Biosynthesis of Polyhydroxyalkanoates (PHAs) by the Valorization of Biomass and Synthetic Waste

Synthetic pollutants are a looming threat to the entire ecosystem, including wildlife, the environment, and human health. Polyhydroxyalkanoates (PHAs) are natural biodegradable microbial polymers with a promising potential to replace synthetic plastics. This research is focused on devising a sustainable approach to produce PHAs by a new microbial strain using untreated synthetic plastics and lignocellulosic biomass. For experiments, 47 soil samples and 18 effluent samples were collected from various areas of Punjab, Pakistan. The samples were primarily screened for PHA detection on agar medium containing Nile blue A stain. The PHA positive bacterial isolates showed prominent orange–yellow fluorescence on irradiation with UV light. They were further screened for PHA estimation by submerged fermentation in the culture broth. Bacterial isolate 16a produced maximum PHA and was identified by 16S rRNA sequencing. It was identified as Stenotrophomonas maltophilia HA-16 ({"type":"entrez-nucleotide","attrs":{"text":"MN240936","term_id":"1712425297","term_text":"MN240936"}}MN240936), reported first time for PHA production. Basic fermentation parameters, such as incubation time, temperature, and pH were optimized for PHA production. Wood chips, cardboard cutouts, plastic bottle cutouts, shredded polystyrene cups, and plastic bags were optimized as alternative sustainable carbon sources for the production of PHAs. A vital finding of this study was the yield obtained by using plastic bags, i.e., 68.24 ± 0.27%. The effective use of plastic and lignocellulosic waste in the cultivation medium for the microbial production of PHA by a novel bacterial strain is discussed in the current study.     

Micellar electrokinetic chromatography of bilirubin, related compounds, and selected drugs with mixtures of binary bile salts

Micellar electrokinetic chromatography was used to study the behavior of quinine, propranolol, bilirubin, biliverdin dimethyl ester, and xanthobilirubin methyl ester in single and binary bile salt micelle systems comprised of glycocholic acid and glycodeoxycholic acid. Micelle systems studied had total bile salt concentrations in the range of 10-33 mM with molar ratios of 1:0, 2:1, 1:1, 1:2, and 0:1 glycocholic acid:glycodeoxycholic acid. A pH 8.5 phosphate-borate buffer system was used. For all analytes except bilirubin, the smallest migration factors were found in glycocholic acid solutions and the largest in glycodeoxycholic acid solutions. Intermediate migration factors were found for all compounds except bilirubin in the binary bile salt systems. Bilirubin behaved uniquely with its largest migration factors in the binary bile salt mixtures.     

Separation of metal cations in acidic solution by capillary electrophoresis with direct and indirect UV detection

Methods for the detectionn of metal cations under acidic conditions, near PH 2, in capillary electrophoresis (CE) were investigatged. Conditions for direct UV detection of UV absorbing metal cations such as Cr³⁺, Cu²⁺, Fe³⁺, UO₂²⁺, VO²⁺, and VO²⁺ were established With aqueous HCl or HClO₄ as the electroyte carrier. The speciation of vanadium(IV) and vanadium(V) at PH 2.3 by CE was achieved with direct detection at 185 nm. With the strong absorbance at 185 nm, no complexation was needed to detect the metal cations. An indirect UV detrection scheme for acidic conditions was also investigated. Several background carrier electolytes (BCES) were studied including 4-methylbenzylamince, nicontinamide, pyridazine, guanidine, 3-picoline, and chromium (III) to determine their effectivencess under very acidic conditions. The efect of ioni c surfactants and the nonionic surfactant, Trition X-100, on the peak heights and N Values was also studied.     

Cyclooxygenase-2 expression in murine and human nonmelanoma skin cancers: implications for therapeutic approaches

Inflammatory stimuli result in the production of cutaneous eicosanoids, which are known to contribute to the process of tumor promotion. Cyclooxygenase (COX), the rate-limiting enzyme for the production of prostaglandins (PG) from arachidonic acid, exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles, whereas increased COX-2 expression is known to occur in several types of epithelial neoplasms. Enhanced PG synthesis is a potential contributing factor in UVB-induced nonmelanoma skin cancers (NMSC). Increased COX-2 staining occurs in murine skin neoplasms after chronic exposure to carcinogenic doses of UVB. In this study, immunohistochemical and Western blot analyses were employed to assess longitudinally COX-2 expression in a standard mouse UVB complete carcinogenesis protocol and in human basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). During UVB irradiation of mice, COX-2 expression consistently increased in the hyperplastic skin, the benign papillomas and the SCC. COX-2 expression was also increased in human actinic keratoses, SCC and BCC as well as in murine SCC and BCC. The pattern of COX-2 expression was quite variable, occurring in a patchy distribution in some lesions with staining confined mainly to suprabasal cell layers. In general, COX-2 expression progressively became more extensive in benign papillomas and well-differentiated murine SCC. The staining was predominantly cytoplasmic and perinuclear in some focal areas in tissue stroma around both murine and human tumors. Western blot analysis confirmed negative COX-2 expression in normal skin, whereas acute UVB exposure resulted in increased enzyme expression, which continued to increase in developing papillomas and SCC. Because of the evidence indicating a pathogenic role for eicosanoids in murine and human skin neoplasms, we performed studies to assess the anti-inflammatory and anticarcinogenic effects of green tea extracts, which are potent antioxidants. Acute exposure of the human skin to UVB (minimum erythema dose x 4) caused a transient enhancement of the COX-2 expression, which reverted to baseline within hours; however, in murine skin the expression persisted for several days. Pretreatment with the topically applied green tea extract (1 mg/cm2) largely abrogated the acute COX-2 response to UVB in mice or humans. In summary, enhanced COX-2 expression serves as a marker of epidermal UVB exposure for murine and human NMSC. These results suggest that COX-2 inhibitors could have potent anticarcinogenic effects in UVB-induced skin cancer.     

Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites

A versatile integrated system has been developed for the automated enrichment and analysis of phosphopeptides by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This system utilizes two independently controlled high-performance liquid chromatography (HPLC) pumps, an autosampler and microvalves to prepare and elute samples into an ion trap mass spectrometer. The use of robust reversed-phase HPLC columns with integrated ESI emitter tips enables the reproducible detection and identification of low-femtomole quantities of phosphopeptides. The entire system is coordinated through a simple user interface by customized software. The ruggedness of the system is demonstrated by highly reproducible analyses of single and multi-protein digests, while its utility is demonstrated by the thorough evaluation of the relative immunoprecipitation efficiencies of several commercially available anti-phosphotyrosine antibodies.     

Comparison of the kinetics and thermodynamics for methyl radical addition to C=C, C=O, and C=S double bonds

The barriers, enthalpies, and rate constants for the addition of methyl radical to the double bonds of a selection of alkene, carbonyl, and thiocarbonyl species (CH(2)=Z, CH(3)CH=Z, and (CH(3))(2)C=Z, where Z = CH(2), O, or S) and for the reverse beta-scission reactions have been investigated using high-level ab inito calculations. The results are rationalized with the aid of the curve-crossing model. The addition reactions proceed via early transition structures in all cases. The barriers for addition of methyl radical to C=C bonds are largely determined by the reaction exothermicities. Addition to the unsubstituted carbon center of C=C double bonds is favored over addition to the substituted carbon center, both kinetically (lower barriers) and thermodynamically (greater exothermicities). The barriers for addition to C=O bonds are influenced by both the reaction exothermicity and the singlet-triplet gap of the substrate. Addition to the carbon center is favored over addition to the oxygen, also both thermodynamically and kinetically. For the thiocarbonyl systems, addition to the carbon center is thermodynamically favored over addition to sulfur. However, in this case, the reaction is contrathermodynamic, addition to the sulfur center having a lower barrier due to spin density considerations. Entropic differences among corresponding addition and beta-scission reactions are relatively minor, and the differences in reaction rates are thus dominated by differences in the respective reaction barriers.     

Conservation of mus-ms enzyme motions in the apo- and substrate-mimicked state

Solution NMR spin-relaxation experiments were used to compare mus-ms dynamics in RNase A in the apo form and as complexed to the substrate-mimic, pTppAp. The crystal structure of the RNase A/pTppAp complex was determined and demonstrates that this ligand binds at the active site and utilizes established substrate binding sites in its interaction with RNase A. Relaxation-compensated CPMG experiments identify flexible residues in and around the active site in both the apo and pTppAp-bound enzyme. Quantitative analysis of the NMR spin-relaxation dispersion curves show that the time scale of motion in RNase A is unchanged when pTppAp binds and is similar to the time scale for the rate-determining step of the catalytic reaction. Temperature-dependent measurements provide an activation barrier for motion of 5.2 +/- 1.0 kcal/mol and 4.5 +/- 1.2 kcal/mol for the apo and pTppAp forms of RNase A, respectively. These data indicate very similar motion exists in the free and bound enzyme. Additionally, chemical shift data suggests that the magnitude of motion is also similar for these two forms and that it is likely that apo enzyme interconverts to a structure that resembles a ligand-bound form. Likewise, it appears that the bound conformation samples the apo enzyme form even when ligand is present. Taken together the data imply that RNase A is in a preexisting dynamic equilibrium between two conformations that represent the open and closed enzyme forms. These data suggest that ligand binding stabilizes the bound conformer but does not induce it.     

Synthesis, structures and rac/meso isomerization behaviour of bisplanar chiral bis(phosphino-η-indenyl)iron(II) complexes

Syntheses of the phosphinoindenes 1-(diphenylphosphino)-3-methylindene (1b), 3-(diphenylphosphino)-2-methylindene (1c), 1-(diphenylphosphino)-2,3-dimethylindene (1d), 4,7-dimethyl-3-(diphenylphosphino)indene (1e), 1-(diphenylphosphino)-3,4,7-trimethylindene (1f) and 3-(diisopropylphosphino)indene (1i) were carried out by treatment of the appropriate indenide with the appropriate chlorophosphine. The silylphosphinoindene 3-(diphenylphosphino)-1-(trimethylsilyl)indene (1h) was prepared by treatment of the indenide of 3-(diphenylphosphino)indene (1a) with trimethylsilylchloride. These indenes, in addition to 1a, were then used, after deprotonation with BuLi, to prepare the corresponding indenyl ferrocenes, 2a-2e, 2h and 2i, by treatment with ferrous chloride in a 2:1 ratio. These compounds were characterized by ¹H, ¹³C, and ³¹P NMR spectroscopy, as well as by mass spectrometry, except for the highly-sensitive diisopropylphosphine 2i that could only be characterized by ³¹P NMR spectroscopy. All of these ferrocene complexes are bisplanar chiral systems that can potentially form rac and meso isomers. In all cases both isomers were observed but for 2b and 2h only one could be isolated. The rac isomers of complexes 2a, 2b, 2d, and 2e, as well as the meso isomer of 2e, were studied by X-ray crystallography. Only complexes 2a and 2i were observed to undergo rac/meso isomerization processes at ambient temperature in THF solvent. We were unable to prepare the sterically congested hexamethylferrocene 2f. Generally, it was found that increasing substitution on the indenyl ring increases the reactivity and sensitivity of the ferrocene.     

Hydrophobicity effects in the condensation of water films on quartz

The surface forces of thin water films condensed onto crystalline quartz plates have been investigated by ellipsometric measurements of film thickness as a function of disjoining pressure. Quartz substrates ranging from fully hydroxylated (contact angle − 0°) to completely dehydroxylated (contact angle − 45°) were used and the results obtained related to the theoretically predicted van der Waals and electrostatic forces present in the system. Water films on fully hydroxylated quartz are much thicker than expected, whereas films on fully dehydroxylated quartz are close to the Lifschitz prediction of dispersion forces. As the extent of dehydroxylation decreases, the adsorption isotherm approaches that obtained on fully hydroxylated quartz.     

Atomic charges derived from electrostatic potentials: A detailed study

A new algorithm for fitting atomic charges to molecular electrostatic potentials is presented. This method is non-iterative and rapid compared to previous work. Results from a variety of gaussian basis sets, including STO-3G, 3-21G and 6-31G*, are presented. Charges for a representative collection of molecules, comprising both first and second row atoms and anions are tabulated. The effects of using experimental and optimized geometries are explored. Charges derived from these fits are found to adequately reproduce SCF dipole moments. A small split valence representation, 3-21G, appears to yield consistently good results in a reasonable amount of time.