Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.     

Novel symmetrical ureas as modulators of protein arginine methyl transferases

Methylation of histone arginine residues is an epigenetic mark related to gene expression that is implicated in a variety of biological processes and can be reversed by small-molecule modulators of protein arginine methyltransferases (PRMTs). A series of symmetrical ureas, designed as analogues of the known PRMT1 inhibitor AMI-1 have been synthesized using Pd-catalyzed Ar–N amide bond formation processes or carbonylation reactions as key steps. Their inhibitory profile has been characterized. The enzymatic assays showed a weak effect on PRMT1 and PRMT5 activity for most of the compounds. The acyclic urea that exhibited the strongest effect on the inhibition of the PRMT1 activity also showed the greatest effect on the expression of some androgen receptor target genes (TMPRSS2 and FKBP5), which may be related with its enzymatic activity. Surprisingly, AMI-1 behaved as an activator of PRMT5 activity, a result not reported so far.     

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia

Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.     

In Vitro Study of Taenia solium Postoncospheral Form

Background

The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages.

Methodology/Principal Findings

T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development–days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15–30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages.

Conclusions/Significance

This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite’s immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.     

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

Tubulin–Na+ ,K +-ATPase interaction: Involvement in enzymatic regulation and cellular function

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na⁺,K ⁺-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin–NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na ⁺. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na ⁺ and K ⁺, and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na ⁺/K ⁺ homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.     

A New Ichnospecies of Arthrophycus from the Upper Cambrian-Lower Tremadocian of Northwest Argentina: Implications for the Arthrophycid Lineage and Potential in Ichnostratigraphy

A new ichnospecies of Arthrophycus Hall 1852, A. minimus , is described from Upper Cambrian-Lower Tremadocian, shallow-marine strata of northwest Argentina. This new ichnospecies consists of small, long, regularly annulated hypichnial elements displaying subcircular to squarish cross-section and a ventral median groove. Side branches are occasionally present, but palmate, fan-like structures and scribbling patterns are absent. We adopt a relatively narrow diagnosis of Arthrophycus , suggesting that roughly annulated, cylindrical structures should not be included in this ichnogenus, unless other diagnostic features (i.e., squarish cross-section, median groove, zipper-like annulations) are also present. Arthrophycus is a common ichnotaxon in Ordovician-Silurian shallow-marine siliciclastic environments. Post-Paleozoic occurrences are removed from Arthrophycus . Arthrophycus has been proposed as a biostratigraphic index fossil in Ordovician-Silurian rocks. The presence of A. minimus in the Santa Rosita Formation of northwest Argentina indicates that Arthrophycus ranges at least from the Upper Cambrian-Lower Tremadocian with probable representatives in the Lower Cambrian and, therefore, its biostratigraphic utility is extended. Arthrophycus minimus represents the first Cambrian occurrence exhibiting not only fine, diagnostic morphologic features, but also the classical Arthrophycus behavioral pattern in dense monoichnospecific assemblages. The exploratory behavioral pattern displayed by A. minimus is simpler than that of the younger ichnospecies, particularly A. brogniartii, A. alleghaniensis, and A. lateralis . This is consistent with the basal position of A. minimus within the arthrophycid lineage.     

Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.