T cell migration requires ion and water influx to regulate actin polymerization.

Migration of T cells is essential for their ability to mount immune responses. Chemokine-induced T cell migration requires WNK1, a kinase that regulates ion influx into the cell. However, it is not known why ion entry is necessary for T cell movement. Here we show that signaling from the chemokine receptor CCR7 leads to activation of WNK1 and its downstream pathway at the leading edge of migrating CD4+ T cells, resulting in ion influx and water entry by osmosis. We propose that WNK1-induced water entry is required to swell the membrane at the leading edge, generating space into which actin filaments can polymerize, thereby facilitating forward movement of the cell. Given the broad expression of WNK1 pathway proteins, our study suggests that ion and water influx are likely to be essential for migration in many cell types, including leukocytes and metastatic tumor cells.

Multi-element analysis of tyre rubber for metal tracers.

The purpose of this study was to identify a characteristic elemental tyre fingerprint that can be utilised in atmospheric source apportionment calculations. Currently zinc is widely used as a single element tracer to quantify tyre wear, however several authors have highlighted issues with this approach. To overcome this, tyre rubber tread was digested and has been analysed for 25 elements by ICP-MS to generate a multielement profile. Additionally, to estimate the percentage of the tyre made up of inert fillers, thermogravimetric analysis was performed on a subset. Comparisons were made between passenger car and heavy goods vehicle tyre composition, and a subset of tyres had both tread and sidewall sampled for further comparison. 19 of the 25 elements were detected in the analysis. The mean mass fraction of zinc detected was 11.17 g/kg, consistent with previous estimates of 1% of the tyre mass. Aluminium, iron, and magnesium were found to be the next most abundant elements. Only one source profile for tyre wear exists in both the US and EU air pollution species profile databases, highlighting the need for more recent data with better coverage of tyre makes and models. This study provides data on new tyres which are currently operating on-road in Europe and is therefore relevant for ongoing atmospheric studies assessing the levels of tyre wear particles in urban areas.

Controlled In-Cell Generation of Active Palladium(0) Species for Bioorthogonal Decaging.

Owing to their bioorthogonality, transition metals have become very popular in the development of biocompatible bond-cleavage reactions. However, many approaches require design and synthesis of complex ligands or formulation of nanoparticles which often perform poorly in living cells. This work reports on a method for the generation of an active palladium species that triggers bond-cleaving reactions inside living cells. We utilized the water-soluble Na2 PdCl4 as a simple source of PdII which can be intracellularly reduced by sodium ascorbate to the active Pd0 species. Once generated, Pd0 triggers the cleavage of allyl ether and carbamate caging groups leading to the release of biologically active molecules. These findings do not only expand the toolbox of available bioorthogonal dissociative reactions but also provide an additional strategy for controlling the reactivity of Pd species involved in Pd-mediated bioorthogonal reactions.

CRYPTOCHROMES promote daily protein homeostasis.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

The metabolic growth limitations of petite cells lacking the mitochondrial genome.

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology.

Between 6-20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl- through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

Inductively Coupled Plasma Mass Spectrometry for Elemental Analysis in Circadian Biology.

Inductively coupled plasma mass spectrometry (ICP-MS) is a sensitive instrumental analysis technique used for multielemental and isotopic determination. Here we provide a sample preparation and circadian ICP-MS analysis protocol for use with mammalian tissues and cells, using mouse fibroblasts as a case study.

Eukaryotic cell biology is temporally coordinated to support the energetic demands of protein homeostasis.

Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H+ export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour.

Comparison of Pain Scores and Medication Usage Between Three Pain Control Strategies for Pediatric Anterior Cruciate Ligament Surgery.

Introduction Assessment and management of postoperative pain in the pediatric population after anterior cruciate ligament (ACL) surgery can be challenging; the optimal approach to pain control remains controversial. Recent studies show that use of intraoperative nerve blocks may reduce the need for opioids to control pain in the postoperative period. However, it is unclear which block type is most beneficial in the pediatric outpatient setting. This study compared effectiveness of pain control among three different pain management strategies. Methods We retrospectively reviewed charts of patients aged 12-17 years who received an elective ACL reconstruction between 2013 and 2017. The three groups were femoral nerve block, combined femoral and sciatic block, and intraarticular injection of bupivacaine (n = 50 per group). The primary variable was postoperative pain scores (visual analog scale 1-10) in the postanesthesia care unit (PACU). Results Less than 50% of patients in the combined nerve block group had opioids intraoperatively or in the PACU compared with nearly 100% of patients in the other two groups (p < 0.0001). Also, for patients receiving opioids, the total intraoperative morphine equivalents and PACU pain scores (all patients) were significantly less in the combined block group (p < 0.001). For patients receiving opioids in the PACU, the total morphine equivalents were significantly higher in the intraarticular injection group compared with the nerve block groups (p < 0.0001). Conclusion Patients in the combined femoral and sciatic nerve block group had significantly better pain scores in the PACU with less cumulative morphine equivalent consumption compared with the femoral nerve block group and the intraarticular injection group.

HIV vasculopathy versus VZV vasculitis in an HIV patient with multiple brain ischaemic infarcts.

We report the case of a 56-year-old man who presented to the emergency department with a 3-day onset of left limb weakness and feeling intoxicated with poor balance. Stroke hospitalisations in the USA decreased from 2000 to 2010, however the number of hospitalised patients with ischaemic stroke and HIV infection has increased significantly. Herein, we discuss the management of this unique case to highlight the importance of a broad differential diagnosis when approaching HIV/AIDS patients presenting with acute or subacute neurological focalisation. Given that HIV vasculopathy is a diagnosis of exclusion, it requires a thoughtful elimination of all possible aetiologies.

Workflow Optimization for Ischemic Stroke in a Community-Based Stroke Center.

BACKGROUND: We analyzed the effect of specific optimization steps to reduce treatment delays in a nonacademic stroke hospital setting. METHODS: The data from patients with ischemic stroke who had been treated with intravenous tissue plasminogen activator or endovascular therapy, or both, were analyzed. The metrics were divided into 2 periods: preoptimization period (October 1, 2015 to September 30, 2016) and postoptimization period (October 1, 2016 to September 30, 2017). The key interventions were 1) notification by the emergency medical service to the emergency department and stroke team; 2) division of the stroke alert between level 1 (intravenous/intra-arterial candidate) and level 2; 3) direct transportation of level 1 patients to brain computed tomography; 4) limitation of nonessential interventions; 5) stroke orientation; 6) 24-hour, 7-day code stroke response by a vascular neurologist; 7) earlier notification of the interventional radiology team; 8) direct transportation from computed tomography to angiography suite for large vessel occlusion; and 9) multidisciplinary monthly meetings to discuss delayed cases. RESULTS: A total of 279 patients were identified. No significant differences in any of the baseline characteristics were documented. Almost all metrics favored the postoptimization period, with remarkable improvement in the door-to-puncture time (median, 64 minutes; interquartile range, 36-86; vs. 47 minutes; interquartile range, 20-62; P = 0.001). We observed an increased percentage of good clinical outcomes in the postoptimization group (60.1% vs. 54.8%; P = 0.500). We found an 8.4% increase in patients with good clinical outcomes in the postoptimization group compared with our previously reported work. CONCLUSIONS: For acute reperfusion therapies, significant reductions in workflow intervals can be achieved after simple optimization methods in a nonacademic community-based hospital.

Daily magnesium fluxes regulate cellular timekeeping and energy balance.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

Deletion of the hemopexin or heme oxygenase-2 gene aggravates brain injury following stroma-free hemoglobin-induced intracerebral hemorrhage.

BACKGROUND: Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury. Hemopexin (Hpx) has the highest binding affinity to heme and participates in its transport, while heme oxygenase 2 (HO2) is the rate-limiting enzyme for the degradation of heme. Microglia are the resident macrophages in the brain; however, the significance and role of HO2 and Hpx on microglial clearance of the toxic heme (iron-protoporphyrin IX) after ICH still remain understudied. Accordingly, we postulated that global deletion of constitutive HO2 or Hpx would lead to worsening of ICH outcomes. METHODS: Intracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induce ICH. Hpx knockout (Hpx(-/-)) or HO2 knockout (HO2(-/-)) mice were injected with 10 µL of SFHb in the striatum. After injection, behavioral/functional tests were performed, along with anatomical analyses. Iron deposition and neuronal degeneration were depicted by Perls' and Fluoro-Jade B staining, respectively. Immunohistochemistry with anti-ionized calcium-binding adapter protein 1 (Iba1) was used to estimate activated microglial cells around the injured site. RESULTS: This study shows that deleting Hpx or HO2 aggravated SFHb-induced brain injury. Compared to wild-type littermates, larger lesion volumes were observed in Hpx(-/-) and HO2(-/-) mice, which also bear more degenerating neurons in the peri-lesion area 24 h postinjection. Fewer Iba1-positive microglial cells were detected at the peri-lesion area in Hpx(-/-) and HO2(-/-) mice, interestingly, which is associated with markedly increased iron-positive microglial cells. Moreover, the Iba1-positive microglial cells increased from 24 to 72 h postinjection and were accompanied with improved neurologic deficits in Hpx(-/-) and HO2(-/-) mice. These results suggest that Iba1-positive microglial cells could engulf the extracellular SFHb and provide protective effects after ICH. We then treated cultured primary microglial cells with SFHb at low and high concentrations. The results show that microglial cells actively take up the extracellular SFHb. Of interest, we also found that iron overload in microglia significantly reduces the Iba1 expression level and resultantly inhibits microglial phagocytosis. CONCLUSIONS: This study suggests that microglial cells contribute to hemoglobin-heme clearance after ICH; however, the resultant iron overloads in microglia appear to decrease Iba1 expression and to further inhibit microglial phagocytosis.

Efficacy of peripheral interventional radiologists performing endovascular stroke therapy guided by CT perfusion triage of patients.

PURPOSE: To assess safety and efficacy of intraarterial mechanical thrombectomy for treatment of ischemic stroke in a community hospital by peripheral interventional radiologists employing computed tomography (CT) perfusion imaging for patient selection. MATERIALS AND METHODS: Forty patients, 11 men (27.5%) and 29 women (72.5%), were treated between February 2008 and October 2011. Eligible patients had a National Institutes of Health Stroke Scale (NIHSS) score greater than 8 and diagnosis of large-vessel ischemic stroke by head CT angiogram, and met previously reported CT perfusion imaging triage criteria. RESULTS: The baseline NIHSS score was 18.0 ± 7.9 (range, 8-35). Sixteen patients (40%) had a baseline NIHSS score greater than 20. Symptom onset was unknown in five patients. Symptom onset to device time in the remaining 35 patients was 254.8 minutes ± 150.9 (range, 75-775 min). A total of 65% of patients showed thrombolysis in cerebral infarction (TICI) 2a, 2b, or 3 flow following the procedure. Symptomatic intracranial hemorrhage was seen in four patients (10.0%). At 90 days, 32 patients (80%) were alive and eight (20%) had died. The modified Rankin scale (mRS) score at 90 days was no more than 2 in 20 patients (50.0%). The mean mRS score at 90 days was 2.9 ± 2.0 (range, 0-6). NIHSS score at 90 days was 5.1 ± 6.1 (range, 0-24). In patients with successful recanalization (ie, TICI 2 or 3 flow), a good clinical outcome (ie, mRS score ≤ 2) was achieved in 65.3% of patients (mean, 2.4 ± 1.9; range, 0-6), and 90-day mortality rate was 15.4%, compared with 28.6% in patients with TICI 0/1 flow. CONCLUSIONS: Peripheral interventional radiologists who use CT perfusion imaging for patient triage can have good neurologic outcomes and provide sustainable, safe, and complete around-the-clock coverage for endovascular stroke treatment.

Delayed catastrophic intracerebral hemorrhage preceded by progressive recovery after carotid stenting for acute ischemic stroke.

BACKGROUND: Hemorrhagic transformation (HT) is a feared complication of reperfusion therapy for treatment of acute ischemic stroke. Generally, HT occurs within 24-36 hours after thrombolysis. SUMMARY OF CASE: We present a case of a fatal symptomatic HT of an infarction that occurred 7 days after acute ischemic stroke which was preceded by a remarkable recovery following a combination of acute revascularization therapies. CONCLUSION: Fatal symptomatic HT is a rare potential complication that can occur after several days of treatment of acute ischemic stroke.

Endovascular stroke therapy: a single-center retrospective review.

OBJECT: Endovascular treatment of acute ischemic stroke delivers direct therapy at the site of an occluded cerebral artery and can be employed beyond the 3-4.5-hour window limit set for intravenous recombinant tissue plasminogen activator. In this paper, the authors report their experience with various endovascular therapies in acute ischemic stroke. METHODS: The authors conducted a retrospective review of their clinical database for acute ischemic stroke in large-vessel cerebral territories that underwent endovascular treatment between May 2005 and February 2009. Endovascular treatment was defined as pharmacological and/or mechanical intervention, angioplasty, stenting, or a combination of these methods. Admission National Institutes of Health Stroke Scale and the modified Rankin Scale scores were recorded. Thrombolysis in Myocardial Infarction (TIMI) scores of 0, 1, 2A, 2B, and 3 were used to define recanalization. RESULTS: Forty procedures were performed in 39 patients, with 1 patient having sequential bilateral strokes. Nine patients were lost to follow-up after discharge. Strokes in the carotid artery circulation occurred in 82.5% of cases, and those in the vertebral-basilar territory occurred in 17.5%. The Merci device was used in 22 (55%) of 40 procedures, and the Penumbra device in 9 (22.5%) of 40. Angioplasty was performed in 15 (37.5%) of 40 procedures, and intraarterial recombinant tissue plasminogen activator was administered in 23 (57.5%) of 40 procedures. In 23 (57.5%) of 40 cases, multiple recanalization methods were used. The recanalization rate for all methods was 60%. The recanalization rate from TIMI Score 0/1 occlusions was 71.4% (20 of 28). An estimated modified Rankin Scale score of ≤ 2 was obtained in 11 (36.7%) of 30 cases. The overall mortality rate was 26.7% (8 of 30). Intracerebral hemorrhage at 24 hours postprocedure was noted in 17 (42.5%) of 40 cases, 3 (7.5%) of which were symptomatic. CONCLUSIONS: The authors' institution performs endovascular stroke treatment with a safety and efficacy profile comparable to those of other major endovascular stroke therapy studies. Recanalization was associated with an improved clinical outcome. Protocols to maximize efficient triage of patients and better documentation of stroke treatments can assist in further studies.

Previous statin use is not associated with an increased prevalence or degree of gradient-echo lesions in patients with acute ischemic stroke or transient ischemic attack.

BACKGROUND AND PURPOSE: Microhemorrhages on gradient-echo T2*-weighted MRI sequences are often found in patients with cerebrovascular disease and are related to intracerebral hemorrhage. Because statin therapy is associated with increased risk of intracerebral hemorrhage, we investigated whether statin use was also associated with microhemorrhages in patients with acute ischemic stroke or transient ischemic attack. METHODS: We performed a retrospective analysis on prospectively collected data from a stroke registry containing patients with acute ischemic stroke or transient ischemic attack. The primary and secondary outcome variables were the prevalence and degree of microhemorrhages as detected on gradient-echo MRI sequences and categorized as mild (1-2), moderate (3-10), or severe (>10). The location of the microhemorrhages was noted and rated by 2 neuroradiologists. Previous use of statins and other covariates were assessed as potential predictors. RESULTS: Three hundred forty-nine patients were admitted from June 2008 to July 2009, and 300 of which were analyzed. Microhemorrhages were detected in 70 subjects (23%); 35 had only lobar lesions, 16 had only deep lesions, and 19 had both lobar and deep lesions. On univariate and multivariate analysis, statin therapy was not associated with the prevalence (OR, 0.73; 95% CI, 0.36-1.51; P=0.40) or degree of microhemorrhages modeled for lesser severity (OR, 2.31; 95% CI, 0.61-8.75; P=0.22). CONCLUSIONS: Previous statin therapy was not associated with the prevalence or degree of microhemorrhages in patients with acute ischemic stroke or transient ischemic attack. The association between statins and intracerebral hemorrhage does not appear to be mediated through microhemorrhages.

Nutrient removal and loading rate analysis of Louisiana forested wetlands assimilating treated municipal effluent.

The relationship between nutrient removal and loading rate was examined using data from five forested wetlands in Louisiana that have received secondarily treated effluent from 3 to 60 years. Loading rates ranged from 0.65 to 26.80 g/m(2)/yr for total nitrogen and 0.18 to 8.96 g/m(2)/yr for total phosphorus. At loading rates below 20 g/m(2)/yr, total nitrogen concentrations in surface waters of Louisiana forested wetlands were reduced to background concentrations (i.e., < or =3 mg/l). Similarly, at loading rates below 2 g/m(2)/yr, total phosphorus concentrations were also generally reduced to background concentrations (i.e., < or =1 mg/l). These data demonstrate that freshwater forested wetlands can reduce nutrient concentrations in treated effluent to background concentrations present in relatively undisturbed wetlands. An understanding of the relationship between loading rates and nutrient removal in natural wetlands is important, particularly in Louisiana where discharges of fresh water are being used in ecosystem restoration.