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Introduction Diagnostic algorithms are used for detection of periprosthetic joint infection (PJI) including sampling for histological evaluation (HE). The purpose was to assess the diagnostic significance of emergency HE of fresh-frozen intraoperative biomaterial as part of preoperative PJI screening of patients undergoing revision total knee arthroplasty (RTKA). Material and methods The prospective study included 83 patients who were admitted to two trauma and orthopaedic centers for RTKA. The European Bone and Joint Infection Society 2021 (EBJIS21) algorithm was used to detect PJI of the knee joint. The diagnostic value of screening PJI with/without regard to the results of an emergency HE was compared with the results of a microbiological study (MBI) of all types of biomaterials obtained from each patient. Subanalysis was additionally performed in patients with aseptic instability and antimicrobial spacer. Results Pathomorphological examination of freshly frozen and paraffin-embedded tissues showed the difference of 7.2 %, which did not significantly affect the interpretation of the results (p > 0.05). Diagnostically significant pathogens were identified in 83.3 % of cases with PJI confirmed by emergency HE (p < 0.001). A positive emergency HE result increased the chances of isolating diagnostically significant organisms by 34 times (95 % confidence interval (CI): 4.721 – 244.859) as compared with negative HE cases. The proportion of detected cases with emergency HE included in the screening increased from 2.4 to 8.4 %. The inclusion of emergency HE in the screening improved the diagnostic value both in the general cohort of patients and in the subanalysis of comparison groups due to a two-fold increase in sensitivity. Conclusion Relevance of the emergency HE results and the PJI criteria should be considered as a significant prognostic factor for an infectious process, however, this technique should be used only in a complex algorithm for PJI detection. Poor outcomes in 18.2 % of cases of probable PJI necessitated a change in the approach to managing this cohort of patients.

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas.

Background: The preservation of placental and fetal tissues will contribute to studying the pathogenesis of high-risk pregnancy diseases. However, few studies have focused on the effects of different preservation methods and cold ischemia time (CIT) on the quality of nucleic acids. An available quality control (QC) strategy will be beneficial to evaluate these effects for high-risk pregnancy biobanks. Methods: We established an evaluation strategy of nucleic acid QC by analyzing total RNA and genomic DNA (gDNA). Through this strategy, the effects of CIT, cryoprotectants (CPAs), and freeze/thaw cycles on the yield and integrity of placental RNA were analyzed. In addition, the effects of CIT on the yield and integrity of fetal DNA were determined. Results: For placental samples, there was no significant difference in RNA integrity (CIT <2 hours). After several freeze/thaw cycles, the RNA quality number values of placental samples in the CPA-free group and in the RNasin (TRIzol) group were decreased. For fetal samples, the DNA integrity of different organs (CIT <24 hours) was completely satisfactory, but it declined with the extension of CIT. Furthermore, different organs had different tolerances to cold ischemia, and the rank was as follows: skin, heart, liver, and placenta. In addition, the content of medium-length (600 bp) and long (1310 bp) fragments of gDNA were mainly reduced with the extension of CIT. Conclusion: The RNA integrity of placental tissue was affected by CIT significantly. It is recommended that placenta should be cryopreserved within 2 hours (4°C) from isolation. To ensure DNA quality of fetal tissues, the samples are suggested to be frozen within 24 hours (4°C) from isolation. On the contrary, if samples have a long CIT, skin is superior to other organs in the aspect of biobanking donor's genetic information. [ABSTRACT FROM AUTHOR]

The single cell RNA sequencing technique has been particularly used during the last years, allowing major discoveries. However, the widespread application of this analysis has showed limitations. Indeed, the direct study of fresh tissues is not always feasible, notably in the case of genetically engineered mouse embryo or sensitive tissues whose integrity is affected by classical digestion methods. To overcome these limitations, single nucleus RNA sequencing offers the possibility to work with frozen samples. Thus, single nucleus RNA sequencing can be performed after genotyping-based selection on samples stocked in tissue bank and is applicable to retrospective studies. Therefore, this technique opens the field to a wide range of applications requiring adapted protocols for nucleus isolation according to the tissue considered. Here we developed a protocol of nucleus isolation from frozen murine placenta and pancreas. These two complex tissues were submitted to a combination of enzymatic and manual dissociation before undergoing different steps of washing and centrifugation. The entire protocol was performed with products usually present in a research lab. Before starting the sequencing process, nuclei were sorted by flow cytometry. The results obtained validate the efficiency of this protocol which is easy to set up and does not require the use of commercial kits. This specificity makes it adaptable to different organs and species. The association of this protocol with single nucleus RNA sequencing allows the study of complex samples that resist classical lysis methods due to the presence of fibrotic or fatty tissue, such as fibrotic kidney, tumors, embryonic tissues or fatty pancreas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Minati, Fages, Dauguet, Zhu and Jacquemin.)

N-glycolyl GM3 ganglioside (NeuGcGM3) expression in lung carcinomas using formalin-fixed and paraffin-embedded (FFPE) samples has been recently demonstrated. In the present work, it was confirmed the tissue expression of NeuGcGM3 in lung carcinomas by mean of two different mAbs, P3 (anti-NeuGc-containing gangliosides and sulfated glycolipids) and 14F7 (a highly specific for NeuGcGM3) using FFPE samples. In addition, it was reported the tissue expression of NeuGcGM3 in frozen lung carcinoma sections, also supported by the chemical extraction of this ganglioside with organic solvents such as: ethanol, methanol and chloroform/methanol. Moreover, the murine, chimeric and humanized versions of 14F7 mAb showed a similar pattern of immunostaining in this kind of samples. It was also demonstrated that formalin fixation prevent the damage and/or extraction of the antigenic determinant recognized by 14F7 mAb. Consequently, the detection of NeuGcGM3 in frozen samples and their FFPE counterparts was comparable. Our data seems to be in agreement with the potential use of chimeric and/or humanized versions of 14F7 mAb for the passive immuno therapy of lung carcinoma expressing NeuGcGM3. The reactivity of 14F7 murine mAb in FFPE tissues permits to consider it as a useful tool in the selection of patients for specific therapies. [ABSTRACT FROM AUTHOR]

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Background: Genome-wide maps of transcription factor binding sites in primary tissues can expand our understanding of genome function, transcriptional regulation, and genetic alterations that contribute to disease risk. However, almost all genome-wide studies of transcription factors have been in cell lines, and performing these experiments in tissues has been technically challenging and limited in throughput. Results: Here we outline a simple strategy for mapping transcription factor binding sites in frozen tissues that utilizes dry pulverization of samples and is scalable for high-throughput analyses. We show that the method leads to accurate and reproducible chromatin immunoprecipitation next-generation sequencing (ChIP-seq) data, and is highly sensitive, identifying high-quality transcription factor binding sites from chromatin corresponding to only 5 mg of liver tissue. Conclusions: The enhanced reproducibility, robustness, and sensitivity of the dry pulverization method, in addition to the ease of implementation and scalability, makes ChIP-seq in primary tissues a widely accessible assay. [ABSTRACT FROM AUTHOR]

Simple Summary: Circular RNAs are a recently appreciated class of regulatory non-coding RNAs. Although a number of high-resolution methods have been developed for the imaging of RNAs in cells and tissues, there is no reliable method for the simultaneous imaging of circular RNAs distinctly from their linear counterparts. Here, we report circFISH as a simple and single-molecule resolution method that will simultaneously image linear and circular RNAs in fixed cells and tissues. We demonstrate that multiple circular RNAs can be imaged using circFISH. We also show the ability of circFISH to work seamlessly with protein and other organelle imaging. We optimized the method to work across sample types, making it a versatile tool for the functional characterization of circular RNAs. Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method 'circular fluorescent in situ hybridization' (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples. [ABSTRACT FROM AUTHOR]