Fluorescence In Situ Hybridization Research Articles

12333 46 XX/XY Chimerism - Positive NIPT And Post-natal Karyotype Is It Real?

Abstract Disclosure: K.L. Del Rosario: None. N. Sheriden: None. N. Vijayakanthi: None. Introduction: Non-Invasive Prenatal Testing(NIPT) using cell free fetal DNA in plasma of pregnant women has become a routine part of prenatal care in screening for fetal aneuploidies including sex chromosome abnormalities in most regions around the world. False positive results are increasingly imposing challenges in pre-natal as well as post-natal care. Case Discussion: We report a neonate born at 32+2 weeks gestation to a 42-year-old G2 P2 mother. Pregnancy was complicated by insulin dependent maternal type 2 diabetes mellitus, maternal chronic hypertension and a positive NIPT for high-risk monosomy X. Prenatal ultrasound showed male anatomy. Interestingly on physical exam after birth, the neonate had typical male external genitalia with bilateral normal male gonads. Chromosome analysis done immediately after birth revealed the presence of two cell lines: 40 of the 50 cells (80%) had a normal female 46 XX karyotype and 10 of the 50 cells (20%) had a normal male 46 XY karyotype. These findings could be due to reabsorbed twin, Klinefelter syndrome with two trisomy rescue events, maternal cell contamination or true chimerism. In addition, fluorescence in situ hybridization (FISH) analysis for X and Y probes revealed the presence of two cell lines. The first cell line (70% of the cells) had two hybridization signals for chromosome X and the second cell line (∼25% of the cells) had one hybridization signal for chromosome X and one for chromosome Y. Postnatal ultrasound showed normal testicles with no evidence of Mullerian structures. Newborn screening was negative for congenital adrenal hyperplasia (CAH). Genetics was consulted and recommended repeat chromosomal analysis, parental testing to determine sex chromosome segregation, and follow-up with pediatric endocrinology. Repeat cytogenetic testing at 2 weeks of life resulted in a normal male chromosome complement of 46,XY. Previous abnormal cytogenetics were thought to be due to maternal cell contamination. Further endocrine labs showed LH(1.16 uIU/ml) and testosterone levels ( 87 ng/dL), appropriate levels for age indicating normal Hypothalamic Pituitary Gonadal (HPG) axis and testicular Leydig cell function. Since the family was previously counseled regarding the concept of maternal cell contamination, they expressed understanding and were happy with the update of the normal karyotype result. Conclusion: False-positive results concerning for rare chimerism on NIPT can occur and should prompt a thorough history, physical exam, ultrasound studies and repeat testing to confirm its presence. Careful consideration should be taken when counseling family about the implications of these results, especially communication about gender assignment of the infant. Presentation: 6/3/2024

Proteomic analysis and experimental validation reveal the blood–brain barrier protective of Huanshaodan in the treatment of SAMP8 mouse model of Alzheimer’s disease

BackgroundHuanshaodan (HSD) is a Chinese Herbal Compound which has a definite clinical effect on Alzheimer’s disease (AD), however, the underlying mechanism remains unclear. The aim of this study is to preliminarily reveal the mechanism of HSD in the treatment of AD model of SAMP8 mice.MethodsChemical composition of HSD and its drug-containing serum were identified by Q-Orbitrap high resolution liquid mass spectrometry. Six-month-old SAMP8 mice were treated with HSD and Donepezil hydrochloride by gavage for 2 months, and Wogonin for 28 days. Behavioral test was performed to test the learning and memory ability of mice. Immunofluorescence (IF) or Western-blot methods were used to detect the levels of pSer404-tau and β-amyloid (Aβ) in the brain of mice. Hematoxylin–eosin (H&E) staining and Transmission electron microscopy (TEM) assay was applied to observe the pathological changes of neurons. Proteomic technology was carried out to analyze and identify the protein network of HSD interventions in AD. Then the pathological process of the revealed AD-related differential proteins was investigated by IF, Q-PCR, Western-blot, Fluorescence in situ hybridization (FISH) and 16S rRNA sequencing methods.ResultsThe results showed that HSD and Wogonin, one of the components in its drug-containing serum, can effectively improve the cognitive impairments of SAMP8 mice, protect hippocampal neurons and synapses, and reduce the expression of pSer404-tau and Aβ. HSD and Wogonin reduced the levels of fibrinogen β chain (FGB) and γ chain (FGG), the potential therapeutic targets revealed by proteomics analysis, reduced the colocalization of FGB and FGG with Aβ, ionized calcium binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), increased level of and myelin basic protein (MBP). Meanwhile, HSD and Wogonin increased ZO-1 and Occludin levels, improved brain microvascular injury, and reduced levels of bacteria/bacterial DNA and lipopolysaccharide (LPS) in the brain of mice. In addition, 16S rRNA sequencing indicated that HSD regulated the structure of intestinal microbiota of mice.ConclusionThe effects of HSD on AD may be achieved by inhibiting the levels of fibrinogen and the interactions on glia cells in the brain, and by modulating the structure of intestinal microbiota and improving the blood–brain barrier function.

Detecting Cholangiocarcinoma in the Setting of Primary Sclerosing Cholangitis: Is Biliary Tract Fluorescence InSitu Hybridization Helpful?

Biliary brushing cytology (BB) to detect cholangiocarcinoma (CCA) is integral in the surveillance of patients with primary sclerosing cholangitis (PSC). Since reactive changes can mimic carcinoma, indeterminant results are frequent. Fluorescence insitu hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of CCA. This study evaluates the performance of FISH for detecting CCA in patients with and without PSC. A query of our pathology database for atypical and suspicious BB with concurrent FISH results was performed from 2014 to 2021. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed to identify patients with PSC and CCA. CCA was confirmed by pathology or clinical impression. Of the 65 patients (103 BB) in the PSC cohort, 59 patients (94 BB) without CCA and 6 patients (9 BB) with CCA were identified. 33 non-PSC patients (41 BB) with CCA were included for comparison. Positive FISH was highest in non-PSC patients with CCA (10/41 BB, 24%). Positive FISH was seen in both PSC with (1/9 BB, 11%) and without (2/94 BB, 2%) CCA. FISH positivity was lower than expected and was positive in PSC patients without CCA. These results question the clinical utility of FISH for CCA surveillance in PSC patients.

Emphasizing the need for preconceptional, prenatal genetic counseling and comprehensive genetic testing in consanguinity: challenges and experience.

Preconception and prenatal genetic counseling is a well-established means of risk assessment in many parts of the world, and in recent years, an emerging concept in India. Likelihood of an offspring having autosomal recessive disorder increases based on the degree of consanguinity. Hence, genetic testing of the couple for the identification of carrier status for disease-causing variants is crucial. The purpose of this study is to understand the frequency of genetic abnormalities in consanguineous marriages by using a comprehensive genetic testing algorithm where in karyotyping, FISH, exome sequencing and microarray are used sequentially to determine the genetic etiology based on the clinical presentation and to evaluate the need and benefits of preconceptional and prenatal genetic counseling. This retrospective study includes 66 couples having consanguinity referred for genetic counseling and testing. Of the 66 couples, 58 underwent comprehensive genetic testing which included Karyotyping, Fluorescence in Situ Hybridization (FISH), Microarray and Exome sequencing based on their clinical presentation. The analyses revealed a genetic abnormality in approximately 31% and chromosomal polymorphic variations & variants of uncertain significance in 17% of the couples. Counseling in these couples helped in identifying the carrier status and enabled them to take an informed decision in subsequent pregnancies. These findings reiterate the acute need for preconception and prenatal genetic counseling services in India.

Biofilm infections of endobronchial valves in COPD patients after endoscopic lung volume reduction: a pilot study with FISHseq

Endoscopic lung volume reduction (ELVR) using endobronchial valves (EBV) is a treatment option for a subset of patients with severe chronic obstructive pulmonary disease (COPD), suffering from emphysema and hyperinflation. In this pilot study, we aimed to determine the presence of bacterial biofilm infections on EBV and investigate their involvement in lack of clinical benefits, worsening symptomatology, and increased exacerbations that lead to the decision to remove EBVs. We analyzed ten COPD patients with ELVR who underwent EBV removal. Clinical data were compared to the microbiological findings from conventional EBV culture. In addition, EBV were analyzed by FISHseq, a combination of Fluorescence in situ hybridization (FISH) with PCR and sequencing, for visualization and identification of microorganisms and biofilms. All ten patients presented with clinical symptoms, including pneumonia and recurrent exacerbations. Microbiological cultures from EBV detected several microorganisms in all ten patients. FISHseq showed either mixed or monospecies colonization on the EBV, including oropharyngeal bacterial flora, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus spp., and Fusobacterium sp. On 5/10 EBV, FISHseq visualized biofilms, on 1/10 microbial microcolonies, on 3/10 single microorganisms, and on 1/10 no microorganisms. The results of the study demonstrate the presence of biofilms on EBV for the first time and its potential involvement in increased exacerbations and clinical worsening in patients with ELVR. However, further prospective studies are needed to evaluate the clinical relevance of biofilm formation on EBV and appropriate treatment options to avoid infections in patients with ELVR.

Cancer-associated fibroblast-secreted exosomes promote prostate cancer cell migration and invasion by the FGL1/SOX5 axis.

Exosomes secreted by cancer-associated fibroblasts (CAFs) play a critical role in cancer progression. This study aimed to explore the effects of CAF exosomes on prostate cancer (PC) cell metastasis. PC cells were treated with these exosomes, and their processes were evaluated using cell-counting kit-8 and Transwell assays. Exosome-regulated mRNAs were explored using quantitative real-time PCR. The relationship between FGL1 and SOX5 was analyzed using co-immunoprecipitation and fluorescence in situ hybridization (FISH) assays. The results of this study showed that exosomes derived from CAFs promoted PC cell viability, migration, and invasion. CAFs promoted PC cell viability and metastasis by releasing exosomes. Exosome treatment increased the levels of FGL1, which interacted with SOX5 and negatively regulated its expression. Rescue experiments demonstrated that CAF exosomes promoted the biological behaviors of PC cells by upregulating FGL1 and downregulating SOX5. Moreover, exosomes accelerated tumor growth by regulating the FGL1 level. In conclusion, CAF-derived exosomes promoted PC cell viability, migration, and invasion by elevating the FGL1/SOX5 axis, suggesting a novel strategy for the treatment of metastatic PC.

Microfluidic Affinity Selection of B-Lineage Cells from Peripheral Blood for Minimal Residual Disease Monitoring in Pediatric B-Type Acute Lymphoblastic Leukemia Patients.

Assessment of minimal residual disease (MRD) is the most powerful predictor of outcome in B-type acute lymphoblastic leukemia (B-ALL). MRD, defined as the presence of leukemic cells in the blood or bone marrow, is used for the evaluation of therapy efficacy. We report on a microfluidic-based MRD (MF-MRD) assay that allows for frequent evaluation of blood for the presence of circulating leukemia cells (CLCs). The microfluidic chip affinity selects B-lineage cells, including CLCs using anti-CD19 antibodies poised on the wall of the microfluidic chip. Affinity-selected cells are released from the capture surface and can be subjected to immunophenotyping to enumerate the CLCs, perform fluorescence in situ hybridization (FISH), and/or molecular analysis of the CLCs' mRNA/gDNA. During longitudinal testing of 20 patients throughout induction and consolidation therapy, the MF-MRD performed 116 tests, while only 41 were completed with multiparameter flow cytometry (MFC-MRD) using a bone marrow aspirate, as standard-of-care. Overall, 57% MF-MRD tests were MRD(+) as defined by CLC numbers exceeding a threshold of 5 × 10-4%, which was determined to be the limit of quantitation. Above a threshold of 0.01%, MFC-MRD was positive in 34% of patients. The MF offered the advantage of the opportunity for efficiently processing small volumes of blood (2 mL), which is important in the care of pediatric patients, especially infants. The minimally invasive means of blood collection are of high value when treating patients whose MRD is typically tested using an invasive bone marrow biopsy. MF-MRD detection can be useful for stratification of patients into risk groups and monitoring of patient well-being after completion of treatment for early recognition of potential impending disease recurrence.

The LINC00319 binding to STAT3 promotes the cell proliferation, migration, invasion and EMT process in oral squamous cell carcinoma

BackgroundLong non-coding RNA LINC00319 has been implicated in the progression of various cancers, including oral squamous cell carcinoma (OSCC). While our previous work has revealed some aspects of LINC00319's role in OSCC, including its upregulation and involvement in a competing endogenous RNA (ceRNA) mechanism, the full extent of its functions and regulatory mechanisms in OSCC progression remain to be fully elucidated. ObjectiveThis study aimed to investigate the function of LINC00319 in OSCC and its potential interaction with the STAT3 signaling pathway, thus uncovering novel regulatory mechanisms and therapeutic targets. MethodsBioinformatics analysis was performed using TCGA data to evaluate LINC00319 expression in OSCC tissues and its correlation with STAT3 signaling. The direct binding between LINC00319 and STAT3 was examined by RNA pull-down, FISH, and RIP assays. Functional experiments, including CCK-8, transwell migration and invasion assays, and western blot analysis of EMT markers and STAT3 pathway activation, were conducted to assess the effects of LINC00319 on OSCC cell behaviors and its interaction with the STAT3 signaling pathway. In vivo xenograft models were established to validate the role of LINC00319 in tumor growth and STAT3 activation. ResultsLINC00319 expression was significantly upregulated in OSCC tissues compared to normal tissues, and high LINC00319 expression correlated with STAT3 signaling activation. Mechanistically, LINC00319 directly bound to STAT3 protein and promoted its phosphorylation at Tyr705. LINC00319 overexpression enhanced, while its knockdown suppressed, the proliferation, migration, invasion, and EMT of OSCC cells. These oncogenic effects were mediated through STAT3 activation and could be reversed by the STAT3 inhibitor stattic. In vivo experiments further confirmed that LINC00319 silencing inhibited tumor growth and STAT3 phosphorylation. ConclusionThis study uncovers that LINC00319 promotes OSCC tumorigenesis by directly binding to and activating STAT3 signaling. These findings provide new insights into the regulatory mechanisms of STAT3 by long non-coding RNAs and highlight the potential of LINC00319 as a biomarker and therapeutic target in OSCC.

Detection, isolation and virulence characterization of Helicobacter suis from pork products aimed to human consumption

Helicobacter suis is the most common non-H. pylori gastric Helicobacter species found in humans. Infection is associated with gastritis, peptic ulcer, gastric MALT lymphoma and neurodegenerative disorders, particularly Parkinson's disease. However, the pathogenicity of this species is still a matter of research, and results of virulence studies and antibiotic susceptibility tests tend to vary between strains. Cholesterol α-glucosyltransferase (αCgT), a known H. pylori virulence factor, appears to be present in most clinical H. suis isolates. The ability to form biofilms also plays a crucial role in the pathogenesis of H. pylori. However, no reports have been published on this ability in H. suis.H. suis is considered an emerging zoonotic pathogen, with pigs being the main source of human infection. However, there is very little information on its presence in pork, mainly due to the difficulties of its culture. Therefore, our aim was to determine the prevalence of H. suis in pork products from our geographical area by PCR and fluorescence in situ hybridisation (FISH), as well as to isolate the bacteria and determine the antibiotic susceptibility patterns, the presence of the αCGT gene and the ability of the isolates to form biofilms.Overall, H. suis was detected in 20 of the 70 (28.6 %) samples analyzed. In 3 of them, H. suis was isolated. The αCGT gene was detected in all isolates and two of them showed a multiresistance pattern. The H. suis reference strain and two of the isolates showed “strong” to “moderate” in vitro biofilm formation ability under optimal growth conditions.Our results seem to indicate that H. suis is significantly prevalent in pork products. The combination of culture with FISH and/or mPCR proved to be a rapid and specific method for the detection, identification and direct visualization of cultivable H. suis cells from pork food products.

Chromosomal mapping of 5S rDNA in two species of the genus Acanthocephalus (Echinorhynchida)

Chromosomal mapping of 5S rDNA in two Acanthocephala species was performed for the first time using fluorescence in situ hybridization (FISH) with a 5S rDNA probe. The 5S rDNA PCR products from the genomes of both species were sequenced and aligned and an identical 141 bp long coding region was determined. The same patterns of 5S rDNA gene cluster distribution were observed, with FISH signal restricted to a single autosomal chromosome pair. A preference for distal positioning on the chromosomes (subtelomeric position) was observed in both species. In addition, two-color FISH was performed to examine the mutual positions of 5S and 18S rDNA on the chromosomes. Our knowledge of the organization of the Acanthocephala genome is extremely limited and its chromosomes are poorly studied. Any new information about the location of chromosomal markers as important features of the respective karyotype may be useful in solving evolutionary questions.

Metformin in combination with chemotherapy increases apoptosis in gastric cancer cells and counteracts senescence induced by chemotherapy.

Gastric cancer (GC) is the fourth leading cause of cancer death in the world, and there is a demand for new therapeutic agents to treat GC. Metformin has been demonstrated to be an antineoplastic agent in some types of cancer; however, it has not been sufficiently valued in treating GC because the effect of metformin in combination with chemotherapy regimens has not yet been evaluated. The present study aimed to evaluate the mechanisms underlying cell death induced by metformin alone or when combined with chemotherapy. The cytogenetic characteristics of the NCI-N87 cell line were determined by fluorescence in situ hybridization (FISH). To determine viability, the cells were treated with metformin, epirubicin, cisplatin, docetaxel and 5-fluorouracil (individually and at different concentrations). Subsequently, the cells were treated with metformin alone, and in combination with the chemotherapeutic drugs and the epirubicin + cisplatin + 5-fluorouracil, docetaxel + cisplatin + 5-fluorouracil, and cisplatin + 5-fluorouracil regimens. Cell viability, proliferation and mitochondrial membrane potential (ΔΨm) were analyzed by spectrophotometry. Apoptosis, caspase activity and cell cycle progression were assessed by flow cytometry. Finally, light microscopy was used to evaluate senescence and clonogenicity. The results revealed that metformin, alone and when combined with chemotherapy, increased the proportion of apoptotic cells, promoted the loss of ΔΨm, and induced apoptosis through caspase activity in GC cells. Moreover, metformin decreased cell proliferation. In addition, metformin alone did not induce senescence and it counteracted the effects of chemotherapy-induced senescence in GC cells. Additionally, metformin, alone and when combined with chemotherapy, decreased the clonogenic capacity of NCI-N87 GC cells. In conclusion, metformin may increase the effects of chemotherapy on NCI-N87 cell death and could represent an option to improve the treatment of GC.

The suppression of OTUD7B by miR-491-5p enhances the ubiquitination of VEGFA to suppress vascular mimicry in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high morbidity and mortality. Vascular mimicry (VM), a distinct microcirculation model in tumors that differs from classical angiogenesis, is strongly associated with poor clinical outcomes in cancer patients. miR-491-5p has been reported to prevent NSCLC progression, including proliferation, metastasis, and angiogenesis. However, the effect and mechanism of miR-491-5p on VM have not been studied in NSCLC. The expression of miR-491-5p was detected by quantitative reverse transcription PCR (qPCR) and fluorescence in situ hybridization (FISH). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays were used to examine cell growth. Tube formation assay was used to assess VM in NSCLC cells. Immunohistochemistry (IHC) and western blot were performed to detect protein expression. Immunoprecipitation was used to confirm the interaction between OTU deubiquitinase 7B (OTUD7B) and vascular endothelial growth factor A (VEGFA), and the level of ubiquitinated VEGFA. A nude mouse tumorigenesis model was used to evaluate the carcinogenic capacity of NSCLC cells in vivo. Luciferase reporter assay was used to identify the potential target of miR-491-5p. MiR-491-5p was found downregulated in NSCLC tissues, and miR-491-5p deficiency was strongly associated with angiogenesis. miR-491-5p mimics suppressed cell viability, migration, and VM. Conversely, an inhibitor of miR-491-5p had the opposite effect. OTUD7B, a deubiquitinase, was identified as a downstream target of miR-491-5p. A luciferase reporter assay indicated that miR-491-5p directly binds to the 3'UTR of OTUD7B. Moreover, mimics of miR-491-5p caused a significant reduction in the OTUD7B protein in NSCLC cells, and an inhibitor of miR-491-5p stabilized the OTUD7B protein. In addition, overexpression of OTUD7B promoted cell proliferation, migration, and VM, similar to the effects of an inhibitor of miR-491-5p. Further exploration revealed that OTUD7B interacts with VEGFA and that the miR-491-5p-OTUD7B axis modulates the ubiquitination of VEGFA. The rescue experiment indicated that OTUD7B compromised the inhibitory effects of miR-491-5p on the cellular function of NSCLC cells. Overall, our study first proved that miR-491-5p impedes VM by suppressing OUTD7B and promoting the ubiquitination of VEGFA. The miR-491-5p/OTUD7B axis may be a novel target for antiangiogenic therapy in NSCLC.

Development of wheat-tetraploid Thinopyrum elongatum 4EL small fragment translocation lines with stripe rust resistance gene Yr4EL.

Two small fragment translocation lines (T4DS·4DL-4EL and T5AS·5AL-4EL) showed high resistance to stripe rust and resistance gene Yr4EL was localized to an about 35 Mb region at the end of chr arm 4EL. Stripe rust, caused by the fungus Puccinia striiformis f. sp. tritici, is a devastating wheat disease worldwide. Deployment of disease resistance (R) genes in wheat cultivars is the most effective way to control the disease. Previously, the all-stage stripe rust R gene Yr4EL from tetraploid Thinopyrum elongatum was introduced into common wheat as 4E(4D) substitution and T4DS·4EL translocation lines. To further map and utilize Yr4EL, Chinese Spring (CS) mutant pairing homoeologous gene ph1b was used in crossing to induce recombination between chromosome (chr) 4EL and wheat chromosomes. Two small fragment translocation lines T4DS·4DL-4EL and T5AS·5AL-4EL with Yr4EL resistance were selected using molecular markers and confirmed by genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and Wheat660K SNP array analyses. We mapped Yr4EL to an about 35Mb region at the end of chr 4EL, corresponding to 577.76-612.97Mb based on the diploid Th. elongatum reference genome. In addition, two competitive allele-specific PCR (KASP) markers co-segregating with Yr4EL were developed to facilitate molecular marker-assisted selection in breeding. The T4DS·4DL-4EL lines were crossed and backcrossed with wheat cultivars SM482 and CM42, and the resulting pre-breeding lines showed high stripe rust resistance and potential for wheat breeding with good agronomic traits. These lines represent new germplasm for wheat stripe rust resistance breeding, as well as providing a solid foundation for Yr4EL fine mapping and cloning.

Advanced Techniques In Oral Oncology: AComprehensiveReview

The most common type of oral cancer, oral squamous cell carcinoma (OSCC), is still amajor global health concern. Due to delayed diagnoses, which are frequently caused by low public awareness and fewscreeningalternatives, the five-year survival rate is still low. Modernmethods in oral oncology have arisen to enhance patient outcomes and early identification, including as molecular and imaging diagnostics. This review showcases several cutting-edge diagnostic techniques, including flow cytometry, next-generation sequencing(NGS), chemiluminescence, toluidine blue, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and nano-diagnostics. By providing insights into tumor behaviourand enabling customized treatment approaches, these techniques advance our knowledge of the genetic and proteomic landscape of oral cancer. Combining these cutting-edge approaches with conventional diagnosticsmight greatly improve the accuracy of OSCC detection and risk assessment, despite drawbacks like false positives and expensive procedures. Improving patient care greatly depends on the creation of quick, noninvasive procedures and the investigation of genetic biomarkers. The present review highlightsthe need for continued research and training in oral oncology to maximize early diagnosis and intervention strategies

Highly divergent satellitomes of two barley species of agronomic importance, Hordeum chilense and H. vulgare

In this paper, we have performed an in-depth study of the complete set of the satellite DNA (satDNA) families (i.e. the satellitomes) in the genome of two barley species of agronomic value in a breeding framework, H. chilense (H1 and H7 accessions) and H. vulgare (H106 accession), which can be useful tools for studying chromosome associations during meiosis. The study has led to the analysis of a total of 18 satDNA families in H. vulgare, 25 satDNA families in H. chilense (accession H1) and 27 satDNA families in H. chilense (accession H7) that constitute 46 different satDNA families forming 36 homology groups. Our study highlights different important contributions of evolutionary and applied interests. Thus, both barley species show very divergent satDNA profiles, which could be partly explained by the differential effects of domestication versus wildlife. Divergence derives from the differential amplification of different common ancestral satellites and the emergence of new satellites in H. chilense, usually from pre-existing ones but also random sequences. There are also differences between the two H. chilense accessions, which support genetically distinct groups. The fluorescence in situ hybridization (FISH) patterns of some satDNAs yield distinctive genetic markers for the identification of specific H. chilense or H. vulgare chromosomes. Some of the satellites have peculiar structures or are related to transposable elements which provide information about their origin and expansion. Among these, we discuss the existence of different (peri)centromeric satellites that supply this region with some plasticity important for centromere evolution. These peri(centromeric) satDNAs and the set of subtelomeric satDNAs (a total of 38 different families) are analyzed in the framework of breeding as the high diversity found in the subtelomeric regions might support their putative implication in chromosome recognition and pairing during meiosis, a key point in the production of addition/substitution lines and hybrids.

Centromeres in cancer: Unraveling the link between chromosomal instability and tumorigenesis.

Centromeres are critical structures involved in chromosome segregation, maintaining genomic stability, and facilitating the accurate transmission of genetic information. They are key in coordinating the assembly and help keep the correct structure, location, and function of the kinetochore, a proteinaceous structure vital for ensuring proper chromosome segregation during cell division. Abnormalities in centromere structure can lead to aneuploidy or chromosomal instability, which have been implicated in various diseases, including cancer. Accordingly, abnormalities in centromeres, such as structural rearrangements and dysregulation of centromere-associated proteins, disrupt gene function, leading to uncontrolled cell growth and tumor progression. For instance, altered expression of CENP-A, CENP-E, and others such as BUB1, BUBR1, MAD1, and INCENP, have been shown to ascribe to centromere over-amplification, chromosome missegregation, aneuploidy, and chromosomal instability; this, in turn, can culminate in tumor progression. These centromere abnormalities also promoted tumor heterogeneity by generating genetically diverse cell populations within tumors. Advanced techniques like fluorescence in situ hybridization (FISH) and chromosomal microarray analysis are crucial for detecting centromere abnormalities, enabling accurate cancer classification and tailored treatment strategies. Researchers are exploring strategies to disrupt centromere-associated proteins for targeted cancer therapies. Thus, this review explores centromere abnormalities in cancer, their molecular mechanisms, diagnostic implications, and therapeutic targeting. It aims to advance our understanding of centromeres' role in cancer and develop advanced diagnostic tools and targeted therapies for improved cancer management and treatment.

Impact of sex chromosome abnormalities in male chronic myeloid leukemia patients

IntroductionChronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) resulting from the reciprocal translocation t(9;22)(q34;q11). Sex chromosomal abnormalities are found rarely in CML patients. Materials and methodsConventional and molecular cytogenetic analysis (Fluorescent In Situ Hybridization (FISH)) were performed to reveal the presence of Ph and additional chromosomal abnormalities (ACA). ResultsThree patients with abnormalities within sex chromosomes, one Klinefelter syndrome (KS) patient and two cases with LOY (Loss of Y) diagnosed with CML CP were included in the study. All three patients initially responded to the targeted therapy, became resistant to their treatment course and switched to 2nd line therapies. In the 15th month, the KS patient abruptly turned into BC and showed clonal evolution with four karyotypic patterns. According to Mitelman databases, this is the first KS-CML patient who showed three major route abnormalities, +8, i(17) and + der(22) in a single clone. Survival analysis showed one patient with LOY chromosome expired on the 48th month of diagnosis. ConclusionThe application of cytogenetic techniques in identifying the constitutional abnormality of KS, ACA at initial CML diagnosis, revealing of extra Ph during the treatment course and clonal evolution in BC (Blast Crisis) assist the proper monitoring of disease transformation and serves as a major diagnostic tool for CML patients thereby switching the treatment protocols. A systematic stratification of patients according to the chromosomal abnormalities is needed in CML patients.

A Novel Urine DNA Predictor for Noninvasive Early Diagnosis and Monitoring Minimal Residual Disease of Upper Tract Urothelial Carcinoma.

For early detection and postoperative monitoring of upper tract urothelial carcinoma (UTUC), the traditional detection method was limited to its invasiveness and insufficient sensitivity. We aim to use urine tumour DNA (utDNA) for detecting minimal residual disease (MRD), early diagnosis and perioperative monitoring in UTUC. We previously established a utDNA multidimensional bioinformatic valuation model, named utLIFE, using low-coverage whole-genome sequencing and targeted deep sequencing. This prospective cohort enrolled 93 patients diagnosed with UTUC without metastasis. We collected morning urine samples on the day of surgery and the discharge day after the operation for utLIFE testing. In addition, we also enrolled 80 healthy controls to further validate the specificity of the utLIFE model in the study. The utLIFE of preoperative samples could discriminate UTUC with high specificity (96.25%, 77/80), and high sensitivity (96.77%, 90/93) regardless of stage and grade. The sensitivity of utLIFE was significantly higher than urine cytology (p < 0.001) and fluorescence insitu hybridisation (FISH) (p < 0.001) (N = 19), especially in early-stage and low-grade UTUC. Postoperative utLIFE scores were significantly decreased compared with those of preoperative samples (79 vs. 36, p < 0.001), indicating its association with tumour burden. For special pathology types, utLIFE performed less well in sensitivity and perioperative alteration. In conclusion, we established a bioinformatic utDNA valuation model, utLIFE, which was validated to be a rapid and noninvasive approach with high sensitivity for early detection and MRD monitoring for UTUC.

Microbial diversity and biogeography across gastrointestinal tracts of Takifugu pufferfish revealed by full-length 16S amplicon sequencing

Fish, which are vital for both aquatic ecosystem functionality and global food supply, rely heavily on their gastrointestinal tract (GIT) microbiota for the digestion that underpins their growth and health. Takifugu pufferfish, which are an example of species evolved through adaptive radiation, possess a GIT that is specialized for antipredator defense and gluttonous feeding behaviors, offering a unique model to explore the effects of GIT compartmentalization and host genetics on gut microbial communities. Here we compiled 78 full and partial-length 16S rRNA amplicon datasets across three anteroposteriorly distinct intestinal sites in a cohort of cohabitating artificial hybrid and purebred Takifugu pufferfishes. Our findings reveal a compositional and functional biogeography of pufferfish gut microbiota along the GIT and between host genetics. Additionally, the differential abundance of specific amplicon sequence variants and their correlation with host genetic backgrounds and intestinal sections highlight the role of environmental filtering in shaping microbial communities, with certain bacterial taxa exhibiting strong preferences for particular intestinal sites or genetic backgrounds, suggesting potential localized adaptation or functional specialization. This study enhances our understanding of the intricate interplay between host genetics, gut anatomy, and microbiota in fish, underscoring the importance of detailed microbial profiling in conservation efforts and aquaculture practices, and emphasizing the necessity of integrating full-length 16S rRNA sequencing with partial-length datasets to comprehensively understand microbial diversity and function, paving the way for improved fish health management and sustainable aquaculture strategies.

Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification.

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.