In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment.

Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998-2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infection in vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancement in vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operating in vivo may modulate ZIKV infection outcomes.IMPORTANCEWe investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998-2020) to neutralize or enhance Zika virus infection in vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, both in vitro and in vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Glutamate binding triggers monomerization of unliganded mGluR2 dimers.

The Metabotropic glutamate receptor 2 (mGluR2) is involved in several neurological and psychiatric disorders and is an attractive drug target. It is believed to form a strict dimer and the dimeric assembly is necessary for glutamate induced activation. Although many studies have focused on glutamate induced conformational changes, the dimerization propensity of mGluR2 with and without glutamate has never been investigated. Also, the role of the unstructured loop in dimerization of mGluR2 is not clear. Here, using Forster Resonance Energy Transfer (FRET) based assay in live cells we show that mGluR2 does not form a "strict dimer" rather it exists in a dynamic monomer-dimer equilibrium. The unstructured loop moderately destabilizes the dimers. Furthermore, binding of glutamate to mGluR2 induces conformational change that promotes monomerization of mGluR2. In the absence of an unstructured loop, mGluR2 neither undergoes conformational change nor monomerizes upon binding to glutamate.

Unstructured loop is essential for the activation of mGluR2.

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or "BC loop") in activation of metabotropic Glutamate receptors is currently unknown. Here, using Förster Resonance Energy Transfer (FRET) based assay in live cells we show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor.

Neonatal Exposure to Endocrine Disrupting Chemicals Impairs Learning Behaviour by Disrupting Hippocampal Organization in Male Swiss Albino Mice.

Hippocampus is highly susceptible to endocrine disrupting chemicals exposure particularly during the critical phase of brain development. In this study, mice offspring were exposed to endocrine disruptors mancozeb (MCZ) and imidacloprid (IMI) individually (40 mg MCZ and 0.65 mg IMI/kg/day) as well as to their equimixture (40 mg MCZ + 0.65 mg IMI/kg/day) through the diet of lactating mothers from post-natal day (PND) 1 to PND 28. Half of the randomly selected male offspring were killed at PND 29, and the rest half were left unexposed and killed at PND 63. Brain weight, histology, plasma hormone profile and working memory performance were the various end-points studied. Brain weight was significantly decreased in the mixture-exposed group at PND 29, which persisted to PND 63. Total thickness of pyramidal cell layers decreased significantly along with misalignment, shrinkage and degeneration of pyramidal neurons in CA1 and CA3 regions of the IMI and mixture-exposed groups. The length and branch points of dendrites of pyramidal neurons were decreased significantly in mixture-exposed group at both PND 29 and PND 63. Dendritic spine density was also reduced in mixture-exposed group offspring. Testosterone level was significantly decreased only at PND 29, but corticosterone level was increased at both PND 29 and PND 63 in mixture-exposed offspring. T-maze task performance revealed significantly increased time duration and reduced path efficiency in mixture-exposed group offspring. The results thus indicate that pesticide mixture exposure could lead to changes in learning behaviour even at doses that individually did not induce any adverse effect on hippocampal organization.

Quaternary structures of opsin in live cells revealed by FRET spectrometry.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

Better innovate than compromise: a novel hepatic outflow reconstruction technique in pediatric living donor liver transplantation.

Pediatric LDLT using donors with unfavorable vascular anatomy is challenging in terms of donor safety, and complexity of reconstruction in the recipient. We describe an innovative technique of hepatic venous outflow reconstruction involving the recipient RHV, in the presence of a rudimentary RHV in the donor. The postoperative course of the donor and recipient was uneventful with satisfactory venous outflow in both. This technique avoided the use of prosthetic material, an important consideration given the recipient age and requirement for growth. This shows that donors previously considered unsuitable for donation can be utilized safely as long as principles of vascular anastomosis are adhered to. Moreover, it highlights that innovation is sometimes necessary to avoid compromise in donor safety.

The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands.

The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s).

Effect of acute hexavalent chromium exposure on pituitary-thyroid axis of a freshwater fish, Channa punctatus (Bloch).

Acute exposure to hexavalent chromium (10 mg L(-1) , 20 mg L(-1) , and 40 mg L(-1) potassium dichromate for 96 h) dose-dependently affected the pituitary-thyroid axis of teleost, Channa punctatus. Significant hypertrophy of the thyroid follicle was observed in 20 mg L(-1) and 40 mg L(-1) groups; the follicular epithelium was however hypertrophied only in 40 mg L(-1) group. The colloid depletion in the lumen of thyroid follicle was evident in 20 mg L(-1) and 40 mg L(-1) groups. Serum thyroid hormones (thyroxine/T4 and triiodothyronine/T3) level increased significantly at both the higher doses. Increased immunointensity and significant hypertrophy of the pituitary thyrotrophs (anti TSHß-immunoreactive cells) was observed in both 20 mg L(-1) and 40 mg L(-1) chromium-exposed fish. The increased thyroid hormones secretion observed in this study might be an adaptive response of the pituitary-thyroid axis under acute chromium-induced stress condition to maintain homeostasis. The long-term Cr(VI) exposures, however, may lead to attenuation/exhaustion of the pituitary-thyroid axis and pose serious threat to fish health and affect their population.

Acute spill-mimicking exposure effect of hexavalent chromium on the pituitary-ovarian axis of a teleost, Channa punctatus (Bloch).

Acute exposure to hexavalent chromium (as 10, 20, and 40 mg/L potassium dichromate for 96 h) adversely affected the pituitary-ovarian axis of a teleost Channa punctatus. The toxic impact of metal exposure on fish ovary was revealed in the form of increased percentage of atretic follicles, significantly in 20 mg/L and 40 mg/L exposure groups. The follicular atresia mostly occurred in vitellogenic (stage II and stage III) oocytes. Reduction of serum level of 17ß-estradiol was also significant in 20 mg/L and 40 mg/L exposure groups. The increase of LH-immunointensity of pituitary gonadotrophs (LHß-immunoreactive cells) and their hypertrophy was evident, significantly in fish of 40 mg/L exposed group. Thus, the present acute metal spill-mimicking laboratory study clearly demonstrated that short-term exposures to high doses of hexavalent chromium may disrupt reproduction of the fish and affect their population.

Effect of sublethal hexavalent chromium exposure on the pituitary-ovarian axis of a teleost, Channa punctatus (Bloch).

Exposure to sublethal hexavalent chromium (as 2 and 4 mg L⁻¹ potassium dichromate for 1 and 2 months) during late preparatory to mid prespawning phase of annual reproductive cycle severely affected the pituitary-ovarian axis of a teleost Channa punctatus. Gonadosomatic index (GSI), ovarian histopathology, immunocytochemistry of the pituitary gonadotrophs (LHß-immunoreactive cells), and serum 17ß-estradiol level revealed distinct dose and duration-dependent effects. Gonadosomatic index was declined. Diameter of ovary as well as ovarian follicles was reduced along with a distinct variation in the percentage of follicles. The greater percentage of previtellogenic follicles (stage I) and decline in vitellogenic ones (stage II and stage III) compared to control indicated the arrest of follicular development. The percentage of atretic follicles was also increased indicating toxic impact of metal on ovary. Decreased serum level of ovarian steroid 17ß-estradiol further indicated the same. Chromium interference of the pituitary was demonstrated as atrophy and less immunointensity of LH-immunoreactive gonadotrophs. Thus, the hexavalent chromium impaired the pituitary-ovarian axis affecting at the sites of both pituitary and ovary.

Chronic exposure to sublethal hexavalent chromium affects organ histopathology and serum cortisol profile of a teleost, Channa punctatus (Bloch).

Effects of chronic exposures (one and two months) to sublethal doses of hexavalent chromium (2 and 4 mg/L potassium dichromate) on organ histopathology and serum cortisol profile were investigated and their overall impact on growth and behavior of a teleost fish, Channa punctatus was elucidated. Histopathological lesions were distinct in the vital organs gill, kidney and liver. The gill lamellae became lifted, fused, and showed oedema. Hyperplasia and hypertrophy of lamellar epithelial cells were distinct with desquamation. Hypertrophy of epithelial cells of renal tubules and reduction in tubular lumens were observed in the trunk kidney. The atrophy of the head kidney interrenal cells and decreased serum cortisol level indicated exhaustion of interrenal activity. Hepatocyte vacuolization and shrinkage, nuclear pyknosis and increase of sinusoidal spaces were observed in the liver. Abnormal behavioral patterns and reduced growth rate were also noticed in the exposed fish. The chronic hexavalent chromium exposure thus by affecting histopathology of gill, kidney (including interrenal tissue) and liver could impair the vital functions of respiration, excretion, metabolic regulation and maintenance of stress homeostasis which in the long-run may pose serious threat to fish health and affect their population.

Effect of hexavalent chromium exposure on the pituitary-interrenal axis of a teleost, Channa punctatus (Bloch).

The immunocytochemistry of pituitary corticotrophs, interrenal histopathology and serum cortisol assay revealed adverse effects of hexavalent chromium [Cr(VI)] exposure (as potassium dichromate; K(2)Cr(2)O(7)) on the pituitary-interrenal axis of a freshwater fish, Channa punctatus. On acute exposure (10, 20 and 40 mg L(-1) for 96 h), corticotrophs showed hypertrophy and intense immunoreactivity to anti-human (h) ACTH(1-29). Cytoplasmic degranulations in some of the cells were also noticed. On the contrary, atrophy and weak anti-h ACTH(1-29) immunoreactivity of corticotrophs was observed with the chronic exposure (2 and 4 mg L(-1) for one month). Exposure-dependent varied effects also reflected in the histopathology of head kidney interrenal tissue and serum cortisol levels. Acute exposure induced hypertrophy and degranulation of interrenal cells along with significant elevation of serum cortisol levels in a concentration-dependent manner (42+/-0.72, 44.6+/-1.34 and 52+/-3.06 ng mL(-1), respectively in 10, 20 and 40 mg L(-1) group, compared to 36.65+/-0.83 of control). Atrophy of interrenal cells and low serum cortisol levels as compared to that of control (34.1+/-0.78 and 29.3+/-0.92 ng mL(-1), respectively in 2 and 4 mg L(-1) groups) in chronic-exposed fish indicates attenuated functioning of the hypothalamic-pituitary-interrenal (HPI) axis. The stress response of the fish (under confinement) further confirmed the impairment of the HPI axis, as the elevation in serum cortisol levels were significantly less (258% and 166% of basal level, respectively in 2 and 4 mg L(-1) group, compared to 374% of control). The long-term interference of this axis by metal exposure therefore, may pose serious threat to fish making them unable to adapt to stress.

Acute toxicity impacts of hexavalent chromium on behavior and histopathology of gill, kidney and liver of the freshwater fish, Channa punctatus (Bloch).

Alteration in behavioral patterns and histopathology of gill, kidney and liver were studied in Channa punctatus, after acute exposure to hexavalent chromium. The 96h LC(50) of chromium salt, potassium dichromate was determined to be 41.75mg/L. The exposed fish displayed erratic swimming and became lethargic. The changes in gills were characterized by epithelial hyperplasia, lamellar fusion, oedema, epithelial lifting, epithelial necrosis and desquamation, aneurism as well as curling of secondary lamellae. Hypertrophy of epithelial cells of renal tubules with reduced lumens, atrophy of the renal tubules, glomeruli contraction in the Bowman's capsules and necrosis of haematopoietic tissues were observed in the trunk kidney of exposed fish. The interrenal cells of the head kidney exhibited distinct hypertrophy and vacuolization. The liver hepatocytes showed cytoplasmic vacuolization with the lateral nuclei arrangement. Hepatocytes atrophy and increase in sinusoidal space were also observed. The result showed that acute chromium toxicity severely affects the vital organs and normal behavior which may be deleterious for fish populations.