Glycyrrhizin Production in Licorice Hairy Roots Based on Metabolic Redirection of Triterpenoid Biosynthetic Pathway by Genome Editing.

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

Regulation of RHOV signaling by interaction with SH3 domain-containing adaptor proteins and phosphorylation by PKA.

RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins.

Procedural characteristics and cardiovascular outcomes in patients undergoing drug-coated balloon angioplasty for de novo lesions in large coronary arteries: an observational study.

Limited data exist regarding drug-coated balloon (DCB) treatment in de novo large coronary arteries. We sought to demonstrate procedural characteristics, residual stenosis, and clinical outcomes following DCB angioplasty for de novo lesions in large versus small coronary arteries. The study included 184 consecutive patients with 223 de novo coronary lesions undergoing paclitaxel DCB angioplasty between January 2019 and August 2020, who were divided according to whether the DCB diameter was ≥ 3.0 mm (large group, n = 58) or < 3.0 mm (small group, n = 125). The large group had a higher proportion of acute coronary syndrome more commonly with ostial, bifurcation, and calcified lesions in large vessels and received lesion preparation with more frequent use of scoring or cutting balloons and atherectomy devices compared to the small group. Postprocedural angiographic diameter stenosis was smaller in the large group compared to the small group (31% [22-37] vs. 35% [26-42], p = 0.032), and intravascular ultrasound revealed no significant difference in postprocedural area stenosis between the groups (66.2 ± 7.7% vs. 67.9 ± 7.8%; p = 0.26). The median follow-up duration was 995 days. The incidence of a composite of all-cause death, myocardial infarction, stroke, or target lesion revascularization was similar between the groups (log-rank p = 0.41) and was influenced by the presence of acute coronary syndrome and anemia but not by DCB diameter. The rate of cardiovascular outcomes after DCB treatment was comparable in de novo large and small coronary arteries. Notably, well-planned lesion preparation with intravascular imaging guidance was prevalent in large vessels.

Creating a Thermostable ß-Glucuronidase Switch for Homogeneous Immunoassay by Disruption of Conserved Salt Bridges at Diagonal Interfaces.

Escherichia coli ß-glucuronidase (GUS) has been used as a reporter enzyme in molecular biology and engineered as an enzyme switch for the development of homogeneous biosensors. In this study, we developed a thermostable GUS enzyme switch based on the thermostable GUS mutant TR3337 by disrupting a conserved salt bridge (H514-E523) between the diagonal subunits of its homotetramer. A combinatorial library (240 variants) was screened using a novel high-throughput strategy, which led to the identification of mutant DLW (H514D/M516L/Y517W) as a functional enzyme switch in a caffeine-recognizing immunosensor. Molecular dynamics simulations were performed to predict the topology change around position 514, and a side-chain flip of D514 (repulsion with E523) was observed in the DLW mutant. Up to 1.8-fold of signal-to-background ratio was confirmed when measured at up to 45 °C, thereby highlighting the DLW mutant as a versatile tool for developing thermostable immunosensors for in vitro and in cellulo applications.

The Rho guanine nucleotide exchange factor PLEKHG1 is activated by interaction with and phosphorylation by Src family kinase member FYN.

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.

Development of a Peptide Sensor Derived from Human ACE2 for Fluorescence Polarization Assays of the SARS-CoV-2 Receptor Binding Domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuing emergence of infectious variants have caused a serious pandemic and a global economic slump since 2019. To overcome the situation and prepare for future pandemic-prone diseases, there is a need to establish a convenient diagnostic test that is quickly adaptable to unexpected emergence of virus variants. Here we report a fluorescent peptide sensor 26-Dan and its application to the fluorescence polarization (FP) assay for the highly sensitive and convenient detection of SARS-CoV-2. The 26-Dan sensor was developed by fluorescent labeling of the 26th amino acid of a peptide derived from the N-terminal α-helix of human angiotensin-converting enzyme 2 (hACE2) receptor. The 26-Dan sensor maintained the α-helical structure and showed FP changes in a concentration-dependent manner of the receptor binding domain (RBD) of the virus. The half maximal effective concentrations (EC50's) for RBD of Wuhan-Hu-1 strain, Delta (B.1.617.2), and Omicron (BA.5) variants were 51, 5.2, and 2.2 nM, respectively, demonstrating that the 26-Dan-based FP assay can be adaptable to virus variants that evade standard diagnostic tests. The 26-Dan-based FP assay could also be applied to model screening of a small molecule that inhibits RBD binding to hACE2 and identified glycyrrhizin as a potential inhibitor. The combination of the sensor with a portable microfluidic fluorescence polarization analyzer allowed for the detection of RBD in a femtomolar range within 3 min, demonstrating the assay could be a promising step toward a rapid and convenient test for SARS-CoV-2 and other possible future pandemic-prone diseases.

Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.

Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 µg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.

Impact of prolonged drug-coated balloon inflation on residual stenosis and clinical outcomes in coronary artery disease patients: A propensity score matched analysis.

BACKGROUND: There is a paucity of data regarding the optimal duration of drug-coated balloon (DCB) inflation for coronary lesions. We sought to explore the effect of DCB angioplasty with versus without long inflation time on residual stenosis and clinical outcomes in patients with coronary artery disease. METHODS: This study included 314 consecutive patients with 445 lesions undergoing paclitaxel DCB angioplasty using different inflation time, divided according to whether the total inflation time of the DCB was ≥180 s (prolonged group) or <180 s (standard group). The primary clinical endpoint, defined as a composite of all-cause death, myocardial infarction, stroke, or target lesion revascularization, was examined in 92 propensity score matched pairs. RESULTS: In the matched cohort, the median clinical follow-up period was 947 days. Postprocedural angiographic diameter stenosis was smaller in the prolonged group than in the standard group (30.0% [22.0-37.0] vs. 33.5% [25.5-40.5]; p = 0.042). Intravascular ultrasound measurements revealed that longer DCB inflation time resulted in smaller area stenosis (66.6 ± 7.8% vs. 69.4 ± 7.0%; p = 0.044) and a less mean increase in percent atheroma volume (-11.2 ± 7.1% vs. -7.4 ± 5.9%; p = 0.004) after angioplasty. The rate of the primary endpoint was lower in the prolonged group than in the standard group (log-rank p = 0.025). The efficacy of prolonged DCB inflation was prominent in patients with in-stent restenosis and longer lesions. CONCLUSION: Prolonged DCB inflation was associated with reduced residual stenosis and improved clinical outcomes in patients with coronary artery disease undergoing percutaneous coronary intervention. Prospective randomized trials are warranted to validate the benefits of DCB angioplasty with long inflation time.

Improvement in Inhalation Properties of Theophylline and Levofloxacin by Co-Amorphization and Enhancement in Its Stability by Addition of Amino Acid as a Third Component.

Co-amorphous systems are amorphous formulations stabilized by the miscible dispersion of small molecules. This study aimed to design a stable co-amorphous system for the co-delivery of two drugs to the lungs as an inhaled formulation. Theophylline (THE) and levofloxacin (LEV) were used as model drugs for treating lung infection with inflammation. Leucine (LEU) or tryptophan (TRP) was employed as the third component to improve the inhalation properties. The co-amorphous system containing THE and LEV in an equal molar ratio was successfully prepared via spray drying where reduction of the particle size and change to the spherical morphology were observed. The addition of LEU or TRP at a one-tenth molar ratio to THE-LEV did not affect the formation of the co-amorphous system, but only TRP acted as an antiplasticizer. The Fourier transform infrared spectroscopy spectra revealed intermolecular interactions between THE and LEV in the co-amorphous system that were retained after the addition of LEU or TRP. The co-amorphous THE-LEV system exhibited better in vitro aerodynamic performance than a physical mixture of these compounds and permitted the simultaneous delivery of both drugs in various stages. The co-amorphous THE-LEV system crystallized at 40 °C, and this crystallization was not prevented by LEU. However, THE-LEV-TRP maintained its amorphous state for 1 month. Thus, TRP can act as a third component to improve the physical stability of the co-amorphous THE-LEV system, while maintaining the enhanced aerodynamic properties.

Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering.

Homogeneous immunosensors integrate the advantages of both biosensors and immunoassays; they include speed, high sensitivity, and accuracy. They have been developed rapidly in the past few years and offer a cost-effective alternative technology with rapidity, sensitivity, and user-friendliness, which has been applied in a wide variety of applications. This review introduces the current directions of immunosensor development, focusing on fluorescent and bioluminescent immunosensors and highlighting the advantages, improvements, and key approaches to overcome the limitations of each.

Efficient and rapid linker optimization with heterodimeric coiled coils improves the response of fluorescent biosensors comprising antibodies and protein M.

We developed a coiled Q-probe (CQ-probe), a fluorescent probe containing a coiled-coil peptide pair E4/K4, to convert antibodies into biosensors for homogeneous immunoassays. This probe consists of an antibody-binding protein, protein M (PM) with the E4 peptide and the K4 peptide with a fluorescent dye. Compared to PM Q-probes, which are generated by modifying the C-terminus of PM with a fluorescent dye, CQ-probe variants with various linkers are easy to prepare and therefore enable the establishment of biosensors with a significant fluorescence response by localizing the fluorescent dye at the optimal position for quenching and antigen-dependent release. The fluorescence changes of biosensors converted from anti-BGP, anti-cortisol, and anti-testosterone antibodies using the rhodamine 6G (or TAMRA)-labeled CQ-probe upon antigen addition were 13 (or 2.6), 9.7 (or 1.5), and 2.1 (or 1.2) times larger than that of the biosensors converted using the PM Q-probe. Furthermore, the CQ-probe converted anti-digoxin IgG into a functional biosensor, whereas the PM Q-probe/antibody complex showed an insufficient response. This technology exhibits a promising capacity to convert antibodies into high-response biosensors, which are expected to be applied in a wide range of fields, including clinical diagnosis, environmental surveys, food analysis, and biological research.

Comparison of the OPTIVUS-Complex PCI Multivessel Cohort With the Historical CREDO-Kyoto Registry Cohort-3.

BACKGROUND: There is a paucity of data on the effect of optimal intravascular ultrasound (IVUS)-guided percutaneous coronary intervention (PCI) compared with standard PCI or coronary artery bypass grafting (CABG) in patients with multivessel disease.Methods and Results: The OPTIVUS-Complex PCI study multivessel cohort was a prospective multicenter single-arm study enrolling 1,021 patients undergoing multivessel PCI including the left anterior descending coronary artery using IVUS aiming to meet the prespecified criteria for optimal stent expansion. We conducted propensity score matching analyses between the OPTIVUS group and historical PCI or CABG control groups from the CREDO-Kyoto registry cohort-3 (1,565 and 899 patients) fulfilling the inclusion criteria for this study. The primary endpoint was a composite of death, myocardial infarction, stroke, or any coronary revascularization. In the propensity score-matched cohort (OPTIVUS vs. historical PCI control: 926 patients in each group; OPTIVUS vs. historical CABG control: 436 patients in each group), the cumulative 1-year incidence of the primary endpoint was significantly lower in the OPTIVUS group than in the historical PCI control group (10.4% vs. 23.3%; log-rank P<0.001) or the historical CABG control group (11.8% vs. 16.5%; log-rank P=0.02). CONCLUSIONS: IVUS-guided PCI targeting the OPTIVUS criteria combined with contemporary clinical practice was associated with superior clinical outcomes at 1 year compared with not only the historical PCI control, but also the historical CABG control.

Impact of COVID-19 on pediatric surgery cancellations: A retrospective study.

BACKGROUND: Prevalence trends and reasons for pediatric surgery cancellation in Japan during the coronavirus disease 2019 (COVID-19) pandemic have not previously been reported. This study aimed to compare the prevalence and reasons for cancellation of pediatric surgeries in Japan before and during the COVID-19 pandemic. METHODS: This single-center retrospective cohort study reviewed the reasons for surgery cancellations scheduled for patients aged <18 years between the prepandemic period (September 2017-December 2019) and the COVID-19 pandemic period (January 2020-April 2022). The cancellation reasons were classified into four major categories: medical, surgical, patient-related, and administrative. RESULTS: Of the 3395 and 3455 surgeries scheduled before and during the COVID-19 pandemic, 305 (9.0%) and 319 (9.2%) surgeries were canceled (p = 0.737), respectively. The proportion of cancellations due to infections or fever in medical reasons decreased from 67.9% to 56.1% (p = 0.003) and that due to patient-related reasons increased from 6.6% to 15.1% (p = 0.001). Further, the proportion of cancellations due to staff shortages in staff administrative reasons increased from 0.3% to 3.1% (p = 0.011). There was no significant difference in the proportion of surgeries canceled due to surgical reasons between the two periods. CONCLUSIONS: The proportion of cancellations due to infections or fever decreased during the COVID-19 pandemic, while that due to staff shortages increased. Infection prevention is an important measure to address the staff shortages. Implementation of national or regional policies and additional strategic interventions may be required to prepare for disasters like the COVID-19 pandemic.

A Diversifiable Synthetic Platform for the Discovery of New Carbasugar SGLT2 Inhibitors Using Azide-Alkyne Click Chemistry.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are clinically available to control blood glucose levels in diabetic patients via an insulin-independent mechanism. It was found that some carbasugar analogs of known SGLT2 inhibitors exert a high inhibiting ability toward SGLT2 and have a prolonged blood glucose lowering effect. In this study, we designed new candidates of carbasugar SGLT2 inhibitor that can be synthesized using copper-catalyzed azide-alkyne cycloaddition (CuAAC) into an aromatic ring, which is a part of the pharmacophore at the final stage in the synthetic protocol for the easier discovery of superior SGLT2 inhibitors. Based on the results of molecular docking studies, some selected compounds have been synthesized. Evaluation of these compounds using a cell-based assay revealed that the majority of these compounds had SGLT2 inhibitory activity in a dose-dependent manner. The SGLT2 inhibitory activity of 7b and 7c was almost equal to that of SGLT2 inhibitors in current use. Furthermore, molecular dynamics simulations also revealed that 7c is a promising novel SGLT2 inhibitor.

VH-Based Mini Q-Body: A Novel Quench-Based Immunosensor.

Quenchbodies (Q-bodies), a type of biosensor, are antibodies labeled with a fluorescent dye near the antigen recognition site. In the absence of an antigen, the dye is quenched by tryptophans in the antibody sequence; however, in its presence, the dye is displaced and therefore de-quenched. Although scFv and Fab are mainly used to create Q-bodies, this is the first report where a single-domain heavy chain VH from a semi-synthetic human antibody library formed the basis. To create a proof of concept "mini Q-body", a human anti-lysozyme single-domain VH antibody C3 was used. Mini Q-bodies were successfully developed using seven dyes. Different responses were observed depending on the dye and linker length; it was concluded that the optimal linker length for the TAMRA dye was C5, and rhodamine 6G was identified as the dye with the largest de-quenching response. Three single-domain antibodies with sequences similar to that of the C3 antibody were chosen, and the results confirmed the applicability of this method in developing mini Q-bodies. In summary, mini Q-bodies are an easy-to-use and time-saving method for detecting proteins.

A Real-Time Learning Analytics Dashboard for Automatic Detection of Online Learners' Affective States.

Students' affective states describe their engagement, concentration, attitude, motivation, happiness, sadness, frustration, off-task behavior, and confusion level in learning. In online learning, students' affective states are determinative of the learning quality. However, measuring various affective states and what influences them is exceedingly challenging for the lecturer without having real interaction with the students. Existing studies primarily use self-reported data to understand students' affective states, while this paper presents a novel learning analytics system called MOEMO (Motion and Emotion) that could measure online learners' affective states of engagement and concentration using emotion data. Therefore, the novelty of this research is to visualize online learners' affective states on lecturers' screens in real-time using an automated emotion detection process. In real-time and offline, the system extracts emotion data by analyzing facial features from the lecture videos captured by the typical built-in web camera of a laptop computer. The system determines online learners' five types of engagement ("strong engagement", "high engagement", "medium engagement", "low engagement", and "disengagement") and two types of concentration levels ("focused" and "distracted"). Furthermore, the dashboard is designed to provide insight into students' emotional states, the clusters of engaged and disengaged students', assistance with intervention, create an after-class summary report, and configure the automation parameters to adapt to the study environment.

Peripheral Beta-2 Adrenergic Receptors Mediate the Sympathetic Efferent Activation from Central Nervous System to Splenocytes in a Mouse Model of Fibromyalgia.

Abnormalities in the peripheral immune system are involved in the pathophysiology of fibromyalgia, although their contribution to the painful symptoms remains unknown. Our previous study reported the ability of splenocytes to develop pain-like behavior and an association between the central nervous system (CNS) and splenocytes. Since the spleen is directly innervated by sympathetic nerves, this study aimed to examine whether adrenergic receptors are necessary for pain development or maintenance using an acid saline-induced generalized pain (AcGP) model (an experimental model of fibromyalgia) and whether the activation of these receptors is also essential for pain reproduction by the adoptive transfer of AcGP splenocytes. The administration of selective ß2-blockers, including one with only peripheral action, prevented the development but did not reverse the maintenance of pain-like behavior in acid saline-treated C57BL/6J mice. Neither a selective α1-blocker nor an anticholinergic drug affects the development of pain-like behavior. Furthermore, ß2-blockade in donor AcGP mice eliminated pain reproduction in recipient mice injected with AcGP splenocytes. These results suggest that peripheral ß2-adrenergic receptors play an important role in the efferent pathway from the CNS to splenocytes in pain development.

Lysophosphatidic acid receptor type-1 mediates brain activation in micro-positron emission tomography analysis in a fibromyalgia-like mouse model.

The intermittent cold stress-induced generalized pain response mimics the pathophysiological and pharmacotherapeutic features reported for fibromyalgia patients, including the presence of chronic generalized pain and female dominance. In addition, the intermittent cold stress-induced generalized pain is abolished in lysophosphatidic acid receptor type-1 knockout mice, as reported in many cases of neuropathic pain models. This study aimed to identify the brain loci involved in the intermittent cold stress generalized pain response and test their dependence on the lysophosphatidic acid receptor type-1. Positron emission tomography analyses using 2-deoxy-2-[18 F]fluoro-d-glucose in the presence of a pain stimulus showed that intermittent cold stress causes a significant increase in uptake in the ipsilateral regions, including the salience networking-related anterior cingulate cortex and insular cortex and the cognition-related hippocampus. A significant decrease was observed in the default mode network-related posterior cingulate cortex. Almost these intermittent cold stress-induced changes were abolished in lysophosphatidic acid receptor type-1 knockout mice. There results suggest that the intermittent cold stress-induced generalized pain response is mediated by the lysophosphatidic acid receptor type-1 in specific brain loci related to salience networking and cognition, which may lead to further developments in the treatment of fibromyalgia.

Design of a Stable Coamorphous System Using Lactose as an Antiplasticizing Agent for Diphenhydramine Hydrochloride with a Low Glass Transition Temperature.

Coamorphous systems comprising small molecules are emerging as counterparts to polymeric solid dispersions. However, the glass transition temperatures (Tgs) of coamorphous materials are relatively low because of the lack of polymeric carriers with higher Tgs. This study aimed to investigate the applicability of lactose (LAC) as an antiplasticizing coformer to a coamorphous system. Diphenhydramine hydrochloride (DPH) was selected as a model drug (Tg = 16 °C). Differential scanning calorimetry showed a comelting point in addition to a decrease in the neat melting points depending on the composition of the physical mixtures, suggesting that the mixture of DPH-LAC was eutectic. The melting point of the eutectic mixture was calculated according to the Schröder-van Laar equation. The heat of fusion of the eutectic mixture was maximized at a 70:30 molar ratio of DPH to LAC; at this point, the melting peaks of the pure components disappeared. The heat flow profiles following the melting and cooling of DPH-LAC physical mixtures at the ratios from 10:90 to 90:10 showed a single Tg, suggesting the formation of a coamorphous system. Lactose showed a Tg of over 100 °C, and the Tg of DPH increased with the molar ratio of LAC; it was 84 °C at a 10:90 molar ratio of DPH to LAC. The Raman image indicated the formation of a homogeneous dispersion of DPH and LAC in the coamorphous system. Peak shifts in the infrared spectra indicated the presence of intermolecular interactions between the amino group of DPH and the hydroxyl group of LAC. Principal component analysis of the infrared spectra revealed a significant change at the 70:30 molar ratio of DPH to LAC, which was in agreement with the results of the thermal analysis. A stability test at 40 °C revealed rapid crystallization of the supercooled liquid DPH. The coamorphous samples containing 10-50% of LAC remained in an amorphous state for 21 days, and no crystallization was observed for the samples containing >60% of LAC for 28 days. The relatively lower Tg (less than 40 °C) of the coamorphous system containing 10-50% of LAC might have caused crystallization during storage. These findings indicate that LAC, which is a safe and widely used pharmaceutical excipient, can be applied to coamorphous systems as an antiplasticizing coformer.