Preliminary Sequence Research Articles

Differences in cell subsets between sun-exposed and unexposed skin: preliminary single-cell sequencing and biological analysis from a single case.

The composition and subsets of skin cells continuously change in a dynamic manner. However, the specific microcosmic alterations of human photoaged skin, independent of chronologic aging, remain unclear and have been infrequently analyzed. This study aimed to evaluate the biological processes and mechanisms underlying cell-subgroup alterations in skin photoaging. We utilized single-cell sequencing and biological analysis from a single case to investigate the effects of photoaging. Skin punch biopsies were taken from sun-exposed forearm skin and unexposed buttock skin from the same individual for comparative analysis. Our analysis identified 25 cell clusters and 12 skin cell types, revealing significant changes in unique gene expressions between the sun-exposed and unexposed skin samples. A comparison of cell numbers within each cluster revealed 9 dominant cell clusters in sun-exposed skin and 16 dominant cell clusters in unexposed skin. Enrichment analysis indicated that PD-L1 expression and the PD-1 checkpoint pathway were more prominent in sun-exposed skin, while MAPK, TNF-alpha, TGF-beta, and apoptosis pathways were more enriched in hair follicle cells of sun-exposed skin. This study reveals changes in cell components in photoaged skin from a single case and provides novel insights into cellular subpopulations and pathology during repeated UVA-induced skin damage. These findings enhance our understanding of the complex interplay between different cells in photoaged skin and offer potential targets for preventing human skin photoaging and UV-induced skin cancers.

The Delicate Situation of Childhood Vaccination: On the Dispreferredness of Soliciting Parents’ Intent to Vaccinate

ABSTRACT In the Netherlands, parents of newborns typically participate in two-, four-, and eight-week medical consultations to monitor their children’s development and discuss vaccinations, which will not be administered before eight weeks. During these visits, healthcare professionals routinely ask parents if they intend to vaccinate their children (i.e. to participate in the National Immunization Program). Using Conversation Analysis, we examine 62 videotaped consultations and present two lines of evidence to argue that the sequence initiated by professionals wherein they solicit parents’ intent to vaccinate is dispreferred. First, this action is routinely deferred by preliminary sequences. Second, when professionals eventually initiate this action (i.e. if it is not preempted by parents during pre-sequences), they orient to its dispreferred status, for example by highlighting benefactive details of vaccination. We discuss the possible existence of asymmetrical (initiator-sided) pre-sequences, why soliciting parents’ vaccination intent might be dispreferred, and implications for the design of communication interventions.

18 Unraveling the Impact of Tumor Microenvironment on Immunotherapy Response/Resistance in Human Sarcomatoid Clear Cell Renal Cell Carcinoma: Insights from a Novel Mouse Model

Abstract Background Renal cell carcinoma (RCC) with histological sarcomatoid de-differentiation (sRCC) has a historically poor prognosis across all RCC subtypes. Recently, immunotherapy with anti-PD1 + anti-CTLA4 combo has significantly improved disease-specific survival, and complete response are seen in upwards of 20% of patients. However, half of sRCC patients still have no response to this immunotherapy, and the mechanism of response remains unclear. Methods Our preliminary bulk RNA sequencing data reveal a distinct tumor microenvironment (TME) in human sRCC tumors, characterized by heightened immune cell infiltration, particularly enriched for regulatory T cells (Tregs). Subsequently, we delineated the spatial gene expression topography within sRCC and clear cell renal cell carcinoma (ccRCC) regions, culminating in the development of the sRCC signature. Concurrently, we established an innovative immunocompetent murine model of sRCC. Leveraging CRISPR technology, we replicated the human sRCC genotype by knocking out Vhl, Bap1, and Cdkn2a/2b genes in a mouse telomerase reverse transcriptase (mTERT) overexpressing renal proximal tubule epithelial cell line. This effort yielded a successfully engineered mouse implantable sRCC cell line bearing VhlMutBap1DelCdkn2aDelCdkn2bDel (HJRCC68N). Results The syngeneic mouse sRCC model we developed faithfully replicates human sRCC histology and tumor microenvironment and recapitulates the sRCC signature identified in human sRCC regions. Importantly, like in humans, mouse sRCC tumors exhibit a heterogeneous response to anti-PD1 + anti-CTLA4 combination, and anti-CTLA4 monotherapy, reflecting the clinical variability observed in human patients. Furthermore, transcriptomic analysis from our mouse model reveals the involvement of type 1 immunity in responder, providing insights into overcoming immunotherapy resistance. Conclusions In conclusion, our study unveiled distinct changes in the TME at the transcriptomic and spatial gene expression levels in human sRCC. Leveraging our novel murine model, which faithfully replicates human sRCC histology, TME characteristics, and mixed response to immunotherapy, we elucidated the involvement of type 1 immunity in treatment response. These insights offer valuable pathways for overcoming immunotherapy resistance and pave the way for the development of tailored treatment strategies aimed at improving outcomes for sRCC patients.

Tumor reactive T cells from the cerebrospinal fluid of patients with leptomeningeal disease from melanoma.

2027 Background: Leptomeningeal disease (LMD) is a devastating complication from cancer, with a median survival of 6-8 weeks. The incidence of LMD is highest in melanoma (5-25%) carrying the worst prognosis. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) showed complete and durable responses in patients with advanced metastatic melanoma. CSF-derived T cell populations in LMD shift to more exhausted phenotypes compared to extracranial and brain metastases. Ex vivo large T cell expansion can overcome this shortfall, with adoptive T cell transfer representing a novel therapeutic approach to treat melanoma LMD (M-LMD). The goal of this study is to optimize rapid CSF-derived tumor reactive T cell expansion in M-LMD, and evaluate the immune microenvironment in CSF compared to peripheral immunity and anti-tumoral response. Methods: CSF was collected from M-LMD patients (n=6) via lumbar puncture or Ommaya. Cells were plated following established TIL culture protocols to determine optimal expansion conditions i.e.: 1) 6000 IU/mL IL-2, 2) IL-2+OKT3, 3) IL-2+anti-4-1BB agonistic antibody, 4) IL-2+IL-7+IL-15+IL-21, 5) IL-2+anti-CD3/CD28 human dynabeads, & 6) IL-2+anti-CD3/CD28/CD137 human dynabeads. After 4 weeks, T cells were propagated via rapid expansion protocol (REP) and phenotyped for CD4+ and CD8+ T cells with flow cytometry. T cells were co-cultured for 18-24 hours with HLA-matched melanoma cell lines to assess functionality, and IFN-y levels evaluated in supernatant. Ongoing studies are evaluating other biomarkers i.e., chemokines via Luminex. To examine the expanded TCR clonotypes, TCR beta seq was done in CSF-derived T cells at different times point of expansion and peripheral T cells. Results: Collected CSF yielded an average 8.12e4 viable cells for expansion (range 5e2-2.43e6). After initial culture in IL-2, 61.9% of samples showed increased cell yield with an average 101.68-fold expansion (range 2.35-1054). PreREP T cells were predominantly CD4+ (45.7%). REP was completed with a 100% success rate (mean 187.47-fold expansion). Post-REP flow cytometry analysis revealed similar results with further expansion of T cells. Additional culture supplements (Methods #2-6) produced similar expansion with a reduced T cell input requirement and demonstrated the potential to enrich for CD8+ T cells. M-LMD T cells were highly functional, producing substantial levels of IFN-y in response to HLA-matched melanoma cell lines. Anti-CD3/CD28 produced a larger expansion of T cells with higher levels of IFN-y. Preliminary TCR beta sequencing data revealed a distinct clonal distribution between peripheral and CSF-derived T cells. Conclusions: Therapies for LMD patients are desperately needed. Results demonstrate successful expansion of T cells ex vivo from CSF in M-LMD. Results raise potential to use autologous CSF-derived T cells as therapeutic strategy for patients with LMD.

SnRNAs from Radical Prostatectomy Specimens Have the Potential to Serve as Prognostic Factors for Clinical Recurrence after Biochemical Recurrence in Patients with High-Risk Prostate Cancer.

In patients with high-risk prostate cancer (HRPC) after radical prostatectomy (RP), biochemical recurrence (BCR) increases the risk of distant metastasis. Accordingly, additional prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence (CR) after BCR. The objective of this study was to identify biomarkers in formalin-fixed paraffin-embedded (FFPE) RP samples that are prognostic for CR in patients with HRPC who experience BCR after RP (post-RP BCR). First, we performed a preliminary RNA sequencing analysis to comprehensively profile RNA expression in FFPE RP samples obtained from patients with HRPC who developed CR after post-RP BCR and found that many snRNAs were very abundant in preserved FFPE samples. Subsequently, we used quantitative polymerase chain reaction (qPCR) to compare the expression levels of highly abundant snRNAs in FFPE RP samples from patients with HRPC with and without CR after post-RP BCR (21 CR patients and 46 non-CR patients who had more than 5 years of follow-up after BCR). The qPCR analysis revealed that the expression levels of snRNA RNU1-1/1-2 and RNU4-1 were significantly higher in patients with CR than in patients without CR. These snRNAs were significantly correlated with clinical recurrence-free survival (RFS) in patients with HRPC who experienced post-RP BCR. Furthermore, snRNA RNU1-1/1-2 could serve as an independent prognostic factor for clinical RFS in post-RP BCR of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between CR and non-CR patients. Our findings provide new insights into the involvement of snRNAs in prostate cancer progression.

Investigating Neutrophil Subpopulations involved in Acute Lung Injury in Pediatric Patients with Congenital Heart Disease using a Single Cell Approach

Abstract Congenital Heart Disease (CHD) is a significant health concern (4-10/1,000 live births). Morbidity in CHD includes a younger age, thrombotic complications, and organ dysfunction. Acute lung injury (ALI) occurs intra/postoperatively in 2–3% of pediatric patients, particularly neonates and infants. Neutrophil recruitment to the lungs during CPB-induced ALI is noted. Recognizing the dual role of neutrophils in both defense and tissue damage, a nuanced strategy is essential. Our preliminary single-cell RNA sequencing (scRNAseq) analysis of blood from pediatric patients undergoing longer (>=3 hours) versus shorter (<3 hours) CPB surgery reveals distinct neutrophil subpopulations to be enriched post-CPB and associated with ALI. Specifically, in patients with a longer CPB, the IFTIM2 gene, associated with type I interferon responses, is enriched. Pathway analysis confirms associations with interferon responses, chemotaxis, cell migration, reactive oxygen species, and cell death. Preliminary data shows that upregulated β2 integrin levels post-longer CPB suggest a predisposition to ALI. This insight aims to guide the design of targeted therapies for specific neutrophil subpopulations while preserving their critical functions. This study will improve the treatment of these patients, addressing a crucial and critical gap in pediatric cardiac intensive care.

CROWD VIDEO SEQUENCES PROCESSING METHODS FOR DETERMINING THE CROWD MOTION PATTERNS

Nowadays, homogeneous objects clusters motion is one of the most important and rapidly developing computer vision and machine learning application. In this paper, we consider the crowd motion patterns determination by using motion maps that we calculate with FlowNet, a neural network examining motion of objects in a video sequence. This approach allows us to get information on the crowd direction and velocity with relation to other objects of scene, which plays the key role in behavior analysis and security establishment. Besides, we consider methods for preliminary video sequence processing, including frame combination, to estimate motion maps more precisely and improve the effectiveness of the dynamic scenes analysis.

Molecular epidemiology and phylogenetic analysis of feline calicivirus in Kunshan, China

Feline calicivirus (FCV) is a highly contagious virus in cats, which typically causes respiratory tract and oral infections. Despite vaccination against FCV being a regular practice in China, new FCV cases still occur. Antigenic diversity of FCV hinders the effective control by vaccination. This is first report which aims to investigate the molecular epidemiology and molecular characteristics of FCV in Kunshan, China. The nasopharyngeal swabs were collected from cats showing variable clinical signs from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and sequencing of the FCV capsid gene were performed to study genetic diversity and evolutionary characteristics. FCV-RNA was identified in 52 (26%) of the samples using RT-PCR. A significant association was found between FCV-positive detection rate, age, gender, vaccination status and living environment, while a non-significant association was found with breed of cats. Nucleotide analysis revealed two genotypes, GI and GII. GII predominated in Kunshan, with diverse strains and amino acid variations potentially affecting vaccination efficacy and FCV detection. Notably, analysis pinpointed certain strains’ association with FCV-virulent systemic disease pathotypes. This investigation sheds light on FCV dynamics, which may aid in developing better prevention strategies and future vaccine designs against circulating FCV genotypes.

System analysis of the ecological distribution of bacteriophages in hospital wastewater

Phage therapy is one of the most important tools for the treatment of infections with multi-drug resistant bacteria. Such phages are usually isolated from hospital effluents, however, no systematic study on the distribution of phages in hospital effluents has been conducted so far. The aim of this study was to isolate the corresponding phages of common pathogenic bacteria isolated in the clinic as hosts, so as to assess the ecological distribution of phages in hospital wastewater and to provide a reference for the isolation and application of phages of drug-resistant bacteria in the clinic. A cross-sectional study design was used in this study. The 125 pathogenic bacteria (belonging to 16 different strains) isolated from the clinical microbiology laboratory of Qilu Hospital of Shandong University from May to June 2023 were selected as the target strains, and the phages corresponding to these strains were isolated and purified from the hospital wastewater by using the double-layer plate sandwich method. At the same time, the distribution of pathogenic bacteria in the same batch of wastewater was analyzed with the help of mNGS sequencing technology, so as to preliminarily investigate the abundance correspondence between pathogenic bacteria and phages in wastewater. The results showed that a total of 56 phage strains were isolated from 125 clinical pathogens as hosts, corresponding to six pathogens, including Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. All six pathogenic bacteria contained strains with different degrees of drug resistance, with a higher percentage of multi-drug resistant strains in A. baumannii, Escherichia coli and P. aeruginosa. The phage acquisition rates of these six pathogens were, in descending order, Escherichia coli (80%), Stenotrophomonas maltophilia (75%), Pseudomonas aeruginosa (70%), Klebsiella pneumoniae (66.67%), Acinetobacter baumannii (36.36%) and Staphylococcus aureus (12.5%). Preliminary mNGS sequencing results showed that the pathogenic bacteria with higher abundance in the batch of effluent were Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Klebsiella aerogenes, Klebsiella michiganensis and Pseudomonas aeruginosa. In conclusion, phages of most common clinical Gram-negative pathogens were isolated from hospital wastewater with high isolation rates; however, phages of Gram-positive pathogens were isolated at lower rates, and only phages corresponding to Staphylococcus aureus were isolated in this study. The corresponding mNGS sequencing results showed that the distribution of Gram-negative pathogens in sewage may had a positive correlation with the ecological distribution of phages.

Abstract B058: Deconvoluting the intra-tumour heterogeneity and subclonal evolution of CDK4/6 inhibitor resistance in ER+ breast cancer

Abstract Subclonal heterogeneity and evolution are characteristics of breast cancer (BC), that play a key role in tumour development, progression, and resistance to current therapies. Preliminary single-cell sequencing studies suggest that subclonal heterogeneity drives therapy response in aggressive BC. Here, we sought to identify whether pre-existing transcriptomically defined cell subpopulations underpin resistance to CDK4/6 inhibitors (CDK4/6i) in ER+ BC, and to identify the distinct or shared resistance mechanisms between different CDK4/6i currently used in the clinic. ER+ BC cell lines, MCF7 and T47D, were infected with a lentiviral barcode library and were long-term exposed to DMSO or CDK4/6i (Abemaciclib or Palbociclib) until drug resistance was achieved. CDK4/6i resistant or DMSO cell lines were then subjected to DNA sequencing for the detection of barcodes coupled with single-cell transcriptomic and barcode sequencing, whole exome sequencing and G1/S cell cycle checkpoint analysis at a protein level. Single-cell lineage tracing identified that resistance to both CDK4/6i in MCF7 cells was stochastic, while in T47D cells, resistance to both CDK4/6i was conserved across all biological replicates. In all cell models, abemaciclib exerted a stronger selective pressure than palbociclib. Detection of barcodes from dead cells highlighted differential selective pressures of the drugs during resistance generation and upon resistance emergence. Single-cell transcriptomic sequencing identified shared and differential mechanisms of resistance between cell lines and specific CDK4/6i, suggestive of different subclonal, non-genetic mechanisms driving resistance. Some of these subclones were pre-defined (i.e. shared transcriptomic features with parental cells), whereas other resistant subclones were demarked by acquisition of pathway up-regulation. For instance, RB1 expression was abolished in the entire palbociclib-resistant T47D population, with copy number loss, indicating RB1 as a genetic driver of resistance. No evidence of RB1 loss was observed in MCF7 palbociclib-resistant clonal population, however a subclonal cluster was identified with loss of RB1 expression. Interestingly, abemaciclib-resistant MCF7 cells had lost RB1 protein expression, suggesting differential non-genetic mechanisms of resistance to CDK4/6i in MCF7 cells. Through single-cell lineage tracing, we have identified that resistance to CDK4/6i can be stochastic or conserved according to the cell model. Additionally, single-cell transcriptomic sequencing identified that in some cell models, cells that have the ability to become resistant are pre-existing, suggesting transcriptomic and epigenomic mechanisms of resistance, where cells exhibit drug-tolerant or drug-induced persistent properties. Citation Format: Ioanna Mavrommati, Fatemeh Ahmadi Moughari, Shefali Thakur, Yu Zhang, Lesley-Ann Martin, Syed Haider, Rachael Natrajan. Deconvoluting the intra-tumour heterogeneity and subclonal evolution of CDK4/6 inhibitor resistance in ER+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B058.

The Diagnostic and Prognostic Value of Plasma Circulating CircNUP98 for Nasopharyngeal Carcinoma.

Our preliminary sequencing analysis revealed increased expression levels of circNUP98 in nasopharyngeal carcinoma (NPC). This study was therefore carried out to explore the role of circNUP98 in NPC. The present study enrolled 56 patients with NPC, 44 patients with cervical lymphadenitis (CL), 50 patients with nose bleeding (NB), 50 patients with chronic sinusitis (CS), 50 patients with lymph node tuberculosis (LNT), and 50 healthy controls (Control). Plasma samples were obtained from all patients and the controls. In addition, NPC and paired non-tumor tissue samples were collected from the 56 NPC patients. The expression of circNUP98 in both tissue and plasma samples was determined by RT-qPCR. The 56 NPC patients were followed up for 5 years to analyze the associations between plasma expression of circNUP98 and the survival of patients. The diagnostic value of circNUP98 for NPC was analyzed through ROC curve analysis. The plasma expression levels of circNUP98 were only increased in NPC, but not in CL, NB, CS and LNT groups compared to that in the Control group. In addition, increased expression levels of circNUP98 were observed in NPC tissues compared to that in non-tumor tissues. Plasma circNUP98 was closely correlated with circNUP98 in NPC tissues, but not circNUP98 in non-tumor tissues. With plasma circNUP98 as a biomarker, NPC patients were separated from CL, NB, CS, LNT, and the Control groups. The Plasma expression of circNUP98 was found to be positively correlated with the poor survival of patients. Moreover, plasma circNUP98 was only closely associated with tumor metastasis, but not tumor size. The expression of circNUP98 in plasma may be used to diagnose NPC and predict patients' survival.

Diversity and novel lineages of black yeasts in Chaetothyriales from freshwater sediments in Spain.

Black yeasts comprise a group of Ascomycota of the order Chaetothyriales with highly variable morphology, a great diversity of ecological niches and life cycles. Despite the ubiquity of these fungi, their diversity in freshwater sediments is still poorly understood. During a survey of culturable Ascomycota from river and stream sediments in various sampling sites in Spain, we obtained 47 isolates of black yeasts by using potato dextrose agar supplemented with cycloheximide. A preliminary morphological study and sequence analyses of the internal transcribed spacer region (ITS) and the large subunit (LSU) of the nuclear rDNA revealed that most of the isolates belonged to the family Herpotrichiellaceae. We have confidently identified 30 isolates representing the following species: Capronia pulcherrima, Cladophialophora emmonsii, Exophiala equina, Exophiala pisciphila, Exophiala radicis, and Phialophora americana. However, we encountered difficulty in assigning 17 cultures to any known species within Chaetothyriales. Combining phenotypic and multi-locus phylogenetic analyses based on the ITS, LSU, β-tubulin (tub2) and translation elongation factor 1-α (tef1-α) gene markers, we propose the new genus Aciculomyces in the Herpotrichiellaceae to accommodate the novel species Aciculomyces restrictus. Other novel species in this family include Cladophialophora denticulata, Cladophialophora heterospora, Cladophialophora irregularis, Exophiala candelabrata, Exophiala dehoogii, Exophiala ramosa, Exophiala verticillata and Phialophora submersa. The new species Cyphellophora spiralis, closely related to Cyphellophora suttonii, is described, and the phylogeny of the genus Anthopsis in the family Cyphellophoraceae is discussed. By utilizing these four markers, we were able to strengthen the phylogenetic resolution and provide more robust taxonomic assessments within the studied group. Our findings indicate that freshwater sediments may serve as a reservoir for intriguing black yeasts, which warrant further investigation to address gaps in phylogenetic relationships, particularly within Herpotrichiellaceae. Citation: Torres-Garcia D, García D, Réblová M, et al. 2023. Diversity and novel lineages of black yeasts in Chaetothyriales from freshwater sediments in Spain. Persoonia 51: 194-228. doi: 10.3767/persoonia.2023.51.05.

Picalm/MLLT10 Expression in Zebrafish Is Associated with an Increase of Hematopoietic Stem Cells and Leads to Leukemia

The PICALM/ MLLT10 (C/A)fusion gene is the result of the (10;11)(p13;q14) translocation, and is found in about 1% of all AML patients, 30% of T-cell ALL with a TCR γ/∂ rearrangement and in some patients with lymphoma. AML patients with a t(10;11)(p13;q14) have poor long-term survival. Mouse models of t(10;11)(p13;q14) show that C/A and the C/A minimal fusion (CA/MF) are strong oncogenic drivers, and that C/A must be expressed in the hematopoietic stem cells (HSCs) to cause leukemia. Patient samples and mouse models of C/A leukemia show that these leukemias harbor additional, cooperating mutations. Zebrafish (ZF) have become an attractive animal model for studying hematological disorders. ZF can be easily genetically modified and kept in large numbers. Although T cell ALL models have been successful in ZF, AML models have not been as successful. Most ZF AML models fail to recapitulate the phenotype, especially transplantability. Here we report a C/A transgenic zebrafish AML model (R:CA/MF). We used the murine Runx1 promoter to direct CA/MF expression to zebrafish HSCs using Tol2-mediated transgenesis. We detected the expression of the CA/MF transcript using RT-PCR in 3 dpf (days post fertilisation) heart marker positive transgenic F0 embryos and the expression of FLAG-tagged CA/MF protein with whole mount in situ immuno histochemistry in stable transgenic R:CA/MF embryos at 3 dpf. We performed whole mount in situ hybridisation (WMISH) and qPCR for hematopoietic genes to examine the impact of CA/MF on zebrafish hematopoiesis. Based on qPCR analysis, we observed that the expression of pu1 (p value <0.0120) and gata1 (p value <0.0004) was significantly decreased in 3 dpf R:CA/MF embryos compared to WT, whereas the expression of mpx at 2 dpf (p value <0.0132) and 3 dpf (p value <0.0378) was significantly increased. Our WMISH analysis showed that the expression of lyz was significantly decreased in R:CA/MF compared to WT embryos at 2 dpf. To investigate the effect of CA/MF expression on HSCs, we injected the R:CA/MF construct into fertilised oocytes of R:GFP;kdrl:RFP fish (R:GFP marks HSCs and Kdrl:RFP marks blood vessels). The results from intravital confocal microscopy showed that the 3 dpf R:GFP larvae that had been injected with the R:CA/MF vector had about a 2.5 fold increase (p value <0.0001) of the GFP expressing cells (HSCs) in both the caudal hematopoietic tissue and circulation compared to control (R:Gal4) and un-injected larvae. These results were also confirmed by flow cytometry. CA/MF transgenic zebrafish developed an aggressive hematological malignancy with a latency of about 1 year and 60 to 70% penetrance. 68% of F1 R:CA/MF (15/22), 64% of F2 R:CA/MF (45/70), 62% of F3 R:CA/MF (22/35) and 62% of F4 R:CA/MF (25/40) adult ZF were euthanised or found dead. The median survival for F1 CA/MF transgenic ZF was 65.6 weeks and 69, 78.4, and 79.1 weeks for F2, F3, and F4, respectively. Histology analysis of R:CA/MF leukemic fish showed hypercellularity in kidney, spleen, heart, gills and eyes. Myeloperoxidase (MPO) staining showed that the number of MPO positive cells in kidney marrow was about 50% higher (p value <0.0001) in R:CA/MF compared to WT. Peripheral blood film analysis of R:CA/MF showed the presence of blast-like cells. In addition, R:CA/MF malignancies were associated with an expansion of either the precursor compartment (N=20/31), the myeloid compartment (N=8/31) or the lymphoid compartment (N=3/31) on forward/side scatter flow cytometry analysis. Transplantation assays showed that R:CA/MF leukemias were serially transplantable. The latency to leukemia development was about six weeks in the first, second, and third recipient fish, which was much shorter than the latency for the primary R:CA/MF leukemias. Our preliminary whole-exome sequencing (WES) analysis identified somatically mutated genes in CA/MF zebrafish leukemia with some of these genes having human orthologues that are known to be somatically mutated in human leukemia suggesting that these are cooperating driver mutations. Preliminary RNA-Seq experiments revealed that genes in the KRAS and MTOR pathways had increased expression in the CA/MF ZF leukemia. R:CA/MF leukemia model is a valuable model for understanding the molecular mechanisms of malignant cell transformation of C/A. It allows us to investigate the effects of C/A on HSCs in vivo and to study the fully developed leukemia via serial transplantation, WES, and RNA-Seq.

A new mammalian universal primer for the mitochondrial cytochrome b locus

We designed a set of amplification primers to target the mitochondrial cytochrome b gene of the rodent genus Oligoryzomys. Combinations of two sets of primers successfully amplified complete mitochondrial and partial cytochrome b (MT-CYB), from not only Oligoryzomys, but also other mammalian species from the orders Rodentia, Didelphimorphia, Primates, Chiroptera, Cetacea, Carnivora, and Lagomorpha. Preliminary sequencing analyses revealed that these primers generate informative sequences for several mammalian species important for phylogenetic and species delimitation analyses.

LncRNA BRCAT54 is downregulated and inhibits cancer cell proliferation by downregulating miR-130b-3p through methylation in prostate cancer.

BRCAT54 and miR-130b-3p are two recently characterized critical players in cancer biology, while their functions in prostate cancer (PC) are unknown. From preliminary sequencing analysis, we observed altered expression of BRCAT54 and miR-130b-3p in PC and an inverse correlation between them. This study was conducted to explore their involvement in PC. A total of 64 PC patients were enrolled to collect paired PC and nontumor tissues. The expression of BRCAT54 and miR-130b-3p were determined by RT-qPCR. Overexpression of BRCAT54 and miR-130b-3p was achieved in PC cells to explore their roles in regulating the expression of each other. Methylation-specific PCR (MSP) was conducted to explore the role of BRCAT54 in regulating promoter methylation of miR-130b-3p. BrdU assay was used to evaluate the role of BRCAT54 and miR-130b-3p in regulating PC cell proliferation. The results showed that PC tissues exhibited downregulation of BRCAT54 and upreglation of miR-130b-3p compared to that in nontumor tissues. They were inversely correlated across PC tissue samples. Overexpression of BRCAT54 decreased RNA accumulation of miR-130b-3p in PC cells. In addition, overexpression of BRCAT54 increased promoter methylation of miR-130b-3p. Moreover, BRCAT54 suppressed the role of miR-130b-3p in promoting PC cell proliferation. In conclusion, BRCAT54 is downregulated in PC and it may inhibit cancer cell proliferation by downregulating miR-130b-3p through methylation.

Big Data in Bioinformatics and Computational Biology: Basic Insights.

The human genome was first sequenced in 1994. It took 10years of cooperation between numerous international research organizations to reveal a preliminary human DNA sequence. Genomics labs can now sequence an entire genome in only a few days. Here, we talk about how the advent of high-performance sequencing platforms has paved the way for Big Data in biology and contributed to the development of modern bioinformatics, which in turn has helped to expand the scope of biology and allied sciences. New technologies and methodologies for the storage, management, analysis, and visualization of big data have been shown to be necessary. Not only does modern bioinformatics have to deal with the challenge of processing massive amounts of heterogeneous data, but it also has to deal with different ways of interpreting and presenting those results, as well as the use of different software programs and file formats. Solutions to these problems are tried to present in this chapter. In order to store massive amounts of data and provide a reasonable period for completing search queries, new database management systems other than relational ones will be necessary. Emerging advance programing approaches, such as machine learning, Hadoop, and MapReduce, aim to provide the capacity to easily construct one's own scripts for data processing and address the issue of the diversity of genomic and proteomic data formats in bioinformatics.

Developing a Generalized Regression Forecasting Network for the Prediction of Human Body Dimensions

With the increasing demand for intelligent custom clothing, the development of highly accurate human body dimension prediction tools using artificial neural network technology has become essential to ensuring high-quality, fashionable, and personalized clothing. Although support vector regression (SVR) networks have demonstrated state-of-the-art (SOTA) performances, they still fall short on prediction accuracy and computation efficiency. We propose a novel generalized regression forecasting network (GRFN) that incorporates kernel ridge regression (KRR) within a multi-strategy multi-subswarm particle swarm optimizer (MMPSO)-SVR nonlinear regression model that applies a residual correction prediction mechanism to enhance prediction accuracy for body dimensions. Importantly, the predictions are generated using only a few basic body size parameters from small-batch samples. The KRR regression model is employed for preliminary residual sequence prediction, and the MMPSO component optimizes the SVR parameters to ensure superior correction of nonlinear relations and noise data, thereby yielding more accurate residual correction value predictions. The GRFN hybrid model is superior to SOTA SVR models and increases the root mean square performance by 91.73–97.12% with a remarkably low mean square error of 0.0054 ± 0.07. This outstanding advancement sets the stage for marketable intelligent apparel design tools for the fast fashion industry.

Ensifer meliloti elite strain U143 used as alfalfa inoculant in Uruguay: Characterization and draft genome sequence

Ensifer (syn. Sinorhizobium) meliloti U143 is an effective nitrogen-fixing strain isolated from Uruguayan soils. For decades, this strain has been used as an inoculant for different alfalfa cultivars. Here we report for the first time a characterization of the U143 elite strain that includes the preliminary genomic sequence, its annotation, and physiological parameters related to its symbiotic efficiency and nitrate respiration capacity. Through Illumina sequencing, the genome of the U143 strain was sequenced. The genome length was 6,801,966 bp, and it contained two megaplasmids, an average GC content of 62.15 %, and 6522 protein-coding sequences. In the symbiotic plasmid, we identified nap, nir, nor, and nos sequences that explain the ability of the U143 strain to respire NO3- in free-living and microaerobic conditions. Field assays performed in two locations for two years showed that alfalfa inoculated with the U143 strain produced 41 % more total shoot dry matter than the non-inoculated control, and between 61.3 % and 66.5 % of shoot N in alfalfa inoculated with strain U143 derived from nitrogen fixation.

Whole-Exome Sequencing Analysis of Idiopathic Hypogonadotropic Hypogonadism: Comparison of Varicocele and Nonobstructive Azoospermia

As a rare disease leading to male infertility, idiopathic hypogonadotropic hypogonadism (IHH) has strong heterogeneity of clinical phenotype and gene mutation. At present, there is no effective diagnosis and treatment method for this disease. This study is to explore the possible new pathogenic gene of idiopathic hypogonadotrophic hypogonadism and the pathological mechanism affecting its occurrence. We performed a whole-exome sequencing on 9 patients with normosmic idiopathic hypogonadotropic hypogonadism (nIHH), 19 varicocele patients with asthenospermia, oligospermia, or azoospermia, 5 patients with simple nonobstructive azoospermia, and 13 normal healthy adult males and carried out comparative analysis, channel analysis, etc. After preliminary sequencing screening, 309–431 genes harbouring variants, including SNPs and indels, were predicted to be harmful per single patient in each group. In genetic variations of nIHH patients’ analysis, variants were detected in 10 loci and nine genes in nine patients. And in co-analysis of the three patient groups, nine nIHH patients, 19 VC patients, and five SN patients shared 116 variants, with 28 variant-harbouring genes detected in five or more patients. We found that the NEFH, CCDC177, and PCLO genes and the Gene Ontology pathways GO:0051301: cell division and GO:0090066: regulation of anatomical structure size may be key factors in the pathogenic mechanism of IHH. Our results suggest that the pathogenic mechanism of IHH is not limited to the central nervous system effects of GnRH but may involve other heterogeneous pathogenic genetic variants that affect peripheral organs.

Transcriptome Sequencing Reveals That Intact Expression of the Chicken Endogenous Retrovirus chERV3 In Vitro Can Possibly Block the Key Innate Immune Pathway.

Endogenous retroviruses (ERVs) are viral sequences that have integrated into the genomes of vertebrates. Our preliminary transcriptome sequencing analysis revealed that chERV3 is active and is located on chromosome 1:32602284-32615631. We hypothesized that chERV3 may have a role in the host innate immune response to viral infection. In this study, using reverse genetics, we constructed the puc57-chERV3 full-length reverse cloning plasmid in vitro. We measured the p27 content in culture supernatant by enzyme-linked immunosorbent assay (ELISA). Finally, transcriptome analysis was performed to analyze the function of chERV3 in innate immunity. The results showed that chERV3 may generate p27 viral particles. We found that compared to the negative control (NC) group (transfected with pMD18T-EGFP), the chERV3 group exhibited 2538 up-regulated differentially expressed genes (DEGs) and 1828 down-regulated DEGs at 24 hours (h) and 1752 up-regulated DEGs and 1282 down-regulated DEGs at 48 h. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the down-regulated DEGs were enriched mainly in immune-related processes such as the inflammatory response, innate immune response, and Toll-like receptor signaling pathway. GSEA showed that the Toll-like receptor signaling pathway was suppressed by chERV3 at both time points. We hypothesized that chERV3 can influence the activation of the innate immune pathway by blocking the Toll-like receptor signaling pathway to achieve immune evasion.