Devices used for photobiomodulation of the brain-a comprehensive and systematic review.

A systematic review was conducted to determine the trends in devices and parameters used for brain photobiomodulation (PBM). The revised studies included clinical and cadaveric approaches, in which light stimuli were applied to the head and/or neck. PubMed, Scopus, Web of Science and Google Scholar databases were used for the systematic search. A total of 2133 records were screened, from which 97 were included in this review. The parameters that were extracted and analysed in each article were the device design, actuation area, actuation site, wavelength, mode of operation, power density, energy density, power output, energy per session and treatment time. To organize device information, 11 categories of devices were defined, according to their characteristics. The most used category of devices was laser handpieces, which relate to 21% of all devices, while 28% of the devices were not described. Studies for cognitive function and physiological characterisation are the most well defined ones and with more tangible results. There is a lack of consistency when reporting PBM studies, with several articles under defining the stimulation protocol, and a wide variety of parameters used for the same health conditions (e.g., Alzheimer's or Parkinson's disease) resulting in positive outcomes. Standardization for the report of these studies is warranted, as well as sham-controlled comparative studies to determine which parameters have the greatest effect on PBM treatments for different neurological conditions.

Influence of Ultrasound Stimulation on the Viability, Proliferation and Protein Expression of Osteoblasts and Periodontal Ligament Fibroblasts.

Among the adjunctive procedures to accelerate orthodontic tooth movement (OTM), ultrasound (US) is a nonsurgical form of mechanical stimulus that has been explored as an alternative to the currently available treatments. This study aimed to clarify the role of US in OTM by exploring different stimulation parameters and their effects on the biological responses of cells involved in OTM. Human fetal osteoblasts and periodontal ligament fibroblasts cell lines were stimulated with US at 1.0 and 1.5 MHz central frequencies and power densities of 30 and 60 mW/cm2 in continuous mode for 5 and 10 min. Cellular proliferation, metabolic activity and protein expression were analyzed. The US parameters that significantly improved the metabolic activity were 1.0 MHz at 30 mW/cm2 for 5 min and 1.0 MHz at 60 mW/cm2 for 5 and 10 min for osteoblasts; and 1.0 MHz at 30 mW/cm2 for 5 min and 1.5 MHz at 60 mW/cm2 for 5 and 10 min for fibroblasts. By stimulating with these parameters, the expression of alkaline phosphatase was maintained, while osteoprotegerin synthesis was induced after three days of US stimulation. The US stimulation improved the biological activity of both osteoblasts and periodontal ligament fibroblasts, inducing their osteogenic differentiation.

Optimization of a Photobiomodulation Protocol to Improve the Cell Viability, Proliferation and Protein Expression in Osteoblasts and Periodontal Ligament Fibroblasts for Accelerated Orthodontic Treatment.

Numerous pieces of evidence have supported the therapeutic potential of photobiomodulation (PBM) to modulate bone remodeling on mechanically stimulated teeth, proving PBM's ability to be used as a coadjuvant treatment to accelerate orthodontic tooth movement (OTM). However, there are still uncertainty and discourse around the optimal PBM protocols, which hampers its optimal and consolidated clinical applicability. Given the differential expression and metabolic patterns exhibited in the tension and compression sides of orthodontically stressed teeth, it is plausible that different types of irradiation may be applied to each side of the teeth. In this sense, this study aimed to design and implement an optimization protocol to find the most appropriate PBM parameters to stimulate specific bone turnover processes. To this end, three levels of wavelength (655, 810 and 940 nm), two power densities (5 and 10 mW/cm2) and two regimens of single and multiple sessions within three consecutive days were tested. The biological response of osteoblasts and periodontal ligament (PDL) fibroblasts was addressed by monitoring the PBM's impact on the cellular metabolic activity, as well as on key bone remodeling mediators, including alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor κ-B ligand (RANK-L), each day. The results suggest that daily irradiation of 655 nm delivered at 10 mW/cm2, as well as 810 and 940 nm light at 5 mW/cm2, lead to an increase in ALP and OPG, potentiating bone formation. In addition, irradiation of 810 nm at 5 mW/cm2 delivered for two consecutive days and suspended by the third day promotes a downregulation of OPG expression and a slight non-significant increase in RANK-L expression, being suitable to stimulate bone resorption. Future studies in animal models may clarify the impact of PBM on bone formation and resorption mediators for longer periods and address the possibility of testing different stimulation periodicities. The present in vitro study offers valuable insights into the effectiveness of specific PBM protocols to promote osteogenic and osteoclastogenesis responses and therefore its potential to stimulate bone formation on the tension side and bone resorption on the compression side of orthodontically stressed teeth.

Internet Addiction, Sleep Habits and Family Communication: The Perspectives of a Sample of Adolescents.

BACKGROUND: With the increase in communication technologies, the internet has become an indispensable tool in the life of the individual. Several studies report on the advantages of this resource; however, there is still a group of individuals who use the internet excessively. The aim of this study was to explore the relationships between internet addiction, daytime sleepiness, and family communication in adolescents. METHODS: A total of 340 adolescents aged between 12 and 17 years participated in this study. All completed the sociodemographic questionnaire, the internet addiction test, the pediatric daytime sleepiness scale, and the family communication scale. RESULTS: The results indicate that 64.1% of the adolescents had mild to moderate addiction to the internet. The main results suggest that internet addiction in adolescents is negatively associated with family communication and positively associated with excessive daytime sleepiness. It was also observed that gender had a significant effect on daytime sleepiness, with female participants having more excessive daytime sleepiness. Regarding age, the results indicate higher values of internet addiction among younger adolescents. CONCLUSIONS: In view of the above, it is considered important to develop preventive actions with a view to healthy family communication, with the adoption of sleep hygiene habits and the promotion of healthy use of the internet.

Accuracy of Invisalign® on Upper Incisors: A Systematic Review.

The current systematic review appraises the effectiveness of the types of tooth movements performed with Invisalign® clear aligner on the maxillary incisors. An electronic literature search of published trials was performed through PubMed, LILACS, Scopus, Cochrane Library, and Web of Science databases, and selected journals, from 2009 to 2020. Out of 291 references, five relevant publications were identified for analysis: four studies were performed retrospectively and one prospectively, all non-randomized. Despite the limited set of selected articles, the sample size is significant, with 148 subjects included in the reviewed studies involving the orthodontic treatment of upper incisors. We concluded that movements with the Invisalign® clear aligner on the upper incisors present distinct accuracy, possibly related with movement complexity; intrusion of the incisors has low accuracy (in some cases, 0% of accuracy was reported when the tooth extruded), while incisor extrusion exhibit some of the highest accuracy values reported in the included studies (45%-142%, when the achieved movement was greater than the predicted). Besides, axial (i.e., torque and tip) and horizontal (i.e., translation and rotation) movements are usually effective, with accuracy values between 39%-156% and 42%-79%, respectively. Overall, we determined that the efficiency of aligner to reach the desired movements in the upper incisors was low, as often refinements were required in the included studies. The use of aligner features must be more often considered to improve movement accuracy.

The Impact of Non-Pathogenic Bacteria on the Spread of Virulence and Resistance Genes.

This review discusses the fate of antimicrobial resistance and virulence genes frequently present among microbiomes. A central concept in epidemiology is the mean number of hosts colonized by one infected host in a population of susceptible hosts: R0. It characterizes the disease's epidemic potential because the pathogen continues its propagation through susceptible hosts if it is above one. R0 is proportional to the average duration of infections, but non-pathogenic microorganisms do not cause host death, and hosts do not need to be rid of them. Therefore, commensal bacteria may colonize hosts for prolonged periods, including those harboring drug resistance or even a few virulence genes. Thus, their R0 is likely to be (much) greater than one, with peculiar consequences for the spread of virulence and resistance genes. For example, computer models that simulate the spread of these genes have shown that their diversities should correlate positively throughout microbiomes. Bioinformatics analysis with real data corroborates this expectation. Those simulations also anticipate that, contrary to the common wisdom, human's microbiomes with a higher diversity of both gene types are the ones that took antibiotics longer ago rather than recently. Here, we discuss the mechanisms and robustness behind these predictions and other public health consequences.

Photobiomodulation and visual stimulation against cognitive decline and Alzheimer's disease pathology: A systematic review.

Introduction: Given the ineffectiveness of the available drug treatment against Alzheimer disease (AD), light-based therapeutic modalities have been increasingly receiving attention with photobiomodulation (PBM) and, more recently, visual stimulation (VS) being among the most promising approaches. However, the PBM and VS light parameters tested so far, as well as their outcomes, vary a lot with conflicting results being reported. Methods: Based on Scopus, PubMed, and Web of Science databases search, this systematic review summarizes, compares, and discusses 43 cell, animal, and human studies of PBM and VS related to cognitive decline and AD pathology. Results: Preclinical work suggests that PBM with 640±30-nm light and VS at 40 Hz attenuates Aß and Tau pathology and improves neuronal and synaptic plasticity with most studies pointing towards enhancement of degradation/clearance mechanisms in the brain of AD animal models. Despite the gap of the translational evidence for both modalities, the few human studies performed so far support the use of PBM at 810-870 nm light pulsing at 40 Hz for improving brain network connectivity and memory in older subjects and AD patients, while 40 Hz VS in humans seems to improve cognition; further clinical investigation is urgently required to clarify the beneficial impact of PBM and VS in AD patients. Discussion: This review highlights PBM and VS as promising light-based therapeutic approaches against AD brain neuropathology and related cognitive decline, clarifying the most effective light parameters for further preclinical and clinical testing and use. Highlights: Light-based brain stimulation produces neural entrainment and reverts neuronal damageBrain PBM and VS attenuate AD neuropathologyPMB and VS are suggested to improve cognitive performance in AD patients and animal modelsLight stimulation represents a promising therapeutic strategy against neurodegeneration.

Interceptive Treatment with Invisalign® First in Moderate and Severe Cases: A Case Series.

The increasing demand for more aesthetic/comfortable orthodontic alternatives fostered the utilization of clear aligners in recent years. However, the efficacy of clear aligners for treating complex malocclusions is often treated with scepticism. This case series aims to evaluate the predictability of the Invisalign® First system in moderate and severe cases requiring interceptive orthodontic treatments in mixed dentition. A total of 23 patients with 102 interceptive orthodontic malocclusion traits were selected for orthodontic treatment with Invisalign® First and were examined over 18 months (Phase 1). Clinical assessments included ClinCheck® predictions, cephalometric measurements, and measuring tools commonly used to quantify tooth movement. Measurements taken at the beginning and end of the treatment were compared. The complexity degree of each case was established based on the set of problems presented by each patient. All treatment objectives were achieved within 18 months, except for two Class II cases, with 69% of them solved with the first set of aligners. Additional aligners were used in the remaining cases. Even though these 23 cases suggest that the Invisalign® First (Phase 1) may be effective in most interceptive problems, controlled randomized clinical trials are required to evaluate movement predictability and how this relates to the problem complexity and additional aligners required.

Genetic overlap between temporomandibular disorders and primary headaches: A systematic review.

Primary headache disorders (PHD), specifically migraine, are strongly associated with temporomandibular disorders (TMD), sharing some patterns of orofacial pain. Both disorders have significant genetic contributions already studied. PRISMA guidelines were followed to conduct this systematic review, which comprehensively summarize and discuss the genetic overlap between TMD and PHD to aid future research in potential therapy targets. This review included eight original articles published between 2015 and 2020, written in English and related to either TMD and/or PHD. The genes simultaneously assessed in PHD and TMD studies were COMT, MTHFR, and ESR1. COMT was proved to play a critical role in TMD pathogenesis, as all studies have concluded about its impact on the occurrence of the disease, although no association with PHD was found. No proof on the impact of MTHFR gene regulation on either TMD or PHD was found. The most robust results are concerning the ESR1 gene, which is present in the genetic profile of both clinical conditions. This novel systematic review highlights not only the need for a clear understanding of the role of ESR1 and COMT genes in pain pathogenesis, but it also evaluates their potential as a promising therapeutic target to treat both pathologies.

Human Interleukin-1ß Profile and Self-Reported Pain Monitoring Using Clear Aligners with or without Acceleration Techniques: A Case Report and Investigational Study.

Introduction: There is a growing demand for more aesthetic, comfortable, and faster orthodontic treatments, and clear aligners emerged as a solution to fulfill this need. However, the effectiveness of clear aligners to treat complex malocclusions is yet contentious. The use of acceleration methods could improve the efficacy of clear aligners by stimulating cells' mechanobiology through numerous pathways, but this hypothesis is still poorly explored. Objective: We aimed to monitor the release profile of an inflammatory marker-the interleukin-1ß-and to evaluate its relationship with self-reported pain scores with and without the use of acceleration techniques during an orthodontic treatment requiring difficult tooth movements with clear aligners. Case Report. Here, we report a case of a 46-year-old female patient who presented functional and aesthetic complaints. Intraoral examination revealed a diminished overjet and overbite, rotation of teeth 45 and 24, absence of teeth 25, 35, and 36, buccolingual dislocation of tooth 21, a tendency to a Class III malocclusion, and a 2 mm left deviation of the lower midline. This study is divided into three stimulation phases: no stimulation, mechanical vibration stimulation, and photobiomodulation. Interleukin-1ß levels in gingival crevicular fluid samples from the pressure side of six selected teeth were evaluated at four time points after the orthodontic treatment onset. Pain monitoring in those teeth was performed using a visual analogue scale at the same time points. Results: Interleukin-1ß protein production peaked 24 h after treatment onset. Complex movements were associated with increased self-reported pain. Conclusion: Clear aligners show limitations in solving complex tooth movements, even when combined with acceleration. The development of customized and programmable stimulation microdevices integrated into "smart aligners," which could be designed to specifically stimulate the direction of movement and stimulation parameters and could constitute a solution to optimize the orthodontic tooth movement with clear aligners.

Harmful behaviour through plasmid transfer: a successful evolutionary strategy of bacteria harbouring conjugative plasmids.

Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a 'weapon' to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Bacterial persistence is essential for susceptible cell survival in indirect resistance, mainly for lower cell densities.

Antibiotic-susceptible bacteria may survive bactericidal antibiotics if other co-inhabiting bacteria detoxify the medium through antibiotic degradation or modification, a phenomenon denominated as indirect resistance. However, it is unclear how susceptible cells survive while the medium is still toxic. One explanation relies on the speed of detoxification, and another, non-exclusive explanation, relies on persistence, a state of bacterial dormancy where cells with low metabolic activity and growth rates are phenotypically tolerant to antibiotics and other cytotoxic substances. Here we simulated the fate of susceptible cells in laboratory experiments in the context of indirect resistance to understand whether persistence is necessary to explain the survival of susceptible cells. Depending on the strain and experimental conditions, the decay of persister populations may follow an exponential or a power-law distribution. Therefore, we studied the impact of both distributions in the simulations. Moreover, we studied the impact of considering that persister cells have a mechanism to sense the presence of a toxic substance-a mechanism that would enable cells to leave the dormant state when the medium becomes nontoxic. The simulations show that surviving susceptible cells under indirect resistance may originate both from persister and non-persister populations if the density of detoxifying cells is high. However, persistence was necessary when the initial density of detoxifying cells was low, although persister cells remained in that dormancy state for just a few hours. Finally, the results of our simulations are consistent both with exponential and power-law decay of the persistence population. Whether indirect resistance involves persistence should impact antibiotic treatments.

Multi-mechanical waves against Alzheimer's disease pathology: a systematic review.

Alzheimer's disease (AD) is the most common cause of dementia, affecting approximately 40 million people worldwide. The ineffectiveness of the available pharmacological treatments against AD has fostered researchers to focus on alternative strategies to overcome this challenge. Mechanical vibrations delivered in different stimulation modes have been associated with marked improvements in cognitive and physical performance in both demented and non-demented elderly. Some of the mechanical-based stimulation modalities in efforts are earlier whole-body vibration, transcranial ultrasound stimulation with microbubble injection, and more recently, auditory stimulation. However, there is a huge variety of treatment specifications, and in many cases, conflicting results are reported. In this review, a search on Scopus, PubMed, and Web of Science databases was performed, resulting in 37 papers . These studies suggest that mechanical vibrations delivered through different stimulation modes are effective in attenuating many parameters of AD pathology including functional connectivity and neuronal circuit integrity deficits in the brains of AD patients, as well as in subjects with cognitive decline and non-demented older adults. Despite the evolving preclinical and clinical evidence on these therapeutic modalities, their translation into clinical practice is not consolidated yet. Thus, this comprehensive and critical systematic review aims to address the most important gaps in the reviewed protocols and propose optimal regimens for future clinical application.

The Perfect Condition for the Rising of Superbugs: Person-to-Person Contact and Antibiotic Use Are the Key Factors Responsible for the Positive Correlation between Antibiotic Resistance Gene Diversity and Virulence Gene Diversity in Human Metagenomes.

Human metagenomes with a high diversity of virulence genes tend to have a high diversity of antibiotic-resistance genes and vice-versa. To understand this positive correlation, we simulated the transfer of these genes and bacterial pathogens in a community of interacting people that take antibiotics when infected by pathogens. Simulations show that people with higher diversity of virulence and resistance genes took antibiotics long ago, not recently. On the other extreme, we find people with low diversity of both gene types because they took antibiotics recently-while antibiotics select specific resistance genes, they also decrease gene diversity by eliminating bacteria. In general, the diversity of virulence and resistance genes becomes positively correlated whenever the transmission probability between people is higher than the probability of losing resistance genes. The positive correlation holds even under changes of several variables, such as the relative or total diversity of virulence and resistance genes, the contamination probability between individuals, the loss rate of resistance genes, or the social network type. Because the loss rate of resistance genes may be shallow, we conclude that the transmission between people and antibiotic usage are the leading causes for the positive correlation between virulence and antibiotic-resistance genes.

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor.

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), we transplanted the B. subtilis FBP-binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

Activation of extracorporeal membrane oxygenation: a therapeutic approach to be considered. / Ativação de oxigenação por membrana extracorpórea: uma atitude terapêutica a ponderar.

OBJECTIVE: To describe the epidemiological profile of victims of cardiac arrest assisted using a nontransporting emergency medical service vehicle and to determine whether these patients met the criteria for the use of extracorporeal membrane oxygenation. METHODS: This study employed a retrospective, cohort, descriptive, and exploratory design. Data were collected in January 2018 in northern Portugal by consulting the records of nontransporting emergency medical service vehicles that provided assistance between 2012 and 2016. An observation grid was prepared that was supported by the instrument used for collecting data from the national registry of out-ofhospital cardiac arrests. RESULTS: After applying the inclusion criteria, the sample consisted of 36 victims. Extracorporeal membrane oxygenation could have been applied to 24 victims during the period analyzed, which might have increased the odds for transplantation, survival, or both, for either the victim or other individuals. CONCLUSION: Nontransporting emergency medical service vehicles have the potential for inclusion in the extracorporeal membrane oxygenation network of the study area.

OBJETIVO: Descrever o perfil epidemiológico das vítimas assistidas pela Viatura Médica de Emergência e Reanimação em paragem cardiorrespiratória e refletir se tinham critérios para utilizar a oxigenação por membrana extracorpórea. MÉTODOS: Estudo retrospectivo, de coorte, descritivo e exploratório. A colheita de dados foi efetuada durante o mês de janeiro de 2018, na região Norte de Portugal, por meio da consulta à base de registos da Viatura Médica de Emergência e Reanimação sobre assistências prestadas no período de 2012 a 2016. Foi elaborada uma grelha de observação suportada pelo instrumento utilizado para a colheita de dados do registo nacional de paragem cardiorrespiratória pré-hospitalar. RESULTADOS: Após aplicar critérios de inclusão, a amostra foi composta por 36 vítimas. Verificou-se que a oxigenação por membrana extracorpórea poderia ter sido aplicada a 24 vítimas no período balizado da colheita de dados, o que resultaria em várias possibilidades de transplantação e/ou sobrevivência, quer da própria vítima quer de outras vidas. CONCLUSÃO: A Viatura Médica de Emergência e Reanimação tem potencial para ser incluída na rede oxigenação por membrana extracorpórea da área em estudo.

Ativação de oxigenação por membrana extracorpórea: uma atitude terapêutica a ponderar / Activation of extracorporeal membrane oxygenation: a therapeutic approach to be considered

RESUMO Objetivo: Descrever o perfil epidemiológico das vítimas assistidas pela Viatura Médica de Emergência e Reanimação em paragem cardiorrespiratória e refletir se tinham critérios para utilizar a oxigenação por membrana extracorpórea. Métodos: Estudo retrospectivo, de coorte, descritivo e exploratório. A colheita de dados foi efetuada durante o mês de janeiro de 2018, na região Norte de Portugal, por meio da consulta à base de registos da Viatura Médica de Emergência e Reanimação sobre assistências prestadas no período de 2012 a 2016. Foi elaborada uma grelha de observação suportada pelo instrumento utilizado para a colheita de dados do registo nacional de paragem cardiorrespiratória pré-hospitalar. Resultados: Após aplicar critérios de inclusão, a amostra foi composta por 36 vítimas. Verificou-se que a oxigenação por membrana extracorpórea poderia ter sido aplicada a 24 vítimas no período balizado da colheita de dados, o que resultaria em várias possibilidades de transplantação e/ou sobrevivência, quer da própria vítima quer de outras vidas. Conclusão: A Viatura Médica de Emergência e Reanimação tem potencial para ser incluída na rede oxigenação por membrana extracorpórea da área em estudo.

ABSTRACT Objective: To describe the epidemiological profile of victims of cardiac arrest assisted using a nontransporting emergency medical service vehicle and to determine whether these patients met the criteria for the use of extracorporeal membrane oxygenation. Methods: This study employed a retrospective, cohort, descriptive, and exploratory design. Data were collected in January 2018 in northern Portugal by consulting the records of nontransporting emergency medical service vehicles that provided assistance between 2012 and 2016. An observation grid was prepared that was supported by the instrument used for collecting data from the national registry of out-ofhospital cardiac arrests. Results: After applying the inclusion criteria, the sample consisted of 36 victims. Extracorporeal membrane oxygenation could have been applied to 24 victims during the period analyzed, which might have increased the odds for transplantation, survival, or both, for either the victim or other individuals. Conclusion: Nontransporting emergency medical service vehicles have the potential for inclusion in the extracorporeal membrane oxygenation network of the study area.

The role of host cell physiology in the productivity of the baculovirus-insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines.

The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five cells. This analysis was conducted in comparison with Sf9 cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five cells. High Five cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 cells, in which the glucose carbon incorporation in the TCA cycle was significantly higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc.

Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles.

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.

Targeted supplementation design for improved production and quality of enveloped viral particles in insect cell-baculovirus expression system.

The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts.