Biology subjects

Gurung, S.

Publications and source records attributed to Gurung, S..

23 records · Page 2Linked to original sources

bioRxiv2020

MATERNAL ZIKA VIRUS (ZIKV) INFECTION FOLLOWING VAGINAL INOCULATION WITH ZIKV-INFECTED SEMEN IN THE TIMED-PREGNANT OLIVE BABOON

ZIKV infection is associated with pregnancy loss, fetal microcephaly and other malformations. While Aedes sp. of mosquito are the primary vector for ZIKV, sexual transmission of ZIKV is a significant route of infection. ZIKV has been documented in human, mouse and non-human primate (NHP) semen. It is critical to establish NHP models of vertical transfer of ZIKV that recapitulate human ZIKV pathogenesis. We hypothesized that vaginal deposition of ZIKV infected baboon semen would lead to maternal infection and vertical transfer in the olive baboon (Papio anubis). Timed pregnant baboons (n=6) were inoculated via vaginal deposition of baboon semen containing 106 ffu ZIKV (n=3, French Polynesian isolate:H/PF/2013, n=3 Puerto Rican isolate:PRVABC59) at mid-gestation (86-95 days gestation [dG]; term 183dG) on day (d) 0 (all dams), and then at 7 day intervals through three weeks. Maternal blood, saliva and cervico-vaginal washes were obtained at select days post-inoculation. Animals were euthanized at 28 days post initial inoculation (dpi; n=5) or 39 dpi (n=1) and maternal/fetal tissues collected. vRNA was quantified by qPCR. Viremia was achieved in 3/3 FP ZIKV infected dams and 2/3 PR ZIKV. ZIKV RNA was detected in cvw (5/6 dams;). ZIKV RNA was detected in lymph nodes, but not ovary, uterus, cervix or vagina in the FP ZIKV dams but was detected in uterus, vagina and lymph nodes. Placenta, amniotic fluid and all fetal tissues were ZIKV RNA negative in the FP infected dams whereas 2/3 PR infected dam placentas were ZIKV RNA positive. We conclude that ZIKV infected semen is a means of ZIKV transmission during pregnancy in primates. The PR isolate appeared more capable of wide spread dissemination to tissues, including placenta compared to the FP strain. IMPORTANCEDue to its established link to pregnancy loss, microcephaly and other major congenital anomalies, Zika virus (ZIKV) remains a worldwide health threat. Although mosquitoes are the primary means of ZIVK transmission, sexual transmission in human populations is well documented and provides a means for widespread dissemination of the virus. Differences in viremia, tissue distribution, immune responses and pregnancy outcome from sexually transmitted ZIKV compared to the subcutaneous route of infection are needed to better clinically manage ZIKV in pregnancy. Through our previous work, we have developed the olive baboon as a non-human primate model of ZIKV infection that is permissible to ZIKV infection via the subcutaneous route of inoculation and transfer of ZIKV to the fetus in pregnancy. The current study evaluated the course of ZIKV infection after vaginal inoculation of ZIKV in pregnant baboons at mid-gestation using baboon semen as the carrier and comparing two isolates of ZIKV, the French Polynesian isolate first associated with microcephaly and the Puerto Rican isolate, associated with an increased risk of microcephaly observed in the Americas.

physiology↗

bioRxiv2019

Preparation and characterization of phospholipid stabilized nanoemulsions in small-scale

Phospholipids have been used to prepare liposomes. The use of phospholipids to stabilize nanoemulsions may cause spontaneous formation of liposomes. The main objective of this study is to develop a method to prepare phospholipid stabilized nanoemulsions in small scale (< 1 mL) and to minimize the formation of liposomes.\n\nA combination of hand extrusion and detergent removal methods was used in this study. Extrusion through polycarbonate membranes was performed in two steps, firstly using membranes of 400 nm followed by 200 nm membranes as the second step. Sodium cholate was used as a detergent to solubilize the formed liposomes which was later removed via dialysis. Nanoemulsions were characterized by measuring their particle size, polydispersity index and zeta-potential using Photon Correlation Spectroscopy and Cryo-TEM pictures. The stability of nanoemulsion stored under refrigeration was also studied.\n\nFifty-one extrusion cycles through polycarbonate membrane of 400 nm pore size followed by one-hundred fifty-three cycles through polycarbonate membrane of 200 nm produced nanoemulsions having particle size below 200 nm (diameter). The nanoemulsions were found to be homogenous as depicted by polydispersity index (PDI) value below 0.1. Similarly, the zeta-potential was measured to be above -30 mV which is sufficient to keep nanoemulsions stable for as long as 7 months when stored under refrigeration. The Cryo-TEM pictures revealed 30 mM to be an optimum concentration of sodium cholate to prepare homogenous nanoemulsions with negligible proportion of liposomes.\n\nIt was concluded that this method could be established as a small scale method of preparing nanoemulsions which will not only reduce the cost of preparation but also the disposal cost of toxic chemicals used for functionalizing nanoemulsions for scientific research.

biophysics↗

bioRxiv2018

ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity

Kainate receptors (KARs) regulate neuronal excitability and network function. Most KARs contain the subunit GluK2 and the properties of these receptors are determined in part by ADAR2-mediated mRNA editing of GluK2 that changes a genomically encoded glutamine (Q) to arginine (R). Suppression of synaptic activity reduces ADAR2-dependent Q/R editing of GluK2 with a consequential increase in GluK2-containing KAR surface expression. However, the mechanism underlying this reduction in GluK2 editing has not been addressed. Here we show that induction of KAR upscaling results in proteasomal degradation of ADAR2, which reduces GluK2 Q/R editing. Because KARs incorporating unedited GluK2(Q) assemble and exit the ER more efficiently this leads to an upscaling of KAR surface expression. Consistent with this, we demonstrate that partial ADAR2 knockdown phenocopies and occludes KAR upscaling. Moreover, we show that although the AMPAR subunit GluA2 also undergoes ADAR2-dependent Q/R editing, this process does not mediate AMPAR upscaling. These data demonstrate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing are key determinants of KAR, but not AMPAR, trafficking and upscaling.\n\nSummary statementSynaptic suppression promotes proteasomal degradation of the mRNA-editing enzyme ADAR2. Decreased ADAR2 levels reduce Q/R editing of the kainate receptor subunit GluK2 leading to enhanced surface expression and homeostatic upscaling.

neuroscience↗

bioRxiv2018

ZIKA VIRUS INFECTION AT MID-GESTATION RESULTS IN FETAL CEREBRAL CORTICAL INJURY AND FETAL DEATH IN THE OLIVE BABOON.

Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to a Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and macaques. While microcephaly has been achieved in mice via direct injection of ZIKV into the fetal brain or via interference with interferon signaling, in macaques the primary fetal CZS outcome are ocular defects. In the present study we develope the olive baboon (Papio anubis), as a model for the vertical transfer of ZIKV during pregnancy. We infected four mid-gestation, timed-pregnant baboons with the French Polynesian ZIKV isolate (104 ffu) and examined the acute phase of vertical transfer by stopping the study of one dam at 7 days post infection (dpi), two at 14 dpi and one at 21 dpi. All dams exhibited mild to moderate rash and conjunctivitis; three of four dams exhibited viremia at 7 dpi. Of the three dams studied to 14 to 21 days, only one still exhibited viremia on day 14. Vertical transfer of ZIKV to the fetus was found in two pregnancies; in one, vertical transfer was associated with fetal death at [~]14 dpi. In the other, vertical transfer was observed at 21 dpi. Both fetuses had ZIKV RNA in the fetal cerebral cortex as well as other tissues. The 21 dpi fetal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, loss and or damage of immature oligodendrocytes and a loss in neuroprogenitor cells (NPCs). In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL-6 expression. The dams studied to 14 dpi (n=2) and 21 dpi (n=1) exhibited a anti-ZIKV IgM response and IgG response (21 dpi) that included transfer of the IgG to the fetal compartment (cord blood). The severity of systemic inflammatory response (cytokines and chemokines) reflected the vertical transfer of ZIKV in the two pregnancies. As such, these events likely represent the early mechanisms that lead to microcephaly and/or other CNS pathologies in a primate infected with ZIKV and are the first to be described in a non-human primate during the acute phase of ZIKV infection with a contemporaneous ZIKV strain. The baboon thus represents a major NHP for advancing as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs such as macaques.\n\nAUTHOR SUMMARYZika virus is endemic in the Americas, primarily spread through mosquitos and sexual contact. Zika virus infection during pregnancy in women is associated with a variety of fetal pathologies now referred to as Congenital Zika Syndrome (CZS), with the most severe pathology being fetal microcephaly. Developing model organisms that faithfully recreate Zika infection in humans is critical for future development of treatments and preventions. In our present study, we infected Olive baboons at mid-gestation with Zika virus and studied the acute period of viremia and transfer of Zika virus to the fetus during the first three weeks after infection to better understand the timing and mechanisms leading to CZS. We observed Zika virus transfer to fetuses resulting in fetal death in one pregnancy and in a second pregnancy, significant damage to the frontal cortex of the fetal brain consistent with development of microcephaly, closely resembling infection in pregnant women. Our baboon model differs from macaque non-human primate models where the primary fetal outcome during pregnancy following infection with contemporary strains of Zika virus is ocular pathology. Thus, the baboon provides a promising new non-human primate model to further compare and contrast the consequences of Zika virus infection in pregnancy to humans and macaques to better understand the disease.

neuroscience↗

bioRxiv2018

ADAR2 mediated Q/R editing of GluK2 regulates homeostatic plasticity of kainate receptors

Kainate receptors (KARs) are heteromeric glutamate-gated ion channels that regulate neuronal excitability and network function in the brain. Most KARs contain the subunit GluK2 and the precise properties of these GluK2-containing KARs are determined by additional factors including ADAR2-mediated mRNA editing of a single codon that changes a genomically encoded glutamine (Q) to arginine (R) in the pore-lining region of GluK2. ADAR2-dependent Q/R editing of GluK2 is dynamically regulated during homeostatic plasticity (scaling) elicited by suppression of synaptic activity with TTX. Here we show that TTX decreases levels of ADAR2 by enhancing its proteasomal degradation. This selectively reduces the numbers of GluK2 subunits that are edited and increases the surface expression of GluK2-containing KARs. Furthermore, we show that partial ADAR2 knockdown phenocopies and occludes TTX-induced scaling of KARs. These data indicate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing provides a mechanism for KAR homeostatic plasticity.

neuroscience↗