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Challenges and Opportunities in Gene Expression

Challenges and Opportunities in Gene Expression

Macedo-Viñas, M. (2023)

Abstract

Background: Pseudomonas aeruginosa is the main pathogen responsible for lung destruction in cystic fibrosis, becoming difficult to eradicate in chronic infection.

Aims: To describe antibiotic resistance among strains of P.aeruginosa isolated from sputa of patients with cystic fibrosis. To investigate in vivo and in vitro expression of genes related to antibiotic resistance and anaerobic growth.

Methods: Sputa (in vivo) and strains (in vitro) from 26 patients were obtained during 17 months. Genotypes were compared by random polymorphic DNA amplification. Expression of nirS (anaerobic respiration) and mexY (MexXY efflux pump) were measured by quantitative real time polymerase chain reaction. Expression levels of nirS in aerobiosis and anaerobiosis were compared to estimate oxygenation status within lungs. Mutations in the regulator gene mexZ were investigated in sputa expressing mexY and were correlated with strains’ antibiotic resistance. 

Results: Nine patients and 56 sputa were finally analysed. Seven patients carried a single genotype. Gene mexY was detected in all the sputa; expression levels were higher in sputa with mexZ mutations. Multi-resistance was frequent. Resistance profiles not always correlated with mexY expression levels or mexZ mutations. Comparison of in vivo and in vitro nirS expression indicated mainly aerobic and microaerophilic environments within sputa.

Discussion: Mutations in e mexZ are frequent in strains of P.aeruginosa colonising patients with cystic fibrosis. Presence of these mutations correlates with increased expression of mexY in vivo and in vitro, but no with in vivo antibiotic resistance. Results of nirS expression suggest that the lungs represent heterogeneous environments regarding oxygenation status. This complexity explains that mechanisms of growth and antibiotic resistance within the lungs of these patients are still largely unknown.

Conclusions: After many years of research few studies, including the present, revealed different aspects of in vivo growth of P. aeruginosa. We determined a cut-off to discriminate between sputa containing mexZ wild type and mutated alleles and showed that comparison of in vivo and in vitro nirS expression allows to predict oxygenation status. So far, none of the studies can explain all the factors influencing the behaviour of P.aeruginosa colonising cystic fibrosis patients making it difficult to design new therapeutic strategies.

Raghavan, P. R. (2023)

Abstract

Endocrine gene expression in PANC-1, a type of pancreatic cancer cell, has been studied in the context of their potential to be reprogrammed toward a normal, differentiated state. Alkaline phosphatase activity has also been shown in pluripotent stem cells to differentiate between feeder and parental cells in reprogramming experiments. Metadichol®-based cell program­ming holds promise as a versatile and potentially safer approach for manipulating cellular behavior without the use of viral vectors, gating, or CRISPR. Using qRT‒PCR the results show multifold increase in the gene expression of CA9GCGINS MAFANEUROD1NGN3NKX2-2PAX6: PDX1SLC2A2FOXO1, and SIRT1. ALP levels increased and this activity is often used to distinguish stem cells from feeder cells as well as from parental cells in reprogramming experiments. Pluripotency was confirmed by the presence of islet-like structures on day eight. Metadichol exhibits anticancer activity with a CC50 value of 5.50 µg/ml compared to standard doxorubicin with a CC50 value of 10.28 µg/ml. At 100 ug/ml Metadichol is 82% cytotoxic.in a MTT assay Anti-tumor gene Klotho’s expression was increased 70fold on day eight. All the genes seen expressed regulate endocrine cell development in the pancreas and are involved in insulin and glucagon secretion. Gene network analysis is presented to show how Metadichol induced expression leads to a closed loop feedback network and biological process that would help in mitigating diabetes and other related disorders.

Tang, M., Sowa, G., Lee, J., Vo, N., & Kang, J. D. (2023)

Abstract

Intervertebral disc degeneration is a pervasive condition contributing to chronic back pain, affecting up to a third of the population, with risk further increasing with age. It is a significant driver of disability for millions of Americans and others worldwide. The standard of care today is reliant on symptomatic treatment rather than addressing the root cause of disease. Surgical interventions alter the structural integrity and biomechanics of the spine, often leading to loss of function and motion, and post-operative complications. This is the basis for innovation in novel biologic treatments, including gene therapy, which aims to reestablish the optimal balance between matrix catabolism and anabolism within pathologically degenerating disc cells. This review will cover the significant advances that have led to identification of target therapeutic genes combined with regulated expression of the therapeutic transgene and successful systems for gene delivery into cells. Recent advances in viral and non-viral vectors for gene transfer, silencing of genes by RNA interference, editing of genes by clustered regularly interspaced short palindromic repeats, and modifying mammalian target of rapamycin signaling pathwayS offer promising treatment avenues. Clinical translation of these approaches, however, will require further investigation of the pathological basis of disc degeneration in addition to systematic safety measures for the adoption of gene therapy.

Mehdipour, P. (2023b)

Abstract

Successful cancer evolution (CE) relies on the sequential molecular and functional events including 1) telomere; 2) sub-telomere; 3) epigenetic; 4-6) hit-episodes; 7) an innovative cell cycle machinery, as the multi-phase, and 8) chromosomal abnormalities. In this regard, eight available, fundamental/evolutionary and strategic key information (Evolutionary- ID) presented.

Telomere length (TL), has the fundamental role in cancer development, with serious challenges in the clinical managements. Breast cancer and brain tumor are an unresolved problem in Science and Medicine. Besides, an early and translatable diagnostic- prognostic-predictive platform, by considering the targets-ID, is required. Diverse TL in two cases affected with astrocytoma with grade IV, revealed to be 12500 and 15000 bp in tumor, and 10000 and 9000 bp at genomic level. Interestingly, TL is declined in the lymph node, i.e., occurrence of evolution.

Sub-telomeres (STs) through the cellular journey, are the neighboring destination at genomic and somatic level. The evolutionary pattern of STs has not been, routinely, decoded to the personalized clinical managements. The STsequences, are diversely predisposed to variety of environmental factors and play influential role in healthy individuals and the patients. An early detection is available by analysis of the ST- hybridized signals in the biopsy of auxiliary lymph nodes (ALN), and/or by circulating tumor cells (CTCs) into the blood stream. Diverse pattern of signal frequency and intensity in individual chromosomes at both somatic (ALN) and genomic (lymphocytes) levels were remarkable. The most common involved targets included chromosomes 5 and 9, 16 and 19; with diverse intensity at p and q chromosomal arms respectively. These findings have the predisposing, and an initial influence through the patients’ course of disease.

ST- signals, by providing the STs-ID, offer periodical and predictive, indices in cancer screening and therapy.

Furthermore, the complementary, cell cycle protein expression (PE) including Ki67, cyclin D1, and cyclin E, accelerates an early clinical management through the period of disease based on the CTCs.

Epigenetics is the next molecular destination by focusing on the genomic/somatic index, as an evolutionary Epigenetics-ID with its impact on the cancer management. The target panel is Ataxia Telangiectasia mutated gene (ATM) as the molecular marker and an initiator of different cancers.


ATM has remarkable roles, including: 1) in DNA double strand break (DSB), 2) to initiate different types of neoplastic disorders, including cancer, and 3), polymorphism, D1853N as a peridisposing marker by initiating the hit process. The influential characteristics include: family history of neoplastic disorders through the pedigree, the key role of ATM promoter methylation, cooperation of ATM/Rb protein expression, D1853N- marker, telomere length (TL) and the clinico-pathological characteristics in different types of brain tumors, and the environmental factors. Interestingly, TL has an independent influence on the progressive cancer evolution. An early detection by CTCs based on the D1853N/Sub-TL/Cell cycle checkpoints based on the PE assay and molecular test facilitate an early detection and therapy, based on the personalized approach.

By highlighting the preventive insight in Medicine, a brief record on the “Methylation in Chorionic villus samples (CVS)” with aim of an early detective strategy is provided. All nine CVS samples were methylated for the MCPH1 gene. An early detection is possible either through CV sampling or by the circulating CV cells in the maternal blood.

Evolutionary Hit includes: presence of D1853N polymorphism of ATM, as the hit-initiator through an evolutionary and progressive molecular based sequential alterations led to discovery of three-hit hypothesis in a patient affected with astrocytoma. More hits include five, and eight- hit hypotheses in primary breast cancer patients. Such platforms are considered as the individualized model in cancer. The pedigrees and details at the molecular follow-up studies and functional alteration at protein level are available in the provided sections.

Novel strategy of Cell cycle phases in breast cancer is the major intersection for cancer therapy.

The novel cell cycle hypothesis (CCH) highlights the mosaic based of dual and/or multi-phases, as minor clones at single cell level in the breast cancer (BC) -patients, escorted by the normal cell population. Such mosaicism provided an archetypal, unique diagnostic and therapeutic model, by applying different mosaic patterns (MPs) as well as “G1/S, S/G2 and G1/S/G2, and accompanied by normal phases, as a sole including G1, S, and G2 at the single cells level.  

Diagnosis is based on the mode of signal copy numbers (SCN) and the related PE. Interestingly MPs were also unmasked in patients with chronic myelogeneous leukemia and other solid tumors.

Finally, the predisposing/predictive/prognostic/preventive square provides an innovate CDKs inhibitor-based therapy in BC and other cancers.


Personalized base cancer therapy is the confusing procedure and requires the pedigree-based data, personalized, evolutionary based information including molecular and functional at both genomic and somatic, at single cell levelThe target territories comprise cell cycle phases, proteins, Telomere length, telomerase, sub-telomere, and Epigenetics. The aim is directing the cell cycle fundamental forces back to normal, by performing:

1) Applying personalized, single cell-based approach, at molecular, functional level, pedigree analysis, and balancing the micro-/macro-environmental factors, including nutrition.

2) Satisfactory high single cell enumeration based on the FISH and protein expression assays;

3) Decoding the required dosage and combined therapeutic regimens accordingly,

4) Unmasking the cell cycle combined (mosaic) phases including different Cyclins; and

5) Bilateral cooperation between Pharmacology, Medicine, and Cancer Genetics/cell biology.


 Let’s combine the evolutionary based strategy by translating the personalized data at molecular/ Functional/ Informative, and pedigree-based level to the personalized therapy.

Chen, Y. (2022)

Abstract

Clinically determined Ki-67 is a well-established marker for assessing proliferation potential in breast and other cancers. However, Ki-67 and the recommended thresholds for clinical decision-making vary systematically across breast cancer subtypes. In this study, an analysis of published gene expression data against Ki-67 in ER+/HER2- and Luminal A ductal carcinomas identified 127 of 14,997 protein-coding genes (elastic-net coefficient ≠ 0). The most upregulated genes associated with high Ki-67 are involved in cancer proliferation and were known in breast cancer studies, while the downregulated genes are involved in diverse signaling transduction processes. Application of the identified gene signature to ER+/HER2- and Luminal A ductal carcinomas consistently stratified two independent, population-based breast cancer cohorts. Although the ER+/HER2- clinical and the Luminal A intrinsic subtypes typically show good prognosis, one subpopulation identified by the signature showed an elevated risk of disease recurrence (hazards ratios 1.59 [95% CI 1.02, 2.47] and 3.80 [95% CI 0.83, 17.27] in two independent application cohorts). The present study identifies a proliferation gene signature specific to ER+/HER2- and Luminal A ductal carcinomas, provides biological insight into the more proliferative cancers, and could be a basis for future therapeutic development.

Linder, K., Watson, R., Ulmer, K., Bender, D., Goodheart, M. J., Devor, E. J., & Bosquet, J. G. (2023)

Abstract

Objective: Early detection of ovarian cancer could lead to improved survival rates, however no method has reliably been able to predict ovarian cancer. The aim of this study is to determine if processing alternative splicing data from high grade serous ovarian cancer patients using machine learning analytics will discriminate high grade serous ovarian cancer from normal fallopian tube samples. The ultimate goal would be to have a model that can predict high grade serous ovarian cancer with a blood test.

Methods: This is a case-control study of patients with confirmed high grade serous ovarian cancer and those undergoing salpingectomy for benign indications. RNA-sequencing was performed on all samples. RNA-sequence data was then put into Deep-learning augmented RNA-seq analysis of transcript splicing software suite. Deep-learning augmented RNA-seq analysis of transcript splicing created a model of differential alternative splicing aimed to discriminate between high grade serous ovarian cancer and normal fallopian tube. DEXSeq analysis was used to determine exon-based expression. Initial results with both analytics were then modelled with multivariate lasso regression to create prediction models (performance determined by area under the curve and 95% CI). Models created were the validated using The Cancer Genome Atlas data sets.

Results: One hundred and twelve high grade serous ovarian cancer and 12 benign samples were successfully sequenced. Deep-learning augmented RNA-sequencing analysis of transcript splicing identified 998 unique differentially expressed exons between high grade serous ovarian cancer and controls. Multivariate lasso regression analysis identified several exons that predicted high grade serous ovarian cancer with high performance. Specifically, ENSG00000182512:E001 from gene GLRX5 was highly predictive of high grade serous ovarian cancer with an area under the curve of 100%.

Conclusions: Application of machine learning analytics to exon differential expression, most likely due to alternative splicing, predicted high grade serous ovarian cancer with high performance. These results were validated in an independent dataset of cases and controls. Differential exon expression from cell-free RNA potentially could be used for early diagnosis of high grade serous ovarian cancer.

Popoola, O., Samuel, T., Popoola, M. A., Akinsola, S., Magbagbeola, O., & Akinloye, O. (2023)

Abstract

Prostate cancer (PCa) is the most prevalent cancer in the Nigerian male population, similar to other black populations. It is postulated that exposure to endogenous or environmental steroids prompts prostatic mediated changes via steroid receptors as well as a decrease in the androgen/estrogen ratio and aging. Thereby contributing to prostatic carcinogenesis and disease progression. This study is aimed to determine the plasma levels of testosterone, 17β-estradiol as well as the pattern of expression of steroid receptors in subjects with prostate cancer, benign prostatic hyperplasia, and controls. Study participants are made up of a total of 195 consented volunteers consisting of 65 Prostate cancer (PCa) and 65 benign prostatic hyperplasia (BPH) treatment naïve participants and 65 apparently healthy subjects as controls. Anthropometric data were measured using standard methods and biochemical parameters determined by enzyme-linked immunosorbent assay (ELISA). The gene expression is quantified by Real-Time PCR with PerfeCTa SYBR Green SuperMix  on CFX96 Bio-Rad, USA. The results of this study showed increased levels of 17β-estradiol, total androgen receptor (AR) and estrogen receptor-beta (Erβ) in prostate cancer participants compared with controls (P<0.05). A Significant reduction in plasma levels of testosterone in prostate cancer subjects compared with BPH and controls was observed (P<0.001). The plasma levels of androgen receptors were significantly increased in PCa and BPH participants (p<0.05). We observed a positive correlation between ERβ levels and PSA levels in the PCa group (r=0.32, p=0.02). There was a differential expression of AR, ESR1 and ESR2 in the studied group. This study shows hypogonadism and an increased 17β-estradiol, ERβ, and AR levels in subjects with prostate cancer. This data suggests that modulation of these hormones and their receptors may be associated with initiation and progression of prostate cancer and could be valuable in the interpretation of PSA kinetics and stratification of cases after screening.

Amano, F. (2023)

Abstract

Macrophage is the immune phagocytic cell, playing variety of immunological and inflammatory reactions. The macrophage activation has been extensively studied in vitro using culture media like Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium (F-12) in response to bacterial infection, tumor development, cytokines and so on. In the study of macrophage activation during co-culture with EL-4 tumor cells, we found different phenotypes of J774.1 macrophage-like cell line in F-12 and DMEM, when treated with lipopolysaccharide (LPS) and interferon-g (IFN- g). Among these phenotypes, nitric oxide (NO) production with corresponding inducible NO synthase (iNOS) expression was remarkable, showing higher in F-12 than in DMEM. Besides, O2generating activity and production of interleukin-1b (IL-1b) were also higher in F-12 than DMEM, although production of tumor necrosis factor-a (TNF- a) was higher in DMEM than F-12. RT-PCR analysis revealed significantly higher expression of mRNA of iNOS, IL-1 b, IL-18, IkBa in F-12, but higher that of p65 and p105 in DMEM after treatment with LPS + IFN-g, suggesting these differences being induced at the transcriptional levels. Through investigation of critical factor(s) in these culture media that influence the activation phenotypes of the macrophages, we found that sodium bicarbonate (NaHCO3) concentrations in these culture media, 14 mM in Ham’s F-12 and 44 mM in DMEM, were the key. Culture medium-induced differences in macrophage activation were also observed in RAW264.7 macrophage-like cell line and in mouse peritoneal macrophages. The recent studies suggested involvement of carrier (SLC) transporter gene expression and subsequent elevation of JAK/STAT signaling cascades in these NaHCO3 responses. Taken together, these results provide evidence for the importance of NaHCO3 in the culture medium in the macrophage activation in vitro, implying important insights to the NaHCO3 concentration in vivo in the body of patients suffering from inflammation, tumor development or immune disorders where macrophage activation is involved.

Cóbar, O. M., & Cóbar, S. (2023)

Abstract

Background: The proteins codified in the Open Reading Frame 1ab -Orf1ab- region of the SARS-CoV-2 genome are the main responsible of the virus transcription, replication, and translation processes inside the human cell.

Once inside the cell, the viral mRNA encode structural and nonstructural proteins, that direct virus assembly, transcription, replication and host control, and the accessory proteins whose function has not been determined.

The largest gene, Orf1ab, contains overlapping open reading frames that encode polyproteins PP1ab and PP1a.

These polyproteins are cleaved to yield 16 nonstructural proteins, NSP1-16.

Production of the longer (PP1ab) or shorter protein (PP1a) depends on a ribosomal frameshifting event.

The proteins, include the papain-like proteinase (NSP3), 3C-like proteinase (NSP5), RNA-dependent RNA polymerase (NSP12, RdRp), helicase (NSP13, HEL), endoRNAse (NSP15), 2′-O-Ribose-Methyltransferase (NSP16) and other nonstructural proteins.

The SARS-CoV-2 nonstructural proteins are responsible for viral transcription, replication, proteolytic processing, suppression of host immune responses, and suppression of host gene expression.

The purpose of the manuscript is to present a systematic review as of September 30, 2023, on the Orf1ab region mutations of the SARS-CoV-2 genome as of September 30, 2023, with the aim to predict, through the mutations profile on that region, the severity of an infection for a new SARS-CoV-2 variant that could emerge in the near future.

Material and Methods: Original scientific articles published in Medline, Pubmed, Science Direct, Web of Science, Scopus, EBSCO and BioMed Central databases, official health organizations (WHO, CDC, ECDEC, NIH) electronic publications, and specialized media in the subject, were electronically searched to accomplish the aim of the study.

Articles published in any language were included from 2020 to present using a variety of keywords in combination.

The studies relevant to our review were analysed and compared.

Results and discussion: The NIH “National Human Genome Research Institute” define the Open Reading Frames (ORFs) as a portion of a DNA sequence that does not include a stop codon.

The Open Reading Frames encode accessory proteins transcribed from the 3′ one-third of the genome to form a set of subgenomic mRNAs (sg mRNAs).

Following entry on the human cell, the viral particle release the genomic RNA molecule that is translated on two large open reading frames, ORF1a and ORF1b.

The resulting polyproteins pp1a and pp1ab are co-translationally and post-translationally processed into the individual non-structural proteins (NSPs) that form the viral replication and transcription complex (RTC).

Translated structural proteins translocate into endoplasmic reticulum (ER) membranes and transit through the ER-to-Golgi intermediate compartment (ERGIC), where interaction with N-encapsidated, newly produced genomic RNA results in budding into the lumen of secretory vesicular compartments.

Two viral proteases, Plpro -NSP3- and 3Cl-pro -NSP5-, process the polyproteins and generate the nonstructural proteins NSP1-NSP16 that directs the transcription, replication and construction of new virions that are secreted by exocytosis from the infected cell.

The evolution of key proteins in viral transcription and replication is clearly observed by carefully studying the structure, function, and evolution of RdRp, Mpro or 3Clpro, and NSP13 proteins directed by the Orf1a and Orf1ab genome mutations.

Conclusions: ORF1ab is cleaved into 16 non-structural proteins involved in SARS-CoV-2 transcription and genome replication.

P323L, P227L, G671S, V776L and A185S are the first five frequent mutations of RdRp (NSP12), the mutations P227L and G671S might have functional consequences in the viral transcription and replication.

Mutations in residues D499 to L514, K545, R555, T611 to M626, G678 to T710, S759 to D761 are directly implicated with the transcription-replication capability of the virus by RdRp.

In Mpro (NSP5) the mutation of residues H41, P132, C145, S145, L226, T234, R298, S301, F305, and Q306 may increase the efficiency of proteolytic cleavage of proteins such as NEMO, thereby improving the ability of the omicron series of viruses to suppress the immune system and accelerate the viral replication.

In Helicase (NSP13) the mutations of residues E261, K218, K288, S289, H290, D374, E375, Q404, K460, R567, and A598 are involved in the separation of the double-stranded RNA or DNA with a 5′→3′ polarity as well as 5′ mRNA capping activity in the virus transcription-replication process.

In the Orf1ab gene, ORF1b:V2354F mutation, corresponding to NSP15:V303F, may induce a conformational change and result in a disruption to a flanking beta-sheet structure.

The premature stop codon ORF7a:Q94*, truncates the transmembrane protein and cytosolic tail used to mediate protein transport, may affect protein localization to the ER-Golgi.

The analyses of Orf1ab genome mutations, allows us to predict, through the mutations profile on that region, the severity of an infection for a new SARS-CoV-2 variant that could emerge in the near future.

Vakulenko, M. (2023)

Abstract

This article presents the results of the clinical significance study of changes in the activity of adenosine deaminase and the content of polyamines in the blood plasma of cats with mammary gland tumors. In our work, we investigated the activity of the adenosine deaminase gene in the tissues of mammary gland tumors in cats and compared the activity of this gene with normal mammary gland tissue. In the course of this work, we also found out the frequency of occurrence of mammary gland tumors in the population of domestic cats in the Rostov region and evaluated the prognosis of survival after surgical treatment of breast cancer.

The incidence of mammary gland tumors was detected, which amounted to 0.4% of the domestic cats who have applied to veterinary clinics in Rostov region, where the cats with mammary gland neoplasms amounted to approximately 400 individuals for every 100,000 animals. These data reflect the importance of the problem of mammary gland tumors and emphasize the need to study the pathophysiological foundations of carcinogenesis in order to create molecular genetic approaches for early diagnosis of mammary gland tumors.

The results of the studies showed that the level of expression of the adenosine deaminase   gene in the tissues of invasive nonspecific carcinoma and in the tissues of fibroepithelial hyperplasia sharply increases compared to the tissue of a healthy mammary gland, 36 times in the tissues of invasive carcinomas and 131 times in the tissues of fibroepithelial hyperplasia. At the same time, there were no significant differences between the activity of adenosine deaminase   in the blood plasma of healthy animals and in the blood plasma of animals with malignant neoplasms of the mammary gland. Measurement of the level of polyamines in the blood of animals showed that the content of putrescine in the erythrocytes of the blood of cats with benign and tumor-like neoplasms of the mammary fibroepithelial hyperplasia significantly increased by 5 times compared with the indicators of the control group. In malignant neoplasms of the mammary gland invasive nonspecific carcinoma, the content of putrescine and spermin in the blood significantly exceeded the control values by 6 and 10 times, respectively.

Alsousi, A., & Igwe, O. J. (2022)

Abstract

Reactive oxygen species (ROS) are implicated in playing a role in initiating and in propagating the pathogenesis of rheumatoid arthritis (RA). We investigated the mechanism(s) by which essential redox-active trace metals (RATM) may activate gene transcription in synovial fibroblasts. The rabbit fibroblast-like synovial cells which express Toll-like receptor 4 (TLR4), were used as a model system for potentially initiating RA through oxidative stress. Potassium peroxychromate (PPC, Cr5+), ferrous chloride (FeCl2, Fe2+), and cuprous chloride (CuCl, Cu+) at the indicated valency states were used as exogenous pro-oxidants.  These trace metals can induce oxidative stress through TLR4 activation to release inflammatory cytokines and high mobility group box 1 protein. We measured the total expression levels of mitogen-activated protein kinase (MAPK) in the synovial cells and examined the effect of the redox-active trace metals on the time-course production of phosphorylated moieties of MAPK by fluorescence cell-sorting flow cytometry. TLR4 siRNA was used to examine the role of TLR4 in the activator protein -1 (AP-1) signalling activity, and western blots were used to measure the time-course phosphorylation levels of AP-1-activation-related proteins. While the redox-active trace metals increased intracellular ROS that can induce oxidative stress, they also induced MAPK kinases to upregulate the expression of AP-1 proteins in synovial cells. Our results show that redox-active trace metal/TLR4-coupled activation may contribute to the pathogenesis of RA. The signaling pathway by which inflammation and its destructive sequel may occur in RA through synovial cells underlies the need for developing therapeutic agents to serve in individualized RA therapy with a consideration for the underlying mechanism(s) of its pathogenesis.

Dasari, V., Bolimera, P., Dokku, S., Gorti, L., & Dravida, S. (2022)

Abstract

The traditional testing requirements for both adult and developmental neurotoxicity evaluations are based on in vivo animal models while the neurotoxic risks associated with molecules or vaccines is mainly determined by neurobehavioral and neuropathological effects in the experimental model chosen. The poor correlation between preclinical in vitro or in vivo data (non-human) with the real time clinical effects leading to severe progressive adverse events is a major concern in general. The employed bioinformatics search tools helped us to short list the affected common genes in neurotoxicity induced by viral, bacterial infections and cytokine storms. Here, we used our group characterized human induced Pluripotent Stem Cell (hiPSC) system developed as an in vitro microphysiological model to record phenotype and genotype perturbations when treated with selected known representative neurotoxins like TEA, Tetanus toxin, MSG, Dopamine, Bungarotoxin etc. The objective was to assess the application qualification of the novel in vitro model that yields human relevant readouts. The recorded phenotype perturbations were barcoded with SOD, BAX, HDAC1, TNFalpha, MAPK14 like gene expressions in generating in vitro patterns to correlate the human functional toxicogenomics information. We showed hiPSC system to be phenotypically responsive and genotypically reactive when treated with neurotoxins. Out of 7 gene expression data sets generated, SOD and BAX were recorded to be downregulated at all the micro-conditions created in the hiPSC system while HDAC was consistently upregulated except in Dopamine treated system. The bioinformatics analysis performed on the selected genes gave insight into their roles in disease specific signalling pathways like JAK-STAT, TNF, Neurotrophin etc. We report configured hiPSC system suitability as an in vitro human surrogate platform/model in generating toxicogenomics signatures to support prediction on the test material in any assay system developed on this well characterized microphysiological base.

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