CT Method Research Articles

Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Leptocybe invasa Fisher and La Salle (Hymenoptera: Eulophidae)

Leptocybe invasa (Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression of L. invasa's target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes' stability (RPS30, ACTR, 18S rRNA, ACT, RPL18, GAPDH, 28S rRNA, and TUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water, Eucalyptus sap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied that ACT and ACTR were the most accurate when comparing sexes. For analyzing different somites, 28S rRNA and RPL18 were ideal; the 28S rRNA and RRS30 were perfect for analyzing at different temperatures. The combination of ACT and GAPDH helped to analyze gene expression in different diets, and GAPDH and 28S rRNA were suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes from L. invasa for precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.

Palaeoenvironmental and palaeoclimatic inferences based on X-ray computer tomography: a case study of alkaline lake deposits in Hungary

Widely distributed freshwater carbonate sediments, i.e., limestone, dolomitic limestone and dolomite, developed in inter-dune alkaline ponds of the Danube-Tisza Interfluve in the centre of the Carpathian Basin during the Holocene. The key parameters that determine the formation of any given type of carbonate mineral (calcite, dolomite) are temperature, evaporation rate, pH and ion concentrations, in addition to CO2 absorption by aquatic plants. CT analysis is capable of recording small-scale density variations attributable to compositional differences of sedimentary rocks. As the type and proportion of rock-forming minerals and other components is an artifact of past environmental and climatic conditions, CT values may act as potential palaeoenvironmental proxies. The present study compares variations in rock-forming components obtained for freshwater carbonates utilizing the CT method with already available geochemical and palaeoecological proxy data. Variations in molluscan ecology and isotope geochemistry, sedimentation times and CTbased rock density values all indicate the relevance of millennial-scale, climate-driven changes in carbonate formation. As previously observed, the emergence of colder conditions in the North Atlantic, which resulted in increased cyclonic activity and heavier rainfall in western Europe and the Danube watershed area between 10.3 and 9.3 kyr cal BP, resulted in the emergence of humid conditions favouring a rise in the groundwater table at our site and precipitation of calcite from pore waters as opposed to high-magnesium calcite. This is clearly reflected in a negative shift in CT density values in our dated rock samples.

Cervical spine trauma – Evaluating the diagnostic power of CT, MRI, X-Ray and LODOX

BackgroundTraumatic cervical spine (c-spine) injuries account for 10% of all spinal injuries. The c-spine is prone to injury by blunt acceleration/deceleration traumas. The Canadian C-Spine rule and NEXUS criteria guide clinical decision-making but lack consensus on imaging modality when necessary. This study aims to evaluate the sensitivity and specificity of CT, MRI, X-Ray, and, for the first time, LODOX-Statscan in identifying c-spine injuries in patients with blunt trauma and neck pain. MethodsWe conducted a retrospective monocenter cohort study using patient data from the emergency department at Inselspital, Bern, Switzerland's largest level one trauma center. We identified patients presenting with trauma and neck pain during the recruitment period from 01.01.2012 to 31.12.2017. We included all patients that required a radiographic c-spine evaluation according to the NEXUS criteria. Certified spine surgeons reviewed each case, analyzed patient demographics, injury classification, trauma mechanism, and emergency management. The retrospective full case review was established as gold standard to decide whether the c-spine was injured. Sensitivity and specificity were calculated for CT, MRI, LODOX, and X-Ray imaging methods. ResultsWe identified 4996 patients, of which 2321 met the inclusion criteria. 91.3% (n = 2120) patients received a CT scan, 8.9% (n = 206) a MRI, 9.3% (n = 215) an X-ray, and 21.5% (n = 498) a LODOX scan. By retrospective case review, 186 participants were classified as injured. The sensitivity of CT was 88.6% (specificity 99%), and 89.8% (specificity 99.2%) with orthopedic surgeon consultation. MRI had a sensitivity of 88.5% (specificity of 96.9%); highlighting 14 cases correctly diagnosed as injured by MRI and misdiagnosed by CT. Projection radiography (36.4% sensitivity, 95.1% specificity) and LODOX (5.3% sensitivity, 100% specificity) were unsuitable for ruling out spinal injury. ConclusionWhile CT offers high sensitivity for detecting traumatic c-spine injury, MRI holds clinical significance in revealing injuries not recognized by CT in symptomatic patients. LODOX and projection radiography are insufficient for accurately ruling out c-spine injury. For patients with neurological symptoms, we recommend extended MRI use when CT scans are negative.

Image-Guided Proton Therapy: A Comprehensive Review.

Image guidance for radiation therapy can improve the accuracy of the delivery of radiation, leading to an improved therapeutic ratio. Proton radiation is able to deliver a highly conformal dose to a target due to its advantageous dosimetric properties, including the Bragg peak. Proton therapy established the standard for daily image guidance as a means of minimizing uncertainties associated with proton treatment. With the increasing adoption of the use of proton therapy over time, image guidance systems for this modality have been changing. The unique properties of proton radiation present a number of differences in image guidance from photon therapy. This paper describes CT and MRI-based simulation and methods of daily image guidance. Developments in dose-guided radiation, upright treatment, and FLASH RT are discussed as well.

Implant Imaging: Perspectives of Nuclear Imaging in Implant, Biomaterial, and Stem Cell Research.

Until now, very few efforts have been made to specifically trace, monitor, and visualize implantations, artificial organs, and bioengineered scaffolds for tissue engineering in vivo. While mainly X-Ray, CT, and MRI methods have been used for this purpose, the applications of more sensitive, quantitative, specific, radiotracer-based nuclear imaging techniques remain a challenge. As the need for biomaterials increases, so does the need for research tools to evaluate host responses. PET (positron emission tomography) and SPECT (single photon emission computer tomography) techniques are promising tools for the clinical translation of such regenerative medicine and tissue engineering efforts. These tracer-based methods offer unique and inevitable support, providing specific, quantitative, visual, non-invasive feedback on implanted biomaterials, devices, or transplanted cells. PET and SPECT can improve and accelerate these studies through biocompatibility, inertivity, and immune-response evaluations over long investigational periods at high sensitivities with low limits of detection. The wide range of radiopharmaceuticals, the newly developed specific bacteria, and the inflammation of specific or fibrosis-specific tracers as well as labeled individual nanomaterials can represent new, valuable tools for implant research. This review aims to summarize the opportunities of nuclear-imaging-supported implant research, including bone, fibrosis, bacteria, nanoparticle, and cell imaging, as well as the latest cutting-edge pretargeting methods.

MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Investigation of Efflux Pump Genes in Resistant Mycobacterium tuberculosis Complex Clinical Isolates Exposed to First Line Antituberculosis Drugs and Verapamil Combination

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRt‑PCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 μg/mL, 1-128 μg/mL, 2-32 μg/mL, 4-16 μg/mL and 15.62-250 μg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.

AXONAL GUIDANCE SIGNALLING IS REGULATED BY MECHANICAL LOADING AND INFLAMMATION IN A 3D MODEL OF HUMAN STEM CELL-DERIVED OSTEOCYTES

Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception.Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception.Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test.All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation.Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression.

Selecting Normalizers for MicroRNA RT-qPCR Expression Analysis in Murine Preimplantation Embryos and the Associated Conditioned Culture Media

Normalizing RT-qPCR miRNA datasets that encompass numerous preimplantation embryo stages requires the identification of miRNAs that may be used as stable reference genes. A need has also arisen for the normalization of the accompanying conditioned culture media as extracellular miRNAs may serve as biomarkers of embryo developmental competence. Here, we evaluate the stability of six commonly used miRNA normalization candidates, as well as small nuclear U6, using five different means of evaluation (BestKeeper, NormFinder, geNorm, the comparative Delta Ct method and RefFinder comprehensive analysis) to assess their stability throughout murine preimplantation embryo development from the oocyte to the late blastocyst stages, both in whole embryos and the associated conditioned culture media. In descending order of effectiveness, miR-16, miR-191 and miR-106 were identified as the most stable individual reference miRNAs for developing whole CD1 murine preimplantation embryos, while miR-16, miR-106 and miR-103 were ideal for the conditioned culture media. Notably, the widely used U6 reference was among the least appropriate for normalizing both whole embryo and conditioned media miRNA datasets. Incorporating multiple reference miRNAs into the normalization basis via a geometric mean was deemed beneficial, and combinations of each set of stable miRNAs are further recommended, pending validation on a per experiment basis.

A Novel Artificial Intelligence Based Denoising Method for Ultra-Low Dose CT Used for Lung Cancer Screening

To assess ultra-low-dose (ULD) computed tomography as well as a novel artificial intelligence-based reconstruction denoising method for ULD (dULD) in screening for lung cancer. This prospective study included 123 patients, 84 (70.6%) men, mean age 62.6 ± 5.35 (55-75), who had a low dose and an ULD scan. A fully convolutional-network, trained using a unique perceptual loss was used for denoising. The network used for the extraction of the perceptual features was trained in an unsupervised manner on the data itself by denoising stacked auto-encoders. The perceptual features were a combination of feature maps taken from different layers of the network, instead of using a single layer for training. Two readers independently reviewed all sets of images. ULD decreased average radiation-dose by 76% (48%-85%). When comparing negative and actionable Lung-RADS categories, there was no difference between dULD and LD (p=0.22 RE, p > 0.999 RR) nor between ULD and LD scans (p=0.75 RE, p > 0.999 RR). ULD negative likelihood ratio (LR) for the readers was 0.033-0.097. dULD performed better with a negative LR of 0.021-0.051. Coronary artery calcifications (CAC) were documented on the dULD scan in 88(74%) and 81(68%) patients, and on the ULD in 74(62.2%) and 77(64.7%) patients. The dULD demonstrated high sensitivity, 93.9%-97.6%, with an accuracy of 91.7%. An almost perfect agreement between readers was noted for CAC scores: for LD (ICC=0.924), dULD (ICC=0.903), and for ULD (ICC=0.817) scans. A novel AI-based denoising method allows a substantial decrease in radiation dose, without misinterpretation of actionable pulmonary nodules or life-threatening findings such as aortic aneurysms.

Validation of stable reference genes in peripheral blood mononuclear cells for expression studies involving vector-borne haemoparasitic diseases in bovines

Normalization of gene expression data using appropriate reference genes is critical to diminish any technical bias in an experiment involving quantitative real-time PCR (qPCR). To the best of our knowledge, this is the first report offering a systematic assessment of 14 potential reference genes (RPLP0, ACTB, RPS28, YWHAZ, SDHA, PPIA, RPS9, RPS15, UXT, GAPDH, B2M, BACH1, HMBS, and PPIB) for the identification of the most stable normalizers for qPCR of target genes in peripheral blood mononuclear cells (PBMCs) of bovines for vector-borne haemoparasitic diseases such as anaplasmosis, babesiosis, theileriosis, and trypanosomiasis. A total of 38 blood samples were collected from healthy as well as diseased cattle and buffaloes representing different haemoparasitic diseases. RNA isolated from the PBMCs was subjected to qPCR for the 14 prospective internal control genes. The comprehensive ranking of the genes was accomplished by the RefFinder tool that integrates the results of three algorithms (geNorm, NormFinder, and BestKeeper) and the comparative CT method. RPS15, B2M, and GAPDH were ranked to be the most stable genes, whereas, PPIA and HMBS emerged to be the least suitable genes. Validation of the selected reference genes by the qPCR analysis of two immunity genes, ISG15 and GPX7 was congruent with the observations of this study. We recommend that a panel of three reference genes including RPS15, B2M, and GAPDH could prove useful in delineating the transcriptional landscape of PBMCs for vector-borne haemoparasitic diseases in bovines.

Research on Microscopic Pore Structure Characteristics and Influencing Factors of Shale Reservoirs: A Case Study of the Second Member of the Permian Lucaogou Formation in Malang Sag, Santanghu Basin

For the second member of the Permian Lucaogou Formation in Malang Sag, Santanghu Basin, we used field emission scanning electron microscopy (SEM), cryogenic nitrogen gas adsorption, and the micro/nano CT method, combined with the fractal theory, to depict the dense reservoir space types of the reservoir and the microcosmic pore structure characteristics, perform the quantitative evaluation of aperture size, pore shape, and connectivity, and to analyze the mineral composition of the micropore structure of the reservoir. The results show that the area is dominated by sandy/argillaceous dolomite, and the reservoir space types mainly develop dissolved intergranular pores and intergranular pores, a few microfractures, and parallel plate and slit nanoscale pores. There is a positive correlation between pore volume and specific surface area, with micropore volume accounting for 14.95%, mesopore volume at 82.47%, and macropore volume at 2.58%. The mesoporous volume provides the main pore storage space. The combined specific surface area of micropores and mesoporous pores accounts for more than 99% of the total specific surface area, providing almost all the pore surface area, which is the main site for shale oil and gas adsorption. The fractal dimension D value of the samples is between 2.39 and 2.49, and the pore distribution of shale is relatively uniform, mainly developing mesoporous pores. The specific surface area and average radius are positively correlated with the content of dolomite in mineral components. The results of the CT experiment also confirm that the pore throat of samples with high dolomite content is mostly a coarse tubular and banded distribution in three-dimensional space, with good connectivity.

Transcriptome-Based Evaluation of Optimal Reference Genes for Quantitative Real-Time PCR in Yak Stomach throughout the Growth Cycle

Efficient nutritional assimilation and energy metabolism in the stomachs of yaks contribute to their adaption to harsh environments. Accurate gene expression profile analysis will help further reveal the molecular mechanism of nutrient and energy metabolism in the yak stomach. RT-qPCR is regarded as an accurate and dependable method for analyzing gene expression. The selection of reference genes is essential to obtain meaningful RT-qPCR results, especially in longitudinal gene expression studies of tissues and organs. Our objective was to select and validate optimal reference genes from across the transcriptome as internal controls for longitudinal gene expression studies in the yak stomach. In this study, 15 candidate reference genes (CRGs) were determined according to transcriptome sequencing (RNA-seq) results and the previous literature. The expression levels of these 15 CRGs were quantified using RT-qPCR in the yak stomach, including the rumen, reticulum, omasum and abomasum at five stages: 0 days, 20 days, 60 days, 15 months and three years old (adult). Subsequently, the expression stabilities of these 15 CRGs were evaluated via four algorithms: geNorm, NormFinder, BestKeeper and the comparative CT method. Furthermore, RefFinder was employed to obtain a comprehensive ranking of the stability of CRGs. The analysis results indicate that RPS15, MRPL39 and RPS23 are the most stable genes in the yak stomach throughout the growth cycle. In addition, to verify the reliability of the selected CRGs, the relative expression levels of HMGCS2 were quantified via RT-qPCR using the three most stable or the three least stable CRGs. Overall, we recommend combining RPS15, MRPL39 and RPS23 as reference genes for the normalization of RT-qPCR data in the yak stomach throughout the growth cycle.

Genetic deviation associated with photodynamic therapy in HeLa cell

Photodynamic therapy (PDT) is a method that is used in cancer treatment. The main therapeutic effect is the production of singlet oxygen (1O2). Phthalocyanines for PDT produce high singlet oxygen with absorbers of about 600–700 nm. AimIt is aimed to analyze cancer cell pathways by flow cytometry analysis and cancer-related genes with q-PCR device by applying phthalocyanine L1ZnPC, which we use as photosensitizer in photodynamic therapy, in HELA cell line. In this study, we investigate the molecular basis of L1ZnPC's anti-cancer activity. Material methodThe cytotoxic effects of L1ZnPC, a phthalocyanine obtained from our previous study, in HELA cells were evaluated and it was determined that it led to a high rate of death as a result. The result of photodynamic therapy was analyzed using q-PCR. From the data received at the conclusion of this investigation, gene expression values were calculated, and expression levels were assessed using the 2−∆∆Ct method to examine the relative changes in these values. Cell death pathways were interpreted with the FLOW cytometer device. One-Way Analysis of Variance (ANOVA) and the Tukey-Kramer Multiple Comparison Test with Post-hoc Test were used for the statistical analysis. ConclusionIn our study, it was observed that HELA cancer cells underwent apoptosis at a rate of 80% with drug application plus photodynamic therapy by flow cytometry method. According to q-PCR results, CT values ​​of eight out of eighty-four genes were found to be significant and their association with cancer was evaluated. L1ZnPC is a new phthalocyanine used in this study and our findings should be supported by further studies. For this reason, different analyses are needed to be performed with this drug in different cancer cell lines. In conclusion, according to our results, this drug looks promising but still needs to be analyzed through new studies. It is necessary to examine in detail which signaling pathways they use and their mechanism of action. For this, additional experiments are required.

Changes in Tophus Composition During Urate-Lowering Therapy: A Dual-Energy Computed Tomography Study.

The gouty tophus is an organized structure composed of monosodium urate (MSU) crystals and chronic inflammatory soft tissue. This dual-energy computed tomography (DECT) study aimed to determine whether the composition of the tophus changes during urate-lowering therapy. Serial DECT scans from 32 people with gout were obtained over 2 years of allopurinol therapy, dose-escalated to serum urate of <0.36 mmoles/liter. Up to 5 index tophi were selected for each patient, with 103 separate tophi included in the analysis. Using manual outlining methods of conventional CT and DECT scans, the same index tophi were serially measured for total tophus volume and urate volume. For each tophus, the soft tissue volume was then calculated by subtracting the urate volume from the total tophus volume. The mean ± SD serum urate reduced from 0.43 ± 0.03 mmoles/liter at baseline to 0.31 ± 0.02 mmoles/liter at year 2. The mean ± SD total tophus volume reduced over the 2-year period from 5.17 ± 5.55 cm3 to 2.61 ± 2.73 cm3 (P < 0.0001). Greater reductions in tophus urate volumes than tophus soft tissue volumes were observed; the tophus urate volume decreased by 70.6%, and tophus soft tissue volume decreased by 37.8% (P < 0.0001). The mean tophus urate:soft tissue ratio reduced from 0.15 at baseline to 0.05 at year 2 (P < 0.001). The composition of the tophus is dynamic and changes during urate-lowering therapy for gout management. The soft tissue component of the tophus is slower to respond and may persist without measurable MSU crystal deposition.

Clinical analysis of 11 patients with neuroendocrine carcinoma in maxillofacial region

Objective: To investigate the clinicopathological features, treatment and prognosis of maxillofacial neuroendocrine carcinoma. Methods: A total of 11 patients with maxillofacial neuroendocrine carcinoma diagnosed in the Department of Pathology of The First Affiliated Hospital of Zhengzhou University from December 2010 to July 2022 were retrospectively enrolled, including 8 males and 3 females, aged (65.2±9.5) years (ranged from 49 to 87 years), with a disease course of 0.5 to 6.0 months. The clinicopathological data including head and neck CT, MRI and treatment methods were analyzed. Results: Submandibular gland and maxilla were involved in 3 cases, parapharynx in 2 cases, and face, tongue root and soft palate in 1 case respectively. Clinically, the initial symptom is a rapidly growing painless or tender mass, which may be accompanied by restricted mouth opening, dysphagia, and local numbness after invasion of masticatory muscles and nerves. The tumors were all invasive and low-density, with unclear boundaries from the surrounding tissues. Among the patients, 9 received surgical treatment, and 5 received adjuvant treatment after surgery (2 received chemotherapy, 3 received radiotherapy+chemotherapy). According to the 5th edition of the World Health Organization classification of head and neck tumors in 2022, there were 1 case (1/11) with poorly differentiated large cells and 10 cases (10/11) with poorly differentiated small cells. Histologically, the macrocell type is composed of large cells with rough chromatin, obvious vacuolar nucleolus, protruding nucleolus, and necrosis. The small cell type is dominated by small blue round cells with neuroendocrine characteristics, with active growth and multifocal necrosis. Immunohistochemical staining showed that cytokeratin (CK), epithelial membrane antigen (EMA) and synaptophysin (Syn) were diffusively expressed, 10 cases expressed CD56, 8 cases expressed p63, 6 cases expressed weakly punctated chromograin-A (CgA), and S-100 was not expressed. The Ki-67 index ranges from 20 to 90 percent. By the end of follow-up (0.5 to 127.0 months), 3 patients were alive, and the mean progression-free survival (21.0 months) of postoperative chemoradiotherapy patients was significantly longer than that of surgery and/or chemotherapy alone (3.3 months). Conclusions: Maxillofacial neuroendocrine carcinoma is characterized by low differentiation of small cells, high degree of malignancy and poor prognosis. Radical surgery combined with chemoradiotherapy has better local control effect.

A body mass index-based method for “MR-only” abdominal MR-guided adaptive radiotherapy

PurposeDose calculation for MR-guided radiotherapy (MRgRT) at the 0.35 T MR-Linac is currently based on deformation of planning CTs (defCT) acquired for each patient. We present a simple and robust bulk density overwrite synthetic CT (sCT) method for abdominal treatments in order to streamline clinical workflows. MethodFifty-six abdominal patient treatment plans were retrospectively evaluated. All patients had been treated at the MR-Linac using MR datasets for treatment planning and plan adaption and defCT for dose calculation. Bulk density CTs (4M-sCT) were generated from MR images with four material compartments (bone, lung, air, soft tissue). The relative electron densities (RED) for bone and lung were extracted from contoured CT structure average REDs. For soft tissue, a correlation between BMI and RED was evaluated. Dose was recalculated on 4M-sCT and compared to dose distributions on defCTs assessing dose differences in the PTV and organs at risk (OAR). ResultsMean RED of bone was 1.17 ± 0.02, mean RED of lung 0.17 ± 0.05. The correlation between BMI and RED for soft tissue was statistically significant (p < 0.01). PTV dose differences between 4M-sCT and defCT were Dmean: −0.4 ± 1.0%, D1%: −0.3 ± 1.1% and D95%: −0.5 ± 1.0%. OARs showed D2%: −0.3 ± 1.9% and Dmean: −0.1 ± 1.4% differences. Local 3D gamma index pass rates (2%/2mm) between dose calculated using 4M-sCT and defCT were 96.8 ± 2.6% (range 89.9–99.6%). ConclusionThe presented method for sCT generation enables precise dose calculation for MR-only abdominal MRgRT.

Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives.

Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (ADF2, CYB5, iPGAM, SCL13, TRXL3-3, and VHA-H) with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend CYB5 to be used as RG for the leaf colour experiment and TRXL3-3 for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in Lactuca spp. under the described conditions.

The potential importance of autophagy genes expression profile dysregulation and ATG polymorphisms in COVID-19 pathogenesis.

Autophagy is one of the important mechanisms in cell maintenance, which is considered associated with different pathological conditions such as viral infections. In this current study, the expression level and polymorphisms in some of the most important genes in the autophagy flux in COVID-19 patients were evaluated. This cross-sectional study was conducted among 50 confirmed COVID-19 patients and 20 healthy controls. The COVID-19 patients were divided into a severe group and a mild group according to their clinical features. The expression levels of ATG5, ATG16L1, LC3, and BECN1 were evaluated by the 2-∆∆CT method and beta-actin as the internal control. The polymorphisms of the ATG5 (rs506027, rs510432) and ATG16L1 (rs2241880 or T300A) were evaluated by the Sanger sequencing following the conventional PCR. The mean age of the included patients was 58.3 ± 17.9 and 22 (44%) were female. The expression levels of the LC3 were downregulated, while BECN1 and ATG16L1 genes represent an upregulation in COVID-19 patients. The polymorphism analysis revealed the ATG16L1 rs2241880 and AGT5 rs506027 polymorphism frequencies are statistically significantly different between COVID-19 and Healthy controls. The autophagy alteration represents an association with COVID-19 pathogenesis and severity. The current study is consistent with the alteration of autophagy elements in COVID-19 patients by mRNA expression-level evaluation. Furthermore, ATG16L1 rs2241880 and AGT5 rs506027 polymorphisms seem to be important in COVID-19 and are highly suggested for further investigations.

Morphometric and volumetric evaluations of orbit using three-dimensional computed tomography in southwestern Iranian population

BackgroundThe aim of the present research was to assess morphometric and volumetric measurements of bony orbit using computed tomography to define the effects of age and gender on orbital anthropometry. Method302 orbital cavities in 151 adult samples (18–90 years with a mean age of 43.10 ± 20.25 years) were investigated (100 males [66.2%] and 51 females [33.8%]). The samples represented no history of orbital trauma, dysplasia, or other diseases with a putative effect on orbital growth. ResultsThe mean orbital height for the right and left sides were 3.75 ± 0.20 and 3.74 ± 0.21 cm, respectively, while the orbital width was 3.44 ± 0.17 and 3.46 ± 0.17 cm, respectively. The mean interorbital-biorbital width was 2.26 ± 0.26 and 9.55 ± 0.41 cm, respectively. The mean OI in both genders was 108.52 ± 6.83. There was no significant difference in OI index between the genders or sides. The study population belonged to the Megaseme category. The mean right/left orbital volume (OV) of both genders was 25.20 and 24.68 cm3, respectively. OV was significantly larger in males than females (P < 0.005) and showed sexual dimorphism. There was a positive, statistically significant association of OV with the samples' age (P < 0.0001) ConclusionThe outcomes of this research showed that the CT and 3D volumetric methods represent useful baseline morphometric data for the southwestern Iranian population using a reliable technique in orbital volumetry. The obtained data can be used for the diagnosis of orbital pathologies and anthropological studies.