Targeting neural circuitry in zebrafish using GAL4 enhancer trapping

We present a pilot enhancer trap screen using GAL4 to drive expression of upstream activator sequence (UAS)-linked transgenes in expression patterns dictated by endogenous enhancers in zebrafish. The patterns presented include expression in small subsets of neurons throughout the larval brain, which in some cases persist into adult. Through targeted photoconversion of UAS-driven Kaede and variegated expression of UAS-driven GFP in single cells, we begin to characterize the cellular components of labeled circuits.     

12S-lipoxygenase protein associates with alpha-actin fibers in human umbilical artery vascular smooth muscle cells

The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to alpha-actin, a component of the cytoplasmic myofilaments. 12-LO/alpha-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to alpha-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein alpha-actin.     

Cytokine storm in COVID-19: from viral infection to immune responses,diagnosis and therapy

The COVID-19 outbreak is emerging as a significant public health challenge. Excessive production of proinflammatory cytokines, also known as cytokine storm, is a severe clinical syndrome known to develop as a complication of infectious or inflammatory diseases. Clinical evidence suggests that the occurrence of cytokine storm in severe acute respiratory syndrome secondary to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely associated with the rapid deterioration and high mortality of severe cases. In this review, we aim to summarize the mechanism of SARS-CoV-2 infection and the subsequent immunological events related to excessive cytokine production and inflammatory responses associated with ACE2-AngII signaling. An overview of the diagnosis and an update on current therapeutic regimens and vaccinations is also provided.     

Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species.

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.     

RNF-121 Is an Endoplasmic Reticulum-Membrane E3 Ubiquitin Ligase Involved in the Regulation of β-Integrin

We report on the characterization of RNF-121, an evolutionarily conserved E3 ligase RING finger protein that is expressed in the endoplasmic reticulum (ER) of various cells and tissues in Caenorhabditis elegans. Inactivation of RNF-121 induced an elevation in BiP expression and increased the sensitivity of worms to ER stress. Genetic analysis placed RNF-121 downstream of the unfolded protein response (UPR) regulator protein kinase-like endoplasmic reticulum kinase (PERK). We identify PAT-3::GFP, the β subunit of the heterodimeric integrin receptors, as an RNF-121 substrate; whereas induction of RNF-121 expression reduced the level of PAT-3::GFP in the gonad distal tip cells, inhibition of RNF-121 led to the accumulation of stably bound PAT-3::GFP inclusions. Correspondingly, overexpression of RNF-121 during early stages of gonad development led to aberrations in germline development and gonad migration that overlap with those observed after PAT-3 inactivation. The formation of these gonad abnormalities required functional ER-associated degradation (ERAD) machinery. Our findings identify RNF-121 as an ER-anchored ubiquitin ligase that plays a specific role in the ERAD pathway by linking it to the regulation of the cell adhesion integrin receptors.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

JAMP optimizes ERAD to protect cells from unfolded proteins

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.     

From Crisis to Emergency: The Shifting Logic of Preparedness

Following the Second Lebanon War (2006), Israeli preparedness exercises were designed in reference to that crisis event. Hold annually for more than a decade, ‘Turning Point’ exercises are now accompanied by a ‘National Emergency Week’. After three years of fieldwork in the National Emergency Management Authority (NEMA) and the Turning Point administration, we came to realise that the conceptualisation of preparedness for such events has morphed. Through an analytical discussion on the concepts of crisis and emergency, we argue that a shift in orientation has occurred and crisis response gave birth to emergency management. That is, preparedness is no longer driven by historical precedent but has become a form of future-oriented emergency practice. Moreover, we argue, an emergency apparatus – a distinct technology of governance – has emerged that, although varying in form and composition, has become the means and ends of civilian-front preparedness.