Spatial tracking of individual fluid dispersed particles via Raman spectroscopy.

We demonstrate a method for the spatial tracking of individual particles, dispersed in a fluid host, via Raman spectroscopy. The effect of moving a particle upon the intensity of different bands within its Raman spectrum is first established computationally through a scattering matrix method. By comparing an experimental spectrum to the computational analysis, we show that the position of the particle can be obtained. We apply this method to the specific cases of molybdenum disulfide and graphene oxide particles, dispersed in a nematic liquid crystal, and contained within a microfluidic channel. By considering the ratio and difference between the intensities of the two Raman bands of molybdenum disulfide and graphene oxide, we demonstrate that an accurate position can be obtained in two dimensions

NEDDylation is essential for Kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-KSHV target.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS) has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs) that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924) is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt). These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

The University of Queensland medical leadership program: a case study

Changes in modern healthcare's provision, complexity, and workforce demands provide a compelling rationale for an increasing emphasis on leadership development at all levels of training within the medical profession. Undergraduate medical education has traditionally focused on the development of clinical acumen with little emphasis on the development of leadership skills or on the operational and systemic issues surrounding healthcare delivery. Incorporating leadership education and competencies presents a number of challenges to medical schools, including defining the subject area, determining the specific skills and knowledge bases that should constitute the basis of the program, and optimizing training to be integrated into the existing clinical curriculum. We present a case study of the Medical Leadership Program at The University of Queensland School of Medicine that runs concurrent to the undergraduate medical degree. We outline the inception of the program, its aims, participant selection, and program components and reflect on the program to date

Differential expression of K4-AP currents and Kv3.1 potassium channel transcripts in cortical neurons that develop distinct firing phenotypes.

Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of cortical neurons. Here we describe the development of cortical neurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a population of immature multiple-spiking (IMS) neurons during the first two postnatal weeks, both in vivo (within layer IV) and in vitro. We report the expression of a slowly inactivating, 4-AP-sensitive potassium current (K4-AP) at significantly higher density in FS compared with RS neurons. The same current is expressed at intermediate levels in IMS neurons. The kinetic, voltage-dependent, and pharmacological properties of the K4-AP current are similar to those observed by heterologous expression of Kv3.1 potassium channel mRNA. Single-cell RT-PCR analysis demonstrates that PCR products representing Kv3.1 transcripts are amplified more frequently from FS than RS neurons, with an intermediate frequency of Kv3.1 detection in neurons with immature firing properties. Taken together, these data suggest that the Kv3.1 gene encodes the K4-AP current and that expression of this gene is regulated in a cell-specific manner during development. Analysis of the effects of 4-AP on firing properties suggests that the K4-AP current is important for rapid action potential repolarization, fast after-hyperpolarization, brief refractory period, and high firing frequency characteristic of FS GABAergic interneurons

Differential expression of K4-AP currents and Kv3.1 potassium channel transcripts in cortical neurons that develop distinct firing phenotypes.

Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of cortical neurons. Here we describe the development of cortical neurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a population of immature multiple-spiking (IMS) neurons during the first two postnatal weeks, both in vivo (within layer IV) and in vitro. We report the expression of a slowly inactivating, 4-AP-sensitive potassium current (K4-AP) at significantly higher density in FS compared with RS neurons. The same current is expressed at intermediate levels in IMS neurons. The kinetic, voltage-dependent, and pharmacological properties of the K4-AP current are similar to those observed by heterologous expression of Kv3.1 potassium channel mRNA. Single-cell RT-PCR analysis demonstrates that PCR products representing Kv3.1 transcripts are amplified more frequently from FS than RS neurons, with an intermediate frequency of Kv3.1 detection in neurons with immature firing properties. Taken together, these data suggest that the Kv3.1 gene encodes the K4-AP current and that expression of this gene is regulated in a cell-specific manner during development. Analysis of the effects of 4-AP on firing properties suggests that the K4-AP current is important for rapid action potential repolarization, fast after-hyperpolarization, brief refractory period, and high firing frequency characteristic of FS GABAergic interneurons

A flexible framework for online collaborative learning

This paper presents a framework for online collaborative learning, also known as telecollaboration. At the centre of this flexible framework are online collaborative educational experiences where knowledge creation and knowledge in action are the nexus of social, teaching and cognitive presence based on the Community of Inquiry model of Garrison, Anderson and Archers (1999). The framework provided should guide educators as they design, develop and implement authentic educational experiences within local, national or international settings in partnership with other educational stakeholders

Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human β2-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPγ35S/GDP exchange of the transducin α subunit (Gαt) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the Gαt C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Gαt C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Gαt-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions