Evaluation of new haematology analyser, XN-31, for malaria detection in blood donors: A single-centre study from India.

BACKGROUND AND OBJECTIVES: Malaria continues to be a significant public health concern in India, with several regions experiencing endemicity and sporadic outbreaks. The prevalence of malaria in blood donors, in India, varies between 0.02% and 0.07%. Common techniques to screen for malaria, in blood donors and patients, include microscopic smear examination and rapid diagnostic tests (RDTs) based on antigen detection. The aim of this study was to evaluate a new fully automated analyser, XN-31, for malaria detection, as compared with current practice of using RDT. MATERIALS AND METHODS: Cross-sectional analytical study was conducted to evaluate clinical sensitivity and specificity of new automated analyser XN-31 among blood donors' samples and clinical samples (patients with suspicion of malaria) from outpatient clinic collected over between July 2021 and October 2022. No additional sample was drawn from blood donor or patient. All blood donors and patients' samples were processed by malaria rapid diagnostic test, thick-smear microscopy (MIC) and the haematology analyser XN-31. Any donor blood unit incriminated for malaria was discarded. Laboratory diagnosis using MIC was considered the 'gold standard' in the present study. Clinical sensitivity and specificity of XN-31 were compared with the gold standard. RESULTS: Fife thousand and five donor samples and 82 diagnostic samples were evaluated. While the clinical sensitivity and specificity for donor samples were 100%, they were 72.7% and 100% for diagnostic samples. CONCLUSION: Automated haematology analysers represent a promising solution, as they can deliver speedy and sensitive donor malaria screening assessments. This method also has the potential to be used for pre-transfusion malaria screening along with haemoglobin estimation.

Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture.

The existence of dynamic cellular phenotypes in changing environmental conditions is of major interest for cell biologists who aim to understand the mechanism and sequence of regulation of gene expression. In the context of therapeutic protein production by Chinese Hamster Ovary (CHO) cells, a detailed temporal understanding of cell-line behavior and control is necessary to achieve a more predictable and reliable process performance. Of particular interest are data on dynamic, temporally resolved transcriptional regulation of genes in response to altered substrate availability and culture conditions. In this study, the gene transcription dynamics throughout a 9-day batch culture of CHO cells was examined by analyzing histone modifications and gene expression profiles in regular 12- and 24-hr intervals, respectively. Three levels of regulation were observed: (a) the presence or absence of DNA methylation in the promoter region provides an ON/OFF switch; (b) a temporally resolved correlation is observed between the presence of active transcription- and promoter-specific histone marks and the expression level of the respective genes; and (c) a major mechanism of gene regulation is identified by interaction of coding genes with long non-coding RNA (lncRNA), as observed in the regulation of the expression level of both neighboring coding/lnc gene pairs and of gene pairs where the lncRNA is able to form RNA-DNA-DNA triplexes. Such triplex-forming regions were predominantly found in the promoter or enhancer region of the targeted coding gene. Significantly, the coding genes with the highest degree of variation in expression during the batch culture are characterized by a larger number of possible triplex-forming interactions with differentially expressed lncRNAs. This indicates a specific role of lncRNA-triplexes in enabling rapid and large changes in transcription. A more comprehensive understanding of these regulatory mechanisms will provide an opportunity for new tools to control cellular behavior and to engineer enhanced phenotypes.

Loss of the canonical spindle orientation function in the Pins/LGN homolog AGS3.

In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαiin vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation.

A CRISPR/Cas9 based engineering strategy for overexpression of multiple genes in Chinese hamster ovary cells.

Manipulation of multiple genes to engineer Chinese Hamster Ovary (CHO) cells for better performance in production processes of biopharmaceuticals has recently become more and more popular. Yet, identification of useful genes and the unequivocally assessment of their effect alone and in combination(s) on the cellular phenotype is difficult due to high variation between subclones. Here, we present development and proof-of-concept of a novel engineering strategy using multiplexable activation of artificially repressed genes (MAARGE). This strategy will allow faster screening of overexpression of multiple genes in all possible combinations. MAARGE, in its here presented installment, comprises four different genes of interest that can all be stably integrated into the genome from one plasmid in a single transfection. Three of the genes are initially repressed by a repressor element (RE) that is integrated between promoter and translation start site. We show that an elongated 5'-UTR with an additional transcription termination (poly(A)) signal most efficiently represses protein expression. Distinct guide RNA (gRNA) targets flanking the REs for each gene then allow to specifically delete the RE by CRISPR/Cas9 and thus to activate the expression of the corresponding gene(s). We show that both individual and multiplexed activation of the genes of interest in a stably transfected CHO cell line is possible. Also, upon transfection of this stable cell line with all three gRNAs together, it was possible to isolate cells that express all potential gene combinations in a single experiment.

ChromaWizard: An open source image analysis software for multicolor fluorescence in situ hybridization analysis.

Multicolor image analysis finds its applications in a broad range of biological studies. Specifically, multiplex fluorescence in situ hybridization (M-FISH) for chromosome painting facilitates the analysis of individual chromosomes in complex metaphase spreads and is widely used to detect both numerical and structural aberrations. While this is well established for human and mouse karyotypes, for which species sophisticated software and analysis tools are available, other organisms and species are less well served. Commercially available software is proprietary and not easily adaptable to other karyotypes. Therefore, a publically available open source software that combines flexibility and customizable functionalities is needed. Here we present such a tool called "ChromaWizard" which is based on popular scientific image analysis libraries (OpenCV, scikit-image, and NumPy). We demonstrate its functionality on the example of primary Chinese hamster (Cricetulus griseus) fibroblasts metaphase spreads and on Chinese Hamster Ovary cell lines known for the large number of chromosomal rearrangements. The application can be easily adapted to any kind of available labeling kits and is independent of the used organism and instrumentation. It allows direct inspection of the original hybridization signals and enables either manual or automatic assignment of colors, making it a functional and versatile tool that can be used also for other multicolor applications.

Arsenic trioxide: insights into its evolution to an anticancer agent.

Arsenic and its various forms have been in use in ancient Chinese medicine for more than 2000 years. Arsenicals have gained importance for having remedial effects for various diseases from syphilis to cancer thus highlighting its role as a therapeutic agent even though it has been labelled as a potential 'poison'. The ability of arsenic, specifically arsenic trioxide, to treat acute promyelocytic leukaemia has radically changed the perception of this poison and has been the main factor for the re-emergence of this candidate to Western medicine for the treatment of leukaemia and other solid tumours. This review highlights the glorious history of arsenic and its various forms with major emphasis on arsenic trioxide as a therapeutic agent. The mechanism of action, pathogenesis, pharmacokinetic profile, safety concerns, ongoing clinical trials and various new forms of arsenic trioxide are discussed. The review also outlines the therapeutic ability of this drug, discusses the latest developments and recent investigations and potential advancement of arsenic trioxide as nanoformulations that has made it emerge as a potential remedial agent.

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting.

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO-S, CHO-K1 8 mM glutamine, and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO-S and CHO-K1, with the diversity increasing and new variants appearing, while CHO-K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.

A reference genome of the Chinese hamster based on a hybrid assembly strategy.

Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read-based assemblies. Here, we completely resequenced C. griseus using single molecule real time sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important Chinese hamster ovary cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.

Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time.

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA-methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA-methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241-2253. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Annotation of additional evolutionary conserved microRNAs in CHO cells from updated genomic data.

MicroRNAs are small non-coding RNAs that play a critical role in post-transcriptional control of gene expression. Recent publications of genomic sequencing data from the Chinese Hamster (CGR) and Chinese hamster ovary (CHO) cells provide new tools for the discovery of novel miRNAs in this important production system. Version 20 of the miRNA registry miRBase contains 307 mature miRNAs and 200 precursor sequences for CGR/CHO. We searched for evolutionary conserved miRNAs from miRBase v20 in recently published genomic data, derived from Chinese hamster and CHO cells, to further extend the list of known miRNAs. With our approach we could identify several hundred miRNA sequences in the genome. For several of these, the expression in CHO cells could be verified from multiple next-generation sequencing experiments. In addition, several hundred unexpressed miRNAs are awaiting further confirmation by testing for their transcription in different Chinese hamster tissues.

A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells.

MicroRNAs (miRNAs) are a novel class of short non-coding RNAs, which negatively regulate target gene expression at post-transcriptional level. They mediate an important layer of control in the global regulation of gene networks, controlling a broad range of physiological as well as patho-physiological pathways including development, cancer, metabolism, proliferation, and stress resistance. So far, more than 365 miRNA genes have been identified in CHO cells. The functional analysis of the physiological effect of such large numbers of miRNAs, however, requires an efficient functional screening method. In the current study, we therefore established and evaluated a protocol to perform miRNA overexpression and to screen their effect on bio-industrially relevant phenotypes, such as growth, viability and productivity, using a recombinant, Epo-Fc producing CHO cell line. For protocol optimization, four CHO miRNAs (cgr-miR-17, cgr-miR-221, cgr-miR-21, and cgr-miR-210) were cloned into small hairpin vectors including a GFP cassette and transfected. After transfection cells were analyzed for growth and productivity over a 4-day period. Even from this small set of four miRNAs, the overexpression of miR-17, one of the members of the oncogenic miR-17-92 cluster, gave proof of principle that this method enables the identification of miRNA engineering candidates as its overexpression increased the speed of cell proliferation without negatively impacting specific productivity. The here presented method is applicable for medium-throughput screening for microRNA, miR-sponge, siRNA, or mRNA overexpression along with detailed functional characterization using the same experimental set up. As the same procedure can be applied to different production cell lines, the protocol can also be used to test for individual, cell line specific responses to microRNAs. Thus our system represents a general platform to functionally screen candidates for rational cell factory design.

Antagomirzymes: oligonucleotide enzymes that specifically silence microRNA function.

Many important cellular processes are regulated by small endogenous noncoding RNAs known as microRNAs (miRNAs). The precise molecular function of many miRNAs is unknown; different loss-of-function methods are required to gain insight into the biology of these small RNA molecules. Nucleic acid enzymes termed antagomirzymes are now shown to be valuable tools for the specific knockdown of miRNA in vitro and in vivo (see scheme).

CRISPR/Cas9-Mediated Knockout of MicroRNA-744 Improves Antibody Titer of CHO Production Cell Lines.

MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L-1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L-1 , pointing towards the potential of miRNA KO for cell line engineering.

A cross-species whole genome siRNA screen in suspension-cultured Chinese hamster ovary cells identifies novel engineering targets.

High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins. Here, we performed a cross-species approach by applying a mouse whole-genome siRNA library to CHO cells, optimized the protocol for suspension cultured cells, as this is the industrial practice for CHO cells, and developed an in silico method to identify functioning siRNAs, which also revealed the limitations of using cross-species libraries. With this method, we were able to identify several genes that, upon knockdown, enhanced the total productivity in the primary screen. A second screen validated two of these genes, Rad21 and Chd4, whose knockdown was tested in additional CHO cell lines, confirming the induced high productivity phenotype, but also demonstrating the cell line/clone specificity of engineering effects.

Interaction between 14mer DNA oligonucleotide and cationic surfactants of various chain lengths.

In the recent genomic era, a novel gene silencing approach has been introduced based on the use of small synthetic oligonucleotides, such as antisense RNAs, siRNAs, to inhibit the expression of a specific target gene. Successful implementation of this methodology calls for the development of efficient systems to deliver small oligonucleotides into the cells using various natural and synthetic cationic agents. While extensive studies have focused on the interaction of various natural and synthetic cationic surfactants with long DNA, less attention has been paid to surfactant interaction with small oligonucleotides. In this study, the interaction between 14mer double stranded DNA and alkyltrimethylammonium bromides of C16 (cetyl, CTAB), C14 (tetradecyl, TTAB), and C12 (dodecyl, DTAB) chain lengths was investigated at different charge ratios by gel electrophoresis, ethidium bromide exclusion, circular dichroism, and UV melting. Our gel studies at 1 microM oligonucleotide concentration showed that CTAB, TTAB, and DTAB neutralize the oligonucleotides at a charge ratio (Z+/-) of 1, 14, and 50, respectively. At lower charge ratios, CTAB and TTAB interact with oligonucleotides, and the complexes show electrophoretic mobility shifts in the gel, while such mobility shifts were completely absent in the case of DTAB. UV melting experiments revealed that interaction with all three surfactants increased the thermostability of the oligonucleotide. The extent of thermal stabilization was highest in the case of CTAB, moderate in the case of TTAB, and extremely low in the case of DTAB. Oligonucleotides within fully neutralized complexes denatured at further higher temperatures, and again, stabilization was the highest in the case of CTAB followed by TTAB and DTAB, hence revealing that the oligonucleotides interacted more strongly with CTAB than with the other two surfactants. Ethidium bromide exclusion studies also supported our UV melting studies, confirming that CTAB binds most strongly to the oligonucleotide. CD titrations of oligonucleotides with increasing amounts of surfactants revealed common spectral patterns consisting of the progressive loss of CD signals for native helical DNA conformations. Overall, our results demonstrate that interaction between oligonucleotides and cationic surfactants, although qualitatively similar to long double stranded DNA, shows subtle differences that need to be understood to improve small oligonucleotide delivery into the cells by using common delivery agents that have been used to deliver long pieces of DNA.

Effect of the head-group geometry of amino acid-based cationic surfactants on interaction with plasmid DNA.

The interaction between DNA and different types of amino acid-based cationic surfactants was investigated. Particular attention was directed to determine the extent of influence of surfactant head-group geometry toward tuning the interaction behavior of these surfactants with DNA. An overview is obtained by gel retardation assay, isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism at different mole ratios of surfactant/DNA; also, cell viability was assessed. The studies show that the surfactants with more complex/bulkier hydrophobic head group interact more strongly with DNA but exclude ethidium bromide less efficiently; thus, the accessibility of DNA to small molecules is preserved to a certain extent. The presence of more hydrophobic groups surrounding the positive amino charge also gave rise to a significantly lower cytotoxicity. The surfactant self-assembly pattern is quite different without and with DNA, illustrating the roles of electrostatic and steric effects in determining the effective shape of a surfactant molecule.

Stem Cells and Progenitors in Human Peripheral Blood Get Activated by Extremely Active Resveratrol (XAR™).

Resveratrol generated enormous interest as it improved functions of multiple organs and could delay aging in animal models. However, basic mechanism of action was not understood and due to poor bioavailability, it has failed to enter the market. A highly active nano-formulation of resveratrol (XAR™) with enhanced bioavailability is now available. Present study was undertaken to evaluate its effects on stem cells biology in the human peripheral blood. Twelve healthy participants were enrolled of which five received XAR™, five were age-matched placebo controls and two were 76 and 85 years old. Peripheral blood was processed to study serum profile to monitor cardiac and pancreatic functions and subjected to density gradient centrifugation to enrich pluripotent (VSELs) and adult stem cells that get enriched along with red blood cells and in the Buffy coat respectively on Day 2 and Day 15 after XAR™ treatment. The XAR™ treatment resulted in an increased expression of pluripotency transcripts specific for VSELs (Oct-4A, Nanog and Sox2) on D2; specific transcripts for differentiation in the progenitors including Oct-4, Ikaros, CD14, CD90 on D15, and anti-ageing and tumor suppressor transcripts NAD, SIRT1, SIRT6 and p53 in both stem cells and progenitors. An improvement of cardiac and pancreatic markers in serum profile was also observed on D15. The decline in VSELs numbers with age and beneficial effects of the XAR™ treatment were evident by up-regulation of specific transcripts and on serum profile. XAR™ is a promising molecule that has the potential to activate pluripotent VSELs and tissue committed adult stem cells 'progenitors' resulting in the rejuvenation of various body tissues and for improved, cancer-free health with advanced age.

Enhanced Genome Editing Tools For Multi-Gene Deletion Knock-Out Approaches Using Paired CRISPR sgRNAs in CHO Cells.

Since the establishment of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, powerful strategies for engineering of CHO cell lines have emerged. Nevertheless, there is still room to expand the scope of the CRISPR tool box for further applications to improve CHO cell factories. Here, the authors demonstrate activity of the alternative CRISPR endonuclease Cpf1 in CHO-K1 for the first time and that it can be used in parallel to CRISPR/Cas9 without any interference. Both, Cas9 and Cpf1, can be effectively used for multi-gene engineering with a strategy based on paired single guide RNAs (sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by removing the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. FUT8 also served as model to verify that deletion efficiency is size-independent (2-150 kb). To test the suitability for multi-gene approaches in combination with gene deletion, clones harboring triple deletions in ß-1,4-Galactosyltransferase (B4GALT) isozymes are identified using solely conventional PCR/qPCR. In addition, two bicistronic transcription strategies are implemented to enable unequivocal pairing of sgRNAs: a CHO-derived tRNA linker that works for both, Cas9 and Cpf1, as well as paired sgRNAs in an array format, which can be used with Cpf1 due to its RNA processing ability. These strategies broaden the range of application of CRISPR for novel gene editing approaches in CHO cells and also enable the efficient realization of a genome-wide deletion library.

Changes in Chromosome Counts and Patterns in CHO Cell Lines upon Generation of Recombinant Cell Lines and Subcloning.

Chinese hamster ovary (CHO) cells are the number one production system for therapeutic proteins. A pre-requirement for their use in industrial production of biopharmaceuticals is to be clonal, thus originating from a single cell in order to be phenotypically and genomically identical. In the present study it was evaluated whether standard procedures, such as the generation of a recombinant cell line in combination with selection for a specific and stable phenotype (expression of the recombinant product) or subcloning have any impact on karyotype stability or homogeneity in CHO cells. Analyses used were the distribution of chromosome counts per cell as well as chromosome painting to identify specific karyotype patterns within a population. Results indicate that subclones both of the host and the recombinant cell line are of comparable heterogeneity and (in)stability as the original pool. In contrast, the rigorous selection for a stably expressing phenotype generated cell lines with fewer variation and more stable karyotypes, both at the level of the sorted pool and derivative subclones. We conclude that the process of subcloning itself does not contribute to an improved karyotypic homogeneity of a population, while the selection for a specific cell property inherently can provide evolutionary pressure that may lead to improved chromosomal stability as well as to a more homogenous population.

microRNAs in viral oncogenesis.

MicroRNAs are a recently discovered class of small noncoding functional RNAs. These molecules mediate post-transcriptional regulation of gene expression in a sequence specific manner. MicroRNAs are now known to be key players in a variety of biological processes and have been shown to be deregulated in a number of cancers. The discovery of viral encoded microRNAs, especially from a family of oncogenic viruses, has attracted immense attention towards the possibility of microRNAs as critical modulators of viral oncogenesis. The host-virus crosstalk mediated by microRNAs, messenger RNAs and proteins, is complex and involves the different cellular regulatory layers. In this commentary, we describe models of microRNA mediated viral oncogenesis.