Molecular Aspects of Medicine最新文献

Over the past decade, novel methods for enrichment and identification of cancer cells circulating in the blood have been established. Blood-based detection of cancer cells and other tumor-associated products can be summarized under the term of Liquid Biopsy. Circulating tumor cells (CTCs) have been used for diagnosis, risk stratification and treatment selection as well as treatment monitoring in several studies over the past years, thus representing a valuable biomarker for cancer patients. A plethora of methods to enrich, detect and analyze CTCs has been established. In contrast to other liquid biopsy analytes (e.g. ctDNA), CTCs represent a viable analyte that provides a unique opportunity to understand the underlaying biology of cancer and the metastatic cascade on the molecular level. In this review, we provide an overview on the current methods used for enrichment, detection, molecular and functional characterization of CTCs.

Single-cell technologies have transformed biomedical research over the last decade, opening up new possibilities for understanding cellular heterogeneity, both at the genomic and transcriptomic level. In addition, more recent developments of spatial transcriptomics technologies have made it possible to profile cells in their tissue context. In parallel, there have been substantial advances in sequencing technologies, and the third generation of methods are able to produce reads that are tens of kilobases long, with error rates matching the second generation short reads. Long reads technologies make it possible to better map large genome rearrangements and quantify isoform specific abundances. This further improves our ability to characterize functionally relevant heterogeneity. Here, we show how researchers have begun to combine single-cell, spatial transcriptomics, and long-read technologies, and how this is resulting in powerful new approaches to profiling both the genome and the transcriptome. We discuss the achievements so far, and we highlight remaining challenges and opportunities.

Massively parallel sequencing technologies have long been used in both basic research and clinical routine. The recent introduction of digital sequencing has made previously challenging applications possible by significantly improving sensitivity and specificity to now allow detection of rare sequence variants, even at single molecule level. Digital sequencing utilizes unique molecular identifiers (UMIs) to minimize sequencing-induced errors and quantification biases. Here, we discuss the principles of UMIs and how they are used in digital sequencing. We outline the properties of different UMI types and the consequences of various UMI approaches in relation to experimental protocols and bioinformatics. Finally, we describe how digital sequencing can be applied in specific research fields, focusing on cancer management where it can be used in screening of asymptomatic individuals, diagnosis, treatment prediction, prognostication, monitoring treatment efficacy and early detection of treatment resistance as well as relapse.

Despite many progresses have been made in the treatment of inflammatory bowel disease, especially due to the increasing number of effective therapies, the development of tissue fibrosis is a very common occurrence along the natural history of this condition. To a certain extent, fibrogenesis is a physiological and necessary process in all those conditions characterised by chronic inflammation. However, the excessive deposition of extracellular matrix within the bowel wall will end up in the formation of strictures, with the consequent need for surgery. A number of mechanisms have been described in this process, but some of them are not yet clear. For sure, the main trigger is the presence of a persistent inflammatory status within the mucosa, which in turn favours the occurrence of a pro-fibrogenic environment. Among the main key players, myofibroblasts, fibroblasts, immune cells, growth factors and cytokines must be mentioned. Although there are no available therapies able to target fibrosis, the only way to prevent it is by controlling inflammation. In this review, we summarize the state of art of the mechanisms involved in gut fibrogenesis, how to diagnose it, and which potential targets could be druggable to tackle fibrosis.

Well-characterized reference materials support harmonization and accuracy when conducting nucleic acid-based tests (such as qPCR); digital PCR (dPCR) can measure the absolute concentration of a specific nucleic acid sequence in a background of non-target sequences, making it ideal for the characterization of nucleic acid-based reference materials. National Metrology Institutes are increasingly using dPCR to characterize and certify their reference materials, as it offers several advantages over indirect methods, such as UV-spectroscopy. While dPCR is gaining widespread adoption, it requires optimization and has certain limitations and considerations that users should be aware of when characterizing reference materials. This review highlights the technical considerations of dPCR, as well as its role when developing and characterizing nucleic acid-based reference materials.

Although significant advances in immunotherapy have revolutionized the treatment of many cancer types over the past decade, the field of vaccine therapy, an important component of cancer immunotherapy, despite decades-long intense efforts, is still transmitting signals of promises and awaiting strong data on efficacy to proceed with regulatory approval. The field of cancer vaccines faces standard challenges, such as tumor-induced immunosuppression, immune response in inhibitory tumor microenvironment (TME), intratumor heterogeneity (ITH), permanently evolving cancer mutational landscape leading to neoantigens, and less known obstacles: neoantigen gain/loss upon immunotherapy, the timing and speed of appearance of neoantigens and responding T cell clonotypes and possible involvement of immune interference/heterologous immunity, in the complex interplay between evolving tumor epitopes and the immune system. In this review, we discuss some key issues related to challenges hampering the development of cancer vaccines, along with the current approaches focusing on neoantigens. We summarize currently well-known ideas/rationales, thus revealing the need for alternative vaccine approaches. Such a discussion should stimulate vaccine researchers to apply out-of-box, unconventional thinking in search of new avenues to deal with critical, often yet unaddressed challenges on the road to a new generation of therapeutics and vaccines.

Current precision cancer medicine is dependent on the analyses of a plethora of clinically relevant genomic aberrations. During the last decade, next-generation sequencing (NGS) has gradually replaced most other methods for precision cancer diagnostics, spanning from targeted tumor-informed assays and gene panel sequencing to global whole-genome and whole-transcriptome sequencing analyses. The shift has been impelled by a clinical need to assess an increasing number of genomic alterations with diagnostic, prognostic and predictive impact, including more complex biomarkers (e.g. microsatellite instability, MSI, and homologous recombination deficiency, HRD), driven by the parallel development of novel targeted therapies and enabled by the rapid reduction in sequencing costs. This review focuses on these sequencing-based methods, puts their emergence in a historic perspective, highlights their clinical utility in diagnostics and decision-making in pediatric and adult cancer, as well as raises challenges for their clinical implementation. Finally, the importance of applying sensitive tools for longitudinal monitoring of treatment response and detection of measurable residual disease, as well as future avenues in the rapidly evolving field of sequencing-based methods are discussed.

Systemic sclerosis (also called scleroderma, SSc) is a chronic autoimmune disorder characterized by excessive collagen deposition leading to skin fibrosis and various internal organ manifestations. The emergent diagnostics and therapeutic strategies for scleroderma focus on early detection and targeted interventions to improve patient outcomes and quality of life. Diagnostics for SSc have evolved significantly in recent years, driven by advancements in serological markers and imaging techniques. Autoantibody profiling, especially antinuclear antibodies (ANA) and specific scleroderma-associated autoantibodies, aids in identifying subsets of scleroderma and predicting disease progression. Furthermore, novel imaging modalities, such as high-frequency ultrasonography and optical coherence tomography, enable early detection of skin fibrosis and internal organ involvement, enhancing the diagnostic precision and allowing for tailored management. Therapeutic strategies for SSc are multifaceted, targeting immune dysregulation, vascular abnormalities, and fibrotic processes. Emerging biologic agents have shown promise in clinical trials, including monoclonal antibodies directed against key cytokines involved in fibrosis, such as transforming growth factor-β (TGF-β) and interleukin-6 (IL-6). Additionally, small-molecule inhibitors that disrupt fibrotic pathways, like tyrosine kinase inhibitors, have exhibited potential in limiting collagen deposition and preventing disease progression. Stem cell therapy, cell ablation and gene editing techniques hold great potential in regenerating damaged tissue and halting fibrotic processes. Early intervention remains crucial in managing SSc, as irreversible tissue damage often occurs in advanced stages. Novel diagnostic methods, such as biomarkers and gene expression profiling, are being explored to identify individuals at high risk for developing progressive severe disease and intervene proactively. Furthermore, patient-tailored therapeutic approaches, employing a combination of immunosuppressive agents and targeted anti-fibrotic therapies, are being investigated to improve treatment efficacy while minimizing adverse effects. The emergent diagnostics and therapeutic strategies in scleroderma are transforming the management of this challenging disease. Nevertheless, ongoing research and clinical trials are needed to optimize the efficacy and safety of these novel approaches in the complex and diverse spectrum of SSc manifestations.

The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and clinical practice. Its theoretical foundations guide the design of experiments, while its practical implications extend to diagnostics, treatment, and research advancements in the life sciences, human and veterinary medicine, agriculture, and forensics. However, the accuracy, reliability and reproducibility of qPCR data face challenges arising from various factors associated with experimental design, execution, data analysis and inadequate reporting details. Addressing these concerns, the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have emerged as a cohesive framework offering a standardised set of recommendations that describe the essential information required for assessing qPCR experiments. By emphasising the importance of methodological rigour, the MIQE guidelines have made a major contribution to improving the trustworthiness, consistency, and transparency of many published qPCR results. However, major challenges related to awareness, resources, and publication pressures continue to affect their consistent application.

Glaucoma is one of the leading causes of visual impairment and blindness worldwide, and is characterized by the progressive damage of retinal ganglion cells (RGCs) and the atrophy of the optic nerve head (ONH). The exact cause of RGC loss and optic nerve damage in glaucoma is not fully understood. The high energy demands of these cells imply a higher sensitivity to mitochondrial defects. Moreover, it has been postulated that the optic nerve is vulnerable towards damage from oxidative stress and mitochondrial dysfunction. To investigate this further, we conducted a pooled analysis of mitochondrial variants related to energy production, specifically focusing on oxidative phosphorylation (OXPHOS) and fatty acid β-oxidation (FAO). Our findings revealed that patients carrying non-synonymous (NS) mitochondrial DNA (mtDNA) variants within the OXPHOS complexes had an almost two-fold increased risk of developing glaucoma. Regarding FAO, our results demonstrated that longer-chain acylcarnitines (AC) tended to decrease, while shorter-chain AC tended to increase in patients with glaucoma. Furthermore, we observed that the knocking down cpt1a (a key rate-limiting enzyme involved in FAO) in zebrafish induced a degenerative process in the optic nerve and RGC, which resembled the characteristics observed in glaucoma. In conclusion, our study provides evidence that genes encoding mitochondrial proteins involved in energy metabolisms, such as OXPHOS and FAO, are associated with glaucoma. These findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and may offer potential targets for therapeutic interventions in the future.