Hasil untuk "Cytology"

Valeria Bica, Veronica Venafra, Giorgia Massacci et al.

Abstract Background About 40% of relapsed or non-responder tumors exhibit therapeutic resistance in the absence of a clear genetic cause, suggesting a pivotal role of intracellular communication. A deeper understanding of signaling pathways rewiring occurring in resistant cells is crucial to propose alternative effective strategies for cancer patients. Methods To achieve this goal, we developed a novel multi-step strategy, which integrates high sensitive mass spectrometry-based phosphoproteomics with network-based analysis. This strategy builds context-specific networks recapitulating the signaling rewiring upon drug treatment in therapy-resistant and sensitive cells. Results We applied this strategy to elucidate the BCR::ABL1-independent mechanisms that drive relapse upon therapy discontinuation in chronic myeloid leukemia (CML) patients. We built a signaling map, detailing - from receptor to key phenotypes - the molecular mechanisms implicated in the control of proliferation, DNA damage response and inflammation of therapy-resistant cells. In-depth analysis of this map uncovered novel therapeutic vulnerabilities. Functional validation in patient-derived leukemic stem cells revealed a crucial role of acquired FLT3-dependency and its underlying molecular mechanism. Conclusions In conclusion, our study presents a novel generally applicable strategy and the reposition of FLT3, one of the most frequently mutated drivers of acute leukemia, as a potential therapeutic target for CML relapsed patients.

Swapnil Galat, Supreeta Nayak, Farheen Tadvi et al.

Introduction: Fine Needle Aspiration (FNA) cytology is a well-established essential, basic diagnostic technique for investigating lumps and lesions at various anatomic sites. While performing FNA of the breast lumps a quick or rapid result is desirable to know the nature of the lump and allay the anxiety of the patient. Rapid On-Site Evaluation (ROSE) is useful to evaluate the cytological smears within a few minutes. This study aims to evaluate the utility of Field’s stain in ROSE of breast lesions in arriving at a diagnosis and its comparison to routine MGG (May Grünwald Giemsa) stain. Materials and methods: This prospective observational study was conducted from July 2023 to December 2023 at a tertiary care center. A total of 89 cases with breast lumps were included. Previously diagnosed cases were excluded. All the cases were evaluated by ROSE using Field’s stain for preliminary diagnosis and compared later with MGG stain for final diagnosis. The categorizaƟon of cases was done as per IAC Yokohama System and further management was advised accordingly. Results: After first and repeat aspiration, 62 (69.6%) cases were categorized as benign, 12 (13.4%) cases were found to be malignant whereas 8 (8.9%), 5 (5.6%), 2 (2.24%) cases were included in the suspicious, atypical and insufficient categories respectively. Categorization of the breast lumps by ROSE using Field stain was in complete agreement with the MGG stained smears and the final FNA diagnosis and showed no discrepancy. Conclusion: The use of Field’s stain for ROSE of breast lumps had comparable staining quality to MGG which resulted in accurate correlaƟon between provisional and final cytologic diagnoses with the added advantage of rapid turnaround Ɵme. Field stain was hence found to be reliable, cost effecƟve and Ɵme saving when used for ROSE of breast lumps. Hence the use of this readily available and affordable stain for ROSE of FNA smears, especially of breast lumps, can prove to be highly beneficial for prompt categorization and triaging of patients. KEYWORDS: Rapid on site evaluation, Breast, Field Stain, Prompt Categorization, Triage

Gonzalo Arturo Medina Bueno, Deyné Maribel Ticona Ramos, Claudia Amparo Mares Cuadros et al.

The objective of this study was to determine the relationship between the prevalence of high-risk human papillomavirus (HRHPV) and age in women with cervical neoplasia or cervical cancer. This retrospective study involved 470 women referred for abnormal cervical cytology between January 2021 and December 2023. The Cobas 4800 test was used to identify HRHPV genotypes; it specifically identified genotypes 16 and 18 and grouped the other high-risk genotypes into another category. The Cobas 4800 test was performed together with colposcopy and biopsies of cervical lesions. From the analysis, we selected 470 women who underwent cervical biopsies and HPV testing. Of them, 208 (44.3%) were HPV-negative. Among the 262 women positive for HPV, 13.0% were positive for genotype 16 only, 1.3% for genotype 18 only, and 35.1% for other HPV genotypes. HPV-16 was found in 58.3% of cases of cervical intraepithelial neoplasia grade 3 (CIN 3) in women under 35 years of age and in 20.9% of cases in women over 35 years of age. Furthermore, 51.9% of patients with cervical cancer tested positive for other high-risk HPV types, whereas 30.8% had HPV-16. Although other HPV genotypes were more frequent than HPV-16 and HPV-18 in individuals with cervical cancer, HPV-16 was the most common individual high-risk genotype in women ≥ 35 years of age with CIN-3.

Aaron K. Olson, Wei Zhong Zhu, Dolena Ledee

Abstract Background The aging heart undergoes physiological changes, many of which are sex dependent and encompass differential responses to cardiac stress. However, much about the molecular changes that occur within the aging heart is still unknown. Thyroid hormone (TH) and the posttranslational modification O-GlcNAcylation (O-GlcNAc) are independently known to regulate cardiac function; therefore, we tested the hypothesis that TH disorders affect cardiac protein O-GlcNAcylation in aged hearts. Results We treated male and female 18–22 month-old aged C57BL/6 mice to create euthyroid, hypothyroid, or hyperthyroid states. Western blots and RT-qPCR from cardiac tissue were used to determine changes in global O-GlcNAc levels along with key regulatory proteins in the O-GlcNAcylation process. Immunoprecipitation and western blotting compared global O-GlcNAc changes to differences on an individual protein. We found increased total O-GlcNAc levels for female hypo- and hyperthyroid mice and male hyperthyroid mice compared to sex-matched euthyroid hearts, with no change for male hypothyroid mice. TH’s O-GlcNAc effect on female mice appears heart specific as liver O-GlcNAc levels were unchanged. The proteins regulating O-GlcNAcylation also demonstrated sex differences. Female hyperthyroid mice had increased protein expression of the O-GlcNAc regulatory proteins GFAT 1, GFAT 2, and OGT, whereas the hyperthyroid male mice showed decreased expression for the regulatory protein OGA. The hypothyroid female mice had increased protein expression for OGT and NAGK, whereas the hypothyroid male mice showed increased protein expression for NAGK alone. Interestingly, the directional changes in these protein levels did not match RNA transcription. We further found O-GlcNAc levels of the mitochondrial thiolase protein ACAA2 diverged from global O-GlcNAc changes. ACAA2 was hyper O-GlcNAcylated in the female hypothyroid group and hypo O-GlcNAcylated in the male hyperthyroid group whereas there was no change in female hyperthyroid or male hypothyroid. Conclusion Protein O-GlcNAcylation is potentially an important mechanism whereby TH perturbations affect the aged heart. We found sex influences O-GlcNAc regulation, global O-GlcNAc levels, and O-GlcNAc protein specificity in response to thyroid hormone perturbations. Our results also suggest the changes in cardiac O-GlcNAc levels are not solely due to TH transcriptional regulation of key O-GlcNAc regulatory enzymes.

Nicol Zielinska, Richard Shane Tubbs, Adrian Balcerzak et al.

The sartorius muscle belongs to the anterior compartment of the thigh. Morphological variations of this muscle are very rare, few cases being described in the literature. An 88-year-old female cadaver was dissected routinely for research and teaching purposes. However, an interesting variation was found during anatomical dissection. The proximal part of the sartorius muscle had the normal course, but the distal part bifurcated into two muscle bellies. The additional head passed medially to the standard head; thereafter, there was a muscular connection between them. This connection then passed into the tendinous distal attachment. It created a pes anserinus superficialis, which was located superficially to the distal attachments of the semitendinosus and gracilis muscles. This superficial layer was very wide and attached to the medial part of the tibial tuberosity and to the crural fascia. Importantly, two cutaneous branches of the saphenous nerve passed between the two heads. The two heads were innervated by separate muscular branches of the femoral nerve. Such morphological variability could be clinically important.

Carme Fàbrega, Núria Gallisà-Suñé, Alice Zuin et al.

The development of novel tools to tackle viral processes has become a central focus in global health, during the COVID-19 pandemic. The spike protein is currently one of the main SARS-CoV-2 targets, owing to its key roles in infectivity and virion formation. In this context, exploring innovative strategies to block the activity of essential factors of SARS-CoV-2, such as spike proteins, will strengthen the capacity to respond to current and future threats. In the present work, we developed and tested novel bispecific molecules that encompass: (i) oligonucleotide aptamers S901 and S702, which bind to the spike protein through its S1 domain, and (ii) hydrophobic tags, such as adamantane and tert-butyl-carbamate-based ligands. Hydrophobic tags have the capacity to trigger the degradation of targets recruited in the context of a proteolytic chimera by activating quality control pathways. We observed that S901-adamantyl conjugates promote the degradation of the S1 spike domain, stably expressed in human cells by genomic insertion. These results highlight the suitability of aptamers as target-recognition molecules and the robustness of protein quality control pathways triggered by hydrophobic signals, and place aptamer-Hytacs as promising tools for counteracting coronavirus progression in human cells.

Raymond Wightman

Movement of cellulose synthase particles have so far been observed on the plant epidermis that are amenable to confocal imaging, yielding appreciable signal and resolution to observe small plasma membrane-localised particles. Presented here is a method, using airyscan confocal microscopy, that permits similar information to be obtained at depth within the developing protoxylem vessels of intact roots.

Morteza Heidarzadeh, Amir Zarebkohan, Reza Rahbarghazi et al.

Abstract Recent advances in extracellular vesicle (EVs) detection and isolation methods have led to the development of novel therapeutic modalities. Among different types of EVs, exosomes (Exos) can transfer different signaling biomolecules and exhibit several superior features compared to whole-cell-based therapies. Therapeutic factors are normally loaded into the Exo lumen or attached to their surface for improving the on-target delivery rate and regenerative outcomes. Despite these advantages, there are several limitations in the application of Exos in in vivo conditions. It was suggested that a set of proteins and other biological compounds are adsorbed around Exos in aqueous phases and constitute an external layer named protein corona (PC). Studies have shown that PC can affect the physicochemical properties of synthetic and natural nanoparticles (NPs) after introduction in biofluids. Likewise, PC is generated around EVs, especially Exos in in vivo conditions. This review article is a preliminary attempt to address the interfering effects of PC on Exo bioactivity and therapeutic effects. Video Abstract Graphical Abstract

Mengqi Gu, Pengzheng Chen, Dongmei Zeng et al.

Abstract Background Foetal renal dysplasia is still the main cause of adult renal disease. Placenta-derived exosomes are an important communication tool, and they may play an important role in placental (both foetal and maternal) function. We hypothesize that in women with preeclampsia, foetal renal dysplasia is impeded by delivering placenta-derived exosomes to glomerular endothelial cells. Methods In the present study, we established a PE trophoblast oxidative stress model to isolate exosomes from supernatants by ultracentrifugation (NO-exo and H/R-exo) and collected normal and PE umbilical cord blood plasma to isolate exosomes by ultracentrifugation combined with sucrose density gradient centrifugation (N-exo and PE-exo), then we investigated their effects on foetal kidney development by in vitro, ex vivo and in vivo models. Results The PE trophoblast oxidative stress model was established successfully. After that, in in vitro studies, we found that H/R-exo and PE-exo could adversely affect glomerular endothelial cell proliferation, tubular formation, migration, and barrier functions. In ex vivo studies, H/R-exo and PE-exo both inhibited the growth and branch formation of kidney explants, along with the decrease of VE-cadherin and Occludin. In in vivo studies, we also found that H/R-exo and PE-exo could result in renal dysplasia, reduced glomerular number, and reduced barrier function in foetal mice. Conclusions In conclusion, we demonstrated that PE placenta-derived exosomes could lead to foetal renal dysplasia by delivering placenta-derived exosomes to foetal glomerular endothelial cells, which provides a novel understanding of the pathogenesis of foetal renal dysplasia. Video Abstract

Zhongyuan Liu, Dalong Xie, Hui Zhang

Filip Vujovic, Neil Hunter, Ramin M. Farahani

Abstract Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in β-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. Video abstract

Ji Yoon Kim, Hayoung Cho, Jung Yoo et al.

Histone deacetylase 8 (HDAC8) is a class I HDAC that catalyzes the deacetylation of histone and non-histone proteins. As one of the best-characterized isoforms, numerous studies have identified interacting partners of HDAC8 pertaining to diverse molecular mechanisms. Consequently, deregulation and overexpression of HDAC8 give rise to diseases. HDAC8 is especially involved in various aspects of cancer progression, such as cancer cell proliferation, metastasis, immune evasion, and drug resistance. HDAC8 is also associated with the development of non-cancer diseases such as Cornelia de Lange Syndrome (CdLS), infectious diseases, cardiovascular diseases, pulmonary diseases, and myopathy. Therefore, HDAC8 is an attractive therapeutic target and various HDAC8 selective inhibitors (HDAC8is) have been developed. Here, we address the pathological function of HDAC8 in cancer and other diseases, as well as illustrate several HDAC8is that have shown anti-cancer effects.

Shuiting Zhang, Chao Liu, Guo Li et al.

Abstract Background Squamous cell carcinoma of the head and neck (SCCHN) is one of the most common types of cancer that cause a substantial number of cancer-related deaths. Our previous study has revealed that makorin ring finger protein 3 (MKRN3) may act as a key regulator of the SCCHN tumorigenesis; however, its specific role in SCCHN progression has not been reported. Methods The Cancer Genome Atlas (TCGA) data analysis and quantitative polymerase chain reaction (qPCR) were used to quantify the MKRN3 mRNA expression levels in SCCHN; immunohistochemical staining or immunoblotting analyses were performed to detect MKRN3 protein expression. Kaplan–Meier plotter was used to assess the prognostic values of MKRN3 in terms of overall survival and disease-free survival. The expression differences based on various clinicopathological features were evaluated using subgroup analysis and forest map analysis. The regulatory mechanism of MKRN3 was further investigated using gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Subsequently, STRING was used to perform a co-expression and enrichment analysis for MKRN3. Homologous modeling, molecular docking, and western blot analyses were performed to investigate the relationship between MKRN3 and its potential target gene P53. Results MKRN3 was ectopically expressed between cancerous and noncancerous SCCHN tissues, and its expression level was tightly associated with high T classifications as well as advanced clinical stages. qPCR analysis revealed that MKRN3 was upregulated in the SCCHN cell line. Moreover, Kaplan–Meier and Cox regression analyses indicated that SCCHN patients with high MKRN3 expression had poorer prognosis and that MKRN3 was a potential prognostic marker for SCCHN. Using gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses, we determined that MKRN3 may be involved in the regulation of synthesis and metabolism and cell growth, death and motility, as well as cancer pathways associated with SCCHN progression. Mechanism investigation further revealed that P53, a potential target of MKRN3, may be involved in the SCCHN tumorigenesis mediated by MKRN3. Conclusions We performed a comprehensive evaluation of the clinical significance of MKRN3 and explored its underlying mechanisms. We concluded that MKRN3 represents a valuable predictive biomarker and potential therapeutic target in SCCHN.

Annina S. Vischer, Gabriela M. Kuster, Raphael Twerenbold et al.

(1) Background: Recently, influences of antihypertensive treatment on the renin–angiotensin–aldosterone system (RAAS) has gained attention, regarding a possible influence on inflammatory and anti-inflammatory pathways. We aimed to study the effects of newly initiated antihypertensive drugs on angiotensin (Ang) II and Ang (1–7) as representers of two counter-regulatory axes. (2) Methods: In this randomized, open-label trial investigating RAAS peptides after the initiation of perindopril, olmesartan, amlodipine, or hydrochlorothiazide, Ang II and Ang (1–7) equilibrium concentrations were measured at 8 a.m. and 12 a.m. at baseline and after four weeks of treatment. Eighty patients were randomized (1:1:1:1 fashion). (3) Results: Between the four substances, we found significant differences regarding the concentrations of Ang II (<i>p</i> < 0.0005 for 8 a.m., 12 a.m.) and Ang (1–7) (<i>p</i> = 0.019 for 8 a.m., <0.0005 for 12 a.m.) four weeks after treatment start. Ang II was decreased by perindopril (<i>p</i> = 0.002), and increased by olmesartan (<i>p</i> < 0.0005), amlodipine (<i>p</i> = 0.012), and hydrochlorothiazide (<i>p</i> = 0.001). Ang (1–7) was increased by perindopril and olmesartan (<i>p</i> = 0.008/0.002), but not measurably altered by amlodipine and hydrochlorothiazide (<i>p</i> = 0.317/ 0.109). (4) Conclusion: The initiation of all first line antihypertensive treatments causes early and distinct alterations of equilibrium angiotensin levels. Given the additional AT1R blocking action of olmesartan, RAAS peptides shift upon initiation of perindopril and olmesartan appear to work in favor of the anti-inflammatory axis compared to amlodipine and hydrochlorothiazide.

Jonas Cornelius, Klemens Rottner, Martin Korte et al.

Postsynaptic structures on excitatory neurons, dendritic spines, are actin-rich. It is well known that actin-binding proteins regulate actin dynamics and by this means orchestrate structural plasticity during the development of the brain, as well as synaptic plasticity mediating learning and memory processes. The actin-binding protein cortactin is localized to pre- and postsynaptic structures and translocates in a stimulus-dependent manner between spines and the dendritic compartment, thereby indicating a crucial role for synaptic plasticity and neuronal function. While it is known that cortactin directly binds F-actin, the Arp2/3 complex important for actin nucleation and branching as well as other factors involved in synaptic plasticity processes, its precise role in modulating actin remodeling in neurons needs to be deciphered. In this study, we characterized the general neuronal function of cortactin in knockout mice. Interestingly, we found that the loss of cortactin leads to deficits in hippocampus-dependent spatial memory formation. This impairment is correlated with a prominent dysregulation of functional and structural plasticity. Additional evidence shows impaired long-term potentiation in cortactin knockout mice together with a complete absence of structural spine plasticity. These phenotypes might at least in part be explained by alterations in the activity-dependent modulation of synaptic actin in cortactin-deficient neurons.

Z. Yang, L. Fan, K. Kwon et al.

BACKGROUND: The aim of this study was to investigate the association between chronological age and the pulp/tooth volume ratio (PTR) of specific teeth using cone-beam computed tomography (CBCT) enhanced with Materialise-Mimics Research software 21.0 in children and young adult population from Eastern China. MATERIALS AND METHODS: CBCT scans of 230 patients (119 males, 111 females), aged 8.18–19.92 years were analysed by two well-trained examiners in this retrospective study. The intraclass correlation coefficient value was calculated to test the intra- and inter-examiner agreement. The volumetric analysis of the pulp and calcified tissues was performed on the maxillary left central incisors and canines. The correlation and regression analyses were then performed. RESULTS: The Pearson correlation analysis showed a strong coefficient of correlation (r) for maxillary left canines (–0.81 for girls and –0.88 for boys) as compared to central incisors (–0.63 for girls and –0.70 for boys). Regarding performance, the canine model was more powerful than the central incisor model. The derived regression equation from maxillary left canines had high coefficients of determination (Age = 21.979 – 105.42 × PTR, R2 = 0.69). CONCLUSIONS: Our study proved that the PTR value of canines had a negative correlation relationship with a subadult’s chronological age and volumetric analysis of CBCT scans using the software may become an efficient method to estimate the chronological age of children and young adults.

Simona-Rebeca Ignat, Sorina Dinescu, Anca Hermenean et al.

Inflammation has been known to be an important driver of fibrogenesis in the liver and onset of hepatic fibrosis. It starts off as a process meant to protect the liver from further damage, but it can become the main promoter of liver fibrosis. There are many inflammation-related pathways activated during liver fibrosis that lead to hepatic stellate cells (HSCs) activation and collagen-deposition in the liver. Such events are mostly modulated upstream of HSCs and involve signals from hepatocytes and innate immune cells. One particular event is represented by cell death during liver injury that generates multiple inflammatory signals that further trigger sterile inflammation and enhancement of inflammatory response. The assembly of inflammasome that responds to danger-associated molecular patterns (DAMPs) stimulates the release of pro-inflammatory cytokines and at the same time, initiates programmed cell death called pyroptosis. This review focuses on cellular and molecular mechanisms responsible for initiation and progress of inflammation in the liver.

Jingjing Duan, Haiyang Zhang, Shuang Li et al.

Abstract Background Cancers can survive the oxidative conditions by upregulating nucleoside diphosphate linked moiety X-type motif 1 (NUDT1). However, the mechanisms underlying gastric carcinogenesis and the dys-regulation of NUDT1 in gastric cancer (GC) remain unknown. Our study aimed to explore the role of NUDT1 and its regulatory pathway by miR-485-5p in GC. Methods Gastric cancer tissues and paired noncancerous tissue samples were collected, and the expression level of NUDT1 and miR-485-5p were detected. Two cohorts from The Cancer Genome Atlas (TCGA) database and another cohort from the Tianjin Medical University Cancer Institute and Hospital were further analyzed. Luciferase assays were performed, and the effects of the miR-485-5p/NUDT1 axis on GC cells and normal gastric cells were determined by subsequent experiments. Results We found that the expression of miR-485-5p was clearly repressed in GC tissues, while NUDT1 expression level was dramatically increased. The overexpression of NUDT1 correlated closely with an increase in invasive depth and a decrease in survival in GC patients. MiR-485-5p could directly bind to the 3′UTR of NUDT1 mRNA and induce its degradation, thus down-regulate its expression. The miR-485-5p/NUDT1 axis could lead to the changes of 8-oxo-dG in GC cells. And the increased expression of NUDT1 resulting from the downregulation of miR-485-5p could accelerate cell proliferation and metastasis in GC. However, the growth and migration of normal gastric cells did not depend on the protection of NUDT1, while the overexpression of NUDT1 could promote malignant transition in normal gastric cells. Conclusions MiR-485-5p acts as a tumor suppressor by targeting NUDT1 in GC. The miR-485-5p/NUDT1 axis is involved in the processes of cell growth and cell motility and plays a key role in the tumorigenesis of GC.

O. D. Rymar

Vladimir A. Zhukov, Alexander I. Zhernakov, Olga A. Kulaeva et al.

The large size and complexity of the garden pea (Pisum sativum L.) genome hamper its sequencing and the discovery of pea gene resources. Although transcriptome sequencing provides extensive information about expressed genes, some tissue-specific transcripts can only be identified from particular organs under appropriate conditions. In this study, we performed RNA sequencing of polyadenylated transcripts from young pea nodules and root tips on an Illumina GAIIx system, followed by de novo transcriptome assembly using the Trinity program. We obtained more than 58,000 and 37,000 contigs from “Nodules” and “Root Tips” assemblies, respectively. The quality of the assemblies was assessed by comparison with pea expressed sequence tags and transcriptome sequencing project data available from NCBI website. The “Nodules” assembly was compared with the “Root Tips” assembly and with pea transcriptome sequencing data from projects indicating tissue specificity. As a result, approximately 13,000 nodule-specific contigs were found and annotated by alignment to known plant protein-coding sequences and by Gene Ontology searching. Of these, 581 sequences were found to possess full CDSs and could thus be considered as novel nodule-specific transcripts of pea. The information about pea nodule-specific gene sequences can be applied for gene-based markers creation, polymorphism studies, and real-time PCR.