Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK+ CD8+ T Cells as Conserved Hallmark of Inflammaging

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Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD

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Transdermal Diagnosis of Malaria Using Vapor Nanobubbles

A fast, precise, noninvasive, high-throughput, and simple approach for detecting malaria in humans and mosquitoes is not possible with current techniques that depend on blood sampling, reagents, facilities, tedious procedures, and trained personnel. We designed a device for rapid (20-second) noninvasive diagnosis of Plasmodium falciparum infection in a malaria patient without drawing blood or using any reagent. This method uses transdermal optical excitation and acoustic detection of vapor nanobubbles around intraparasite hemozoin. The same device also identified individual malaria parasite–infected Anopheles mosquitoes in a few seconds and can be realized as a low-cost universal tool for clinical and field diagnoses.     

AMP-activated protein kinase regulates hERG potassium channel

Besides their role in cardiac repolarization, human ether-a-go-go-related gene potassium (hERG) channels are expressed in several tumor cells including rhabdomyosarcoma cells. The channels foster cell proliferation. Ubiquitously expressed AMP-dependent protein kinase (AMPK) is a serine-/threonine kinase, stimulating energy-generating and inhibiting energy-consuming processes thereby helping cells survive periods of energy depletion. AMPK has previously been shown to regulate Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Ca²⁺ channels and K⁺ channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1β1γ1), constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), or catalytically inactive ^αK45RAMPK (α1(K45R)β1γ1) were expressed in Xenopus oocytes with hERG. Tail currents were determined as a measure of hERG channel activity by two-electrode-voltage clamp. hERG membrane abundance was quantified by chemiluminescence and visualized by immunocytochemistry and confocal microscopy. Moreover, hERG currents were measured in RD rhabdomyosarcoma cells after pharmacological modification of AMPK activity using the patch clamp technique. Coexpression of wild-type AMPK and of constitutively active ^γR70QAMPK significantly downregulated the tail currents in hERG-expressing Xenopus oocytes. Pharmacological activation of AMPK with AICAR or with phenformin inhibited hERG currents in Xenopus oocytes, an effect abrogated by AMPK inhibitor compound C. ^γR70QAMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active ^γR70QAMPK decreased membrane expression of hERG in Xenopus oocytes. Compound C significantly enhanced whereas AICAR tended to inhibit hERG currents in RD rhabdomyosarcoma cells. AMPK is a powerful regulator of hERG-mediated currents in both, Xenopus oocytes and RD rhabdomyosarcoma cells. AMPK-dependent regulation of hERG may be particularly relevant in cardiac hypertrophy and tumor growth.     

Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase

The 5′-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca²⁺ activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na⁺ channel ENaC and the voltage-gated K⁺ channel KCNE1/KCNQ1. It is partially effective by decreasing PIP₂ formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K⁺ channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK^γR70Q (AMPK^α1-HA+AMPK^β1-Flag+AMPKγ1^R70Q), or of inactive AMPK^αK45R (AMPK^α1K45R+AMPK^β1-Flag+AMPK^γ1-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na⁺ and K⁺ excretion were determined in AMPK^α1-deficient mice (ampk ^?/? ) and wild-type mice (ampk ^+/+ ) prior to and following an acute K⁺ load (111 mM KCl, 30 mM NaHCO₃, 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 μl/h. As a result, coexpression of AMPK^γR70Q but not of AMPK^αK45R significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI_(4,5)P₂ significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK^γR70Q. Under control conditions, no significant differences between ampk ^?/? and ampk ^+/+ mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na⁺, and K⁺ concentrations as well as absolute and fractional Na⁺ and K⁺ excretion. Following an acute K⁺ load, GFR, urinary flow rate, serum aldosterone, plasma Na⁺, and K⁺ concentration were again similar in both genotypes, but renal absolute and fractional Na⁺ and K⁺ excretion were higher in ampk ^?/? than in ampk ^+/+ mice. According to micropuncture following a K⁺ load, delivery of Na⁺ to the early distal tubule but not delivery of K⁺ to late proximal and early distal tubules was increased in ampk ^?/? mice. The upregulation of renal ROMK1 protein expression by acute K⁺ load was more pronounced in ampk ^?/? than in ampk ^+/+ mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K⁺ following an acute K⁺ load.     

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O₂), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O₂. Specifically, we demonstrate that hypoxic Tsc2^−/− (tuberous sclerosis complex 2^−/−) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.     

Mapping of multiple muscles with transcranial magnetic stimulation: absolute and relative test–retest reliability

The spatial accuracy of transcranial magnetic stimulation (TMS) may be as small as a few millimeters. Despite such great potential, navigated TMS (nTMS) mapping is still underused for the assessment of motor plasticity, particularly in clinical settings. Here, we investigate the within‐limb somatotopy gradient as well as absolute and relative reliability of three hand muscle cortical representations (MCRs) using a comprehensive grid‐based sulcus‐informed nTMS motor mapping. We enrolled 22 young healthy male volunteers. Two nTMS mapping sessions were separated by 5–10 days. Motor evoked potentials were obtained from abductor pollicis brevis (APB), abductor digiti minimi, and extensor digitorum communis. In addition to individual MRI‐based analysis, we studied normalized MNI MCRs. For the reliability assessment, we calculated intraclass correlation and the smallest detectable change. Our results revealed a somatotopy gradient reflected by APB MCR having the most lateral location. Reliability analysis showed that the commonly used metrics of MCRs, such as areas, volumes, centers of gravity (COGs), and hotspots had a high relative and low absolute reliability for all three muscles. For within‐limb TMS somatotopy, the most common metrics such as the shifts between MCR COGs and hotspots had poor relative reliability. However, overlaps between different muscle MCRs were highly reliable. We, thus, provide novel evidence that inter‐muscle MCR interaction can be reliably traced using MCR overlaps while shifts between the COGs and hotspots of different MCRs are not suitable for this purpose. Our results have implications for the interpretation of nTMS motor mapping results in healthy subjects and patients with neurological conditions.     

Neurochemical underpinning of hemodynamic response to neuropsychiatric drugs: A meta- and cluster analysis of preclinical studies

Functional magnetic resonance imaging (fMRI) is an extensively used method for the investigation of normal and pathological brain function. In particular, fMRI has been used to characterize spatiotemporal hemodynamic response to pharmacological challenges as a non-invasive readout of neuronal activity. However, the mechanisms underlying regional signal changes are yet unclear. In this study, we use a meta-analytic approach to converge data from microdialysis experiments with relative cerebral blood volume (rCBV) changes following acute administration of neuropsychiatric drugs in adult male rats. At whole-brain level, the functional response patterns show very weak correlation with neurochemical alterations, while for numerous brain areas a strong positive correlation with noradrenaline release exists. At a local scale of individual brain regions, the rCBV response to neurotransmitters is anatomically heterogeneous and, importantly, based on a complex interplay of different neurotransmitters that often exert opposing effects, thus providing a mechanism for regulating and fine tuning hemodynamic responses in specific regions.     

Multiparametric MR evaluation of uterine leiomyosarcoma and STUMP versus leiomyoma in symptomatic women planned for high frequency focussed ultrasound: accuracy of imaging parameters and interobserver agreement for identification of malignancy

Objective:To assess accuracy of and interobserver agreement on multiparametric MR findings to distinguish uterine leiomyoma (LM) from uterine leiomyosarcoma (LMS) and soft tissue tumour of unknown malignant potential.Methods:Inclusion criteria: All females over 18 years with least one uterine mass measuring 5 cm or more in at least one of the three standard orthogonal dimensions on MR with histopathological confirmation of LM, LMS, or soft tissue tumour of unknown malignant potential (STUMP) in the 3 months following MR. Patients with LMS were drawn from a larger cohort being assessed for MR-guided focussed ultrasound (MRgFUS) suitability. Image evaluation: Assessed variables were: lesion margin, margin definition, T2 signal homogeneity, >50% of lesion with T2 signal brighter than myometrium, haemorrhage, restricted diffusion, contrast enhancement (CE), CE pattern, local lymphadenopathy and ascites.Results:32 LM, 10 LMS and 1 STUMP were evaluated. Ill-defined (p-value = 0.0003–0.0004) or irregular (p = 0.003–0.004) lesion margin, T2 hyperintensity >50% (p = 0.001–0.004), and peripheral CE (p = 0.02–0.05) were significantly more common in LMS/STUMP than LM for both radiologists. 10/11 (Reader 2) and 11/11 (Reader 1) LMS/STUMP displayed restricted diffusion but so did 63–80% of LM. Agreement was greatest for margin characteristics (κ = 0.73–0.81).Conclusion:Irregular/ill-defined lesion margin best distinguished LMS/STUMP from LM with good interrater reliability.Advances in knowledge:Assessment of agreement regarding MR parameters distinguishing LM from LMS and STUMP has not previously been undertaken in a cohort including a large number of patients with LMS. This will help inform evaluation of females considering minimally invasive LM treatment.