Thermal stability of DNA with interstrand crosslinks

Although many anticancer drugs exert their biological activity by forming DNA interstrand crosslinks (ICLs), the thermodynamics of biologically relevant long crosslinked DNAs has not been intensively studied in contrast to short duplexes. Here, we carry out computer modeling of the shift of melting temperature of long DNAs caused by ICLs taking into account crosslinking effect in itself and concomitant local alterations in the free energy (δG) of the helix-coil transition at sites of ICLs. Depending on δG, DNA interstrand crosslinks at per nucleotide concentration r = 0.05 can change the melting temperature by value from -17 to +47°C, and the influence weakly depends on DNA sequence and GC content. A change in melting temperature caused by introduction of interstrand crosslinking in modified DNA at sites of modifications also depends on δG and varies from 0 to +12°C. Comparison with experiment for the three platinum crosslinking compounds demonstrates utility of the theoretical method for understanding how crosslinking compounds can influence the melting behavior. On the basis of the method, interdependence of local distortions and crosslinking in itself was studied for thermal effect of ICLs. A method for evaluating the nature of the structural alteration that produces a change in thermal stability for short crosslinked DNA is also proposed. The methods can be used for comparative thermodynamic characterization of various DNA crosslinking agents.     

Correlation Between Thermal Aggregation and Stability of Lysozyme with Salts Described by Molar Surface Tension Increment: An Exceptional Propensity of Ammonium Salts as Aggregation Suppressor

Protein aggregation is a critical problem for biotechnology and pharmaceutical industries. Despite the fact that soluble proteins have been used for many applications, our understanding of the effect of the solution chemistry on protein aggregation still remains to be elucidated. This paper investigates the process of thermal aggregation of lysozyme in the presence of various types of salts. The simple law was found; the aggregation rate of lysozyme increased with increasing melting temperature of the protein (T _m) governed by chemical characteristics of additional salts. Ammonium salts were, however, ruled out; the aggregation rates of lysozyme in the presence of the ammonium salts were smaller than the ones estimated from T _m. Comparing with sodium salts, ammonium salts increased the solubility of the hydrophobic amino acids, indicating that ammonium salts adsorb the hydrophobic region of proteins, which leads to the decrease in aggregation more effectively than sodium salts. The positive relation between aggregation rate and T _m was described by another factor such as the surface tension of salt solutions. Fourier transform infrared spectral analysis showed that the thermal aggregates were likely to form β-sheet in solutions that give high molar surface tension increment. These results suggest that protein aggregation is attributed to the surface free energy of the solution.     

Determination of figures of merit for near-infrared and raman spectrometry by net analyte signal analysis for a 4-component solid dosage system

Process analytical technology has elevated the role of sensors in pharmaceutical manufacturing. Often the ideal technology must be selected from many suitable candidates based on limited data. Net analyte signal (NAS) theory provides an effective platform for method characterization based on multivariate figures of merit (FOM). The objective of this work was to demonstrate that these tools can be used to characterize the performance of 2 dissimilar analyzers based on different underlying spectroscopic principles for the analysis of pharmaceutical compacts. A fully balanced, 4-constituent mixture design composed of anhydrous theophylline, lactose monohydrate, microcrystalline cellulose, and starch was generated; it consisted of 29 design points. Six 13-mm tablets were produced from each mixture at 5 compaction levels and were analyzed by near-infrared and Raman spectroscopy. Partial least squares regression and NAS analyses were performed for each component, which allowed for the computation of FOM. Based on the calibration error statistics, both instruments were capable of accurately modeling all constituents. The results of this work indicate that these statistical tools are a suitable platform for comparing dissimilar analyzers and illustrate the complexity of technology selection.     

Challenges and Strategies in Thermal Processing of Amorphous Solid Dispersions: A Review

Thermal processing of amorphous solid dispersions continues to gain interest in the pharmaceutical industry, as evident by several recently approved commercial products. Still, a number of pharmaceutical polymer carriers exhibit thermal or viscoelastic limitations in thermal processing, especially at smaller scales. Additionally, active pharmaceutical ingredients with high melting points and/or that are thermally labile present their own specific challenges. This review will outline a number of formulation and process-driven strategies to enable thermal processing of challenging compositions. These include the use of traditional plasticizers and surfactants, temporary plasticizers utilizing sub- or supercritical carbon dioxide, designer polymers tailored for hot-melt extrusion processing, and KinetiSol® Dispersing technology. Recent case studies of each strategy will be described along with potential benefits and limitations.     

Production of monomeric phenols by thermochemical conversion of biomass: a review

Biomass is a renewable and alternative source for the production of fuels and chemicals. This paper provides a brief survey of lignin precursors as well as thermogravimetric and pyrolysis studies of lignin with special reference to the production of phenols. Thermogravimetric analysis provides information on pyrolysis kinetics while thermogravimetry in combination with mass or infrared spectrometers allowed a rapid characterization of the vapours produced by thermal treatment. Pyrolysis enabled even greater insight into the thermal behaviour of lignin. Pyrolysis of single, dimeric and trimeric model lignin compounds can determine the thermal stability of the intermediate compounds formed and the origin of the pyrolysis products. A free radical mechanism has been suggested as a major route during the early lignin degradation stages followed by a combined free radical and concerted pathway at elevated temperatures. Pyrolysis of lignin in the presence of catalysts as additives was investigated. Significant differences in terms of yields of pyrolysis products and phenolic compounds were observed. The addition of salts resulted in a high weight loss at low temperature and yielded more char than untreated wood. Some metal catalysts such as transition metals and metal oxides such as Fe2O3 and Cu exhibited a better activity in terms of selectivity for the degradation of lignin.     

Supercritical CO2 Generation of Nanometric Structure from Ocimum basilicum Mucilage Prepared for Pharmaceutical Applications

Plant-derived polymers are widely used in the pharmaceutical industry due to their emollient, lack of toxicity, and irritating nature and low cost. In this work, basil seed mucilage was dried using supercritical carbon dioxide phase inversion technique to form a nanometric structure. The obtained polymeric structures were characterized by scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET) method, and Fourier transform infrared spectroscopy (FTIR) and compared with the oven-derived sample group. It was demonstrated that the product morphology could be controlled by altering the composition of methanol which functioned as the co-solvent in the nonsolvent stream. The most homogeneous product (60-nm mean pore size diameter, 78 m²/g BET surface area with no agglomeration) was obtained with 2.5% methanol. The FTIR data showed that the presence of hydroxyl and carboxyl groups suggested the bioadhesive property of basil seed mucilage was good and many active pharmaceutical compounds might be loaded to the resultant nanometric structure to enhance drug release. Furthermore, the FTIR analyses indicated that the nature of the final product did not change during the supercritical drying procedure.KEY WORDS: basil seed gum, drug delivery, FTIR characterization, nanostructure, natural polymer, supercritical phase inversion     

Conformational stability of HPr: the histidine-containing phosphocarrier protein from Bacillus subtilis.

The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1). Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.     

Thermal Stability and Decomposition Kinetic Studies of Acyclovir and Zidovudine Drug Compounds

Investigations on thermal behavior of drug samples such as acyclovir and zidovudine are interesting not only for obtaining stability information for their processing in pharmaceutical industry but also for predicting their shelf lives and suitable storage conditions. The present work describes thermal behaviors and decomposition kinetics of acyclovir and zidovudine in solid state, studied by some thermal analysis techniques including differential scanning calorimetry (DSC) and simultaneous thermogravimetry–differential thermal analysis (TG/DTA). TG analysis revealed that thermal degradation of the acyclovir and zidovudine is started at the temperatures of 400°C and 190°C, respectively. Meanwhile, TG–DTA analysis of acyclovir indicated that this drug melts at about 256°C. However, melting of zidovudine occurred at 142°C, which is 100°C before starting its decomposition (242°C). Different heating rates were applied to study the DSC behavior of drug samples in order to compute their thermokinetic and thermodynamic parameters by non-isothermal kinetic methods. Thermokinetic data showed that both drugs at the room temperature have slow degradation reaction rates and long shelf lives. However, acyclovir is considerably more thermally stable than zidovudine.     

Differential Scanning Calorimetry (DSC): A Tool to Study the Thermal Behavior of Lipid Bilayers and Liposomal Stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process.

The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis.

Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. ΔH and Tm), which affect the stability of the liposomal suspension under given storage conditions.     

Biophysics and Thermodynamics: The Scientific Building Blocks of Bio-inspired Drug Delivery Nano Systems

Biophysics and thermodynamics are considered as the scientific milestones for investigating the properties of materials. The relationship between the changes of temperature with the biophysical variables of biomaterials is important in the process of the development of drug delivery systems. Biophysics is a challenge sector of physics and should be used complementary with the biochemistry in order to discover new and promising technological platforms (i.e., drug delivery systems) and to disclose the ‘silence functionality’ of bio-inspired biological and artificial membranes. Thermal analysis and biophysical approaches in pharmaceuticals present reliable and versatile tools for their characterization and for the successful development of pharmaceutical products. The metastable phases of self-assembled nanostructures such as liposomes should be taken into consideration because they represent the thermal events can affect the functionality of advanced drug delivery nano systems. In conclusion, biophysics and thermodynamics are characterized as the building blocks for design and development of bio-inspired drug delivery systems.KEY WORDS: biophysics, drug delivery nano systems, pharmaceutics, thermal analysis, thermodynamics     

Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models

The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.     

The characteristics of interactions of pharmaceuticals and their active ingredients with lipid membranes

A comparative study of the effects of the active pharmaceutical ingredients of amixin, aspirin, metronidazole, phenibut, and fenspiride, as well as corresponding pharmaceuticals on lipid membranes was carried out. Lipid membranes of L-α-dipalmitoylphosphatidylcholine and native carp spermatozoa were used as models. A decrease in the melting temperature of L-α-dipalmitoylphosphatidylcholine membranes in the presence of all active ingredients and pharmaceuticals was found using differential scanning calorimetry. The only exception was the group of phenibut, where a splitting of the melting peak was observed. It was found that in the pharmaceuticals studied the active pharmaceutical ingredients exhibit a determinative membranotropic effect, whereas excipients play a modulating role. Changes in the parameters of water ν_ОН bands in the L-α-dipalmitoylphosphatidylcholine membranes containing active pharmaceutical ingredients were quantitatively characterized by Fourier transform infrared spectroscopy. According to these parameters, the effect of the phenibut group differed in these parameters from that of the other ingredients. The changes of ν_ОН bands caused by excipients were elucidated. An increase in the permeability of carp sperm cell membranes to water was observed in vitro for pharmaceuticals that induced a decrease in the phase transition temperature of a model membrane (amixin) and lipid lateral phase separation (phenibut).     

Accurate detection of dairy cow mastitis with deep learning technology: a new and comprehensive detection method based on infrared thermal images

Mastitis is one of the most common diseases in dairy cows and has a negative impact on their welfare and life, causing significant economic losses to the dairy industry. Many attempts have been made to develop a detection method for mastitis using thermal infrared thermography. However, the use of this detection technique to determine the health of the cow's udder is susceptible to external factors, resulting in inaccurate detection of dairy cow mastitis. Therefore, this study explored a new and comprehensive detection method of dairy cow mastitis based on infrared thermal images. This method combined the left and right udder skin surface temperature (USST) difference detection method with the ocular surface temperature and USST difference detection method with improvements. The effect of external factors on dairy cow USST was effectively reduced. In addition, after comparing different target localisation algorithms, this paper used the You Only Look Once v5 (YOLOv5) deep learning network model to obtain the temperature information of eyes and udders, and mastitis detection of dairy cows was performed. A total of 105 dairy cows passing through a passage were randomly selected from the thermal infrared video and detected by the new and comprehensive detection method, and the results of cow mastitis detection were compared with somatic cell count. The results showed that the accuracy, specificity, and sensitivity of mastitis detection were 87.62, 84.62, and 96.30%, respectively. Using the YOLOv5 deep learning network model to locate the key parts of the cow had a good effect, with an average accuracy of 96.1%, and an average frame rate of 116.3f/s. The detection accuracy of dairy cow mastitis by deep learning technology combined with the detection method in this paper reached 85.71%. The results showed that the new and comprehensive detection method based on infrared thermal images can be used for the detection of dairy cow mastitis with high detection accuracy. This method can reduce the influence of external factors and can be integrated into the automatic identification system of dairy mastitis based on YOLOv5 to realise on-site monitoring of dairy mastitis.     

Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima.

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.     

1,2,4-benzothiadiazine linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates: synthesis, DNA-binding affinity and cytotoxicity

Benzothiadiazine-pyrrolobenzodiazepine conjugates linked through different alkane spacers have been prepared. These new classes of hybrid molecules exhibit cytotoxicity against many cancer cell lines. Their DNA thermal denaturation studies have been carried out and one of the compounds (4b) elevates the DNA helix melting temperature of the CT-DNA by 6.7 degrees C after incubation for 36 h.     

Recent advances in applications of liquid chromatography-tandem mass spectrometry to the analysis of reactive drug metabolites

Biotransformation of chemically stable compounds to reactive metabolites which can bind covalently to macromolecules, such as proteins and DNA, is considered as an undesirable feature of drug candidates. As part of an overall assessment of absorption, distribution, metabolism and excretion (ADME) properties, many pharmaceutical companies have put methods in place to screen drug candidates for their tendency to generate reactive metabolites and as well characterize the nature of the reactive metabolites through in vitro and in vivo studies. After identification of the problematic compounds, steps can be taken to minimize the potential of bioactivation through appropriate structural modifications. For these reasons, detection, structural characterization and quantification of reactive metabolites by mass spectrometry have become an important task in the drug discovery process. Triple quadrupole mass spectrometry is traditionally employed for the analysis of reactive metabolites. In the past 3 years, a number of new mass spectrometry methodologies have been developed to improve the sensitivity, selectivity and throughput of the analysis. This review focuses on the recent advances in the detection and characterization of reactive metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in drug discovery and development, especially through the use of linear ion trap (LTQ), hybrid triple quadrupole-linear ion trap (Q-trap) and the high resolution LTQ-Orbitrap instruments.     

Differential Scanning Calorimetry (DSC): a tool to study the thermal behavior of lipid bilayers and liposomal stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process. The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis. Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. DeltaH and Tm), which affect the stability of the liposomal suspension under given storage conditions.     

The value of infrared thermography for research on mammals: previous applications and future directions

• 1 Infrared thermography (IRT) involves the precise measurement of infrared radiation which allows surface temperature to be determined according to simple physical laws. This review describes previous applications of IRT in studies of thermal physiology, veterinary diagnosis of disease or injury and population surveys on domestic and wild mammals.
• 2 IRT is a useful technique because it is non‐invasive and measurements can be made at distances of <1 m to examine specific sites of heat loss to >1000 m to count large mammals. Detailed measurements of surface temperature variation can be made where large numbers of temperature sensors would otherwise be required and where conventional solid sensors can give false readings on mammal coats. Studies need to take into account sources of error due to variation in emissivity, evaporative cooling and radiative heating of the coat.
• 3 Recent advances in thermal imaging technology have produced lightweight, portable systems that store digital images with high temperature and spatial resolution. For these reasons, there are many further opportunities for IRT in studies of captive and wild mammals.

     

In silico analysis of squalene synthase in Fabaceae family using bioinformatics tools

Triterpenoid saponins are a diverse group of bioactive compounds, which are used for possessing of many biomedical and pharmaceutical products. Generally, squalene synthase (SQS) is defined as an emerging and essential branch point enzyme far from the major pathway of isoprenoids biosynthetic and a latent adjusting point, which manages carbon flux into triterpenes biosynthesis and sterols. The present study deals with the detailed characterization of SQS by bioinformatics approaches to evaluate physicochemical properties, structural characteristics including secondary and 3D structure prediction and functional analysis from eight plants related to Fabaceae family and Arabidopsis thaliana. Bioinformatics analysis revealed that SQS proteins have two transmembrane regions in the C-terminal. The predicted motifs were used to design universal degenerate primers for PCR analysis and other molecular applications. Phylogenetic analysis showed conserved regions at different stretches with maximum homology in amino acid residues within all SQSs. The secondary structure prediction results showed that the amino acid sequence of all squalene synthases had α helix and random coil as the main components. The reliability of the received model was confirmed using the ProSA and RAMPAGE programs. Determining of active site by CASTp proposes the possibility of using this protein as probable medication target. The findings of the present study may be useful for further assessments on characterization and cloning of squalene synthase.