Celecoxib and sulindac sulfide elicit anticancer effects on PIK3CA-mutated head and neck cancer cells through endoplasmic reticulum stress, reactive oxygen species, and mitochondrial dysfunction.

Gain-of-function mutation in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) is a significant factor in head and neck cancer (HNC). Patients with HNC harboring PIK3CA mutations receive therapeutic benefits from the use of non-steroidal anti-inflammatory drugs (NSAIDs). However, the molecular mechanisms underlying these effects remain unknown. Here, we examined the Detroit562 and FaDu cell lines as HNC models with and without a hyperactive PIK3CA mutation (H1047R), respectively, regarding their possible distinct responses to the NSAIDs celecoxib and sulindac sulfide (SUS). Detroit562 cells exhibited relatively high PI3K/Akt pathway-dependent cyclooxygenase-2 (COX-2) expression, associated with cell proliferation. Celecoxib treatment restricted cell proliferation and upregulated endoplasmic reticulum (ER) stress-related markers, including GRP78, C/EBP-homologous protein, activating transcription factor 4, death receptor 5, and reactive oxygen species (ROS). These effects were much stronger in Detroit562 cells than in FaDu cells and were largely COX-2-independent. SUS treatment yielded similar results. Salubrinal (an ER stress inhibitor) and N-acetyl-L-cysteine (a ROS scavenger) prevented NSAID-induced ROS generation and ER stress, respectively, indicating crosstalk between ER and oxidative stress. In addition, celecoxib and/or SUS elevated cleaved caspase-3 levels, Bcl-2-associated X protein/Bcl-2-interacting mediator of cell death expression, and mitochondrial damage, which was more pronounced in Detroit562 than in FaDu cells. Salubrinal and N-acetyl-L-cysteine attenuated celecoxib-induced mitochondrial dysfunction. Collectively, our results suggest that celecoxib and SUS efficiently suppress activating PIK3CA mutation-harboring HNC progression by inducing ER and oxidative stress and mitochondrial dysfunction, leading to apoptotic cell death, further supporting NSAID treatment as a useful strategy for oncogenic PIK3CA-mutated HNC therapy.

Investigating the effects of Carpesii fructus extract on the liver transcriptome of olive flounder (Paralichthys olivaceus) as a potential antiparasitic agent.

Olive flounder (Paralichthys olivaceus), a popular aquaculture species, is plagued by the disease scuticociliatosis caused by Miamiensis avidus, which has a high mortality rate and is typically treated with chemicals such as formalin and hydrogen peroxide. However, Carpesii fructus extract has shown potential as a natural therapeutic agent by reducing the motility of M. avidus. However, despite its potential importance, the effect of the extract on fish metabolism remains unknown. In this study, the effect of Carpesii fructus extract and formalin on fish metabolism was analysed by whole transcriptome analysis in the liver of P. olivaceus. A total of 37,796 transcripts were generated and differential expression genes (DEGs) were identified in the liver of P. olivaceus treated with Carpesii fructus extract or formalin. In addition, functional analysis of DEGs between treatment groups was presented using Gene Ontology. These results will be crucial for the study of scuticociliatosis in various fish species, including P. olivaceus, and for the development of therapeutic agents for other diseases.

A study on the estimation of area of origin of swing cast-off pattern.

In bloodstain pattern analysis (BPA), a field of forensic science, there has been active discussion on the estimation of the area of origin of impact spatter. However, there is no established methodology to quantitatively analyze the area of origin of a swing cast-off pattern. To quantitatively analyze the methodology of previous research on estimation of area of origin, a device for generating uniform swing cast-off patterns was produced. Using artificial blood, 10 swing cast-off patterns were generated on porous paper; in each, 10 blood drops were selected for the calculation of the impact angle. Hemospat software was used for individual bloodstain analysis, and an open source code was used for estimation of area of origin. Under the same conditions, an additional 10 swing cast-off patterns were generated, and quantitative analysis was performed using trigonometric functions and an adjustment formula that minimized errors in calculating the impact angle. The adjustment formula was corrected to calculate the impact angle for the bloodstains on the porous surface. As uncertainty decreases, the error increases, and the point at which both uncertainty and error can be minimized is calculated as 75%. The existing formula included the trajectory in the estimated likelihood range in 75% of samples. When the adjustment formula was applied, the accuracy was improved, with the trajectory included in the area with a 90% likelihood.

Dataset for selection of stable reference genes for accurate quantitative gene expression analysis in silvertip tetra (Hasemania nana): Implications for sex differentiation and determination.

Real-time quantitative PCR (RT-qPCR) is a widely used method for accurate quantitative gene expression analysis. For accurate quantitative verification of RT-qPCR, it is essential to select a reference gene with high expression stability depending on the experimental environment or the different tissues. In this study, we evaluated the stability of nine candidate reference genes, labeled elongation factor 1-alpha (EF1A), ERBB receptor feedback inhibitor 1-like isoform x2 (ERRFI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), integrin beta2 like (ITGB2), phosphatidylinositol-binding clathrin assembly protein-like isoform x3 (PICALM), 60 s ribosomal protein L5 (RPL5), 60 s ribosomal protein L7 (RPL7), tubulin beta chain (TUBB), and ubiquitin-conjugating enzyme E2A (UBE2A), in the brain (including pituitary gland) gonads and caudal fins of silvertip tetra (Hasemania nana) males and females. The stability evaluation of the reference gene was analyzed using a program based on the geNorm, NormFinder, BestKeeper, and RankAggreg algorithms. As a result, RPL5 (brain, caudal fin), EF1A (gonad), and PICALM (three tissue types) genes were evaluated as the most stable genes in silvertip tetra females. In males, TUBB (brain, caudal fin) and ITGB2 (gonads, three tissue types) genes were the most stable, and in both sexes, TUBB (brain), ITGB2 (caudal fin), RPL5 (gonads), and PICALM (three tissue types) genes are considered appropriate as reference genes for qRT-PCR analysis. However, the GAPDH gene was judged to be inappropriate for use as a reference gene because gene stability in the brain, caudal fin, and gonads was evaluated to be low in all males and females. As an introductory study on silvertip tetra, a new research model fish, the results of this study are expected to provide helpful information regarding sex differentiation and determination in fish.

Preliminary analysis of predicting the first recurrence in patients with neovascular age-related macular degeneration using deep learning.

BACKGROUND: To predict, using deep learning, the first recurrence in patients with neovascular age-related macular degeneration (nAMD) after three monthly loading injections of intravitreal anti-vascular endothelial growth factor (anti-VEGF). METHODS: Optical coherence tomography (OCT) images were obtained at baseline and after the loading phase. The first recurrence was defined as the initial appearance of a new retinal hemorrhage or intra/subretinal fluid accumulation after the initial resolution of exudative changes after three loading injections. Standard U-Net architecture was used to identify the three retinal fluid compartments, which include pigment epithelial detachment, subretinal fluid, and intraretinal fluid. To predict the first recurrence of nAMD, classification learning was conducted to determine whether the first recurrence occurred within three months after the loading phase. The recurrence classification architecture was built using ResNet50. The model with retinal regions of interest of the entire region and fluid region on OCT at baseline and after the loading phase is presented. RESULTS: A total of 1,444 eyes of 1,302 patients were included. The mean duration until the first recurrence after the loading phase was 8.20 ± 15.56 months. The recurrence classification system revealed that the model with the fluid region of OCT after the loading phase provided the highest classification performance, with an area under the receiver operating characteristic curve (AUC) of 0.725 ± 0.012. Heatmap analysis revealed that three pathological fluids, subsided choroidal neovascularization lesions, and hyperreflective foci were important areas for the first recurrence. CONCLUSIONS: The deep learning algorithm allowed for the prediction of the first recurrence for three months after the loading phase with adequate feasibility. An automated prediction system may assist in establishing patient-specific treatment plans and the provision of individualized medical care for patients with nAMD.

Acupuncture for patients with insomnia and predictors of treatment response: a chart review.

BACKGROUND: Acupuncture is a potentially effective non-pharmacological treatment for insomnia. OBJECTIVE: We observed the responses of patients with insomnia to acupuncture in routine clinical practice. In addition, we explored patient characteristics that might affect the treatment response to acupuncture for insomnia. METHODS: Medical records of patients with insomnia in a Korean medicine clinic with baseline Insomnia Severity Index (ISI) scores ⩾8 and Pittsburgh Sleep Quality Index (PSQI) scores ⩾5 were reviewed. Acupuncture was applied at ST43, GB41, ST41, SI5, HT3, KI10, HT7 and ST3, for 1-2 months. The ISI and PSQI were measured monthly to assess insomnia severity. The effect of acupuncture over time was analyzed using a multilevel linear model for repeated measures. In addition, logistic regression was used to explore predictors of treatment response. RESULTS: A total of 91 patients with insomnia aged 59.2 ± 12.5 years (mean ± standard deviation (SD)) (90.1% female) were included in the analysis. After the acupuncture treatment, ISI scores were significantly reduced by -3.75 (95% confidence interval (CI) = -4.99, -2.50) and -4.69 (95% CI = -6.22, -3.16) after the first and second month, respectively. The PSQI global scores also improved, and sleep duration showed a tendency to increase by 0.35 h (95% CI = -0.17, 0.86) after acupuncture treatment. Three cases of mild fatigue were reported. In addition, higher baseline pain/discomfort predicted a greater likelihood of response after acupuncture treatment (odds ratio (OR) = 1.66, 95% CI = 1.10, 2.60). CONCLUSION: In a real-world setting, the insomnia of outpatients in a clinic was slightly alleviated after acupuncture treatment. These findings require validation by randomized controlled trials.

PIP5Kγ Mediates PI(4,5)P2/Merlin/LATS1 Signaling Activation and Interplays with Hsc70 in Hippo-YAP Pathway Regulation.

The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family produces the critical lipid regulator phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM). Here, we investigated the potential role of PIP5Kγ, a PIP5K isoform, in the Hippo pathway. The ectopic expression of PIP5Kγ87 or PIP5Kγ90, two major PIP5Kγ splice variants, activated large tumor suppressor kinase 1 (LATS1) and inhibited Yes-associated protein (YAP), whereas PIP5Kγ knockdown yielded opposite effects. The regulatory effects of PIP5Kγ were dependent on its catalytic activity and the presence of Merlin and LATS1. PIP5Kγ knockdown weakened the restoration of YAP phosphorylation upon stimulation with epidermal growth factor or lysophosphatidic acid. We further found that PIP5Kγ90 bound to the Merlin's band 4.1/ezrin/radixin/moesin (FERM) domain, forming a complex with PI(4,5)P2 and LATS1 at the PM. Notably, PIP5Kγ90, but not its kinase-deficient mutant, potentiated Merlin-LATS1 interaction and recruited LATS1 to the PM. Consistently, PIP5Kγ knockdown or inhibitor (UNC3230) enhanced colony formation in carcinoma cell lines YAP-dependently. In addition, PIP5Kγ90 interacted with heat shock cognate 71-kDa protein (Hsc70), which also contributed to Hippo pathway activation. Collectively, our results suggest that PIP5Kγ regulates the Hippo-YAP pathway by forming a functional complex with Merlin and LATS1 at the PI(4,5)P2-rich PM and via interplay with Hsc70.

Rural-urban differences of sarcopenia and spinal health in the older women: a comparative observational study.

INTRODUCTION: We aimed to investigate the correlation between spinal sarcopenia, spinal sagittal balance (SSB), and spinal function in older women living in rural areas versus those of the older urban women in our previous study. METHODS: Twenty-five older rural-dwelling women aged more than 70 years were compared with 24 older urban-dwelling women from our previous study. Demographic variables, conventional and spinal sarcopenic indices, variable functional outcome parameters, occupational state, and exercise participation rate were evaluated. We also measured the isometric back extensor strength, radiological parameters for SSB on whole-spine radiography, and volumetric parameters of the lumbar extensor muscle on computed tomography. RESULTS: There were no significant intergroup differences in demographic variables or the prevalence of sarcopenia. Older women in rural areas had greater handgrip strength than those in urban areas (22.7±3.7 kg v 20.0±3.4 kg, p=0.010). However, their mean lumbar lordosis angle was lower (31.7±15.3° v 42.3±11.2°, p=0.012). Isometric back extensor strength was lower in rural women than in urban women. The vocational activity participation rate of rural women was significantly higher (84% v 12.5%, p<0.001), whereas their exercise participation rate was significantly lower (60% v 92%, p<0.001). CONCLUSION: Older women in rural areas had greater handgrip strength and vocational participation rates but lower back extensor strength and exercise participation rates. Therefore, more attention is needed for healthcare services to support their spinal health and exercise habits.

Analysis of the Factors of Wound Healing Problems After Transtibial Amputation in Diabetic Patients.

Diabetes mellitus has a global impact, necessitating surgical intervention when conservative methods fail. Transtibial amputation (TTA) is commonly performed on diabetic patients, yet surgical site complications can lead to more procedures. This study aimed to identify factors linked to wound healing issues post-TTA in diabetics.A total of 181 patients who underwent TTA between 2004 and 2021 at a single hospital were included in the study. Exclusion criteria comprised trauma, non-diabetic mellitus, follow-up duration of less than 1 year, incomplete medical records, and surgeries performed by different surgeons. The comparison focused on underlying diseases other than diabetes between the group with wound problems and the group without. Additionally, factors impacting blood flow, such as presurgery hemoglobin levels, intraoperative blood transfusion, the use of antithrombotic or anticoagulant drugs, and the presence of procedures like percutaneous transluminal angioplasty (PTA) and bypass surgery, were analyzed.Among the 181 cases, 22.1% experienced problems at the surgical site while 77.9% did not. Statistical analysis revealed that age was a significant variable affecting wound healing problems after TTA in diabetic patients (p = .007). However, there were no significant differences in wound problems based on comorbidities other than diabetes (p = .209), gender (p = .677), preoperative anemia (p = .102), intraoperative blood transfusion (p = .633), the use of antithrombotic or anticoagulant medications (p = .556), and the performance of PTA or bypass surgery (p = .6).In conclusion, this study found that age was a significant variable affecting wound healing problems after TTA in diabetic patients. Although no association was observed between underlying diseases and wound healing problems, further investigation and cautious management of factors such as preoperative anemia, intraoperative blood transfusion, the use of antithrombotic or anticoagulant drugs, and the performance of PTA or bypass surgery are warranted to prevent complications and optimize wound healing outcomes in diabetic patients undergoing TTA.

Effects of the APOEɛ4 Allele on the Relationship Between Tau and Amyloid-ß in Early- and Late-Onset Alzheimer's Disease.

BACKGROUND: Little is known regarding the differential effects of the apolipoprotein E (APOE) ɛ4 on the regional topography of amyloid and tau in patients with both early-onset (EOAD) and late-onset Alzheimer's disease (LOAD). OBJECTIVE: To compare the distribution and association of tau, amyloid, and cortical thickness among groups classified by the presence of APOEɛ4 allele and onset age. METHODS: A total of 165 participants including 54 EOAD patients (29 ɛ4-; 25 ɛ4+), 45 LOAD patients (21 ɛ4-; 24 ɛ4+), and 66 age-matched controls underwent 3T MRI, 18F-THK5351 (THK) and 18F-flutemetamol (FLUTE) PET scans, APOE genotyping, and neuropsychological tests. Data for voxel-wise and standardized uptake values from PET scans were analyzed in the context of APOE and age at onset. RESULTS: EOAD ɛ4- patients showed greater THK retention in the association cortices, whereas their EOAD ɛ4+ counterparts had more retention in medial temporal areas. THK topography of LOAD ɛ4+ was similar to EOAD ɛ4 + . THK correlated positively with FLUTE and conversely with mean cortical thickness, being lowest in EOAD ɛ4-, highest in LOAD ɛ4-, and modest in ɛ4+ groups. Even in the APOEɛ4+ groups, THK tended to correlate with FLUTE and mean cortical thickness in the inferior parietal region in EOAD and in the medial temporal region in LOAD. LOAD ɛ4- manifested with prevalent small vessel disease markers and the lowest correlation between THK retention and cognition. CONCLUSION: Our observations suggest the differential effects of the APOEɛ4 on the relationship between tau and amyloid in EOAD and LOAD.

Effectiveness and clinical application of multidisciplinary combined exercise and nutrition intervention for sarcopenic older adults with metabolic syndrome: study protocol for a multicentre randomised controlled trial.

INTRODUCTION: Among chronic diseases affecting older adults, metabolic syndrome (MetS) is known to be closely related to sarcopenia. Insulin resistance may play a key role in the increased frequency of sarcopenia associated with metabolic disorders. To date, an exercise-nutrition combined intervention has been the treatment of choice for sarcopenia. However, trials of combined interventions for individuals with sarcopenia and MetS are still lacking. This study aims to develop and conduct a standardised intervention, named the Multidisciplinary combined Exercise and Nutrition inTervention fOR Sarcopenia (MENTORS), for sarcopenic older patients with MetS. METHODS AND ANALYSIS: This multicentre, randomised controlled trial includes 168 community-dwelling older adults with sarcopenia and MetS. The 12-week MENTORS comprises an exercise intervention consisting of an introductory phase (3 weeks; twice-weekly visits), an expanded phase (3 weeks; twice-weekly visits) and a maintenance phase (6 weeks; once-weekly visits); and a nutrition intervention tailored to the nutritional status of individual subjects. Outcomes will be measured at 0-week, 12-week and 24-week postintervention. The data will be analysed using the intention-to-treat and per-protocol principle. ETHICS AND DISSEMINATION: Before screening, all participants will be provided with oral and written information. Ethical approval has already been obtained from all participating hospitals. The study results will be disseminated in peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT04948736.

Prognostic Impact of Operator Experience and IVUS Guidance on Long-Term Clinical Outcomes After Complex PCI.

BACKGROUND: Although consistent clinical data support intravascular ultrasound (IVUS) use during complex percutaneous coronary intervention (PCI), long-term follow-up outcomes on differential effects of IVUS according to operator experience are scarce. OBJECTIVES: The current study aimed to evaluate the influence of operator experience on lesion complexity, long-term clinical outcomes, and the interactions of IVUS guidance in patients undergoing complex PCI. METHODS: A total of 6,005 patients who underwent PCI with drug-eluting stents for complex lesions were recruited from the institutional registry of Samsung Medical Center. The study population was stratified by the use of IVUS and operator experience (less experienced operator [lifetime independent experience with PCI ≤5 years] vs experienced operator). The primary endpoint was a composite of cardiac death or target vessel myocardial infarction (TVMI) up to 10 years. RESULTS: Compared with less experienced operators, experienced operators used IVUS more frequently (29.6% [1,128/3,805] vs 24.8% [546/2,200]; P < 0.001) and achieved a lower risk of cardiac death or TVMI (experienced vs less experienced, adjusted HR: 0.779; 95% CI: 0.663-0.915; P = 0.002). IVUS use was associated with a significantly lower risk of cardiac death or TVMI than angiography alone for less experienced operators (23.5% vs 11.4%; adjusted HR: 0.477; 95% CI: 0.337-0.673; P < 0.001) as well as experienced operators (18.0% vs 13.5%; adjusted HR: 0.747; 95% CI: 0.559-0.998; P = 0.048). There were significant interactions for the risk of cardiac death or TVMI between IVUS use and operator experience (P = 0.037). CONCLUSIONS: PCI by experienced operators and the use of IVUS during complex PCI were independently associated with lower long-term risks of cardiac death or TVMI. The beneficial effects of IVUS were more prominent for less experienced operators. (Prospective Percutaneous Coronary Intervention Registry [SMC-PCI]; NCT05624905).

Truncation or proteolysis of α-synuclein in Parkinsonism.

Posttranslational modifications of α-synuclein, such as truncation or abnormal proteolysis, are implicated in Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). A key focus of this article includes the proteases responsible for inducing truncation, the specific sites susceptible to truncation, and the resultant influence of these truncated species on the seeding and aggregation of endogenous α-synuclein. We also shed light on the unique structural attributes of these truncated species, and how these modifications can lead to distinctive forms of synucleinopathies. In addition, we explore the comparative toxic potentials of various α-synuclein species. An extensive analysis of available evidence of truncated α-synuclein species in human-synucleinopathy brains is also provided. Lastly, we delve into the detrimental impact of truncated species on key cellular structures such as the mitochondria and endoplasmic reticulum. Our article discusses enzymes involved in α-synuclein truncation, including 20 S proteasome, cathepsins, asparagine endopeptidase, caspase-1, calpain-1, neurosin/kallikrein-6, matrix metalloproteinase-1/-3, and plasmin. Truncation patterns impact α-synuclein aggregation - C-terminal truncation accelerates aggregation with larger truncations correlated with shortened aggregation lag times. N-terminal truncation affects aggregation differently based on the truncation location. C-terminally truncated α-synuclein forms compact, shorter fibrils compared to the full-length (FL) protein. N-terminally truncated monomers form fibrils similar in length to FL α-synuclein. Truncated forms show distinct fibril morphologies, increased ß-sheet structures, and greater protease resistance. Misfolded α-synuclein can adopt various conformations, leading to unique aggregates and distinct synucleinopathies. Fibrils, with prion-like transmission, are potentially more toxic than oligomers, though this is still debated. Different α-synuclein variants with N- and C-terminal truncations, namely 5-140, 39-140, 65-140, 66-140, 68-140, 71-140, 1-139, 1-135, 1-133, 1-122, 1-119, 1-115, 1-110, and 1-103 have been found in PD, DLB, and MSA patients' brains. In Parkinsonism, excess misfolded α-synuclein overwhelms the proteasome degradation system, resulting in truncated protein production and accumulation in the mitochondria and endoplasmic reticulum.

Combined exercise and nutrition intervention for older women with spinal sarcopenia: an open-label single-arm trial.

PURPOSE: Spinal sarcopenia is a multifactorial disorder associated with atrophy and fatty changes in paraspinal muscles. Interventional studies for spinal sarcopenia are limited. We aimed to evaluate the effectiveness of a combined exercise and nutrition intervention for the treatment of spinal sarcopenia. METHODS: 35 community-dwelling older women diagnosed with spinal sarcopenia in a previous cohort study were included. The 12-week combined intervention consisted of back extensor strengthening exercises and protein supplementation. The following outcomes were measured at baseline (week 0), after the intervention (week 12), and follow-up (week 24): conventional variables of sarcopenia (appendicular skeletal muscle mass, handgrip strength, 6-meter gait speed, and short physical performance battery); lumbar extensor muscle mass; lumbar extensor muscle volume and signal intensity; back extensor isokinetic strength; and back performance scale. We used the intention-to-treat analysis method, and repeated measures analysis of variance was used to analyze the data. RESULTS: Of the total 35 potential participants, 26 older women participated in the study (mean age 72.5 ± 4.0 years old). After 12 weeks of combined exercise and nutrition intervention, there were no changes in the appendicular skeletal muscle mass, lumbar extensor muscle mass, volume, or signal intensity. Handgrip strength and back extensor isokinetic strength did not change significantly. Short physical performance battery significantly increased (P = 0.042) from 11.46 ± 0.86 to 11.77 ± 0.53 at week 12 and 11.82 ± 0.40 at week 24. The back performance scale sum score also significantly improved (P = 0.034) from 2.68 ± 1.81 to 1.95 ± 1.21 at week 12 and 2.09 ± 1.34 at week 24. CONCLUSION: The combined exercise and nutrition intervention for community-dwelling older women with spinal sarcopenia could be feasible and helpful in improving the physical performance as well as back performance.

Lipid kinase PIP5Kα contributes to Hippo pathway activation via interaction with Merlin and by mediating plasma membrane targeting of LATS1.

BACKGROUND: The Hippo pathway plays a critical role in controlled cell proliferation. The tumor suppressor Merlin and large tumor suppressor kinase 1 (LATS1) mediate activation of Hippo pathway, consequently inhibiting the primary effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid present in the plasma membrane (PM), binds to and activates Merlin. Phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) is an enzyme responsible for PIP2 production. However, the functional role of PIP5Kα in regulation of Merlin and LATS1 under Hippo signaling conditions remains unclear. METHODS: PIP5Kα, Merlin, or LATS1 knockout or knockdown cells and transfected cells with them were used. LATS1, YAP, and TAZ activities were measured using biochemical methods and PIP2 levels were evaluated using cell imaging. Low/high cell density and serum starvation/stimulation conditions were tested. Colocalization of PIP5Kα and PIP2 with Merlin and LATS1, and their protein interactions were examined using transfection, confocal imaging, immunoprecipitation, western blotting, and/or pull-down experiments. Colony formation and adipocyte differentiation assays were performed. RESULTS: We found that PIP5Kα induced LATS1 activation and YAP/TAZ inhibition in a kinase activity-dependent manner. Consistent with these findings, PIP5Kα suppressed cell proliferation and enhanced adipocyte differentiation of mesenchymal stem cells. Moreover, PIP5Kα protein stability and PIP2 levels were elevated at high cell density compared with those at low cell density, and both PIP2 and YAP phosphorylation levels initially declined, then recovered upon serum stimulation. Under these conditions, YAP/TAZ activity was aberrantly regulated by PIP5Kα deficiency. Mechanistically, either Merlin deficiency or LATS1 deficiency abrogated PIP5Kα-mediated YAP/TAZ inactivation. Additionally, the catalytic domain of PIP5Kα directly interacted with the band 4.1/ezrin/radixin/moesin domain of Merlin, and this interaction reinforced interaction of Merlin with LATS1. In accordance with these findings, PIP5Kα and PIP2 colocalized with Merlin and LATS1 in the PM. In PIP5Kα-deficient cells, Merlin colocalization with PIP2 was reduced, and LATS1 solubility increased. CONCLUSIONS: Collectively, our results support that PIP5Kα serves as an activator of the Hippo pathway through interaction and colocalization with Merlin, which promotes PIP2-dependent Merlin activation and induces local recruitment of LATS1 to the PIP2-rich PM and its activation, thereby negatively regulating YAP/TAZ activity. Video Abstract.

Impact of Atrial Fibrillation on Patients Undergoing Transcatheter Aortic Valve Implantation (TAVI): The K-TAVI Registry.

PURPOSE: The incidence and prognostic implications of atrial fibrillation (AF) in patients with severe aortic stenosis (AS) undergoing transcatheter aortic valve implantation (TAVI) are controversial, especially for Korean patients. Furthermore, the pattern of antithrombotic therapy for these patients is unknown. The present study sought to identify the impact of AF on Korean patients undergoing TAVI and demonstrate the status of antithrombotic therapy for these patients. MATERIALS AND METHODS: A total of 660 patients who underwent TAVI for severe AS were recruited from the nationwide K-TAVI registry in Korea. The enrolled patients were stratified into sinus rhythm (SR) and AF groups. The primary endpoint was all-cause death at 1-year. RESULTS: AF was recorded in 135 patients [pre-existing AF 108 (16.4%) and new-onset AF 27 (4.1%)]. The rate of all-cause death at 1 year was significantly higher in patients with AF than in those with SR [16.2% vs. 6.4%, adjusted hazard ratio (HR): 2.207, 95% confidence interval (CI): 1.182-4.120, p=0.013], regardless of the onset timing of AF. The rate of new pacemaker insertion at 1 year was also significantly higher in patients with AF than in those with SR (14.0% vs. 5.5%, adjusted HR: 3.137, 95%CI: 1.621-6.071, p=0.001). Among AF patients, substantial number of patients received the combination of multiple antithrombotic agents (77.8%), and the most common combination was that of aspirin and clopidogrel (38.1%). CONCLUSION: AF was an independent predictor of 1-year mortality and new pacemaker insertion in Korean patients undergoing TAVI.

BBT-176, a Novel Fourth-Generation Tyrosine Kinase Inhibitor for Osimertinib-Resistant EGFR Mutations in Non-Small Cell Lung Cancer.

PURPOSE: Resistance to third-generation EGFR inhibitors including osimertinib arises in part from the C797S mutation in EGFR. Currently, no targeted treatment option is available for these patients. We have developed a new EGFR tyrosine kinase inhibitor (TKI), BBT-176, targeting the C797S mutation. PATIENTS AND METHODS: Recombinant EGFR proteins and Ba/F3 cell lines, patient-derived cells, and patient-derived xenografts expressing mutant EGFRs were used to test the inhibitory potency and the anticancer efficacy of BBT-176 both in vitro and in vivo. Patient case data are also available from an ongoing phase I clinical trial (NCT04820023). RESULTS: The half maximal inhibitory concentration (IC50) of BBT-176 against EGFR 19Del/C797S, EGFR 19Del/T790M/C797S, and EGFR L858R/C797S proteins were measured at 4.36, 1.79, and 5.35 nmol/L, respectively (vs. 304.39, 124.82, and 573.72 nmol/L, for osimertinib). IC50 values of BBT-176 against Ba/F3 cells expressing EGFR 19Del/C797S, EGFR 19Del/T790M/C797S, EGFR L858R/C797S, and EGFR L858R/T790M/C797S were 42, 49, 183, and 202 nmol/L, respectively (vs. 869, 1,134, 2,799, and 2,685 nmol/L for osimertinib). N-ethyl-N-nitrosourea mutagenesis suggested that BBT-176 treatment does not introduce any secondary mutations in the EGFR gene but increases EGFR expression levels. Combined with the EGFR antibody cetuximab, BBT-176 effectively suppressed the growth of BBT-176-resistant clones. BBT-176 strongly inhibited the tumor growth, and in some conditions induced tumor regression in mouse models. In the clinical trial, two patients harboring EGFR 19Del/T790M/C797S in blood showed tumor shrinkage and radiologic improvements. CONCLUSIONS: BBT-176 is a fourth-generation EGFR inhibitor showing promising preclinical activity against NSCLC resistant to current EGFR TKI, with early clinical efficacy and safety.

Biomechanical microenvironmental stimulating effect of pulsed electromagnetic field on the regeneration of crush injured rat sciatic nerve.

This study evaluated the biomechanical microenvironmental stimulating effect of pulsed electromagnetic field (PEMF) on the regeneration of crush-injured rat sciatic nerve, when combined with bone marrow mesenchymal stem cells (BMSCs) and recombinant human nerve growth factor (rhNGF-ß), in the form of an adenoviral vector-mediated NGF. Sprague-Dawley rats were equally distributed into six groups; PBS, BMSC, NGF-Ad + BMSC, PEMF + PBS, PEMF + BMSC and PEMF + NGF-Ad + BMSC. The PBS group received PBS (volume: 10µL/rat), the BMSC group with BMSCs (1 × 106 cell/10 µL/rat) and NGF-Ad group with the rhNGF-ß Ad infected BMSCs (1 × 106 cell/10 µL/rat) immediate after right sciatic nerve crush injury. The PEMF groups were exposed to PEMF of 1mT, 50 Hz, 1 h/day. The rats were observed for 3 weeks. PEMF alone did not showed the positive effect compared with negative control group. The groups transplanted with BMSCs showed higher axonal regeneration compared with the groups without transplantation of the cells whether BMSC was infected with NGF-Ad or not and whether the animals received PEMF. PEMF + NGF-Ad + BMSC group showed the significantly highest number of axons than the other groups. Functionally, all groups showed marked improvement at 3 weeks postoperatively although the difference was not statistically significant among the groups. PEMF showed the positive effect when combined with BMSC and NGF-ad in aspect of number of axons. Therefore, combining the microenvironment stimulation methods of PEMF and conventional methods such as transplantation of stem cells and growth factor could be considered for the regeneration methods in the nerve damage.

Synthesis, In Silico and In Vitro Characterization of Novel N,N-Substituted Pyrazolopyrimidine Acetamide Derivatives for the 18KDa Translocator Protein (TSPO).

The translocator protein (TSPO) is an interesting biological target for molecular imaging and therapy because the overexpression of TSPO is associated with microglial activation caused by neuronal damage or neuroinflammation, and these activated microglia are involved in various central nervous system (CNS) diseases. The TSPO is a target for neuroprotective treatment, which is used with the aim of reducing microglial cell activation. The novel N,N-disubstituted pyrazolopyrimidine acetamides scaffold (GMA 7-17), which bears a fluorine atom and is directly linked to the phenyl moiety, was synthesized, and each of the novel ligands was characterized in vitro. All of the newly synthesized ligands displayed picomolar to nanomolar affinity for the TSPO. Particularly, an in vitro affinity study led to the discovery of 2-(5,7-diethyl-2-(4-fluorophenyl)pyrazolo [1,5-a]pyrimidin-3-yl)-N-ethyl-N-phenylacetamide GMA 15 (Ki = 60 pM), a novel TSPO ligand that exhibits a 61-fold enhancement in affinity compared to the reference standard DPA-714 (Ki = 3.66 nM). Molecular dynamic (MD) studies of the highest affinity binder, GMA 15, were carried out to check its time-dependent stability with the receptor compared to DPA-714 and PK11195. The hydrogen bond plot also indicated that GMA 15 formed higher hydrogen bonds compared to DPA-714 and PK11195. We anticipate that further optimization to enhance the potency in a cellular assay needs to be followed, but our strategy of identifying potential TSPO binding novel scaffolds may open up a new avenue to develop novel TSPO ligands suited for potential molecular imaging and a wide range of therapeutic applications.

Fracture Liaison Service in Korea: 2022 Position Statement of the Korean Society for Bone and Mineral Research.

Osteoporosis and osteoporotic fractures cause socioeconomic concerns, and medical system and policies appear insufficient to prepare for these issues in Korea, where the older adult population is rapidly increasing. Many countries around the world are already responding to osteoporosis and osteoporotic fractures by adopting fracture liaison service (FLS), and such an attempt has only begun in Korea. In this article, we introduce the operation methods for institutions implementing FLS and characteristics of services, and activities of the FLS Committee for FLS implementation in the Korean Society for Bone and Mineral Research. In addition, we hope that the current position statement will contribute to the implementation of FLS in Korea and impel policy changes to enable a multidisciplinary and integrated FLS operated under the medical system.