Multiplexed RNA-FISH-guided Laser Capture Microdissection RNA Sequencing Improves Breast Cancer Molecular Subtyping, Prognostic Classification, and Predicts Response to Antibody Drug Conjugates.

On a retrospective cohort of 1,082 FFPE breast tumors, we demonstrated the analytical validity of a test using multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing (mFISHseq), which showed 93% accuracy compared to immunohistochemistry. The combination of these technologies makes strides in i) precisely assessing tumor heterogeneity, ii) obtaining pure tumor samples using LCM to ensure accurate biomarker expression and multigene testing, and iii) providing thorough and granular data from whole transcriptome profiling. We also constructed a 293-gene intrinsic subtype classifier that performed equivalent to the research based PAM50 and AIMS classifiers. By combining three molecular classifiers for consensus subtyping, mFISHseq alleviated single sample discordance, provided near perfect concordance with other classifiers (κ > 0.85), and reclassified 30% of samples into different subtypes with prognostic implications. We also use a consensus approach to combine information from 4 multigene prognostic classifiers and clinical risk to characterize high, low, and ultra-low risk patients that relapse early (< 5 years), late (> 10 years), and rarely, respectively. Lastly, to identify potential patient subpopulations that may be responsive to treatments like antibody drug-conjugates (ADC), we curated a list of 92 genes and 110 gene signatures to interrogate their association with molecular subtype and overall survival. Many genes and gene signatures related to ADC processing (e.g., antigen/payload targets, endocytosis, and lysosome activity) were independent predictors of overall survival in multivariate Cox regression models, thus highlighting potential ADC treatment-responsive subgroups. To test this hypothesis, we constructed a unique 19-feature classifier using multivariate logistic regression with elastic net that predicted response to trastuzumab emtansine (T-DM1; AUC = 0.96) better than either ERBB2 mRNA or Her2 IHC alone in the T-DM1 arm of the I-SPY2 trial. This test was deployed in a research-use only format on 26 patients and revealed clinical insights into patient selection for novel therapies like ADCs and immunotherapies and de-escalation of adjuvant chemotherapy.

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system.

Sensitive and rapid point-of-care assays have been crucial in the global response to SARS-CoV-2. Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool given its simplicity and minimal equipment requirements, although limitations exist regarding sensitivity and the methods used to detect reaction products. We describe the development of Vivid COVID-19 LAMP, which leverages a metallochromic detection system utilizing zinc ions and a zinc sensor, 5-Br-PAPS, to circumvent the limitations of classic detection systems dependent on pH indicators or magnesium chelators. We make important strides in improving RT-LAMP sensitivity by establishing principles for using LNA-modified LAMP primers, multiplexing, and conducting extensive optimizations of reaction parameters. To enable point-of-care testing, we introduce a rapid sample inactivation procedure without RNA extraction that is compatible with self-collected, non-invasive gargle samples. Our quadruplexed assay (targeting E, N, ORF1a, and RdRP) reliably detects 1 RNA copy/µl of sample (=8 copies/reaction) from extracted RNA and 2 RNA copies/µl of sample (=16 copies/reaction) directly from gargle samples, making it one of the most sensitive RT-LAMP tests and even comparable to RT-qPCR. Additionally, we demonstrate a self-contained, mobile version of our assay in a variety of high-throughput field testing scenarios on nearly 9,000 crude gargle samples. Vivid COVID-19 LAMP can be an important asset for the endemic phase of COVID-19 as well as preparing for future pandemics.

Ejaculatory Abstinence Affects the Sperm Quality in Normozoospermic Men-How Does the Seminal Bacteriome Respond?

This study was designed to describe bacterial profiles of ejaculates collected following a long and short ejaculatory abstinence set in the context of changes in the conventional, oxidative, and immunological characteristics of semen. Two specimens were collected in succession from normozoospermic men (n = 51) following 2 days and 2 h, respectively. Semen samples were processed and analyzed according to the World Health Organization (WHO) 2021 guidelines. Afterwards, sperm DNA fragmentation, mitochondrial function, levels of reactive oxygen species (ROS), total antioxidant capacity, and oxidative damage to sperm lipids and proteins were evaluated in each specimen. Selected cytokine levels were quantified using the ELISA method. Bacterial identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that samples collected following two days of abstinence presented with a higher bacterial load and diversity, and a greater prevalence of potentially uropathogenic bacteria including Escherichia coli, Staphylococcus aureus and Enterococcus faecalis. Only staphylococci and Escherichia coli remained present in specimens obtained after 2 h of abstinence. Whilst all samples accomplished the criteria set by WHO, a significantly higher motility (p < 0.05), membrane integrity (p < 0.05), mitochondrial membrane potential (p < 0.05), and DNA integrity (p < 0.0001) were detected following 2 h of ejaculatory abstinence. On the other hand, significantly higher ROS levels (p < 0.001), protein oxidation (p < 0.001), and lipid peroxidation (p < 0.01) accompanied by significantly higher concentrations of tumor necrosis factor alpha (p < 0.05), interleukin-6 (p < 0.01), and interferon gamma (p < 0.05) were observed in specimens collected after two days of abstinence. It may be summarized that shorter ejaculatory abstinence does not compromise sperm quality in normozoospermic men, while it contributes to a decreased occurrence of bacteria in semen which is accompanied by a lower probability of damage to spermatozoa by ROS or pro-inflammatory cytokines.

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men.

This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis, Staphylococcus capitis and Micrococcus luteus. The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.

The Effect of Non-Thermal Plasma on the Structural and Functional Characteristics of Human Spermatozoa.

Significant antibacterial properties of non-thermal plasma (NTP) have converted this technology into a promising alternative to the widespread use of antibiotics in assisted reproduction. As substantial data available on the specific in vitro effects of NTP on male reproductive cells are currently missing, this study was designed to investigate selected quality parameters of human spermatozoa (n = 51) exposed to diffuse coplanar surface barrier discharge NTP for 0 s, 15 s, 30 s, 60 s and 90 s. Sperm motility characteristics, membrane integrity, mitochondrial activity, production of reactive oxygen species (ROS), DNA fragmentation and lipid peroxidation (LPO) were investigated immediately following exposure to NTP and 2 h post-NTP treatment. Exposure to NTP with a power input of 40 W for 15 s or 30 s was found to have no negative effects on the sperm structure or function. However, a prolonged NTP treatment impaired all the sperm quality markers in a time- and dose-dependent manner. The most likely mechanism of action of high NTP doses may be connected to ROS overproduction, leading to plasma membrane destabilization, LPO, mitochondrial failure and a subsequent loss of motility as well as DNA integrity. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully selected in order to preserve the sperm vitality, should NTP be used in the practical management of bacteriospermia in the future.