The Intracellular Consequences of the Interaction between Epstein-Barr Virus Protein BZLF1 and the Human Protein Pax5

Epstein-Barr virus (EBV) is a human herpes virus that is the cause of infectious mononucleosis and is associated with several types of cancers. These cancers include Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and a variety of leukemias. EBV is found in two states; latent (dormant state) and lytic (the replicating state). A protein essential for the switch and establishment of the lytic state is the immediate-early protein BZLF1. BZLF1 is known to physically associate with a variety of host cellular proteins leading to changes in cellular environment. One such association is with the human B cell protein Pax5. Pax5 plays an important role in determining B cell differentiation as well as promoting the latent state in EBV infected cells through the activation of the EBV latent promoter Wp. The BZLF1/Pax5 complex was examined to determine if this interaction produces specific cellular changes. Pax5 protein levels in B cells before, during and after EBV infection were established. I also examined the effect the BZLF1/Pax5 complex has on the histone methylation state of chromatin during EBV infection. My final goal was to determine if the BZLF1/Pax5 complex affects Pax5 ability to transcriptionally activate two of its target genes, CD19 and CD79a. My results show that during lytic replication the expression of the EBV protein BZLF1 results in increased levels of Pax5. These increased levels of Pax5 result in increased levels of Pax5 transcriptional targets CD19 and CD79a. Also the increased levels of Pax5 and the BZLF1/Pax5 complex resulted in hypermethylation of histone 3 lysine 9

Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

The essential immediate early transcriptional activator RTA, encoded by gene 50, is conserved among all characterized gammaherpesviruses. Analyses of a recombinant murine gammaherpesvirus 68 (MHV68) lacking both of the known gene 50 promoters (G50DblKo) revealed that this mutant retained the ability to replicate in the simian kidney epithelial cell line Vero but not in permissive murine fibroblasts following low-multiplicity infection. However, G50DblKo replication in permissive fibroblasts was partially rescued by high-multiplicity infection. In addition, replication of the G50DblKo virus was rescued by growth on mouse embryonic fibroblasts (MEFs) isolated from IFN-α/βR(−/−) mice, while growth on Vero cells was suppressed by the addition of alpha interferon (IFN-α). 5′ rapid amplification of cDNA ends (RACE) analyses of RNAs prepared from G50DblKo and wild-type MHV68-infected murine macrophages identified three novel gene 50 transcripts initiating from 2 transcription initiation sites located upstream of the currently defined proximal and distal gene 50 promoters. In transient promoter assays, neither of the newly identified gene 50 promoters exhibited sensitivity to IFN-α treatment. Furthermore, in a single-step growth analysis RTA levels were higher at early times postinfection with the G50DblKo mutant than with wild-type virus but ultimately fell below the levels of RTA expressed by wild-type virus at later times in infection. Infection of mice with the MHV68 G50DblKo virus demonstrated that this mutant virus was able to establish latency in the spleen and peritoneal exudate cells (PECs) of C57BL/6 mice with about 1/10 the efficiency of wild-type virus or marker rescue virus. However, despite the ability to establish latency, the G50DblKo virus mutant was severely impaired in its ability to reactivate from either latently infected splenocytes or PECs. Consistent with the ability to rescue replication of the G50DblKo mutant by growth on type I interferon receptor null MEFs, infection of IFN-α/βR(−/−) mice with the G50DblKo mutant virus demonstrated partial rescue of (i) acute virus replication in the lungs, (ii) establishment of latency, and (iii) reactivation from latency. The identification of additional gene 50/RTA transcripts highlights the complex mechanisms involved in controlling expression of RTA, likely reflecting time-dependent and/or cell-specific roles of different gene 50 promoters in controlling virus replication. Furthermore, the newly identified gene 50 transcripts may also act as negative regulators that modulate RTA expression. IMPORTANCE The viral transcription factor RTA, encoded by open reading frame 50 (Orf50), is well conserved among all known gammaherpesviruses and is essential for both virus replication and reactivation from latently infected cells. Previous studies have shown that regulation of gene 50 transcription is complex. The studies reported here describe the presence of additional alternatively initiated, spliced transcripts that encode RTA. Understanding how expression of this essential viral gene product is regulated may identify new strategies for interfering with infection in the setting of gammaherpesvirus-induced diseases

Microbial arms race: Ballistic "nematocysts" in dinoflagellates represent a new extreme in organelle complexity

We examine the origin of harpoon-like secretory organelles (nematocysts) in dinoflagellate protists. These ballistic organelles have been hypothesized to be homologous to similarly complex structures in animals (cnidarians); but we show, using structural, functional, and phylogenomic data, that nematocysts evolved independently in both lineages. We also recorded the first high-resolution videos of nematocyst discharge in dinoflagellates. Unexpectedly, our data suggest that different types of dinoflagellate nematocysts use two fundamentally different types of ballistic mechanisms: one type relies on a single pressurized capsule for propulsion, whereas the other type launches 11 to 15 projectiles froman arrangement similar to a Gatling gun.Despite their radical structural differences, these nematocysts share a single origin within dinoflagellates and both potentially use a contraction-based mechanism to generate ballistic force. The diversity of traits in dinoflagellate nematocysts demonstrates a stepwise route by which simple secretory structures diversified to yield elaborate subcellular weaponry

Malignant melanotic nerve sheath tumor with PRKAR1A, KMT2C and GNAQ mutations

Malignant melanotic nerve sheath tumor (MMNST) is a rare and potentially aggressive lesion defined in the 2021 WHO Classification of Tumors of the Central Nervous System. MMNST demonstrate overlapping histologic and clinical features of schwannoma and melanoma. MMNST often harbor PRKAR1A mutations, especially within the Carney Complex. We present a case of aggressive MMNST of the sacral region in a 48-year-old woman. The tumor contained PRKAR1A frameshift pR352Hfs*89, KMT2C splice site c.7443-1G>T and GNAQ p.R183L missense mutations, as well as BRAF and MYC gains. Genomic DNA methylation analysis using the Illumina 850K EpicBead chip revealed that the lesion did not match an established methylation class; however, uniform manifold approximation and projection (UMAP) placed the tumor very near, or with, schwannomas. The tumor expressed PD-L1, and the patient was treated with radiation and immune checkpoint inhibitors following en bloc resection. Although she had symptomatic improvement, she suffered early disease progression with local recurrence, and distant metastases, and died 18 months after resection. It has been suggested that the presence of GNAQ mutations can differentiate leptomeningeal melanocytic neoplasms and uveal melanoma from MMNST. This case and others demonstrate that GNAQ mutations may exist in malignant nerve sheath tumors; that GNAQ and PRKAR1A mutations are not always mutually exclusive and that neither can be used to differentiate MMNST or MPNST from all melanocytic lesions

Dinoflagellate nucleus contains an extensive endomembrane network, the nuclear net

Dinoflagellates are some of the most common eukaryotic cells in the ocean, but have very unusual nuclei. Many exhibit a form of closed mitosis (dinomitosis) wherein the nuclear envelope (NE) invaginates to form one or more trans-nuclear tunnels. Rather than contact spindles directly, the chromatids then bind to membrane-based kinetochores on the NE. To better understand these unique mitotic features, we reconstructed the nuclear architecture of Polykrikos kofoidii in 3D using focused ion beam scanning electron microscopy (FIB-SEM) in conjunction with high-pressure freezing, freeze-substitution, TEM, and confocal microscopy. We found that P. kofoidii possessed six nuclear tunnels, which were continuous with a reticulating network of membranes that has thus far gone unnoticed. These membranous extensions interconnect the six tunnels while ramifying throughout the nucleus to form a "nuclear net." To our knowledge, the nuclear net is the most elaborate endomembrane structure described within a nucleus. Our findings demonstrate the utility of tomographic approaches for detecting 3D membrane networks and show that nuclear complexity has been underestimated in Polykrikos kofoidii and, potentially, in other dinoflagellates

Assessment of subpatent Plasmodium infection in northwestern Ethiopia.

BACKGROUND: Ethiopia has set a goal for malaria elimination by 2030. Low parasite density infections may go undetected by conventional diagnostic methods (microscopy and rapid diagnostic tests) and their contribution to malaria transmission varies by transmission settings. This study quantified the burden of subpatent infections from samples collected from three regions of northwest Ethiopia. METHODS: Sub-samples of dried blood spots from the Ethiopian Malaria Indicator Survey 2015 (EMIS-2015) were tested and compared using microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (nPCR) to determine the prevalence of subpatent infection. Paired seroprevalence results previously reported along with gender, age, and elevation of residence were explored as risk factors for Plasmodium infection. RESULTS: Of the 2608 samples collected, the highest positive rate for Plasmodium infection was found with nPCR 3.3% (95% CI 2.7-4.1) compared with RDT 2.8% (95% CI 2.2-3.5) and microscopy 1.2% (95% CI 0.8-1.7). Of the nPCR positive cases, Plasmodium falciparum accounted for 3.1% (95% CI 2.5-3.8), Plasmodium vivax 0.4% (95% CI 0.2-0.7), mixed P. falciparum and P. vivax 0.1% (95% CI 0.0-0.4), and mixed P. falciparum and Plasmodium malariae 0.1% (95% CI 0.0-0.3). nPCR detected an additional 30 samples that had not been detected by conventional methods. The majority of the nPCR positive cases (61% (53/87)) were from the Benishangul-Gumuz Region. Malaria seropositivity had significant association with nPCR positivity [adjusted OR 10.0 (95% CI 3.2-29.4), P < 0.001]. CONCLUSION: Using nPCR the detection rate of malaria parasites increased by nearly threefold over rates based on microscopy in samples collected during a national cross-sectional survey in 2015 in Ethiopia. Such subpatent infections might contribute to malaria transmission. In addition to strengthening routine surveillance systems, malaria programmes may need to consider low-density, subpatent infections in order to accelerate malaria elimination efforts

Cosmopolitan distribution of Endozoicomonas-like organisms and other intracellular microcolonies of bacteria causing infection in marine molluscs

Intracellular microcolonies of bacteria, in some cases developing large extracellular cysts, have been historically reported infecting a wide diversity of economically important mollusc species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterise those organisms, traditionally named as Rickettsia or Chlamydia -like organisms (RLO/CLO), via international collaboration, 98 samples comprising 20 mollusc species were collected over 10 countries and examined using histology and phylogenetic analysis. A 16S rRNA gene amplicon library-based sequencing showed the presence of different species of Endozoicomonas-like organisms (ELO) in all the mollusc species analysed, infecting primarily gill and digestive glands. Co-infections of ELOs with other intracellular bacteria were also observed. Subsequent phylogenetic analysis of Operational Taxonomic Units (OTU) revealed a novel microbial diversity associated with molluscan RLO/CLOs infection distributed along different taxa, including Spirochaetes phyla, Rickettsiales order, Simkaniaceae family, Mycoplasma and Francisella genera, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained. The presence of ELO sequences in the RLO/CLO infection was confirmed by standard PCR, Sanger sequencing, and by in situ hybridisation in a selection of samples. The phylogenetic analysis conducted in this study will allow for further characterization of the microbial community associated with Rickettsia and Chlamydia-like infection in marine molluscs and their correlation with severity of the lesions in order to reveal their role as endosymbionts, commensals or true pathogens.info:eu-repo/semantics/publishedVersio

Multiplex serology demonstrate cumulative prevalence and spatial distribution of malaria in Ethiopia.

BACKGROUND: Measures of malaria burden using microscopy and rapid diagnostic tests (RDTs) in cross-sectional household surveys may incompletely describe the burden of malaria in low-transmission settings. This study describes the pattern of malaria transmission in Ethiopia using serological antibody estimates derived from a nationwide household survey completed in 2015. METHODS: Dried blood spot (DBS) samples were collected during the Ethiopian Malaria Indicator Survey in 2015 from malarious areas across Ethiopia. Samples were analysed using bead-based multiplex assays for IgG antibodies for six Plasmodium antigens: four human malaria species-specific merozoite surface protein-1 19kD antigens (MSP-1) and Apical Membrane Antigen-1 (AMA-1) for Plasmodium falciparum and Plasmodium vivax. Seroprevalence was estimated by age, elevation and region. The seroconversion rate was estimated using a reversible catalytic model fitted with maximum likelihood methods. RESULTS: Of the 10,278 DBS samples available, 93.6% (9622/10,278) had valid serological results. The mean age of participants was 15.8 years and 53.3% were female. National seroprevalence for antibodies to P. falciparum was 32.1% (95% confidence interval (CI) 29.8-34.4) and 25.0% (95% CI 22.7-27.3) to P. vivax. Estimated seroprevalences for Plasmodium malariae and Plasmodium ovale were 8.6% (95% CI 7.6-9.7) and 3.1% (95% CI 2.5-3.8), respectively. For P. falciparum seroprevalence estimates were significantly higher at lower elevations (< 2000 m) compared to higher (2000-2500 m) (aOR 4.4; p < 0.01). Among regions, P. falciparum seroprevalence ranged from 11.0% (95% CI 8.8-13.7) in Somali to 65.0% (95% CI 58.0-71.4) in Gambela Region and for P. vivax from 4.0% (95% CI 2.6-6.2) in Somali to 36.7% (95% CI 30.0-44.1) in Amhara Region. Models fitted to measure seroconversion rates showed variation nationally and by elevation, region, antigen type, and within species. CONCLUSION: Using multiplex serology assays, this study explored the cumulative malaria burden and regional dynamics of the four human malarias in Ethiopia. High malaria burden was observed in the northwest compared to the east. High transmission in the Gambela and Benishangul-Gumuz Regions and the neglected presence of P. malariae and P. ovale may require programmatic attention. The use of a multiplex assay for antibody detection in low transmission settings has the potential to act as a more sensitive biomarker

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties