B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Ultrastructure and histochemistry of the periplasm in oocytes of sturgeons during egg envelope formation.

In the cytoplasm of oocytes (ooplasm) located in ovarian follicles with diameters 2000 microm and 2150 microm in Acipenser gueldenstaedtii, and 2000 microm and 2350 microm in A. baerii, periplasm containing a basophilic compartment and endoplasm containing reserve materials was formed. Vesicles involved in polyspermy blocking and in the formation of the embryo were located in the periplasm. These included compact (cCGs), low-electron-dense cortical granules (lCGs), and lamellar bodies. The cCGs were bounded by a membrane, comprised fibrillar material, fibrils and rod-shaped components. The lCGs were membrane-bounded and contained fibrillar material and granular inclusions. Endoplasmic reticulum (ER) and Golgi complexes were involved in the formation of cCG and lCG. The basophilic compartment, ER and Golgi vesicles participated in the formation of lamellar bodies. They comprised numerous membranes and fibrillar material. It is assumed that they transfer membranes and their precursors to the growing furrow during cleavage and release their content to organize the extracellular matrix. The location of compounds in the developing egg envelope of A. gueldenstaedtii was presented and discussed. Ovaries of both investigated species represented the first pubertal stages of development. Such fish should not be used for reproduction.

Secondary growth ovarian follicles of the pigmented sterlet sturgeon Acipenser ruthenus L. 1758 (Acipenseriformes, Chondrostei, Actinopterygii, Osteichthyes) - Microscopic study of oocytes, egg envelope and diversification of follicular cells.

The individual ovarian follicle of sturgeons (Acipenseriformes, Acipenseridae) contains an oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. The late stages of the secondary growth of follicles (mid- and advanced vitellogenic) are not fully explained in Acipenseriformes. To explore and discuss the ultrastructure of oocytes, FCs, an egg envelope, and explain how micropylar cells differentiate and the canals of a multiple micropyle are formed, the samples of ovaries of the mature sterlet sturgeon Acipenser ruthenus were examined. The oocytes are polarized, the nucleus is located in the animal hemisphere, contains lampbrush chromosomes and multiple nucleoli. In the ooplasm three regions are present: a perinuclear (contains the mitochondria), an endoplasm (contains the lipid droplets and yolk platelets), and a periplasm (contains the cortical granules, melanosomes, endocytotic and exocytotic vesicles). The melanosomes in animal hemisphere form two concentric rings separated by a lighter region between them. The FCs are differentiated into bright and dark cells that are both translationally and secretory active. Diversification of FCs involves repeated and cytoskeleton-dependent change of shape. In the advanced follicles the FCs are diversified into micropylar, the animal and vegetal regions cells, and the cells that delaminated from the epithelium in the animal region. The egg envelope is present in the perioocytic space and consists of three layers: (1) an inner layer or vitelline envelope, (2) a middle layer, and (3) an outer layer. The inner layer consists of four sublayers: (a) a filamentous sublayer composed of filaments released from the oocytes, (b) a trabecular 1 sublayer and (c) a trabecular 2 sublayer named due to the sequence of the deposition, and composed of filaments, fibres and trabecules, (d) a homogeneous sublayer located between the trabecular 1 and trabecular 2 sublayers composed of filaments that adhere to each other closely. The middle layer contains two sublayers: a porous 1 and a porous 2 (composed of granular material) which are released by the oocyte and FCs. The outer layer consists of fibrillar material released by the FCs. The egg envelope is pierced by radial canals formed around the microvilli of the oocyte and the microvilli-like processes of FCs. A micropylar field in the egg envelope that covers the animal pole of the oocyte contains 1 - 4 micropylar canals. Micropylar cells are involved in their formation. The shape of these cells is icicle-like and the cytoplasm is differentiated into two regions (a basal and apical bearing a projection) equipped with different sets of organelles.

UNG-RPA interaction governs the choice between high-fidelity and mutagenic uracil repair.

Mammalian Uracil DNA glycosylase (UNG) removes uracils and initiates high-fidelity base excision repair to maintain genomic stability. During B cell development, activation-induced cytidine deaminase (AID) creates uracils that UNG processes in an error-prone fashion to accomplish immunoglobulin (Ig) somatic hypermutation (SHM) or class switch recombination (CSR). The mechanism that governs high-fidelity versus mutagenic uracil repair is not understood. The B cell tropic gammaherpesvirus (GHV) encodes a functional homolog of UNG that can process AID induced genomic uracils. GHVUNG does not support hypermutation, suggesting intrinsic properties of UNG influence repair outcome. Noting the structural divergence between the UNGs, we define the RPA interacting motif as the determinant of mutation outcome. UNG or RPA mutants unable to interact with each other, only support high-fidelity repair. In B cells, transversions at the Ig variable region are abated while CSR is supported. Thus UNG-RPA governs the generation of mutations and has implications for locus specific mutagenesis in B cells and deamination associated mutational signatures in cancer.

Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species.

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.

Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.

The Longitudinal Analysis of Convergent Antibody VDJ Regions in SARS-CoV-2-Positive Patients Using RNA-Seq.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an ongoing pandemic that continues to evolve and reinfect individuals. To understand the convergent antibody responses that evolved over the course of the pandemic, we evaluated the immunoglobulin repertoire of individuals infected by different SARS-CoV-2 variants for similarity between patients. We utilized four public RNA-seq data sets collected between March 2020 and March 2022 from the Gene Expression Omnibus (GEO) in our longitudinal analysis. This covered individuals infected with Alpha and Omicron variants. In total, from 269 SARS-CoV-2-positive patients and 26 negative patients, 629,133 immunoglobulin heavy-chain variable region V(D)J sequences were reconstructed from sequencing data. We grouped samples based on the SARS-CoV-2 variant type and/or the time they were collected from patients. Our comparison of patients within each SARS-CoV-2-positive group found 1011 common V(D)Js (same V gene, J gene and CDR3 amino acid sequence) shared by more than one patient and no common V(D)Js in the noninfected group. Taking convergence into account, we clustered based on similar CDR3 sequence and identified 129 convergent clusters from the SARS-CoV-2-positive groups. Within the top 15 clusters, 4 contain known anti-SARS-CoV-2 immunoglobulin sequences with 1 cluster confirmed to cross-neutralize variants from Alpha to Omicron. In our analysis of longitudinal groups that include Alpha and Omicron variants, we find that 2.7% of the common CDR3s found within groups were also present in more than one group. Our analysis reveals common and convergent antibodies, which include anti-SARS-CoV-2 antibodies, in patient groups over various stages of the pandemic.

Divergent structures of Mammalian and gammaherpesvirus uracil DNA glycosylases confer distinct DNA binding and substrate activity.

Uracil DNA glycosylase (UNG) removes mutagenic uracil base from DNA to initiate base excision repair (BER). The result is an abasic site (AP site) that is further processed by the high-fidelity BER pathway to complete repair and maintain genome integrity. The gammaherpesviruses (GHVs), human Kaposi sarcoma herpesvirus (KSHV), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68) encode functional UNGs that have a role in viral genome replication. Mammalian and GHVs UNG share overall structure and sequence similarity except for a divergent amino-terminal domain and a leucine loop motif in the DNA binding domain that varies in sequence and length. To determine if divergent domains contribute to functional differences between GHV and mammalian UNGs, we analyzed their roles in DNA interaction and catalysis. By utilizing chimeric UNGs with swapped domains we found that the leucine loop in GHV, but not mammalian UNGs facilitates interaction with AP sites and that the amino-terminal domain modulates this interaction. We also found that the leucine loop structure contributes to differential UDGase activity on uracil in single- versus double-stranded DNA. Taken together we demonstrate that the GHV UNGs evolved divergent domains from their mammalian counterparts that contribute to differential biochemical properties from their mammalian counterparts.

A Recombinant Antibody For Tracking Murine Gammaherpesvirus 68 Uracil DNA Glycosylase Expression.

Antibodies are powerful tools to detect expressed proteins. However off-target recognition can confound their use. Therefore, careful characterization is needed to validate specificity in distinct applications. Here we report the sequence and characterization of a mouse recombinant antibody that specifically detects ORF46 of murine gammaherpesvirus 68 (MHV68). This ORF encodes the viral uracil DNA glycosylase (vUNG). The antibody does not recognize murine uracil DNA glycosylase and is useful in detecting vUNG expressed in virally infected cells. It can detect expressed vUNG in cells via immunostaining and microscopy or flow cytometry analysis. The antibody can detect vUNG from lysates of expressing cells via immunoblot under native conditions but not denaturing conditions. This suggests it recognizes a confirmational based epitope. Altogether this manuscript describes the utility of the anti-vUNG antibody and suitability for use in studies of MHV68 infected cells.

B cell-intrinsic STAT3-mediated support of latency and interferon suppression during murine gammaherpesvirus 68 infection revealed through an in vivo competition model.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak latency approximately 7-fold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.

Molecular phylogeny and species delimitation in the section Longibrachiatum of Trichoderma.

The phylogenetically most derived group of the genus Trichoderma - section Longibrachiatum, includes some of the most intensively studied species, such as the industrial cellulase producer T. reesei (teleomorph Hypocrea jecorina), or the facultative opportunistic human pathogens T. longibrachiatum and H. orientalis. At the same time, the phylogeny of this clade is only poorly understood. Here we used a collection of 112 strains representing all currently recognized species and isolates that were tentatively identified as members of the group, to analyze species diversity and molecular evolution. Bayesian phylogenetic analyses based on several unlinked loci in individual and concatenated datasets confirmed 13 previously described species and 3 previously recognized phylogenetic species all of which were not yet described formally. When the genealogical concordance criterion, the K/θ method and comparison of frequencies of pairwise nucleotide differences were applied to the data sample, 10 additional new phylogenetic species were recognized, seven of which consisted only of a single lineage. Our analysis thus identifies 26 putative species in section Longibrachiatum, what doubles the currently estimated taxonomic diversity of the group, and illustrates the power of combining genealogical concordance and population genetic analysis for dissecting species in a recently diverged group of fungal species.

Microscopic study of the primary growth ovarian follicles of the pike-perch Sander lucioperca (Linnaeus 1758) (Actinopterygii, Perciformes).

The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle is composed of an oocyte surrounded by flattened follicular cells, the basal lamina, and thecal cells. The early stages of oocyte development (primary growth = previtellogenesis) are not fully explained in this species. The results of research with the use of stereoscopic, light, fluorescence, and transmission electron microscopes on ovarian follicles containing developing primary oocytes of S. lucioperca are presented. The polarization and ultrastructure of oocytes are described and discussed. The deposition of egg envelopes during the primary growth and the ultrastructure of the eggshell in maturing follicles of S. lucioperca are also presented. Nuclei in primary oocytes comprise lampbrush chromosomes, nuclear bodies, and nucleoli. Numerous additional nucleoli arise in the nucleoplasm during primary growth and locate close to the nuclear envelope. The Balbiani body in the cytoplasm of oocytes (ooplasm) is composed of nuage aggregations of nuclear origin and mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. The presence of the Balbiani body was reported in oocytes of numerous species of Actinopterygii; however, its ultrastructure was investigated in a limited number of species. In primary oocytes of S. lucioperca, the Balbiani body is initially located in the perinuclear ooplasm on one side of the nucleus. Next, it surrounds the nucleus, expands toward the plasma membrane of oocytes (oolemma), and becomes fragmented. Within the Balbiani body, the granular nuage condenses in the form of threads, locates near the oolemma, at the vegetal oocyte pole, and then dissolves. Mitochondria and cisternae of the rough endoplasmic reticulum (RER) are present between the threads. During primary growth micropylar cells differentiate in the follicular epithelium. They contain cisternae and vesicles of the RER and Golgi apparatus as well as numerous dense vesicles suggesting high synthetic and secretory activity. During the final step of primary growth several follicular cells delaminate from the follicular epithelium, migrate toward the oocyte and submerge in the most external egg envelope. In the ooplasm, three regions are distinguished: perinuclear, endoplasm, and periplasm. Cortical alveoli arise in the perinuclear ooplasm and in the endoplasm as a result of the fusion of RER vesicles with Golgi ones. They are evenly distributed. Lamellar bodies in the periplasm store the plasma membrane and release it into a space between the follicular cells and the oocyte. The developing eggshell in this space is made up of two egg envelopes (the internal one and the external) that are pierced by canals formed around the microvilli of oocytes and the processes of follicular cells. In the deposition of egg envelopes the oocyte itself and follicular cells are engaged. In maturing ovarian follicles the eggshell is solid and the internal egg envelope is covered with protuberances.

Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis.

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool.

Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture.

The scarcity of fresh water in the Mediterranean region necessitates the search for halotolerant agents of biological control of plant diseases that can be applied in arid-zone agriculture irrigated with saline water. Among 29 Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges, the greatest number of isolates belong to the Trichoderma longibrachiatum-Hypocrea orientalis species pair (9), H. atroviridis/T. atroviride (9), and T. harzianum species complex (7), all of which are known for high mycoparasitic potential. In addition, one isolate of T. asperelloides and two putative new species, Trichoderma sp. O.Y. 14707 and O.Y. 2407, from Longibrachiatum and Strictipilosa clades, respectively, have been identified. In vitro salinity assays showed that the ability to tolerate increasing osmotic pressure (halotolerance) is a strain- or clade-specific property rather than a feature of a species. Only a few isolates were found to be sensitive to increased salinity, while others either were halotolerant or even demonstrated improved growth in increasingly saline conditions. In vitro antibiosis assays revealed strong antagonistic activity toward phytopathogens due to the production of both soluble and volatile metabolites. Two marine-derived Trichoderma isolates, identified as T. atroviride and T. asperelloides, respectively, effectively reduced Rhizoctonia solani damping-off disease on beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans. This is the first inclusive evaluation of marine fungi as potential biocontrol agents.

Genetic variation in gsα protein as a new indicator in screening test for vasovagal syncope.

BACKGROUND: A quantitative history using Calgary syncope syndrome score (CSSS) is able to define the likely cause of syncope, but there is still a lack of diagnostic screening tests for vasovagal syncope (VVS). The aim of the present study was to develop a screening test for VVS on the basis of CSSS and the relationship between polymorphic variants of the G-system signaling protein genes and tilting results. METHODS AND RESULTS: From 730 syncopal patients, 307 consecutive subjects without structural and electrical abnormalities were genotyped and examined on blood pressure (BP) and tilt testing. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism in genes encoding Gsα-protein GNAS1 (rs7121), G-protein ß 3 subunit (rs5443) and the cardiac regulator of G-protein signaling RGS2 (rs4606). The control group consisted of 100 healthy volunteers with a negative history of syncope. From multivariate regression analysis, being a carrier of 393T GNAS1 (odds ratio [OR], 2.29) and systolic BP (OR, 0.98) remained as independent factors associated with positive tilt results. The resultant screening test for VVS consisted of the following: carrier of 393T GNAS1; systolic BP < 131 mm Hg (from the receiver operating characteristic [ROC] curve); and CSSS ≥-2. Using ROC curve analysis for systolic BP and CSSS, 2 final models for the screening test were constructed: highest sensitivity (89%) and highest specificity (99%). CONCLUSIONS: The novel screening test including the variation of Gsα protein gene seems to be helpful to identify tilt-induced vasovagal patients.

The Trichoderma harzianum demon: complex speciation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages.

BACKGROUND: The mitosporic fungus Trichoderma harzianum (Hypocrea, Ascomycota, Hypocreales, Hypocreaceae) is an ubiquitous species in the environment with some strains commercially exploited for the biological control of plant pathogenic fungi. Although T. harzianum is asexual (or anamorphic), its sexual stage (or teleomorph) has been described as Hypocrea lixii. Since recombination would be an important issue for the efficacy of an agent of the biological control in the field, we investigated the phylogenetic structure of the species. RESULTS: Using DNA sequence data from three unlinked loci for each of 93 strains collected worldwide, we detected a complex speciation process revealing overlapping reproductively isolated biological species, recent agamospecies and numerous relict lineages with unresolved phylogenetic positions. Genealogical concordance and recombination analyses confirm the existence of two genetically isolated agamospecies including T. harzianum sensu stricto and two hypothetical holomorphic species related to but different from H. lixii. The exact phylogenetic position of the majority of strains was not resolved and therefore attributed to a diverse network of recombining strains conventionally called 'pseudoharzianum matrix'. Since H. lixii and T. harzianum are evidently genetically isolated, the anamorph - teleomorph combination comprising H. lixii/T. harzianum in one holomorph must be rejected in favor of two separate species. CONCLUSIONS: Our data illustrate a complex speciation within H. lixii - T. harzianum species group, which is based on coexistence and interaction of organisms with different evolutionary histories and on the absence of strict genetic borders between them.

Clonal species Trichoderma parareesei sp. nov. likely resembles the ancestor of the cellulase producer Hypocrea jecorina/T. reesei.

We have previously reported that the prominent industrial enzyme producer Trichoderma reesei (teleomorph Hypocrea jecorina; Hypocreales, Ascomycota, Dikarya) has a genetically isolated, sympatric sister species devoid of sexual reproduction and which is constituted by the majority of anamorphic strains previously attributed to H. jecorina/T. reesei. In this paper we present the formal taxonomic description of this new species, T. parareesei, complemented by multivariate phenotype profiling and molecular evolutionary examination. A phylogenetic analysis of relatively conserved loci, such as coding fragments of the RNA polymerase B subunit II (rpb2) and GH18 chitinase (chi18-5), showed that T. parareesei is genetically invariable and likely resembles the ancestor which gave raise to H. jecorina. This and the fact that at least one mating type gene of T. parareesei has previously been found to be essentially altered compared to the sequence of H. jecorina/T. reesei indicate that divergence probably occurred due to the impaired functionality of the mating system in the hypothetical ancestor of both species. In contrast, we show that the sexually reproducing and correspondingly more polymorphic H. jecorina/T. reesei is essentially evolutionarily derived. Phenotype microarray analyses performed at seven temperature regimens support our previous speculations that T. parareesei possesses a relatively high opportunistic potential, which probably ensured the survival of this species in ancient and sustainable environment such as tropical forests.

Asymmetry in the cytoplasm of oocytes of largescale yellowfish Labeobarbus marequensis Smith 1841 (Teleostei: Cypriniformes: Cyprinidae).

The ovaries of the largescale yellowfish, Labeobarbus marequensis (Teleostei: Cypriniformes: Cyprinidae), are made up of the germinal epithelium, nests of late chromatin nucleolus stage oocytes, and ovarian follicles. Each follicle is composed of a single oocyte, which is surrounded by somatic follicular cells and a basal lamina covered by thecal cells. We describe polarization and ultrastructure of oocytes during the primary growth stage. The oocyte nucleus contains lampbrush chromosomes, nuclear bodies and fibrillar material in which multiple nucleoli arise. Nuage aggregations composed of material of a nuclear origin are present in the perinuclear cytoplasm. The Balbiani body (Bb) contains aggregations of nuage, rough endoplasmic reticulum, individual mitochondria and complexes of mitochondria with nuage (cement). Some mitochondria in the Bb come into close contact with endoplasmic reticulum cisternae and vesicles that contain granular material. At the start of primary growth, the Bb is present in the cytoplasm close to the nucleus. Next, it expands towards the oocyte plasma membrane. In these oocytes, a spherical structure, the so-called yolk nucleus, arises in the Bb. It consists of granular nuage in which mitochondria and vesicles containing granular material are immersed. Later, the Bb becomes fragmented and a fully grown yolk nucleus is present in the vegetal region. It contains numerous threads composed of granular nuage, mitochondria, lysosome-like organelles and autophagosomes. We discuss the formation of autophagosomes in the cytoplasm of primary growth oocytes. During the final step of primary growth, the cortical alveoli arise in the cytoplasm and are distributed evenly. The eggshell is deposited on the external surface of the oocyte plasma membrane and is made up of two egg envelopes that are pierced by numerous pore canals. The external egg envelope is covered in protuberances. During primary growth no lipid droplets are synthesized or stored in the oocytes.

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire.

A common biologic property of the gammaherpesviruses Epstein-Barr Virus and Kaposi sarcoma herpesvirus is their use of B lymphocytes as a reservoir of latency in healthy individuals that can undergo oncogenic transformation later in life. Gammaherpesviruses (GHVs) employ an impressive arsenal of proteins and non-coding RNAs to reprogram lymphocytes for proliferative expansion. Within lymphoid tissues, the germinal center (GC) reaction is a hub of B cell proliferation and death. The goal of a GC is to generate and then select for a pool of immunoglobulin (Ig) genes that will provide a protective humoral adaptive immune response. B cells infected with GHVs are detected in GCs and bear the hallmark signatures of the mutagenic processes of somatic hypermutation and isotype class switching of the Ig genes. However, data also supports extrafollicular B cells as a reservoir engaged by GHVs. Next-generation sequencing technologies provide unprecedented detail of the Ig sequence that informs the natural history of infection at the single cell level. Here, we review recent reports from human and murine GHV systems that identify striking differences in the immunoglobulin repertoire of infected B cells compared to their uninfected counterparts. Implications for virus biology, GHV-associated cancers, and host immune dysfunction will be discussed.