ALL-tRNAseq enables robust tRNA profiling in tissue samples.

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.

In vivo assessment of TiO2 based wear nanoparticles in periprosthetic tissues.

A multimodal approach combining inductively coupled plasma mass spectrometry (ICP-MS), single-particle ICP-MS (spICP-MS), scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman spectroscopy enabled a deeper insight into the balance between total titanium (Ti), the soluble titanium fraction and titanium dioxide based particle fraction levels in periprosthetic tissues collected from patients undergoing revision surgery. Hydrofluoric acid usage in the sample digestion allowed for complete digestion of TiO2 particles, thus enabling accurate estimation of total Ti levels. The TiO2 fraction represents 38-94% of the titanium load in the six samples where particles were detected, and the fraction is present mainly in samples from patients with aseptically loosened total hip arthroplasty. Further attention was given to this fraction determining the elemental composition, particle count, particle size and modification of TiO2. The spICP-MS analysis confirmed the presence of the TiO2-derived (nano)particles (NPs) with a 39- to 187-nm median size and particle count up to 2.3 × 1011 particles per gram of tissue. On top of that, the SEM-EDS confirmed the presence of the TiO2 nanoparticles with 230-nm median size and an anatase crystal phase was determined by Raman spectroscopy. This study presents a novel multimodal approach for TiO2 particle determination and characterization in tissue samples and is the first in vivo study of this character.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of the acetyl-coenzyme A competitive p300/CBP catalytic inhibitor A-485 in biological samples.

The small molecule A-485 competitively inhibits the histone acetyltransferase domain of CBP (cyclic-adenosine monophosphate response element-binding protein)/p300. Apart from its antineoplastic activity, researchers are exploring its potential benefits in treating osteoporosis and its impact on energy metabolism. However, so far, only limited pharmacokinetic data are available, and the crucial determination of A-485 concentration in various biological materials with small sample volumes remains unpublished. A rapid and sensitive LC-tandem mass spectrometry method has been developed and validated to quantify A-485 in mouse serum and tissue. In this method, serum samples underwent precipitation with acetonitrile, while cell lysates were appropriately diluted. The determination of A-485 utilized a reversed-phase column with a mobile phase gradient, and detection was carried out in multiple reaction monitoring mode. The lower standard sample, with a concentration of 7.8 ng/mL, served as the lower limit of quantification, while the upper standard was established at 1000 ng/mL. A-485 concentrations were assessed in both serum samples and the lysate of all examined tissues, revealing swift metabolic clearance. The analytical method outlined here is deemed appropriate for subsequent studies. The ability to measure the active ingredient in various compartments facilitates the determination of accurate pharmacokinetic parameters. In the event of human use of A-485, the analysis method can be seamlessly transferred to human samples.

Development and validation of a one-tube, nested real-time PCR method suitable for routine detection of Mycobacterium bovis in animal tissue.

AIMS: Development and validation of a real-time PCR test for high-throughput routine screening of animal tissue for Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) members. METHODS AND RESULTS: A preliminary study compared the results of a combination of five tissue preparation/DNA extraction methods and nine PCR assays on a panel of 92 cattle tissue samples of known M. bovis culture status (55 positive and 37 negative). The combination of DNA extraction and PCR was found to be important in achieving optimal detection of M. bovis. The optimal combination of a simple tissue preparation/DNA extraction method and a one-tube, nested real-time PCR to maximize the sensitivity of detection of an M. bovis-specific RD4 deletion and an IS1081 MTBC-specific target was selected for further evaluation. In total, tissue samples collected from 981 cattle and 366 non-bovine animals and submitted for routine TB culture were parallel tested with the selected method, as well as tissue samples obtained from 156 animals in certified TB-free cattle herds. CONCLUSION: For cattle, the optimized RD4-IS1081 PCR test exhibited a diagnostic sensitivity of 96% (95% CI: 94-97%) and specificity of 97% (95% CI: 95-98%) compared to culture. Specificity was 100% when testing the 156 samples from known TB-free cattle. For non-bovine species, the PCR had a diagnostic sensitivity of 93% (95% CI: 83-98%) and a specificity of 99% (95% CI: 97-100%).

Test of the FlashFREEZE unit in tissue samples freezing for biobanking purposes.

Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.

Tissue sampling is non-inferior in comparison to sonication in orthopedic revision surgery.

BACKGROUND: The aim of this study was to assess the role of sonication fluid cultures in detecting musculoskeletal infections in orthopedic revision surgery in patients suspected of having peri-prosthetic joint infection (PJI), fracture-related infections (FRI), or postoperative spinal implant infections (PSII). METHODS: Between 2016 and 2019, 149 cases with a data set including sonication fluid cultures and tissue specimen and histological analysis were included. Accuracy of each diagnostic tool as well as the influence of antibiotic therapy was analyzed. Pathogens identified in the sonication cultures and in the associated tissue samples were compared based on the matching of the antibiograms. Therapeutic benefits were then assessed. RESULTS: Of 149 cases, 43.6% (n = 65) were identified as PJI, 2.7% (n = 4) as FRI, 12.8% (n = 19) as PSII, 6.7% (n = 10) as aseptic non-union, and 34.2% (n = 51) as aseptic implant loosening. The sensitivity and specificity of tissue and synovial specimens showed no significant difference with respect to sonication fluid cultures (sensitivity/specificity: tissue: 68.2%/96.7%; sonication fluid cultures: 60.2%/98.4%). The administration of antibiotics over 14 days prior to microbiological sampling (n = 40) resulted in a lower sensitivity of 42.9% each. Histological analysis showed a sensitivity 86.3% and specificity of 97.4%. In 83.9% (n = 125) of the cases, the results of sonication fluid cultures and tissue specimens were identical. Different microorganisms were found in only four cases. In 17 cases, tissue samples (n = 5) or sonication (n = 12) were false-negatives. CONCLUSION: Sonication fluid culture showed no additional benefit compared to conventional microbiological diagnostics of tissue and synovial fluid cultures. Preoperative administration of antibiotics had a clearly negative effect on microbiologic test accuracy. In over 83.9% of the cases, sonication fluid and tissue cultures showed identical results. In the other cases, sonication fluid culture did not further contribute to the therapy decision, whereas other factors, such as fistulas, cell counts, or histological analysis, were decisive in determining therapy.

[The use of immunohistochemical slides for performing FISH as a useful method of conserving tissue samples]. / Ré-utilisation de lames d'immunohistochimie pour la réalisation de FISH : une solution pertinente d'épargne tissulaire.

Diagnostic updates, an increased precision of tumor sub-type classification and the development of new diagnostic biomarkers (immunohistochemistry (IHC), Fluorescence in situ hybridization (FISH) and other molecular pathology techniques), have a significant impact on pathologists' management of tissue samples. The objective of this work was to test and validate the FISH technique on detached IHC slides. An IHC technique was first performed on 30 tissue samples. After detachment of the lamella, a FISH technique was then performed according to the usual protocol with a centromeric probe. A validation cohort (n=10) with duplicate testing using a traditional FISH technique and an IHC slide with a detached lamella was then carried out. Finally, a cohort of 20 "old" cases (IHC carried out over 2years ago) was also tested. Different types of probes (specific locus, break apart) have been used. All the slides were interpreted by a technician and a pathologist. Evaluation criteria were: the general interpretability of the slide ; the percentage of labeled nuclei; intensity of the signal and the presence or absence of autofluorescence. FISH was interpretable in 100% of recently treated cases and 90% of "old" cases with a satisfactory intensity and a high percentage of labeled nuclei, without autofluorescence. The results of our study show that the reuse of IHC slides for performing FISH is a powerful means of economizing tissue samples, especially for small samples and in the absence of archived representative material.

WITHDRAWN: lncRNA Linc00173 modulates glucose metabolism and multidrug chemoresistance in SCLC: Potential molecular panel for targeted therapy.

This article has been withdrawn at the request of the editor-in-chief. Following publication of this article, the editor-in-chief discovered evidence of image duplication in Figures 1I, 1J, 3F, S5B, and S6B. Given the duplication of several western blots representing several gene products, the editor-in-chief has lost faith in the findings presented in this article. The authors maintain that these image duplications were the result of errors in file management and do not affect the conclusions of the study. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

[Pitfalls in dermatohistology : Stumbling blocks and problems in routine dermatopathology]. / Fallstricke in der Dermatohistologie : Von Stolpersteinen und Problemen im dermatopathologischen Alltag.

Excisions and biopsies are firmly anchored in everyday dermatology. The biopsy, excision or diagnostic-therapeutic confirmation of the clinical diagnosis of neoplasms or inflammatory diseases is decisive for the dermatopathological diagnosis of tissue samples. Dermatopathology, however, is not a magic box into which a tissue sample can be placed without comment or information and receive-within 24 h at the latest-a complete, high-quality diagnosis. The present article describes problems, hurdles, and challenges in everyday dermatopathology that occur on the way to the microscope, even before the actual dermatopathological diagnosis takes place.

Direct PCR on Tissue Samples To Detect Mycobacterium tuberculosis Complex: an Alternative to the Bacteriological Culture.

Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union. Microbiological culture is the official confirmation technique for the presence of Mycobacterium tuberculosis complex (MTBC) members in bovine tissues, but several methodological issues, such as moderate sensitivity and long incubation times, require the development of more sensitive and rapid techniques. This study evaluates the analytical and diagnostic performance, comparative to culture, of a real-time PCR targeting the MTBC-specific IS6110 transposon using a panel of bovine tissue samples sourced from the Spanish bTB eradication campaign. Robustness and repeatability were evaluated in an interlaboratory trial between European Union National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic equivalents. Diagnostic sensitivity (Se) and specificity (Sp) were, respectively, 96.45% and 93.66%, and the overall agreement (κ) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as Mycobacterium marinum and "Mycobacterium avium subsp. hominissuis," and whole-genome sequencing (WGS) analysis of the latter isolate revealed an IS6110-like sequence with 83% identity. An identical IS-like element was found in other Mycobacterium avium complex species in the NCBI nucleotide and WGS databases. Despite this finding, this methodology is considered a valuable alternative to culture, and the strategy of use should be defined depending on the control or eradication programs.

Post-mortem persistence of SARS-CoV-2: a preliminary study.

Since the beginning of March 2020, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been the cause of millions of deaths worldwide. The need to better define the pathogenesis of coronavirus disease 19 (Covid-19) as well as to provide the correct statistical records concerning deaths related to this virus, inevitably involves the role of forensic pathology and routine autopsy practice. Currently, some data on macroscopic and microscopic features in autopsies performed in suspected Covid-19 cases are reported in the literature. The persistence of SARS-CoV-2 in cadavers has not yet been elucidated and only a few reports have emphasized the importance of evaluating the Virus RNA in post-mortem tissues. In this preliminary study, we observed that SARS-CoV-2 survives in multiple cadaver tissues many days after death despite some extreme conditions of post-mortem body preservation. The results of this on-going analysis could help improve the safety of working practices for pathologists as well as understanding the possible interaction between microbiological agents and the cadaver tissue's supravital reactions.

Data-dependent normalization strategies for untargeted metabolomics-a case study.

Despite the recent advances in the standardization of untargeted metabolomics workflows, there is still a lack of attention to specific data treatment strategies that require deep knowledge of the biological problem and need to be applied after a well-thought out process to understand the effect of the practice. One of those strategies is data normalization. Data-driven assumptions are critical especially addressing unwanted variation present in the biological model as it can be the case in heterogeneous tissues, cells with different sizes or biofluids with different concentrations. Chronic kidney disease (CKD) is a widespread disorder affecting kidney structure and function. Animal models are being developed to be able to get valuable insights into the etiopathogenesis of the condition and effect of the treatments. Moreover, diagnosis and disease staging still require defining appropriate biomarkers. Untargeted metabolomics has the potential to deal with those challenges. Renal fibrosis is one of the consequences of kidney injury which greatly affects the concentration of metabolites in the same quantity of sample. To overcome this challenge, several data normalization strategies have been applied, following a multilevel normalization method with the overall aim of focussing on the relevant biological information and reducing the influence of disturbing factors. A comprehensive evaluation of the performance of the normalization strategies, both on methods assessing the intragroup variation and on the impact on differential analysis, is provided. Finally, we present evidence of the importance of biological-model-driven guided normalization methods and discuss multiple criteria that need to be taken into consideration to obtain robust and reliable data. Special concern is transmitted on the misleading conclusions that might be the consequence of inappropriate data pre-treatment solutions applied for untargeted methods. Graphical abstract.

Pleural mesothelioma and lung cancer: the role of asbestos exposure and genetic variants in selected iron metabolism and inflammation genes.

Two of the major cancerous diseases associated with asbestos exposure are malignant pleural mesothelioma (MPM) and lung cancer (LC). In addition to asbestos exposure, genetic factors have been suggested to be associated with asbestos-related carcinogenesis and lung genotoxicity. While genetic factors involved in the susceptibility to MPM were reported, to date the influence of individual genetic variations on asbestos-related lung cancer risk is still poorly understood. Since inflammation and disruption of iron (Fe) homeostasis are hallmarks of asbestos exposure affecting the pulmonary tissue, this study aimed at investigating the association between Fe-metabolism and inflammasome gene variants and susceptibility to develop LC or MPM, by comparing an asbestos-exposed population affected by LC with an "asbestos-resistant exposed population". A retrospective approach similar to our previous autopsy-based pilot study was employed in a novel cohort of autoptic samples, thus giving us the possibility to corroborate previous findings obtained on MPM by repeating the analysis in a novel cohort of autoptic samples. The protective role of HEPH coding SNP was further confirmed. In addition, the two non-coding SNPs, either in FTH1 or in TF, emerged to exert a similar protective role in a new cohort of LC exposed individuals from the same geographic area of MPM subjects. No association was found between NLRP1 and NLRP3 polymorphisms with susceptibility to develop MPM and LC. Further research into a specific MPM and LC "genetic signature" may be needed to broaden our knowledge of the genetic landscape attributed to result in MPM and LC.

DNA extraction from FFPE tissue samples - a comparison of three procedures.

AIM OF THE STUDY: One of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA. MATERIAL AND METHODS: Slides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies). RESULTS: The results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method. CONCLUSIONS: To our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA.

In situ detection of CD73+ CD90+ CD105+ lineage: Mesenchymal stromal cells in human placenta and bone marrow specimens by chipcytometry.

Mesenchymal stromal cells (MSCs) support endogenous regeneration and present therefore promising opportunities for in situ tissue engineering. They can be isolated and expanded from various tissues, for example, bone marrow, adipose tissue, or placenta. The minimal consensus definition criteria of ex vivo expanded MSCs requires them to be positive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, CD14, CD19, and HLA-DR. This study aimed to compare the in situ phenotype of MSCs with that of their culture-expanded progeny. We report for the first time in situ detection of cells expressing this marker combination in human placenta cryosections as well as in bone marrow aspirates using multiplex-immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell. © 2018 International Society for Advancement of Cytometry.

Muscle fiber diameter assessment in cleft lip using image processing.

OBJECTIVE: To pilot investigation of muscle fiber diameter (MFD) on medial and lateral sides of the cleft in 18 infants with cleft lip with or without cleft palate (CL/P) using image processing. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue samples from the medial and lateral sides of the cleft were analyzed for MFD using an image-processing program (ImageJ). For within-case comparison, a paired Student's t test was performed. For comparisons between classes, an unpaired t test was used. RESULTS: Image processing enabled rapid measurement of MFD with majority of fibers showing diameter between 6 and 11 µm. There was no significant difference in mean MFD between the medial and lateral sides, or between CL and CLP. However, we found a significant difference on the medial side (p = .032) between males and females. CONCLUSION: The image processing on FFPE tissues resulted in easy quantification of MFD with finding of a smaller MFD on the medial side in males suggesting possible differences in orbicularis oris (OO) muscle between the two sexes in CL that warrants replication using larger number of cases. Moreover, this finding can aid subclinical phenotyping and potentially in the restoration of the anatomy and function of the upper lip.

MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples.

Histone post-translational modifications (PTMs), histone variants and enzymes responsible for the incorporation or the removal of the PTMs are being increasingly associated with human disease. Combinations of histone PTMs and the specific incorporation of variants contribute to the establishment of cellular identity and hence are potential markers that could be exploited in disease diagnostics and prognostics and therapy response prediction. Due to the scarcity of suitable antibodies and the pre-requirement of tissue homogenization for more advanced analytical techniques, comprehensive information regarding the spatial distribution of these factors at the tissue level has been lacking. MALDI imaging mass spectrometry provides an ideal platform to measure histone PTMs and variants from tissues while maintaining the information about their spatial distribution. Discussed in this review are the relevance of histones in the context of human disease and the contribution of MALDI imaging mass spectrometry in measuring histones in situ.

Whirling agitated single drop microextraction technique for the simultaneous analysis of Paraquat and Maneb in tissue samples of treated mice.

A new microextraction technique, whirling agitated single drop microextraction, has been proposed for the simultaneous analysis of Paraquat and Maneb in tissue samples before liquid chromatography with tandem mass spectrometry. This technique is based on the idea that the escalatory motion of the sample solution along with the extraction solvent increases the movement of molecules into the extraction solvent. In this technique, a simple handheld rotator was utilized to rapidly agitate the biphasic extraction system for the instantaneous extraction of targeted analytes. After extraction, the extracted phase was directly solidified by cooling in crushed ice and easily collected using a micro-spatula. The method showed good performance by achieving sensitive detection limits at 4.81 ng g(-1) (Paraquat) and 9.12 ng g(-1) (Maneb). Mean recoveries and enrichment factors were obtained >91.21% and up to 114 that ensured the preconcentration capacity of the method. The method precision was verified by evaluating intraday variation (n = 10) ≤4.57 (Paraquat) and ≤4.68 (Maneb) in terms of percent relative standard deviation. Additionally, method efficacy was assured by obtaining very little matrix interferences (≤3.11%). Moreover, the method suitability was also checked with its application on tissue samples of intraperitoneally treated mice with Paraquat and Maneb.

Ultrafast extraction of proteins from tissues using desorption by impulsive vibrational excitation.

A picosecond IR laser (PIRL) can be used to blast proteins out of tissues through desorption by impulsive excitation (DIVE) of intramolecular vibrational states of water molecules in the cell in less than a millisecond. With PIRL-DIVE proteins covering a range of a few kDa up to several MDa are extracted in high quantities compared to conventional approaches. The chemical composition of extracted proteins remains unaltered and even enzymatic activities are maintained.

Forensic utility of carboxyhemoglobin levels in postmortem spleen specimens in South Korea.

In order to determine whether CO poisoning was the definitive cause of death, the concentration of carboxyhemoglobin (COHb) in spleen specimens was analyzed using a gas chromatography-thermal conductivity detector. 125 cases of forensic autopsy reports containing COHb analysis requests were analyzed and subdivided into two groups, improbable and highly probable of CO intoxication. In the first group which consists of 100 cases, the results of COHb analysis were negative, and the circumstances of death, as well as the postmortem findings could not validate the exposure to CO. In the second group which consists of 25 cases, the results of COHb were positive, and both postmortem findings and circumstances of death confirmed the exposure to CO. In the cases of indoors and vehicle fires or those including the use of briquettes, COHb levels reached 43.1-97.5 %, whereas in individuals without any feature of CO poisoning had COHb level high as 29.8 %. However, certain cases without any connection to fire nor CO exposure also contained significant amount of CO based on post-mortem analysis. This study focuses on cases without any relationship to fire or CO and proves that COHb levels below 30 % may be considered as a contributing factor to but not exclusively as the cause of death.