Masters Degrees (Medical Biochemistry)

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1,4,7,10,13,16-Hexaazacyclooctadecane (Hexacyclen) Induced Nitrosative Stress and Downregulated NF-κB Cell Survival Pathway in Human Embryonic Kidney (Hek293) and Colorectal Adenocarcinoma (Caco2) Cells.
(2022) Nxumalo, Mthokozisi Bongani.; Khan, Rene Bernadette.; Khumalo, H.
Colorectal cancer (CRC) is the third most common malignancy detected and the second leading cause of cancer-related mortality. Mammalian cells require metals for the physiological process as they are part of the structure or co-factor of many proteins. However, excessive accumulation may manifest in toxicity. In addition, the promotion of oncogenesis and tumour growth has been associated with an increased presence of metals. Promising anticancer compounds that disrupt the onset and progression of carcinogenesis are currently being intensely investigated by the scientific community. Hexacyclen, a nitrogen electron donor and a potent metal ion chelator that binds various metal and transition metal cations, is one such anticancer drug. The cytotoxic effects of Hexacyclen on human colorectal adenocarcinoma cells (Caco2) and normal embryonic kidney cells (Hek293) were investigated in this work after acute exposure (48 hours). The toxicity of Hexacyclen was studied in Hek293 and Caco2 cells at different concentration ranges [(0-500 μM) and (0-50 μM), respectively]. The MTT (to determine IC20 and IC50), ATP and mitochondrial membrane potential (ΔΨM) assays were used to assess metabolic activity, while TBARS, NOS and GSH assays were used to assess oxidative activity. Caspase activity (-8, -9, -3/7), phosphatidylserine externalisation and LDH leakage were used to assess cell death by apoptosis. In addition, western blotting was used to examine the expression of antioxidant (SOD2, GPx, catalase), pro-and anti-apoptotic (p-p53, Bcl-2, HSP70, PARP, cPARP) and inflammatory (NF-κB, STAT3 and p-STAT3) proteins. From the dose-dependent MTT curve, an IC20 and IC50 of 6μM and 38μM (Hek293) and 1.2μM and 5μM (Caco2 cells) were determined. The decreased ATP concentration in Hek293 (p<0.05) and Caco2 (p>0.05) cells for both treatments was consistent with altered ΔΨM in both cell lines, indicating reduced metabolic activity. Elevated RNS was implied by increased iNOS particularly at the Caco2 IC50 (p<0.05) that promoted nitric oxide production at the IC20 (p>0.05) and IC50 (p<0.05) for Hek293 and Caco2 cells respectively. The decreased MDA in Hek293 cells (p>0.05) was associated with increased SOD2 (p<0.05) and GPx (p<0.05), while slightly increased MDA in Caco2 cells (p>0.05) accompanied increased SOD2 (p>0.05) and GPx (p<0.05 at the IC50 only). Furthermore, GSH levels were increased significantly in IC50-treated Hek293 and Caco2 cells (p<0.05), but downregulation of catalase in Hek293 and Caco2 cells was not significant. In this study, apoptosis was initiated by an increase in caspase-9 (IC50, p<0.05) but not caspase 8, which was decreased for both treatments in Hek293 cells (p<0.05). In Caco2 cells, caspase-8 (p<0.05) and caspase 9 (p>0.05) were increased. Anti-apoptotic Bcl-2 (p<0.05) and HSP70 (p<0.05 for Caco2 cells) were downregulated in both cell lines. The activity of p-p53 was not affected in IC20, whereas it was significantly reduced in IC50-treated (p<0.05) in Hek293 and Caco2 cells. Apoptosis was executed as caspase 3/7 was increased in all treatments (p<0.05), albeit non-significantly for IC20-treated Hek293 cells. Moreover, phosphatidylserine externalisation, an early apoptosis marker, was increased in both cell lines (p<0.05 for IC50-treated Hek293 cells), while LDH (a late marker) was increased for Hek293 cells (p<0.05) but not Caco2 cells (p>0.05). Interestingly, decreased cPARP/PARP activity was observed for IC50-treated cells (p<0.05) in both cell lines. Finally, the inflammatory markers NF-κB (p>0.05 for IC20-treated Hek293 cells) and p-STAT3/STAT3 (p>0.05 for IC20-treated Caco2 cells) were downregulated in this study. Hexacyclen induced apoptosis in Hek293 and Caco2 cells via an RNS-mediated mechanism. Intrinsic apoptosis was noted in Hek293 cells, while both pathways facilitated apoptosis in Caco2 cells. Interestingly, apoptosis proceeded concurrently with a reduction in the NF-κB cell survival pathway.
Aflatoxin B1 modulates oxidative stress and apoptosis in human embryonic kidney cells.
(2019) Dlamini, Nomali Zanele.; Khan, Rene Bernadette.
Introduction: Aflatoxin B1 (AFB1) is produced by filamentous fungal strains of Aspergillus flavus and Aspergillus parasiticus that infect field crops, therefore AFB1 is a frequent contaminant of dietary staples such as rice, maize and peanuts. Humans and animals are exposed to AFB1 through consumption of contaminated foods, predisposing them to various diseases. AFB1 is a potent hepatotoxin that has been classified by the International Agency of Research on cancer (IARC) as a group1 carcinogen. The carcinogenic effects of AFB1 have been attributed to the metabolism of this toxin to an epoxide that promotes the production of free radicals, mitochondrial toxicity and induction of cell death. With the increasing prevalence of kidney associated diseases in humans, and the AFB1-associated kidney toxicity observed in animals, this study investigated the cytotoxic effects/mechanism of AFB1 in human embryonic kidney (Hek293) cells. Methods: Hek293 cells were exposed to AFB1 (0-100μM) for 24hrs. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. Results and discussion: The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1-treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalisation of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated Hek293 cells. Conclusion: The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in Hek293 cells. Keywords : Aflatoxin B1 (AFB1), oxidative stress, apoptosis, Hek293 cells
Allicin ameliorates some deoxynivalenol-induced cytotoxic effects in human embryonic kidney (Hek293) cells, but also elicits synergistic and potentiating adverse effects.
(2020) Mamane, Yandisa Zintle.; Khan, Rene Bernadette.
Introduction: Deoxynivalenol (DON), a type B trichothecene produced by plant pathogenic fungi, especially Fusarium graminearum and F. culmorum, is a highly toxic mycotoxin found throughout South Africa. DON is consumed unintentionally through maize derived products and is rapidly becoming a potential health risk to humans and animals. It is a known immunosuppressant that induces apoptosis and oxidative stress and may cause liver lesions and kidney problems. Recently, dietary therapeutics have demonstrated a role against mycotoxin-induced cytotoxicity. Garlic (Allium sativum) is part of the Alliaceae family. The garlic bulb is used for medicine and as food consumption. The aqueous extract has recently demonstrated the potential to protect against mycotoxin-induced cell death and decrease reactive oxygen species (ROS). Aim: This study investigated the induction of apoptosis and oxidative stress by DON in Hek293 cells, and the ability of allicin to ameliorate these effects. Methods: Hek293 cells were treated with a range of allicin concentrations (0-150mM) over 24hrs. An EC50 of 1.7mM was obtained from the MTT assay and used in all subsequent assays. Hek293 cells were treated with 5μM DON, 1.7mM allicin (A), or a combination (DON+A) for 24hrs; untreated cells served as the control. Lipid peroxidation [malondialdehyde (MDA) and lactate dehydrogenase (LDH) assays] were used to indirectly quantify reactive oxygen species (ROS) and oxidative stress; reactive nitrogen species (RNS) were quantified using the nitrates assay. Apoptotic induction was determined by the detection of phosphatidylserine (annexin V) and DNA fragmentation. Necrotic cells were distinguished by propidium iodide uptake. Luminometric quantification of ATP, reduced glutathione (GSH), and caspase 9, 3/7, were used to verify these events. In addition, antioxidant enzymes protein expression of superoxide dismutase (SOD2), catalase and glutathione peroxidase (GPx1); as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and heat shock protein (Hsp70), and apoptotic markers associated protein expression of p53, Bax, and poly (ADP-ribose) polymerase (PARP) were detected by western blotting. Results: DON-induced ROS production was suggested by the depletion of antioxidants including SOD2 (p < 0.0001), catalase (p < 0.0001) and GSH (p = 0.0886). Decreased lipid peroxidation indicated by the decreased MDA concentration (p < 0.0001) and reduced LDH (p = 0.0342) imply that the Hek293 cells were spared from the membrane-damaging effect of oxidative stress. A reduction in Hsp70 (p = 0.0056) and Nrf2 (p < 0.0001), and upregulation of GPx1 (p = 0.0362) protein expression was noted. In addition, increased nitrate concentration in all treatments compared to the control (p < 0.0001) suggested a shift to RNS production. Notably, allicin maintained Nrf2 protein expression similar to the control. The decrease in MDA concentration (p = 0.0109) by allicin was concurrent with depleted GSH (p = 0.0504)and increased SOD2, catalase and GPx1 (p < 0.0001), and suggests allicin induced an oxidative stress response. Allicin also protected DON-treated cells from oxidative stress by upregulating Hsp70 (p < 0.0001), catalase (p = 0.0006) and GPx1 (p = 0.0018), with concurrent decreased GSH (p = 0.0342) and ATP (p = 0.2028) concentration, which were also decreased by DON. In addition, allicin increased MDA (p < 0.0001) and LDH (p = 0.1267) towards control levels in the combined treatment. Apoptosis was reduced in the DON (p = 0.4631) and DON+A (p < 0.0488) treated cells in comparison to the control, necrosis was not evident in any treatment. The slight induction of p53 (p = 0.0008) and PARP-1 (p = 0.4036) by DON implies an attempt at DNA repair, but the Hek293 cells experienced reduced levels of apoptosis. Indeed, Bax expression was slightly reduced (p = 0.1071), caspases 9 (p = 0.0705) and 3/7 (p = 0.4431) activities were diminished, phosphatidylserine was not externalized, and PARP-1 was not cleaved. A non-fragmented DNA profile in allicin-treated and DON+A-treated Hek293 cells may be explained by increased expression of DNA repair proteins, PARP-1 (p = 0.0048 and p = 0.0004 respectively) and p53 (p < 0.0001). The upregulation of p53 is associated with an increase in Bax expression (p < 0.0001 and p = 0.0026 respectively). However, caspases 9 (p = 0.0596) and 3/7 (p = 0.0311) were not activated and apoptosis did not occur. Conclusion: DON treatment induced oxidative stress but not apoptosis in Hek293 cells at the concentration tested. In addition, its mechanism of toxicity in Hek293 cells appears to be more related to nitrosative stress and induction of DNA damage. Oxidative stress and not apoptosis is the possible mechanism of allicin-induced effects in Hek293 cells. Although allicin ameliorated some of the effects of DON in Hek293 cells, it also elicited synergistically or potentiating adverse effects that require further investigation.
Artemisia afra crude aqueous leaf extract indices oxidative stress and inflammation in human colon adenocarcinoma cells via the upregulation of the TNF-a,p38 and STAT3 pathway.
(2022) Mposula, Slindelo.; Khan, Rene Bernadette.
ABSTRACT Introduction: Artemisia afra (A. afra) is a widely used medicinal plant located in the southern African region. It is traditionally used to alleviate medical conditions such as coughs. Literature indicates a protective role by improving antioxidant capacity and reducing cell proliferation, which suggests anti-cancer potential. Colorectal carcinoma (CRC) is a global public health crisis and the second common cause of cancer-related fatalities. Current cancer treatment is deemed effective but not easily accessible and expensive in the southern African region. Therefore, the need for naturally derived anti-cancer agents remains to be investigated for accessible and affordable treatment. This study investigates the antiproliferative and antioxidant effects of A. afra crude aqueous leaf extract in the Caco-2 cell line. Materials and Methods: Caco-2 cells were treated with a range of A. afra concentrations (0-5000 μg/ml) for 48 hours. An IC50 was derived from the MTT assay and all subsequent assays compared the IC50 -treatement to an untreated control. Mitochondrial integrity was luminometrically assessed by measuring JC-10 fluorescence and ATP. Free radical production (TBARS, NOS) and membrane damage (LDH cytotoxicity), together with GSH quantitation were used to infer the presence of oxidative stress; antioxidant enzymes (SOD2, GPx-1, catalase, Nrf2) were also detected by western blotting. Apoptotic induction was verified by measuring phosphatidylserine externalisation, quantifying caspase activities and detecting pro- and anti-apoptotic proteins (Bax, Bcl2, cIAP, xIAP) by western blotting. Single strand DNA fragmentation was evaluated via the comet assay. Additionally, relative expression of DNA repair, inflammation and stress markers were determined using western blotting and qPCR. Results: Crude aqueous leaf extract of A. afra induced a dose-dependent reduction in cell viability, yielding an IC50 of 250 μg/ml. Decreased mitochondrial integrity (p = 0.697) was associated with significant depletion of intracellular ATP (p = 0.0043) and increased ROS production as validated by increased lipid peroxidation (p = 0.1638) and DNA oxidation (amplified OGG1). In addition, increased iNOS contributed to the production of RNS. Artemisia afra induced an antioxidant response that elevated Nrf2 at the mRNA and protein level, causing increased GSH (p = 0.0001), GPx-1 (p = 0.5067) and catalase, but SOD2 was decreased. Heightened levels of heatshock proteins (HSP27 and HSP70) correlate with increased ROS and upregulated phosphorylated p38 protein, but ERK and JNK protein expression was downregulated. Significant downregulation caspase-8 (p = 0.0252), caspase-9 (p = 0.0099) and caspases-3/7 (p = 0.0232) was associated with reduced Annexin-V) and extracellular LDH. In addition, the Bax/Bcl-2 ratio (p = 0.0033) and protein expression of inhibitors of apoptosis protein such as cIAP-1 and xIAP indicated reduced apoptotic activity in this study. Comet tail analysis indicated intact DNA, in congruence with decreased OGG1. Both TNF-α (p = 0.2323) and STAT-3 were upregulated, but NF-ĸB was decreased. In addition, cellular Myc and phosphorylated retinoblastoma were upregulated. Conclusion: The crude aqueous leaf extract of A. afra induced mitochondrial toxicity and ROS production. Despite a heightened antioxidant defense, ROS-mediated upregulation of TNF-, p38 and STAT3 promoted cell proliferation and inhibited apoptosis in Caco-2 cells. Taken together, A. afra is a cytotoxic and genotoxic agent that may induce cancer in human colorectal cells.
Momordica foetida facilitates glucose uptake independent of AMPK2 and PI3K to attenuate hyperglycemia-induced oxidative stress via a JNK-STAT3 mediated pathway in HepG2 cells.
(2022) Netshitangani, Tshamano Fulufhelo.; Khan, Rene Bernadette.
Introduction: The exponential rise in the global prevalence and incidence of type 2 diabetes is concerning. Hyperglycemia is a hallmark of type 2 diabetes that induces oxidative stress, leading to impairment of vital liver metabolic pathways. Metformin is the first-line treatment for type 2 diabetes mellitus. However, Momordica foetida has been used in folk medicine for the treatment and management of diabetes mellitus in various parts of the world including South Africa. Aim: In the present study, the cytoprotective effects of M. foetida on liver impaired glucose metabolism and oxidative stress damage were investigated on high glucose induced HepG2 cells, with Metformin as a positive drug control. Methods: The M. foetida leaves were used to prepare an aqueous lyophilized extract. The HepG2 cells were serum starved for 1 hour, then exposed to hyperglycemic conditions (30mM D-glucose) for 24 hours. Cells were treated with various concentrations (125 - 1000 μg/ml) of the lyophilized M. foetida aqueous extract for 24 hours, and the 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay evaluated the effects of high glucose and M. foetida on the metabolic activity of HepG2 cells. Antioxidants and prooxidants were assessed and quantified using luminometry, thiobarbituric acid reactive substances (TBARS) and nitric oxide synthase (NOS) assays. Western blot and quantitative real-time (qPCR) were used to observe the effects high glucose and M. foetida on signaling pathways and antioxidant response. Results: Glucose uptake in hyperglycemic conditions was mediated by increased gene expression of adenosine monophosphate-activated protein kinase alpha 2 (AMPKα2) (p˂0.05) and phosphatidylinositol 3‑kinase (PI3K) (p˂0.05), but glucose transporter 2 (GLUT2), glucokinase (GK) and glycogen synthase (GS) were downregulated (p˂0.05). Interestingly, an opposing response was noted for Metformin and M. foetida treatments, where AMPKα2 (p˂0.05) and PI3K (p˂0.05) were downregulated, whereas GLUT2, GK and GS were upregulated (p˂0.05) compared to the hyperglycemic control. When compared to the hyperglycemic conditions control, M. foetida treatments and Metformin showed an increase in glucose uptake. Hyperglycemic conditions induced toxicity indicated by increased extracellular lactate dehydrogenase (LDH) and decreased adenosine triphosphate (ATP), but Metformin and M. foetida decreased LDH activity back to xvi normoglycemic levels, indicating reduced cytotoxicity. Increased mitochondrial membrane potential (m) in hyperglycemic conditions was accompanied by increased lipid peroxidation (p˂0.05) and reactive nitrogen species (RNS) (p˂0.05). The m was increased further by M. foetida, with minimal effect on reactive oxygen species (ROS) production but effectively increasing RNS (p˂0.05). Oxidative damage was reduced in the hyperglycemic control but was increased by Metformin and M. foetida treatments prompting the activation of p53 in these cells (p˂0.05). Effective oxidative stress response was mounted by NRF2 (p˂0.05) and antioxidants SOD2 (p˂0.05) and GSH, but GPx1 and CAT (p˂0.05) were decreased. Interestingly, Metformin and M. foetida induced CAT (p˂0.05) and GPx1 (p˂0.05) in the antioxidant response, consequently decreasing GSH. Metformin decreased NRF2 (p˂0.05) and SOD2, while M. foetida increased NRF2 significantly and had no effect on SOD2 relative to the hyperglycemic control. Hyperglycemic conditions downregulated the oxidative stress response by MAPK (p-p38, pJNK and pERK1/2) (p˂0.05). However, Metformin upregulated pJNK (p˂0.05) and pERK1/2 (p˂0.05), but p-p38 (p˂0.05) was downregulated. Interestingly, M. foetida upregulated pJNK (p˂0.05), downregulated pERK1/2 (p˂0.05) and had no effect on p-p38. Hyperglycemic conditions also increased pSTAT3, which was downregulated by Metformin and M. foetida treatments (p˂0.05). Conclusion: Taken together, the results demonstrated that M. foetida enhanced the metabolic activity and reduced cell cytotoxicity in HepG2 cells. Furthermore, M. foetida facilitated glucose uptake independent of AMPK2 and PI3K. The main source of oxidative stress was increased RNS, which was alleviated by an effective MAPK/JNK and antioxidant response involving CAT.
N, N Bis (2-Pyridylmethyl)-1, 2-Ethylenediamine Tetrahydrochloride Stimulates Intrinsic Apoptosis Mediated by Oxidative and Nitrosative Stress Induction of the NF-B/STAT3 Pathway in Human Hepatocellular Carcinoma (HepG2) Cells.
(2022) Ntanzi, Nosipho.; Khan, Rene Bernadette.
Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Its incidence is rising, and this trend is expected to continue for decades. Several cancer therapeutics have already been discovered and are being used to treat HCC, however, most of them cause severe side effects which decrease the treatment's effectiveness. Metal chelators such as ethylenediaminetetraacetic acid (EDTA) have previously demonstrated anti-cancer potential. N, Nbis (2-pyridylmethyl)-ethylenediamine tetrahydrochloride (H2pmen) is a tetradentate ligand that forms stable complexes with Fe, Cr, Cu(II), and Zn (II), and it has been shown to be a potentially effective reagent for metal chelation. This study investigated the antiproliferative and cytotoxic effects of H2pmen in the HepG2 cell line. Methods: The cell viability was determined by treating HepG2 cells with different concentrations (0–1000 μM) of H2pmen over 24h. MTT assay was used to obtain an IC50, which was then used in all subsequent assays. The cells were then assayed for oxidative stress and membrane damage (TBARS, NOS, GSH, and LDH cytotoxicity), apoptotic induction (ATP assay, JC-10 assay, Annexin v, Caspases), cytochrome P450 3A4 activity (Luminometry). Protein expression of iNOS, SOD2, Bax, Caspase-2, and STAT3 were identified using western blot analysis. The gene expression of GPx1, Nrf2, NF-κB, p53, and OGG1 was determined using qPCR. Results: H2pmen induced a dose-dependent decrease in cell viability (IC50 of 209 μg/ml), a significant increase in CYP34A activity (p0.05 at IC20 and IC50), a decrease in ATP production (at IC20 p0.05 and at IC50), a significant decrease in m (p0.05 at IC20 and at IC50). The ROSassociated membrane was induced, indicated by an increase in lipid peroxidation (p0.05 at IC20 and p≥0.05 at IC50), an increase in RNS production (p≥0.05 at IC20 and at IC50), an upregulation in iNOS protein expression (at IC20 where p0.05 and at IC50) and NF-κB gene expression (at IC20 where p0.05 and at IC50). Oxidative stress occurred due to a decrease in GSH levels (at IC20 and p≥0.05 at IC50), a significant downregulation in SOD2 protein expression, and upregulation in gene expression of GPx-1 (at IC20 where p≥0.05 and at IC50) and Nrf2 (at IC20 and at IC50 where p0.05). H2pmen initiated caspase-dependent apoptosis that was indicated by a decrease in Caspase-2 (p0.05at IC20 and at IC50), caspase-8 (at IC20 and p≥0.05 at IC50), a slight insignificant decrease at IC20 and an increase at the IC50 in caspase-9, a significant upregulation in Bax (p0.05 at IC20 and at IC50) protein expression and p53 (at IC20 where p0.05 and at IC50) gene expression. The significant increase in caspase-3/7 (p≥0.05 at IC20 and IC50), Annexin V levels (p≥0.05 at IC20 and at IC50), LDH (p≥0.05 at xviii IC20 and IC50), STAT3 (p0.05at IC20 at IC50), PARP1 (p0.05 at IC20 and at IC50), and OGG1 (p0.05 at IC20 and at IC50) shows that apoptosis was executed by H2pmen in HepG2 cells. Conclusion: H2pmen reduced cell viability of HepG2 cells, exerting a cytotoxic effect associated with decreased m and ATP, and increased LDH leakage. The chelating properties of H2pmen was linked to the induction of oxidative and nitrosative stress that affected lipids and DNA. The HepG2 cells mounted an antioxidant defense involving Nrf2 to counteract the depletion of SOD2 and GSH, with evidence of its effect associated with upregulation of GPx. The prevailing oxidative stress activated DNA repair enzymes (PARP1 and p53), while NF-κB and STAT3 pathways were also induced. Bax-induced MOMP and caspase-2 invoked VDAC triggered caspase-dependant apoptosis via the intrinsic pathway.
Terminalia phanerophlebia crude aqueous leaf extract activates the NRF2-mediated antioxidant defence to prevent oxidative stress in human hepatocellular carcinoma cells.
(2021) Nyahada, Marcilyn Rutendo.; Khan, Rene Bernadette.
Abstract available in PDF.

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