RESUMO
In early 2020, a global emergency was upon us in the form of the coronavirus disease 2019 (COVID-19) pandemic. While horrific in its health, social and economic devastation, one silver lining to this crisis has been a rapid mobilization of cross-institute, and even cross-country teams that shared common goals of learning as much as we could as quickly as possible about the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and how the immune system would respond to both the virus and COVID-19 vaccines. Many of these teams were formed by women who quickly realized that the classical model of "publish first at all costs" was maladaptive for the circumstances and needed to be supplanted by a more collaborative solution-focused approach. This review is an example of a collaboration that unfolded in separate countries, first Canada and the United States, and then also Israel. Not only did the collaboration allow us to cross-validate our results using different hands/techniques/samples, but it also took advantage of different vaccine types and schedules that were rolled out in our respective home countries. The result of this collaboration was a new understanding of how mucosal immunity to SARS-CoV-2 infection vs COVID-19 vaccination can be measured using saliva as a biofluid, what types of vaccines are best able to induce (limited) mucosal immunity, and what are potential correlates of protection against breakthrough infection. In this review, we will share what we have learned about the mucosal immune response to SARS-CoV-2 and to COVID-19 vaccines and provide a perspective on what may be required for next-generation pan-sarbecoronavirus vaccine approaches.
Assuntos
COVID-19 , Vacinas Virais , Anticorpos Antivirais , Vacinas contra COVID-19 , Feminino , Humanos , Imunoglobulina A , SARS-CoV-2 , VacinaçãoRESUMO
Membrane fusion during the entry of herpesviruses is carried out by the viral fusogen gB that is activated by its partner protein gH in some manner. The fusogenic activity of gB is controlled by its cytoplasmic (or intraviral) domain (gBCTD) and, according to the current model, the gBCTD is a trimeric, inhibitory clamp that restrains gB in the prefusion conformation. But how the gBCTD clamp is released by gH is unclear. Here, we identified two new regulatory elements within gB and gH from the prototypical herpes simplex virus 1: a surface pocket within the gBCTD and residue V831 within the gH cytoplasmic tail. Mutagenesis and structural modeling suggest that gH V831 interacts with the gB pocket. The gB pocket is located above the interface between adjacent protomers, and we hypothesize that insertion of the gH V831 wedge into the pocket serves to push the protomers apart, which releases the inhibitory clamp. In this manner, gH activates the fusogenic activity of gB. Both gB and gH are conserved across all herpesviruses, and this activation mechanism could be used by other gB homologs. Our proposed mechanism emphasizes a central role for the cytoplasmic regions in regulating the activity of a viral fusogen.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , Herpesvirus Humano 1/fisiologia , Humanos , Fusão de Membrana/fisiologia , Subunidades Proteicas/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do VírusRESUMO
Extravasation of T cells from the bloodstream into inflamed tissues requires interactions between T cells and vascular endothelial cells, a necessary step that allows T cells to exert their effector function during the immune response to pathogens and to sterile insults. This cellular cross talk involves adhesion molecules on both the vascular endothelium and the T cells themselves that function as receptor-ligand pairs to slow down circulating T cells. These will eventually extravasate into sites of inflammation when they receive the correct chemokine signals. Accumulation of T cells within the vascular wall can lead to vessel thickening and vascular disease, whereas T-cell extravasation into the myocardium often leads to cardiac chronic inflammation and adverse cardiac remodeling, hallmarks of heart failure. On the flip side, T-cell trafficking is required for pathogen clearance and to promote tissue repair after injury resulting from cardiac ischemia. Thus, a better understanding of the central players mediating these interactions may help develop novel therapeutics to modulate vascular and cardiac inflammation. Here, we review the most recent literature on pathways that regulate T-cell transendothelial migration, the last step leading to T-cell infiltration into tissues and organs in the context of vascular and cardiac inflammation. We discuss new potential avenues to therapeutically modulate these pathways to enhance or prevent immune cell infiltration in cardiovascular disease.
Assuntos
Células Endoteliais , Linfócitos T , Humanos , Adesão Celular/fisiologia , Leucócitos/metabolismo , Inflamação/metabolismo , Endotélio Vascular/metabolismoRESUMO
Coronavirus disease-2019 (COVID-19) was declared a global pandemic on 11 March 2020. Scientists and clinicians must acknowledge that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the potential to attack the human body in multiple ways simultaneously and exploit any weaknesses of its host. A multipronged attack could potentially explain the severity and extensive variety of signs and symptoms observed in patients with COVID-19. Understanding the diverse tactics of this virus to infect the human body is both critical and incredibly complex. Although patients diagnosed with COVID-19 have primarily presented with pulmonary involvement, viral invasion, and injury to diverse end organs is also prevalent and well documented in these patients, but has been largely unheeded. Human organs known for angiotensin-converting enzyme 2 (ACE2) expression including the gastrointestinal tract, kidneys, heart, adrenals, brain, and testicles are examples of extra pulmonary tissues with confirmed invasion by SARS-CoV-2. Initial multiple organ involvement may present with vague signs and symptoms to alert health care professionals early in the course of COVID-19. Another example of an ongoing, yet neglected element of the syndromic features of COVID-19, are the reported findings of loss of smell, altered taste, ataxia, headache, dizziness, and loss of consciousness, which suggest a potential for neural involvement. In this review, we further deliberate on the neuroinvasive potential of SARS-CoV-2, the neurologic symptomology observed in COVID-19, the host-virus interaction, possible routes of SARS-CoV-2 to invade the central nervous system, other neurologic considerations for patients with COVID-19, and a collective call to action.
Assuntos
COVID-19/complicações , Doenças do Sistema Nervoso/virologia , Humanos , SARS-CoV-2/patogenicidadeRESUMO
N-methyl-d-aspartate receptors (NMDARs) at excitatory synapses are central to activity-dependent synaptic plasticity and learning and memory. NMDARs act as ionotropic and metabotropic receptors by elevating postsynaptic calcium concentrations and by direct intracellular protein signaling. In the forebrain, these properties are controlled largely by the auxiliary GluN2 subunits, GluN2A and GluN2B. While calcium conductance through NMDAR channels and intracellular protein signaling make separate contributions to synaptic plasticity, it is not known if these properties individually influence learning and memory. To address this issue, we created chimeric GluN2 subunits containing the amino-terminal domain and transmembrane domains from GluN2A or GluN2B fused to the carboxy-terminal domain of GluN2B (termed ABc) or GluN2A ATD (termed BAc), respectively, and expressed these mutated GluN2 subunits in transgenic mice. Expression was confirmed at the mRNA level and protein subunit translation and translocation into dendrites were observed in forebrain neurons. In the spatial version of the Morris water maze, BAc mice displayed signs of a learning deficit. In contrast, ABc animals performed similarly to wild-types during training, but showed a more direct approach to the goal location during a long-term memory test. There was no effect of ABc or BAc expression in a nonspatial water escape task. Since background expression is predominantly GluN2A in mature animals, the results suggest that spatial learning is more sensitive to manipulations of the amino-terminal domain and transmembrane domains (calcium conductance) and long-term memory is regulated more by the carboxy-terminal domain (intracellular protein signaling).
Assuntos
Aprendizagem em Labirinto/fisiologia , Memória de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Deficiências da Aprendizagem/metabolismo , Transtornos da Memória/metabolismo , Camundongos Transgênicos , Neurônios/metabolismo , Prosencéfalo/metabolismo , Domínios Proteicos , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Comportamento Espacial/fisiologiaRESUMO
A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism.
Assuntos
Osso e Ossos/efeitos dos fármacos , Ossificação Heterotópica/prevenção & controle , Osteogênese/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Traumatismos por Explosões/complicações , Osso e Ossos/patologia , Modelos Animais de Doenças , Masculino , Ossificação Heterotópica/patologia , Osteogênese/genética , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Microtomografia por Raio-X/métodosRESUMO
Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.
Assuntos
Evolução Molecular , Complexo Principal de Histocompatibilidade/genética , Urocordados/genética , Urocordados/imunologia , Alelos , Sequência de Aminoácidos , Animais , Western Blotting , Perfilação da Expressão Gênica , Variação Genética , Células HEK293 , Haplótipos , Humanos , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Vermelha FluorescenteRESUMO
UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.
Assuntos
Família Multigênica , Micobacteriófagos/classificação , Micobacteriófagos/genética , Mycobacterium smegmatis/virologia , RNA de Transferência/genética , RNA Viral , Composição de Bases , Sequência de Bases , Códon , Sequência Conservada , Ordem dos Genes , Tamanho do Genoma , Genoma Viral , Sequências Repetidas Invertidas , Lisogenia/genética , Micobacteriófagos/ultraestrutura , Fases de Leitura Aberta , Filogenia , RNA de Transferência/química , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Vírion/genética , Vírion/ultraestrutura , Montagem de Vírus/genéticaRESUMO
The neural mechanisms that support the late postnatal development of spatial navigation are currently unknown. We investigated this in rats and found that an increase in the duration of AMPAR-mediated synaptic responses in the hippocampus was related to the emergence of spatial navigation. More specifically, spontaneous alternation rate, a behavioral indicator of hippocampal integrity, increased at the end of the third postnatal week in association with increases in AMPAR response duration at SC-CA1 synapses and synaptically driven postsynaptic discharge of CA1 pyramidal neurons. Pharmacological prolongation of glutamatergic synaptic transmission in juveniles increased the spontaneous alternation rate and CA1 postsynaptic discharge and reduced the threshold for the induction of activity-dependent synaptic plasticity at SC-CA1 synapses. A decrease in GluA1 and increases in GluA3 subunit and transmembrane AMPAR regulatory protein (TARP) expression at the end of the third postnatal week provide a molecular explanation for the increase in AMPAR response duration and reduced efficacy of AMPAR modulators with increasing age. A shift in the composition of AMPARs and increased association with AMPAR protein complex accessory proteins at the end of the third postnatal week likely "turns on" the hippocampus by increasing AMPAR response duration and postsynaptic excitability and reducing the threshold for activity-dependent synaptic potentiation.
Assuntos
Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/fisiologia , Aprendizagem em Labirinto/fisiologia , Receptores de AMPA/fisiologia , Percepção Espacial/fisiologia , Fatores Etários , Animais , Canais de Cálcio/fisiologia , Eletrofisiologia , Potenciais Evocados/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Oxazinas/farmacologia , Ratos , Ratos Long-Evans , Receptores de AMPA/agonistas , Sinapses/fisiologiaRESUMO
BACKGROUND: Gonad differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. The colonial ascidian, Botryllus schlosseri is a chordate model organism which offers two unique traits that can be utilized to characterize the genes underlying germline development: a colonial life history and variable fertility. These properties allow individual genotypes to be isolated at different stages of fertility and gene expression can be characterized comprehensively. RESULTS: Here we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis (asexual development) using differential expression analysis. We identified genes (as few as 7 and as many as 647) regulating fertility in Botryllus at each stage of blastogenesis. Several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. Spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads. CONCLUSION: We have identified several genes expressed in developing and mature gonads in B. schlosseri. Analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates.
Assuntos
Fertilidade/genética , Urocordados/genética , Urocordados/fisiologia , Animais , Feminino , Humanos , Infertilidade/genética , Masculino , Ovário/metabolismo , Ovário/fisiologia , Ovário/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Testículo/metabolismo , Testículo/fisiologia , Testículo/fisiopatologiaRESUMO
Lyme disease is a tick-borne disease caused by bacteria of the genus Borrelia. The host factors that modulate susceptibility for Lyme disease have remained mostly unknown. Using epidemiological and genetic data from FinnGen and Estonian Biobank, we identify two previously known variants and an unknown common missense variant at the gene encoding for Secretoglobin family 1D member 2 (SCGB1D2) protein that increases the susceptibility for Lyme disease. Using live Borrelia burgdorferi (Bb) we find that recombinant reference SCGB1D2 protein inhibits the growth of Bb in vitro more efficiently than the recombinant protein with SCGB1D2 P53L deleterious missense variant. Finally, using an in vivo murine infection model we show that recombinant SCGB1D2 prevents infection by Borrelia in vivo. Together, these data suggest that SCGB1D2 is a host defense factor present in the skin, sweat, and other secretions which protects against Bb infection and opens an exciting therapeutic avenue for Lyme disease.
Assuntos
Borrelia burgdorferi , Ixodes , Doença de Lyme , Secretoglobinas , Animais , Humanos , Camundongos , Borrelia burgdorferi/genética , Ixodes/microbiologia , Doença de Lyme/microbiologiaRESUMO
Heart failure with preserved ejection fraction (HFpEF) is a widespread syndrome with limited therapeutic options and poorly understood immune pathophysiology. Using a 2-hit preclinical model of cardiometabolic HFpEF that induces obesity and hypertension, we found that cardiac T cell infiltration and lymphoid expansion occurred concomitantly with cardiac pathology and that diastolic dysfunction, cardiomyocyte hypertrophy, and cardiac phospholamban phosphorylation were T cell dependent. Heart-infiltrating T cells were not restricted to cardiac antigens and were uniquely characterized by impaired activation of the inositol-requiring enzyme 1α/X-box-binding protein 1 (IRE1α/XBP1) arm of the unfolded protein response. Notably, selective ablation of XBP1 in T cells enhanced their persistence in the heart and lymphoid organs of mice with preclinical HFpEF. Furthermore, T cell IRE1α/XBP1 activation was restored after withdrawal of the 2 comorbidities inducing HFpEF, resulting in partial improvement of cardiac pathology. Our results demonstrated that diastolic dysfunction and cardiomyocyte hypertrophy in preclinical HFpEF were T cell dependent and that reversible dysregulation of the T cell IRE1α/XBP1 axis was a T cell signature of HFpEF.
Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Animais , Camundongos , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Insuficiência Cardíaca/metabolismo , Hipertrofia , Inflamação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Volume Sistólico/fisiologia , Linfócitos T/patologia , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.
Assuntos
Divisão Celular , Forma Celular , Células Epiteliais/fisiologia , Mecanotransdução Celular , Animais , Animais Geneticamente Modificados , Comunicação Celular , Segregação de Cromossomos , Simulação por Computador , Cães , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Células Madin Darby de Rim Canino , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Estresse Mecânico , Fatores de Tempo , Imagem com Lapso de TempoRESUMO
The 2019 novel coronavirus disease (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a zoonotic disease that is dominated by pulmonary symptoms. However, recent reports of isolation of the virus from cerebrospinal fluid (CSF) coupled with radiological evidence of zones of necrosis in the brain, have elucidated the neurotropic potential of SARS-CoV-2. The acute respiratory failure seen in patients with COVID-19 is alarming and could be due to the effects of SARS-CoV-2 on the central respiratory regulatory centers in the brainstem. Appropriate interventions can be implemented to prevent severe outcomes of neurological invasion by SARS-CoV-2 to reduce the morbidity and mortality of patients with COVID-19. It is of paramount importance that the scientific community alerts the healthcare professionals of the pieces of evidence that can herald them on the covert neurological deficits in progress in COVID-19.
Assuntos
Infecções por Coronavirus/complicações , Disgeusia/etiologia , Doenças do Sistema Nervoso/etiologia , Transtornos do Olfato/etiologia , Pneumonia Viral/complicações , Betacoronavirus/fisiologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Encéfalo/virologia , COVID-19 , Infecções por Coronavirus/fisiopatologia , Disgeusia/fisiopatologia , Osso Etmoide , Humanos , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Transtornos do Olfato/fisiopatologia , Pandemias , Pneumonia Viral/fisiopatologia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/fisiopatologia , SARS-CoV-2 , Tropismo ViralRESUMO
Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [-] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.
Assuntos
DNA Viral/isolamento & purificação , Dependovirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Linhagem Celular , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Genoma Viral , Células HEK293 , Humanos , Plasmídeos , TransgenesRESUMO
Despite the conserved function of aggression across taxa in obtaining critical resources such as food and mates, serotonin's (5-HT) modulatory role on aggressive behavior appears to be largely inhibitory for vertebrates but stimulatory for invertebrates. However, critical gaps exist in our knowledge of invertebrates that need to be addressed before definitively stating opposing roles for 5-HT and aggression. Specifically, the role of 5-HT receptor subtypes are largely unknown, as is the potential interactive role of 5-HT with other neurochemical systems known to play a critical role in aggression. Similarly, the influence of these systems in driving sex differences in aggressive behavior of invertebrates is not well understood. Here, we investigated these questions by employing complementary approaches in a novel invertebrate model of aggression, the stalk-eyed fly. A combination of altered social conditions, pharmacological manipulation and 5-HT2 receptor knockdown by siRNA revealed an inhibitory role of this receptor subtype on aggression. Additionally, we provide evidence for 5-HT2's involvement in regulating neuropeptide F activity, a suspected inhibitor of aggression. However, this function appears to be stage-specific, altering only the initiation stage of aggressive conflicts. Alternatively, pharmacologically increasing systemic concentrations of 5-HT significantly elevated the expression of the neuropeptide tachykinin, which did not affect contest initiation but instead promoted escalation via production of high intensity aggressive behaviors. Notably, these effects were limited solely to males, with female aggression and neuropeptide expression remaining unaltered by any manipulation that affected 5-HT. Together, these results demonstrate a more nuanced role for 5-HT in modulating aggression in invertebrates, revealing an important interactive role with neuropeptides that is more reminiscent of vertebrates. The sex-differences described here also provide valuable insight into the evolutionary contexts of this complex behavior.
Assuntos
Agressão/fisiologia , Comportamento Animal/fisiologia , Dípteros/fisiologia , Caracteres Sexuais , 5-Hidroxitriptofano/administração & dosagem , 5-Hidroxitriptofano/farmacologia , Agressão/efeitos dos fármacos , Animais , Técnicas de Observação do Comportamento/métodos , Comportamento Animal/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Masculino , Modelos Animais , Neuropeptídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores 5-HT2 de Serotonina/genética , Receptores 5-HT2 de Serotonina/metabolismo , Serotonina/metabolismo , Taquicininas/metabolismoRESUMO
We sought to test a hypothesis that systemic blind spots in active learning are a barrier both for instructors-who cannot see what every student is actually thinking on each concept in each class-and for students-who often cannot tell precisely whether their thinking is right or wrong, let alone exactly how to fix it. We tested a strategy for eliminating these blind spots by having students answer open-ended, conceptual problems using a Web-based platform, and measured the effects on student attrition, engagement, and performance. In 4 years of testing both in class and using an online platform, this approach revealed (and provided specific resolution lessons for) more than 200 distinct conceptual errors, dramatically increased average student engagement, and reduced student attrition by approximately fourfold compared with the original lecture course format (down from 48.3% to 11.4%), especially for women undergraduates (down from 73.1% to 7.4%). Median exam scores increased from 53% to 72-80%, and the bottom half of students boosted their scores to the range in which the top half had scored before the pedagogical switch. By contrast, in our control year with the same active-learning content (but without this "zero blind spots" approach), these gains were not observed.
Assuntos
Desempenho Acadêmico , Biologia Computacional/educação , Currículo , Aprendizagem Baseada em Problemas , Estudantes , Avaliação Educacional , Feminino , Humanos , MasculinoRESUMO
Fusarium is a complex genus of ascomycete fungi that consists of plant pathogens of agricultural relevance. Controlling Fusarium infection in crops that leads to substantial yield losses is challenging. These economic losses along with environmental and human health concerns over the usage of chemicals in attaining disease control are shifting focus toward the use of biocontrol agents for effective control of phytopathogenic Fusarium spp. In the present study, an analysis of the plant-growth promoting (PGP) and biocontrol attributes of four bacilli (Bacillus simplex 30N-5, B. simplex 11, B. simplex 237, and B. subtilis 30VD-1) has been conducted. The production of cellulase, xylanase, pectinase, and chitinase in functional assays was studied, followed by in silico gene analysis of the PGP-related and biocontrol-associated genes. Of all the bacilli included in this study, B. subtilis 30VD-1 (30VD-1) demonstrated the most effective antagonism against Fusarium spp. under in vitro conditions. Additionally, 100 µg/ml of the crude 1-butanol extract of 30VD-1's cell-free culture filtrate caused about 40% inhibition in radial growth of Fusarium spp. Pea seed bacterization with 30VD-1 led to considerable reduction in wilt severity in plants with about 35% increase in dry plant biomass over uninoculated plants growing in Fusarium-infested soil. Phase contrast microscopy demonstrated distortions and abnormal swellings in F. oxysporum hyphae on co-culturing with 30VD-1. The results suggest a multivariate mode of antagonism of 30VD-1 against phytopathogenic Fusarium spp., by producing chitinase, volatiles, and other antifungal molecules, the characterization of which is underway.
RESUMO
Neural networks that undergo acute insults display remarkable reorganization. This injury related plasticity is thought to permit recovery of function in the face of damage that cannot be reversed. Previously, an increase in the transmission strength at Schaffer collateral to CA1 pyramidal cell synapses was observed after long-term activity reduction in organotypic hippocampal slices. Here we report that, following acute preparation of adult rat hippocampal slices and surgical removal of area CA3, input to area CA1 was reduced and Schaffer collateral synapses underwent functional strengthening. This increase in synaptic strength was limited to Schaffer collateral inputs (no alteration to temporoammonic synapses) and acted to normalize postsynaptic discharge, supporting a homeostatic or compensatory response. Short-term plasticity was not altered, but an increase in immunohistochemical labeling of GluA1 subunits was observed in the stratum radiatum (but not stratum moleculare), suggesting increased numbers of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and a postsynaptic locus of expression. Combined, these data support the idea that, in response to the reduction in presynaptic activity caused by removal of area CA3, Schaffer collateral synapses undergo a relatively rapid increase in functional efficacy likely supported by insertion of more AMPARs, which maintains postsynaptic excitability in CA1 pyramidal neurons. This novel fast compensatory plasticity exhibits properties that would allow it to maintain optimal network activity levels in the hippocampus, a brain structure lauded for its ongoing experience-dependent malleability.
Assuntos
Região CA3 Hipocampal/fisiologia , Potenciais Pós-Sinápticos Excitadores , Hipocampo/fisiologia , Plasticidade Neuronal , Células Piramidais/fisiologia , Sinapses/fisiologia , Animais , Estimulação Elétrica , Masculino , Ratos Long-Evans , Receptores de AMPA/fisiologiaRESUMO
Organ renewal is governed by the dynamics of cell division, differentiation and loss. To study these dynamics in real time, we present a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem-cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12-16 hr and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. By applying a pipeline for movie processing and analysis, we uncover new and intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells use slow kinetics of Notch activation to reach a fate-specifying threshold, and that enterocytes extrude via ratcheted constriction of a junctional ring. By enabling real-time study of midgut phenomena that were previously inaccessible, our platform opens a new realm for dynamic understanding of adult organ renewal.