Fluorescence lifetime imaging microscopy (FLIM) has been rapidly developed over the past 30 years and widely applied in biomedical engineering. Recent progress in fluorophore-dyed probe design has widened the application prospects of fluorescence. Because fluorescence lifetime is sensitive to microenvironments and molecule alterations, FLIM is promising for the detection of pathological conditions. Current cancer-related FLIM applications can be divided into three main categories: (i) FLIM with autofluorescence molecules in or out of a cell, especially with reduced form of nicotinamide adenine dinucleotide, and flavin adenine dinucleotide for cellular metabolism research; (ii) FLIM with Förster resonance energy transfer for monitoring protein interactions; and (iii) FLIM with fluorophore-dyed probes for specific aberration detection. Advancements in nanomaterial production and efficient calculation systems, as well as novel cancer biomarker discoveries, have promoted FLIM optimization, offering more opportunities for medical research and applications to cancer diagnosis and treatment monitoring. This review summarizes cutting-edge researches from 2015 to 2020 on cancer-related FLIM applications and the potential of FLIM for future cancer diagnosis methods and anti-cancer therapy development. We also highlight current challenges and provide perspectives for further investigation.
Epigenetic regulation, particularly RNA n6 methyl adenosine (m6A) modification, plays an important role in the immune response. However, the regulatory role of m6A in the immune microenvironment in osteoarthritis (OA) remains unclear. Accordingly, we systematically studied RNA modification patterns mediated by 23 m6A regulators in 38 samples and discussed the characteristics of the immune microenvironment modified by m6A. Next, we constructed a novel OA m6A nomogram, an m6A-transcription factor-miRNA network, and a drug network. Healthy and OA samples showed distinct m6A regulatory factor expression patterns. YTHDF3 expression was upregulated in OA samples and positively correlated with type II helper cells and TGFb family member receptors. Furthermore, three different RNA modification patterns were mediated by 23 m6A regulatory factors; in Mode 3, the expression levels of YTHDF3, type II T helper cells, and TGFb family member receptors were upregulated. Pathways related to endoplasmic reticulum oxidative stress and mitochondrial autophagy showed a strong correlation with the regulatory factors associated with Mode 3 and 23 m6A regulatory factors. Through RT-qPCR we validated that SREBF2 and EGR1 as transcription factors of YTHDF3 and IGF2BP3 are closely associated with the development of OA, hsa-miR-340 as a miRNA for YTHDF3 and IGF2BP3 was involved in the development of OA, we also detected the protein expression levels of IGF2BP3, YTHDF3, EGR1 and SREBF2 by western blotting, and the results were consistent with PCR. Overall, the constructed nomogram can facilitate the prediction of OA risk.
In China, a 9-year-old boy was transferred to the hospital with fever, vomiting, and headache. The disease rapidly deteriorated into vague consciousness. Applying conventional clinical examinations such as blood and cerebrospinal fluid (CSF) tests, the diagnosis of bacterial meningoencephalitis was first drawn, and expectant treatments were adopted immediately. However, the symptoms did not alleviate, adversely, this boy died 3 days after admission. Considering the skeptical points of the duration, such as the unknown infectious bacteria and the pathogen invasion path, blood and CSF samples were then sent for metagenomic next-generation sequencing (mNGS) to ascertain the cause of death. The 42,899 and 1,337 specific sequences of N. fowleri were detected by mNGS in the CSF sample and the blood sample, respectively. PCR results and pathological smear subsequently confirmed the mNGS detection. The patient was finally diagnosed as primary amoebic meningoencephalitis. Besides, in this article, 15 similar child infection cases in the past 10 years are summarized and analyzed to promote the early diagnosis of this rare disease.
BackgroundAn increasing number of observational studies have revealed an association among the gut microbiota, gut metabolites, and epilepsy. However, this association is easily influenced by confounders such as diet, and the causality of this association remains obscure.MethodsAiming to explore the causal relationship and ascertain specific gut microbe taxa for epilepsy, we conducted a bi-directional Mendelian randomization (MR) study based on the genome-wide association study (GWAS) data of epilepsy from the International League Against Epilepsy, with the gut microbiota GWAS results from MiBioGen, and summary-level GWAS data of gut microbiota-dependent metabolites trimethylamine N-oxide and its predecessors.ResultsNine phyla, 15 classes, 19 orders, 30 families, and 96 genera were analyzed. A suggestive association of host-genetic-driven increase in family Veillonellaceae with a higher risk of childhood absence epilepsy (odds ratio [OR]: 1.033, confidential interval [CI]: 1.015–1.051, PIVW = 0.0003), class Melainabacteria with a lower risk of generalized epilepsy with tonic-clonic seizures (OR = 0.986, CI = 0.979–0.994, PIVW = 0.0002), class Betaproteobacteria (OR = 0.958, CI = 0.937–0.979, PIVW = 0.0001), and order Burkholderiales (OR = 0.960, CI = 0.937–0.984, PIVW = 0.0010) with a lower risk of juvenile myoclonic epilepsy were identified after multiple-testing correction. Our sensitivity analysis revealed no evidence of pleiotropy, reverse causality, weak instrument bias, or heterogeneity.ConclusionThis is the first MR analysis to explore the potential causal relationship among the gut microbiota, metabolites, and epilepsy. Four gut microbiota features (two class levels, one order level, and one family level) were identified as potential interventional targets for patients with childhood absence epilepsy, generalized epilepsy with tonic-clonic seizures, and juvenile myoclonic epilepsy. Previous associations in numerous observational studies may had been interfered by confounders. More rigorous studies were needed to ascertain the relationship among the gut microbiota, metabolites, and epilepsy.
ObjectiveAlbeit the gene of PCDH19-FE was ascertained, the correlation of gene mutation, PCDH19 protein structure, and phenotype heterogeneity remained obscure. This study aimed to report a five-generation pedigree of seven female patients of PCDH19-FE and tried to explore whether two variants were correlated with PCDH19 protein structure and function alteration, and PCDH19-FE phenotype.MethodsWe analyzed the clinical data and genetic variants of a PCDH19-FE pedigree, to explore the phenotype heterogeneity of PCDH19-FE and underlying mechanisms. In addition to the clinical information of family members, next-generation sequencing was adopted to detect the variant sites of probands with validation by sanger sequencing. And the sanger sequencing was conducted in other patients in this pedigree. The biological conservation analysis and population polymorphism analysis of variants were also performed subsequently. The structure alteration of mutated PCDH19 protein was predicted by AlphaFold2.ResultsBased on a five-generation pedigree of PCDH19-FE, missense variants of c.695A>G and c.2760T>A in the PCDH19 gene were found in the heterozygous proband (V:1), which resulted in the change of amino acid 232 from Asn to Ser (p.Asn232Ser) and amino acid 920 from Asp to Glu (p.Asp920Glu) influencing PCDH19 function. The other six females in the pedigree (II:6, II:8, IV:3, IV:4, IV:5, IV:11) exhibited different clinical phenotypes but shared the same variant. Two males with the same variant have no clinical manifestations (III:3, III:10). The biological conservation analysis and population polymorphism analysis demonstrated the highly conservative characteristics of these two variants. AlphaFold2 predicted that the variant, p.Asp920Glu, led to the disappearance of the hydrogen bond between Asp at position 920 and His at position 919. Furthermore, the hydrogen bond between Asp920 and His919 also disappeared when the Asn amino acid mutated to Ser at position 232.ConclusionA strong genotype-phenotype heterogeneity was observed among female patients with the same genotype in our PCDH19-FE pedigree. And two missense variants, c.695A > G and c.2760T>A in the PCDH19 gene, have been identified in our pedigree. The c.2760T>A variant was a novel variant site probably related to the PCDH19-FE.
ObjectiveIt is widely acknowledged that central nervous system (CNS) infection is a serious infectious disease accompanied by various complications. However, the accuracy of current detection methods is limited, leading to delayed diagnosis and treatment. In recent years, metagenomic next-generation sequencing (mNGS) has been increasingly adopted to improve the diagnostic yield. The present study sought to evaluate the value of mNGS in CNS infection diagnosis.MethodsFollowing the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2022 guidelines, we searched relevant articles published in seven databases, including PubMed, Web of Science, and Cochrane Library, published from January 2014 to January 2022. High-quality articles related to mNGS applications in the CNS infection diagnosis were included. The comparison between mNGS and the gold standard of CNS infection, such as culture, PCR or serology, and microscopy, was conducted to obtain true positive (TP), true negative (TN), false positive (FP), and false negative (FN) values, which were extracted for sensitivity and specificity calculation.ResultsA total of 272 related studies were retrieved and strictly selected according to the inclusion and exclusion criteria. Finally, 12 studies were included for meta-analysis and the pooled sensitivity was 77% (95% CI: 70–82%, I2 = 39.69%) and specificity was 96% (95% CI: 93–98%, I2 = 72.07%). Although no significant heterogeneity in sensitivity was observed, a sub-group analysis was conducted based on the pathogen, region, age, and sample pretreatment method to ascertain potential confounders. The area under the curve (AUC) of the summary receiver operating characteristic curve (SROC) of mNGS for CNS infection was 0.91 (95% CI: 0.88–0.93). Besides, Deek's Funnel Plot Asymmetry Test indicated no publication bias in the included studies (Figure 3, p > 0.05).ConclusionOverall, mNGS exhibits good sensitivity and specificity for diagnosing CNS infection and diagnostic performance during clinical application by assisting in identifying the pathogen. However, the efficacy remains inconsistent, warranting subsequent studies for further performance improvement during its clinical application.Study registration numberINPLASY202120002
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