Jewelry utilizes lead either directly or as a base metal. Costume jewelry requires lead before molding and plating the product with valuable metals. Therefore, such ornaments have a great potential to release heavy metals having health hazards. Also, jewelry makers engaged in preparing German silver, an alloy, apply lead in smelting, alloying, rolling and milling silver wires and pieces. The metal is taken up by blood, soft tissues and bone. The biological effects of lead are dependent upon the level and duration of exposure. Lead inhibits three enzymes of heme biosynthesis- δ-amino-levulinic-acid dehydratase (ALAD), coproporphyrin oxidase, and ferrochelatase, impairing heme synthesis and depressing serum level of erythropoietin resulting in decreased hemoglobin synthesis. Lead exposure also affects calcium metabolism and impair the synthesis of Calcitriol. In the present study, jewelry makers from Dhaka, Bangladesh, were shown to have significantly high levels of lead, protein, albumin, and parathormone in their blood, and significantly high amount of zinc-protoporphyrin and δ-amino-levulinic-acid in their urine. The control group, on the other hand showed significantly higher amounts of calcium (both total and ionized form) Vitamin D3and non-activated erythrocyte ALAD in their blood, along with hemoglobin. It might be due to inhibition of 1-α-hydroxylase enzyme in renal tubules. Lead causes nephro-toxicity and inhibits 1-α- hydroxylase enzyme leading to decreased calcitriol synthesis resulting in impaired calcium absorption across gastro-intestinal tract and renal tubules. Low Vitamin D3and significantly increased Parathyroid hormone (PTH) in study group has been found.
Power electronics and fuel cell technologies play an important role in the field of renewable energy. The demand for fuel cells will increase as fuel cells become the main power source for portable applications. In this application, a high-efficiency converter is an essential requirement and a key parameter of the overall system. This is because the size, cost, efficiency, and reliability of the overall system for portable applications primarily depend on the converter. Therefore, the selection of an appropriate converter topology is an important and fundamental aspect of designing a fuel cell system for portable applications as the converter alone plays a major role in determining the overall performance of the system. This paper presents a review of power electronics applications in fuel cell systems, which include various topology combinations of DC converters and AC inverters and which are primarily used in fuel cell systems for portable or stand-alone applications. This paper also reviews the switching techniques used in power conditioning for fuel cell systems. Finally, this paper addresses the current problem encountered with DC converters and AC inverter.
This paper reports the wireless Shape-Memory-Polymer actuator operated by external radio frequency magnetic fields and its application in a drug delivery device. The actuator is driven by a frequency-sensitive wireless resonant heater which is bonded directly to the Shape-Memory-Polymer and is activated only when the field frequency is tuned to the resonant frequency of heater. The heater is fabricated using a double-sided Cu-clad Polyimide with much simpler fabrication steps compared to previously reported methods. The actuation range of 140 μm as the tip opening distance is achieved at device temperature 44 °C in 30 s using 0.05 W RF power. A repeatability test shows that the actuator's average maximum displacement is 110 μm and standard deviation of 12 μm. An experiment is conducted to demonstrate drug release with 5 μL of an acidic solution loaded in the reservoir and the device is immersed in DI water. The actuator is successfully operated in water through wireless activation. The acidic solution is released and diffused in water with an average release rate of 0.172 μL/min.
This paper reports a method that enables real-time displacement monitoring and control of micromachined resonant-type actuators using wireless radiofrequency (RF). The method is applied to an out-of-plane, spiral-coil microactuator based on shape-memory-alloy (SMA). The SMA spiral coil forms an inductor-capacitor resonant circuit that is excited using external RF magnetic fields to thermally actuate the coil. The actuation causes a shift in the circuit's resonance as the coil is displaced vertically, which is wirelessly monitored through an external antenna to track the displacements. Controlled actuation and displacement monitoring using the developed method is demonstrated with the microfabricated device. The device exhibits a frequency sensitivity to displacement of 10 kHz/µm or more for a full out-of-plane travel range of 466 µm and an average actuation velocity of up to 155 µm/s. The method described permits the actuator to have a self-sensing function that is passively operated, thereby eliminating the need for separate sensors and batteries on the device, thus realizing precise control while attaining a high level of miniaturization in the device.
Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2 expression, showing the highest expression when glucose was depleted and ethanol-acetic acid was increased. Meanwhile, S. cerevisiae showed a constitutive ADH2 expression throughout the fermentation process. Discussion. ADH2 expression in L. fermentati may be subjected to changes in the presence of non-fermentative carbon source. The nucleotide sequence showed that ADH2 transcription could be influenced by other transcription genes of glycolysis oriented due to the lack of specific activation sites for Adr1. Our study suggests that if Adr1 is not capable of promoting LfeADH2 activation, the transcription can be controlled by Rap1 and Sp1 due to their inherent roles. Therefore in future, it is interesting to observe ADH2 gene being highly regulated by these potential transcription factors and functioned as a promoter for yeast under high volume of ethanol and organic acids.
In industrial application, immobilized lipase are typically not reused and served as industrial waste after a certain process is completed. The capacity on the reusability of the spent lipase is not well studied. This current study embarks on reusing the remaining lipase from the spent immobilized enzyme. Active lipases were recovered using a simple reverse micellar extraction (RME). RME is the extraction process of targeted biomolecules using an organic solvent and a surfactant. This method was the first attempt reported on the recovery of the lipase from the used immobilized lipase. RME of the spent lipase was done using the nonionic Triton X-100 surfactant and toluene. Various parameters were optimized to maximize the lipase recovery from the used immobilized lipase. The optimum forward extraction condition was 0.075 M KCl, and backward conditions were at 0.15 M Triton X-100/toluene (pH 6, 2 M KCl) with recovery of 66%. The extracted lipase was immobilized via simple adsorption into the ethanol pretreated carrier. The optimum conditions of immobilization resulted in 96% of the extracted lipase was reimmobilized. The reimmobilized lipase was incubated for 20 h in pH 6 buffer at 50 °C of water bath shaker. The reimmobilized lipase still had 27% residual activity after 18 h of incubation, which higher thermal stability compared to the free lipase. In conclusion, the free lipase was successfully extracted from the spent immobilized lipase and reimmobilized into the new support. It exhibited high thermal stability, and the reusability of the spent lipase will promote continued use of industrial lipase and reduce the cost of the manufacturing process.
Stillbirth is a devastating event to the parents, relatives, friends, and families. The role of anticoagulants in the prevention of unexplained stillbirths is uncertain. An open-label interventional prospective cohort study was conducted on 144 women with a history of unexplained stillbirths. The intervention group had a high umbilical artery resistance index (RI) and received bemiparin. The nonintervention group had a normal RI and did not receive any intervention. We measured the adjusted odds ratio (OR) and 95% confidence interval (CI) of the main outcome for these variables using logistic regression analysis. Fresh stillbirth and early neonatal death rates were lower (P = .005, OR = 11.949 and 95% CI = 2.099-68.014) and newborn weight was higher (P = .015, OR = 0.048, 95% CI = 0.004-0.549) in the group that received bemiparin. Bemiparin is effective in decreasing the rate of stillbirth in women with a history of previous unexplained stillbirths.
The flow of unprocessed sewage through municipal sewers is a great source of water contamination. This study aims to observe the pollutants removal efficiencies of walnut shells as an efficient low-cost adsorbent material compared to gravel materials as an anaerobic filter medium. Two models of the De-Centralized Wastewater Treatment System (DEWATS) were constructed. The wastewater flowing from toilets and handwashing places was connected to anaerobic filters filled with walnut shells and gravel. The efficiency of both filter media in the removal of biological oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS), total dissolved solids (TDS), nitrate (NO3), and phosphate (PO43), pH and temperature were observed at the influent of the settler tank and then at the effluent of the collection tank (CT). Temperature and pH were within the acceptable limit of wastewater discharge. The results also indicated that the walnut shells filter media was more efficient at removing organic pollutants (TSS 94%, BOD5 88%, COD 85%, Nitrate 57%, phosphate 46%, and TDS 29%) than the gravel (TSS 81%, BOD5 82%, COD 84%, Nitrate 35%, phosphate 38%, and TDS 26%) at the successive stages. The average removal efficiency of the walnut shell was 88% while in the gravel case, it was 83%. The removal efficiency of walnut shell filters was extensively better over the complete experiment compared to gravel filters for the removal of pollutants, representing the high sorption capability of the walnut shell material. The results of this study show that the walnut shells may be a very useful substitute for other conventional fillers for anaerobic treatment in the anaerobic filter of DEWATS.
Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications.
Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.
This paper elaborates on the novel intelligence assessment method using the brainwave sub-band power ratio features. The study focuses only on the left hemisphere brainwave in its relaxed state. Distinct intelligence quotient groups have been established earlier from the score of the Raven Progressive Matrices. Sub-band power ratios are calculated from energy spectral density of theta, alpha and beta frequency bands. Synthetic data have been generated to increase dataset from 50 to 120. The features are used as input to the artificial neural network. Subsequently, the brain behaviour model has been developed using an artificial neural network that is trained with optimized learning rate, momentum constant and hidden nodes. Findings indicate that the distinct intelligence quotient groups can be classified from the brainwave sub-band power ratios with 100% training and 88.89% testing accuracies.
The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilic Pseudomonas sp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases from Pseudomonas sp. MIS38 and Serratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0°C and 5°C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future.
Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
The gene encoding for a novel cold-adapted enzyme from family II of bacterial classification (GDSL family) was cloned from the genomic DNA of Photobacterium sp. strain J15 in an Escherichia coli system, yielding a recombinant 36 kDa J15 GDSL esterase which was purified in two steps with a final yield and purification of 38.6 and 15.3 respectively. Characterization of the biochemical properties showed the J15 GDSL esterase had maximum activity at 20 °C and pH 8.0, was stable at 10 °C for 3 h and retained 50 % of its activity after a 6 h incubation at 10 °C. The enzyme was activated by Tween-20, -60 and Triton-X100 and inhibited by 1 mM Sodium dodecyl sulphate (SDS), while β-mercaptoethanol and Dithiothreitol (DTT) enhanced activity by 4.3 and 5.4 fold respectively. These results showed the J15 GDSL esterase was a novel cold-adapted enzyme from family II of lipolytic enzymes. A structural model constructed using autotransporter EstA from Pseudomonas aeruginosa as a template revealed the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine which was verified with site directed mutagenesis on active serine.
A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.
The utilization of cold active lipases in organic solvents proves an excellent approach for chiral synthesis and modification of fats and oil due to the inherent flexibility of lipases under low water conditions. In order to verify whether this lipase can function as a valuable synthetic catalyst, the mechanism concerning activation of the lid and interacting solvent residues in the presence of organic solvent must be well understood. A new alkaline cold-adapted lipase, AMS8, from Pseudomonas fluorescens was studied for its structural adaptation and flexibility prior to its exposure to non-polar, polar aprotic and protic solvents. Solvents such as ethanol, toluene, DMSO and 2-propanol showed to have good interactions with active sites. Asparagine (Asn) and tyrosine (Tyr) were key residues attracted to solvents because they could form hydrogen bonds. Unlike in other solvents, Phe-18, Tyr-236 and Tyr-318 were predicted to have aromatic-aromatic side-chain interactions with toluene. Non-polar solvent also was found to possess highest energy binding compared to polar solvents. Due to this circumstance, the interaction of toluene and AMS8 lipase was primarily based on hydrophobicity and molecular recognition. The molecular dynamic simulation showed that lid 2 (residues 148-167) was very flexible in toluene and Ca(2+). As a result, lid 2 moves away from the catalytic areas, leaving an opening for better substrate accessibility which promotes protein activation. Only a single lid (lid 2) showed the movement following interactions with toluene, although AMS8 lipase displayed double lids. The secondary conformation of AMS8 lipase that was affected by toluene observed a reduction of helical strands and increased coil structure. Overall, this work shows that cold active lipase, AMS8 exhibits distinguish interfacial activation and stability in the presence of polar and non-polar solvents.
In this work, advanced nanoscale surface characterization of CuO Nanoflowers synthesized by controlled hydrothermal approach for significant enhancement of catalytic properties has been investigated. The CuO nanoflower samples were characterized by field-emission scanning electron microscopy (FE-SEM), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, high-resolution transmission electron microscopy (HR-TEM), selected-area electron diffraction (SAED), high-angular annular dark field scanning transmission electron microscopy (HAADF-STEM) with elemental mapping, energy dispersive spectroscopy (STEM-EDS) and UV-Vis spectroscopy techniques. The nanoscale analysis of the surface study of monodispersed individual CuO nanoflower confirmed the fine crystalline shaped morphology composed of ultrathin leaves, monoclinic structure and purified phase. The result of HR-TEM shows that the length of one ultrathin leaf of copper oxide nanoflower is about ~650-700 nm, base is about ~300.77 ± 30 nm and the average thickness of the tip of individual ultrathin leaf of copper oxide nanoflower is about ~10 ± 2 nm. Enhanced absorption of visible light ~850 nm and larger value of band gap energy (1.68 eV) have further supported that the as-grown material (CuO nanoflowers) is an active and well-designed surface morphology at the nanoscale level. Furthermore, significant enhancement of catalytic properties of copper oxide nanoflowers in the presence of H2O2 for the degradation of methylene blue (MB) with efficiency ~96.7% after 170 min was obtained. The results showed that the superb catalytic performance of well-fabricated CuO nanoflowers can open a new way for substantial applications of dye removal from wastewater and environment fields.