Current Advances in Chemistry and Biochemistry Vol. 1

Synthesis of bimetallic nanoparticles has employed many applications especially as larvicidal agents, these bimetallic nanoparticles therefore need to be produced via a cost-effective and eco-friendly route. Here, green synthesis of Ag-Co hybrid nanoparticles obtained from aqueous root extract of Palmyra palm is reported. The hybrid nanoparticles formation was noticed by a colour change from light pink to light brown and further studied using UV-Vis and FT-IR spectrophotometers. The maximum absorption wavelength, ?_max as determined by the UV-Visible Spectrophotometer was found to be 420 nm. The FT-IR showed the formation and stabilization of the BMNPs. The nanolarvicidal potency was evaluated by the application of varying concentration ranging from 5 to 50 mg/L against first to fourth instars of larvae and recording the percentage mortality after 24 hours. Probit analysis showed the LC₅₀ and LC₉₀ for 1^st instar to be 5.237 mg/L and 49.240 mg/L, 9.310 mg/L and 94.969 mg/L for 2^nd instar, 13.626 mg/L and 105.542 mg/L for 3^rd/4^th instars respectively. This result therefore suggests that the nanoparticles can be used as potential control for larval population growth. These plant-mediated nanoparticles were active against Culex quinquefasciatus mosquito larvae.

Progesterone is considered as the pregnancy hormone.  Progesterone was measured in the saliva of pregnant women at the end of gestation. Samples were collected two times daily (morning and afternoon) during the last two week before the expected delivery date and 2 days after delivery. Analysis was performed using commercial ELISA kits after simple freezing, thawing and centrifugation of the samples. It appeared that during the period before delivery the level of progesterone was in a steady state, until about 2 days before delivery, at which point a rapid decrease could be detected. Immediately after delivery, levels decreased again to non- pregnant values. For comparison reasons, two different ELISA kits were used and evaluated for suitability. In addition, in a preliminary experiment, the applicability and usefulness of the additional determination of salivary estriol was investigated. In conclusion, salivary progesterone may be used as indicator for imminent delivery in pregnant women. Salivary estriol may be an additional parameter that supports the usefulness of salivary progesterone. Further, estriol is said to be a trigger to delivery and is also known to give information about the wellbeing of the fetus. Consequently, its analysis may further contribute to predict the time of delivery, to optionally take measures in the case of premature delivery or complicated pregnancies and give information about the health of the fetus. There is additional research required in order to statistically assess the findings of this study by including more subjects, both having natural deliveries and complicated pregnancies. The results of this study are only intended to indicate the possibility to develop a biosensor for home-use that would greatly contribute to the health of mother and newborn.

Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using Streptococcus pyogenes CRISPR-Cas9 as the model enzyme.

Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data, available on the CRISPR-Cas9 system, were also analyzed.

Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013.

Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were used for analysis. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic studies of this group of enzymes to confirm the possible amino acids involved in the active sites and catalysis.

Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. The HNH endonuclease domains are also found in DNA polymerase and reverse transcriptase genes of group I and group II introns, respectively. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, MSA of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. From the catalytic amino acids identified from them, they are grouped either -DH---N or -HH--N types. There are at least two types of metal-binding sites identified and they bind Mg²⁺ or Zn²⁺ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a catalytic Mg²⁺ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs.

Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor. The N in –HNH- Cas9 confers specificity by interacting with the nucleotide at the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and zinc finger nucleases have already found applications in genome editing.

Kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP.  Although the general features of the kinesin walking mechanism are becoming increasingly clear, some key questions remain unanswered, such as how they convert the chemical energy of ATP into mechanical energy and walk processively. In this study, through molecular simulations and free energy calculations, we found that in aqueous solution, kinesin favors an extended form with its microtubule-binding interface (MTBI) motif unfolded, as seen in a recent x-ray structure of kinesin-8.  Through the flexible fitting of two newly released cryo-electron microscopy (cryo-EM) maps, we derived atomic structures of the kinesin dimer-microtubule complexes in both two-head-bound and one-head bound states.  Free energy calculations showed that kinesin bound to microtubules has a lower free energy than the extended form and that the free energy difference is in the range of the free energy released by ATP hydrolysis.  The transition between the extended and compact forms, the structural differences of the leading and trailing heads, and atomic force simulations suggest a completely new mechanism by which kinesin dimers walk on microtubules. A structure cycle and energy cycle are presented to describe kinesin dimers walking on microtubules. Identifying the extended form of kinesin provides a new target for the regulation of kinesins.

The mineral composition, surface morphology, dispersion and specific surface of dispersed clay fillers of various chemical nature have been experimentally determined. Natural clay disperse fillers are widely used in the development of new polymer composites and other composite materials of various applications. As materials for research used clay mineral disperse fillers of various chemical and mineral composition: Bentonite clay, kaolinite clay and diabase flour. Using the thermogravimetric (TG) analysis and the differential thermal analysis (DTA), the structure of the hydroxyl-hydrated surface layer of the filler surface has been studied. The amount and acidity of the surface active centers of clay fillers have been investigated using pK-metric and pH-metric methods. It was established that the mineral composition is the determining factor in the acid-base surface properties of clay fillers. The thickness of the hydroxyl-hydrated surface layer affects the acidity of the surface, at certain large sizes. It has been shown that clay fillers, which include montmorillonite as the dominant minerals, are characterized by higher acidity than the fillers that consist of kaolinite. In the mixed clay fillers, the acid-base properties of the surface depend on the chemical nature of the impurities and related minerals.

Aquatic reservoirs remain the ultimate sink of chemical pollutants emanating from anthropogenic activities such as agricultural, industrial and mining processes. Chemicals such as pesticides, heavy metals and polycyclic hydrocarbons are generated during the various processes carried out by man and these chemicals find their way to aquatic bodies via runoff, leaching and aerial drifts. Freshwater biota undoubtedly is at risk from the adverse effects of these water pollutants and there is therefore, a need to monitor effects of these chemical pollutants in order to safeguard the health of aquatic life. We investigated the oxidative stress effects of chlorpyrifos and lead on the freshwater snail Helisoma duryi to assess the potential of using this enzyme system as a biondicator of exposure to environmental pollutants. Groups of snails were exposed to 5 ppb lead acetate and 25 ppb chlorpyrifos for 7 days after which half of the snails were sacrificed and the other half allowed to recover in clean water and sacrificed after another 7 days. Post mitochondrial fractions were used to measure the activities of the following antioxidant enzymes; superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and diphosphotriphosphodiaphorase. Both pollutants enhanced the activities of all the antioxidant enzymes suggesting a defensive mechanism by the snail to combat the oxidative stress due to the organophosphate chlopryrifos and metal pollutant lead. There was a significant recovery of the antioxidant defense system of the snails allowed to recover in clean water shown by the reduced alteration of the antioxidant enzyme activities of the snails allowed to recover for 7 days. This supports the need to minimize exposure of aquatic biota to chemical pollutants and if polluted the need remediate the polluted water reservoirs in order to safe guard the health of aquatic organisms.

A series of Lycopodium alkaloids, namely lycosquarosine A (1), acetylaposerratinine (2), huperzine A (3), huperzine B (4), 8?-hydrophlemariurine B (5) and huperzinine (6), has been isolated from Vietnamese Huperzia squarrosa. Among them, lycosquarosine A (1) is the new metabolite of the natural source. Lycosquarosine A completely inhibited AChE activity in a dose dependent manner with an IC₅₀ value of 54.3 ?g/mL, while acetylaposerratinine (2) showed stronger inhibitory activity than 1 with an IC₅₀ value of 15.2 µg/mL. This result indicates that these alkaloids may be a potent source of AChE inhibitors. This is the first report on the alkaloid constituents of H. squarrosa from Vietnam and the potential cholinesterase inhibitory activity of these compounds might suggest new sources of anti-Alzheimer disease agents.

In Vietnam Traditional Medicine, the KNC remedy includes 14 herbs and has been used to treat rheumatism. However, the anti-inflammatory and anti-osteoarthritis studies of KNC have been reported detail so far. The present study aimed to explore the antinociceptive and anti-inflammatory effects of an aqueous extract of the Vietnamese traditional formulation, KNC, in experimental animal models of nociception and inflammation, in vivo. The KNC extract showed anti-inflammatory activity, significantly reducing the development of carrageenan-induced oedema and granuloma weights. The anti-OA effects of KNC resulted in the significant recovery of hind limb weight-bearing, the inhibition of serum cytokine production, include tumour necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6, prostaglandin E2 (PGE₂) and the inhibition of damage and synovial hyperplasia in the knees of a rat OA model. Therefore, KNC extract demonstrated anti-inflammatory and anti-OA activity in Wistar rats.

Thin films have attracted huge interest because of could be used in wide applications including optoelectronic, solar cells, laser devices and gas sensor. In this work, X-ray photoelectron spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, UV-visible spectrophotometer and energy dispersive x-ray have been used to characterize the properties of the obtained thin films. Generally, combinatorial characterization approach is needed to determine good quality of obtained films. The main aim of this book chapter is to summarize the advantages, disadvantages and highlighted experimental results of these tools that are available for the characterization of thin films.  

Seven pimarane diterpenes (1–7) were isolated from Orthosiphon stamineus Benth. by assay-guided isolation. All of the isolates possessed a 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose uptake effect in 3T3-L1 adipocytes at concentrations of 5 and 10 ?M. Most of them showed potent inhibition against protein tyrosine phosphatase 1B with IC₅₀ values ranging from 0.33 to 9.84 ?M. In the kinetic study, all inhibition types were exposed for the examined potencies, including mixed-competitive (1), non-competitives (3 and 5), competitive (6), and uncompetitive (7). The results suggested that O. stamineus and its pimarane diterpenes might exert the hypoglycemic effect via the insulin signaling pathway targeting inhibition of protein tyrosine phosphatase 1B (PTP1B) activity. The results suggest that these active constituents from O. stamineus may be the potential natural products for the development of anti-hypoglycemic agents.

The assay of quinine sulfate, a drug that is administered for the clinical treatment of Plasmodium falciparum, can be accomplished utilizing isocratic solvent conditions and with high performance liquid chromatography (HPLC). Plasmodium falciparum (P. falciparum) will cause the most severe form of human malaria and has been found to be responsible for 85% of all malaria cases. In addition, this study intends to show that quinine can be identified and assayed from raw human urine. The drug quinine is detected after elution with an ultraviolet light detector, which is set at 222 nm (or alternatively at 254 nm). The stock solution of quinine sulfate was prepared in solvent consisting of 64% ethanol (v/v) and 36% water (v/v), at a concentration of 1.3155 x 10^-2 molar. The test samples injected into HPLC instrument were predominantly in a solvent that consisted of 95% (v/v) water and 5% ethanol (v/v). The column solvent can have a range from 5% ethanol to 20% ethanol (v/v), with 1% to 4% glacial acetic acid (v/v) and the remaining volume being distilled water. The limit of detection (LOD) was found to be 2.2628 x 10^-5 molar and the limit of quantitation (LOQ) was found to be 7.542 x 10^-5 molar. This highly sensitive methodology for analysis of quinine from water mixtures, 5% glucose, cellulose based tablets and directly from urine will be useful in determining patient compliance and regimen maintenance, as well as quality control in manufacturing.

ICP-OES emission spectrophotometry is an effective technique for the detection of metals in environmental samples. However, the sensitivity of ICP-OES for lead requires improvement. Studies were done in Atomic absorption spectroscopy, with usage of oxidizers and other additives to improve lead by hydride formation but this present study found the influence of an oxidizing agent without any hydride reaction especially in emission spectroscopy, for the first time to the best of our knowledge. In this attempt, four different oxidizers comprising hydrogen peroxide -nitric acid, vanadium pentoxide-nitric acid, potassium permanganate-nitric acid, ammonium persulphate-nitric acid were compared.  Then the influencing oxidizer and its reaction conditions were optimized. Then the proposed method was applied for the detection of lead in various real samples like old paint scraps, plastic toys and food packaging material. The most noticeable observation of the additives on detectability of lead is only from the combination of hydrogen peroxide and nitric acid. Therefore the detection of lead with the proposed additives using ICP-OES is concluded to be an improvised and simple method. The results revealed that the maximum amount of lead (23.84 mg/g) was present in old paint scraps among the analyzed samples.