The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C.
Smoking, passive smoking, and nonsmoking are conditions that give different degrees of stress to the body. In this study, a proteomic technique was used to analyze differentially urinary protein expression between these three groups of subjects. Urinary proteins were precipitated using ammonium sulfate followed by separation according to molecular weights using SDS-PAGE. The gel was stained by Coommassie blue, and the image of the gel was captured for the comparison study. The protein bands that were consistently detected but expressed at different intensity between the smokers and nonsmokers were targeted for further analysis. Three targeted protein bands were excised from the gel, consisting of a unique protein band of smokers and a pair of differentially expressed protein bands from smokers and nonsmokers. The proteins were digested in gel by trypsin. The tryptic peptides were analyzed with ultra performance liquid chromatography-tandem mass spectrometry. Protein identity was determined by the product ion spectrum in the MS/MS scan. Four unique proteins from the smokers, namely, pancreatic alpha amylase, proepidermal growth factor, protein 4.1, and prostatic acid phosphatase, were found to be potential urinary biomarkers to indicate smoking status of a person.
Major concern about the presence of fluoranthene, which consists of four fused benzene rings, in the environment has been raised in the past few years due to its toxic, mutagenic, and persistent organic pollutant properties. In this study, we investigated the removal of fluoranthene under static and agitated conditions. About 89% fluoranthene was removed within 30 days under the agitated condition, whereas under the static condition, only 54% fluoranthene was removed. We further investigated the behavior and mechanism of fluoranthene biosorption and biotransformation by Pleurotus eryngii F032 to accelerate the elimination of fluoranthene. The optimum conditions for the elimination of fluoranthene by P. eryngii F032 included a temperature of 35 °C, pH 3, 0.2% inoculum concentration, and a C/N ratio of 16. Under these conditions at the initial fluoranthene concentration of 10 mg/L, more than 95% of fluoranthene was successfully removed within 30 days. Of those factors influencing the biodegradation of fluoranthene, salinity, glucose, and rhamnolipid content were of the greatest importance. Degradation metabolites identified using gas chromatography-mass spectrometry were 1-naphthalenecarboxylic acid and salicylic acid, suggesting possible metabolic pathways. Finally, it can be presumed that the major mechanism of fluoranthene elimination by white-rot fungi is to mineralize polycyclic aromatic hydrocarbons via biotransformation enzymes like laccase.
Mycobacterium tuberculosis is a causative agent of tuberculosis (TB). The ability of M. tuberculosis to be quiescent in the cell has caused the emergence of latent infection. A comprehensive proteomic analysis of M. tuberculosis H37Rv over three growth phases, namely mid-log (14-day culture), early stationary (28-day culture), and late stationary (50-day culture), was performed in order to study the change in proteome from the mid-log phase to late-stationary phase. Combination methods of two-dimensional electrophoresis (2-DE) and tandem mass spectrometry were used to generate proteome maps of M. tuberculosis at different growth phases. Ten proteins were detected differentially expressed in the late-stationary phase compared with the other two phases. These proteins were SucD, TrpD, and Rv2161c, which belong to metabolic pathway proteins; FadE5, AccD5, DesA1, and Rv1139c are proteins involved in cell wall or lipid biosynthesis, whereas TB21.7 and Rv3224 are conserved hypothetical proteins with unknown function. A surface antigen protein, DesA1, was not detectable in the late-stationary phase, although present in both log and early-stationary phases. The changes in the expression levels of these proteins were in line with the growth environment changes of the bacteria from mid-log phase to late-stationary phase. The information gathered may be valuable in the intervention against latent TB infection.
A white-rot fungus of Polyporus sp. S133 was isolated from an oil-polluted soil. The metabolism of pyrene by this fungus was investigated in liquid medium with 5 mg of the compound. Depletion of pyrene was evident during the 30-day growth period and was 21% and 90%, respectively, in cometabolism and metabolism of pyrene alone. Pyrene was absorbed to fungal cells or biodegraded to form simpler structural compounds. Seventy-one percent of eliminated pyrene was transformed by Polyporus sp. S133 into other compounds, whereas only 18% was absorbed in the fungal cell. The effects of pH and temperature on biomass production of Polyporus sp. S133 for pyrene were examined; the properties of laccase and 1,2-dioxygenase produced by Polyporus sp. S133 during pyrene degradation were investigated. The optimal values of pH were 3, 5, and 4 for laccase, 1,2-dioxygenase, and biomass production, respectively, whereas the optimal values of temperature were 25 °C for laccase and 50 °C for 1,2-dioxygenase and biomass production. Under optimal conditions, pyrene was mainly metabolized to 1-hydroxypyrene and gentisic acid. The structure of 1-hydroxypyrene and gentisic acid was determined by gas chromatography-mass spectrometry after identification using thin-layer chromatography.
Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances, including polysaccharides, polyphenols, and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein content among the four methods. The chloroform-trichloroacetic acid-acetone method using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer provided the best results in terms of protein content, pellets, spot resolution, and intensity of unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2-DE gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies of Andrographis paniculata, which may also be applied to other recalcitrant medicinal plant tissues.
One of the advantages of human adipose-derived stem cells (ASCs) in regenerative medicine is that they can be harvested in abundance. However, the stemness biomarkers, which marked the safety and efficacy of ASCs in accordance with the good manufacturing practice guidelines, is not yet well established. This study was designed to investigate the effect of long-term culture on the stemness properties of ASCs using quantitative real-time polymerase chain reaction and flow cytometry. Results showed the growth rate of ASCs was at its peak when they reached P10 (population doubling; PD = 26) but started to decrease when they were expanded to P15 (PD = 36) and P20 (PD = 46). The ASCs can be culture expanded with minimal alteration in the stemness genes and cluster of differentiation (CD) markers expression up to P10. Expression level of Sox2, Nestin, and Nanog3 was significantly decreased at later passage. CD31, CD45, CD117, and human leukocyte antigen DR, DQ, and DP were lowly expressed at P5 and P10 but their expressions increased significantly at P15 or P20. The differentiation ability of ASCs (adipogenesis, osteogenesis, and neurogenesis) also decreased in long-term culture. Our findings suggested that P10 (PD = 26) should be the "cutoff point" for clinical usage because ASCs at passage 15 onward showed significant changes in the stemness genes, CD markers expression, and differentiation capability.
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.
The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.
Breast cancer is the leading cause of cancer-related mortality and morbidity among women worldwide and IDC (infiltrating ductal carcinoma) is the most common type of invasive breast cancer. The changes in the biological behaviour of cancer tissue can be predicted by measuring the differential protein expression of normal and cancerous tissues. Using a combination of SDS/PAGE and LC (liquid chromatography)-MS/MS (tandem MS), we identified 82 common and differentially expressed proteins from normal and cancerous breast tissues in 20 Malaysian Chinese patients with IDC. These proteins are extracted from the normal and cancerous tissue of patients and therefore represent the actual proteins involved in cancer development. Proteins identified possibly have significant roles in the development of breast cancer in Malaysian Chinese patients in view of their consistent expression in most of the patients, although some of the proteins had not been detected in earlier studies that were mostly carried out in Western countries. This observation suggests that molecular mechanisms leading to breast cancer development in this region may not be identical with those leading to IDC in Western regions.
HBcAg (hepatitis B core antigen) is a nanoplex bioproduct that has a great potential in the development of therapeutic drugs and vaccines. In the present study, a continuous-flow bead milling for the disruption of Escherichia coli was optimized and a direct recovery protocol to isolate the recombinant HBcAg from the unclarified E. coli disruptate was developed. The optimal condition for continuous-flow bead milling for the release of HBcAg from E. coli was achieved at a feed flow rate of 15 litres/h, biomass concentration of 10% [ww/v (wet weight/vol.)] and impeller tip speed of 14 m/s. The sucrose-density-gradient analysis showed that the particulate form of the HBcAg released by this optimal condition is still preserved. In the direct purification of HBcAg from the unclarified disruptate, the AE-EBAC (anion-exchange expanded-bed adsorption chromatography) technique was employed. A 54% adsorption and 50.7% recovery of HBcAg were achieved in this direct recovery process. The purity of HBcAg recovered was 49.8%, which corresponds to a purification factor of 2.0. ELISA showed that the HBcAg recovered is functionally active.
Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002, isolated from palm oil mill effluent, accumulated poly(3-hydroxyalkanoates) (PHAs) when grown on aliphatic fatty acids, sugars, and glycerol. The substrates were supplied at 20:1 C/N mole ratio. Among C-even n-alkanoic acids, myristic acid gave the highest PHA content 26 and 28 wt% in P. putida and D. tsuruhatensis, respectively. Among C-odd n-alkanoic acids, undecanoic gave the highest PHA content at 40 wt% in P. putida and 46 wt% in D. tsuruhatensis on pentadecanoic acid. Sugar and glycerol gave <10 wt% of PHA content for both bacteria. Interestingly, D. tsuruhatensis accumulated both short- and medium-chain length PHA when supplied with n-alkanoic acids ranging from octanoic to lauric, sucrose, and glycerol with 3-hydroxybutyrate as the major monomer unit. In P. putida, the major hydroxyalkanoates unit was 3-hydroxyoctanoate and 3-hydroxydecanoate when grown on C-even acids. Conversely, 3-hydroxyheptanoate, 3-hydrxoynonanoate, and 3-hydroxyundecanoate were accumulated with C-odd acids. Weight-averaged molecular weight (Mw ) was in the range of 53-81 kDa and 107-415 kDa for P. putida and D. tsuruhatensis, respectively. Calorimetric analyses indicated that both bacteria synthesized semicrystalline polymer with good thermal stability with degradation temperature (Td ) ranging from 178 to 282 °C.
The gene encoding a cellobiohydrolase 7B (CBH7B) of the thermophilic fungus Thielavia terrestris was identified, subcloned, and expressed in Pichia pastoris. CBH7B encoded 455 amino acid residues with a molecular mass of 51.8 kDa. Domain analysis indicated that CBH7B contains a family 7 glycosyl hydrolase catalytic core but lacks a carbohydrate-binding module. Purified CBH7B exhibited optimum catalytic activity at pH 5.0 and 55 °C with 4-methylumbelliferryl-cellobioside as the substrate and retained 85% of its activity following 24 H incubation at 50 °C. Despite the lack of activity toward microcrystalline substrates, this enzyme worked synergistically with the commercial enzyme cocktail Cellic(®) CTec2 to enhance saccharification by 39% when added to a reaction mixture containing 0.25% alkaline pretreated oil palm empty fruit bunch (OPEFB). Attenuated total reflectance Fourier transform infrared spectroscopy suggested a reduction of lignin and crystalline cellulose in OPEFB samples supplemented with CBH7B. Scanning electron microscopy revealed greater destruction extent of OPEFB strands in samples supplemented with CBH7B as compared with the nonsupplemented control. Therefore, CBH7B has the potential to complement commercial enzymes in hydrolyzing lignocellulosic biomass.
Results from the present study have shown that the ionic species of buffers, pH values and reaction temperature can affect the enzyme unit activities and product specificity of Toruzyme (Novo Nordisk A/S Bagsvaerd, Denmark) CGTase (cyclodextrin glucanotransferase). Applying a similar reaction environment (acetate buffer, pH 6.0; temperature, 60 degrees C), the CGTase was found to be capable of producing pre dominantly beta-cyclodextrin from either raw or gelatinized sago (Cycas revoluta) starch. Changing the buffer from acetate to phosphate reduced the yield of beta-cyclodextrin from 2.48 to 1.42 mg/ml and also affected the product specificity, where production of both alpha- and beta-cyclodextrins were more pronounced. The decrease in the production of cyclodextrins in phosphate buffer was significant at both pH 6.0 and 7.0. However, changing the buffer to Tris/HCl (pH 7.0) showed a significant increase in beta-cyclodextrin production. Increasing the ionic strength of sodium acetate and Tris/HCl buffers at pH 6.0 and 7.0 to equivalent ionic strength of phosphate buffers showed no significant effects on cyclodextrin production. Higher yield of cyclodextrins at pH 7.0 when Tris/HCl was used might be due to the binding of chloride ions at the calcium-binding sites of the CGTase, resulting in the shift of the optimum pH close to physiological environment, leading to an increase in the activities and specificity.
Ovarian cancer starts in the ovaries in its earlier stages and then spreads to the pelvis, uterus, and abdominal region. The success of an ovarian cancer treatment depends on the stage of the cancer and the diagnostic system. Squamous cell carcinoma antigen (SCC-Ag) is one of the most efficient cancer biomarkers, and elevated levels of SCC-Ag in ovarian cancer cells have been used to identify ovarian cancer. Carbon is a potential material for biosensing applications due to its thermal, electrical, and physical properties. Multiwalled carbon nanotubes (MWCNTs) are carbon-based materials that can be used here to detect SCC-Ag. Anti-SCC-Ag antibody was immobilized on the amine-modified MWCNT dielectric sensing surface to detect SCC-Ag. The uniformity of the surface structure was measured with a 3D nanoprofiler, and the results confirmed the detection of SCC-Ag at ∼80 pM. The specific detection of SCC-Ag was confirmed with two control proteins (factor IX and human serum albumin), and the system did not show biofouling. This experimental set-up with MWCNTs a dielectric sensing surface can lead to the detection of ovarian cancer in its initial stages.
Phytosynthesis of gold nanoparticles (AuNPs) has achieved an indispensable significance due to the diverse roles played by biomolecules in directing the physiochemical characteristics of biosynthesized nanoparticles. Therefore, the precise identification of key bioactive compounds involved in producing AuNPs is vital to control their tunable characteristics for potential applications. Herein, qualitative and quantitative determination of key biocompounds contributing to the formation of AuNPs using aqueous Elaeis guineensis leaves extract is reported. Moreover, roles of phenolic compounds and flavonoids in reduction of Au3+ and stabilization of AuNPs have been elucidated by establishing a reaction mechanism. Fourier-transform infrared spectroscopy (FTIR) showed shifting of O─H stretching vibrations toward longer wavenumbers and C═O toward shorter wavenumbers due to involvement of polyphenolic compounds in biosynthesis and oxidation of polyphenolic into carboxylic compounds, respectively, which cape nanoparticles to inhibit the aggregation. Congruently, pyrolysis-gas chromatography-mass spectrometry revealed the major contribution of polyphenolic compounds in the synthesis of AuNPs, which was further endorsed by reduction of total phenolic and total flavonoids contents from 48.08 ± 1.98 to 9.59 ± 0.92 mg GAE/g and 32.02 ± 1.31 to 13.8 ± 0.97 mg CE/g within 60 Min, respectively. Based on experimental results, reaction mechanism explained the roles of phenolic compounds and flavonoids in producing spherical-shaped AuNPs.
Homocysteine [HSCH2 CH2 CH(NH2 )COOH] (Hcy) is a sulfur-containing amino acid of 135.18 Da of molecular weight, generated during conversion of methionine to cysteine. If there is a higher accumulation of Hcy in the blood, that is usually above 15 µmol/L, it leads to a condition referred to as hyperhomocysteinemia. A meta-analysis of observational study suggested an elevated concentration of Hcy in blood, which is termed as the risk factors leading to ischemic heart disease and stroke. Further experimental studies stated that Hcy can lead to an increase in the proliferation of vascular smooth muscle cells and functional impairment of endothelial cells. The analyses confirmed some of the predictors for Hcy presence, such as serum uric acid (UA), systolic blood pressure, and hematocrit. However, angiotensin-converting enzyme inhibitors angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) alone are inadequate for controlling UA and creatinine level, although the addition of folic acid may be beneficial in hypertensive patients who are known to have a high prevalence of elevated Hcy. We hypothesized that combination therapy with an ARB (olmesartan) and folic acid is a promising treatment for lowering the UA and creatinine level in hyperhomocysteinemia-associated hypertension.
Non-small cell lung cancer (NSCLC) incited by epidermal growth factor receptor (EGFR) mutation makes up ∼85% of lung cancer diagnosed and death cases worldwide. The presented study introduced an alternative approach in detecting EGFR mutation using nano-silica integrated with polydimethylsiloxane (PDMS) polymer on interdigitated electrode (IDE) sensor. A 400 μm gap-sized aluminum IDE was modified with nano-polymer layer, which was made up of silica nanoparticles and PDMS polymer. IDE and PDMS-coated IDE (PDMS/IDE) were imaged using electron microscopes that reveals its smooth and ideal sensor morphology. The nano-silica-integrated PDMS/IDE surface was immobilized with EGFR probe and target to specify the lung cancer detection. The sensor specificity was justified through the insignificant current readouts with one-base mismatch and noncomplementary targets. The sensitivity of nano-silica-integrated PDMS/IDE was examined with mutant target spiked in human serum, where the resulting current affirms the detection of EGFR mutation. Based on the slope of the calibration curve, the sensitivity of nano-silica-integrated PDMS/IDE was 2.24E-9 A M-1 . The sensor recognizes EGFR mutation lowest at 1 aM complementary mutant target; however, the detection limit obtained based on 3σ calculation is 10 aM with regression value of 0.97.
Overuse of antibiotics has led to the development of multi drug resistant strains. Antibiotic resistance is a major drawback in the biomedical field since medical implants are prone to infection by biofilms of antibiotic resistant strains of bacteria. With increasing prevalence of antibiotic resistant pathogenic bacteria, the search for alternative method is utmost importance. In this regard, magnetic nanoparticles are commonly used as a substitute for antibiotics that can circumvent the problem of biofilms growth on the surface of biomedical implants. Iron oxide nanoparticles (IONPs) have unique magnetic properties that can be exploited in various ways in the biomedical applications. IONPs are engineered employing different methods to induce surface functionalization that include the use of polyethyleneimine and oleic acid. IONPs have a mechanical effect on biofilms when in presence of an external magnet. In this review, a detailed description of surface engineered magnetic nanoparticles as ideal antibacterial agents is provided, accompanied by various methods of literature review. This article is protected by copyright. All rights reserved.
This study demonstrated the terminated sialo-sugar chains (Neu5Acα2,6Gal and Neu5Acα2,3Gal) mediated specificity enhancement of influenza virus and chicken red blood cell (RBC) by hemagglutination assay. These glycan chains were immobilized on the gold nanoparticle (GNP) to withhold the higher numbers. With the preliminary optimization, a clear button formation with 0.5% RBC was visualized. On the other hand, intact B/Tokio/53/99 with 750 nM hemagglutinin (HA) displayed a nice hemagglutination. The interference on the specificity of RBC and influenza virus was observed by anti-influenza aptamer at the concentration 31 nM, however, there is no hemagglutination prevention was noticed in the presence of complementary aptamer sequences. Spiking GNP conjugated Neu5Acα2,6Gal or Neu5Acα2,3Gal or a mixture of these two to the reaction promoted the hemagglutination to 63 folds higher with 12 nM virus, whereas under the same condition the heat inactivated viruses were lost the hemagglutination. Neuraminidases from Clostridium perfringens and Arthrobacter ureafaciens at 0.0025 neuraminidase units are able to abolish the hemagglutination. Other enzymes, Glycopeptidase F (Elizabethkingia meningoseptica) and Endoglycosidase H (Streptomyces plicatus) did not show the changes with agglutination. Obviously, sialyl-Gal-terminated glycan conjugated GNP amendment has enhanced the specificity of erythrocyte-influenza virus and able to be controlled by aptamer or neuraminidases. This article is protected by copyright. All rights reserved.