Personalized evolutionary hypothesis of breast neoplasm based on differential hybridization signals of subtelomeric probes

The limited availability of data on somatic gene evolution in breast cancer is challenging item to apply an appropriate clinical management for each individual. As human subtelomeric sequences are diverse‐prone and variable, they can be considered as hot spots for analysis of the health or disease status. We compared the hybridization signals of subtelomeric sequences in normal cells with those in auxiliary lymph nodes (ALNs) isolated from a single patient. Distinct signal intensities were found in all chromosomes: weak (5, 6, 9–12, and 16–19), medium (1, 5–9, 19, and X), and strong (2, 5, 9, 10, 16, and 18) intensities. Signal intensities in the patient's ALN and lymphocytes were higher than in normal tissues. The absence and presence of one or more hybridization signals and the presence of signals in the p and q arms were also variable. Whereas chromosomes 2, 3, 5, 7, 8, 10, 16, 18, 20, and X showed three hybridization spots, chromosomes 1, 4, 9, 12, 17, and 18–19 presented a reduced signal in the ALN and lymphocytes. In addition, signal intensity in the p arm was higher than in the q arm in most patients’ chromosomes. Therefore, we propose that subtelomeric hybridization be followed periodically in individuals with breast neoplasm to provide specific patterns. Such profiling could be considered as a prediction marker throughout the patients’ life. Together with the Ki67, cyclin D1, and cyclin E expression profiles, the subtelomeric hybridization profile could provide complementary information for cancer management.     

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis,gating pyruvate generation,and increasing pyruvate flux into the TCA cycle

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

Bax and Bak knockout apoptosis-resistant Chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed-batch culture process

In the field of therapeutic protein production, process intensification strategies entailing higher starting cell seeding densities, can potentially increase culture productivity, lower cost of goods and improve facility utilization. However, increased cell densities often trigger apoptotic cell death at the end of the cell culture process and thus reduce total viable cell count. Apoptosis-resistant Chinese hamster ovary cell lines may offer the possibility to diminish this undesired outcome of the intensified production process. In this study, we have generated and tested Bax/Bak double-knock-out (DKO) apoptosis resistant hosts to express standard and bispecific antibodies, as well as complex molecules in intensified production processes both as pools and single cell clones, and at different scales. In all cases, therapeutic proteins expressed from clones or pools generated from the Bax/Bak DKO hosts showed not only better viability but also enabled extended productivity in the later stages of the 14-day intensified production process. The product qualities of the produced molecules were comparable between Bax/Bak DKO and wild type cells. Overall, we showed that Bax/Bak DKO apoptosis-resistant host cell lines significantly improve viability and volumetric productivity of the intensified production cultures without altering product qualities.     

Parasitoids of the ladybird beetles (Coleoptera: Coccinellidae) in Iran: an update

Summary

Over the past few decades, many records of the parasitoids parasitizing Coccinellidae have been reported from Iran. Some of them, presented in hard to access conference proceedings, theses, older books and journal articles, were then inaccurately quoted in other papers. Furthermore, some other records were subsequently considered misidentifications and the corrected identity of the parasitoids was proposed in later published papers. Due to the accumulation of that misleading information, we decided to gather and critically evaluate all available Iranian data on the parasitoids of Coccinellidae. To the review presented here we also added new data on the parasitism of Coccinella septempunctata L. by a braconid Dinocampus coccinellae (Schrank) in the Lorestan province and on the parasitism of Hyperaspis pseudopustulata Mulsant by an encyrtid Homalotylus flaminius (Dalman) in the Kermanshah province. Hyperaspis pseudopustulata is a new host for H. flaminius, and is also a species new to the fauna of Iran. Altogether, our review revealed one species of dipteran and 16 species of hymenopteran parasitoids associated with Coccinellidae in Iran. The identity of some of those species, however, seems uncertain and should be re-examined.