Possible Role of Accessory Proteins in the Viral Replication for the 20I/501Y.V1 (B.1.1.7) SARS CoV-2 Variant

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) has been a global concern. The B.1.1.7 variant of SARS CoV-2 is reported to cause higher transmission. The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 to hypothesis the possible role of different genes in viral replication. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher transmissibility. The experimental evidence published linked ORF6 and ORF8 to play important role in replication and we also observed their higher expression. This leads us to hypothesis the possible role of ORF6 and ORF8 in B.1.1.7 higher replication which causes higher transmission.

TbasCO: Trait-based Comparative ’Omics Identifies Ecosystem-Level and Niche- Differentiating Adaptations of an Engineered Microbiome

ABSTRACTA grand challenge in microbial ecology is disentangling the traits of individual populations within complex communities. Various cultivation-independent approaches have been used to infer traits based on the presence of marker genes. However, marker genes are not linked to traits with complete fidelity, nor do they capture important attributes, such as the timing of expression or coordination among traits. To address this, we present an approach for assessing the trait landscape of microbial communities by statistically defining a trait attribute as shared transcriptional pattern across multiple organisms. Leveraging the KEGG pathway database as a trait library and the Enhanced Biological Phosphorus Removal (EBPR) model microbial ecosystem, we demonstrate that a majority (65%) of traits present in 10 or more genomes have niche-differentiating expression attributes. For example, while 14 genomes containing the high-affinity phosphorus transporter pstABCS display a canonical attribute (e.g. up-regulation under phosphorus starvation), we identified another attribute shared by 11 genomes where transcription was highest under high phosphorus conditions. Taken together, we provide a novel framework for revealing hidden metabolic versatility when investigating genomic data alone by assigning trait-attributes through genome-resolved time-series metatranscriptomics.

Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas

In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.

Single-Cell RNA Sequencing Suggests Novel Drivers of Chronic Lymphocytic Leukemia Patients with Ibrutinib Resistance

Objective: Ibrutinib is currently the most widely used BTK inhibitor that approved for the treatment of both initially diagnosed and relapsed and refractory chronic lymphocytic leukemia (CLL) patients. Although ibrutinib shows high response rates in clinical practice, it has certain limitations. There are still a certain number of patients who have to discontinue treatment due to drug-resistance or side effects. The ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. Methods: Here, we examined the heterogeneity of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-sensitive (IBS) and -resistant (IBR) CLL by analyzing bulk and single-cell level gene expression profiles, clinical features, biological properties, and phenotypes. Seven distinct ibrutinib-resistant subpopulations were identified and two candidate genes LGALS1 (galectin 1, Gal-1) and LAG3 (lymphocyte-activating gene 3, CD223) were screened that contribute toward ibrutinib-resistance and poor survival in CLL patients. These results were validated in primary cells from CLL patients and also in ibrutinib-resistant CLL cell line (MEC1-IR) which was generated by culturing the parental cell line in vitro with progressively increasing concentrations of ibrutinib. Marker-gene expression was detected using qRT-PCR, western blotting, and ELISA, while functional analyses including CCK8, flow cytometry and trypan blue staining were conducted with or without OTX008, a selective Gal-1 inhibitor. Results: ScRNA-seq revealed that cells from IBR and IBS samples were distributed in different clusters and suggested that IBR cells display a unique transcriptional pattern (Fig A). IBR-B cells have higher stemness scores and are enriched in some energy metabolism Pathways (Fig B). According to the proportion of B cells from IBR samples, we classified each B-cell cluster into three main subgroups, i.e., IBR, IBS, and shared cluster (Fig C). IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS B cells, suggesting that IBR B cells may actively build connections with other immune cells to reshape the protective niche (Fig D). A close correlation between LGALS1 and LAG3 expression was observed and both of them were found to be highly expressed in IBR CLL patients (Fig E), their expression level gradually increased along the trajectory of B cells from IBS to IBR (Fig F). Diagnosis and prognosis stratification of CLL with receiver operating characteristic (ROC) curves revealed that patients with higher expression of both LGALS1 and LAG3 showed the poorest overall survival, indicating that LGALS1 and LAG3 are associated with ibrutinib-resistance and poor prognosis in CLL (Fig G). Concordantly, acquired resistance following chronic exposure to ibrutinib leads to upregulation of LGALS1 and LAG3 (Fig H). LGALS1 inhibitor OTX008 effectively inhibits the growth of ibrutinib-resistant CLL cells, particularly for IBR patients (Fig I). Conclusion: In conclusion, our findings demonstrate that ibrutinib-resistant CLL cells exhibit a unique transcriptional pattern. The combination of LGALS1 and LAG3 expression could serve as an indicator of the sensitivity of ibrutinib and prognosis of CLL patients. LGALS1 inhibitor OTX008 helps to overcome ibrutinib-resistance of CLL cells. Our findings may expand the current knowledge regarding ibrutinib-resistant CLL patients, identify improved biomarkers for patient selection, and offer a promising combinatorial therapeutic strategy for IBR CLL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Haemonchus contortus Transthyretin-Like Protein TTR-31 Plays Roles in Post-Embryonic Larval Development and Potentially Apoptosis of Germ Cells

Transthyretin (TTR)-like proteins play multi-function roles in nematode and are important component of excretory/secretory product in Haemonchus contortus. In this study, we functionally characterised a secretory transthyretin-like protein in the barber’s pole worm H. contortus. A full-length of transthyretin-like protein-coding gene (Hc-ttr-31) was identified in this parasitic nematode, representing a counterpart of Ce-ttr-31 in Caenorhabditis elegans. High transcriptional levels of Hc-ttr-31 were detected in the egg and early larval stages of H. contortus, with the lowest level measured in the adult stage, indicating a decreased transcriptional pattern of this gene during nematode development. Localisation analysis indicated a secretion of TTR-31 from the intestine to the gonad, suggesting additional roles of Hc-ttr-31 in nematode reproduction. Expression of Hc-ttr-31 and Ce-ttr-31 in C. elegans did not show marked influence on the nematode development and reproduction, whereas Hc-ttr-31 RNA interference-mediated gene knockdown of Ce-ttr-31 shortened the lifespan, decreased the brood size, slowed the pumping rate and inhibited the growth of treated worms. Particularly, gene knockdown of Hc-ttr-31 in C. elegans was linked to activated apoptosis signalling pathway, increased general reactive oxygen species (ROS) level, apoptotic germ cells and facultative vivipary phenotype, as well as suppressed germ cell removal signalling pathways. Taken together, Hc-ttr-31 appears to play roles in regulating post-embryonic larval development, and potentially in protecting gonad from oxidative stress and mediating engulfment of apoptotic germ cells. A better knowledge of these aspects should contribute to a better understanding of the developmental biology of H. contortus and a discovery of potential targets against this and related parasitic worms.

STEM-17. THE GLIOMA STEM CELL PLATELET INTERACTION DRIVES GBM ONCOGENESIS IDENTIFYING A NOVEL THERAPEUTIC APPROACH

The effect of platelets on oncogenesis has been studied extensively in cancer metastasis, but not in glioblastoma (GBM), where metastasis is rare. Here we identify the unique crosstalk between glioma stem cells (GSCs) and platelets within GBM solid tumors that enhance disease progression. Targeting GSCs is considered a promising therapeutic approach; however, no clear method has been identified. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Immunofluorescence, qPCR, and western blot were used to evaluate the presence of GSCs and platelets and their colocalization in GBM patient tissue at University Hospitals-Seidman Cancer Center. Functional assays followed by RNA sequencing were conducted to determine the functional effect of healthy and GBM platelets on growth of patient derived, autologous GSCs. Our clinical studies suggest elevated platelet counts positively correlate with GSC proliferation and negatively correlate with overall survival in patients with GBM. Patients with high platelet counts ( >350k/µl) had a median survival time of 9 months compared to 16 months median survival for patients with normal platelet count (150-350/µl) (p<0.05). We demonstrate platelet and GSC co-localization in GBM solid tissue and platelet exposure to patient derived GSCs cell lines results in a ≥ 3-fold increase in GSC proliferation compared to GSCs not exposed to platelets (p<0.0005). Similarly we found that platelets increased the self-renewing capacity by enhancing the average sphere size (p < 0.005), and increasing the GSC “Stem-like” transcriptional pattern (P< 0.05). Conversely, pharmacologic inhibition of platelet activation reversed the effect of platelets on GSC proliferation (p ranging from 0.05-0.005). These studies suggests the platelet-GSC interactions appear to stimulate GBM oncogenesis, identifying a potential therapeutic target for the treatment of GBM.

Differential Allelic Expression among Long Non-Coding RNAs

Long non-coding RNAs (lncRNA) comprise a diverse group of non-protein-coding RNAs >200 bp in length that are involved in various normal cellular processes and disease states, and can affect coding gene expression through mechanisms in cis or in trans. Since the discovery of the first functional lncRNAs transcribed by RNA Polymerase II, H19 and Xist, many others have been identified and noted for their unusual transcriptional pattern, whereby expression from one chromosome homolog is strongly favored over the other, also known as mono-allelic or differential allelic expression. lncRNAs with differential allelic expression have been observed to play critical roles in developmental gene regulation, chromosome structure, and disease. Here, we will focus on known examples of differential allelic expression of lncRNAs and highlight recent research describing functional lncRNAs expressed from both imprinted and random mono-allelic expression domains.

The prevalence and density of asymptomatic Plasmodium falciparum infections among children and adults in three communities of western Kenya

Background Further reductions in malaria incidence as more countries approach malaria elimination require the identification and treatment of asymptomatic individuals who carry mosquito-infective Plasmodium gametocytes that are responsible for furthering malaria transmission. Assessing the relationship between total parasitaemia and gametocytaemia in field surveys can provide insight as to whether detection of low-density, asymptomatic Plasmodium falciparum infections with sensitive molecular methods can adequately detect the majority of infected individuals who are potentially capable of onward transmission. Methods In a cross-sectional survey of 1354 healthy children and adults in three communities in western Kenya across a gradient of malaria transmission (Ajigo, Webuye, and Kapsisywa–Kipsamoite), asymptomatic P. falciparum infections were screened by rapid diagnostic tests, blood smear, and quantitative PCR of dried blood spots targeting the varATS gene in genomic DNA. A multiplex quantitative reverse-transcriptase PCR assay targeting female and male gametocyte genes (pfs25, pfs230p), a gene with a transcriptional pattern restricted to asexual blood stages (piesp2), and human GAPDH was also developed to determine total parasite and gametocyte densities among parasitaemic individuals. Results The prevalence of varATS-detectable asymptomatic infections was greatest in Ajigo (42%), followed by Webuye (10%). Only two infections were detected in Kapsisywa. No infections were detected in Kipsamoite. Across all communities, children aged 11–15 years account for the greatest proportion total and sub-microscopic asymptomatic infections. In younger age groups, the majority of infections were detectable by microscopy, while 68% of asymptomatically infected adults (> 21 years old) had sub-microscopic parasitaemia. Piesp2-derived parasite densities correlated poorly with microscopy-determined parasite densities in patent infections relative to varATS-based detection. In general, both male and female gametocytaemia increased with increasing varATS-derived total parasitaemia. A substantial proportion (41.7%) of individuals with potential for onward transmission had qPCR-estimated parasite densities below the limit of microscopic detection, but above the detectable limit of varATS qPCR. Conclusions This assessment of parasitaemia and gametocytaemia in three communities with different transmission intensities revealed evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers of P. falciparum. Experimental studies are needed to definitively determine whether the low-density infections in communities such as Ajigo and Webuye contribute significantly to malaria transmission.

Plasmodium falciparum transcription in different clinical presentations of malaria associates with circulation time of infected erythrocytes

Following Plasmodium falciparum infection, individuals can remain asymptomatic, present with mild fever in uncomplicated malaria cases, or show one or more severe malaria symptoms. Several studies have investigated associations between parasite transcription and clinical severity, but no broad conclusions have yet been drawn. Here, we apply a series of bioinformatic approaches based on P. falciparum’s tightly regulated transcriptional pattern during its ~48-hour intraerythrocytic developmental cycle (IDC) to publicly available transcriptomes of parasites obtained from malaria cases of differing clinical severity across multiple studies. Our analysis shows that within each IDC, the circulation time of infected erythrocytes without sequestering to endothelial cells decreases with increasing parasitaemia or disease severity. Accordingly, we find that the size of circulating infected erythrocytes is inversely related to parasite density and disease severity. We propose that enhanced adhesiveness of infected erythrocytes leads to a rapid increase in parasite burden, promoting higher parasitaemia and increased disease severity.

Integrating Genome-Wide Association Analysis With Transcriptome Sequencing to Identify Candidate Genes Related to Blooming Time in Prunus mume

Prunus mume is one of the most important woody perennials for edible and ornamental use. Despite a substantial variation in the flowering phenology among the P. mume germplasm resources, the genetic control for flowering time remains to be elucidated. In this study, we examined five blooming time-related traits of 235 P. mume landraces for 2 years. Based on the phenotypic data, we performed genome-wide association studies, which included a combination of marker- and gene-based association tests, and identified 1,445 candidate genes that are consistently linked with flowering time across multiple years. Furthermore, we assessed the global transcriptome change of floral buds from the two P. mume cultivars exhibiting contrasting bloom dates and detected 617 associated genes that were differentially expressed during the flowering process. By integrating a co-expression network analysis, we screened out 191 gene candidates of conserved transcriptional pattern during blooming across cultivars. Finally, we validated the temporal expression profiles of these candidates and highlighted their putative roles in regulating floral bud break and blooming time in P. mume. Our findings are important to expand the understanding of flowering time control in woody perennials and will boost the molecular breeding of novel varieties in P. mume.