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Purpose: To investigate bone regeneration among three different bone graft materials in a rat calvarum model. Materials and Methods: A total of 24 rats had two 5-mm defects placed per calvarial. Rats were divided into four groups: bovine xenograft (XG), demineralized bone matrix (DBM), mineralized bone graft (MBG), and collagen membrane control (CC). Within each group, samples were collected at two time points: 4 weeks (T4) and 8 weeks (T8). Bone regeneration was assessed by microcomputed tomography (micro-CT) imaging and was analyzed using MATLAB software. Additionally, the fixed samples were subsequently demineralized for immunohistochemistry and histomorphometry. Slides were mounted and stained with hematoxylin and eosin (H&E) stain as well as bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (RUNX2) markers. The numbers of positive cells/area were calculated for each group and analyzed. Results: At 4 weeks, DBM showed low mineral density (7.7%) compared to the control (25.2%), but increased dramatically at 8 weeks (DBM, T8 = 27.6%; CC, T8 = 27.2%). Xenograft material showed an increase in mineral desnity between T4 and T8 (XG, T4 = 25.0%; XG, T8 = 32.3%). MBG remained consistent over the 8-week trial period (MBG, T4 = 30.4%; MBG, T8 = 30.4%). BMP-2 expression was present in cells adherent to all graft materials. RUNX2 expression was also observed in cells adherent to all graft materials, indicating that during the 4- to 8-week healing period, all materials supported osteogenesis. Conclusions: Compared to other materials, the DBM had high osteoinductive properties during the 4- to 8-week time period based on increased mineral content. All materials were associated with immunohistologic evidence of osteogenesis in the rat calvarial defect model. [ABSTRACT FROM AUTHOR]

Background: Most anticancer drugs possess the capacity to control precise molecular processes and gene-regulated protein expressions, which could enhance the efficacy of cancer treatments. Hirudinaria manillensis is prevalent in South Asia and has been employed for several decades in medical conditions based on its curative benefits. Methods and results: The present study aimed to explore the molecular regulation of the target EGFR gene, and the protein expression of EGFR on the HeLa cell line. To achieve our aim, quantitative real-time PCR and immunocytochemistry assays were conducted. Interestingly, qRT-PCR results confirmed the downregulation of the EGFR gene on the HeLa cells in comparison with the β- actin internal control gene. Furthermore, immunocytochemistry assay results confirmed the decreasing level of EGFR gene expression in the HeLa cells. Conclusion: Therefore, Hirudinaria manillensis could be a promising anticancer candidate for cervical cancer treatment. [ABSTRACT FROM AUTHOR]

Radiotherapy is one of the risk factors for radiation-induced premature ovarian failure and infertility in cancer patients. The development of methods for ovarian radioprotection remains relevant. Moreover, electrons are a little-studied and promising method of radiation with the least toxic effect on normal tissues. The assessment of intracellular mechanisms regulating the protective effects of leukocyte-poor platelet-rich plasma in a model of radiation-induced premature ovarian failure caused by electron irradiation. Wistar rats were divided into four groups, namely a control group, irradiation group (electron exposure), irradiation + leukocyte-poor platelet-rich plasma group, and only leukocyte-poor platelet-rich plasma group. Fragments of ovaries were removed and hormonal, oxidant, histological, and morphometric studies were carried out. The cell cycle of ovarian follicles and the inflammatory and vascular response were assessed using immunohistochemistry. The activity of MAPK, ERK, and PI3K pathways was also assessed using the RT-qPCR. We found that electron irradiation causes a decrease in the functional activity of the ovaries and the death of follicular cells through apoptosis. The administration of LP-PRP led to a partial restoration of the cytokine balance. In addition, minor ovarian damage and mild inflammation were observed in this group. Leukocyte-poor platelet-rich plasma components have anti-inflammatory, angiogenetic, and radioprotective effects, reducing the activation of the NOX4, caspase and cytokine cascades, and inflammatory response severity through the MAPK/p38/JNK signaling pathway. This leads to the induction of endogenous antioxidant protection, the repair of post-radiation follicular damage, and slowing down the development of radiation-induced premature ovarian failure after electron irradiation. [ABSTRACT FROM AUTHOR]

This article discusses a rare case of amelanotic melanoma arising from a giant congenital melanocytic nevus (GCMN) in an infant. GCMN is a type of birthmark that can increase the risk of developing melanoma. The article highlights the challenges in diagnosing amelanotic melanoma and emphasizes the importance of fine needle aspiration (FNA) in providing an accurate diagnosis. The authors recommend close monitoring of GCMN and early treatment for better outcomes. They also discuss the differential diagnoses and the use of immunocytochemistry to confirm the diagnosis. [Extracted from the article]

Liquid biopsy for circulating tumour cell (CTC) detection is generally unexplored in veterinary medicine. Dogs with highly aggressive and heterogeneous tumours, such as oral malignant melanoma (OMM), could benefit from studies involving size‐based isolation methods for CTCs, as they do not depend on specific antibodies. This pilot study aimed to detect CTCs from canine OMM using Isolation by Size of Epithelial Tumor Cells (ISET), a microfiltration methodology, followed by immunocytochemistry (ICC) with Melan‐A, PNL2, and S100 antibodies. Ten canine patients diagnosed by histopathology and confirmed as OMM by immunohistochemistry were enrolled, their prognostic data was assessed, and blood samples were collected for CTC analysis. Results have shown the detection of intact cells in 9/10 patients. ICC has shown 3/9 Melan‐A‐positive, 3/9 PNL2‐positive, and 8/9 S100‐positive patients, confirming the importance of opting for a multimarker assay. A significant number of negative‐stained CTCs were found, suggesting their high heterogeneity in circulation. Microemboli stained with either PNL2 or S100 were found in a patient with a high isolated cell count and advanced clinical stage. Preliminary statistical analysis shows a significant difference in CTC count between patients with and without lymph node metastasis (p <.05), which may correlate with tumour metastatic potential. However, we recommend further studies with more extensive sampling to confirm this result. This pilot study is the first report of intact CTC detection in canine OMM and the first application of ISET in veterinary medicine, opening new possibilities for liquid biopsy studies in canine OMM and other tumours. [ABSTRACT FROM AUTHOR]

A 7‐year‐old female spayed Bernese Mountain dog was presented for evaluation of hematuria. Incidentally, a right stifle sarcoma was diagnosed via cytology, which raised concern for histiocytic sarcoma (given the patient's signalment) versus another joint‐associated sarcoma. Histopathology and immunohistochemistry revealed a CD18‐negative, non‐histiocytic origin cell population. Findings were consistent with a joint‐associated grade II soft tissue sarcoma (STS). The patient's hematuria was progressive over 5 months, and urinary bladder transitional cell carcinoma (TCC) was diagnosed via cystoscopy and histopathology. An enlarged right medial iliac lymph node was identified on routine restaging via abdominal ultrasound 3 months later. Cytology of the lymph node revealed a markedly pleomorphic cell population, again raising concern for histiocytic sarcoma (HS). Other differentials included an anaplastic metastatic population from the joint‐associated STS or the TCC. Immunocytochemistry revealed a cytokeratin‐positive, CD18‐, CD204‐, and vimentin‐negative cell population, consistent with a carcinoma. DNA was extracted from cytology slides to sequence cells for BRAF mutation status. Sequencing revealed a homozygous V596E (transcript ENSCAFT00845055173.1) BRAF mutation, consistent with the known biology of TCC. In neither case was HS truly present in this patient, but immunocytochemistry provided information that helped to optimize the patient's chemotherapy recommendations. [ABSTRACT FROM AUTHOR]

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5 nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75–85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies. [ABSTRACT FROM AUTHOR]

The muscle system and the FMRFamide immunopositive components of the nervous system of flatworms Acrolichanus auriculatus (Digenea, Allocreadiidae), an intestine parasite of acipenserid fishes, were studied for the first time by immunocytochemical, histochemical methods and confocal laser scanning microscopy. Scanning electron microscopy in A. auriculatus revealed a consistent pattern in the number and arrangement of ciliated and unciliated sensory papillae along the digenean body. Morphological data demonstrated the presence of the circular, longitudinal and diagonal muscles in different body regions of A. auriculatus. The musculature of the oral and ventral suckers, the digestive system, as well as the muscles extending radially from the genital pore are described. The study revealed the presence of the FMRFamide immunopositive staining in the central and peripheral nervous systems of A. auriculatus: in the neurons and neurites of the brain, brain commissure, in three pairs of the longitudinal nerve cords and commissures. FMRFamide immunopositive nerve elements innervate the attachment organs and the compartments of the digestive, reproductive and excretory systems. [ABSTRACT FROM AUTHOR]

The quantitative real-time reverse polymerase chain reaction (RRT-PCR) is the preferred test method for the diagnosis of avian influenza (AI), but can be performed only in specialized laboratories. Different antigen detection methods for the diagnosis of AI were previously reported to be specific and sensitive in field outbreaks. These tests can be performed in basic countryside labs. Brain smears of domestic birds (n = 105) collected during AI field outbreaks were examined with immunocytochemistry (IC). The results were statistically analysed by comparing IC to brain histology (BH), and immunohistochemistry (IHC), to gross pathological examination (GP) (n = 105), and RRT-PCR (n = 91). AI was diagnosed with RRT-PCR in 66 cases. IC and IHC were positive in 59/66 (90%) and 60/66 (91%) cases, respectively. Lesions suspicious for AI were detected with GP and HP in 66/66 (100%) and 61/66 (92%) cases, respectively. An almost perfect agreement was found between RRT-PCR, IC, IHC, and HP. Substantial agreement was found between IC and GP, between IHC and GP, between HP and GP, and between RRT-PCR and GP. The chromogen-based IC test presented in this study produces durable staining, which can be evaluated using a simple brightfield microscope. The test is rapid (can be completed in 2 h), sensitive (90%), specific (100%), and cost-effective, which makes the method suitable for routine diagnostic tests in AI epidemics. RESEARCH HIGHLIGHTS Avian influenza virus (AIV) antigen detection was examined in field outbreaks. Bird brain smears were tested using immunocytochemistry (IC). IC results strongly correlated with real-time RT-PCR results. The IC method was rapid, specific, sensitive, and cost-effective in AIV field outbreaks. [ABSTRACT FROM AUTHOR]

Regulation of autophagy through the 62 kDa ubiquitin-binding protein/autophagosome cargo protein sequestosome 1 (p62/SQSTM1), whose level is generally inversely proportional to autophagy, is crucial in microglial functions. Since autophagy is involved in inflammatory mechanisms, we investigated the actions of pro-inflammatory lipopolysaccharide (LPS) and anti-inflammatory rosuvastatin (RST) in secondary microglial cultures with or without bafilomycin A1 (BAF) pretreatment, an antibiotic that potently inhibits autophagosome fusion with lysosomes. The levels of the microglia marker protein Iba1 and the autophagosome marker protein p62/SQSTM1 were quantified by Western blots, while the number of p62/SQSTM1 immunoreactive puncta was quantitatively analyzed using fluorescent immunocytochemistry. BAF pretreatment hampered microglial survival and decreased Iba1 protein level under all culturing conditions. Cytoplasmic p62/SQSTM1 level was increased in cultures treated with LPS+RST but reversed markedly when BAF+LPS+RST were applied together. Furthermore, the number of p62/SQSTM1 immunoreactive autophagosome puncta was significantly reduced when RST was used but increased significantly in BAF+RST-treated cultures, indicating a modulation of autophagic flux through reduction in p62/SQSTM1 degradation. These findings collectively indicate that the cytoplasmic level of p62/SQSTM1 protein and autophagocytotic flux are differentially regulated, regardless of pro- or anti-inflammatory state, and provide context for understanding the role of autophagy in microglial function in various inflammatory settings. [ABSTRACT FROM AUTHOR]

Background: The Ki-67 proliferative index (PI) is part of the diagnosis of nodal B-cell lymphoma (nBCL), but its determination in cytological samples is not standardized. We aimed to establish an approach for the accurate determination of the Ki-67 PI in cytological slides to differentiate between indolent and aggressive nBCLs. Methods: Patients diagnosed with nBCL by fine-needle aspiration biopsy and subsequent excision biopsy were included. Cell suspensions were prepared from biopsy samples for CD3/Ki-67 double immunocytochemical staining and flow-cytometric verification of lymphoma B-cell counts. The Ki-67 PI was assessed by manual counting and eyeballing in cytology and eyeballing in histology. The cut-off values for the differentiation between aggressive and indolent lymphomas were determined for each method. Results: A strong correlation between manual and flow-cytometric counting of lymphoma B cells was confirmed (interclass correlation coefficient (IC coef.) = 0.78). The correlation of the Ki-67 PI determined in cytological and histological slides was also strong (IC coef. > 0.80). Histologically, 55 cases were classified as indolent and 31 as aggressive nBCLs. KI-67 PI cut-off values of 28.5%, 27.5%, and 35.5% were established for manual counting and eyeballing in cytology and eyeballing in histology, respectively, with high sensitivity and specificity. Conclusions: The Ki-67 PI, assessed by manual counting and eyeballing in cytological samples, accurately differentiates between indolent and aggressive nBCLs. [ABSTRACT FROM AUTHOR]

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), β(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and postsynaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2β2γ2-GABAARs versus extrasynaptic type α4β3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2β2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2β2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4β3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2β2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit. [ABSTRACT FROM AUTHOR]

Objective: Immunocytochemistry (ICC) is essential for enhancing diagnostic accuracy and identifying markers for diagnosis, prognosis and targeted therapies. While cell blocks (CBs) are preferred for standardization and optimized staining, cytological smears are an alternative when CBs are unavailable. However, the literature on ICC protocols for smears is sparse. This review addresses preparation, fixation and protocols for nuclear and cytoplasmic antibodies on smears, drawing from our laboratory's experience. Methods: We reviewed procedures for ICC on cytological smears using existing literature and practical insights from our laboratory. Results: Commercially available antibodies were found to be reliable for ICC on smears if specimens are properly prepared and fixed. Protocols developed in our laboratory maintained antigenicity and provided clear staining results. Conclusions: Although ICC on CBs is the gold standard for standardization, cytological smears are a viable alternative when CBs are unavailable. Success in ICC on smears depends on proper preparation and fixation. This review offers practical protocols and insights to help laboratories optimize ICC on cytological smears. Further research and standardization are necessary to enhance reproducibility and reliability of ICC on smears. The practical information provided is based on personal experience in our laboratory. [ABSTRACT FROM AUTHOR]

Abstract PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

This article discusses a case study of a rare type of ovarian tumor called Sertoli-Leydig cell tumor (SLCT). SLCTs are composed of Sertoli and Leydig cells and make up less than 0.5% of all ovarian tumors. The article describes the cytomorphologic features of an SLCT found in an uncommon metastatic site in a young female. The study highlights the importance of accurate cytologic diagnosis and clinical correlation in determining the prognosis and appropriate treatment for SLCTs. [Extracted from the article]

This article discusses the detection of primary peritoneal low-grade serous carcinoma in cervical smears. The study reports three cases of asymptomatic women who had abnormal glandular cells in routine Pap smears. Further investigations revealed raised serum CA125 levels and mild ascitic fluid collection in the pelvis. The patients underwent exploratory laparotomy, which confirmed the presence of low-grade serous carcinoma. The article emphasizes the diagnostic value of cellblock immunocytochemistry in confirming the diagnosis. The document also discusses the presence of low-grade serous papillary cell clusters with psammoma bodies in the tubal lumen, which are characteristic of primary peritoneal serous tumors. The document highlights the importance of considering various factors to distinguish primary peritoneal serous carcinomas from other types of serous tumors. The shedding of peritoneal tumor cells into the uterine cavity is proposed as a possible explanation for abnormal Pap tests. [Extracted from the article]

LCH has a distinct site predilection and cytological feature. However, it can cause diagnostic difficulties at unusual sites or deviated cytomorphology. FNAC is a minimally invasive procedure that can be performed in any organ (parotid and thyroid where biopsy is not commonly indicated) and multiple sites/organs simultaneously for diagnosis. FNA sampling can also help with cell block preparation for performing immunohistochemistry and molecular studies if necessary. [ABSTRACT FROM AUTHOR]

Background: Thyroid fine needle aspiration cytology (FNAC) is an important diagnostic tool for thyroid disorders. The Bethesda technique for Reporting Thyroid Cytopathology (TBSRTC) is an unambiguous, six-category reporting system for thyroid fine needle aspiration (FNA) that is widely acknowledged as a standard reporting guideline for institutes across continents which can minimize needless surgeries. Material and Methods: The study was carried out at the Pathology Department of the Assam Medical College and Hospital, Dibrugarh, Assam, India, from March 2023 to February 2024. The aim is to study the different cytological pattern in thyroid lesion by FNAC The results are interpreted using the features and categories specified in the TBSRTC. A total of 182 cases were studied. The distribution of the various categories from evaluated thyroid FNAs are as follows: nondiagnostic/unsatisfactory-8.2%, benign-75.9%, atypia of undetermined significance-6.0%,follicular neoplasm (FN) or suspicious for a FN-4.3%,suspicious for malignancy - 1%, malignant - 4.3%.Conclusion: The results of a FNA according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) give a standard and systematic approach to managing these nodules. Its standardized nomenclature enhanced thyroid FNAC reporting by facilitating communication and interlaboratory agreement among cytopathologists and clinicians, potentially leading to more standardized patient care management. [ABSTRACT FROM AUTHOR]

Our research explores the interplay between Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) and the host's inflammatory response in molar/incisor pattern periodontitis (MIPP). Cdt disrupts phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling, influencing cytokine expression through canonical and non-canonical inflammasome activation as well as nuclear factor-κB (NF-κB) activation, leading to inflammation in MIPP. THP-1 differentiated macrophages (TDMs) exposed to Cdt exhibited an upregulation of pro-inflammatory genes and subsequent cytokine release. We analyzed the ability of a small molecule therapeutic, LGM2605, known for its anti-inflammatory properties, to reduce pro-inflammatory gene expression and cytokine release in Cdt-exposed and Aa-inoculated TDMs. LGM2605's mechanism of action involves inhibiting NF-κB while activating the Nrf2–transcription factor and antioxidants. Herein, we show that this small molecule therapeutic mitigates Cdt-induced pro-inflammatory cytokine expression and secretion. Our study also further defines Cdt's impact on osteoclast differentiation and maturation in MIPP. Cdt promotes increased TRAP+ cells, indicating heightened osteoclast differentiation, specific to Cdt's phosphatase activity. Cathepsin K levels rise during this process, reflecting changes in TRAP distribution between control and Cdt-treated cells. Exploring LGM2605's effect on Cdt-induced osteoclast differentiation and maturation, we found TRAP+ cells significantly reduced with LGM2605 treatment compared to Cdt alone. Upon LGM2605 treatment, immunocytochemistry revealed a decreased TRAP intensity and number of multinucleated cells. Moreover, immunoblotting showed reduced TRAP and cathepsin K levels, suggesting LGM2605's potential to curb osteoclast differentiation and maturation by modulating inflammatory cytokines, possibly involving Nrf2 activation. In summary, our research reveals the intricate connections between Cdt, pro-inflammatory cytokines, and osteoclast differentiation, offering novel therapeutic possibilities for managing these conditions. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to explore the efficacy of immunocytochemistry in diagnosing abdominopelvic washings (APWs) and evaluate the superiority of cytology combined with immunocytochemistry over cytology alone. Material and Methods: Data on APW cytology and available cell blocks from patients who underwent radical surgery for endometrial cancer between January 2021 and December 2022 were reviewed. Cytology was re-evaluated according to a five-tier system. Immunocytochemistry analysis for targets such as Sry box transcription factor 1(SOX17), Paired box gene 2 (Pax-2) protein, Phosphatase and tensin (PTEN), and β-catenin was performed on each case with non-negative cytology. Mismatch repair (MMR) protein and P53 immunocytochemistry analyses were performed using cell blocks from cases with abnormal MMR or P53 expression in their primary lesion. The accuracies of cytology combined with immunocytochemistry and cytology alone were calculated. Results: Overall, 126 patients were included in this study, 18 of whom demonstrated non-negative cytology of APW. Cell blocks were successfully prepared for 16 cases. SOX17 positivity was observed in 16 cases, including 1 of serous carcinoma, 1 of clear cell carcinoma, and 14 of endometrioid carcinoma (EC). Loss of Pax-2 and PTEN expression was observed in the APWs of the 14 patients with EC. MMR deficiency was noted in two patients with EC, and P53 mutation was noted in another two patients with EC. Compared with 10 metastatic carcinomas (10/18, 55.56%) diagnosed by cytology alone, 15 malignant APWs (15/18, 83.33%) were confirmed through combination cytology and immunocytochemistry. APWs were more likely to be observed in cases with more than half myometrial invasion than those with no or less than half myometrial invasion (P = 0.0067). The probability of malignant APW occurrence was slightly elevated in cases of EC exhibiting microcystic, elongated, and fragmented(MELF) infiltrative growth (P = 0.039). Conclusion: SOX17 is a useful Müllerian marker for distinguishing endometrial epithelium in APW. Loss of Pax-2 and PTEN expression offers evidence of metastatic endometrial carcinoma. Furthermore, positive APWs retained molecular features similar to primary lesions. The use of multiple immunocytochemical markers can effectively enhance the diagnostic efficiency of APWs. [ABSTRACT FROM AUTHOR]

Introduction: Breast cancer is the second most diagnosed cancer, as well as the primary cause of cancer death in women worldwide. Of the different breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly aggressive and is associated with poor prognosis. Black women are two to three times more likely to be diagnosed with TNBCs than white women. Recent experimental evidence suggests that basal-like TNBCs may derive from luminal cells which acquire basal characteristics through phenotypic plasticity, a newly recognized hallmark of cancer. Whether chemical exposures can promote phenotypic plasticity in breast cells is poorly understood. Methods: To investigate further, we developed a high-content immunocytochemistry assay using normal human breast cells to test whether chemical exposures can impact luminal/basal plasticity by unbiased quantification of keratin 14 (KRT14), a basal-myoepithelial marker; keratin 8 (KRT8), a luminal-epithelial marker; and Hoechst 33342, a DNA marker. Six cell lines established from healthy tissue from donors to the Susan G. Komen Normal Tissue Bank were exposed for 48 hours to three different concentrations (0.1mM, 1mM, and 10mM) of eight ubiquitous chemicals (arsenic, BPA, BPS, cadmium, copper, DDE, lead, and PFNA), with documented exposure disparities in US Black women, in triplicate. Automated fluorescence image quantification was performed using Cell Profiler software, and a random-forest classifier was trained to classify individual cells as KRT8 positive, KRT14 positive, or hybrid (both KRT8 and KRT14 positive) using Cell Profiler Analyst. Results and discussion: Results demonstrated significant concentrationdependent increases in hybrid populations in response to BPA, BPS, DDE, and PFNA. The increase in hybrid populations expressing both KRT14 and KRT8 is indicative of a phenotypically plastic progenitor-like population in line with known theories of carcinogenesis. Furthermore, BPA, BPS, DDE, and copper produced significant increases in cell proliferation, which could be indicative of a more malignant phenotype. These results further elucidate the relationship between chemical exposure and breast phenotypic plasticity and highlight potential environmental factors that may impact TNBC risk. [ABSTRACT FROM AUTHOR]

Introduction: Mechanical sensitive channels expressed in mammalian retinas are effectors of elevated pressure stresses, but it is unclear how their activation affects visual function in pressure-related retinal disorders. Methods: This study investigated the role of the transient potential channel vanilloid TRPV4 in photoreceptors and rod bipolar cells (RBCs) with immunohistochemistry, confocal microscopy, electroretinography (ERG), and patch-clamp techniques. Results: TRPV4 immunoreactivity (IR) was found in the outer segments of photoreceptors, dendrites and somas of PKCa-positive RBCs and other BCs, plexiform layers, and retinal ganglion cells (RGCs) in wild-type mice. TRPV4-IR was largely diminished in the retinas of homozygous TRPV4 transgenic mice. Genetically suppressing TRPV4 expression moderately but significantly enhanced the amplitude of ERG a- and b-waves evoked by scotopic and mesopic lights (0.55 to 200 Rh*rod-1 s-1) and photopic lights (105-106 Rh*rod-1 s-1) compared to wildtype mice in fully dark-adapted conditions. The implicit time evoked by dim lights (0.55 to 200 Rh*rod-1 s-1) was significantly decreased for b-waves and elongated for a-waves in the transgenic mice. ERG b-wave evoked by dim lights is primarily mediated by RBCs, and under voltage-clamp conditions, the latency of the lightevoked cation current in RBCs of the transgenic mice was significantly shorter compared to wild-type mice. About 10% of the transgenic mice had one eye undeveloped, and the percentage was significantly higher than in wild-type mice. Conclusions: The data indicates that TRPV4 involves ocular development and is expressed and active in outer retinal neurons, and interventions of TRPV4 can variably affect visual signals in rods, cones, RBCs, and cone ON BCs. [ABSTRACT FROM AUTHOR]

PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane. [ABSTRACT FROM AUTHOR]

Objectives: The presence of prostate‐specific antigen (PSA) in saliva and salivary glands has been reported. Nevertheless, its release pathway in these glands remains to be elucidated. Here, we showed PSA subcellular distribution focusing on its plausible route in human salivary parenchyma. Materials and Methods: Sections of parotid and submandibular glands were subjected to the immunohistochemical demonstration of PSA by the streptavidin–biotin method revealed by alkaline phosphatase. Moreover, ultrathin sections were collected on nickel grids and processed for immunocytochemical analysis, to visualize the intracellular distribution pattern of PSA through the observation by transmission electron microscopy. Results: By immunohistochemistry, in both parotid and submandibular glands PSA expression was detected in serous secretory acini and striated ducts. By immunocytochemistry, immunoreactivity was retrieved in the cytoplasmic compartment of acinar and ductal cells, often associated with small cytoplasmic vesicles. PSA labeling appeared also on rough endoplasmic reticulum and in the acini's lumen. A negligible PSA labeling appeared in most of the secretory granules of both glands. Conclusions: Our findings clearly support that human parotid and submandibular glands are involved in PSA secretion. Moreover, based on the immunoreactivity pattern, its release in oral cavity would probably occur by minor regulated secretory or constitutive‐like secretory pathways. [ABSTRACT FROM AUTHOR]

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin‐fixed paraffin‐embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale‐up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non‐TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics. [ABSTRACT FROM AUTHOR]

The cell wall rebuilding is one of the first stage of protoplast development that enables further mitotic divisions and differentiation. Therefore, this work focuses on the comparison of the cell wall regeneration in the parental protoplasts of Fagopyrum tataricum, F. esculentum and the F. tataricum (+) F. esculentum hybrids, which are promising materials in terms of future breeding and research programmes. It is worth emphasizing that the preparation of buckwheat hybrids using electrofusion was described for the first time. The results indicate that cell wall rebuilding exhibited a common mechanism for parent protoplasts and the heterokaryon as all analysed cell wall components recognising arabinogalactan proteins (JIM13, JIM16), extensin (JIM20), xyloglucan (LM25) and pectins (LM20, LM5, LM6) were detected during the process of wall regeneration. However, there were certainly differences in the spatio-temporal appearance or disappearance of individual epitopes during the 72 h of the cell culture, which have been discussed in the paper. Key message: The hybrid protoplasts similarly restore their wall to F. tataricum and F. esculentum protoplasts despite some qualitative and quantitative differences in epitope distribution. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Angiosarcoma, a very rare malignant tumor constituting 2%–4% of soft tissue sarcomas, manifest in diverse organs including skin, soft tissues, and bones. Histologically, angiosarcoma presents a wide range of morphologies, with epithelioid angiosarcoma (EAS) resemblance to carcinoma. The difficulty arises from the shared epithelial‐like morphology and expression of epithelial markers in immunohistochemistry. Case This study reports a case where EAS diagnosis was achieved through a combination of gross findings in a lung resection sample, imprint cytology, and immunocytochemistry. Imprint cytology revealed clusters of epithelioid cells, while immunocytochemistry showed positive results for CD31, ERG, Fli‐1, and AE1/AE3, proving instrumental in diagnosing EAS. The described immunocytochemical protocol facilitates prompt diagnosis exclusively through cytology samples. Conclusion This report emphasizes the potential for diagnosing EAS using cytological specimens, which is especially useful in cases where obtaining tissue samples proves challenging.

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Background: There are numerous methods and procedures described for the preparation of cell blocks (CBs) from cytological samples. The objective of this study was to determine current practices and issues with CBs in European laboratories. Methods: A link to an online survey, with 11 questions about CB practices, was distributed to cytology laboratories via participants of United Kingdom National External Quality Assurance Service for Cellular Pathology Techniques and national representatives in the European Federation of Cytology Societies. Results: A total of 402 laboratories responded completely (337/402, 84%) or partially (65/402, 16%) to the survey by February 4, 2022. The most common CB practice is embedding cell pellets using plasma and thrombin (23.3%), agar (17.1%), Shandon/Epredia Cytoblock (11.4%), HistoGel (7.9%), and Cellient (3.5%). Other methods such as CytoFoam, albumin, gelatin, Cytomatrix, and collodion bags are rarely used (1.0%, 0.7%, 0.7%, 0.3%, and 0.2%, respectively). CBs are also prepared from naturally occurring clots or tissue fragments (29.5%) and cells scraped from unstained or prestained smears (4.4%). The most frequent issues with the CBs in a daily cytology practice are low cellularity (248/402, 62%) and dispersed cells (89/402, 22%), regardless of the CBs preparation method or how the samples for embedding were selected. Conclusions: There is a great variability in CB practices in European laboratories with low cellular CBs as the main issue. Additional studies are mandatory to evaluate and improve performance and cellular yield of CBs. There is great variability in cell block (CBs) practices in European laboratories with low cellular CBs as the main issue. Additional studies are needed in order to evaluate and improve performance and cellular yield of CBs. [ABSTRACT FROM AUTHOR]

The withdrawal of the iView detection system (iV) forced many cytopathology laboratories, including ours, to substitute immunocytochemical (ICC) staining protocols for routine practice with other detection systems. Our objective was to optimize, validate, and implement ICC protocols using OptiView (OV) and EnVision FLEX (EnV) detection systems, comparing the results with those obtained using iV. Residual cytologic samples with known diagnoses were used, testing antibodies for the ten most common markers in routine cytopathology diagnostics (calretinin, Ber-EP4, MOC-31, CKAE1/AE3, CK5/6, CD68, LCA, desmin, HBME-1, and WT1). Different staining parameters were tested using OV on BenchMark ULTRA and EnV on Dako Omnis immunostainer, respectively. Optimal staining protocols were then selected and validated on 10 positive and 10 negative cases. The staining results were compared with iV protocols through evaluation of UK NEQAS and internal scores. The optimal staining protocols with OV and EnV demonstrated similar or superior results compared to the existing iV protocols, with slightly stronger intensity regarding positive cells. We have successfully established and validated optimal ICC staining protocols for commonly used markers in routine cytopathology practice. These protocols may benefit other laboratories using similar staining platforms. However, the challenge regarding standardizing ICC protocols across different cytopathology laboratories remains unresolved. [ABSTRACT FROM AUTHOR]

Tanycytes are stem/progenitor cells that reside in the hypothalamus of the adult vertebrate brain. Tanycytes can be cultured as free-floating neurospheres in vitro but tend to spontaneously differentiate over time. Here we asked whether morphological cues provided by engineered polymer scaffolds can modify spontaneous differentiation. Tanycyte-derived neurospheres were cultured on electrospun scaffolds, prepared with either random or aligned fiber morphologies. Cells dispersed widely on the scaffolds, and - on aligned scaffolds - were highly organized, orientated parallel to the fibers. Immunocytochemical analysis showed that cells cultured on aligned scaffolds showed significantly greater expression of the neural stem/progenitor cell marker, NrCAM and reduced expression of differentiated cell markers in comparison to those cultured on random scaffolds. Together this shows that tanycytes respond to local engineered cues, and that a morphologically constrained environment can better maintain tanycytes as stem cells. The aligned scaffold culture system provides a powerful tool to better investigate this novel stem/progenitor cell population. [ABSTRACT FROM AUTHOR]

Fine‐needle aspiration cytology (FNAC) is a versatile diagnostic procedure uniquely suited for tissue biopsy of breast carcinomas and axillary metastases and/or recurrences. With the expanding treatment options and accompanying theragnostic tests, it is crucial to recognize the developments on ancillary testing and digital cytopathology techniques related to aspiration cytology of metastatic breast carcinoma. In this review, we aim to summarize and update the evidence of immunocytochemistry, for the detection of carcinoma cells (epithelial markers), confirmation of breast primary (breast‐specific markers), assessment of surrogate immunostains (hormone receptors, ki‐67 proliferative index and HER2) and theragnostic biomarkers, with discussion on potential diagnostic pitfalls, followed by the application of molecular tests, and digital cytopathologic techniques for assessing metastatic breast carcinoma in cytology. [ABSTRACT FROM AUTHOR]

Colorectal adenocarcinoma is an aggressive malignant tumor in cats that frequently metastasizes to the lymph nodes and/or distant organs. However, research on feline colorectal adenocarcinoma is limited, and experimental models have not been established. A novel cell line, FeLeco-G7, was established from the lymph node of a 12-year-old spayed female Maine Coon cat with metastatic colorectal adenocarcinoma. FeLeco-G7 cells were polygonal with abundant cytoplasm and adherent growth. The population-doubling time was approximately 28.3 hours, and the mean number of chromosomes was 37.6±0.1 per cell (ranging between 32 and 41). Consistent with the original tumor, FeLeco-G7 cells were immunopositive for cytokeratin (CK) 20 and CDX2, and immunonegative for CD10 and CK7. Nuclear accumulation of β-catenin was rarely observed. Mutation analysis suggested TP53 gene alterations. A subcutaneous injection of FeLeco-G7 cells into immunodeficient mice resulted in the formation of a mass at the injection site without the development of metastatic lesions. An orthotopic (intrarectal) transplantation of FeLeco-G7 cells caused cachexia and diffuse involvement of the rectal mucosa in one of the 3 mice and the formation of masses around the rectum in the other 2 mice. Metastases to the regional lymph nodes and lungs were detected in three of the 3 and one of the 3 mice, respectively. The histological findings and immunohistochemical features of these masses were similar to those of the original tumor. These results suggest that FeLeco-G7 cells and the orthotopically transplanted mouse model are valuable tools for further molecular and therapeutic research on feline colorectal adenocarcinoma. [ABSTRACT FROM AUTHOR]

Bangkok chicken, renowned for its exceptional characteristics, presents a valuable option for crossbreeding to enhance the local chicken varieties in Indonesia. Cryopreservation is widely considered a promising approach for protecting the genetic diversity of the Bangkok chicken. This research aims to investigate the biological characteristics of embryonic fibroblast cells from Bangkok chickens, with the goal of preserving the chicken strain's genetic diversity through cryopreservation methods. The fibroblast cells were derived from 8-10-day-old Bangkok chicken embryos and cultivated in DMEM media. Growth curves and Population Doubling Time were observed to assess the rate of fibroblast cell growth. Immunocytochemistry staining was employed to examine the protein expression associated with fibroblast cell adhesion. The results showed that fibroblast cells predominantly exhibited a fusiform morphology rather than an elongated cytoplasmic morphology. The cells proliferated continuously with an average population doubling time of 204 h and the maximum cell count was 7.83×104±1.7 cells/ml. The cells exhibited normal detection of protein expression, including integrin, cadherin, actin, and microtubules. It was also noted that after cryopreservation in liquid nitrogen, fibroblast cells took 4-6 days to grow and reach 80% confluency. The data collectively verifies the successful establishment of cell culture and cryopreservation for Bangkok chicken embryonic fibroblast cells. [ABSTRACT FROM AUTHOR]

EWSR1::NFATC2 sarcoma, a rare round cell sarcoma constituting the majority of EWSR1::non-ETS sarcomas, has recently been defined in the latest WHO classification. To date, the cytological findings of EWSR1::NFATC2 sarcoma remain undocumented. We present the case of a 25-year-old man with a history of polyostotic fibrous dysplasia in the right leg, referred to our hospital with left thigh pain. Cytological findings included metachromasia, minimally pleomorphic round cells, and eosinophilic infiltration. There was no precursor fibrous dysplasia and the initial diagnosis was undifferentiated pleomorphic sarcoma. Following histologic review, we successfully performed immunocytochemistry and fluorescence in situ hybridization (FISH) on archival cytology specimens. The tumor cells were positive for NKX2-2, NKX3-1, and PAX7 and showed amplified 5ʹ single signals of EWSR1 gene. Reverse transcriptase-polymerase chain reaction revealed an in-frame fusion of EWSR1 and NFATC2. This report describes the cytological features of EWSR1::NFATC2 sarcoma and highlights the diagnostic utility of archival cytology specimens. [ABSTRACT FROM AUTHOR]

In the present study, an insulin-resistant cell model (human hepatocellular carcinoma cell line: HepG2) was chosen to investigate the efficacy of two compound classes and their common molecular motif for glycemic control and insulin sensitization. The two compounds' classes were flavonoid extracts from Rourea cuspidata and quassinoid extracts from Picrasma crenata. The flavonoid-like hydroxyhydroquinone (HHQ) was synthesized. HepG2 cells were tested in a high-glucose environment (HepG2/IRM) by monitoring ROS activity, the concentration of adenosine triphosphate (ATP), and the measurement of mitochondrial membrane potential (MMP). The expression of forkhead box O1 (FOXO1) protein, which mediates gluconeogenesis and insulin resistance, was also investigated using indirect immunocytochemistry and Western blot techniques. A significant increase in glucose uptake and well-regulated ATP concentrations were observed in the treated cells. The downregulation of FOXO1 expression was seen in cells treated with HHQ and quassinoids in comparison to cells treated with flavonoids. This study provides a pharmacological basis for the application of HHQ, quassinoids from P. crenata, and flavonoids from R. cuspidata in the treatment of metabolic diseases such as type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Agarose-based cell block (CB) technique can be modified to be combined with the frozen section technique for the preparation of a high-quality frozen-embedded CB (F-CB) from an effusion or fine-needle aspiration (FNA) cytology sample. This combined technique can be effectively used for the immunocharacterization of the hematolymphoid cells on F-CB. To demonstrate the applicability of performing diagnostic ICC on F-CB, we have analyzed the immunophenotype of the hematolymphoid cells in a series of eight cases of effusions and eight cases of FNA cytology specimens by using CB-ICC on sections cut from frozen-embedded CBs. The SurePathTM residue or cytologic material scraped off from the FNA cytology smear that was diagnostic for or suspicious of hematolymphoid malignancy was pelleted and pre-embedded in agarose. Half of the agarose-embedded pellet was frozen-embedded in OCT compound for the preparation of F-CB, while the other half was processed for the preparation of paraffin-embedded CB. Sections cut from the F-CB and P-CB were used for CB-ICC. Panels of ICC on the F-CBs could enable the immunocytochemical differential diagnosis of large cell hematologic malignancies that encompass anaplastic large cell lymphoma and other forms of large-cell hematolymphoid malignancies such as large B-cell lymphomas, anaplastic plasma cell myeloma, myeloid sarcoma, and T-lymphoblastic lymphoma. It also appeared that the small B-cell lymphomas in the effusions or FNAs could be differentially diagnosed with the aid of CB-ICC on the F-CB. A modified agarose-based CB technique can be combined with the frozen-embedded CB method for the preparation of F-CB that can be directly used for the immunocytochemical differential diagnosis of hematolymphoid cytology samples. [ABSTRACT FROM AUTHOR]

Simple Summary: Muscular degeneration is a prevalent disease with a challenging prognosis. Mesenchymal stem/stromal cells (MSCs) show promise in treating muscular degeneration due to their myogenic differentiation potential and immunomodulatory capabilities. Adipose tissues have emerged as a promising source of MSCs as a result of their easy accessibility and superior quality compared to other sources. However, their characteristics and differentiation ability vary among the sources and species of origin and remain controversial. Understanding these features is crucial for effectively treating muscular diseases using adipose-derived MSCs (ADP MSCs). To deepen our comprehension, we used the rat model to investigate the myogenic differentiation potential and immunomodulatory characteristics of ADP MSCs isolated from periscapular fat. ADP MSCs expressed key genes involved in immunomodulation. They also differentiated into myogenic cells and expressed myogenic markers. These findings suggest the promising clinical potential of ADP MSCs for muscle regeneration. The myogenic differentiation potential of MSCs is a key factor in their potential use as a cell source for muscle tissue repair and regeneration. Additionally, evaluating the immunomodulatory properties of MSCs is important to highlight their potential for regulating inflammation and supporting tissue regeneration. Given the limited literature on muscle differentiation potential and immunomodulatory properties, this study aims to characterize rat ADP MSCs for treating muscle disease. We isolated MSCs from adipose tissues around the periscapular region of the rats. We used a monoculture method for the myogenic differentiation and modified the myogenic induction medium by supplementing it with the growth factors FGF, HGF, and IGF. In rat ADP MSCs, expression of the MSC-specific marker, CD90, was 87.7%, while CD44 was 42.8%. For genes involved in immunomodulation, IGF1 and TGFB1 were highly expressed, while IL6 was poorly expressed. In addition to their trilineage differentiation potential, ADP MSCs exhibited the capacity to differentiate into myogenic cell lines, as evidenced by changes in cell morphology, leading to elongated and aligned structures and the expression of the MyoD and MYOG antibodies. The study found that ADP MSCs show great clinical promise for muscle regeneration. [ABSTRACT FROM AUTHOR]

Wound healing requires the coordinated interaction of dermis cells, the proper deposition of extracellular matrix, re-epithelialization, and angiogenesis. Extracorporeal shock wave (ESW) is a promising therapeutic modality for chronic wounds. This study determined the biological mechanisms activated under ESW, facilitating the healing of pressure ulcers (PUs). A group of 10 patients with PUs received two sessions of radial ESW (300 + 100 pulses, 2.5 bars, 0.15 mJ/mm2, 5 Hz). Histomorphological and immunocytochemical assessments were performed on tissue sections obtained from the wound edges before the ESW (M0) and after the first (M1) and second (M2) ESW. The proliferation index of keratinocytes and fibroblasts (Ki-67), the micro-vessels' density (CD31), and the number of myofibroblasts (α-SMA) were evaluated. The involvement of the yes-associated protein (YAP1) in sensing mechanical strain, and whether the nuclear localization of YAP1, was shown. The increased proliferative activity of epidermal cells and skin fibroblasts and the increased number of myofibroblasts, often visible as integrated cell bands, were also demonstrated as an effect of wound exposure to an ESW. The results indicate that the major skin cells, keratinocytes, and fibroblasts are mechanosensitive. They intensify proliferation and extracellular matrix remodeling in response to mechanical stress. A significant improvement in clinical wound parameters was also observed. [ABSTRACT FROM AUTHOR]

IntroductionBreast cancer is the second most diagnosed cancer, as well as the primary cause of cancer death in women worldwide. Of the different breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly aggressive and is associated with poor prognosis. Black women are two to three times more likely to be diagnosed with TNBCs than white women. Recent experimental evidence suggests that basal-like TNBCs may derive from luminal cells which acquire basal characteristics through phenotypic plasticity, a newly recognized hallmark of cancer. Whether chemical exposures can promote phenotypic plasticity in breast cells is poorly understood. MethodsTo investigate further, we developed a high-content immunocytochemistry assay using normal human breast cells to test whether chemical exposures can impact luminal/basal plasticity by unbiased quantification of keratin 14 (KRT14), a basal-myoepithelial marker; keratin 8 (KRT8), a luminal-epithelial marker; and Hoechst 33342, a DNA marker. Six cell lines established from healthy tissue from donors to the Susan G. Komen Normal Tissue Bank were exposed for 48 hours to three different concentrations (0.1μM, 1μM, and 10μM) of eight ubiquitous chemicals (arsenic, BPA, BPS, cadmium, copper, DDE, lead, and PFNA), with documented exposure disparities in US Black women, in triplicate. Automated fluorescence image quantification was performed using Cell Profiler software, and a random-forest classifier was trained to classify individual cells as KRT8 positive, KRT14 positive, or hybrid (both KRT8 and KRT14 positive) using Cell Profiler Analyst. Results and discussionResults demonstrated significant concentration-dependent increases in hybrid populations in response to BPA, BPS, DDE, and PFNA. The increase in hybrid populations expressing both KRT14 and KRT8 is indicative of a phenotypically plastic progenitor-like population in line with known theories of carcinogenesis. Furthermore, BPA, BPS, DDE, and copper produced significant increases in cell proliferation, which could be indicative of a more malignant phenotype. These results further elucidate the relationship between chemical exposure and breast phenotypic plasticity and highlight potential environmental factors that may impact TNBC risk.

Abstract Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin‐fixed paraffin‐embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale‐up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non‐TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

The purpose of the research is to analyze the literature devoted to the study of the physiological role and functional significance of biologically active substances: FMRFamide-like neuropeptides in the locomotion of root-knot nematodes Meloidogyne incognita, M. minor, M. hapla and M. graminicola using immunological, phylogenetic, molecular and bioinformatic research methods.Results and discussion. The present work shows the importance of endogenous FMRFamide-like neuropeptides (FLPs) in such behavioral reactions of plant nematodes as locomotion, which ensures the vital activity of plant parasites; the functional significance of flp genes in the neurobiology of root-knot nematodes is discussed. It was especially noted that the main physiological and functional characteristics of endogenous FLP in root-knot nematodes were obtained as a result of studies of the functional role of the flp genes encoding these neuropeptides. In the nematodes M. incognita and M. graminicola, components of the peptidergic nervous system were identified in the nervous structures: FMRFamide-like positive immunoreactivity, FLP, flp genes encoding neuropeptides, and G-protein-coupled receptors (GPCR) activated by these neuropeptides. It was shown that the main functional characteristics of endogenous FLPs in nematodes were obtained using one of the methods of reverse genetics, i.e., flp genes knockdown in shadow by means of RNA-interference. It has been established that FLP cause two types of physiological effects on the somatic muscles of root-knot nematodes – stimulation of the locomotor activity of the muscles and its inhibition. In most works, the data obtained on the physiological effects of neuropeptides on the locomotor activity of phytonematodes are considered with a view to their possible use in the development of new targeted anthelmintic drugs.

Abstract Background Arabinogalactan proteins (AGPs) are plant cell components found in the extracellular matrix that play crucial roles in fruit growth and development. AGPs demonstrate structural diversity due to the presence of a protein domain and an expanded carbohydrate moiety. Considering their molecular structure, the modification of glycosylation is a primary factor contributing to the functional variety of AGPs. Main body Immunocytochemical methods are used for qualitative and quantitative analyses of AGPs in fruit tissues. These include in situ techniques such as immunofluorescence and immunogold labelling for visualising AGP distribution at different cellular levels and ex situ methods such as Western blotting and enzyme-linked immunoenzymatic assays (ELISA) for molecular characterisation and quantitative detection of isolated AGPs. The presented techniques were modified by considering the structure of AGPs and the changes that occur in fruit tissues during the development and ripening processes. These methods are based on antibodies that recognise carbohydrate chains, which are the only commercially available highly AGP-specific tools. These probes recognise AGP epitopes and identify structural modifications and changes in spatio-temporal distribution, shedding light on their functions in fruit. Conclusion This paper provides a concise overview of AGP research methods, emphasising their use in fruit tissue analysis and demonstrating the accessibility gaps in other tools used in such research (e.g. antibodies against protein moieties). It underscores fruit tissue as a valuable source of AGPs and emphasises the potential for future research to understand of AGP synthesis, degradation, and their roles in various physiological processes. Moreover, the application of advanced probes for AGP visualisation is a milestone in obtaining more detailed insights into the localisation and function of these proteins within fruit.

Radiotherapy is one of the risk factors for radiation-induced premature ovarian failure and infertility in cancer patients. The development of methods for ovarian radioprotection remains relevant. Moreover, electrons are a little-studied and promising method of radiation with the least toxic effect on normal tissues. The assessment of intracellular mechanisms regulating the protective effects of leukocyte-poor platelet-rich plasma in a model of radiation-induced premature ovarian failure caused by electron irradiation. Wistar rats were divided into four groups, namely a control group, irradiation group (electron exposure), irradiation + leukocyte-poor platelet-rich plasma group, and only leukocyte-poor platelet-rich plasma group. Fragments of ovaries were removed and hormonal, oxidant, histological, and morphometric studies were carried out. The cell cycle of ovarian follicles and the inflammatory and vascular response were assessed using immunohistochemistry. The activity of MAPK, ERK, and PI3K pathways was also assessed using the RT-qPCR. We found that electron irradiation causes a decrease in the functional activity of the ovaries and the death of follicular cells through apoptosis. The administration of LP-PRP led to a partial restoration of the cytokine balance. In addition, minor ovarian damage and mild inflammation were observed in this group. Leukocyte-poor platelet-rich plasma components have anti-inflammatory, angiogenetic, and radioprotective effects, reducing the activation of the NOX4, caspase and cytokine cascades, and inflammatory response severity through the MAPK/p38/JNK signaling pathway. This leads to the induction of endogenous antioxidant protection, the repair of post-radiation follicular damage, and slowing down the development of radiation-induced premature ovarian failure after electron irradiation.