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Journal of Visualized Experiments : JoVE Sep 2022Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to...
Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.
Topics: Anaphase-Promoting Complex-Cyclosome; Aneuploidy; Animals; Cell Cycle Proteins; Kinetochores; M Phase Cell Cycle Checkpoints; Mice; Nocodazole; Oocytes; Securin; Spindle Apparatus
PubMed: 36190266
DOI: 10.3791/64459 -
Mucosal Immunology Jun 2024Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils...
Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils to the lung is linked to increased bacterial burden and aggravated pathology in TB. The basis for the permissiveness of neutrophils for Mtb and the ability to be pathogenic in TB has been elusive. Here, we identified metabolic and functional features of neutrophils that contribute to their permissiveness in Mtb infection. Using single-cell metabolic and transcriptional analyses, we found that neutrophils in the Mtb-infected lung displayed elevated mitochondrial metabolism, which was largely attributed to the induction of activated neutrophils with enhanced metabolic activities. The activated neutrophil subpopulation was also identified in the lung granulomas from Mtb-infected non-human primates. Functionally, activated neutrophils harbored more viable bacteria and displayed enhanced lipid uptake and accumulation. Surprisingly, we found that interferon-γ promoted the activation of lung neutrophils during Mtb infection. Lastly, perturbation of lipid uptake pathways selectively compromised Mtb survival in activated neutrophils. These findings suggest that neutrophil heterogeneity and metabolic diversity are key to their permissiveness for Mtb and that metabolic pathways in neutrophils represent potential host-directed therapeutics in TB.
PubMed: 38844208
DOI: 10.1016/j.mucimm.2024.05.007 -
Cureus Apr 2024Nutritional support is a critical component of care for critically ill patients, impacting their recovery and overall prognosis. Traditional approaches to feeding in the... (Review)
Review
Nutritional support is a critical component of care for critically ill patients, impacting their recovery and overall prognosis. Traditional approaches to feeding in the intensive care unit (ICU) have focused on meeting estimated energy requirements, often resulting in unintended consequences such as overfeeding and associated complications. Permissive underfeeding, a concept gaining attention recently, offers a more controlled approach by intentionally providing fewer calories than traditionally recommended. This comprehensive review explores the rationale, evidence, and practical considerations surrounding permissive underfeeding in critically ill patients. We discuss the physiological basis of permissive underfeeding, its potential benefits in mitigating the risks of overfeeding, and the challenges associated with implementation in clinical practice. Through an analysis of critical studies and clinical trials, we evaluate the comparative effectiveness of permissive underfeeding versus traditional feeding methods and examine its impact on patient outcomes. Recommendations for patient selection, monitoring, and future research directions are provided to guide clinicians in optimizing nutritional support strategies for critically ill individuals. By considering the role of permissive underfeeding alongside traditional feeding approaches, healthcare professionals can tailor nutritional interventions to individual patient needs, ultimately improving outcomes in the ICU.
PubMed: 38741818
DOI: 10.7759/cureus.58083 -
The American Journal of Clinical... Oct 2021This is an extension of "The Future Orientation of Constructive Memory" published in 2008 (Rossi, Erickson-Klein & Rossi). In a context of neuroscience, questions were...
This is an extension of "The Future Orientation of Constructive Memory" published in 2008 (Rossi, Erickson-Klein & Rossi). In a context of neuroscience, questions were raised regarding retrospective functions of dreaming. This paper summarizes the ideas from the original article and provides updates relevant to studies that have been conducted in the interim. The 4-Stage Creative Process model is used to conceptualize the manner in which activity-dependent genomic stimulation contributes to future adaptive behaviors. Neurophysiological constructs are discussed with their relevance to clinical practice. Two case summaries illustrate the application of the 4-Stage Creative Process as a framework for therapeutic hypnosis and permissive suggestion. The collaboration between Ernest Rossi and Milton Erickson offers an increasingly relevant and nuanced understanding about the interface of behavior, genomic expression, and activity-dependent gene expression.
Topics: Humans; Hypnosis; Neurosciences; Retrospective Studies; Suggestion
PubMed: 34723777
DOI: 10.1080/00029157.2021.1941744 -
Vaccines Nov 2022The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased...
The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased viral load. Omicron exhibited a shift in tropism infecting the upper respiratory tract compared to other variants of concern which have tropism for the lower respiratory tract. The tropism of omicron variants in cell lines of different hosts and tissue origins still remains unclear. Considering this, we assessed the susceptibility of different cell lines to the SARS-CoV-2 omicron BA.1.1 variant and permissiveness among different cell lines for omicron replication. Susceptibility and permissiveness of a total of eleven cell lines, including six animal cell lines and five human cell lines for omicron BA.1.1 infection, were evaluated by infecting individual cell lines with omicron BA.1.1 isolate at a 0.1 multiplicity of infection. Virus replication was assessed by observation of cytopathic effects followed by viral load determination by real-time PCR assay and virus infectivity determination by TCID50 assay. The characteristic cytopathic effect, increased viral load, and productive omicron replication was detected in Vero CCL-81, Vero E6, Vero/hSLAM, MA-104, and Calu-3 cells. Although LLC MK-2 cells showed an increased TCID50 titer at the second infection, the viral load did not show much difference in both infections. Caco-2 cells did not show evident CPE, but they supported omicron replication at a low level. A549, RD, MRC-5, and BHK-21 cells supported omicron BA.1.1 replication without the CPE. This is the first study on the comparison of susceptibility of different cell lines to Omicron variant BA.1.1, which might be useful for future studies on emerging SARS-CoV-2 variants.
PubMed: 36423057
DOI: 10.3390/vaccines10111962 -
Frontiers in Cellular and Infection... 2022The mosquito-borne Usutu virus (USUV) is a zoonotic flavivirus and an emerging pathogen. So far therapeutical options or vaccines are not available in human and...
The mosquito-borne Usutu virus (USUV) is a zoonotic flavivirus and an emerging pathogen. So far therapeutical options or vaccines are not available in human and veterinary medicine. The bioenergetic profile based on extracellular flux analysis revealed an USUV infection-associated significant increase in basal and stressed glycolysis on Vero and with a tendency for basal glycolysis on the avian cell line TME-R derived from Eurasian blackbirds. On both cell lines this was accompanied by a significant drop in the metabolic potential of glycolysis. Moreover, glycolysis contributed to production of virus progeny, as inhibition of glycolysis with 2-deoxy-D-glucose reduced virus yield on Vero by one log step. Additionally, the increase in glycolysis observed on Vero cells after USUV infection was lost after the addition of exogenous type I interferon (IFN) β. To further explore the contribution of the IFN response pathway to the impact of USUV on cellular metabolism, USUV infection was characterized on human A549 respiratory cells with a knockout of the type I IFN receptor, either solely or together with the receptor of type III IFN. Notably, only the double knockout of types I and III IFN receptor increased permissiveness to USUV and supported viral replication together with an alteration of the glycolytic activity, namely an increase in basal glycolysis to an extent that a further increase after injection of metabolic stressors during extracellular flux analysis was not noted. This study provides evidence for glycolysis as a possible target for therapeutic intervention of USUV replication. Moreover, presented data highlight type I and type III IFN system as a determinant for human host cell permissiveness and for the infection-associated impact on glycolysis.
Topics: Animals; Chlorocebus aethiops; Flavivirus; Flavivirus Infections; Glycolysis; Humans; Interferons; Vero Cells
PubMed: 35186796
DOI: 10.3389/fcimb.2022.823181 -
Aesthetic Plastic Surgery Dec 2023Permissive hypotension, defined as mean arterial pressure (MAP) of 60-70 mm Hg, has been regarded as favorable among surgeons performing rhinoplasty. Furthermore,... (Review)
Review
BACKGROUND/PURPOSE
Permissive hypotension, defined as mean arterial pressure (MAP) of 60-70 mm Hg, has been regarded as favorable among surgeons performing rhinoplasty. Furthermore, management of blood pressure has been shown to promote greater visualization of the surgical field and decrease postoperative complications, such as ecchymosis and edema. While multiple therapies have been utilized to achieve permissive hypotension, it remains unclear how modalities compare in terms of safety and efficacy. The purpose of this study was to conduct a systematic review to better understand the specific modalities and associated outcomes in managing blood pressure during rhinoplasty.
METHODS
A systematic literature review was conducted in order to identify and assess therapeutics utilized in achieving permissive hypotension during rhinoplasty. Variables collected included year of publication, journal, article title, organization of study, patient sample, treatment modality, associated outcomes (i.e., intraoperative bleeding, edema, and ecchymosis), adverse events, complications, and satisfaction. Articles were then categorized by the level of evidence as set forth by the American Society of Plastic Surgeons. Any conflicts were resolved through discussion and full-text review among co-authors. Of note, the search was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. No funding was required to conduct this review of the literature.
RESULTS
Initial review yielded sixty-five articles. Title and abstract review followed by standardized application of inclusion and exclusion criteria resulted in a total of ten studies for analysis. Articles discussed multiple therapies for management of blood pressure during rhinoplasty, including dexmedetomidine, dexamethasone, gabapentin, labetalol, nitroglycerine, remifentanil, magnesium sulfate, clonidine, and metoprolol. Overall, intraoperative bleeding, as well as postoperative ecchymosis and edema were reduced when MAP was controlled.
CONCLUSION
Given its intra- and postoperative benefits, permissive hypotension can be leveraged to improve outcomes in rhinoplasty. This study presents an updated comprehensive review of various modalities used to achieve permission hypotension in rhinoplasty. Future studies should explore how comorbidities may impact choice of treatment regimen among patients undergoing rhinoplasty.
LEVEL OF EVIDENCE III
This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
Topics: Humans; Hemorrhage; Hypotension; Rhinoplasty; Treatment Outcome; Postoperative Complications
PubMed: 36877227
DOI: 10.1007/s00266-023-03298-y -
MBio Jun 2022Chikungunya virus (CHIKV) is an arthritogenic reemerging virus replicating in plasma membrane-derived compartments termed "spherules." Here, we identify the human...
Chikungunya virus (CHIKV) is an arthritogenic reemerging virus replicating in plasma membrane-derived compartments termed "spherules." Here, we identify the human transmembrane protein CD81 as host factor required for CHIKV replication. Ablation of CD81 results in decreased CHIKV permissiveness, while overexpression enhances infection. CD81 is dispensable for virus uptake but critically required for viral genome replication. Likewise, murine CD81 is crucial for CHIKV permissiveness and is expressed in target cells such as dermal fibroblasts, muscle and liver cells. Whereas related alphaviruses, including Ross River virus (RRV), Semliki Forest virus (SFV), Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV), also depend on CD81 for infection, RNA viruses from other families, such as coronaviruses, replicate independently of CD81. Strikingly, the replication-enhancing function of CD81 is linked to cholesterol binding. These results define a mechanism exploited by alphaviruses to hijack the membrane microdomain-modeling protein CD81 for virus replication through interaction with cholesterol. In this study, we discover the tetraspanin CD81 as a host factor for the globally emerging chikungunya virus and related alphaviruses. We show that CD81 promotes replication of viral genomes in human and mouse cells, while virus entry into cells is independent of CD81. This provides novel insights into how alphaviruses hijack host proteins to complete their life cycle. Alphaviruses replicate at distinct sites of the plasma membrane, which are enriched in cholesterol. We found that the cholesterol-binding ability of CD81 is important for its function as an alphavirus host factor. This discovery thus broadens our understanding of the alphavirus replication process and the use of host factors to reprogram cells into virus replication factories.
Topics: Animals; Chikungunya Fever; Chikungunya virus; Cholesterol; Humans; Mice; Tetraspanins; Virus Replication; Viruses
PubMed: 35612284
DOI: 10.1128/mbio.00731-22 -
Critical Care (London, England) Feb 2020Despite sound basis to suspect that aggressive and early administration of nutritional support may hold therapeutic benefits during sepsis, recommendations for... (Review)
Review
Despite sound basis to suspect that aggressive and early administration of nutritional support may hold therapeutic benefits during sepsis, recommendations for nutritional support have been somewhat underwhelming. Current guidelines (ESPEN and ASPEN) recognise a lack of clear evidence demonstrating the beneficial effect of nutritional support during sepsis, raising the question: why, given the perceived low efficacy of nutritionals support, are there no high-quality clinical trials on the efficacy of permissive underfeeding in sepsis? Here, we review clinically relevant beneficial effects of permissive underfeeding, motivating the urgent need to investigate the clinical benefits of delaying nutritional support during sepsis.
Topics: Critical Illness; Energy Intake; Enteral Nutrition; Humans; Nutritional Requirements; Nutritional Support; Sepsis
PubMed: 32059698
DOI: 10.1186/s13054-020-2771-4 -
Emerging Microbes & Infections Dec 2023Rift Valley fever (RVF) is an arboviral disease of zoonotic origin that causes recurrent epidemics in Africa, the Arabic Peninsula, and islands of the South West of the...
Rift Valley fever (RVF) is an arboviral disease of zoonotic origin that causes recurrent epidemics in Africa, the Arabic Peninsula, and islands of the South West of the Indian Ocean. RVF occurs mainly in livestock but also affects humans with severe clinical manifestations, including neurological disorders. However, human neuropathogenesis of Rift Valley fever virus (RVFV) is still poorly characterized. To study the interactions between RVFV and the central nervous system (CNS), we focused on RVFV infection of astrocytes, the major glial cells of the CNS that have several supporting roles including immune response regulation. We confirmed the permissiveness of astrocytes to RVFV infection and highlighted a strain-dependent infectivity. We showed that RVFV infection of astrocytes induced cell apoptosis and observed that the RVFV Non-Structural protein NSs, a known virulence factor, potentially delayed apoptosis by sequestrating activated-caspase 3 in the nucleus. Our study also showed that RVFV-infected astrocytes upregulated expression of genes associated with inflammatory and type I interferon responses at the mRNA level, but not at the protein level. This inhibition of immune response is potentially due to a NSs-dependent mechanism of mRNA nuclear export inhibition. Together, these results highlighted the direct impact of RVFV infection on the human CNS through the induction of apoptosis and a possible inhibition of early-onset immune responses that are crucial for the host survival.
Topics: Animals; Humans; Rift Valley fever virus; Astrocytes; Rift Valley Fever; Immunity; RNA, Messenger
PubMed: 37306630
DOI: 10.1080/22221751.2023.2207672