Autophagy accompanying the developmental process of male germline stem cells.

Germline stem cells are a crucial type of stem cell that can stably pass on genetic information to the next generation, providing the necessary foundation for the reproduction and survival of organisms. Male mammalian germline stem cells are unique cell types that include primordial germ cells and spermatogonial stem cells. They can differentiate into germ cells, such as sperm and eggs, thereby facilitating offspring reproduction. In addition, they continuously generate stem cells through self-renewal mechanisms to support the normal function of the reproductive system. Autophagy involves the use of lysosomes to degrade proteins and organelles that are regulated by relevant genes. This process plays an important role in maintaining the homeostasis of germline stem cells and the synthesis, degradation, and recycling of germline stem cell products. Recently, the developmental regulatory mechanism of germline stem cells has been further elucidated, and autophagy has been shown to be involved in the regulation of self-renewal and differentiation of germline stem cells. In this review, we introduce autophagy accompanying the development of germline stem cells, focusing on the autophagy process accompanying the development of male spermatogonial stem cells and the roles of related genes and proteins. We also briefly outline the effects of autophagy dysfunction on germline stem cells and reproduction.

Bisdemethoxycurcumin Augments Docetaxel Efficacy for Treatment of Prostate Cancer.

Bisdemethoxycurcumin (BDMC) is one of major forms of curcuminoids found in the rhizomes of turmeric. Docetaxel (DTX) is the standard of care for men diagnosed with androgen-independent prostate cancers. Here we report for the first time that BDMC could reinforce the effect of DTX against prostate cancer in vitro and in vivo. In vitro study, PC3 and LNCaP cells were cultured and treated with BDMC and DTX alone or in combination. The effects on cell viability were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by annexin V/propidium iodide (PI) staining, while cell cycle was assessed by PI staining. Bax, Bcl-2, caspase, poly(ADP-ribose)polymerase (PARP), cyclin B1 and CDK1 expression were assayed by Western blot. We found that a combination treatment of BDMC (10 µM) with DTX (10 nM) was more effective in the inhibition of PC3 and LNCaP cell growth and induction of apoptosis as well as G2/M arrest, which is accompanied with the significant inhibition of Bcl-2, cyclin B1, CDK1 expression and significant increase of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, than those by treatment of BDMC or DTX alone. Moreover, in vivo evaluation further demonstrated the superior anticancer efficacy of BDMC and DTX compared to DTX alone in a murine prostate cancer model. These results suggest that BDMC can be an attractive therapeutic candidate in enhancing the efficacy of DTX in prostate cancer treatment.

Study on Rapid Non-Destructive Detection Method of Corn Freshness Based on Hyperspectral Imaging Technology.

(1) Background: To achieve the rapid, non-destructive detection of corn freshness and staleness for better use in the storage, processing and utilization of corn. (2) Methods: In this study, three varieties of corn were subjected to accelerated aging treatment to study the trend in fatty acid values of corn. The study focused on the use of hyperspectral imaging technology to collect information from corn samples with different aging levels. Spectral data were preprocessed by a convolutional smoothing derivative method (SG, SG1, SG2), derivative method (D1, D2), multiple scattering correction (MSC), and standard normal transform (SNV); the characteristic wavelengths were extracted by the competitive adaptive reweighting method (CARS) and successive projection algorithm (SPA); a neural network (BP) and random forest (RF) were utilized to establish a prediction model for the quantification of fatty acid values of corn. And, the distribution of fatty acid values was visualized based on fatty acid values under the corresponding optimal prediction model. (3) Results: With the prolongation of the aging time, all three varieties of corn showed an overall increasing trend. The fatty acid value of corn can be used as the most important index for characterizing the degree of aging of corn. SG2-SPA-RF was the quantitative prediction model for optimal fatty acid values of corn. The model extracted 31 wavelengths, only 12.11% of the total number of wavelengths, where the coefficient of determination RP2 of the test set was 0.9655 and the root mean square error (RMSE) was 3.6255. (4) Conclusions: This study can provide a reliable and effective new method for the rapid non-destructive testing of corn freshness.

Design, synthesis and evaluation of heterocyclic 2-phenylacetate derivatives as water-soluble rapid recovery hypnotics.

In this work, a series of novel heterocyclic 2-phenylacetate derivatives were designed and synthesized as water-soluble and rapid recovery hypnotic agents. After introducing heterocyclic ring to the amide group of propanidid, the obtained propanidid derivatives showed greatly improved hydrophilicity and good anesthetic activity. In three animal experiments (mice, rats, and rabbits), compounds 13-15 showed potent hypnotic potency (HD50 = 7.6, 6.5, 7.4 mg/kg in rabbits, respectively) and higher therapeutic indexes (TI = 17.3, 16.6, 15.2 in rabbits, respectively) than propanidid (TI = 14.7 in rabbits) or propofol (TI = 5.4 in rabbits). Moreover, the recovery time of compounds 13-15 (time to walk, 96.6, 79.6, 81.4 s in rabbits, respectively) were shorter than that of propanidid (124.5 s in rabbits) or propofol (425.3 s in rabbits). The experimental results suggested the potential of compounds 13-15 as water-soluble anesthetics with rapid recovery profile.

COVIDX-LwNet: A Lightweight Network Ensemble Model for the Detection of COVID-19 Based on Chest X-ray Images.

Recently, the COVID-19 pandemic coronavirus has put a lot of pressure on health systems around the world. One of the most common ways to detect COVID-19 is to use chest X-ray images, which have the advantage of being cheap and fast. However, in the early days of the COVID-19 outbreak, most studies applied pretrained convolutional neural network (CNN) models, and the features produced by the last convolutional layer were directly passed into the classification head. In this study, the proposed ensemble model consists of three lightweight networks, Xception, MobileNetV2 and NasNetMobile as three original feature extractors, and then three base classifiers are obtained by adding the coordinated attention module, LSTM and a new classification head to the original feature extractors. The classification results from the three base classifiers are then fused by a confidence fusion method. Three publicly available chest X-ray datasets for COVID-19 testing were considered, with ternary (COVID-19, normal and other pneumonia) and quaternary (COVID-19, normal) analyses performed on the first two datasets, bacterial pneumonia and viral pneumonia classification, and achieved high accuracy rates of 95.56% and 91.20%, respectively. The third dataset was used to compare the performance of the model compared to other models and the generalization ability on different datasets. We performed a thorough ablation study on the first dataset to understand the impact of each proposed component. Finally, we also performed visualizations. These saliency maps not only explain key prediction decisions of the model, but also help radiologists locate areas of infection. Through extensive experiments, it was finally found that the results obtained by the proposed method are comparable to the state-of-the-art methods.

Hb Huadu [α124(H7)Ser→Thr (TCC>ACC), HBA2: c.373T>A]: A Novel Variant of the α-Globin Gene.

Here, we report a novel α chain hemoglobin (Hb) variant found during routine thalassemia screening. This Hb variant can be detected by capillary electrophoresis (CE) but cannot be recognized by high performance liquid chromatography (HPLC). Sanger sequencing revealed a heterozygous missense substitution at nucleotide 373 on the HBA2 gene, which results in the replacement of serine by threonine at codon 124 [α124(H7)Ser→Thr (TCC>ACC), HBA2: c.373T>A]. It is the first report of this variant, named Hb Huadu for the birthplace of the proband. In addition, the proband coinherited the heterozygous codons 41/42 (-TTCT) (HBB: c126_129delCTTT) on the ß-globin gene.

A-kinase-interacting protein 1 promotes EMT and metastasis via PI3K/Akt/IKKß pathway in cervical cancer.

Overexpression of A-kinase-interacting protein 1 (AKIP1) has been reported in prostate and breast cancers. Nevertheless, the clinical potential of AKIP1 during the development of cervical cancer (CC) remains unclear. A series of experiments involving BdU, colony formation, wound healing and cell invasion assays were performed to determine cell proliferation, migration and invasion, respectively. Gene expression changes were validated by qRT-PCR, Western blotting and immunocytochemistry. We found that AKIP1 expression is increased in CC cell lines and tissue specimens from CC patients. The elevated AKIP1 expression in primary tumours was related to lymph node metastasis in CC patients. In addition, we observed that overexpression of AKIP1 promotes CC cell proliferation. Enhanced expression of AKIP1 facilitated the migration and invasion of CC cells by inducing NF-κB-dependent epithelial-mesenchymal transition (EMT). Moreover, mechanistic investigations revealed that AKIP1 induced nuclear translocation and phosphorylation of the p65 NF-κB subunit through the PI3K/Akt/IKKß pathway. Conversely, enhanced expression of phosphatase and tensin homologue (PTEN) inhibited this signalling pathway and restored an epithelial phenotype to CC cells in the presence of overexpressed AKIP1. Our results indicate that AKIP1 promotes the EMT and metastasis in CC by activating NF-κB signalling through the PI3K/Akt/IKKß pathway, suggesting that AKIP1 could be a pivotal regulator of an EMT axis in CC. SIGNIFICANCE OF THE STUDY: AKIP1 expression in the samples of CC patients and in in vitro tumour cell lines provides evidence that expression of AKIP1 in CC contributes to cancer progression. Our findings indicate that AKIP1 promotes the epithelial-mesenchymal transition and metastasis in CC by activating NF-κB signalling through the PI3K/Akt/IKKß pathway, suggesting that AKIP1 is a pivotal regulator of an EMT axis in CC. AKIP1 could be implicated as a potential therapeutic target as well as a valuable biomarker for evaluating prognosis for patients with CC.

Recognition of Common Non-Normal Walking Actions Based on Relief-F Feature Selection and Relief-Bagging-SVM.

Action recognition algorithms are widely used in the fields of medical health and pedestrian dead reckoning (PDR). The classification and recognition of non-normal walking actions and normal walking actions are very important for improving the accuracy of medical health indicators and PDR steps. Existing motion recognition algorithms focus on the recognition of normal walking actions, and the recognition of non-normal walking actions common to daily life is incomplete or inaccurate, resulting in a low overall recognition accuracy. This paper proposes a microelectromechanical system (MEMS) action recognition method based on Relief-F feature selection and relief-bagging-support vector machine (SVM). Feature selection using the Relief-F algorithm reduces the dimensions by 16 and reduces the optimization time by an average of 9.55 s. Experiments show that the improved algorithm for identifying non-normal walking actions has an accuracy of 96.63%; compared with Decision Tree (DT), it increased by 11.63%; compared with k-nearest neighbor (KNN), it increased by 26.62%; and compared with random forest (RF), it increased by 11.63%. The average Area Under Curve (AUC) of the improved algorithm improved by 0.1143 compared to KNN, by 0.0235 compared to DT, and by 0.04 compared to RF.

Characterization of the molecular mechanism of the autophagy-related Atg8-Atg3 protein interaction in Toxoplasma gondii.

Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Toxoplasma gondii Discovery of novel drugs against T. gondii infection could circumvent the toxicity of existing drugs and T. gondii resistance to current treatments. The autophagy-related protein 8 (Atg8)-Atg3 interaction in T. gondii is a promising drug target because of its importance for regulating Atg8 lipidation. We reported previously that TgAtg8 and TgAtg3 interact directly. Here we validated that substitutions of conserved residues of TgAtg8 interacting with the Atg8 family-interacting motif (AIM) in Atg3 disrupt the TgAtg8-TgAtg3 interaction and reduce TgAtg8 lipidation and autophagosome formation. These findings were consistent with results reported previously for Plasmodium Atg8, suggesting functional conservation of Atg8 in Toxoplasma and Plasmodium. Moreover, using peptide and AlphaScreen assays, we identified the AIM sequence in TgAtg3 that binds TgAtg8. We determined that the core TgAtg3 AIM contains a Phe239-Ala240-Asp241-Ile242 (239FADI242) signature distinct from the 105WLLP108 signature in the AIM of Plasmodium Atg3. Furthermore, an alanine-scanning assay revealed that the TgAtg8-TgAtg3 interaction in T. gondii also depends strongly on several residues surrounding the core TgAtg3 AIM, such as Asn238, Asp243, and Cys244 These results indicate that distinct AIMs in Atg3 contribute to differences between Toxoplasma and Plasmodium Atg8-Atg3 interactions. By elucidating critical residues involved in the TgAtg8-TgAtg3 interaction, our work paves the way for the discovery of potential anti-toxoplasmosis drugs. The quantitative and straightforward AlphaScreen assay developed here may enable high-throughput screening for small molecules disrupting the TgAtg8-TgAtg3 interaction.

Paper-based cell impedance sensor and its application for cytotoxic evaluation.

Disposable analytical devices for developing sensitive and label-free monitoring of cancerous cells would be attractive for cancer research. Here, paper-based electroanalytical devices based on impedance spectrometry were applied for the study of K562 cells and the toxic effect of anticancer drugs. The proposed device integrating gold nanorods modified ITO electrodes could provide a biocompatible surface for immobilization of living cells maintaining their bioactivity. The impedance results exhibited good correlation to the logarithmic value of cell numbers ranging from 7.5 × 10(2) to 3.9 × 10(6) cells mL(-1) with a detection limit of 500 cells mL(-1). Furthermore, this strategy was used to evaluate the cytotoxic effects of arsenic trioxide and cyclophosphamide. Results obtained by the impedimetric method correlate well with the conventional cell viability assay. Cells exposed to drugs exhibited a prominent reduction of impedance data, showing an inverse dose-dependent relationship. This simple, cost-effective and portable paper-based electrochemical analytical device could provide a new impedance platform for applications in monitoring cell behavior, pharmacological studies and toxicological analyses.

Carbon dots as a luminescence sensor for ultrasensitive detection of phosphate and their bioimaging properties.

Highly blue fluorescence carbon dots were synthesized by one-step hydrothermal treatment of potatoes. The as-obtained C-dots have been applied to bioimaging of HeLa cells, which shows their excellent biocompatibility and low cytotoxicity. The results reveal that C-dots are promising for real cell imaging applications. In addition, the carbon dots can be utilized as a probe for sensing phosphate.

'Imperfect' conjugated polymer nanoparticles from MEH-PPV for bioimaging and Fe(III) sensing.

A simple and effective method was reported for the preparation from MEH-PPV of conjugated polymer nanoparticles (Pdots) that are water-soluble and well dispersed. The as-prepared Pdots show bright orange fluorescence at a quantum yield up to 32.37%. The fluorescence intensity of Pdots can be quenched with good selectively by the successive addition of Fe(3+) . In addition, the as-obtained Pdots were applied to the imaging of HeLa cells, and exhibited low cytotoxicity and excellent biocompatibility.

Synthesis of highly luminescent mercaptosuccinic acid-coated CdSe nanocrystals under atmospheric conditions.

Here we report a facile one-pot method for the preparation of high-quality CdSe nanocrystals (NCs) in aqueous solution under an air atmosphere. Compared with the traditional use of NaHSe or H2 Se, the more stable sodium selenite is utilized as the Se source for preparing highly luminescent CdSe nanocrystals. By using mercaptosuccinic acid (MSA) as the capping agent and borate-citrate acid as the buffering solution, CdSe nanocrystals with high quantum yield (up to 70%) have been synthesized conveniently. The influence of different experimental parameters, such as the pH of the precursor solution, the molar ratio of Cd(2+) to Na2 SeO3 and Cd(2+) to MSA on the CdSe nanocrystals, has been systematically investigated. The prepared CdSe NCs were spherical with a size of ~ 5 nm.

Physical-Entanglements-Supported Polymeric Form Stable Phase Change Materials with Ultrahigh Melting Enthalpy.

With the rapid development of new energy and the upgrading of electronic devices, structurally stable phase change materials (PCMs) have attracted widespread attentions from both academia and industries. Traditional cross-linking, composites, or microencapsulation methods for preparation of form stable PCMs usually sacrifice part of the phase change enthalpy and recyclability. Based on the basic polymer viscoelasticity and crystallization theories, here, a kind of novel recyclable polymeric PCM is developed by simple solution mixing ultrahigh molecular weight of polyethylene oxide (UHMWPEO) with its chemical identical oligomer polyethylene glycol (PEG). Rheological and leakage-proof experiments confirm that, even containing 90% of phase change fraction PEG oligomers, long-term of structure stability of PCMs can be achieved when the molecular weight of UHMWPEO is higher than 7000 kg mol-1 due to their ultralong terminal relaxation time and large number of entanglements per chain. Furthermore, because of the reduced overall entanglement concentration, phase change enthalpy of PCMs can be greatly promoted, even reaching to ≈185 J g-1, which is larger than any PEG-based form stable PCMs in literatures. This work provides a new strategy and mechanism for designing physical-entanglements-supported form stable PCMs with ultrahigh phase change enthalpies.

Study on the interaction between agglutinin and chondroitin sulfate and dermatan sulfate using multiple methods.

In this work, the interaction of chondroitin sulfate (CS) and dermatan sulfate (DS) with plant lectins was studied by affinity capillary electrophoresis (ACE), surface plasmon resonance (SPR) technology, molecular docking simulation, and circular dichroism spectroscopy. The ACE method was used for the first time to study the interaction of Ricinus Communis Agglutinin I (RCA I), Wisteria Floribunda Lectin (WFA), and Soybean Agglutinin (SBA) with CS and DS, and the results were in good agreement with those of the SPR method. The results of experiments indicate that RCA I has a strong binding affinity with CS, and the sulfated position does not affect the relationship, but the degree of sulfation can affect the combination of RCA I with CS to some extent. However, the binding affinity with DS is very weak. This study lays the foundation for developing more specialized analysis methods for CS and DS based on RCA I.

The synergistic anti-nociceptive effects of nefopam and gabapentinoids in inflammatory, osteoarthritis, and neuropathic pain mouse models.

Pain is a common public health problem and remains as an unmet medical need. Currently available analgesics usually have limited efficacy or are accompanied by many adverse side effects. To achieve satisfactory pain relief by multimodal analgesia, new combinations of nefopam and gabapentinoids (pregabalin/gabapentin) were designed and assessed in inflammatory, osteoarthritis and neuropathic pain. Isobolographic analysis was performed to analyze the interactions between nefopam and gabapentinoids in carrageenan-induced inflammatory pain, mono-iodoacetate-induced osteoarthritis pain and paclitaxel-induced peripheral neuropathic pain in mice. The anti-inflammatory effect and motor performance of monotherapy or their combinations were evaluated in the carrageenan-induced inflammatory responses and rotarod test, respectively. Nefopam (1, 3, 5, 10, 30 mg/kg, p.o.), pregabalin (3, 6, 12, 24 mg/kg, p.o.) or gabapentin (25, 50, 75, 100 mg/kg, p.o.) dose-dependently reversed mechanical allodynia in three pain models. Isobolographic analysis indicated that the combinations of nefopam and gabapentinoids exerted synergistic anti-nociceptive effects in inflammatory, osteoarthritis, and neuropathic pain mouse models, as evidenced by the experimental ED50 (median effective dose) falling below the predicted additive line. Moreover, the combination of nefopam-pregabalin/gabapentin alleviated carrageenan-induced inflammation and edema, and also prevented gabapentinoids-related sedation or ataxia by lowering their effective doses. Collectively, the co-administration of nefopam and gabapentinoids showed synergistic analgesic effects and may result in improved therapeutic benefits for treating pain.

Recent Advances in Nanobiotechnology for the Treatment of Non-Hodgkin's Lymphoma.

Lymphoma is the eighth most common type of cancer worldwide. Currently, lymphoma is mainly classified into two main groups: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), with NHL accounting for 80% to 90% of the cases. NHL is primarily divided into B, T, and natural killer (NK) cell lymphoma. Nanotechnology is developing rapidly and has made significant contributions to the field of medicine. This review summarizes the advancements of nanobiotechnology in recent years and its applications in the treatment of NHL, especially in diffuse large B cell lymphoma (DLBCL), central nerve cell lymphoma, and follicular lymphoma. The technologies discussed include clinical imaging, targeted drug delivery, photodynamic therapy (PDT), and thermodynamic therapy for lymphoma. This review aims to provide a better understanding of the use of nanotechnology in the treatment of non-Hodgkin's lymphoma.

Lyoprotectant Formulation and Optimization of the J-Aggregates Astaxanthin/BSA/Chitosan Nanosuspension.

Astaxanthin is a carotenoid with excellent antioxidant activity. However, this small lipid-soluble molecule is insoluble in water and has low stability. Although this situation can be improved when astaxanthin is prepared as a nanosuspension, the aqueous form is still not as convenient and safe as the dry powder form for storage, transport, and use. The lyophilization process provides better protection for thermosensitive materials, but this leads to collapse and agglomeration between nanoparticles. To improve this situation, appropriate lyophilization protectants are needed to offer support between the nanoparticles, such as sugars, amino acids, and hydroxy alcohols. The purpose of this work is to screen lyophilization protectants by single-factor experiments and response surface optimization experiments and then explore the optimal ratio of compound lyophilization protectants, and finally, make excellent astaxanthin/BSA/chitosan nanosuspension (ABC-NPs) lyophilized powder. The work shows that the optimal ratio of the compounding lyophilization protectant is 0.46% oligomeric mannose, 0.44% maltose, and 0.05% sorbitol (w/v). The ABC-NPs lyophilized powder prepared under the above conditions had a re-soluble particle size of 472 nm, with a ratio of 1.32 to the particle size of the sample before lyophilization. The lyophilized powder was all in the form of a pink layer. The sample was fluffy and dissolved entirely within 10 s by shaking with water. Consequently, it is expected to solve the problem of inconvenient storage and transportation of aqueous drugs and to expand the application of nanomedicine powders and tablets.

Vimentin as a potential target for diverse nervous system diseases.

Vimentin is a major type III intermediate filament protein that plays important roles in several basic cellular functions including cell migration, proliferation, and division. Although vimentin is a cytoplasmic protein, it also exists in the extracellular matrix and at the cell surface. Previous studies have shown that vimentin may exert multiple physiological effects in different nervous system injuries and diseases. For example, the studies of vimentin in spinal cord injury and stroke mainly focus on the formation of reactive astrocytes. Reduced glial scar, increased axonal regeneration, and improved motor function have been noted after spinal cord injury in vimentin and glial fibrillary acidic protein knockout (GFAP-/-VIM-/-) mice. However, attenuated glial scar formation in post-stroke in GFAP-/- VIM-/- mice resulted in abnormal neuronal network restoration and worse neurological recovery. These opposite results have been attributed to the multiple roles of glial scar in different temporal and spatial conditions. In addition, extracellular vimentin may be a neurotrophic factor that promotes axonal extension by interaction with the insulin-like growth factor 1 receptor. In the pathogenesis of bacterial meningitis, cell surface vimentin is a meningitis facilitator, acting as a receptor of multiple pathogenic bacteria, including E. coli K1, Listeria monocytogenes, and group B streptococcus. Compared with wild type mice, VIM-/- mice are less susceptible to bacterial infection and exhibit a reduced inflammatory response, suggesting that vimentin is necessary to induce the pathogenesis of meningitis. Recently published literature showed that vimentin serves as a double-edged sword in the nervous system, regulating axonal regrowth, myelination, apoptosis, and neuroinflammation. This review aims to provide an overview of vimentin in spinal cord injury, stroke, bacterial meningitis, gliomas, and peripheral nerve injury and to discuss the potential therapeutic methods involving vimentin manipulation in improving axonal regeneration, alleviating infection, inhibiting brain tumor progression, and enhancing nerve myelination.

[Genotype and Phenotype of α-Thalassemia Fusion Gene in Huadu District of Guangzhou, Guangdong Province of China].

OBJECTIVE: To explore the carrier rate, genotype and phenotype of α-thalassemia fusion gene in Huadu district of Guangzhou, Guangdong province of China, and provide data reference for the prevention and control of thalassemia. METHODS: A total of 10 769 samples who were screened for thalassemia in Maternal and Child Health Hospital of Huadu District from July 2019 to November 2020 were analyzed retrospectively. Blood cell analysis and hemoglobin (Hb) electrophoresis were performed. Thalassemia genes were analyzed by gap-PCR and PCR-reverse dot blot hybridization (PCR-RDB). RESULTS: A total of 9 cases with α-thalassemia fusion gene were detected in 10 769 samples (0.08%). There were 7 cases with fusion gene heterozygote, 1 case with compound of α-thalassemia fusion gene and Hb G-Honolulu, 1 case with compound of α-thalassemia fusion gene and Hb QS. The MCV results of 4 samples of blood cell analysis were within the reference range, the Hb A2 value of 1 case was decreased, and there were no other abnormalities found. CONCLUSION: The α-thalassemia fusion gene is common in Huadu district of Guangzhou, and heterozygotes are more common, and current screening methods easily lead to misdiagnosis.