Functional Access to Neuron Subclasses in Rodent and Primate Forebrain

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Investigating the combination of single-pass tangential flow filtration and anion exchange chromatography for intensified mAb polishing

There is growing interest within the biopharmaceutical industry to improve manufacturing efficiency through process intensification, with the goal of generating more product in less time with smaller equipment. In monoclonal antibody (mAb) purification, a unit operation that can benefit from intensification is anion exchange (AEX) polishing chromatography. Single-pass tangential flow filtration (SPTFF) technology offers an opportunity for process intensification by reducing intermediate pool volumes and increasing product concentration without recirculation. This study evaluated the performance of an AEX resin, both in terms of host cell protein (HCP) purification and viral clearance, following concentration of a mAb feed using SPTFF. Results show that preconcentration of AEX feed material improved isotherm conditions for HCP binding, resulting in a fourfold increase in resin mAb loading at the target HCP clearance level. Excellent clearance of minute virus of mouse and xenotropic murine virus was maintained at this higher load level. The increased mAb loading enabled by SPTFF preconcentration effectively reduced AEX column volume and buffer requirements, shrinking the overall size of the polishing step. In addition, the suitability of SPTFF for extended processing time operation was demonstrated, indicating that this approach can be implemented for continuous biomanufacturing. The combination of SPTFF concentration and AEX chromatography for an intensified mAb polishing step which improves both manufacturing flexibility and process productivity is supported.     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor

Breast cancer cells experience a range of shear stresses in the tumor microenvironment (TME). However most current in vitro three-dimensional (3D) models fail to systematically probe the effects of this biophysical stimuli on cancer cell metastasis, proliferation, and chemoresistance. To investigate the roles of shear stress within the mammary and lung pleural effusion TME, a bioreactor capable of applying shear stress to cells within a 3D extracellular matrix was designed and characterized. Breast cancer cells were encapsulated within an interpenetrating network hydrogel and subjected to shear stress of 5.4 dynes cm⁻² for 72 hr. Finite element modeling assessed shear stress profiles within the bioreactor. Cells exposed to shear stress had significantly higher cellular area and significantly lower circularity, indicating a motile phenotype. Stimulated cells were more proliferative than static controls and showed higher rates of chemoresistance to the anti-neoplastic drug paclitaxel. Fluid shear stress-induced significant upregulation of the PLAU gene and elevated urokinase activity was confirmed through zymography and activity assay. Overall, these results indicate that pulsatile shear stress promotes breast cancer cell proliferation, invasive potential, chemoresistance, and PLAU signaling.     

Study of oxidative stress in isoniazid-induced hepatic injury in young rats with and without protein-energy malnutrition

The role of oxidative stress as a mechanism of hepatic injury caused by isoniazid (INH) was investigated in young growing rats. The interaction of moderate and severe degree of protein-energy malnutrition (PEM) was also investigated. Hepatic injury was produced by giving 50 mg/kg/day of INH for 2 weeks. Liver showed kupffer cell hyperplasia along with patchy sinusoidal congestion in hematoxylin (H) and eosin (E) staining. However, diffuse microglobules of oil red O′ positive fat globules could be demonstrated in frozen sections stained with oil red O′. The concomitant elevation of serum ALT/AST added support to the histopathologic injury. Electronmicroscopic analysis revealed the proliferation of rough endoplasmic reticulum in INH-treated groups. The glutathione and related thiols were decreased significantly by INH both in blood and liver tissues, indicating a decrease in protective mechanism. Glutathione reductase activity was elevated concomitantly in both the tissues. A significant decrease in the activity of glutathione peroxidase and catalase is again indicative of diminished capacity to handle the disposal of hydrogen peroxide (H₂O₂) and lipid peroxides. All these alterations indicated that the damage to the liver cell could well be operating through the inefficient disposal of superoxides (O⁻₂) and H₂O₂. A profound decrease in the protective mechanism further aggravated the picture in moderate and severe PEM, which was observed with INH alone. © 1997 John Wiley & Sons, Inc.     

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6–RACK1–ERK1/2–FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.     

Focused ultrasound stimulation on meibomian glands for the treatment of evaporative dry eye

Current treatments for meibomian gland dysfunction have several limitations, creating a necessity for other advanced treatment options. The purpose of this study is to determine the effectiveness of focused ultrasound stimulation for the treatment of dry eye disease caused by meibomian gland dysfunction. An in vivo study of nine Dutch Belted rabbits was conducted with focused ultrasound stimulation of the meibomian glands. A customized line-focused ultrasonic transducer was designed for treatment. Fluorescein imaging, Schirmer’s test, and Lipiview II ocular interferometer were used to quantify outcomes from three aspects: safety, tear production, and lipid layer thickness. Both tear secretion and lipid layer thickness improved following ultrasound treatment. Five to 10 min after the ultrasound treatment, the mean values of lipid layer thickness increased from 55.33 ± 11.15 nm to 95.67 ± 22.77 nm (p < 0.05), while the mean values measured with the Schirmer’s test increased from 2.0 ± 2.3 to 7.2 ± 4.3 (p < 0.05). Positive effects lasted more than three weeks. Adverse events such as redness, swelling, and mild burn, occurred in two rabbits in preliminary experiments when the eyelids sustained a temperature higher than 42°C. No serious adverse events were found. The results suggest that ultrasound stimulation of meibomian glands can improve both tear production and lipid secretion. Ultimately, ultrasound stimulation has the potential to be an option for the treatment of evaporative dry eye disease caused by meibomian gland dysfunction.     

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.