Tyrosine kinase inhibitor insensitivity of non-cycling CD34+ human acute myeloid leukaemia cells with FMS-like tyrosine kinase 3 mutations.

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Improved clonality assessment in germinal centre/post-germinal centre non-Hodgkin's lymphomas with high rates of somatic hypermutation.

BACKGROUND: PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR. AIMS: To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies. METHODS: DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig kappa/lambda rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1. RESULTS: Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5-13.5)) and FL (median (range) 5.3% (2.3-11.9)) with a clonal rearrangement. CONCLUSIONS: Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.

BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was

A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer

INTRODUCTION: The presence of ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) rearrangements in lung cancers confers sensitivity to ROS kinase inhibitors, including crizotinib. However, they are rare abnormalities (in ∼1% of non-small cell lung carcinomas) that are typically identified by fluorescence in situ hybridization (FISH), and so screening using immunohistochemical (IHC) staining would be both cost- and time-efficient.

METHODS: A cohort of lung tumors negative for other common mutations related to targeted therapies were screened to assess the sensitivity and specificity of IHC staining in detecting ROS1 gene rearrangements, enriched by four other cases first identified by FISH. A review of published data was also undertaken.

RESULTS: IHC staining was 100% sensitive (95% confidence interval: 48-100) and 83% specific (95% confidence interval: 86-100) overall when an h-score higher than 100 was used. Patients with ROS1 gene rearrangements were younger and typically never-smokers, with the tumors all being adenocarcinomas with higher-grade architectural features and focal signet ring morphologic features (two of five). Four patients treated with crizotinib showed a partial response, with three also showing a partial response to pemetrexed. Three of four patients remain alive at 13, 27, and 31 months, respectively.

CONCLUSION: IHC staining can be used to screen for ROS1 gene rearrangements, with patients herein showing a response to crizotinib. Patients with tumors that test positive according to IHC staining but negative according to FISH were also identified, which may have implications for treatment selection.

Rheology and Microfluidic flows of a cationic surfactant and hydrotropic salt mixture

Thesis (Ph.D.)--University of Washington, 2016-08

Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency, and the severity of the resulting clinical symptoms varies according to the level of 21-hydroxylase activity. 21-Hydroxylase deficiency is usually caused by mutations in the CYP21A2 gene, which is located on the RCCX module, a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements, such as gene duplications, gross deletions and gene conversions of variable extensions. Molecular genotyping of CYP21A2 and the RCCX module has proved useful for a more accurate diagnosis of the disease, and prenatal diagnosis. This article summarises the clinical features of 21-hydroxylase deficiency, explains current understanding of the disease at the molecular level, and highlights recent developments, particularly in diagnosis.

Guided Migration and Collective Behavior: Cell Dynamics during Cancer Progression

This dissertation focuses on gaining understanding of cell migration and collective behavior through a combination of experiment, analysis, and modeling techniques. Cell migration is a ubiquitous process that plays an important role during embryonic development and wound healing as well as in diseases like cancer, which is a particular focus of this work. As cancer cells become increasingly malignant, they acquire the ability to migrate away from the primary tumor and spread throughout the body to form metastatic tumors. During this process, changes in gene expression and the surrounding tumor environment can lead to changes in cell migration characteristics. In this thesis, I analyze how cells are guided by the texture of their environment and how cells cooperate with their neighbors to move collectively. The emergent properties of collectively moving groups are a particular focus of this work as collective cell dynamics are known to change in diseases such as cancer. The internal machinery for cell migration involves polymerization of the actin cytoskeleton to create protrusions that---in coordination with retraction of the rear of the cell---lead to cell motion. This actin machinery has been previously shown to respond to the topography of the surrounding surface, leading to guided migration of amoeboid cells. Here we show that epithelial cells on nanoscale ridge structures also show changes in the morphology of their cytoskeletons; actin is found to align with the ridge structures. The migration of the cells is also guided preferentially along the ridge length. These ridge structures are on length scales similar to those found in tumor microenvironments and as such provide a system for studying the response of the cells' internal migration machinery to physiologically relevant topographical cues. In addition to sensing surface topography, individual cells can also be influenced by the pushing and pulling of neighboring cells. The emergent properties of collectively migrating cells show interesting dynamics and are relevant for cancer progression, but have been less studied than the motion of individual cells. We use Particle Image Velocimetry (PIV) to extract the motion of a collectively migrating cell sheet from time lapse images. The resulting flow fields allow us to analyze collective behavior over multiple length and time scales. To analyze the connection between individual cell properties and collective migration behavior, we compare experimental flow fields with the migration of simulated cell groups. Our collective migration metrics allow for a quantitative comparison between experimental and simulated results. This comparison shows that tissue-scale decreases in collective behavior can result from changes in individual cell activity without the need to postulate the existence of subpopulations of leader cells or global gradients. In addition to tissue-scale trends in collective behavior, the migration of cell groups includes localized dynamic features such as cell rearrangements. An individual cell may smoothly follow the motion of its neighbors (affine motion) or move in a more individualistic manner (non-affine motion). By decomposing individual motion into both affine and non-affine components, we measure cell rearrangements within a collective sheet. Finally, finite-time Lyapunov exponent (FTLE) values capture the stretching of the flow field and reflect its chaotic character. Applying collective migration analysis techniques to experimental data on both malignant and non-malignant human breast epithelial cells reveals differences in collective behavior that are not found from analyzing migration speeds alone. Non-malignant cells show increased cooperative motion on long time scales whereas malignant cells remain uncooperative as time progresses. Combining multiple analysis techniques also shows that these two cell types differ in their response to a perturbation of cell-cell adhesion through the molecule E-cadherin. Non-malignant MCF10A cells use E-cadherin for short time coordination of collective motion, yet even with decreased E-cadherin expression, the cells remain coordinated over long time scales. In contrast, the migration behavior of malignant and invasive MCF10CA1a cells, which already shows decreased collective dynamics on both time scales, is insensitive to the change in E-cadherin expression.

Managerial experiences related to corporate demerger

In the current economic situation very few business operations are static. In some cases, organizations feel that the most rational solution is not expanding but focusing on their core competencies or certain areas of business through corporate restructuring. The focus in this thesis is on corporate demerger, a process that involves a complete separation of units within a company. Despite the vast amount of research considering corporate demergers, there is still a lack of knowledge about how top managers experience these kinds of organizational rearrangements. In this qualitative case study, the aim is to bring forward the thoughts of executives in two companies that emerged from a single company through corporate demerger a few years ago. By conducting dyadic semi-structured interviews with the management group and board members in both the parent company and the demerger company, I pursue to understand the impressions that they had during the demerger process as well as during the change that followed afterwards. Additionally, their retrospective sense-making patterns are briefly examined. The findings suggest that the way operations are managed before plays an important role in the demerger process. In this case the pre-demerger organization was quite distinctly divided into two business areas, which allowed a somewhat natural allocation of resources. In addition, apart from the top management, every employee knew their future company and role, thus decreasing the amount of uncertainty in lower organizational levels. Consequently, the ambiguity and change faced by the top executives was evidently more powerful compared to the operational actors. The fragmented characteristics also enabled certain points of reference in the unfolding demerger process. Along with the information about future management groups, the pre-demerger re-grouping took place. This sparked up the mental division, where both groups started to act separately. On a personal level, the managers involved in the demerger planning have made retrospective sense of the repercussions related to the restructuring process. For them, it was an extremely demanding, ambiguous and burdensome project that provided them with managerial experiences they are likely not to have again.

Temporal Control of Ion Channel Activation

Ion channels are a large class of integral membrane proteins that allow for the diffusion of ions across a cellular membrane and are found in all forms of life. Pentameric ligand-gated ion channels (pLGICs) comprise a large family of proteins that include the nicotinic acetylcholine receptor (nAChR) and the γ-aminobutyric acid (GABA) receptor. These ion channels are responsible for the fast synaptic transmission that occurs in humans and as a result are of fundamental biological importance. pLGICs bind ligands (neurotransmitters), and upon ligand-binding undergo activation. The activation event causes an ion channel to enter a new physical state that is able to conduct ions. Ion channels allow for the flux of ions across the membrane through a pore that is formed upon ion channel activation. For pLGICs to function properly both ligand-binding and ion channel activation must occur. The ligand-binding event has been studied extensively over the past few decades, and a detailed mechanism of binding has emerged. During activation the ion channel must undergo structural rearrangements that allow the protein to enter a conformation in which ions can flow through. Despite this great and ubiquitous importance, a fundamental understanding of the ion channel activation mechanism and kinetics, as well as concomitant structural arrangements, remains elusive.

This dissertation describes efforts that have been made to temporally control the activation of ligand-gated ion channels. Temporal control of ion channel activation provides a means by which to activate ion channels when desired. The majority of this work examines the use of light to activate ion channels. Several photocages were examined in this thesis; photocages are molecules that release a ligand under irradiation, and, for the work described here, the released ligand then activates the ion channel. First, a new water-soluble photoacid was developed for the activation of proton-sensitive ion channels. Activation of acid-sensing ion channels, ASIC2a and GLIC, was observed only upon irradiation. Next, a variety of Ru²⁺ photocages were also developed for the release of amine ligands. The Ru²⁺ systems interacted in a deleterious manner with a representative subset of biologically essential ion channels. The rapid mixing of ion channels with agonist was also examined. A detection system was built to monitor ion channels activation in the rapid mixing experiments. I have shown that liposomes, and functionally-reconstituted ELIC, are not destroyed during the mixing process. The work presented here provides the means to deliver agonist to ligand-gated ion channels in a controlled fashion.

Guided Migration and Collective Behavior: Cell Dynamics during Cancer Progression

This dissertation focuses on gaining understanding of cell migration and collective behavior through a combination of experiment, analysis, and modeling techniques. Cell migration is a ubiquitous process that plays an important role during embryonic development and wound healing as well as in diseases like cancer, which is a particular focus of this work. As cancer cells become increasingly malignant, they acquire the ability to migrate away from the primary tumor and spread throughout the body to form metastatic tumors. During this process, changes in gene expression and the surrounding tumor environment can lead to changes in cell migration characteristics. In this thesis, I analyze how cells are guided by the texture of their environment and how cells cooperate with their neighbors to move collectively. The emergent properties of collectively moving groups are a particular focus of this work as collective cell dynamics are known to change in diseases such as cancer. The internal machinery for cell migration involves polymerization of the actin cytoskeleton to create protrusions that---in coordination with retraction of the rear of the cell---lead to cell motion. This actin machinery has been previously shown to respond to the topography of the surrounding surface, leading to guided migration of amoeboid cells. Here we show that epithelial cells on nanoscale ridge structures also show changes in the morphology of their cytoskeletons; actin is found to align with the ridge structures. The migration of the cells is also guided preferentially along the ridge length. These ridge structures are on length scales similar to those found in tumor microenvironments and as such provide a system for studying the response of the cells' internal migration machinery to physiologically relevant topographical cues. In addition to sensing surface topography, individual cells can also be influenced by the pushing and pulling of neighboring cells. The emergent properties of collectively migrating cells show interesting dynamics and are relevant for cancer progression, but have been less studied than the motion of individual cells. We use Particle Image Velocimetry (PIV) to extract the motion of a collectively migrating cell sheet from time lapse images. The resulting flow fields allow us to analyze collective behavior over multiple length and time scales. To analyze the connection between individual cell properties and collective migration behavior, we compare experimental flow fields with the migration of simulated cell groups. Our collective migration metrics allow for a quantitative comparison between experimental and simulated results. This comparison shows that tissue-scale decreases in collective behavior can result from changes in individual cell activity without the need to postulate the existence of subpopulations of leader cells or global gradients. In addition to tissue-scale trends in collective behavior, the migration of cell groups includes localized dynamic features such as cell rearrangements. An individual cell may smoothly follow the motion of its neighbors (affine motion) or move in a more individualistic manner (non-affine motion). By decomposing individual motion into both affine and non-affine components, we measure cell rearrangements within a collective sheet. Finally, finite-time Lyapunov exponent (FTLE) values capture the stretching of the flow field and reflect its chaotic character. Applying collective migration analysis techniques to experimental data on both malignant and non-malignant human breast epithelial cells reveals differences in collective behavior that are not found from analyzing migration speeds alone. Non-malignant cells show increased cooperative motion on long time scales whereas malignant cells remain uncooperative as time progresses. Combining multiple analysis techniques also shows that these two cell types differ in their response to a perturbation of cell-cell adhesion through the molecule E-cadherin. Non-malignant MCF10A cells use E-cadherin for short time coordination of collective motion, yet even with decreased E-cadherin expression, the cells remain coordinated over long time scales. In contrast, the migration behavior of malignant and invasive MCF10CA1a cells, which already shows decreased collective dynamics on both time scales, is insensitive to the change in E-cadherin expression.

Karyotype and genome size in Euterpe Mart. (Arecaceae) species.

Euterpe (Martius, 1823), a genus from Central and South America, has species with high economic importance in Brazil, because of their palm heart and fruits, known as açaí berries. Breeding programs have been conducted to increase yield and establish cultivation systems to replace the extraction of wild material. These programs need basic information about the genome of these species to better explore the available genetic variability. The aim of this study was to compare E. edulis (Martius, 1824), E. oleracea (Martius, 1824) and E. precatoria (Martius, 1842), with regard to karyotype, type of interphase nucleus and nuclear DNA amount. Metaphase chromosomes and interphase nuclei from root tip meristematic cells were obtained by the squashing technique and solid stained for microscope analysis. The DNA amount was estimated by flow cytometry. There were previous reports on the chromosome number of E. edulis and E. oleracea, but chromosome morphology of these two species and the whole karyotype of E. precatoria are reported for the first time. The species have 2n=36, a number considered as a pleisomorphic feature in Arecoideae since the modern species, according to floral morphology, have the lowest chromosome number (2n=28 and 2n=30). The three Euterpe species also have the same type of interphase nuclei, classified as semi-reticulate. The species differed on karyotypic formulas, on localization of secondary constriction and genome size. The data suggest that the main forces driving Euterpe karyotype evolution were structural rearrangements, such as inversions and translocations that alter chromosome morphology, and either deletion or amplification that led to changes in chromosome size.

An updated evolutionary classification of CRISPR–Cas systems

The evolution of CRISPR–cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR–cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

Mitigating effects of Jambul against lead induced toxicity in epididymis and vas deferens of mice

Background: Precious fruits like jambul are neglected and wasted while environmental pollutants like lead intake remain overlooked. Objective: The aim of this study was to investigate the effects of the Jambul pulp extract on lead detrimental effects in pseudostratified epithelium and the stereocilia of mice epididymis and vas deferens. Materials and Methods: Thirty young males mice (Mus musculus) were distributed randomly in 3 groups (n= 10) called control, Pb (Lead) and Pb-J (Lead-Jambul). The Pb and Pb-J were provided 50ppm Pb in drinking water ad libitum for 15 days and Pb free water for the next 5 days. The Pb-J group received 0.2ml jambul pulp extract on 12 hourly bases. Control group was not given any treatment. Organs (epididymis and vas deference) were recovered on 21st day after euthanasia. The organs were finally processed for histological and micrometric studies. Results: Marked histologic and micrometric changes in both organs were noted in Pb group. These include significant (P ≤ 0.05) decrease in cross sectional area of caput and cauda epididymis folding tubing along with evident alterations of their endothelial thickness. Prominent signs of apoptosis (vacuolations) in the corpus pseudostratified endothelium and the destruction of stereocilia of the epididymis and vas deferens in Pb compared to control group were observed. Evident signs of recovery, in both organs, such as proliferation and rearrangements in pseudostratified endothelium and the stereocilia along with convincing recovery in micrometric parameters were observed in Pb-J group. Conclusion: The results indicate that epididymis and vas deferens are highly sensitive to Pb exposure while Jambul pulp extract has shown rich mitigating potentials against such histopathologies.