoot regenerated between the types of extracts juices determined by ANOVA test. Comparing number of shoots regenerated that were cultured in control media, it showed higher than all of experimental medium composition. There were no big different in cost required in preparation of control media and the experimental media. Applications of five kinds of local fruit in tissue culture media should be considered since it responsive in shoot regeneration.
Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.
An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.
Agitation speed was found to influence the tannase production and fungal growth of Aspergillus niger FETL FT3. The optimal agitation speed was at 200 rpm which produced 1.41 U/ml tannase and 3.75 g/l of fungal growth. Lower or higher agitation speeds than 200 rpm produced lower enzyme production and fungal growth. Based on the SEM and TEM micrograph observation, there was a significant correlation between agitation speed and the morphology of the fungal mycelia. The results revealed an increase of the enzyme production with the change of the fungal growth morphology from filamentous to pelleted growth forms. However, the exposure to higher shear stress with an increasing agitation speed of the shaker also resulted in lower biomass yields as well as enzyme production.
Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.
Skin substitutes are one of the main treatments for skin loss, and a skin substitute that is readily available would be the best treatment option. However, most cell-based skin substitutes require long production times, and therefore, patients endure long waiting times. The proteins secreted from the cells and tissues play vital roles in promoting wound healing. Thus, we aimed to develop an acellular three-dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Fibroblasts from human skin samples were cultured using serum-free keratinocyte-specific media (KM1 or KM2) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. The acellular 3D skin patch was soft, semi-solid, and translucent. Collagen mixed with DFCM-KM1 and DFCM-KM2 showed higher protein release compared to collagen plus DFCM-FM. In vitro and in vivo testing revealed that DFCM and collagen hydrogel did not induce an immune response. The implantation of the 3D skin patch with or without DFCM on the dorsum of BALB/c mice demonstrated a significantly faster healing rate compared to the no-treatment group 7 days after implantation, and all groups had complete re-epithelialization at day 17. Histological analysis confirmed the structure and integrity of the regenerated skin, with positive expression of cytokeratin 14 and type I collagen in the epidermal and dermal layer, respectively. These findings highlight the possibility of using fibroblast secretory factors together with collagen hydrogel in an acellular 3D skin patch that can be used allogeneically for immediate treatment of full-thickness skin loss.
Scientists working in assisted reproduction [members of Scientists in Reproductive Technology (SIRT) Australia, and subscribers of the online forums EmbryoMail and Quartec] were invited to complete an online questionnaire on the use of human blood products in assisted reproductive technologies (ART). A total of 260 started the questionnaire, with 208 (80%) completing it. A total of 62% of respondents had worked in human ART ≥8 years and 68% had post-graduate qualifications. The majority (82%) reported using products of animal or human origin, with 75% knowing why protein was added to culture media and 41% not worried by this. Almost half (49%) of respondents were unaware of regulations surrounding the use of human blood products in health care and 70% were unaware of adverse events involving human blood products in human ART. Most respondents (70%) indicated that they were not concerned about infections such as hepatitis, but agents such as prions were a cause for concern (57%). A total of 57% of respondents were unaware of alternatives, but 77% would use a suitable alternative. Using blood products in human ART is surrounded by a lack of awareness, often independent of respondents' qualifications or experience. A better understanding of these products and possible alternatives is required if informed decisions about their suitability are to be made.
Acacia honey is a natural product which has proven to have therapeutic effects on skin wound healing, but its potential healing effects in corneal wound healing have not been studied. This study aimed to explore the effects of Acacia honey (AH) on corneal keratocytes morphology, proliferative capacity, cell cycle, gene and protein analyses. Keratocytes from the corneal stroma of six New Zealand white rabbits were isolated and cultured until passage 1. The optimal dose of AH in the basal medium (FD) and medium containing serum (FDS) for keratocytes proliferation was identified using MTT assay. The morphological changes, gene and protein expressions of aldehyde dehydrogenase (ALDH), marker for quiescent keratocytes and vimentin, marker for fibroblasts were detected using q-RTPCR and immunocytochemistry respectively. Flowcytometry was performed to evaluate the cell cycle analysis of corneal keratocytes.
An efficient and low cost optical method for directly measuring the concentration of homogenous biological solutes is proposed and demonstrated. The proposed system operates by Fresnel reflection, with a flat-cleaved single-mode fiber serving as the sensor probe. A laser provides a 12.9 dBm sensor signal at 1,550 nm, while a computer-controlled optical power meter measures the power of the signal returned by the probe. Three different mesenchymal stem cell (MSC) lines were obtained, sub-cultured and trypsinized daily over 9 days. Counts were measured using a haemocytometer and the conditioned media (CM) was collected daily and stored at -80 °C. MSCs release excretory biomolecules proportional to their growth rate into the CM, which changes the refractive index of the latter. The sensor is capable of detecting changes in the number of stem cells via correlation to the change in the refractive index of the CM, with the measured power loss decreasing approximately 0.4 dB in the CM sample per average 1,000 cells in the MSC subculture. The proposed system is highly cost-effective, simple to deploy, operate, and maintain, is non-destructive, and allows reliable real-time measurement of various stem cell proliferation parameters.
Cupriavidus sp. USMAA1020, a local isolate was able to biosynthesis poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer with various 4HB precursors as the sole carbon source. Manipulation of the culture conditions such as cell concentration, phosphate ratio and culture aeration significantly affected the synthesis of P(3HB-co-4HB) copolymer and 4HB composition. P(3HB-co-4HB) copolymer with 4HB compositions ranging from 23 to 75 mol% 4HB with various mechanical and thermal properties were successfully produced by varying the medium aeration. The physical and mechanical properties of P(3HB-co-4HB) copolymers were characterized by NMR spectroscopy, gel-permeation chromatography, tensile test, and differential scanning calorimetry. The number-average molecular weights (M (n)) of copolymers ranged from 260 x 10(3) to 590 x 10(3)Da, and the polydispersities (M (w)/M (n)) were between 1.8 and 3.0. Increases in the 4HB composition lowered the molecular weight of these copolymers. In addition, the increase in 4HB composition affected the randomness of copolymer, melting temperature (T (m)), glass transition temperature (T (g)), tensile strength, and elongation to break. Enzymatic degradation of P(3HB-co-4HB) films with an extracellular depolymerase from Ochrobactrum sp. DP5 showed that the degradation rate increased proportionally with time as the 4HB fraction increased from 17 to 50 mol% but were much lower with higher 4HB fraction. Degradation of P(3HB-co-4HB) films with lipase from Chromobacterium viscosum exhibited highest degradation rate at 75 mol% 4HB. The biocompatibility of P(3HB-co-4HB) copolymers were evaluated and these copolymers have been shown to support the growth and proliferation of fibroblast cells.
The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.
Infectious diseases, in particular bacterial infections, are the leading cause of morbidity and mortality posing a global threat to human health. The emergence of antibiotic resistance has exacerbated the problem further. Hence, there is a need to search for novel sources of antibacterials. Herein, we explored gut bacteria of a variety of animals living in polluted environments for their antibacterial properties against multi-drug resistant pathogenic bacteria. A variety of species were procured including invertebrate species, Blaptica dubia (cockroach), Gromphadorhina portentosa (cockroach), Scylla serrata (crab), Grammostola rosea (tarantula), Scolopendra subspinipes (centipede) and vertebrate species including Varanus salvator (water monitor lizard), Malayopython reticulatus (python), Cuora amboinensis (tortoise), Oreochromis mossambicus (tilapia fish), Rattus rattus (rat), Gallus gallus domesticus (chicken) and Lithobates catesbeianus (frog). Gut bacteria of these animals were isolated and identified using microbiological, biochemical, analytical profiling index (API) and through molecluar identification using 16S rRNA sequencing. Bacterial conditioned media (CM) were prepared and tested against selected Gram-positive and Gram-negative pathogenic bacteria as well as human cells (HaCaT). The results revealed that CM exhibited significant broad-spectrum antibacterial activities. Upon heat inactivation, CM retained their antibacterial properties suggesting that this effect may be due to secondary metabolites or small peptides. CM showed minimal cytotoxicity against human cells. These findings suggest that gut bacteria of animals living in polluted environments produce broad-spectrum antibacterial molecule(s). The molecular identity of the active molecule(s) together with their mode of action is the subject of future studies which could lead to the rational development of novel antibacterial(s).
Mangrove-dwelling microalgae are well adapted to frequent encounters of salinity fluctuations across their various growth phases but are lesser studied. The current study explored the adaptive changes (in terms of biomass, oil content and fatty acid composition) of mangrove-isolated C. vulgaris UMT-M1 cultured under different salinity levels (5, 10, 15, 20, 30 ppt). The highest total oil content was recorded in cultures at 15 ppt salinity (63.5% of dry weight) with uncompromised biomass productivity, thus highlighting the 'trigger-threshold' for oil accumulation in C. vulgaris UMT-M1. Subsequently, C. vulgaris UMT-M1 was further assessed across different growth phases under 15 ppt. The various short, medium and long-chain fatty acids (particularly C20:0), coupled with a high level of C18:3n3 PUFA reported at early exponential phase represents their physiological importance during rapid cell growth. Accumulation of C18:1 and C18:2 at stationary growth phase across all salinities was seen as cells accumulating substrate for C18:3n3 should the cells anticipate a move from stationary phase into new growth phase. This study sheds some light on the possibility of 'triggered' oil accumulation with uninterrupted growth and the participation of various fatty acid types upon salinity mitigation in a mangrove-dwelling microalgae.
The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.
Mesenchymal stem cells (MSCs) transplantation seems to be a promising new therapy for diabetic wound healing (DWH), and currently, arrays of MSCs from various sources ranging from umbilical, adipose to dental sources are available as a treatment modality for this disease. However, it now appears that only a fraction of transplanted cells actually assimilate and survive in host tissues suggesting that the major mechanism by which stem cells participate in tissue repair are most likely related to their secretome level. These include a wide range of growth factors, cytokines, and chemokines, which can be found from the conditioned medium (CM) used to culture the cells. Basic studies and preclinical work confirm that the therapeutic effect of CMs are comparable with the application of stem cells. This review describes in detail the wound healing process in diabetes and the cellular and biological factors that influence the process. Subsequently, through a comprehensive literature search of studies related to wound healing in diabetics, we aim to provide an overview of scientific merits of using MSCs-CM in the treatment of diabetic wound as well as the significant caveats, which restricts its potential use in clinical set-ups. To our best knowledge, this is one of the first review papers that collect the importance of stem cells as an alternative treatment to the DWH. We anticipate that the success of this treatment will have a significant clinical impact on diabetic wounds.
The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 106 spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.