Structural Transformation of Bovine Serum Albumin Induced by Dimethyl Sulfoxide and Probed by Fluorescence Correlation Spectroscopy and Additional Methods

Determining the structure of a protein and its transformation under different conditions is key to understanding its activity. The structural stability and activity of proteins in aqueous–organic solvent mixtures, which is an intriguing topic of research in biochemistry, is dependent on the nature of the protein and the properties of the medium. Herein, the effect of a commonly used cosolvent, dimethyl sulfoxide (DMSO), on the structure and conformational dynamics of bovine serum albumin (BSA) protein is studied by fluorescence correlation spectroscopy (FCS) measurements on fluorescein isothiocyanate (FITC)‐labeled BSA. The FCS study reveals a change of the hydrodynamic radius of BSA from 3.7 nm in the native state to 7.0 nm in the presence of 40 % DMSO, which suggests complete unfolding of the protein under these conditions. Fluorescence self‐quenching of FITC has been exploited to understand the conformational dynamics of BSA. The time constant of the conformational dynamics of BSA is found to change from 35 μs in its native state to 50 μs as the protein unfolds with increasing DMSO concentration. The FCS results are corroborated by the near‐UV circular dichroism spectra of the protein, which suggest a loss of its tertiary structure with increasing concentration of DMSO. The intrinsic fluorescence of BSA and the fluorescence response of 1‐anilinonaphthalene‐8‐sulfonic acid, used as a probe molecule, provide information that is consistent with the FCS measurements, except that aggregation of BSA is observed in the presence of 40 % DMSO in the ensemble measurements.     

An Efficient Procedure for the Preparation of 3′-Acetyl-2′,3′-dihydrobenzothiazoles by Ring Contraction of 2′,3′-Dihydro-1,5-Benzothiazepines Under Acetylating Conditions

Reaction of 2-benzoyl-6-hydroxy-3-methyl-5-(2′-substituted-2′,3′-dihydro-1,5-benzothiazein-4′-yl) benzofurans (4a-f) with a mixture of acetic anhydride and pyridine afforded 6-acetoxy-2-benzoyl-3-methyl-5-(3′-acetyl-2′-substitutedstyryl-2′,3′-dihydrobenzothiazole-2′-yl) benzofurans (5a-f) as sole products in good yields. A reaction mechanism for the ring contraction is proposed. All the compounds (5a-f) were screened for their antifeedant activity by the “Non-Choice test method” using 6 h prestarved fourth instar larvae of Spodoptera litura F. Compounds 5a, 5c and 5d exhibited highest antifeedant activity.     

An Unusual Reaction of Dimethyl Sulfonium Methylide with Fhenyl Alkyl Ketones Having p-Electron Donating Substituents

A recent report by El Feraly et. al ³ describing the direct conversion of 5, 7-dimethoxy - 2, 3 - dihydro - 1H-benzcyclopenten- 1 -one(1) to the homologated aldehyde (3) by dimethyl sulfonium 4 methylide⁴ (2), prompted us to present results obtained from our investigation of the reaction of various aromatic aldehydes and ketones, possessing electron releasing p-substituent, with (2). Our interest in examining this particular reaction arose from a need to prepare the oxirane (5) and the aldehyde (6), required in connection with the synthesis and biological evaluation of semi-rigid analogues of catecholamines as probes for the study 5 of adrenergic and dopaminergic receptors.⁵     

A Novel Chemo‐selective Epoxidation across Acrylate Ester: Synthesis of New Water‐Soluble Forskolin Analogues

While removing the TBDMS group from OH protection, a novel epoxidation reaction occurred across acrylate ester attached to a forskolin fragment. Besides spectroscopic data, the epoxide formation was confirmed by ring opening with a secondary amine. This unique epoxidation reaction, to our knowledge, is not known in the literature. This reaction led us to discover a simple deblocking protocol. The epoxide and the desired Michael substrates were used to introduce imidazole into forskolin.     

A Facile Synthesis of 2-Benzoyl-6-Hydroxy-3-Methyl-5-(2-Substituted-2,3-Dihydro-1H-1,5-Benzodiazepin-4-YL) Benzo[b]Furans

The synthesis and characterisation of some new 1,5-benzodiazepines prepared from 4,6-diacetylresorcinol is reported.     

Total Synthesis of Pulchellalactam via an RCM Strategy

Total synthesis of (Z) pulchellalactam, a CD protein tyrosine phosphatase inhibitor, from commercially available methallyl chloride employing ring‐closure metathesis (RCM) as a key step is described.     

rmpM Genosensor for Detection of Human Brain Bacterial Meningitis in Cerebrospinal Fluid

Human brain bacterial meningitis is a life-threatening disease caused mainly by Neisseria meningitidis, lead to damage of the outer membrane covering (meninges) of brain or even death. The usual methods of diagnosis are either time-consuming or have some limitations. The specific rmpM (reduction-modifiable protein M) virulent gene based genosensor is more sensitive, specific, and can detect N. meningitidis directly from the patient cerebrospinal fluid in 30 min including 1-min response time. 5′-Thiol-labeled single-stranded DNA (ssDNA) probe was immobilized onto screen-printed gold electrode (SPGE) and hybridized with denatured (95 °C) single-stranded genomic DNA (ssG-DNA) for 10 min at 25 °C. The electrochemical response was measured by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance using redox indicators. The sensitivity of the genosensor was 9.5087?(μA/cm²)/ng with DPV and limit of detection was 3 ng/6 μL ssG-DNA. The immobilization of the ssDNA probe and hybridization with ssG-DNA from N. meningitidis was characterized by atomic force microscopy and Fourier transform infrared spectroscopy. The rmpM genosensor was stable for 6 months at 4 °C with 10 % loss in initial DPV current. The advantage of rmpM genosensor is to detect bacterial meningitis simultaneously in multiple patients using SPGE array during an outbreak of the disease.     

Carbon Nanotubes-Based Label-Free Affinity Sensors for Environmental Monitoring

Nanostructures, such as nanowires, nanobelts, nanosprings, and nanotubes, are receiving growing interest as transducer elements of bio/chemical sensors as they provide high sensitivity, multiplexing, small size, and portability. Single-walled carbon nanotubes (SWNTs) are one such class of nanostructure materials that exhibit superior sensing behavior due to its large-surface carbon atoms that are highly responsive to surface adsorption events. Further, their compatibility with modern microfabrication technologies and facile functionalization with molecular recognition elements make them promising candidates for bio/chemical sensors applications. Here, we review recent results on nanosensors based on SWNTs modified with biological receptors such as aptamers, antibodies, and binding proteins, to develop highly sensitive, selective, rapid, and cost-effective label-free chemiresistor/field-effect transistor nanobiosensors for applications in environmental monitoring.     

Discarded Oranges and Brewer’s Spent Grains as Promoting Ingredients for Microbial Growth by Submerged and Solid State Fermentation of Agro-industrial Waste Mixtures

The exploitation of various agro-industrial wastes for microbial cell mass production of Kluyveromyces marxianus, kefir, and Saccharomyces cerevisiae is reported in the present investigation. Specifically, the promotional effect of whole orange pulp on cell growth in mixtures consisting of cheese whey, molasses, and potato pulp in submerged fermentation processes was examined. A 2- to 3-fold increase of cell mass was observed in the presence of orange pulp. Likewise, the promotional effect of brewer’s spent grains on cell growth in solid state fermentation of mixtures of whey, molasses, potato pulp, malt spent rootlets, and orange pulp was examined. The cell mass was increased by 3-fold for K. marxianus and 2-fold for S. cerevisiae in the presence of these substrates, proving their suitability for single-cell protein production without the need for extra nutrients. Cell growth kinetics were also studied by measurements of cell counts at various time intervals at different concentrations of added orange pulp. The protein content of the fermented substrates was increased substantially, indicating potential use of mixed agro-industrial wastes of negligible cost, as protein-enriched livestock feed, achieving at the same time creation of added value and waste minimization.     

Use of NAD(P)H Fluorescence Measurement for On-Line Monitoring of Metabolic State of Azohydromonas australica in Poly(3-hydroxybutyrate) Production

Culture fluorescence measurement is an indirect and non-invasive method of biomass estimation to assess the metabolic state of the microorganism in a fermentation process. In the present investigation, NAD(P)H fluorescence has been used for on-line in situ characterization of metabolic changes occurring during different phases of batch cultivation of Azohydromonas australica in growth associated poly(3-hydroxybutyrate) or PHB production. A linear correlation between biomass concentration and net NAD(P)H fluorescence was obtained during early log phase (3–12 h) and late log phase (24–39 h) of PHB fermentation. After 12 h (mid log phase) cultivation PHB accumulation shot up and a drop in culture fluorescence was observed which synchronously exhibited continuous utilization of NAD(P)H for the synthesis of biomass and PHB formation simultaneously. A decrease in the observed net fluorescence value was observed again towards the end of fermentation (at 39 h) which corresponded very well with the culture starvation and substrate depletion towards the end of cultivation inside the bioreactor. It was therefore concluded that NAD(P)H fluorescence measurements could be used for indication of the time of fresh nutrient (substrate) feed during substrate limitation to further enhance the PHB production.