Anandamide produced by Ca2+-insensitive enzymes induces excitation in primary sensory neurons

The endogenous lipid agent N-arachidonoylethanolamine (anandamide), among other effects, has been shown to be involved in nociceptive processing both in the central and peripheral nervous systems. Anandamide is thought to be synthesised by several enzymatic pathways both in a Ca²⁺-sensitive and Ca²⁺-insensitive manner, and rat primary sensory neurons produce anandamide. Here, we show for the first time, that cultured rat primary sensory neurons express at least four of the five known Ca²⁺-insensitive enzymes implicated in the synthesis of anandamide, and that application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl, the common substrate of the anandamide-synthesising pathways, results in anandamide production which is not changed by the removal of extracellular Ca²⁺. We also show that anandamide, which has been synthesised in primary sensory neurons following the application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl induces a transient receptor potential vanilloid type 1 ion channel-mediated excitatory effect that is not inhibited by concomitant activation of the cannabinoid type 1 receptor. Finally, we show that sub-populations of transient receptor potential vanilloid type 1 ion channel-expressing primary sensory neurons also express some of the putative Ca²⁺-insensitive anandamide-synthesising enzymes. Together, these findings indicate that anandamide synthesised by primary sensory neuron via a Ca²⁺-insensitive manner has an excitatory rather than an inhibitory role in primary sensory neurons and that excitation is mediated predominantly through autocrine signalling. Regulation of the activity of the Ca²⁺-insensitive anandamide-synthesising enzymes in these neurons may be capable of regulating the activity of these cells, with potential relevance to controlling nociceptive processing.     

Surgical repair of atrial septal defect with severe pulmonary hypertension during pregnancy: a case report with literature review

We are reporting a case of a 37-year-old pregnant woman with a large secundum atrial septal defect with left-to-right shunt and severe pulmonary hypertension. Her atrial septal defect was undiagnosed before this pregnancy. After carefully considering all the options, we repaired her atrial septal defect with an open heart surgical closure at 20 weeks of gestation. A substantial and consistent reduction in pulmonary arterial pressure after the surgery and subsequent uneventful delivery indicate that surgical repair of atrial septal defects is a viable option that should be considered for such patients.     

Characterization of trisomy 8 in pediatric undifferentiated sarcomas using advanced molecular cytogenetic techniques

Pediatric undifferentiated soft tissue sarcomas (USTS) are a rare group of neoplasms that are unclassifiable despite the application of immunohistochemical, cytogenetic, and molecular techniques. To date, there is a dearth of studies looking at the cytogenetic and molecular genetic alterations in such tumors. Trisomy 8, a frequent molecular alteration in neoplasia, is seen in several soft tissue sarcomas, including Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET), synovial sarcoma, and leiomyosarcoma. Because USTS share several clinicobiological features with the aforementioned tumors, the occurrence of alterations in chromosome 8 was studied in 11 pediatric USTS using a combination of interphase fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and genomic profiling with oligonucleotide array comparative genomic hybridization (aCGH). The copy number status of MYC was also assessed on the same tumors using dual-color FISH, with the aim of delineating the degree and intratumoral distribution of MYC amplification in this tumor. A near-uniform presence of an increase in MYC copy number was observed, along with an increase in chromosome 8 copy number in all the tumors. SKY and aCGH analysis of tumors exhibiting trisomy 8 confirmed the numerical imbalances. The occurrence of trisomy 8 in a subset of pediatric USTS confirms a shared genomic alteration with several other soft tissue sarcomas. Further studies are required to determine the clinical implications of such a finding.     

Wide geographic spread of diverse acquired AmpC beta-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland

OBJECTIVES: To determine the distribution of acquired AmpC beta-lactamases in 173 isolates of Escherichia coli and Klebsiella spp. submitted to the UK's national reference laboratory for antibiotic resistance. METHODS: MICs were determined and interpreted according to BSAC guidelines. Candidate isolates were those resistant to cefotaxime and/or ceftazidime, irrespective of addition of clavulanic acid. Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought by PCR. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE), and one bla(AmpC) amplicon was sequenced. RESULTS: Genes encoding acquired AmpC enzymes were detected in 67 (49%) candidate E. coli and 21 (55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance of the remaining isolates (n = 85; 49%) was inferred to result from reduced permeability or, in E. coli, from hyperexpression of chromosomal ampC. Twenty-four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these additionally producing a group 1 CTX-M extended-spectrum beta-lactamase. PFGE indicated that these isolates belonged to UK epidemic strain A, which normally produces CTX-M-15, but no acquired AmpC. Sequencing a representative bla(AmpC) amplicon indicated that in one centre this strain had acquired a novel CMY-2 variant, designated CMY-23. CONCLUSIONS: Diverse acquired AmpC enzymes occur in E. coli and Klebsiella spp. isolates in the UK and Ireland, with CIT types the most common. Producers are geographically scattered, but with some local outbreaks. Acquisition of a CMY-2-like enzyme by E. coli epidemic strain A suggests that these enzymes may be poised to become an important public health issue.     

Trends of Stroke Incidence and 28-Day All-Cause Mortality after a Stroke in Malaysia: A Linkage of National Data Sources

Background:Data on nationwide trends for stroke metrics are crucial to understand the extent of the disease burden to a country’s health system. Yet, this information remains scarce in low- and middle-income countries.Objectives:This study investigated trends of stroke incidence and 28-day all-cause mortality after a stroke from 2008 to 2016 in Malaysia, through linkage across national data sources.Methods:Hospital admissions with a principal diagnosis of stroke or transient ischemic attack were included. Cases with first stroke were identified through linkage of hospital admission registers where age and sex-standardized trends of stroke incidence and its subtypes were calculated. By linking hospital registers to the National Death Register, the 28-day all-cause mortality rates after a stroke were estimated. Mann-Kendall’s test was used for trend evaluation.Results:From 243,765 records, the trend of stroke incidence showed an increase of 4.9% in men and a drop of 3.8% among women. Incidences were higher in men, at 99.1 per 100,000 population in 2008 and 103.9 per 100,000 in 2016 than women (80.3 per 100,000 in 2008 and 77.2 per 100,000 in 2016). There was a substantial increase in stroke incidence among those below 65 years old, with the largest increase of 53.3% in men aged between 35–39 years and 50.4% in women of similar age group. The trend for 28-day all-cause mortality showed a decline for men at –13.1% and women, –10.6%. Women had higher mortality from stroke (22.0% in 2008 and 19.7% in 2016) than men (19.4% in 2008 to 17.2% in 2016).Conclusion:This first empirical study on stroke trends in Malaysia revealed a worrying increase in stroke incidence among the younger population. Despite a declining trend, mortality rates remained moderately high especially in women. Comprehensive strategies to strengthen the prevention and management of stroke care are warranted.     

Comparison of the Framingham Risk Score,SCORE and WHO/ISH cardiovascular risk prediction models in an Asian population

Background

Cardiovascular risk-prediction models are used in clinical practice to identify and treat high-risk populations, and to communicate risk effectively. We assessed the validity and utility of four cardiovascular risk-prediction models in an Asian population of a middle-income country.

Methods

Data from a national population-based survey of 14,863 participants aged 40 to 65 years, with a follow-up duration of 73,277 person-years was used. The Framingham Risk Score (FRS), SCORE (Systematic COronary Risk Evaluation)-high and -low cardiovascular-risk regions and the World Health Organization/International Society of Hypertension (WHO/ISH) models were assessed. The outcome of interest was 5-year cardiovascular mortality. Discrimination was assessed for all models and calibration for the SCORE models.

Results

Cardiovascular risk factors were highly prevalent; smoking 20%, obesity 32%, hypertension 55%, diabetes mellitus 18% and hypercholesterolemia 34%. The FRS and SCORE models showed good agreement in risk stratification. The FRS, SCORE-high and -low models showed good discrimination for cardiovascular mortality, areas under the ROC curve (AUC) were 0.768, 0.774 and 0.775 respectively. The WHO/ISH model showed poor discrimination, AUC = 0.613. Calibration of the SCORE-high model was graphically and statistically acceptable for men (χ² goodness-of-fit, p = 0.097). The SCORE-low model was statistically acceptable for men (χ² goodness-of-fit, p = 0.067). Both SCORE-models underestimated risk in women (p < 0.001).

Conclusions

The FRS and SCORE-high models, but not the WHO/ISH model can be used to identify high cardiovascular risk in the Malaysian population. The SCORE-high model predicts risk accurately in men but underestimated it in women.     

Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

The cytoskeleton is a highly dynamic structure composed of multiple types of filamentous proteins. In eukaryotic cells, actin, microtubules, and intermediate filaments (IFs) each form intricate networks of entangled and cross-linked filaments. The organization of each individual network is precisely controlled to enable essential cellular functions. However, many core processes also require interactions among the different cytoskeletal components. For example, filamentous-actin (F-actin) and microtubules work together to control cell shape and polarity, which are critical for development, cell migration, and division. Close associations between microtubules and vimentin IFs (VIFs) have also been proposed based on similarities in their spatial distributions and the dependence of the organization of VIF networks on the microtubule-associated motors, kinesin and dynein (1–3). Indeed, there is some experimental evidence that microtubules can template VIF assembly and that VIFs can guide microtubules (4, 5), while VIFs stabilize microtubules in vitro (6). In addition, in stratified epithelial cells, a subplasmalemmal rim of keratin IFs can be localized just below the actin cortex, suggesting cooperativity of keratin and actin networks in regulating cell mechanics (7). Despite such interactions, VIFs and F-actin are generally thought to form two coexisting but separate networks. For example, fluorescence microscopy typically reveals the strongest signals for F-actin in the cell periphery, whereas the strongest signals for VIFs are near the nucleus in the bulk cytoplasm, suggesting that the two networks have little or no interaction. Furthermore, the functions of F-actin and VIFs appear to be largely contrasting: F-actin generates forces, whereas VIFs provide stability against these forces. Nevertheless, some evidence suggests there may be connections between vimentin and actin: for example, vimentin knockout cells are less motile and less contractile than their wild-type (WT) counterparts (8). Furthermore, some interactions have been observed between F-actin and VIFs (9–11) as well as the precursors to keratin, another IF system (12). These findings suggest that direct interactions or connections may exist between VIFs and F-actin. However, there have been no reports of direct observations of these interactions through imaging or other means, which would provide conclusive evidence of their significance. Such connections would belie our current understanding of the two independent cytoskeletal networks but could have a profound effect on the mechanical properties of cells. The possibility of such connections demands a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks.Here we present evidence that VIFs and F-actin do work synergistically and form an interpenetrating network (IPN) structure within the cell cortex, defined as the cortical cytoplasm adjacent to the cell surface. We combine high-resolution structured illumination microscopy (SIM) and cryo-electron tomography (cryo-ET) to image mouse embryonic fibroblasts (MEFs) and observe coupling between F-actin and VIF structures within the cortex, contrary to the widely accepted view that they are each spatially segregated. In fact, the association of VIFs with cortical arrays of F-actin stress fibers occurs at multiple length scales. For example, VIFs run through and frequently appear to interconnect with adjacent stress fibers, forming meshworks that surround them. These organizational states are consistent with the formation of an IPN. We show that this IPN structure has important functional consequences in cells and can result in enhanced contractile forces. Moreover, our results indicate that specific associations exist between actin and vimentin proteins in the cytoplasmic environment, which may facilitate the formation of an IPN; the results also show that the VIF network can influence the diffusive behavior of actin monomers, which may, in turn, have downstream effects on other actin-driven processes. Thus, vimentin has a far more comprehensive role in cellular function than previously thought. These findings confirm the importance of the interplay between VIFs and F-actin, especially as it relates to the formation of IPNs and their consequences on the contractile nature of cells.