Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

<p>Abstract</p> <p>Background</p> <p>Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)<it>P</it>₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)<it>P</it>₂also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)<it>P</it>₂levels in living cells is critical to understanding the biology of PtdIns(4,5)<it>P</it>₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)<it>P</it>₂imaging tools.</p> <p>Results</p> <p>All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)<it>P</it>₂showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)<it>P</it>₂. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)<it>P</it>₂binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P₃. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)<it>P</it>₂and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.</p> <p>Conclusion</p> <p>These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)<it>P</it>₂changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P₃binding may compromise its accuracy to measure PtdIns(4,5)<it>P</it>₂changes. The Tubby domain is more accurate to report on PtdIns(4,5)<it>P</it>₂but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)<it>P</it>₂levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)<it>P</it>₂.</p

Isolated Double-Chambered Right Ventricle in a Young Adult

Double-chambered right ventricle (DCRV) is a rare congenital heart disorder in which the right ventricle is divided by an anomalous muscle bundle into a high pressure inlet portion and a low pressure outlet portion. We report a case of isolated DCRV without symptoms in adulthood, diagnosed through echocardiography, cardiac catheterization and cardiac magnetic resonance imaging

Incidence and Risk Factors Associated with Superior Mesenteric Artery Syndrome following Surgical Correction of Scoliosis

STUDY DESIGN: Retrospective study. PURPOSE: To more accurately determine the incidence and clarify risk factors. OVERVIEW OF LITERATURE: Superior mesenteric artery syndrome is one of the possible complications following correctional operation for scoliosis. However, when preliminary symptoms are vague, the diagnosis of superior mesenteric artery syndrome may be easily missed. METHODS: We conducted a retrospective study using clinical data from 118 patients (43 men and 75 women) who underwent correctional operations for scoliosis between September 2001 and August 2007. The mean patient age was 15.9 years (range 9~24 years). The risk factors under scrutiny were the patient body mass index (BMI), change in Cobb's angle, and trunk length. RESULTS: The incidence of subjects confirmed to have obstruction was 2.5%. However, the rate increased to 7.6% with the inclusion of the 6 subjects who only showed clinical symptoms of obstruction without confirmative study. The BMI for the asymptomatic and symptomatic groups were 18.4+/-3.4 and 14.6+/-3, respectively. The change in Cobb's angle for the asymptomatic and symptomatic groups were 24.8+/-13.6 degrees and 23.4+/-9.1 degrees , respectively. The change in trunk length for the asymptomatic and symptomatic groups were 2.3+/-2.1 cm and 4.5+/-4.8 cm, respectively. Differences in Cobb's angle and the change in trunk length between the two groups did not reach statistical significance, although there was a greater increase in trunk length for the symptomatic group than for the asymptomatic group. CONCLUSIONS: Our study shows that the incidence of superior mesenteric artery syndrome may be greater than the previously accepted rate of 4.7%. Therefore, in the face of any early signs or symptoms of superior mesenteric artery syndrome, prompt recognition and treatment are necessaryope

Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-golgi junctions

Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in the PTDSS1 gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism

Adiponectin is Associated with Impaired Fasting Glucose in the Non-Diabetic Population

OBJECTIVES: Adiponectin is strongly associated with diabetes in the Western population. However, whether adiponectin is independently associated with impaired fasting glucose (IFG) in the non-obese population is unknown. METHODS: The serum adiponectin, insulin resistance (IR), and waist circumference (WC) of 27,549 healthy Koreans were measured. Individuals were then classified into tertile groups by gender. IFG was defined as a fasting serum glucose of 100-125 mg/dL without diabetes. IR was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR). The association of adiponectin and IFG was determined using logistic regression analysis. RESULTS: WC and adiponectin were associated with IFG in both men and women. However, the association of WC with IFG was attenuated in both men and women after adjustment for the HOMA-IR. Adiponectin was still associated with IFG after adjustment for and stratification by HOMA-IR in men and women. Strong combined associations of IR and adiponectin with IFG were observed in men and women. Multivariate adjusted odds ratios (ORs) (95% confidence interval [CI]) among those in the highest tertile of IR and the lowest tertile of adiponectin were 9.8 (7.96 to 12.07) for men and 24.1 (13.86 to 41.94) for women. CONCLUSION: These results suggest that adiponectin is strongly associated with IFG, and point to adiponectin as an additional diagnostic biomarker of IFG in the non-diabetic population.ope

Effect of blood pressure and glycemic control on the plasma cell-free DNA in hemodialysis patients

AbstractBackgroundThe plasma levels of cell-free DNA (cfDNA) are known to be elevated under inflammatory or apoptotic conditions. Increased cfDNA levels have been reported in hemodialysis (HD) patients. The aim of this study was to investigate the clinical significance of cfDNA in HD patients.MethodsA total of 95 patients on HD were enrolled. We measured their predialysis cfDNA levels using real-time EIF2C1 gene sequence amplification and analyzed its association with certain clinical parameters.ResultsThe mean plasma cfDNA level in the HD patients was 3,884 ± 407 GE/mL, and the mean plasma cfDNA level in the control group was 1,420 ± 121 GE/mL (P < 0.05). Diabetic patients showed higher plasma cfDNA levels compared with nondiabetic patients (P < 0.01). Patients with cardiovascular complications also showed higher plasma cfDNA levels compared with those without cardiovascular complication (P < 0.05). In univariable analysis, the cfDNA level was associated with 3-month mean systolic blood pressure (SBP), white blood cell, serum albumin, creatinine (Cr), normalized protein catabolic rate in HD patients. In diabetic patients, it was significantly correlated with SBP, hemoglobin A1c, and serum albumin. In multivariate analysis, SBP was the independent determinant for the cfDNA level. In diabetic patients, cfDNA level was independently associated with hemoglobin A1c and SBP.ConclusionsIn patients with HD, cfDNA is elevated in diabetic patients and patients with cardiovascular diseases. Uncontrolled hypertension and poor glycemic control are independent determinants for the elevated cfDNA. Our data suggest that cfDNA might be a marker of vascular injury rather than proinflammatory condition in HD patients

Isolated Double-Chambered Right Ventricle in a Young Adult

Double-chambered right ventricle (DCRV) is a rare congenital heart disorder in which the right ventricle is divided by an anomalous muscle bundle into a high pressure inlet portion and a low pressure outlet portion. We report a case of isolated DCRV without symptoms in adulthood, diagnosed through echocardiography, cardiac catheterization and cardiac magnetic resonance imaging

Management of Complex Regional Pain Syndrome Type 1 With Total Spinal Block

Complex regional pain syndrome (CRPS) is a painful and disabling disorder that can affect one or more extremities. Unfortunately, the knowledge concerning its natural history and mechanism is very limited and many current rationales in treatment of CRPS are mainly dependent on efficacy originated in other common conditions of neuropathic pain. Therefore, in this study, we present a case using a total spinal block (TSB) for the refractory pain management of a 16-year-old male CRPS patient, who suffered from constant stabbing and squeezing pain, with severe touch allodynia in the left upper extremity following an operation of chondroblastoma. After the TSB, the patient's continuous and spontaneous pain became mild and the allodynia disappeared and maintained decreased for 1 month

Development and validation of a machine learning-based fall-related injury risk prediction model using nationwide claims database in Korean community-dwelling older population

Background Falls impact over 25% of older adults annually, making fall prevention a critical public health focus. We aimed to develop and validate a machine learning-based prediction model for serious fall-related injuries (FRIs) among community-dwelling older adults, incorporating various medication factors. Methods Utilizing annual national patient sample data, we segmented outpatient older adults without FRIs in the preceding three months into development and validation cohorts based on data from 2018 and 2019, respectively. The outcome of interest was serious FRIs, which we defined operationally as incidents necessitating an emergency department visit or hospital admission, identified by the diagnostic codes of injuries that are likely associated with falls. We developed four machine-learning models (light gradient boosting machine, Catboost, eXtreme Gradient Boosting, and Random forest), along with a logistic regression model as a reference. Results In both cohorts, FRIs leading to hospitalization/emergency department visits occurred in approximately 2% of patients. After selecting features from initial set of 187, we retained 26, with 15 of them being medication-related. Catboost emerged as the top model, with area under the receiver operating characteristic of 0.700, along with sensitivity and specificity rates around 65%. The high-risk group showed more than threefold greater risk of FRIs than the low-risk group, and model interpretations aligned with clinical intuition. Conclusion We developed and validated an explainable machine-learning model for predicting serious FRIs in community-dwelling older adults. With prospective validation, this model could facilitate targeted fall prevention strategies in primary care or community-pharmacy settings.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2020R1A2C110097111) and the Creative-Pioneering Researchers Program through Seoul National Universit