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Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.

Competing Interests: Declaration of Competing Interest No conflicts of interest to disclose.

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2 nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14 th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2 nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen Candida albicans in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of "trained immunity." Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Sequencing-based microbiome profiling aims at detecting and quantifying individual members of a microbial community in a culture-independent manner. While amplicon-based sequencing (ABS) of bacterial or fungal ribosomal DNA is the most widely used technology due to its low cost, it suffers from PCR amplification biases that hinder accurate representation of microbial population structures. Shotgun metagenomics (SMG) conversely allows unbiased microbiome profiling but requires high sequencing depth. Here we report the development of a meta-total RNA sequencing (MeTRS) method based on shotgun sequencing of total RNA and benchmark it on a human stool sample spiked in with known abundances of bacterial and fungal cells. MeTRS displayed the highest overall sensitivity and linearity for both bacteria and fungi, the greatest reproducibility compared to SMG and ABS, while requiring a ~20-fold lower sequencing depth than SMG. We therefore present MeTRS as a valuable alternative to existing technologies for large-scale profiling of complex microbiomes., Competing Interests: The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Candida albicans is responsible for ~400,000 systemic fungal infections annually, with an associated mortality rate of 46-75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of Candida species and is a major source of systemic fungal infections. However, the factors that control GI colonization by Candida species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of Candida species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic C. albicans cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly-related Candida species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of Candida strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI-mimicking and stressful environments or with in vitro and in vivo virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed β-glucan on the surface of fungal cells, we found this parameter, unlike total β-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed β-glucan by Dectin-1 receptor appears to severely limit Candida GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis.

Myasthenia gravis (MG) is a T-cell dependent autoimmune disorder of the neuromuscular junction, characterised by muscle weakness and fatigability. Autoimmunity is thought to initiate in the thymus of acetylcholine receptor (AChR)-positive MG patients; however, the molecular mechanisms linking intra-thymic MG pathogenesis with autoreactivity via the circulation to the muscle target organ are poorly understood. Using whole-transcriptome sequencing, we compared the transcriptional profile of peripheral blood mononuclear cells from AChR-early onset MG (AChR-EOMG) patients with healthy controls: 178 coding transcripts and 229 long non-coding RNAs, including 11 pre-miRNAs, were differentially expressed. Among the 178 coding transcripts, 128 were annotated of which 17% were associated with the 'infectious disease' functional category and 46% with 'inflammatory disease' and 'inflammatory response-associated' categories. Validation of selected transcripts by qPCR indicated that of the infectious disease-related transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were upregulated, whereas CLC and IL4 were downregulated in AChR-EOMG patients; in the 'inflammatory' categories, ABCA1, FUS, and RELB were upregulated, suggesting a contribution of these molecules to immunological dysfunctions in MG. Data selection and validation were also based on predicted microRNA-mRNA interactions. We found that miR-612, miR-3654, and miR-3651 were increased, whereas miR-612-putative AKAp12 and HRH4 targets and the miR-3651-putative CRISP3 target were downregulated in AChR-EOMG, also suggesting altered immunoregulation. Our findings reveal a novel peripheral molecular signature in AChR-EOMG, reflecting a critical involvement of inflammatory- and infectious disease-related immune responses in disease pathogenesis., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. We used a novel IL-21eGFP reporter mouse strain to analyze the diversity and role of Tfh cells. Through the analysis of GFP expression in lymphoid organs of IL-21eGFP mice, we identified a subpopulation of GFP(+), high IL-21 producing Tfh cells present only in Peyer's Patches. GFP(+)Tfh cells were found to be polyclonal and related to GFP(-)Tfh cells of Peyer's Patches in TCR repertoire composition and overall gene expression. Studies on the mechanisms of induction of GFP(+)Tfh cells demonstrated that they required the intestinal microbiota and a diverse repertoire of CD4(+) T cells and B cells. Importantly, ablation of GFP(+) cells resulted in a reduced frequency of Peyer's Patches IgG1 and germinal center B cells in addition to small but significant shifts in gut microbiome composition. Our work highlights the diversity among IL-21 producing CD4(+) Tfh cells, and the interrelationship between the intestinal bacteria and Tfh cell responses in the gut.

Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Isolated carotid artery agenesis is not generally recognized as a cause of congenital oculomotor nerve palsy. We report this rare association in 2 children and examine the underlying mechanism., (Copyright © 2014 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)

Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape., Competing Interests: The authors have declared that no competing interests exist.

Background: Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained., Methods: To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers., Results: We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death., Conclusions: Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.

Maternally deposited mRNAs direct early development before the initiation of zygotic transcription during mid-blastula transition (MBT). To study mechanisms regulating this developmental event in zebrafish, we applied mRNA deep sequencing technology and generated comprehensive information and valuable resources on transcriptome dynamics during early embryonic (egg to early gastrulation) stages. Genome-wide transcriptome analysis documented at least 8000 maternal genes and identified the earliest cohort of zygotic transcripts. We determined expression levels of maternal and zygotic transcripts with the highest resolution possible using mRNA-seq and clustered them based on their expression pattern. We unravel delayed polyadenylation in a large cohort of maternal transcripts prior to the MBT for the first time in zebrafish. Blocking polyadenylation of these transcripts confirms their role in regulating development from the MBT onward. Our study also identified a large number of novel transcribed regions in annotated and unannotated regions of the genome, which will facilitate reannotation of the zebrafish genome. We also identified splice variants with an estimated frequency of 50%-60%. Taken together, our data constitute a useful genomic information and valuable transcriptome resource for gene discovery and for understanding the mechanisms of early embryogenesis in zebrafish.

We describe the eye-of-the-tiger sign on magnetic resonance imaging (MRI) of the brain in a 40-year-old man presenting with extra pyramidal symptoms like chorea, flexion neck dystonia, tongue tremors, dysarthria and postural instability as the sequelae of organophosphorus poisoning six months previously. This typical radiological sign has been described in extrapyramidal parkinsonian disorders including cortical-basal ganglionic degeneration, early onset levodopa-responsive parkinsonism and Steele-Richardson-Olszewski syndrome (progressive supranuclear palsy) but hitherto has not been reported in insecticide poisoning. T2-weighted scans showed low signal intensity surrounding a central region of high signal intensity in the anteromedial globus pallidus (gliosis), producing an eye-of-the-tiger appearance with the central hyperintense signal intensity better appreciated in T2W and fluid attenuated inversion recovery (FLAIR) sequences.

Mineralizing microangiopathy is a late complication of radiation-induced brain injury that occurs during the treatment of CNS malignancies in children and adults. Early diagnosis of the radiation-induced brain injury helps to tailor the radiation dose and prevent further complications. Here we describe an operated oatient with craniopharyngioma who underwent external beam radiotherapy. The patient developed gradual loss of vision after two years and a review CT scan revealed the local recurrence of tumour with dystrophic calcification of the basal ganglia, frontal and temporal subcortical regions representing recurrent craniopharyngioma with radiation-induced mineralizing microangiopathy.

Hypopituitarism can be the sequela of a variety of causes like postpartum pituitary necrosis or Sheehan's syndrome, lymphocytic hypophysitis, trauma and encephalitis. A very rare cause is envenomation by a bite of a Russell's viper. Very few cases with documented imaging findings of chronic pituitary failure resulting from snake bite have been reported. We describe a case of hypopituitarism with clinical, endocrine and magnetic resonance (MR) imaging studies occurring as a delayed complication of snake bite.

Maple syrup urine disease (MSUD) is a rare inherited metabolic disorder. It is due to decreased de-carboxylation of branched-chain amino acids (leucine, valine, and isoleucine) so their accumulation gives a characteristic maple syrup odour in urine and leads to severe neurological deterioration. Early diagnosis and dietary intervention prevent complications and may allow for normal intellectual development. Various types of MSUD exist, classical MSUD being the most common and severe form of this disease. We describe a case of classical MSUD in a four- month-old infant where the MR imaging suggested the diagnosis of MSUD prior to the clinical diagnosis which was further confirmed by laboratory techniques.

The development of extrapyramidal syndrome characterised by rigidity, bradykinesia, dysphagia and dysarthria in a male individual with four distinct episodes of (mania like) behavioural disturbances with fairly good remission in a time frame of five years, in a male individual, was suspected to develop the neurological manifestations of Wilson's disease and was investigated. In the absence of Kayser-Fleischer ring by slit-lamp examination and with normal copper and ceruloplasmin serum levels, the diagnosis was possible because of the positive findings of the magnetic resonance imaging (MRI) studies and increased 24 hours urinary copper levels with the penicillamine challenge test. The findings and its implications are highlighted and discussed.

Two cases of isolated superior mesenteric artery dissection diagnosed by contrast enhanced 64 slice CT are reported. In both, the dissection was seen extending along the entire length of the artery with one of them showing partially thrombosed false lumen. One case was associated with dissection of left renal artery with consequent renal infarcts. Although superior mesenteric artery dissection is a rare phenomenon, it should be considered in the differential diagnosis of abdominal angina.

Identification of lineage-specific innovations in genomic control elements is critical for understanding transcriptional regulatory networks and phenotypic heterogeneity. We analyzed, from an evolutionary perspective, the binding regions of seven mammalian transcription factors (ESR1, TP53, MYC, RELA, POU5F1, SOX2, and CTCF) identified on a genome-wide scale by different chromatin immunoprecipitation approaches and found that only a minority of sites appear to be conserved at the sequence level. Instead, we uncovered a pervasive association with genomic repeats by showing that a large fraction of the bona fide binding sites for five of the seven transcription factors (ESR1, TP53, POU5F1, SOX2, and CTCF) are embedded in distinctive families of transposable elements. Using the age of the repeats, we established that these repeat-associated binding sites (RABS) have been associated with significant regulatory expansions throughout the mammalian phylogeny. We validated the functional significance of these RABS by showing that they are over-represented in proximity of regulated genes and that the binding motifs within these repeats have undergone evolutionary selection. Our results demonstrate that transcriptional regulatory networks are highly dynamic in eukaryotic genomes and that transposable elements play an important role in expanding the repertoire of binding sites.

The most serious complication of Paget's disease is sarcomatous degeneration of pagetic bone. Multifocal sarcomatous degeneration occurs mainly in polyostotic Paget's disease. Paget sarcomas are the most threatening sarcomas of bone irrespective of the histopathology, their prognosis being far more negative than primary sarcomas. Paget's disease is a common entity but osteosarcoma arising in Paget's disease is an infrequent complication. The skull involvement is even rarer. We report a case of malignant transformation of a skull lesion in a patient of polyostotic Paget's disease. Skull radiographs, computed tomography and magnetic resonance imaging scans of the tumour area show interesting results.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Identification of unconventional functional features such as fusion transcripts is a challenging task in the effort to annotate all functional DNA elements in the human genome. Paired-End diTag (PET) analysis possesses a unique capability to accurately and efficiently characterize the two ends of DNA fragments, which may have either normal or unusual compositions. This unique nature of PET analysis makes it an ideal tool for uncovering unconventional features residing in the human genome. Using the PET approach for comprehensive transcriptome analysis, we were able to identify fusion transcripts derived from genome rearrangements and actively expressed retrotransposed pseudogenes, which would be difficult to capture by other means. Here, we demonstrate this unique capability through the analysis of 865,000 individual transcripts in two types of cancer cells. In addition to the characterization of a large number of differentially expressed alternative 5' and 3' transcript variants and novel transcriptional units, we identified 70 fusion transcript candidates in this study. One was validated as the product of a fusion gene between BCAS4 and BCAS3 resulting from an amplification followed by a translocation event between the two loci, chr20q13 and chr17q23. Through an examination of PETs that mapped to multiple genomic locations, we identified 4055 retrotransposed loci in the human genome, of which at least three were found to be transcriptionally active. The PET mapping strategy presented here promises to be a useful tool in annotating the human genome, especially aberrations in human cancer genomes.

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200,000 to 300,000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.

We have sequenced the complete genome of a hibiscus-infecting tobamovirus, Hibiscus latent Singapore virus (HLSV). The experimental host range of HLSV is similar to that of another distinct species of hibiscus infecting tobamovirus, Hibiscus latent Fort Pierce virus (HLFPV). The genomic structure of HLSV is similar to other tobamoviruses in general. It consists of a 5' untranslated region (UTR), followed by ORFs encoding for a 128 kDa protein and a 186 kDa readthrough protein, a 30 kDa movement protein (MP), 18 kDa coat protein (CP) and a 3' UTR. The unique feature of HLSV is the presence of a poly(A) tract within its 3' UTR. In our previous work, we have reported MP and CP sequences of HLSV and its phylogenetic analysis. Here we report the complete nucleotide sequence of HLSV, phylogenetic analysis of the nucleotide and amino acid sequences of 128/186 kDa ORFs and the presence of a uniquely located poly(A) tract within the 3' UTR.