Next generation biotherapeutics production system Trichoderma reeseiChristopher Landowski
The filamentous fungus Trichoderma reesei is an important production organism used by industrial enzyme companies world-wide. It is a low cost production system that secretes its native enzymes at levels exceeding 100 g/L of culture medium. Several T. reesei produced enzymes have obtained the generally recognized as safe status by the U.S. Food and Drug Administration. T. reesei has tremendous prospects to be a cost efficient and high yield system for producing therapeutic proteins. We have adapted the fungus to become more suitable for biotherapeutic production by reducing secreted protease activity and altering glycosylation pathways needed for adding mammalian glycoforms.
Expression strains for monoclonal antibodies, Fab antibody fragments, interferon alpha-2b, insulin-like growth factor 1, and fibroblast growth factor 21 were constructed, cultivated in bioreactors, and expression levels were measured from the culture medium. After deleting 13 of the most critical protease genes, the general secreted protease activity was reduced over 30-fold. Monoclonal antibodies could be produced up to 7.6 g/L, Fab antibody fragments up to 8.2 g/L, interferon alpha-2b at 7.9 g/L, and insulin-like growth factor fusion protein at 8 g/L. With protease inhibitor treatment interferon alpha-2b could be produced at over 10 g/L, insulin-like growth factor fusion protein at 19 g/L, and full length fibroblast growth factor 21 at 200 mg/L in addition to a shorter form at 3.5 g/L. Human glycoforms such as G0 and FG0 were produced on monoclonal antibodies.
Expression levels and product quality improved dramatically after multiple protease deletions and optimization of culture conditions. While the production levels achieved are already relatively high, the strains could be developed further to reach the 100 g/L potential of the organism. This study demonstrates the excellent prospects of T. reesei as a host for therapeutic protein production.
Standardized cells with completely predictable and controllable behavior is a key focus of synthetic biology. Recent advances such as the implantation of a synthesized genome into a DNA-free bacterial shell at the J. Craig Venter Institute marked the dawn of these whole-genome scale projects. A first step towards predictable synthetic minimal cells is made through further reduction of the cellular complexity by minimizing the bacterial genome. This has lead to a short list of essential genes for cells to still be considered living entities. Although these minimal cells increase our fundamental understanding of key cellular processes, there are other important criteria to consider for synthetic cells to become biotechnologically relevant.
In this colloquium I will address the current state of research concerning biotechnological useful cells that can be used as a platform for operating precisely designed and independent biological systems. Top-down methods for the design of these organisms will be reviewed, highlighting remarkable experiments.
Objective: To investigate the changes in the retina due to deltamethrin toxicity and the process in cell inflammation and apoptosis.
Study Design: Sixteen Wistar albino rats were randomly divided into two groups as control (n=8) and deltamethrin (n=8) groups. Saline was given to the control group, and 0.5 mL of 5 mg/kg deltamethrin was given to the deltamethrin group for 14 days each. Blood was collected for biochemical analysis. Retinal tissue was processed for histological examination.
Results: Compared to the control group, MDA levels were high while GSH and CAT levels were low in the deltamethrin group. Histopathological analysis showed spaces between the pigment epithelium, irregularity in the delimiting membrane, degenerated ganglion, cone and bacillus cell, pyknotic nuclei, thinned inner limitation membrane, and thickened vascular wall. The control group showed FAS expression in the pigment layer limiting membranes, in the nuclei of many cone and bacillus cells, and ganglion cells in the control group sections. In the deltamethrin group, FAS expression was observed in the inner and outer limiting membranes of the pigment epithelium, cone and bacillus cells, and ganglion cell nuclei. In the control group, negative NOS expression in the pigment epithelium and outer limiting membranes, internal limitation membrane, and ganglion cells in the cone and bacillus cell nuclei were observed. In the deltamethrin group, NOS expression was positive in the pigment epithelium, cone and bacillus, and ganglion cell nuclei.
Conclusion: We suggest that deltamethrin toxicity induced apoptotic process due to increased inflammation in the retina and may cause visual impairment as a result of neural damage.
Keywords: deltamethrin, FAS, insecticides, NOS, nitric oxide synthase, retina
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
Next generation biotherapeutics production system Trichoderma reeseiChristopher Landowski
The filamentous fungus Trichoderma reesei is an important production organism used by industrial enzyme companies world-wide. It is a low cost production system that secretes its native enzymes at levels exceeding 100 g/L of culture medium. Several T. reesei produced enzymes have obtained the generally recognized as safe status by the U.S. Food and Drug Administration. T. reesei has tremendous prospects to be a cost efficient and high yield system for producing therapeutic proteins. We have adapted the fungus to become more suitable for biotherapeutic production by reducing secreted protease activity and altering glycosylation pathways needed for adding mammalian glycoforms.
Expression strains for monoclonal antibodies, Fab antibody fragments, interferon alpha-2b, insulin-like growth factor 1, and fibroblast growth factor 21 were constructed, cultivated in bioreactors, and expression levels were measured from the culture medium. After deleting 13 of the most critical protease genes, the general secreted protease activity was reduced over 30-fold. Monoclonal antibodies could be produced up to 7.6 g/L, Fab antibody fragments up to 8.2 g/L, interferon alpha-2b at 7.9 g/L, and insulin-like growth factor fusion protein at 8 g/L. With protease inhibitor treatment interferon alpha-2b could be produced at over 10 g/L, insulin-like growth factor fusion protein at 19 g/L, and full length fibroblast growth factor 21 at 200 mg/L in addition to a shorter form at 3.5 g/L. Human glycoforms such as G0 and FG0 were produced on monoclonal antibodies.
Expression levels and product quality improved dramatically after multiple protease deletions and optimization of culture conditions. While the production levels achieved are already relatively high, the strains could be developed further to reach the 100 g/L potential of the organism. This study demonstrates the excellent prospects of T. reesei as a host for therapeutic protein production.
Standardized cells with completely predictable and controllable behavior is a key focus of synthetic biology. Recent advances such as the implantation of a synthesized genome into a DNA-free bacterial shell at the J. Craig Venter Institute marked the dawn of these whole-genome scale projects. A first step towards predictable synthetic minimal cells is made through further reduction of the cellular complexity by minimizing the bacterial genome. This has lead to a short list of essential genes for cells to still be considered living entities. Although these minimal cells increase our fundamental understanding of key cellular processes, there are other important criteria to consider for synthetic cells to become biotechnologically relevant.
In this colloquium I will address the current state of research concerning biotechnological useful cells that can be used as a platform for operating precisely designed and independent biological systems. Top-down methods for the design of these organisms will be reviewed, highlighting remarkable experiments.
Objective: To investigate the changes in the retina due to deltamethrin toxicity and the process in cell inflammation and apoptosis.
Study Design: Sixteen Wistar albino rats were randomly divided into two groups as control (n=8) and deltamethrin (n=8) groups. Saline was given to the control group, and 0.5 mL of 5 mg/kg deltamethrin was given to the deltamethrin group for 14 days each. Blood was collected for biochemical analysis. Retinal tissue was processed for histological examination.
Results: Compared to the control group, MDA levels were high while GSH and CAT levels were low in the deltamethrin group. Histopathological analysis showed spaces between the pigment epithelium, irregularity in the delimiting membrane, degenerated ganglion, cone and bacillus cell, pyknotic nuclei, thinned inner limitation membrane, and thickened vascular wall. The control group showed FAS expression in the pigment layer limiting membranes, in the nuclei of many cone and bacillus cells, and ganglion cells in the control group sections. In the deltamethrin group, FAS expression was observed in the inner and outer limiting membranes of the pigment epithelium, cone and bacillus cells, and ganglion cell nuclei. In the control group, negative NOS expression in the pigment epithelium and outer limiting membranes, internal limitation membrane, and ganglion cells in the cone and bacillus cell nuclei were observed. In the deltamethrin group, NOS expression was positive in the pigment epithelium, cone and bacillus, and ganglion cell nuclei.
Conclusion: We suggest that deltamethrin toxicity induced apoptotic process due to increased inflammation in the retina and may cause visual impairment as a result of neural damage.
Keywords: deltamethrin, FAS, insecticides, NOS, nitric oxide synthase, retina
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
MilliporeSigma's Jennifer Pratt recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating the utility of HepaRG MRP2 Knockout cells for investigating drug-transporter interactions in the liver involving MRP2.
1. Chronic Restraint Stress Modulates Expression of Genes
Y. Wang1, Y. Zhang2, D. Yin2, J. Moorman2, Xiao Zeng1
1SuperArray Bioscience Corporation, Frederick, MD,
2Department of Internal Medicine, East Tennessee State University, Johnson City, TN.
Abstract
Materials and Methods
Restraint stress mouse model: 6-8 week old male Balb/c mice from Charles
River Laboratories were maintained in the Division of Laboratory Animal Resources
at East Tennessee State University (ETSU). The mice were subjected to a chronic
physical restraint protocol (3). Briefly, mice were placed in a 50-ml conical centrifuge
tube filled with multiple punctures to allow ventilation. Mice were held horizontally in
the tubes for a continuous 12 h followed by a 12-h rest, while food and water were
provided ad libitum. Control mice were kept in their original cages and in the same
diet schedule as the experimental group. Mice were physically restrained for two
cycles. After physical restraint, the spleens were rapidly frozen in liquid nitrogen and
stored at -80ºC for later isolation of total RNA. Genomic DNA interference was
eliminated by DNase I treatment.
Results
Figure 3: Validation of Microarray Data Using Real-Time PCR
RealFigure 1: Microarray Analysis of Apoptotic Genes in Restraint Stress
Stress
B
A
Control
Stress
Physical restraint stress alters apoptotic
gene expression profiles in the spleen.
Panel A displays representative images
of the Oligo GEArray® Mouse Apoptosis
Microarrays. The experiments were
repeated three times. As an example, the
up-regulation of Bnip3 is circled in red.
Panel B lists the 23 genes whose
expression levels are significantly
changed in stressed versus unstressed
mice.
RNA using the TrueLabeling-AMP™ 2.0 Kit (Catalog No. GA-030). The resulting
biotin-labeled cRNA probe was allowed to hybridize overnight to the Oligo GEArray®
microarray in a standard hybridization oven at 60ºC. After blocking, washing and
chemiluminescent detection steps, images were acquired by a Chemi-Doc imaging
system (CCD camera). Raw signal intensity for individual genes on the microarray
was extracted using web-based GEArray® Expression Analysis Suite software and
normalized against signal intensities from housekeeping genes.
Real-time RT-PCR: cDNA was synthesized from total RNA using the
ReactionReady™ First Strand cDNA Synthesis Kit (Catalog No. C-01). Real-time
PCR was performed using RT2 Real-Time™ SYBR Green Fluorescein PCR Master
Mix (Catalog No. PA-011) on the Bio-Rad iCycler real-time PCR detection system.
GAPDH and β-Actin were chosen as housekeeping genes for normalization.
Threshold cycle numbers (Ct) were used for “∆∆Ct” analysis. Fold-changes between
samples were then calculated as 2^(-∆∆Ct). The statistical significance was
determined by one-way ANOVA and Bonferroni tests (p<0.05).
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A
Control
Stress
Physical restraint stress alters the p53signaling pathway in the spleen.
Panel A displays representative images
of the Oligo GEArray® Mouse p53
Signaling Pathway Microarrays. The
experiments were repeated three times.
As an example, the up-regulation of p21
is shown in red circles. Panel B lists the
24 genes whose expression levels are
significantly increased in stressed versus
unstressed mice, as well as Stat1 whose
gene expression decreased.
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2.5
2.0
1.5
1.0
0.5
0.0
FAS
FADD
p53
p21
Up-regulation of Fas, FADD,
p53, and p21 genes in
spleens from mice with
physical restraint stress was
confirmed by quantitative
real-time PCR. The data are
presented as the relative
fold-changes normalized to
GAPDH as the internal
control. The data are
representative of three
experiments where the
asterisk (*) denotes p < 0.01
when compared to control.
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Conclusions
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By focusing on apoptosis and p53 signaling pathways, we have
found that 22.3% of the examined genes showed significant upregulated expression in splenocytes between stressed and
unstressed mice.
The up-regulation of FADD, FAS, p53 and p21 is further confirmed
through real time PCR.
B
Position
Description
3
Apaf1: Apoptotic peptidase activating factor 1
4
Apex1: Apurinic/apyrimidinic endonuclease 1
7
Aurkb: Aurora kinase B
8
Wdr39: WD repeat domain 39
10
Bak1: BCL2-antagonist/killer 1
11
Bap1: Brca1 associated protein 1
12
Bax: Bcl2-associated X protein
15
Bid: BH3 interacting domain death agonist
16
Birc5: Baculoviral IAP repeat-containing 5
18
Brca1: Breast cancer 1
19
Brca2: Breast cancer 2
20
Btg2: B-cell translocation gene 2, anti-proliferative
25
Ccng2: Cyclin G2
26
Ccnh: Cyclin H
Cdc25a: Cell division cycle 25 homolog A (S.
27
cerevisiae)
28
Cdc25c: Cell division cycle 25 homolog C (S. cerevisiae)
29
Cdc2a: Cell division cycle 2 homolog A (S. pombe)
Cdk7: Cyclin-dependent kinase 7 (homolog of Xenopus
31
MO15 cdk-activating kinase)
32
Cdkn1a: Cyclin-dependent kinase inhibitor 1A (P21)
34
Chek1: Checkpoint kinase 1 homolog (S. pombe)
35
Chek2: CHK2 checkpoint homolog (S. pombe)
39
Cyr61: Cysteine rich protein 61
41
Daxx: Fas death domain-associated protein
Gadd45a: Growth arrest and DNA-damage-inducible 45
53
alpha
95
Stat1: Signal transducer and activator of transcription 1
Control
Stress
4.9
Figure 2: Microarray Analysis of p53-Mediated Signaling in Restraint Stress
p53-
Oligo GEArray® System: Two cataloged Oligo GEArray® microarrays from
SuperArray were used in this study. The Oligo GEArray® Mouse Apoptosis
Microarray (Catalog No. OMM-012) contains 112 genes involved in apoptosis. The
Oligo GEArray® Mouse p53 Signaling Pathway Microarray (Catalog No. OMM-027) is
designed to profile the expression of 113 key genes involved in the p53 signaling
pathway.
Microarray processing: Biotinylated cRNA target was synthesized from total
Position
Description
2
Akt1: Thymoma viral proto-oncogene 1
5
Api5: Apoptosis inhibitor 5
14
Bax: Bcl2-associated X protein
Bnip3l: BCL2/adenovirus E1B interacting protein 325
like
28
Birc1e: Baculoviral IAP repeat-containing 1e
31
Birc2: Baculoviral IAP repeat-containing 2
Bnip2: BCL2/adenovirus E1B interacting protein 1,
35
NIP2
Bnip3: BCL2/adenovirus E1B interacting protein 1,
36
NIP3
37
Casp3: Caspase 3
38
Bok: Bcl-2-related ovarian killer protein
48
Casp2: Caspase 2
52
Casp8: Caspase 8
53
Dsip1: TSC22 domain family 3
Cradd: CASP2 and RIPK1 domain containing adaptor
58
with death domain
65
Fadd: Fas (TNFRSF6)-associated via death domain
71
Ltbr: Lymphotoxin B receptor
Nfkb1: Nuclear factor of kappa light chain gene
74
enhancer in B-cells 1, p105
75
Zc3hc1: Zinc finger, C3HC type 1
87
Rnf7: Ring finger protein 7
Tnfrsf21: Tumor necrosis factor receptor superfamily,
95
member 21
98
Fas: Fas (TNF receptor superfamily member)
109 Trp53: Transformation related protein 53
Trp53inp1: Transformation related protein 53
111
inducible nuclear protein 1
F o ld C h a n g e
Psychological and physical stress can alter the immune system in both
human and animals (1). It has been reported that mice subjected to
chronic 12-hour daily physical restraint for two days showed dramatic
apoptosis in splenocytes (2). To identify genes that contribute to the
splenocyte apoptosis, we compared gene expression in the spleens of
restraint stressed mice with that in the spleens of unstressed mice
using oligo microarrays consisting of 225 genes. We report here that
mice subjected to chronic 12-hour daily physical restraint for two days
exhibited significantly altered expression of 48 out of 225 genes. These
genes included pro-apoptotic genes. We selected four genes of
interest, and confirmed the microarray results by real time PCR.
Although these findings are not specific for restraint stress-induced
apoptosis in splenocytes, they identify a potentially important
component of pro-apoptotic activity in restraint stress and suggest a
possible target for anti-apoptotic therapy in stress-induced apoptosis
in splenocytes. Our mouse model and identified biomarkers would be
very useful for further study of the intercommunication of the immune
and the nervous systems.
Fold
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2.2
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Our data provide additional evidence that apoptotic machinery
underlines lymphopenia during stress.
It remains to be further investigated whether the apoptotic genes
provide a marker for individuals with chronic stress.
Pathway-focused microarray analysis is a simple and easy
approach to screen for genes of interest.
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3.6
References
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5.3
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1. Padgett,D.A. and Glaser,R. (2003). How stress influences the immune response. Trends
Immunol. 24, 444-448.
3.0
2. Yin,D., Tuthill,D., Mufson,R.A., and Shi,Y. (2000). Chronic restraint stress promotes
lymphocyte apoptosis by modulating CD95 expression. J. Exp. Med. 191, 1423-1428.
0.5
3. Sheridan,J.F., Dobbs,C., Jung,J., Chu,X., Konstantinos,A., Padgett,D., and Glaser,R.
(1998). Stress-induced neuroendocrine modulation of viral pathogenesis and immunity.
Ann. N. Y. Acad. Sci. 840, 803-808.