Comparative analysis of fixation techniques for signal detection in avian embryos.

The choice of fixation method significantly impacts tissue morphology and visualization of gene expression and proteins after in situ hybridization chain reaction (HCR) or immunohistochemistry (IHC), respectively. In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation techniques prior to HCR and IHC on chicken embryos. Our findings underscore the importance of optimizing fixation methods for accurate visualization and subsequent interpretation of HCR and IHC results, with implications for probe and antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei and neural tubes compared to PFA fixation. Additionally, TCA fixation altered the subcellular fluorescence signal intensity of various proteins, including transcription factors, cytoskeletal proteins, and cadherins. Notably, TCA fixation revealed protein signals in tissues that may be inaccessible with PFA fixation. In contrast, TCA fixation proved ineffective for mRNA visualization. These results highlight the need for optimization of fixation protocols depending on the target and model system, emphasizing the importance of methodological considerations in biological analyses.

A new generation of nanobody research tools using improved mass spectrometry-based discovery methods.

Single-domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, therapeutic, and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research-i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat immunoglobulin G. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

Detection of dendritic cell subsets in the tumor microenvironment by multiplex immunohistochemistry.

Dendritic cells (DCs) are essential in antitumor immunity. In humans, three main DC subsets are defined: two types of conventional DCs (cDC1s and cDC2s) and plasmacytoid DCs (pDCs). To study DC subsets in the tumor microenvironment (TME), it is important to correctly identify them in tumor tissues. Tumor-derived DCs are often analyzed in cell suspensions in which spatial information about DCs which can be important to determine their function within the TME is lost. Therefore, we developed the first standardized and optimized multiplex immunohistochemistry panel, simultaneously detecting cDC1s, cDC2s, and pDCs within their tissue context. We report on this panel's development, validation, and quantitative analysis. A multiplex immunohistochemistry panel consisting of CD1c, CD303, X-C motif chemokine receptor 1, CD14, CD19, a tumor marker, and DAPI was established. The ImmuNet machine learning pipeline was trained for the detection of DC subsets. The performance of ImmuNet was compared with conventional cell phenotyping software. Ultimately, frequencies of DC subsets within several tumors were defined. In conclusion, this panel provides a method to study cDC1s, cDC2s, and pDCs in the spatial context of the TME, which supports unraveling their specific roles in antitumor immunity.

MIF as a potential diagnostic and prognostic biomarker for triple-negative breast cancer that correlates with the polarization of M2 macrophages.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a crucial role in antitumor immunity. However, the role of MIF in influencing the tumor microenvironment (TME) and prognosis of triple-negative breast cancer (TNBC) remains to be elucidated. Using R, we analyzed single-cell RNA sequencing (scRNA-seq) data of 41 567 cells from 10 TNBC tumor samples and spatial transcriptomic data from two patients. Relationships between MIF expression and immune cell infiltration, clinicopathological stage, and survival prognosis were determined using samples from The Cancer Genome Atlas (TCGA) and validated in a clinical cohort using immunohistochemistry. Analysis of scRNA-seq data revealed that MIF secreted by epithelial cells in TNBC patients could regulate the polarization of macrophages into the M2 phenotype, which plays a key role in modulating the TME. Spatial transcriptomic data also showed that epithelial cells (tumor cells) and MIF were proximally located. Analysis of TCGA samples confirmed that tumor tissues of patients with high MIF expression were enriched with M2 macrophages and showed a higher T stage. High MIF expression was significantly associated with poor patient prognosis. Immunohistochemical staining showed high MIF expression was associated with younger patients and worse clinicopathological staging. MIF secreted by epithelial cells may represent a potential biomarker for the diagnosis and prognosis of TNBC and may promote TNBC invasion by remodeling the tumor immune microenvironment.

Characterization of HOXB13 expression patterns in localized and metastatic castration-resistant prostate cancer.

HOXB13 is a key lineage homeobox transcription factor that plays a critical role in the differentiation of the prostate gland. Several studies have suggested that HOXB13 alterations may be involved in prostate cancer development and progression. Despite its potential biological relevance, little is known about the expression of HOXB13 across the disease spectrum of prostate cancer. To this end, we validated a HOXB13 antibody using genetic controls and investigated HOXB13 protein expression in murine and human developing prostates, localized prostate cancers, and metastatic castration-resistant prostate cancers. We observed that HOXB13 expression increases during later stages of murine prostate development. All localized prostate cancers showed HOXB13 protein expression. Interestingly, lower HOXB13 expression levels were observed in higher-grade tumors, although no significant association between HOXB13 expression and recurrence or disease-specific survival was found. In advanced metastatic prostate cancers, HOXB13 expression was retained in the majority of tumors. While we observed lower levels of HOXB13 protein and mRNA levels in tumors with evidence of lineage plasticity, 84% of androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers (NEPCs) retained detectable levels of HOXB13. Notably, the reduced expression observed in NEPCs was associated with a gain of HOXB13 gene body CpG methylation. In comparison to the commonly used prostate lineage marker NKX3.1, HOXB13 showed greater sensitivity in detecting advanced metastatic prostate cancers. Additionally, in a cohort of 837 patients, 383 with prostatic and 454 with non-prostatic tumors, we found that HOXB13 immunohistochemistry had a 97% sensitivity and 99% specificity for prostatic origin. Taken together, our studies provide valuable insight into the expression pattern of HOXB13 during prostate development and cancer progression. Furthermore, our findings support the utility of HOXB13 as a diagnostic biomarker for prostate cancer, particularly to confirm the prostatic origin of advanced metastatic castration-resistant tumors. © 2023 The Pathological Society of Great Britain and Ireland.

Characterization of discordance between mismatch repair deficiency and microsatellite instability testing may prevent inappropriate treatment with immunotherapy.

In the Drug Rediscovery Protocol (DRUP), patients with cancer are treated based on their tumor molecular profile with approved targeted and immunotherapies outside the labeled indication. Importantly, patients undergo a tumor biopsy for whole-genome sequencing (WGS) which allows for a WGS-based evaluation of routine diagnostics. Notably, we observed that not all biopsies of patients with dMMR/MSI-positive tumors as determined by routine diagnostics were classified as microsatellite-unstable by subsequent WGS. Therefore, we aimed to evaluate the discordance rate between routine dMMR/MSI diagnostics and WGS and to further characterize discordant cases. We assessed patients enrolled in DRUP with dMMR/MSI-positive tumors identified by routine diagnostics, who were treated with immune checkpoint blockade (ICB) and for whom WGS data were available. Patient and tumor characteristics, study treatment outcomes, and material from routine care were retrieved from the patient medical records and via Palga (the Dutch Pathology Registry), and were compared with WGS results. Initially, discordance between routine dMMR/MSI diagnostics and WGS was observed in 13 patients (13/121; 11%). The majority of these patients did not benefit from ICB (11/13; 85%). After further characterization, we found that in six patients (5%) discordance was caused by dMMR tumors that did not harbor an MSI molecular phenotype by WGS. In six patients (5%), discordance was false due to the presence of multiple primary tumors (n = 3, 2%) and misdiagnosis of dMMR status by immunohistochemistry (n = 3, 2%). In one patient (1%), the exact underlying cause of discordance could not be identified. Thus, in this group of patients limited to those initially diagnosed with dMMR/MSI tumors by current routine diagnostics, the true assay-based discordance rate between routine dMMR/MSI-positive diagnostics and WGS was 5%. To prevent inappropriate ICB treatment, clinicians and pathologists should be aware of the risk of multiple primary tumors and the limitations of different tests. © 2024 The Pathological Society of Great Britain and Ireland.

Dissecting the immune infiltrate of primary luminal B-like breast carcinomas in relation to age.

The impact of aging on the immune landscape of luminal breast cancer (Lum-BC) is poorly characterized. Understanding the age-related dynamics of immune editing in Lum-BC is anticipated to improve the therapeutic benefit of immunotherapy in older patients. To this end, here we applied the 'multiple iterative labeling by antibody neo-deposition' (MILAN) technique, a spatially resolved single-cell multiplex immunohistochemistry method. We created tissue microarrays by sampling both the tumor center and invasive front of luminal breast tumors collected from a cohort of treatment-naïve patients enrolled in the prospective monocentric IMAGE (IMmune system and AGEing) study. Patients were subdivided into three nonoverlapping age categories (35-45 = 'young', n = 12; 55-65 = 'middle', n = 15; ≥70 = 'old', n = 26). Additionally, depending on localization and amount of cytotoxic T lymphocytes, the tumor immune types 'desert' (n = 22), 'excluded' (n = 19), and 'inflamed' (n = 12) were identified. For the MILAN technique we used 58 markers comprising phenotypic and functional markers allowing in-depth characterization of T and B lymphocytes (T&B-lym). These were compared between age groups and tumor immune types using Wilcoxon's test and Pearson's correlation. Cytometric analysis revealed a decline of the immune cell compartment with aging. T&B-lym were numerically less abundant in tumors from middle-aged and old compared to young patients, regardless of the geographical tumor zone. Likewise, desert-type tumors showed the smallest immune-cell compartment and were not represented in the group of young patients. Analysis of immune checkpoint molecules revealed a heterogeneous geographical pattern of expression, indicating higher numbers of PD-L1 and OX40-positive T&B-lym in young compared to old patients. Despite the numerical decline of immune infiltration, old patients retained higher expression levels of OX40 in T helper cells located near cancer cells, compared to middle-aged and young patients. Aging is associated with important numerical and functional changes of the immune landscape in Lum-BC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer.

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Automated tumor immunophenotyping predicts clinical benefit from anti-PD-L1 immunotherapy.

Cancer immunotherapy has transformed the clinical approach to patients with malignancies, as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. We developed approaches to categorize solid tumors into 'desert', 'excluded', and 'inflamed' types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach, we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. We show that categorization based on 'manual' observation is predictive for clinical benefit from anti-programmed death ligand 1 therapy in two large cohorts of patients with non-small cell lung cancer or triple-negative breast cancer. For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based readouts and the patient's response to therapy. Our findings suggest that tumor immunophenotype generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. © 2024 The Pathological Society of Great Britain and Ireland.

Clinical Proteomics for Solid Organ Tissues.

The evaluation of biopsied solid organ tissue has long relied on visual examination using a microscope. Immunohistochemistry is critical in this process, labeling and detecting cell lineage markers and therapeutic targets. However, while the practice of immunohistochemistry has reshaped diagnostic pathology and facilitated improvements in cancer treatment, it has also been subject to pervasive challenges with respect to standardization and reproducibility. Efforts are ongoing to improve immunohistochemistry, but for some applications, the benefit of such initiatives could be impeded by its reliance on monospecific antibody-protein reagents and limited multiplexing capacity. This perspective surveys the relevant challenges facing traditional immunohistochemistry and describes how mass spectrometry, particularly liquid chromatography-tandem mass spectrometry, could help alleviate problems. In particular, targeted mass spectrometry assays could facilitate measurements of individual proteins or analyte panels, using internal standards for more robust quantification and improved interlaboratory reproducibility. Meanwhile, untargeted mass spectrometry, showcased to date clinically in the form of amyloid typing, is inherently multiplexed, facilitating the detection and crude quantification of 100s to 1000s of proteins in a single analysis. Further, data-independent acquisition has yet to be applied in clinical practice, but offers particular strengths that could appeal to clinical users. Finally, we discuss the guidance that is needed to facilitate broader utilization in clinical environments and achieve standardization.

Interneurons in the CA1 stratum oriens expressing αTTP may play a role in the delayed-ageing Pol µ mouse model.

Neurodegeneration associated with ageing is closely linked to oxidative stress (OS) and disrupted calcium homeostasis. Some areas of the brain, like the hippocampus - particularly the CA1 region - have shown a high susceptibility to age-related changes, displaying early signs of pathology and neuronal loss. Antioxidants such as α-tocopherol (αT) have been effective in mitigating the impact of OS during ageing. αT homeostasis is primarily regulated by the α-tocopherol transfer protein (αTTP), which is widely distributed throughout the brain - where it plays a crucial role in maintaining αT levels within neuronal cells. This study investigates the distribution of αTTP in the hippocampus of 4- and 24-month-old Pol µ knockout mice (Pol µ-/-), a delayed-ageing model, and the wild type (Pol µ+/+). We also examine the colocalisation in the stratum oriens (st.or) of CA1 region with the primary interneuron populations expressing calcium-binding proteins (CBPs) (calbindin (CB), parvalbumin (PV), and calretinin (CR)). Our findings reveal that αTTP immunoreactivity (-IR) in the st.or of Pol µ mice is significantly reduced. The density of PV-expressing interneurons (INs) increased in aged mice in both Pol µ genotypes (Pol µ-/- and Pol µ+/+), although the density of PV-positive INs was lower in the aged Pol µ-/- mice compared to wild-type mice. By contrast, CR- and CB-positive INs in Pol µ mice remained unchanged during ageing. Furthermore, double immunohistochemistry reveals the colocalisation of αTTP with CBPs in INs of the CA1 st.or. Our study also shows that the PV/αTTP-positive IN population remains unchanged in all groups. A significant decrease of CB/αTTP-positive INs in young Pol µ-/- mice has been detected, as well as a significant increase in CR/αTTP-IR in older Pol µ-/- animals. These results suggest that the differential expression of αTTP and CBPs could have a crucial effect in aiding the survival and maintenance of the different IN populations in the CA1 st.or, and their coexpression could contribute to the enhancement of their resistance to OS-related damage and neurodegeneration associated with ageing.

Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue.

BACKGROUND: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. RESULTS: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. CONCLUSIONS: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.

Disruption of Fuz in mouse embryos generates hypoplastic hindbrain development and reduced cranial nerve ganglia.

BACKGROUND: The brain and spinal cord formation is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Environmental or genetic interferences can impair neurulation, resulting in clinically significant birth defects known collectively as neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. RESULTS: We demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent reduction of ventral neuroepithelial stiffness in a notochord adjacent area at the level of the rhombomere 5. The formation of cranial and paravertebral ganglia is also impaired in these embryos. CONCLUSIONS: This study reveals that hypoplastic hindbrain development, identified by abnormal rhombomere morphology and persistent loss of ventral neuroepithelial stiffness, precedes exencephaly in Fuz ablated murine mutants, indicating that the gene Fuz has a critical function sustaining normal neural tube development and neuronal differentiation.

Single-nucleotide polymorphism array and fluorescence in situ hybridization analysis to decode the cytogenetic profile of atypical partial hydatidiform moles diagnosed by short tandem repeat polymorphism analysis.

Accurate diagnosis of partial hydatidiform moles (PHMs) is crucial for improving outcomes of gestational trophoblastic neoplasia. The use of short tandem repeat (STR) polymorphism analysis to distinguish between PHM and hydropic abortuses is instrumental; however, its diagnostic power has not been comprehensively assessed. Herein, we evaluated the diagnostic efficacy of STR in differentiating between PHM and hydropic abortus, thus providing an opportunity for early measurement of human chorionic gonadotropin for PHMs. We reviewed charts of STR polymorphism analysis performed on fresh villous specimens and patient blood samples using a commercial kit for 16 loci. The genetic classification of 79 PHMs was confirmed. STR was reliable in differentiating PHMs when at least 15 loci were available. Typically, PHMs are characterized by their triploidy, including two paternal and one maternal haploid contribution. In our sample, seven PHMs lacked the three-allelic loci, requiring fluorescence in situ hybridization (FISH) analysis to investigate imbalanced biparental conceptus and single-nucleotide polymorphism array analysis to reveal cytogenetic details. Of these PHMs, two, three, and one were identified as androgenetic/biparental mosaics (diploids), monospermic diandric monogynic triploids, and a typical dispermic diandric monogynic triploid, respectively. The remaining case was monospermic origin, but its ploidy details could not be available. Therefore, STR differentiated PHM from a biparental diploid abortus in most cases. However, PHM diagnosis may be compromised when STR is used as the sole method for cases displaying distinct cytogenetic patterns lacking the three-allelic loci, including androgenetic/biparental mosaicism. Therefore, FISH should be considered to confirm the diagnosis.

Transcriptomics and Spatial Proteomics for Discovery and Validation of Missing Proteins in the Human Ovary.

Efforts to understand the complexities of human biology encompass multidimensional aspects, with proteins emerging as crucial components. However, studying the human ovary introduces unique challenges due to its complex dynamics and changes over a lifetime, varied cellular composition, and limited sample access. Here, four new RNA-seq samples of ovarian cortex spanning ages of 7 to 32 were sequenced and added to the existing data in the Human Protein Atlas (HPA) database www.proteinatlas.org, opening the doors to unique possibilities for exploration of oocyte-specific proteins. Based on transcriptomics analysis of the four new tissue samples representing both prepubertal girls and women of fertile age, we selected 20 protein candidates that lacked previous evidence at the protein level, so-called "missing proteins" (MPs). The proteins were validated using high-resolution antibody-based profiling and single-cell transcriptomics. Fourteen proteins exhibited consistent single-cell expression patterns in oocytes and granulosa cells, confirming their presence in the ovary and suggesting that these proteins play important roles in ovarian function, thus proposing that these 14 proteins should no longer be classified as MPs. This research significantly advances the understanding of MPs, unearthing fresh avenues for prospective exploration. By integrating innovative methodologies and leveraging the wealth of data in the HPA database, these insights contribute to refining our understanding of protein roles within the human ovary and opening the doors for further investigations into missing proteins and human reproduction.

Inhibition and potentiation of the exercise pressor reflex by pharmacological modulation of TRPC6 in male rats.

We determined the role played by the transient receptor potential canonical 6 (TRPC6) channel in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. TRPC6 channels were identified by quadruple-labelled (DiI, TRPC6, neurofilament-200 and peripherin) immunohistochemistry in dorsal root ganglion (DRG) cells innervating the triceps surae muscles (n = 12). The exercise pressor reflex was evoked by statically contracting the triceps surae muscles before and after injection of the TRPC6 antagonist BI-749327 (n = 11; 12 µg kg-1 ) or SAR7334 (n = 11; 7 µg kg-1 ) or the TRPC6 positive modulator C20 (n = 11; 18 µg kg-1 ). Similar experiments were conducted while the muscles were passively stretched (n = 8-12), a manoeuvre that isolated the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded. Of the DRG cells innervating the triceps surae muscles, 85% stained positive for the TRPC6 antigen, and 45% of those cells co-expressed neurofilament-200. Both TRPC6 antagonists decreased the reflex pressor responses to static contraction (-32 to -42%; P < 0.05) and to passive stretch (-35 to -52%; P < 0.05), whereas C20 increased these responses (55-65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05), whereas C20 increased the RSNA to passive stretch only. The onset latency of the decrease or increase in RSNA occurred within 2 s of the onset of the manoeuvres (P < 0.05). Collectively, our results show that TRPC6 plays a key role in evoking the mechanical component of the exercise pressor reflex. KEY POINTS: The exercise pressor reflex plays a key role in the sympathetic and haemodynamic responses to exercise. This reflex is composed of two components, namely the mechanoreflex and the metaboreflex. The receptors responsible for evoking the mechanoreflex are poorly documented. A good candidate for this function is the transient receptor potential canonical 6 (TRPC6) channel, which is activated by mechanical stimuli and expressed in dorsal root ganglia of rats. Using two TRPC6 antagonists and one positive modulator, we investigated the role played by TRPC6 in evoking the mechanoreflex in decerebrated rats. Blocking TRPC6 decreased the renal sympathetic and the pressor responses to both contraction and stretch, the latter being a manoeuvre that isolates the mechanoreflex. In contrast, the positive modulator increased the pressor reflex to contraction and stretch, in addition to the sympathetic response to stretch. Our results provide strong support for a role played by the TRPC6 channel in evoking the mechanoreflex.

Neutrophil Extracellular Traps and Thrombolysis Resistance: New Insights for Targeting Therapies.

BACKGROUND: Thrombosis is linked to neutrophil release of neutrophil extracellular traps (NETs). NETs are proposed as a mechanism of resistance to thrombolysis. This study intends to analyze the composition of thrombi retrieved after mechanical thrombectomy, estimate the age and organization of thrombi, and evaluate associations with the use of thrombolysis, antiplatelets, and heparin. METHODS: This retrospective observational study involved 72 samples (44 from cerebral and 28 coronary arteries), which were stained with hematoxylin and eosin, anti-NE (neutrophil elastase) antibody, and anti-histone H2B (histone H2B) antibody, representing different components in NET formation, all detectable during the later stages of NETosis, for histochemical and digital quantification of NET content. The histological and morphological evaluations of the specimens were correlated, through univariate and mediation analyses, with clinical information and therapy administered before intervention. RESULTS: The results demonstrated that the composition of cerebral and coronary thrombi differs, and there were significantly more lytic cerebral thrombi than coronary thrombi (66% versus 14%; P=0.005). There was a considerably higher expression of NETs in the cerebral thrombi as testified by the higher expression of H2B (P=0.031). Thrombolysis was remarkably associated with higher NE positivity (average marginal effect, 6.461 [95% CI, 0.7901-12.13]; P=0.02555), regardless of the origin of thrombi. There was no notable association between the administration of antiaggregant therapy/heparin and H2B/NE amount when adjusted for the thrombus location. Importantly, the age of the thrombus was the only independent predictor of NET content without any mediation of the thrombolytic treatment (P=0.014). CONCLUSIONS: The age of the thrombus is the driving force for NET content, which correlates with impaired clinical outcomes. The therapy that is currently administered does not modify NET content. This study supports the need to investigate new pharmacological approaches added to thrombolysis to prevent NET formation or enhance their disruption, such as recombinant human DNase I (deoxyribonuclease I).

Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.