Divergence of 10 satellite repeats in Artemisia (Asteraceae: Anthemideae) based on sequential fluorescence in situ hybridization analysis: evidence for species identification and evolution.

Artemisia is a large genus encompassing about 400 diverse species, many of which have considerable medicinal and ecological value. However, complex morphological information and variation in ploidy level and nuclear DNA content have presented challenges for evolution studies of this genus. Consequently, taxonomic inconsistencies within the genus persist, hindering the utilization of such large plant resources. Researchers have utilized satellite DNAs to aid in chromosome identification, species classification, and evolutionary studies due to their significant sequence and copy number variation between species and close relatives. In the present study, the RepeatExplorer2 pipeline was utilized to identify 10 satellite DNAs from three species (Artemisia annua, Artemisia vulgaris, Artemisia viridisquama), and fluorescence in situ hybridization confirmed their distribution on chromosomes in 24 species, including 19 Artemisia species with 5 outgroup species from Ajania and Chrysanthemum. Signals of satellite DNAs exhibited substantial differences between species. We obtained one genus-specific satellite from the sequences. Additionally, molecular cytogenetic maps were constructed for Artemisia vulgaris, Artemisia leucophylla, and Artemisia viridisquama. One species (Artemisia verbenacea) showed a FISH distribution pattern suggestive of an allotriploid origin. Heteromorphic FISH signals between homologous chromosomes in Artemisia plants were observed at a high level. Additionally, the relative relationships between species were discussed by comparing ideograms. The results of the present study provide new insights into the accurate identification and taxonomy of the Artemisia genus using molecular cytological methods.

Revisiting the role of Acinetobacter spp. in side-stream enhanced biological phosphorus removal (S2EBPR) systems.

We piloted the incorporation of side-stream enhanced biological phosphorus removal (S2EBPR) with A/B stage short-cut nitrogen removal processes to enable simultaneous carbon-energy-efficient nutrients removal. This unique configuration and system conditions exerted selective force on microbial populations distinct from those in conventional EBPR. Interestingly, effective P removal was achieved with the predominance of Acinetobacter (21.5 ± 0.1 %) with nearly negligible level of known conical PAOs (Ca. Accumulibacter and Tetrasphaera were 0.04 ± 0.10 % and 0.47 ± 0.32 %, respectively). Using a combination of techniques, such as fluorescence in situ hybridization (FISH) coupled with single cell Raman spectroscopy (SCRS), the metabolic tracing of Acinetobacter-like cells exerted PAO-like phenotypic profiling. In addition, comparative metagenomics analysis of the closely related Acinetobacter spp. revealed the EBPR relevant metabolic pathways. Further oligotyping analysis of 16s rRNA V4 region revealed sub-clusters (microdiversity) of the Acinetobacter and revealed that the sub-group (oligo type 1, identical (100 % alignment identity) hits from Acinetobacter_midas_s_49494, and Acinetobacter_midas_s_55652) correlated with EBPR activities parameters, provided strong evidence that the identified Acinetobacter most likely contributed to the overall P removal in our A/B-shortcut N-S2EBPR system. To the best of our knowledge, this is the first study to confirm the in situ EBPR activity of Acinetobacter using combined genomics and SCRS Raman techniques. Further research is needed to identify the specific taxon, and phenotype of the Acinetobacter that are responsible for the P-removal.

Neonatal di-(2-ethylhexyl)phthalate exposure induces permanent alterations in secretory CRH neuron characteristics in the hypothalamus paraventricular region of adult male rats.

Corticotrophin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) play a critical role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis. Early-life exposure to di-(2-ethylhexyl) phthalate (DEHP) has been associated with an increased risk of developing psychiatric disorders in adulthood. The present work was designed to explore the impact of neonatal exposure to DEHP on adult PVN CRH neuronal activity. DEHP or vehicle was given to male rat pups from PND16 to PND22. Then, anxiety-like behaviors, serum corticosterone and testosterone, immunohistochemistry, western blotting, fluorescence in situ hybridization and acute ex vivo slice electrophysiological recordings were used to evaluate the influence of DEHP on adult PVN secretory CRH neurons. Neonatal DEHP-exposed rats exhibited enhanced anxiety-like behaviors in adults, with an increase in CORT. Secretory CRH neurons showed higher spontaneous firing activity but could be inhibited by GABAAR blockers. CRH neurons displayed fewer firing spikes, prolonged first-spike latency, depolarizing shifts in GABA reversal potential and strengthened GABAergic inputs, as indicated by increases in the frequency and amplitude of sIPSCs. Enhancement of GABAergic transmission was accompanied by upregulated expression of GAD67 and downregulated expression of GABABR1, KCC2 and GAT1. These findings suggest that neonatal exposure to DEHP permanently altered the characteristics of secretory CRH neurons in the PVN, which may contribute to the development of psychiatric disorders later in life.

Primary pulmonary adenoid cystic carcinoma: A clinicopathological study of 64 patients.

BACKGROUND: This study aimed to investigate the clinicopathological features and prognostic indicators of primary pulmonary adenoid cystic carcinoma (PACC). METHODS: Clinical data were collected from 64 primary PACC patients and analyzed retrospectively at the Tianjin Medical University General Hospital, the West China Hospital of Sichuan University, the First Affiliated Hospital of Guangxi Medical University, and the Bishan Hospital of Chongqing Medical University from January 2003 to August 2023. The 64 patients (28 males and 36 females) were aged from 20 to 73 years, with a median age of 49 years and an average age of 49.3 years. RESULTS: Immunohistochemical staining showed that the tumors expressed CK7, S-100 protein, CK5/6, CD117, and p63. Seven patients underwent fluorescence in situ hybridization (FISH) testing and three were found to have myeloblastosis (MYB) gene translocation. In total, 53 patients underwent surgery, among whom 31 received only surgery and 22 received both surgery and postoperative chemoradiotherapy. In addition, 10 patients received chemoradiotherapy only, while one patient underwent treatment with traditional Chinese medicine. The overall survival rates in the first, third, and fifth years were 98.4%, 95.3%, and 87.5%, respectively. CONCLUSION: Prognostic analysis revealed that age, tumor size, lymph node metastasis status, margin status, and choice of treatment modality significantly influenced the patients' prognosis.

Mechanistic insights into aniline-induced liver injury: Role of the mmu_circ_26984/Myh9/NLRP3 axis and modulation by N-acetylcysteine.

Aniline is a widely used chemical. Chronic or high-dose exposure to aniline can lead to hepatocellular damage. Although the hepatic pathogenicity of aniline has been established in previous studies, studies involving pathogenic genes during aniline-induced liver injury are limited. Our study first discovered and identified the role and mechanism underlying a new circRNA mmu_circ_26984 in aniline-induced chemical liver injury. Further, we discuss the protective effect of N-acetylcysteine (NAC) in this pathway. After constructing in vitro and in vivo models of aniline treatment, we screened the circRNA with significant differences in expression in AML12 cells from control and aniline-treated groups by circRNA microarray analysis. Next, using RNA pulldown, liquid chromatography-mass spectrometry (LC-MS), and RNA immunoprecipitation, we analyzed the relationship between mmu_circ_26984 and myosin heavy chain 9 (Myh9). Subsequently, we determined the specific mechanism of action of mmu_circ_26984 and Myh9 in aniline-induced liver injury and the protective effect of NAC against aniline-induced liver injury process using Cell Counting Kit-8, Western blot, RNA extraction, a reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence in situ hybridization, immunohistochemistry, and immunofluorescence. The expression of mmu_circ_26984 was significantly increased in liver tissues and AML12 cells of aniline-treated mice compared with the control group. This high expression of mmu_circ_26984 increased the expression of injury-related inflammatory factors, such as NLRP3, Caspase-1, IL-18, and IL-1ß in vivo and ex vivo, which exacerbated the level of liver injury. The interaction of mmu_circ_26984 with Myh9 also affected the course of liver injury. Mmu_circ_26984 overexpression and reduced treatment affected the levels of Myh9 expression in AML12 cells, as well as downstream inflammatory factors associated with injury, such as NLRP3. In addition, NAC reduced the process of liver injury mediated by the mmu_circ_26984/Myh9/NLRP3 axis. In conclusion, mmu_circ_26984 is a potential molecular marker and therapeutic target in the process of aniline-induced liver injury that can mediate aniline-exposure-induced liver injury via modulation of the mmu_circ_26984/Myh9/NLRP3 axis, and NAC can effectively attenuate the effect of this liver injury.

Pairwise biosynthesis of ion channels stabilizes excitability and mitigates arrhythmias.

Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.

Retrieval of Trophoblast Cells from the Cervical Canal: A Promising Non-invasive Method for Prenatal Diagnosis.

Background: Extravillous trophoblasts (EVTs) cells have shown promise for their application in non-invasive prenatal diagnosis during the first trimester. The Trophoblast Retrieval and Isolation from the Cervix (TRIC) method allows for the isolation of homogeneous trophoblast cells from pregnant women as early as 5 weeks gestation. Objective: This study aimed to explore the potential value of extravillous trophoblast cells collected from the cervix, enriched, and purified using the TRIC method for first-trimester prenatal diagnosis. Methods: A prospective observational study was conducted, and we collected extravillous trophoblast cells from the cervixes of 100 pregnant women between 5-7 weeks gestation before an induced abortion. Subsequently, these cells underwent STR analysis and fluorescence in situ hybridization (FISH). Results: Out of the 100 cases, trophoblast cells were successfully collected from 96 cases. Among them, STR analysis revealed maternal cell contamination in 13 cases. Gender determination using FISH showed 44 male cases (including one case with 47, XY, +21) and 39 female cases (including one case with 47, XXX). The results of the FISH examination of these 83 cases were in concordance with those of the villi FISH examination. Conclusions: The collection of fetal trophoblast cells from the cervix represents a feasible and non-invasive approach for first-trimester prenatal diagnosis. The TRIC method enables efficient enrichment and purification of trophoblast cells, which can be of significant benefit for subsequent diagnosis using the FISH method.

Expanding the Diversity of Accumulibacter with a Novel Type and Deciphering the Transcriptional and Morphological Features among Co-Occurring Strains.

"Candidatus Accumulibacter" is the major polyphosphate-accumulating organism (PAO) in global wastewater treatment systems, and its phylogenetic and functional diversity have expanded in recent years. In addition to the widely recognized type I and II sublineages, we discovered a novel type enriched in laboratory bioreactors. Core gene and machine learning-based gene feature profiling supported the assertion that type III "Ca. Accumulibacter" is a potential PAO with the unique function of using dimethyl sulfoxide as an electron acceptor. Based on the correlation between ppk1 and genome similarity, the species-level richness of Accumulibacter was estimated to be over 100, suggesting that the currently recognized species are only the tip of the iceberg. Meanwhile, the interstrain transcriptional and morphological features of multiple "Ca. Accumulibacter" strains co-occurring in a bioreactor were investigated. Metatranscriptomics of seven co-occurring strains indicated that the expression level and interphasic dynamics of PAO phenotype-related genes had minimal correlation with their phylogeny. In particular, the expression of denitrifying and polyphosphate (poly-P) metabolism genes exhibited higher interstrain and interphasic divergence than expression of glycogen and polyhydroxyalkanoate metabolic genes. A strategy of cloning rRNA genes from different strains based on similar genomic synteny was successfully applied to differentiate their morphology via fluorescence in situ hybridization. Our study further expands the phylogenetic and functional diversity of "Ca. Accumulibacter" and proposes that deciphering the function and capability of certain "Ca. Accumulibacter" should be tailored to the environment and population in question. IMPORTANCE In the last 2 decades, "Ca. Accumulibacter" has garnered significant attention as the core functional but uncultured taxon for enhanced biological phosphorus removal due to its phylogenetic and functional diversity and intragenus niche differentiation. Since 2002, it has been widely known that this genus has two sublineages (type I and II). However, in this study, a metagenomic approach led to the discovery of a novel type (type III) with proposed novel functional features. By comparing the average nucleotide identity of "Ca. Accumulibacter" genomes and the similarity of ppk1, a phylogenetic biomarker largely deposited in databases, the global species-level richness of "Ca. Accumulibacter" was estimated for the first time to be over 100. Furthermore, we observed the co-occurrence of multiple "Ca. Accumulibacter" strains in a single bioreactor and found the simultaneous transcriptional divergence of these strains intriguing with regard to their niche differentiation within a single community. Our results indicated a decoupling feature between transcriptional pattern and phylogeny for co-occurring strains.

Cryptic KMT2A/MLLT10 fusion detected by next-generation sequencing in a case of pediatric acute megakaryoblastic leukemia.

KMT2A (11q23.3) gene rearrangements are found in acute leukemia and are associated with a poor or intermediate prognosis. MLLT10 is the fourth most common gene fusion partner for KMT2A. A reciprocal translocation t(10;11) is insufficient to produce an in-frame KMT2A/MLLT10 fusion, because the genes involved in the rearrangement have opposite transcriptional orientations. In order to bring KMT2A and MLLT10 into juxtaposition, complex rearrangements are required. Until now, conventional chromosome, fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) studies have been used to detect KMT2A/MLLT10 fusions. However, conventional studies have limitations, such as poor and inconsistent resolution, when compared to next-generation sequencing (NGS). In this study, we report a pediatric patient with acute megakaryoblastic leukemia, in whom the cryptic KMT2A/MLLT10 fusion was not detected by KMT2A break-apart probe FISH and chromosome analysis, but detected by NGS. In this patient, NGS showed cryptic insertion of MLLT10 exons 9-24 into intron 9 of KMT2A, resulting in a KMT2A/MLLT10 fusion. Therefore, NGS is a valuable complementary option for the evaluation of structural aberrations, especially those with a cryptic size.

A Case of Maternal Uniparental Disomy of Chromosome 6 with Intrauterine Growth Restriction.

Background: Uniparental disomy (UPD) is a well-known epigenomic anomaly characterized by the inheritance of both copies of a homologous pair of chromosomes (or part thereof) from the same parent. This genetic condition can have significant implications for prenatal diagnosis and management. Case Presentation: We present a case of a 29-year-old gravida 1 para 0 female who underwent amniocentesis at pregnancy Week 19 due to a high possibility of trisomy chromosome 6, as indicated by noninvasive prenatal testing (NIPT). However, fluorescence in situ hybridization (FISH) and whole-exome sequencing (WES) revealed no abnormalities. Subsequently, chromosomal microarray analysis (CMA) detected uniparental disomy of chromosome 6. Additionally, an ultrasound examination at 28 weeks of gestation revealed intrauterine growth restriction (IUGR). Given these findings, the parents made the decision to terminate the pregnancy. Conclusions: The combination of genetic counseling, FISH, karyotype analysis, WES, CMA, NIPT, and prenatal ultrasound can provide valuable insights for the prenatal diagnosis of UPD. These diagnostic approaches play a crucial role in identifying and managing cases of UPD, primarily when associated with intrauterine growth restrictions.

Genome sequence analysis provides insights into the mode of 2n egg formation in Solanum malmeanum.

KEY MESSAGE: Our genomic investigation confirms the mechanism of 2n eggs formation in S. malmeanum and aid in optimizing the use of wild germplasm. Wild potatoes are a valuable source of agronomic traits. However, substantial reproductive barriers limit gene flow into cultivated species. 2n gametes are instrumental in preventing endosperm abortion caused by genetic imbalances in the endosperm. However, little is known about the molecular mechanisms underlying the formation of 2n gametes. Here, the wild species Solanum malmeanum Bitter (2x, 1EBN, endosperm balance number) was used in inter- and intrapoloid crosses with other Solanum species, with viable seeds being produced only when S. malmeanum was used as the female parent to cross the 2EBN Solanum genus and with the likely involvement of 2n gametes. Subsequently, we substantiated the formation of 2n eggs in S. malmeanum using fluorescence in situ hybridization (FISH) and genomic sequencing technology. Additionally, the transmission rate of maternal heterozygous polymorphism sites was assessed from a genomic perspective to analyze the mode of 2n egg formation in S. malmeanum × S. tuberosum and S. malmeanum × S. chacoense crosses; each cross acquired an average of 31.12% and 22.79% maternal sites, respectively. This confirmed that 2n egg formation in S. malmeanum attributed to second-division restitution (SDR) coupled with the occurrence of exchange events. The high-throughput sequencing technology used in this study has strong advantages over traditional cytological analyses. Furthermore, S. malmeanum, which has a variety of excellent traits not available from present cultivated potato genepool, has received little research attention and has successfully achieved gene flow in cultivated species in the current study. These findings will facilitate the understanding and optimization of wild germplasm utilization in potatoes.

Regenerative plantlets with improved agronomic characteristics caused by anther culture of tetraploid potato (Solanum tuberosum L.).

Objective: As the primary means of plant-induced haploid, anther culture is of great significance in quickly obtaining pure lines and significantly shortening the potato breeding cycle. Nevertheless, the methods of anther culture of tetraploid potato were still not well established. Methods: In this study, 16 potato cultivars (lines) were used for anther culture in vitro. The corresponding relation between the different development stages of microspores and the external morphology of buds was investigated. A highly-efficient anther culture system of tetraploid potatoes was established. Results: It was shown in the results that the combined use of 0.5 mg/L 1-Naphthylacetic acid (NAA), 1.0 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), and 1.0 mg/L Kinetin (KT) was the ideal choice of hormone pairing for anther callus. Ten of the 16 potato cultivars examined could be induced callus with their respective anthers, and the induction rate ranged from 4.44% to 22.67% using this hormone combination. According to the outcome from the orthogonal design experiments of four kinds of appendages, we found that the medium with sucrose (40 g/L), AgNO3 (30 mg/L), activated carbon (3 g/L), potato extract (200 g/L) had a promotive induction effect on the anther callus. In contrast, adding 1 mg/L Zeatin (ZT) effectively facilitated callus differentiation. Conclusion: Finally, 201 anther culture plantlets were differentiated from 10 potato cultivars. Among these, Qingshu 168 and Ningshu 15 had higher efficiency than anther culture. After identification by flow cytometry and fluorescence in situ hybridization, 10 haploid plantlets (5%), 177 tetraploids (88%), and 14 octoploids (7%) were obtained. Some premium anther-cultured plantlets were further selected by morphological and agronomic comparison. Our findings provide important guidance for potato ploidy breeding.

Insights into simultaneous nitrogen and phosphorus removal in biofilm: The overlooked comammox Nitrospira and the positive role of glycogen-accumulating organisms.

Simultaneous nitrogen and phosphorus removal (SNPR) biofilm system is an effective wastewater treatment process. However, the understanding on the mechanism of functional microorganisms driving SNPR is still limited, especially the role of complete ammonia oxidation (comammox) Nitrospira and glycogen-accumulating organisms (GAO). In this study, a sequencing batch biofilm reactor (SBBR) performing SNPR was operated for 249 d. Based on the 16S rRNA gene, comammox amoA amplicon sequencing, metagenomics and batch experiment, we found that comammox Nitrospira was the main ammonia-oxidizing microorganisms (AOM) and provided nitrite for anaerobic ammonia oxidation (anammox) bacteria (AnAOB). Besides, GAO was dominated by the bacteria of genus Defluviicoccus and played a primary role in reducing nitrate rather than nitrite. Fluorescent in situ hybridization (FISH) analysis confirmed that Nitrospira was enriched in the inner layer of the biofilm. Thus, we put forward a novel insight into the mechanism of SNPR biofilm system. Comammox Nitrospira was responsible for nitrite and nitrate production in the inner biofilm, and AnAOB consumed the produced nitrite during the anammox process. While GAO reduced nitrate to nitrite and polyphosphate-accumulating organisms (PAO) converted nitrite to dinitrogen via denitrifying phosphorus removal in the outer biofilm. These findings provide a new understanding in SNPR biofilm system.

An Endocrinological Perspective on 22q11.2 Deletion Syndrome: A Single-center Experience

Objective: 22q11.2 deletion syndrome (22q11.2 DS) is the most common chromosomal microdeletion disorder. Associated problems in 22q11.2 DS may include cardiac abnormalities, immune dysfunction, facial dysmorphism, with endocrine, genitourinary and gastrointestinal problems, and developmental delay. The aim of this study was to evaluate and present all endocrinological findings of patients with 22q11.2 DS from a single center. Methods: All participants had confirmed 22q11.2 DS by fluorescence in situ hybridization with hypoparathyroidism. Data were retrieved by retrospective review of patient records. Results: A total of 17 patients were reviewed. On physical examination, all patients had similar dysmorphic features. The median age at diagnosis was 45 days (1 day-13 years). Most cases (64.7%, 11/17) were diagnosed with hypoparathyroidism incidentally after routine tests. At the time of diagnosis, mean calcium was 7.04±0.80 mg/dL, phosphorus was 6.2±1.1 mg/dL, and median parathyroid hormone (PTH) was 11.5 (3.7-47.6) ng/L. Transient hypoparathyroidism was detected in five cases (29.4%). There was no significant difference between patients with permanent or transient hypoparathyroidism regarding gender, age at diagnosis, calcium, phosphorus, and PTH levels. However, vitamin D levels were significantly lower in the transient group (p=0.036). During follow-up, short stature, obesity, and type 2 diabetes mellitus were absent. Thyroid autoantibodies were detected in two patients with normal thyroid function tests. Despite there being no pathological short stature, final stature was shorter than the general population (mean height standard deviation score: -0.94±0.83). Conclusion: Hypocalcemia may be detected during acute illness in some cases where hypocalcemia appears at later ages. There was no significant difference between permanent and transient hypoparathyroidism cases in terms of PTH level. Recognition of the more specific facial findings is important to trigger investigation of genetic variants, additional anomalies, and for follow-up.

Molecular and spatial profiling of the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) is known to regulate various cognitive and behavioral processes. However, while functional diversity among PVT circuits has often been linked to cellular differences, the molecular identity and spatial distribution of PVT cell types remain unclear. To address this gap, here we used single nucleus RNA sequencing (snRNA-seq) and identified five molecularly distinct PVT neuronal subtypes in the mouse brain. Additionally, multiplex fluorescent in situ hybridization of top marker genes revealed that PVT subtypes are organized by a combination of previously unidentified molecular gradients. Lastly, comparing our dataset with a recently published single-cell sequencing atlas of the thalamus yielded novel insight into the PVT's connectivity with the cortex, including unexpected innervation of auditory and visual areas. This comparison also revealed that our data contains a largely non-overlapping transcriptomic map of multiple midline thalamic nuclei. Collectively, our findings uncover previously unknown features of the molecular diversity and anatomical organization of the PVT and provide a valuable resource for future investigations.

HER2 IHC Expression and Gene Amplification in p53-aberrant High-grade Endometrial Endometrioid Carcinoma Suggests That This Population May Benefit From HER2 Testing and Targeted Therapy.

Among gynecologic cancers, uterine serous carcinoma (USC) has been shown to be human epidermal growth factor receptor 2 (HER2) amplified and trastuzumab has been included in the recent National Comprehensive Cancer Network (NCCN) guidelines for treatment of advanced stage or recurrent USC with HER2 overexpression/amplification. There is limited literature suggesting that a subset of high-grade endometrioid carcinomas with aberrant p53 expression may also be HER2 amplified and these patients could benefit from the addition of targeted therapy. We identified 59 p53-aberrant (mismatch repair proficient) FIGO 3 endometrioid carcinomas of the uterus. HER2 immunohistochemistry was performed in all 59 tumors and HER2 fluorescence in situ hybridization (FISH) was performed in 52 of the 59 cases. Four of the 59 cases were HER2 3+ by immunohistochemistry (6.7%), using the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2007, 2013, and 2018 criteria. HER2 FISH was performed in 3 of the 4 cases and was amplified in all 3. Nine, 8, and 7 tumors showed 2+ HER2 staining when applying 2018, 2013, and 2007 criteria, respectively, FISH was performed in 7 tumors and none were amplified. An additional 4 cases did not perfectly meet the 2018 ASCO/CAP criteria but were assigned a score of 2+, none were amplified by HER2 FISH. The remaining 42 cases showed 1+ or no staining for HER2, FISH was successfully performed in 38 tumors and none showed amplification. Approximately half of the tumors fulfilled criteria for HER2-low or HER2-very low (10 HER2-low and 20 HER2-very low). Our data shows that a subset of p53-aberrant high-grade endometrial endometrioid carcinoma express HER2 and these patients may benefit from the addition of targeted therapy. The role of targeted therapy in HER2-low gynecologic carcinoma is currently unexplored.

Shaoyao decoction restores the mucus layer in mice with DSS-induced colitis by regulating Notch signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Restoring the mucus layer is a potential strategy for treating ulcerative colitis (UC). Previous studies reported that a Chinese medicine formula Shaoyao Decoction (SYD) effectively improved UC. However, the role and mechanism of SYD in restoring the mucus layer are still vague. AIM OF THE STUDY: This study aimed to research the therapeutical effects and unravel the involved mechanism of SYD on DSS-evoked UC. MATERIALS AND METHODS: First, the constituents of SYD were detected by UPLC-QTOF-MS/MS. Then, the DSS-induced UC model was introduced to investigate the pharmacologic action and molecular mechanism of SYD on UC. Pharmacodynamic indicators were assessed including body weight, colon length, ulcerations, disease activity index (DAI), inflammatory cytokines and histological parameters. To investigate the integrality and functions of the mucous layer, AB-PAS stain and UEA-1 stain were used to evaluate the completeness of mucous layer, as well as the maturation of goblet cells (GCs). The bacterial invasion was detected by fluorescence in situ hybridization. As to mechanism exploration, the expressions of Notch/Hes1 pathway were investigated by using agonists in lipopolysaccharides (LPS) -stimulated LS174T cell. RESULTS: After modeling in mice, SYD remarkedly ameliorated the symptoms of mouse colitis, the expression of pro-inflammatory factors declined, and increased IL-10 expression was observed in SYD-treated mice. Besides, SYD repaired the structure of the mucus layer and prevented bacterial invasion. Mechanism investigation discovered that SYD promoted GCs differentiation by inhibiting the Notch pathway, which was consistent with the results in LPS-challenged LS174 cell. CONCLUSIONS: These findings demonstrated that SYD could restore the mucus layer to prevent UC via suppressing the Notch signaling pathway, which provided evidences for the UC treatment of SYD in the clinic.

Folic acid protects against age-associated apoptosis and telomere attrition of neural stem cells in senescence-accelerated mouse prone 8.

Folic acid (FA) could improve cognitive performance and attenuate brain cell injury in the aging brain; FA supplementation is also associated with inhibiting neural stem cell (NSC) apoptosis. However, its role in age-associated telomere attrition remains unclear. We hypothesized that FA supplementation attenuates age-associated apoptosis of NSCs in mice via alleviating telomere attrition in senescence-accelerated mouse prone 8 (SAMP8). In this study, 4-month-old male SAMP8 mice were assigned equal numbers to four different diet groups (n = 15). Fifteen age-matched senescence-accelerated mouse resistant 1 mice, fed with the FA-normal diet, were used as the standard aging control group. After FA treatment for 6 months, all mice were sacrificed. NSC apoptosis, proliferation, oxidative damage, and telomere length were evaluated by immunofluorescence and Q-fluorescent in situ hybridization. The results showed that FA supplementation inhibited age-associated NSC apoptosis and prevented telomere attrition in the cerebral cortex of SAMP8 mice. Importantly, this effect might be explained by the decreased levels of oxidative damage. In conclusion, we demonstrate it may be one of the mechanisms by which FA inhibits age-associated NSC apoptosis by alleviating telomere length shortening.

Spectrum of Histopathological, Immunohistochemical, Molecular and Radiological Features in 12 Cases of BCOR::CCNB3-positive Sarcomas With Literature Review.

Introduction BCOR::CCNB3-positive undifferentiated sarcomas are rare. Herein, we present clinicopathological features including immunohistochemical and molecular data, along with the radiological profile of 12 such tumors. Methods Tumors were tested for BCOR::CCNB3 fusion by reverse transcription polymerase chain reaction (RT-PCR) technique. Eight tumors were tested for EWSR1 and three for SS18 gene rearrangements by fluorescence in situ hybridization, and two for SS18::SSX fusion by fragment analysis. Results Ten of 12 patients were male with ages ranging between 4 and 17 years (median = 13, average = 14.4). Nine tumors occurred in bones and three in soft tissues (median size = 8 cm). Four of five tumors within the appendicular bones were metadiaphyseal and appeared as permeative lesions, invariably associated with cortical thickening. Three tumors displayed mineralization. Histopathologically, the tumors comprised round to epithelioid cells with round to oval to spindle-shaped nuclei, mostly diffusely arranged in a myxoid stroma with intervening thin-walled vessels. Immunohistochemically, tumor cells were positive for BCOR (10/11), SATB2 (8/9), TLE1 (5/6), cyclinD1 (4/4), and EMA (3/8). All tumors revealed BCOR::CCNB3 fusion transcript. Nine patients underwent neoadjuvant chemotherapy, including five who underwent surgical resection, with two patients, who received adjuvant radiation therapy. A single patient, each, underwent palliative chemotherapy and palliative radiotherapy, respectively. Four patients developed pulmonary metastasis and three developed local recurrences. Four patients were alive-with-disease and two were free-of-disease. Conclusions It is crucial to identify BCOR::CCNB3 fusion-positive sarcomas, given significant treatment-associated implications. Certain clinicoradiological, histopathological features, absent EWSR1 rearrangement and BCOR, SATB2, and TLE1 immunoexpression are useful for triaging these tumors for molecular testing. A review of the literature on these ultra-rare tumors, including their diagnostic mimics is presented.

A novel method for making cell blocks with higher cellular yield.

INTRODUCTION: Cytopathology is an important part of pathology that is used to diagnose disease on the cellular level. The application of the cell block (CB) technique plays a vital role in cytological diagnosis, as blocks and slides can be further used for special stains, immunohistochemistry (IHC), and molecular pathological analysis. Several methods for making CBs have been reported, but their procedures and cellular yield are still deemed unsatisfactory. In this article, we used gellan gum (GG) as an adjuvant for CBs, which resulted in higher cellular yield with simpler procedures. METHODS: CBs were prepared by using GG, copper sulfate, plasma/thrombin, or pregelatinized starch methods. The procedures of each of these four methods were then compared. CB sections were stained with hematoxylin and eosin (H&E), and the background and morphological features seen by H&E staining were compared. A preliminary IHC and fluorescence in situ hybridization (FISH) study was performed using cytology specimens from eleven and five cases, respectively. The expression of immunocomplex by IHC and the molecular signals detected by FISH were compared in CB sections made by the four methods and a section derived from the biopsy specimen block from the same patient. Feulgen staining, Alcian blue staining, and Masson trichrome staining were performed on the CB sections from 3 cases of pleural fluid. The cellular yield of CB sections from 83 cases according to the four methods was compared using NDP analysis software. RESULTS: The results demonstrated that sections derived from CBs made with GG had a clear background and good morphological features by H&E staining. The expression of immunocomplex by IHC and the molecular signals of FISH detection in the sections from CBs made by GG were accurately located just as those in biopsy sections from the same patient. The DNA, acidic mucus, and fibrin could be clearly identified through special stains in the CB sections. The procedures involved in the GG method were easily controllable and the coagulated gel increased the ease by which the CB was embedded and sectioned. Specifically, sections from CBs made by the GG method contained higher cellular yield because cells could be concentrated on the bottom of the gel after centrifugation. CONCLUSION: This novel method for making CBs is a practical, simple method that can result in higher cellular yield. This method is therefore worth promoting in clinical applications.