Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line

Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/CRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/CRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.     

Characterization and distribution of bombesin binding sites in the goldfish hypothalamic feeding center and pituitary

Bombesin (BBS)/gastrin-releasing peptide (GRP) binding sites were characterized and their distribution examined in the goldfish brain and pituitary by radioligand binding and autoradiography. Binding of ¹²⁵I-[Tyr⁴]-BBS-14 to tissue sections was found to be saturable, reversible, time-dependent and displaceable by BBS/GRP-like peptides. Analysis of saturable equilibrium binding revealed a one-site model fit with a K_d of 0.665 ± 0.267 nM. This binding site displayed high affinity for members of the BBS subfamily of peptides, including GRP10 (K_i; 0.292 ± 0.038 nM) and GRP27 (K_i; 2.034 ± 1.597 nM), but showed no affinity for the BBS_8–14 fragment. While an approximate 100-fold lower binding affinity was displayed by the binding site for neuromedin B (K_i; 61.5 ± 28.2 nM), litorin was highly effective in displacing radiolabeled BBS binding (K_i; 1.469 ± 0.427 nM). The localization of saturable and high affinity BBS/GRP binding sites in specific areas of the goldfish brain and pituitary generally revealed a similar anatomical distribution to BBS/GRP-like immunoreactive material reported previously by our laboratory. Quantitative densitometric analysis of radiolabeled BBS binding to brain nuclei and the pituitary revealed a moderate concentration of BBS/GRP binding sites in the hypothalamic feeding area, including the nucleus diffusus lobi inferioris, nucleus recessus lateralis, nucleus lateral tuberis, and nucleus anterior tuberis. Other brain nuclei known to influence the brain feeding center which contained a high density of BBS/GRP binding sites included nuclei of the dorsal and ventro-medial telencephalon, the preoptic hypothalamus, and the optic tectum. High densities of BBS/GRP binding sites were also localized in the dorsal cerebellum, and nucleus habenularis. In the pituitary, BBS/GRP binding sites were present in high concentration in the neurointermediate lobe, with a relatively lower density localized in the pars distalis. The present study further supports a role for BBS/GRP-like peptides in the regulation of feeding behavior and anterior pituitary hormone secretion in teleosts.     

Synthesis and evaluation of azaproline peptides as potential inhibitors of dipeptidyl peptidase IV and prolyl oligopeptidase

Summary A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range.     

Phage P22 helps phage MB78 to overcome blockage of DNA maturation in rifampicin-resistant host

Bacteriophage MB78, a virulent phage ofSalmonella typhimurium cannot grow in rifampicin-resistant mutant (rif-39) of the host having altered RNA polymerase. The temperate phage P22 which cannot multiply in presence of the virulent phage MB78 can, however, help MB78 to overcome replication inhibition in rif-39. The processing of concatemeric phage DNA to monomer is blocked in this nonpermissive host. Superinfection with P22 induces synthesis of at least five P22 specific polypeptides which help phage MB78 in the processing of the concatemeric DNA and maturation of phage particles.     

Bombesin-like peptides stimulate phosphatidylinositol turnover in rat brain slices

The ability of bombesin (BN)-like peptides to stimulate phosphatidylinositol turnover in rat brain slices was investigated. BN (1 μM) significantly stimulated inositol-1-phosphate (IP₁) but not inositol-4,5-biphosphate (IP₂) or inositol-1,4,5-trisphosphate (IP₃) production using frontal cortex slices in the presence of LiCl (7.5 mM); BN had no effect on cAMP or cGMP levels. BN and the structurally-related gastrin releasing peptide (GRP) elevated IP₁ levels in a dose-dependent manner. Similarly, nanomolar concentrations of the GRP fragment (Ac-GRP^20–27) significantly elevated IP₁ levels, whereas micromolar concentrations of the inactive GRP^1–16 did not. BN significantly elevated IP₁ levels in those brain regions enriched in BN receptors such as the olfactory bulb, hippocampus, striatum, thalamus and frontal cortex, whereas IP₁ levels were not significantly increased in areas which have a low density of BN receptors such as the cerebellum, medulla/pons and midbrain. These data suggest that CNS BN receptors may utilize phosphatidylinositol as a second messenger.     

Cleavage and degradation of EDEM1 promotes coxsackievirus B3 replication via ATF6a‐mediated unfolded protein response signalling

Our previous study of coxsackievirus B3 (CVB3)‐induced unfolded protein responses (UPR) found that overexpression of ATF6a enhances CVB3 VP1 capsid protein production and increases viral particle formation. These findings implicate that ATF6a signalling benefits CVB3 replication. However, the mechanism by which ATF6a signalling is transduced to promote virus replication is unclear. In this study, using a Tet‐On inducible ATF6a HeLa cell line, we found that ATF6a signalling downregulated the protein expression of the endoplasmic reticulum (ER) degradation‐enhancing α‐mannosidase‐like protein 1 (EDEM1), resulting in accumulation of CVB3 VP1 protein; in contrast, expression of a dominant negative ATF6a had the opposite effect. Furthermore, we found that EDEM1 was cleaved by both CVB3 protease 3C and virus‐activated caspase and subsequently degraded via the ubiquitin‐proteasome pathway. However, overexpression of EDEM1 caused VP1 degradation, likely via a glycosylation‐independent and ubiquitin‐lysosome pathway. Finally, we demonstrated that CRISPR/Cas9‐mediated knockout of EDEM1 increased VP1 accumulation and thus CVB3 replication. This is the first study to report the ER protein quality control of non‐enveloped RNA virus and reveals a novel mechanism by which CVB3 evades host ER quality control pathways through cleavage and degradation of the UPR target gene EDEM1, to ultimately benefit its own replication.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.     

Fabrication,Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.