Hemocytes are immune cells that patrol the mosquito hemocoel and mediate critical cellular defense responses against pathogens. However, despite their importance, a comprehensive transcriptome of ...these cells was lacking because they constitute a very small fraction of the total cells in the insect, limiting the study of hemocyte differentiation and immune function.
In this study, an in-depth hemocyte transcriptome was built by extensive bulk RNA sequencing and assembly of hemocyte RNAs from adult A. gambiae female mosquitoes, based on approximately 2.4 billion short Illumina and about 9.4 million long PacBio high-quality reads that mapped to the A. gambiae PEST genome (P4.14 version). A total of 34,939 transcripts were annotated including 4,020 transcripts from novel genes and 20,008 novel isoforms that result from extensive differential splicing of transcripts from previously annotated genes. Most hemocyte transcripts identified (89.8%) are protein-coding while 10.2% are non-coding RNAs. The number of transcripts identified in the novel hemocyte transcriptome is twice the number in the current annotation of the A. gambiae genome (P4.14 version). Furthermore, we were able to refine the analysis of a previously published single-cell transcriptome (scRNAseq) data set by using the novel hemocyte transcriptome as a reference to re-define the hemocyte clusters and determine the path of hemocyte differentiation. Unsupervised pseudo-temporal ordering using the Tools for Single Cell Analysis software uncovered a novel putative prohemocyte precursor cell type that gives rise to prohemocytes. Pseudo-temporal ordering with the Monocle 3 software, which analyses changes in gene expression during dynamic biological processes, determined that oenocytoids derive from prohemocytes, a cell population that also gives rise to the granulocyte lineage.
A high number of mRNA splice variants are expressed in hemocytes, and they may account for the plasticity required to mount efficient responses to many different pathogens. This study highlights the importance of a comprehensive set of reference transcripts to perform robust single-cell transcriptomic data analysis of cells present in low abundance. The detailed annotation of the hemocyte transcriptome will uncover new facets of hemocyte development and function in adult dipterans and is a valuable community resource for future studies on mosquito cellular immunity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent work has established associations between elevated p21, the accumulation of senescent cells, and skeletal muscle dysfunction in mice and humans. Using a mouse model of p21 overexpression ...(p21OE), we examined if p21 mechanistically contributes to cellular senescence and pathological features in skeletal muscle. We show that p21 induces several core properties of cellular senescence in skeletal muscle, including an altered transcriptome, DNA damage, mitochondrial dysfunction, and the senescence-associated secretory phenotype (SASP). Furthermore, p21OE mice exhibit manifestations of skeletal muscle pathology, such as atrophy, fibrosis, and impaired physical function when compared to age-matched controls. These findings suggest p21 alone is sufficient to drive a cellular senescence program and reveal a novel source of skeletal muscle loss and dysfunction.
•p21 induces a transcriptional program in skeletal muscle consistent with a senescence program.•p21 induces core properties of senescence in skeletal muscle, including DNA damage, mitochondrial dysfunction, and the SASP.•Mice that overexpress p21 exhibit signs of muscle pathology, such as atrophy, fibrosis, and impaired physical function.
Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with ...lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes,
PSMB8
and
PSMB10
, whereas one patient showed additive loss-of-function mutations in
PSMB8
. Variants in two previously not associated proteasome genes,
PSMA5
and
PSMC5
, were found in a patient who also carried the
PSMB8
founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.
BackgroundCancer neoantigens arise from protein-altering somatic mutations in tumor and rank among the most promising next-generation immuno-oncology agents when used in combination with immune ...checkpoint inhibitors. We previously developed a computational framework, REAL-neo, for identification, quality control, and prioritization of both class-I and class-II human leucocyte antigen (HLA)-presented neoantigens resulting from somatic single-nucleotide mutations, small insertions and deletions, and gene fusions. In this study, we developed a new module, SPLICE-neo, to identify neoantigens from aberrant RNA transcripts from two distinct sources: (1) DNA mutations within splice sites and (2) de novo RNA aberrant splicings.MethodsFirst, SPLICE-neo was used to profile all DNA splice-site mutations in 11,892 tumors from The Cancer Genome Atlas (TCGA) and identified 11 profiles of splicing donor or acceptor site gains or losses. Transcript isoforms resulting from the top seven most frequent profiles were computed using novel logic models. Second, SPLICE-neo identified de novo RNA splicing events using RNA sequencing reads mapped to novel exon junctions from either single, double, or multiple exon-skipping events. The aberrant transcripts from both sources were then ranked based on isoform expression levels and z-scores assuming that individual aberrant splicing events are rare. Finally, top-ranked novel isoforms were translated into protein, and the resulting neoepitopes were evaluated for neoantigen potential using REAL-neo. The top splicing neoantigen candidates binding to HLA-A*02:01 were validated using in vitro T2 binding assays.ResultsWe identified abundant splicing neoantigens in four representative TCGA cancers: BRCA, LUAD, LUSC, and LIHC. In addition to their substantial contribution to neoantigen load, several splicing neoantigens were potent tumor antigens with stronger bindings to HLA compared with the positive control of antigens from influenza virus.ConclusionsSPLICE-neo is the first tool to comprehensively identify and prioritize splicing neoantigens from both DNA splice-site mutations and de novo RNA aberrant splicings. There are two major advances of SPLICE-neo. First, we developed novel logic models that assemble and prioritize full-length aberrant transcripts from DNA splice-site mutations. Second, SPLICE-neo can identify exon-skipping events involving more than two exons, which account for a quarter to one-third of all skipping events.
The extraordinarily long stigmatic silks of corn (Zea mays L.) are critical for grain production but the biology of their growth and emergence from husk leaves has remained underexplored. ...Accordingly, gene expression was assayed for inbreds ‘B73’ and ‘Mo17’ across five contiguous silk sections. Half of the maize genes (∼20,000) are expressed in silks, mostly in spatiotemporally dynamic patterns. In particular, emergence triggers strong differential expression of ∼1,500 genes collectively enriched for gene ontology terms associated with abiotic and biotic stress responses, hormone signaling, cell–cell communication, and defense metabolism. Further, a meta‐analysis of published maize transcriptomic studies on seedling stress showed that silk emergence elicits an upregulated transcriptomic response that overlaps strongly with both abiotic and biotic stress responses. Although the two inbreds revealed similar silk transcriptomic programs overall, genotypic expression differences were observed for 5,643 B73–Mo17 syntenic gene pairs and collectively account for >50% of genome‐wide expression variance. Coexpression clusters, including many based on genotypic divergence, were identified and interrogated via ontology‐term enrichment analyses to generate biological hypotheses for future research. Ultimately, dissecting how gene expression changes along the length of silks and between husk‐encased and emerged states offers testable models for silk development and plant response to environmental stresses.
Plants utilize sophisticated mechanisms and morphologies to mitigate stress. The epidermis, a single-cell boundary layer to the external environment, offers protection against stresses and is ...comprised of diverse specialized cell-types. In grasses, bulliform cells, which are highly vacuolated and positioned in the adaxial leaf epidermis, are utilized to mitigate drought stress by mechanically mediating the leaf rolling response, thereby reducing transpirational water losses. Considering that leaf rolling intensity directly relates to plant water status, other plant features that enhance water capture and utilization may also influence the leaf rolling response. As such, the structure-function relationships between bulliform cells, leaf rolling, and drought-stress mitigation is complex and not well understood, especially in diverse collections of germplasm used in today’s crop breeding programs. To elucidate these relationships, phenomic and genomic analyses were undertaken on bulliform cell architectures, leaf rolling, and a variety of morphological traits on a diverse panel of maize inbred lines. Surprisingly, leaf width had the greatest effect on leaf rolling propensity. However, genetic and physiological evidence indicated that reductions in bulliform cell size under drought are also distinctly associated to leaf rolling. In addition to mitigating drought stress, epidermal cells sense environmental conditions and accordingly regulate responses and metabolite secretions. Due to their extraordinary lengths and dense nutrient contents, maize’s stigmatic silks are quite affected by water deficits, pathogens, and pests. These vulnerabilities are particularly high following emergence from husk-leaves that protectively encase the ear. To examine molecular responses elicited after silk emergence into the external environment, gene expression was assayed across five contiguous silk sections that span both husk-encased and emerged portions of silks. The examined expression patterns revealed dramatic activation of a diverse set of stress pathways, including defense hormone signaling, cellular secretion pathways, and defense metabolite biosynthesis in emerged silks, providing intriguing molecular insights into the abiotic and biotic stress responses employed by this tissue. In conclusion, these results advance our current understanding of the mechanisms and morphologies utilized by the epidermis to mitigate stress.
Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other ...host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.
Root hairs are tubular extensions of the epidermis. Root hairs of the monogenic recessive maize mutant roothairless 6 (rth6) are arrested after bulge formation during the transition to tip growth and ...display a rough cell surface. BSR-Seq in combination with Seq-walking and subsequent analyses of four independently generated mutant alleles established that rth6 encodes CSLD5 a plasma membrane localized 129 kD D-type cellulose synthase with eight transmembrane domains. Cellulose synthases are required for the biosynthesis of cellulose, the most abundant biopolymer of plant cell walls. Phylogenetic analyses revealed that RTH6 is part of a monocot specific clade of D-type cellulose synthases. D-type cellulose synthases are highly conserved in the plant kingdom with five gene family members in maize and homologs even among early land plants such as the moss Physcomitrella patens or the clubmoss Selaginella moellendorffii. Expression profiling demonstrated that rth6 transcripts are highly enriched in root hairs as compared to all other root tissues. Moreover, in addition to the strong knock down of rth6 expression in young primary roots of the mutant rth6, the gene is also significantly down-regulated in rth3 and rth5 mutants, while it is up-regulated in rth2 mutants, suggesting that these genes interact in cell wall biosynthesis.
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