Coral reef ecosystems are the most productive and biodiverse marine ecosystems, with their productivity levels highly dependent on the symbiotic dinoflagellates belonging to the family Symbiodiniaceae. As a unique life history strategy, resting cyst production is of great significance in the ecology of many dinoflagellate species, those HABs-causing species in particular, however, there has been no confirmative evidence for the resting cyst production in any species of the family Symbiodiniaceae. Based on morphological and life history observations of cultures in the laboratory and morpho-molecular detections of cysts from the marine sediments via fluorescence in situ hybridization (FISH), cyst photography, and subsequent singe-cyst PCR sequencing, here we provide evidences for the asexual production of resting cysts by Effrenium voratum, the free-living, red tide-forming, and the type species of the genus Effrenium in Symbiodiniaceae. The evidences from the marine sediments were obtained through a sequential detections: Firstly, E. voratum amplicon sequence variants (ASVs) were detected in the cyst assemblages that were concentrated with the sodium polytungstate (SPT) method from the sediments collected from different regions of China Seas by high-throughput next generation sequencing (NGS); Secondly, the presence of E. voratum in the sediments was detected by PCR using the species-specific primers for the DNA directly extracted from sediment; Thirdly, E. voratum cysts were confirmed by a combined approach of FISH using the species-specific probes, light microscopic (LM) photography of the FISH-positive cysts, and a subsequent single-cyst PCR sequencing for the FISH-positive and photographed cysts. The evidences from the laboratory-reared clonal cultures of E. voratum include that: 1) numerous cysts formed in the two clonal cultures and exhibited a spherical shape, a smooth surface, absence of ornaments, and a large red accumulation body; 2) cysts could maintain morphologically intact for a storage of two weeks to six months at 4 °C in darkness and of which 76-92 % successfully germinated through an internal development processes within a time period of 3-21 days after being transferred back to the normal culturing conditions; 3) two or four germlings were released from each cyst through the cryptopylic archeopyle in all cysts with continuous observations of germination processes; and 4) while neither sexual mating of gametes nor planozygote (cells with two longitudinal flagella) were observed, the haploidy of cysts was proven with flow cytometric measurements and direct LM measurements of fluorescence from cells stained with either propidium iodide (PI) or DAPI, which together suggest that the cysts were formed asexually. All evidences led to a conclusion that E. voratum is capable of producing asexual resting cysts, although its sexuality cannot be completely excluded, which guarantees a more intensive investigation. This work fills a gap in the knowledge about the life cycle, particularly the potential of resting cyst formation, of the species in Symbiodiniaceae, a group of dinoflagellates having unique life forms and vital significance in the ecology of coral reefs, and may provide novel insights into understanding the recovery mechanisms of coral reefs destructed by the global climate change and suggest various forms of resting cysts in the cyst assemblages of dinoflagellates observed in the field sediments, including HABs-causing species.

BACKGROUND: Alveolar soft part sarcoma (ASPS) occurs most often in the deep muscles or fascia of the extremities in adults, with only 3.4% of these tumours originating from the head, face and neck. To date, only 17 cases of buccal ASPS have been reported, including the case presented here. Only one case of ASPS recurrence at the primary site, similar to our case, has been reported thus far. Immune checkpoint inhibitors (ICPis)-associated diabetes, with an estimated incidence of 0.43%, is usually seen in older cancer patients and has not been reported in younger people or in patients with ASPS.
METHODS: A 24-year-old male patient presented with a slowly progressing right cheek mass with a clinical history of approximately 28 months. Sonographic imaging revealed a hypoechoic mass, which was considered a benign tumour. However, a pathological diagnosis of ASPS was made after excision of the mass. Five days later, functional right cervical lymph node dissection was performed. No other adjuvant therapy was administered after surgery. In a periodic follow-up of the patient six months later, blood-rich tumour growth was noted at the primary site, and Positron emission tomography-computedtomography (PET-CT) ruled out distant metastasis in other areas. The patient was referred to the Ninth People\'s Hospital of Shanghai Jiaotong University. Due to the large extent of the mass, the patient received a combination of a Programmed Cell Death Ligand 1(PD-L1) inhibitor and a targeted drug. Unfortunately, the patient developed three episodes of severe diabetic ketoacidosis after the administration of the drugs. A confirmed diagnosis of ICPis-associated diabetes was confirmed. After the second operation, the postoperative pathological diagnosis was ASPS, and the margins were all negative. Therefore, we made a final clinical diagnosis of ASPS recurrence at the primary site. Currently in the follow-up, the patient is alive, has no distant metastases, and undergoes multiple imaging examinations every 3 months for the monitoring of their condition.
CONCLUSIONS: In analysing the characteristics of all previously reported cases of buccal ASPS, it was found that the clinical history ranged from 1 to 24 months, with a mean of approximately 3 to 9 months. Tumour recurrence at the primary site has been reported in only one patient with buccal ASPS, and the short-term recurrence in our patient may be related to the extraordinarily long 28-month history. ICPis-associated diabetes may be noted in young patients with rare tumours, and regular insulin level monitoring after use is necessary.

Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon sequencing are commonly used for microbial ecological analyses in biological enhanced phosphorus removal (EBPR) systems, the successful application of which was governed by the oligonucleotides used. We performed a systemic evaluation of commonly used probes/primers for known polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). Most FISH probes showed blind spots and covered nontarget bacterial groups. Ca. Competibacter probes showed promising coverage and specificity. Those for Ca. Accumulibacter are desirable in coverage but targeted out-group bacteria, including Ca. Competibacter, Thauera, Dechlorosoma, and some polyphosphate-accumulating Cyanobacteria. Defluviicoccus probes are good in specificity but poor in coverage. Probes targeting Tetrasphaera or Dechloromonas showed low coverage and specificity. Specifically, DEMEF455, Bet135, and Dech453 for Dechloromonas covered Ca. Accumulibacter. Special attentions are needed when using these probes to resolve the PAO/GAO phenotype of Dechloromonas. Most species-specific probes for Ca. Accumulibacter, Ca. Lutibacillus, Ca. Phosphoribacter, and Tetrasphaera are highly specific. Overall, 1.4% Ca. Accumulibacter, 9.6% Ca. Competibacter, 43.3% Defluviicoccus, and 54.0% Dechloromonas in the MiDAS database were not covered by existing FISH probes. Different 16S rRNA amplicon primer sets showed distinct coverage of known PAOs and GAOs. None of them covered all members. Overall, 520F-802R and 515F-926R showed the most balanced coverage. All primers showed extremely low coverage of Microlunatus (<36.0%), implying their probably overlooked roles in EBPR systems. A clear understanding of the strength and weaknesses of each probe and primer set is a premise for rational evaluation and interpretation of obtained community results.

Numerous studies have investigated the effects of stannous ions on specific microbes and their efficacy in reducing dental plaque. Nonetheless, our understanding of their impact on the oral microbiome is still a subject of ongoing exploration. Therefore, this study sought to evaluate the effects of a stannous-containing sodium fluoride dentifrice in comparison to a zinc-containing sodium fluoride dentifrice and a control group on intact, healthy oral biofilms. Utilizing the novel 2bRAD-M approach for species-resolved metagenomics, and FISH/CLSM with probes targeting periodontal and caries associated species alongside Sn2+ and Zn2+ ions, we collected and analyzed in situ biofilms from 15 generally healthy individuals with measurable dental plaque and treated the biofilms with dentifrices to elucidate variations in microbial distribution. Although significant shifts in the microbiome upon treatment were not observed, the use of a stannous-containing sodium fluoride dentifrice primarily led to an increase in health-associated commensal species and decrease in pathogenic species. Notably, FISH/CLSM analysis highlighted a marked reduction in representative species associated with periodontitis and caries following treatment with the use of a stannous-containing sodium fluoride dentifrice, as opposed to a zinc-containing sodium fluoride dentifrice and the control group. Additionally, Sn2+ specific intracellular imaging reflected the colocalization of Sn2+ ions with P. gingivalis but not with other species. In contrast, Zn2+ ions exhibited non-specific binding, thus suggesting that Sn2+ could exhibit selective binding toward pathogenic species. Altogether, our results demonstrate that stannous ions could help to maintain a healthy oral microbiome by preferentially targeting certain pathogenic bacteria to reverse dysbiosis and underscores the importance of the continual usage of such products as a preventive measure for oral diseases and the maintenance of health.

A comparative karyotype analysis of four species of yellow-flowered Eranthis sect. Eranthis, i.e., E. bulgarica, E. cilicica, E. hyemalis, and E. longistipitata from different areas, has been carried out for the first time. All the studied specimens had somatic chromosome number 2n = 16 with basic chromosome number x = 8. Karyotypes of the investigated plants included five pairs of metacentric chromosomes and three pairs of submetacentric/subtelocentric chromosomes. The chromosome sets of the investigated species differ mainly in the ratio of submetacentric/subtelocentric chromosomes, their relative lengths, and arm ratios. A new oligonucleotide probe was developed and tested to detect 45S rDNA clusters. Using this probe and an oligonucleotide probe to 5S rDNA, 45S and 5S rDNA clusters were localized for the first time on chromosomes of E. cilicica, E. hyemalis, and E. longistipitata. Major 45S rDNA clusters were identified on satellite chromosomes in all the species; in E. cilicica, minor clusters were also identified in the terminal regions of one metacentric chromosome pair. The number and distribution of 5S rDNA clusters is more specific. In E. cilicica, two major clusters were identified in the pericentromeric region of a pair of metacentric chromosomes. Two major clusters in the pericentromeric region of a pair of submetacentric chromosomes and two major clusters in the interstitial region of a pair of metacentric chromosomes were observed in E. longistipitata. E. hyemalis has many clusters of different sizes, localized mainly in the pericentromeric regions. Summarizing new data on the karyotype structure of E. sect. Eranthis and previously obtained data on E. sect. Shibateranthis allowed conclusions to be formed about the clear interspecific karyological differences of the genus Eranthis.

UNASSIGNED: Intrahepatic cholangiocarcinoma (ICC) is a subtype of primary liver cancer for which effective therapeutic agents are lacking. Fibroblast growth factor receptor 2 (FGFR2) has become a promising therapeutic target in ICC; however, its incidence and optimum testing method have not been fully assessed. This study investigated the rearrangement of FGFR2 in intrahepatic cholangiocarcinoma using multiple molecular detection methods.
UNASSIGNED: The samples and clinical data of 167 patients who underwent surgical resection of intrahepatic cholangiocarcinoma in Zhongshan hospital, Fudan university were collected. The presence of FGFR2 gene rearrangement was confirmed using fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (NGS). FGFR2 protein expression was determined using immunohistochemistry (IHC). The concordance between the methods was statistically compared. PD-L1 expression was also assessed in this cohort. The clinicopathological characteristics and genomic profile related to FGFR2 rearrangements were also analyzed to assist candidate-screening for targeted therapies.
UNASSIGNED: FGFR2 rearrangement was detected in 21 of the 167 ICC cases (12.5%) using FISH. NGS analysis revealed that FGFR2 rearrangement was present in 16 of the 20 FISH-positive cases, which was consistent with the FISH results (kappa value=0.696, p<0.01). IHC showed that 80 of the 167 cases (48%) were positive for FGFR2 expression, which was discordant with both FISH and NGS results. By comparison, FGFR2-positivity tended to correlate with unique clinicopathological subgroups, featuring early clinical stage, histologically small duct subtype, and reduced mucus production (P<0.05), with improved overall survival (p<0.05). FGFR2-positivity was not associated with PD-L1 expression in ICCs. In genome research, we identified eight partner genes fused with FGFR2, among which FGFR2-BICC1 was the most common fusion type. BAP1, CDKN2A, and CDKN2B were the most common concomitant genetic alterations of FGFR2, whereas KRAS and IDH1 mutations were mutually exclusive to FGFR2 rearrangements.
UNASSIGNED: FISH achieved satisfactory concordance with NGS, has potential value for FGFR2 screening for targeted therapies. FGFR2 detection should be prioritized for unique clinical subgroups in ICC, which features a histological small duct subtype, early clinical stage, and reduced mucus production.

UNASSIGNED: Transurethral resection of bladder tumor (TURBT) combined with intravesical Bacillus Calmette-Guérin (BCG) perfusion is a widely accepted treatment for moderate or high risk non-muscular-invasive bladder cancer (BCa). Despite its effectiveness, the recurrence and progression rate of tumor are still high. We evaluated the predictive role of fluorescence in situ hybridization (FISH) for the response to BCG perfusion in BCa.
UNASSIGNED: Patients with BCa who underwent BCG perfusion and FISH test in our hospital were selected. Logistic regression and Kaplan-Meier methods were used to evaluate the relationship between FISH results and tumor recurrence or progression. COX proportional hazards regression analysis was used to identify risk factors for recurrence or progression. SPSS (version 24.0, IBM Corporation, USA) was used for statistical analysis.
UNASSIGNED: Seventy-six patients were included in this study, with a median age of 63.0 (55.0-70.0) years, and a median follow-up time of 19.0 (7.5-29.0) months. Fifteen patients relapsed after BCG perfusion. Before TURBT, 39 patients were positive for FISH and 20 were negative. There was no significant difference in the recurrence rate of BCG after perfusion predicted by preoperative FISH positive (10.3% vs. 10%, P=0.675). No association was found between preoperative FISH and tumor recurrence (P=0.955) or disease progression (P=0.186). After BCG perfusion, 10 patients were FISH positive and 14 patients were FISH negative. There was significant difference in recurrence rate of BCa predicted by positive FISH (100% vs. 7.1%, P<0.001). FISH results were significantly associated with tumor recurrence (P<0.001) and disease progression (P=0.001). Kaplan-Meier and univariate COX proportional hazards regression analysis clarified that FISH positive after BCG perfusion, tumor-node-metastasis (TNM) stage, and multiple tumors were risk factors for tumor recurrence and progression (P<0.05). Tumor TNM stage and FISH positive after BCG perfusion were independent risk factors for recurrence.
UNASSIGNED: Positive FISH after BCG perfusion can well predict the risk of recurrence and progression of BCa, and recurrence within six months is more likely. After BCG perfusion, it is better to recommend close follow up in patients with positive FISH.

Molecular combing technology (MCT) is an effective means for stretching DNA molecules and making them thus accessible for in situ studies. MCT uses the force exerted in the process of liquid flow via surface tension to stretch DNA molecules and spread them on solid surfaces, i.e. glass cover slips. Many DNA molecules can be stretched at the same time in parallel and neatly arranged side-by-side, making the approach convenient for statistical analysis. Accordingly, DNA replication and transcription can be studied at the single molecule level. In this paper, the principle, experimental methods, important applications, advantages and shortcuts of MCT in medical field are presented and discussed.

UNASSIGNED: 1q21 gain/amplification (1q21+) is a common abnormal karyotype in multiple myeloma, and its proportion in Chinese patients is much higher. If 1q21+ is included as one of the poor prognostic factors, it will greatly increase the proportion of high-risk patients in newly diagnosed multiple myelome (NDMM) patients. Therefore, the poor prognostic significance of 1q21+ is still controversial. This study mainly analyzed the clinical characteristics, treatment response and prognostic significance of 1q21+ in NDMM patients.
UNASSIGNED: 248 NDMM patients admitted in The First Affiliated Hospital of Soochow University from September 01, 2018 to August 31, 2021 of a VRD registration study, were retrospectively analyzed. 135 cases (54.4%) had 1q21+ by CD38-sorted fluorescence in situ hybridization (FISH). The clinical characteristics, treatment response and prognosis of the general population and subgroups were analyzed, among which 153 patients were compared for the involved genes by CytoScan.
UNASSIGNED: Compared with negative patients, 1q21+ patients were more likely to have anemia, hypoalbuminemia, renal insufficiency, high lactate dehydrogenase and high proportion of R-ISS-III stage. The patients with 1q21+ involving CKS1B detected by Cytoscan had a higher proportion of complex karyotypes and abnormal CNVs, and all at middle-risk or high-risk groups defined by Prognostic Index. Multivariate analysis showed that 1q21+ was an independent adverse prognostic factor (PFS HR=2.358, 95%CI 1.286-4.324, P=0.006; OS HR=2.598, 95%CI 1.050-6.425, P=0.039). 1q21+ subgroup had an inferior outcome (PFS P=0.0133, OS P=0.0293). Furthermore 1q21 amplification had a shorter PFS than 1q21 gain (24 months vs not reached, P=0.0403), but the OS difference was not clinically significant. The proportion of 1q had no effects on prognosis. In addition, 1q21+ in main clone rather than subclone was an adverse factor affecting the prognosis (PFS P=0.0172, OS P=0.1260). Autologous stem cell transplantation can effectively improve the survival of 1q21+ patients (P<0.05).
UNASSIGNED: Patients with 1q21+ have clinically significant end-stage organ damage and higher tumor burden, more likely to combine 13q14-, t(4;14), 1p32- and other cytogenetic abnormalities. 1q21+ is an independent high-risk cytogenetic factor for poor prognosis in NDMM patients, of which 4 or more copy numbers and main clone position significantly associated with prognosis results.

Garden asparagus (Asparagus officinalis, 2n = 2x = 20 chromosomes) is an important dioecious vegetable crop and a model species for studying sex chromosome formation and evolution. However, few molecular cytogenetic studies on garden asparagus have been reported because of its small metaphase chromosomes, the scarcity of distinguished cytogenetic markers, and the high content of repetitive sequences. In this study, a set of single copy genes free of repetitive sequences with sizes ranging from 4.3 kb to 8.2 kb were screened and used as probes for fluorescence in situ hybridization (FISH) to identify individual chromosomes of garden asparagus. The chromosome-specific signal distribution patterns of these probes enabled the distinguishment of each pair of chromosomes. The sequence assembly and cytogenetic map were successfully integrated, and the results confirmed that the chromosome 1 representing the sex chromosome in the genome assembly is chromosome 5 in the karyotype analysis. The cytogenetic identification of the male-specific region of the Y chromosome (MSY) was implemented using a mixed probe derived from a number of MSY-specific single copy sequences. In addition, the chromosome orthologous relationship between garden asparagus (A1-A10, karyotypic analysis) and its hermaphrodite close relative, A. setaceus (B1-B10, karyotypic analysis), was analyzed using this collection of chromosome-specific cytological markers. The results showed that B3 is the ortholog of sex chromosome A5 and thus may represent the ancestral autosome of the current sex chromosome in garden asparagus. Chromosomes B5, B4, B1, B8, B7, and B9 are the orthologs of A2, A3, A4, A7, A8, and A10, respectively. The chromosome identification, cytogenetic recognition of MSY, and the orthologous relationship analysis between garden asparagus and A. setaceus are valuable for the further investigation of the sex chromosome emergence and evolutionary mechanism of garden asparagus and genome structure evolution in the Asparagus genus.