As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short c U 50 T is introduced. Analyzing a set of ene reductases, c U 50 T is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and c U 50 T reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of c U 50 versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.

Silver iodide is a prototype compound of superionic conductors that allows ions to flow through its structure. It exhibits a first-order phase transition at 420 K, characterized by an abrupt change in its ionic conductivity behavior, and above this temperature, its ionic conductivity increases by more than three orders of magnitude. Introducing small concentrations of carbon into the silver iodide structure produces a new material with a mixed conductivity (ionic and electronic) that increases with increasing temperature. In this work, we report the experimental results of the ionic conductivity as a function of the reciprocal temperature for the (AgI)x - C(1-x) mixture at low carbon concentrations (x = 0.99, 0.98, and 0.97). The ionic conductivity behavior as a function of reciprocal temperature was well fitted using a phenomenological model based on a random variable theory with a probability distribution function for the carriers. The experimental data show a proximity effect between the C and AgI phases. As a consequence of this proximity behavior, carbon concentration or temperature can control the conductivity of the (AgI)x - C(1-x) mixture.

The Non-tuberculous mycobacterial (NTM) isolates should be distinguished from tuberculosis and identified at the species level for choosing an appropriate treatment plan. In this study, two molecular methods were used to differentiate NTM species, including a new designed High Resolution Melting (HRM) and Multilocus Sequence Analysis (MLSA). Seventy-five mycobacterial isolates were evaluated by sequencing four genes ( MLSA) and a HRM assay specifically targeting atpE was designed to rapidly and accurately identify and differentiate mycobacterium species. Out of 70 NTM isolates, 66 (94.3%), 65 (92.9%), 65 (92.9%) and 64 (91.4%) isolates were identified to the species level by PCR of atpE, tuf, rpoB and dnaK genes. We could identify 100% of the isolates to the species level (14 different species) by MLSA. By using HRM assay, all NTM isolates were identified and classified into eight groups, in addition, Mycobacterium tuberculosis and Nocardia were also detected simultaneously. The MLSA technique was able to differentiate all 14 species of NTM isolates. According to the results, the HRM assay is a rapid and beneficial method for identifying NTM, M. tuberculosis (MTB), and Nocardia isolates without sequencing.

BACKGROUND: Determination of DPYD and UGT1A1 polymorphisms prior to 5-fluorouracil and irinotecan therapy is crucial for avoiding severe adverse drug effects. Hence, there is a pressing need for accurate and reliable genotyping methods for the most common DPYD and UGT1A1 polymorphisms. In this study, we introduce a novel polymerase chain reaction (PCR) melting curve analysis method for discriminating DPYD c.1236G > A, c.1679 T > G, c.2846A > T, IVS14 + 1G > A and UGT1A1*1, *28, *6 (G71R) genotypes.
METHODS: Following protocol optimization, this technique was employed to genotype 28 patients, recruited between March 2023 and October 2023, at the First Affiliated Hospital of Xiamen University. These patients included 20 with UGT1A1 *1/*1, 8 with UGT1A1 *1/*28, 4 with UGT1A1 *28/*28, 22 with UGT1A1*6 G/G, 6 with UGT1A1*6 G/A, 4 with UGT1A1*6 A/A, 27 with DPYD(c.1236) G/G, 3 with DPYD(c.1236) G/A, 2 with DPYD(c.1236) A/A, 27 with DPYD(c.1679) T/T, 2 with DPYD(c.1679) T/G, 3 with DPYD(c.1679) G/G, 28 with DPYD(c.2846A/T) A/A, 2 with DPYD(c.2846A/T) A/T, 2 with DPYD(c.2846A/T) T/T, 28 with DPYD(c.IVS14 + 1) G/G, 2 with DPYD(c.IVS14 + 1) G/G, and 2 with DPYD(c.IVS14 + 1) G/G, as well as 3 plasmid standards. Method accuracy was assessed by comparing results with those from Sanger sequencing or Multiplex quantitative PCR(qPCR). Intra- and inter-run precision of melting temperatures (Tms) were calculated to evaluate reliability, and sensitivity was assessed through limit of detection examination.
RESULTS: The new method accurately identified all genotypes and exhibited higher accuracy than Multiplex qPCR. Intra- and inter-run coefficients of variation for Tms were both ≤1.97 %, with standard deviations ≤0.95 °C. The limit of detection was 0.09 ng/μL of input genomic DNA.
CONCLUSIONS: Our developed PCR melting curve analysis offers accurate, reliable, rapid, simple, and cost-effective detection of DPYD and UGT1A1 polymorphisms. Its application can be easily extended to clinical laboratories equipped with a fluorescent PCR platform.

Fenofibrate (FNF) is used to treat hyperlipidemia. However, FNF is a poorly water-soluble drug, and the dosage of commercial products is relatively high at 160 mg in a Lipidil® tablet. Therefore, this study aimed to develop an FNF-solid dispersion (SD) that solubilizes and stabilizes FNF. The melting method that uses the low melting point of FNF was employed. The dissolution percentage of FNF in the optimal formulation (SD2) increased by 1.2-, 1.3-, and 1.3-fold at 5 min compared to that of Lipidil® and increased by 2.0-, 2.1-, and 2.0-fold compared to the pure FNF in pH 1.2 media, distilled water, and pH 6.8 buffer, which included 0.025 M sodium lauryl sulfate, respectively. The SD2 formulation showed a dissolution percentage of nearly 100 % in all dissolution media after 60 min. The physicochemical properties of the SD2 formulation exhibited slight changes in the melting point and crystallinity of FNF. Moreover, the stability of the SD2 formulation was maintained for six months. In particular, it was challenging to secure stability when starch#1500 was excluded from the SD2 formulation. In conclusion, the dissolution percentage of FNF in the SD2 formulation was improved owing to the weak binding force between FNF and the excipients, stability was secured, and favorable results are expected in future animal experiments.

BACKGROUND: Molecular syndromic panels can improve rapidity of results and ease clinical laboratory workflow, although caution has been raised for potential false-positive results. Upon implementation of a new panel for infectious diarrhea (BioFire® FilmArray® Gastrointestinal [GI] Panel, bioMérieux) in our clinical laboratory, a higher than expected number of stool samples with norovirus were detected.
OBJECTIVE: The goal of this study was to investigate positive percent agreement and the false-positive rate of norovirus detected by the multiplex BioFire GI panel compared to a singleplex commercial assay.
METHODS: From October 2023 to January 2024, all prospective stool samples with a positive norovirus result by BioFire had melting curves reviewed manually using the BioFire FilmArray Torch System. Stool samples further underwent testing by a supplementary real-time RT-PCR assay (Xpert® Norovirus, Cepheid) for comparative analysis.
RESULTS: Of the 50 stool samples with norovirus detected by BioFire, 18 (36 %) tested negative by Xpert (deemed \"false-positives\"). Furthermore, melting curve analysis revealed nearly all of these samples had atypical melting curve morphologies for the \"Noro-1\" target on BioFire (16/18, 89 %), which was statistically significant (Odds Ratio 173.2, 95 % CI [22.2, 5326.9], p < 0.0001). Stool samples with multiple pathogens detected by BioFire including norovirus were not more likely to produce false-positive norovirus results (Odds Ratio 1, 95 % CI [0.3, 3.3], p = 1).
CONCLUSIONS: Although not described in the manufacturer\'s Instructions for Use, we propose routine manual review of melting curves for the BioFire GI panel prior to reporting, to mitigate potential false-positive norovirus results.

The nearest-neighbor (NN) model is a general tool for the evaluation for oligonucleotide thermodynamic stability. It is primarily used for the prediction of melting temperatures but has also found use in RNA secondary structure prediction and theoretical models of hybridization kinetics. One of the key problems is to obtain the NN parameters from melting temperatures, and VarGibbs was designed to obtain those parameters directly from melting temperatures. Here we will describe the basic workflow from RNA melting temperatures to NN parameters with the use of VarGibbs. We start by a brief revision of the basic concepts of RNA hybridization and of the NN model and then show how to prepare the data files, run the parameter optimization, and interpret the results.

BACKGROUND: The clinically significant Factor V Leiden (FVL) point mutation (1691 G/A) causes replacement of Arg with Gln (glutamine), preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe high-resolution melting analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay (fluorogenic 5\' nuclease PCR hydrolysis assay). HRMA is a post-PCR, homogenous, closed-tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant. Therefore, HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate.
METHODS: Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagNaPure. Reactions of both HRMA and TaqMan were carried out on 3 controls (1691 G/G, 1691 G/A, and 1691 G/G and G/A) and 20 samples.
RESULTS: The genotypes for 3 reference controls purchased from Coriell (F5 1691 G/G, FVL 1691 G/A, and Heterozygote 1691 G/G and G/A) were confirmed by both the HRMA and TaqMan FVL assays. All 20 samples were confirmed to be F5 1691 G/G by both HRMA and TaqMan assays.
CONCLUSIONS: Comparing the results of the unlabeled probe HRMA FVL assay with a real-time TaqMan probe end point genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2═O stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this \"sub-molecular scale thermometer\", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is ∼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL ∼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is ∼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL ∼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.

Sucrose and trehalose pharmaceutical excipients are employed to stabilize protein therapeutics in a dried state. The mechanism of therapeutic protein stabilization is dependent on the sugars being present in an amorphous solid-state. Colyophilization of sugars with high glass transition polymers, polyvinylpyrrolidone (PVP), and poly(vinylpyrrolidone vinyl acetate) (PVPVA), enhances amorphous sugar stability. This study investigates the stability of colyophilized sugar-polymer systems in the frozen solution state, dried state postlyophilization, and upon exposure to elevated humidity. Binary systems of sucrose or trehalose with PVP or PVPVA were lyophilized with sugar/polymer ratios ranging from 2:8 to 8:2. Frozen sugar-PVPVA solutions exhibited a higher glass transition temperature of the maximally freeze-concentrated amorphous phase (Tg\') compared to sugar-PVP solutions, despite the glass transition temperature (Tg) of PVPVA being lower than PVP. Tg values of all colyophilized systems were in a similar temperature range irrespective of polymer type. Greater hydrogen bonding between sugars and PVP and the lower hygroscopicity of PVPVA influenced polymer antiplasticization effects and the plasticization effects of residual water. Plasticization due to water sorption was investigated in a dynamic vapor sorption humidity ramping experiment. Lyophilized sucrose systems exhibited increased amorphous stability compared to trehalose upon exposure to the humidity. Recrystallization of trehalose was observed and stabilized by polymer addition. Lower concentrations of PVP inhibited trehalose recrystallization compared to PVPVA. These stabilizing effects were attributed to the increased hydrogen bonding between trehalose and PVP compared to trehalose and PVPVA. Overall, the study demonstrated how differences in polymer hygroscopicity and hydrogen bonding with sugars influence the stability of colyophilized amorphous dispersions. These insights into excipient solid-state stability are relevant to the development of stabilized biopharmaceutical solid-state formulations.