Ingredients (n = 5887) were classified according to the extension and purpose of their industrial processing. The recipes’ titles were content analyzed to identify the health-related words and phrases used. Recipes contained ultra-processed foods and not enough unprocessed or minimally processed foods, such as legumes , nuts and seeds , and fruits . The recipes’ titles mentioned 564 health-related terms, appealing to physical characteristics, including weight loss, and fads, such as gluten-free, dukan, low-carb, detox, fitness, ripped body, and belly burner. Therefore, the ‘healthy’ recipes available on the Brazilian recipe-sharing website presented many aspects not in accordance with national dietary guidelines.Cooking is crucial to the achievement of healthy eating habits, and the internet, as host of culinary recipes websites, is a medium for the dissemination of cooking-related content. Research has revealed that most recipes available on internet sites do not have healthy characteristics when compared to recommendations for healthy eating, even the ones promoted as ‘healthy’. This study investigated culinary recipes available on the ‘healthy eating’ section of a popular Brazilian recipe-sharing website. Recipes ( Food is one of the basic requirements for the promotion and protection of health, acting as a determining factor in the prevention of chronic non-communicable diseases . The firConversely, a healthy diet is one based on unprocessed or minimally processed foods and on the culinary preparations they are made with, seasoned and cooked in moderation with the addition of processed culinary ingredients, such as salt, sugar, and fats ,3. Therehow to cook dishes can be learned and transmitted amongst people [Specifically, the Dietary Guidelines for the Brazilian Population recommend looking up recipes on the internet as one way to develop cooking skills . Culinart people , and thet people ,16. The t people , who spet people .Some of the biggest recipe websites on the World Wide Web contain not only recipes posted by the website designers, but also personal recipes uploaded by subscribed users . ResearcThe use of the internet by different population groups to obtain information on health, food, and nutrition has increased in recent decades ,27,28,29If individuals are to obtain knowledge about cooking that enhances health promotion through healthy eating, arguably the content made available must meet guidelines for healthy eating. The concept of healthy eating, however, is not always clear to individuals and is not understood and interpreted in the same way by all . This caOne of the main challenges in health promotion actions and programs is the development of individuals’ skills to translate dietary information received from a variety of sources into practical information on food selection to achieve healthy eating habits . This isAs part of the food environment, the internet is increasingly identified as an important influencer of food choices. Health problems related to unbalanced diets can be tackled with initiatives that involve the food environment of individuals, but effective and feasible strategies to facilitate the decision making of individuals must come after identifying the contents conveyed in the food environment, whether physical or digital .®, San Bruno, CA, USA) [Studies on the quality of recipes shared on the internet are scarce  and show that most recipes do not have healthy characteristics when compared to recommendations for healthy eating ,43, inclTo our knowledge, this is the first paper that aims to analyze the healthiness of culinary recipes available on the ‘healthy eating’ section of a popular Brazilian recipe-sharing website.® platform. Google® was identified as the most accessed search engine in Brazil, according to Amazon® ranking [n = 13 excluded); being characterized as collaborative, i.e., user-created content (posted recipes), based on their own personal experiences (n = 6 excluded); being solely devoted to sharing recipes and no other type of content (n = 3 excluded); not being sponsored by the food industry (n = 19 excluded); and not being advertised at the top of the search results (n = 8 excluded).For this cross-sectional study, one website specializing in recipe sharing was purposely selected in October 2019, after a systematic search conducted on the Google ranking . Navigat ranking , but exp ranking . We condshared recipe notebook that brought together traditional family recipes and new discoveries in the kitchen”. There are more than 196 thousand recipes available, which are accessed by 30 million individuals monthly and by more than 1 million Brazilians daily [Only one website met the eligibility criteria. TudoGostoso (tudogostoso.com.br) was launched in 2005 and, at the time of the manuscript write-up, claimed to be the largest recipe website in Brazil. The page was created as a collaborative website, a kind of “ns daily .The third author accessed and downloaded all recipes available on the ‘healthy eating’ section of the website until March 2020 as PDF files. Data collection took place from October 2019 to March 2020. A database in Microsoft Excel 2016 was created to register the following information for each recipe: title, link, ingredients, preparation instructions, and preparation time. Only one recipe was excluded from analysis because it was a homemade cough syrup, i.e., not a culinary recipe. Ingredients were gathered from the recipes’ ingredients list and preparation instructions. The final database was composed of 814 recipes.The variables used to characterize the recipes were the recipes’ titles, ingredients, and preparation time. The variables used to assess the recipes’ healthiness were ingredients and preparation instructions.The recipes’ titles and ingredients were submitted to content analysis for the categorization of the type of culinary preparation  (first author). Words and phrases were coded according to similarity, and then categorized into groups. Weak and general categories were regrouped until strong or terminal categories resulted . AnalysiThe recipes’ titles were also content analyzed  to identThe recipes’ ingredients were classified according to the extension and purpose of industrial processing into one of four groups: unprocessed/minimally processed (U/MP); processed culinary ingredient (PCI); processed (P); or ultra-processed foods (UP) ,3,50  were canned foods, breads, and cheese. Foods formulated with the addition of substances not usually present in culinary preparations, such as flavoring agents, dyes, sweeteners, emulsifiers, and other additives, were classified as UP ,3,50.Ingredients that did not have their preparation described in the recipe  but were available for purchase in industrialized versions were classified as P or UP, according to the predominant characteristics of products available in Brazilian retail outlets. In case of no agreement between the first two researchers who conducted the analysis about the extension and purpose of industrial processing, the ingredient was classified in the group of lower processing .The recipes’ ingredients and preparation instructions were assessed with the Qualitative Framework for the Assessment of Culinary Recipes’ Healthiness  . Ingredients not mentioned in the ingredients’ list but cited in the preparation instructions section were included in the analysis. When more than one option of ingredient was mentioned, only the first was considered for analysis . Ingredients cited as ‘optional’ were not assessed. Alcoholic beverages mentioned as ingredients of recipes were excluded from the analysis.Two researchers experienced in the adopted methods were involved in all steps of the analyses. For each analysis, 10% of the recipes from the dataset were randomly selected (randomizer.org (accessed on 18 August 2021)) to be independently checked for agreement (first and second authors). The weighted kappa test of agreement between researchers on the assessment of the ingredients’ extension and purpose of industrial processing was 0.96. On the application of the Qualitative Framework for the Assessment of Culinary Recipes’ Healthiness, agreement ranged between 0.90 and 1.0 (kappa and weighted kappa) between researchers, indicating an almost perfect agreement . DivergeQualitative dichotomous and polytomous variables are presented in absolute and relative frequencies. Quantitative variables are presented as median and interquartile range (IQR), as they were not normally distributed when assessed by the Shapiro–Wilk test . Stata SE version 13.0  was used for the analysis.n = 814). The most frequent category of recipes was ‘cakes and baked goods’ ; appeal to body image and physical exercise ; appeal to the absence or reduction in ingredients or nutrients ; production practices and cooking methods ; vegetarian and vegan (n = 51 codes); whole grain (n = 42 codes); health and nutrition ; appeal to the presence or increase in ingredients or nutrients ; and natural (n = 4 codes).Regarding the recipes’ titles, 564 terms referring to health were coded  and organ = 3673) and PCI . Ingredients classified as P  and UP  were less frequent. The ten most frequent U/MP ingredients in the sample were, in decreasing order of appearance: egg, onion, water, oat, garlic, banana, pepper, tomato, carrot, and cinnamon. The ten most frequent UP ingredients in the sample were, in decreasing order of appearance: artificial sweetener, UHT cream, margarine, bottled coconut milk, cheese spread, whey protein, industrialized tomato sauce, textured soy protein, shoyu sauce, and vanilla essence.From the total 5887 ingredients analyzed, the majority were U/MP , and the use of either lean cuts of meat or eggs . The exclusive use of industrialized seasonings  and of frying as a cooking method , as well as the presence of foods with a high sugar concentration , were not frequent. Negative aspects to be highlighted are the low presence of fruits , legumes , nuts and seeds , and the presence of margarine  in the recipes. The categories that presented the most evenly distributed positive and negative criteria were the presence of vegetables and different types of sauces  in the main recipe category shared (cakes and baked goods). This explains the low presence of foods with a high sugar concentration observed in the analysis with the framework, which, in theory, should be a positive result. Artificial sweeteners were developed by the food industry in the context of a dramatic increase in the incidence of obesity to reduce calorie and, specifically, added sugars intake ,58. HoweThe framework analysis identified the presence of margarine in some recipes. It was also one of the most frequent UP ingredients in the sample. Margarine was developed by the food industry in the 19th century as a cheap substitute for butter, but it was later marketed as an option to lower saturated fat intake because of its vegetable origin . As an UThe common use of artificial sweeteners and margarine by users of the investigated website may indicate that reductionist conceptions—‘butter and sugar are unhealthy’—are still leading to the substitution of processed culinary ingredients by UP foods. If used in moderation and in recipes based on U/MP foods, PCI can indeed be part of a healthy diet ,3,64. AwMany recipes’ titles alluded to health by using words or expressions from diet trends , or appealing to body image and physical exercise . Whey protein was one of the most frequent UP foods present in the sample. This suggests that the individuals posting recipes on the website may associate healthiness to weight loss and physical appearance, which does not resonate with the perspective of national dietary guidelines . BeliefsIt is in this scenario that restrictive diet trends, which have no proven long-term benefits or sustainability ,68,69, cLastly, although U/MP ingredients were highly frequent in the sample analyzed, the application of the framework evidenced that fruits, legumes, nuts, and seeds were rather absent in the recipes. Therefore, some key food groups for healthy eating are not being as valued as other aspects, such as favoring whole cereals and lean cuts of meat. This result mirrors the results of epidemiological studies of Brazilians’ diets, which is reportedly low in fruits, legumes, nuts, and seeds , and may® and not focused on ‘healthy recipes’, also identified the presence of UP foods as ingredients and a scarcity of fruits, legumes, nuts, and seeds [Comparisons with previous research on the healthiness of recipes shared on websites devoted to this are complex, because this is a relatively new field of research with only a few studies published. Additionally, such studies addressed nutrient content to assess recipes’ healthiness , while ond seeds . Our resIn a nutshell, interventions and health education activities are needed to tackle specific points when encouraging individuals to cook and use the internet as a source of information. Firstly, people need to be alerted that even recipes branded as ‘healthy’ are not necessarily following dietary guidelines for healthy eating. Secondly, it is important to promote cooking in a healthy way, without the addition of UP foods as ingredients to  enhance flavor or as shortcuts to shorten preparation time. It is also relevant to demonstrate how it is possible to incorporate more fruits, legumes, nuts, and seeds in recipes to improve their quality. As stated earlier, a negative criterion was given with the aim of raising awareness about the critical characteristics of recipes, and therefore promote healthier eating habits. Each negative criterion is considered a point of possible improvement through the substitution, addition, or exclusion of ingredients, and/or the alteration of the cooking method. We highlight that to make an original recipe healthier, not all the negatively assessed components should be changed. Finally, as proposed by Marsola et al., it is necessary not only to disseminate information about the composition, degree of processing, and origin of foods, but to take into consideration beliefs and values individuals are giving to healthy eating . In the One of the study’s limitations is the adoption of a conservative criterion to classify ingredients by the extension and purpose of industrial processing. This criterion was used because ingredients were identified from recipes, and not from information on labels. Although minimally employed, this strategy may have led to an underestimation of the number of UP ingredients . Data weThe recipes’ titles appealed to diet trends, body image, and physical exercise. Recipes employed UP foods as ingredients, but the use of fruits, legumes, nuts, and seeds was not encouraged. Therefore, the ‘healthy’ recipes available on the Brazilian recipe-sharing website presented many aspects not in accordance with national dietary guidelines. Findings are relevant to inform health professionals and policymakers to act towards educating individuals on how to choose proper healthy recipes to effectively promote cooking as a healthy eating strategy.


Biofouling is the major factor that limits long-term monitoring studies with automated optical instruments. Protection of the sensing areas, surfaces, and structural housing of the sensors must be considered to deliver reliable data without the need for cleaning or maintenance. In this work, we present the design and field validation of different techniques for biofouling protection based on different housing materials, biocides, and transparent coatings. Six optical turbidity probes were built using polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), PLA with copper filament, ABS coated with PDMS, ABS coated with epoxy and ABS assembled with a system for in situ chlorine production. The probes were deployed in the sea for 48 days and their anti-biofouling efficiency was evaluated using the results of the field experiment, visual inspections, and calibration signal loss after the tests. The PLA and ABS were used as samplers without fouling protection. The probe with chlorine production outperformed the other techniques, providing reliable data during the in situ experiment. The copper probe had lower performance but still retarded the biological growth. The techniques based on transparent coatings, epoxy, and PDMS did not prevent biofilm formation and suffered mostly from micro-biofouling. Adsorption of organic and inorganic macromolecules immediately after immersion, forming the primary film.Transport of microbial cells to the surface and the immobilization of bacteria on the surface.Bacterial attachment to the substratum through extracellular polymer production, forming a microbial film on the surface.Development of a more complex community with the presence of multicellular species, microalgae, debris, sediments, etc., on the surface.Attachment of macroalgae and marine invertebrates such as barnacles or mussels.Biofouling, or biological fouling, is the accumulation of biological material from molecules to metazoans on surfaces. Soon after a structure is immersed in water  it is gradually covered by organisms which may compromise the operation of the devices. In some cases, this biofouling formation is virtually instantaneous . These oHowever, the fouling formation is not standard, but a combination of different physical, chemical, and biological factors. The water properties , hydraulic conditions, depth, season, and local fauna and flora species composition play a major role in the fouling development. Thus, while the formation of biofouling in the surfaces is certain, its development is very difficult to predict .Biofouling is of extreme importance for monitoring instruments. Even a small amount of biofilm in the sensing area can produce undesired interferences in the readings of the sensor, which can no longer provide reliable information. These interferences can result from the clogging of sections, membranes, or filters in mechanical sensors ; contamination in chemical sensors ; increasing resistance to heat exchange in thermal sensors; increasing of acoustic absorption and consequent decrease in receiving power in acoustic sensors; and increasing of optical absorption and consequent decrease in receiving power in optical sensors ,9,10,11.Additional problems associated with biofouling emerge in the case of marine optical sensors where high optical transparency is required. Before deployment, calibrations are needed for the good operation of most monitoring instruments. Long-term monitoring can result in biological attachments in the sensor surface that may require hard mechanical cleaning methods, such as high-pressure water jets or brushes, or chemical methods. These methods can modify the initial status of the sensitive sensor area, and consequently, make the comparison of the metrological response of the sensor before and after the deployment difficult. Moreover, bad cleaning will have the same result .Some commercial anti-fouling techniques are available and applied to oceanographic instruments. The most known are the biocide generation systems based on copper corrosion mechanisms or TBT (tributyl tin) leaching . Even thNevertheless, copper biocide properties can still be used to protect the monitoring instruments. This protection, usually called “copper shutter”, uses a copper housing with a mechanism with a motor driver and a shutter that only opens when taking measurements. The sensor is kept inside the copper shutter that remains closed while the sensor is not taking measurements, in the darkness, and allows biocide concentration to increase, thus preventing biofilm formation .Another mechanical anti-fouling technique, and the most common in use for commercial instruments, is the use of wipers or scrapers in the sensing area ,17. ThisNew technologies have been presented in the recent literature to reduce the impact of biofilm, as also ways of controlling its presence on the surface of sensors. Different active techniques based on ultraviolet ,19,20, lAnother active technique is based on the use of chlorine, both by in situ production by salty water electrolysis or by bleach injection ,30. PrimFinally, nano-coatings of the glass surface, coatings of the sensing area of the sensors or bio-mimetics shapes and materials are emerging techniques that have shown their potential mostly in laboratory experiments ,35,36,37Even though biofouling is far from being a solved problem, many techniques have been presented in the last decades to extend the deployment time of these instruments. However, many of them are also still restricted to laboratory conditions and sea truth validation of their reliability is still needed.We have been developing several cost-effective oceanographic optical instruments for in situ continuous monitoring ,39,40,41Sensors that make use of optical techniques are usually more susceptible to biofouling, decreasing their sensitivity and reliable lifetime. Even a very thin biofilm on the surface of the optical regions may produce interference in the measurements due to absorption and/or total or partial light scatter. If we consider that biofouling starts its formation the moment that the instrument is submerged, optical sensors can have a very short time of reliable operation.We presented an optical sensor to measure turbidity and suspended particulate matter using transmitted light, backscattering, and nephelometric light techniques . For theTo reduce the complexity of the biofouling effects in the measurements, we narrowed the problem to the use of just the transmitted light technique, using infrared wavelength, as used by many optical oceanographic sensors.Six probes were built using different anti-biofouling techniques or housing properties. The probes were designed in a tube shape with an optical measuring area in its inside. This shape was chosen to create a sensing area with low fluid flow and turbulence so that some of the techniques  can be more effective rather than in an open design, unprotected from the normal stream flow.Each one of the probes uses an infrared 980 nm light emitting diode (LED) and one infrared 980 nm phototransistor that measures light at 180° to the LED (transmitted light detection technique). The sensor housing presented in the image above was used as standard and was slightly changed to adapt to the different anti-biofouling techniques tested. These techniques can be divided into four different groups: structural material, copper biocide, transparent coating, and chlorine production.The characteristics of the surface which houses the sensor play a major role in the attachment of biofilm. Smooth faces are less susceptible to attaching biological organisms when compared to rough surfaces . In addHowever, the most effective materials against biofouling are also more expensive. Since the main objective of this work is to test the application of anti-biofouling techniques in low-cost instruments, we tested the two cheapest and wider in-use 3D printing materials: PLA and ABS.It is important to notice that the PLA and ABS probes are not anti-biofouling techniques but rather the use of different structural materials. In both probes, the optical transducer surfaces are in contact with the water without any protection. However, both ABS and PLA materials are often catalyzed with organotin which can leach out and act as antifoulants ,44.The performance of ABS and PLA probes in the field is not expected to differ significantly, which would allow their use as control samples to be compared with the other techniques.The PLA and ABS probes are shown in This technique, PLA with copper filament (FlashForge PLA Copper 1.75 mm), is also based on 3D printed material for the structural housing. As before, the surfaces of the optical transducers are in contact with water. However, as discussed in The advantage of the use of this technique is that it is easy to apply  and it is still a cost-effective material. The main disadvantages are that the copper protection has a limited time since it loses its biocide properties over time and, as discussed before, the biocide is not environmentally friendly.The PLA with copper filament probe is presented in ® 184 Silicone Elastomer Kit). Both probes were built in ABS and the optical transducers were coated with transparent materials. The idea of using both PDMS and epoxy as anti-biofouling coatings has been presented before [Contrasting with the previous techniques, the coating techniques are used to protect the transducer surfaces, which are no longer in contact with water. In this technique, the formation of biofilm will happen on the surface of the coating that is supposed to have better anti-biofouling properties. However, optical sensors present an enhanced challenge since the coatings must ensure the passage of light. Attending to this need, we selected two easy-to-apply transparent materials: transparent epoxy resin (HB EPOSURF2—HBQUIMICA) and polydimethylsiloxane , dimensionally stable anodes (DSAs) of a Ti-support coated by noble metal oxides such as ruthenium, iridium, tantalum, zirconium, and doped diamond electrodes. Although this technique is very effective to produce chlorine biocide, the opacity of the electrodes has always been an obstacle to its application in optical instruments.2 area. The production was set to be on all the time and only turns off when the sensor is collecting data. With this configuration, the sensor is achieving a production rate of approximately 4 mg of chlorine per hour.We presented a new approach to chlorine biocide production combining the advantageous features of the platinum material with transparent conductive oxides ,51. In tThe main disadvantage of this technique is the need for electric power which reduces the operation time of the sensors. However, it allows controlling the amount of chlorine biocide to the minimum required to avoid biofouling, maintaining the sensor area cleaned without negative impact on the marine environment. This technique is only suited for marine environments since it does not work in freshwater.The chlorine production sensor probe is presented in The six probes were assembled on one board with a minimum distance of 1 cm from each other. The probes based on biocides production are not expected to affect other probes through the spread of the biocides due to the confined shape of the probes. As demonstrated by Pinto et al. , chlorinBefore deployment at sea, the probes were calibrated to correlate the electrical output to the corresponding value of turbidity. As in previous work from the authors , a standAn initial solution of 4000 NTU was diluted in distilled water to obtain solutions with lower turbidity values. The ratio proportion of the initial and final solutions is given by the following equation:The probes were submerged in a container with different turbidity solutions. Different dilution factors were used to calibrate the sensors for 1000, 500, 250, 125, 62, 31, 15, 7.5, and 3.8 NTU.Each one of the curves was used to convert the electrical output of the probes to turbidity values during the in situ test. After the experiment and cleaning of the sensors, a new calibration was conducted to check if the probes kept the same output as before the experiment, or if there was a decrease in the signal resulting from the biofouling.−2 d−1) [The probes were deployed, from 23 May to 9 July 2022, in the dock of the Marine Biological station of the University of Vigo, located at Toralla Island, Ria of Vigo, Spain (42°12′07.1″ N 8°47′54.6″ W). The Ria of Vigo is a highly productive coastal ecosystem (primary production rates of up to 3 g cm−2 d−1) , charactThe instrument was moored attached to a post of the floating dock, at 1.5 m depth from the surface, with the opening of the probes faced to the sea bottom (less influence of the daylight in the measurements). This location has a depth variability of 5 to 7 m during tides.The probes were connected by an electric cable, that shares power and communications, to a data logger that recorded the measurements of the six anti-biofouling techniques with a sample rate of one reading per hour. The whole system was supplied by the electrical grid available on the dock.Two days before the end of the in situ experiment, the probes were taken out of the water for soft cleaning. This cleaning process, referred to as in situ cleaning hereafter, consisted of the removal of the macro biofouling attached to the sensors and the cleaning of the inside of the probes with fresh water and a soft cloth. After the in situ cleaning, the probes were deployed again for two more days to confirm the results.After the in situ experiment, the sensors were taken to the laboratory for hard cleaning. The probes were submerged for five days in a water tank with a high concentration of chlorine. This process killed and detached the algae from the instrument, leaving just the cirripeds, which were then removed. Finally, and with the probes clean of macro biofouling, the inside of the probes was cleaned with fresh water and a soft cloth.The efficacy of the developed techniques was evaluated by three different metrics: the analysis of the turbidity measurements of the probes during the in situ experiment; visual inspections of the probes; and the comparison of calibrations before and after the in situ test.The ABS and PLA, which were not expected to provide biofouling protection, took about five days to present a significant drift in the signal. The turbidity measurement delivered by the PLA probe increased during the experiment, a signal of biofouling interference. For the ABS, turbidity increased till the 28 July and then the values decreased to about 100 NTU, which suggests that macro-fouling attached to the sensor, for example, algae, blocked the passage of light in the sensing area of the sensor (turbidity increases). Measurement gets back to normal values once algae are detached . Previous field tests conducted with similar optical instruments ,39,40,41The PLA with copper filament presents a significant drift from the 8 June. Compared with the ABS and PLA probes, which also have optical transducers in contact with water, the releasing of copper biocide into the water seems to increase the operation time of the sensor without interferences of biofouling. While this technique was also affected by biofouling, the measurements are more constant when compared to the other material techniques.The probes with epoxy and PDMS coatings did not demonstrate effectiveness against biofouling. The epoxy measurements started with values of 56 NTU and in the first 3 days of the experiment, it increased to values around 100 NTU. From the beginning of the experiment to the 29 June, this probe produced measurements of around 100 NTU but showed a high variability (about 40 NTU in amplitude). From this day to the in situ cleaning, the sensor registered an increase in the turbidity values, probably caused by macro fouling.The PDMS probe produced very similar results. In the first 3 days of the experiments, the turbidity measurements increased from 50 NTU to 150 NTU, and this value slowly increased to around 200 NTU on the 30 June. As for the epoxy technique, the recorded turbidity measurements increased the days before in situ cleaning.The last technique, chlorine production, produced the best results among the six probes. Since the first day of the experiment, the probe presented values of 50 NTU with a maximum 5 NTU variation in its measurements. On the 5 July, an increase in the turbidity values was observed, probably associated with the beginning of the drift. The measurements produced by this probe are the expected without biofouling interference.On 7 July, the probes were cleaned and deployed again on the water to check if the in situ cleaning process was sufficient to eliminate the biofouling interference. Good results were obtained for the PLA, ABS, and chlorine probes since the turbidity measurements after the cleaning dropped to similar values at the beginning of the experiment.For the epoxy and PDMS techniques, the cleaning also produced positive effects decreasing the recorded turbidity values. However, these values were still higher than the 50 NTU at the beginning of the tests, which indicates that the micro-biofouling was not eliminated.Finally, the cleaning did not produce any effect on the copper probe and the turbidity values of the measurements kept increasing till the end of the experiment.The objective of the visual inspections was to provide qualitative comparisons between the probes with respect to the quantity of biofouling at different stages of the experiment (mostly macro-biofouling).Photographs of each probe were taken before the in situ test, before and after the in situ cleaning on the 7 July and after the hard cleaning when the experiment ended. When the instrument was taken out of the water on the 7 July for the in situ cleaning, the external housing of all probes was covered with macro-fouling, namely algae, barnacles, and some small mussels. However, there was a visible difference in the amount of macro-biofouling between the outside and the inside (sensing area) of the probes. The inside of the probes had fewer traces of biofouling, even for the PLA and ABS substrates, probably because the instrument was deployed facing the seafloor and the inside of the probes was protected from direct sunlight, which contributes to the biological growth.The in situ cleaning procedure removed the algae, and the interior of the probes was cleaned with fresh water and a soft cloth. The observation of the probes after the in situ cleaning . However, the sensing areas were clean and in good condition. After the hard cleaning, all the probes had similar inside conditions as before the experiment, except for the PDMS coating, which was not as transparent as before, presenting a dark blur.It is important to notice that both coating techniques (PDMS and epoxy) and the chlorine production probe did not have any trace of macro-fouling in their surfaces at any stage of the visual inspections. Additionally, the copper technique was the probe that visually had fewer biofouling traces on its inside. However, during the hard cleaning, we noticed that a barnacle was growing on the surface of the LED, which was probably the reason why the in situ cleaning did not result in lower turbidity values (see data presented in the previous sub-section).After the in situ test and the hard cleaning, the sensors were calibrated again to check if the probes kept the same output as before the field experiment. The PLA, chlorine, and copper probes presented the lowest performance loss from the first to the second calibration. The turbidity measurements using the formazine solutions in the second calibration were practically the same as for the first one. The ABS probe presented an average relative performance of 96%, which is lower than the previous ones, but still acceptable.The coating materials are the techniques that presented lower performances, displaying the PDMS with the worst results with a relative performance of only 59%. This result is consistent with the visual inspections after the hard cleaning when it was noticed that this coating was with a dark blur on its surface. Even with the cleaning process, these techniques did not present similar calibration results as before the field test.Considering the three metrics used to evaluate the operation of the six anti-biofouling techniques, we conclude that chlorine production outperformed the other tested techniques. The measurements carried out during the field test presented a signal without drift and the interference of biofouling was only detected after 41 days of testing, during the visual inspections the inside of the probe always appeared clean, and the calibration of the probe did not show changes after the deployment at sea.The PLA with copper filament also produced satisfactory results in the field tests. Compared with the other techniques (except the chlorine), the drift originated by biofouling interference was detected later, and the probe provided 15 days of reliable data. This was the only technique where the in situ cleaning did not have positive effects, possibly due to the growth of barnacles on the sensors LED surface that was only detected during the hard cleaning. This is one disadvantage of this technique, since the copper biocide, although satisfactorily protected the probe housing , failed to protect the transducer surface.The results for the other four probes did not provide insights into good protection against biofouling. During the field tests, the epoxy and PDMS presented biofouling interference from the day of deployment. However, both stabilized on 100 NTU and 150 NTU, respectively, till almost the end of the experiment. Even the in situ cleaning was not sufficient to obtain turbidity measurements similar to those of the first deployment day. In the second calibration, these techniques presented the worst results, with the PDMS having a significant signal loss resulting from the blur of its coatings. All these factors suggest that these coatings are very susceptible to micro-biofouling, even to an irreversible state.Finally, the PLA and ABS substrates, which were used for comparison with the other techniques and were not as effective anti-biofouling protections, presented the worst results during the field tests. The PLA probe produced satisfactory measurements till the 5th of June, but after that day the drift in the measures increased significantly. For the ABS probe, the drift was visible since the day of deployment, and contrary to the epoxy and PDMS that suffered mostly from micro-biofouling, macro-biofouling interference was constant throughout the experiment. On the positive side, the in situ cleaning provided good results and the hard cleaning left the probes in the same conditions as before the experiment.The results obtained in this investigation are consistent with those theoretically expected. Chlorine is a strong oxidant that destroys primary biofilms and microbes, and propagules of macro-fouling. The anti-fouling efficiency against the primary film is crucial for a long-term operation without biofouling interference in the measurements. The other techniques tested, based on the release of organotin and toxic compounds into the water, may also be harmful to macro-fouling organisms but are not as effective as chlorine biocide against biofilm formation. The anti-fouling efficiency of copper biocide has been previously validated, as shown in the scientific literature and, consequently, satisfactory results would be expected from this experiment.In this work, we present the design and field experiment of different anti-biofouling techniques to be applied to optical instruments. Six different probes were built based on different materials: the housing of the probes in PLA and ABS; copper biocide due to the housing of the probe in PLA with copper filament; chlorine biocide production by the electrolysis of seawater; and transparent coatings using epoxy and PDMS.All the probes were calibrated before and after the field experiment using formazine solutions. The system was deployed in a coastal area with low sedimentary dynamics from the 23 May 2022 to the 9 June 2022 with the probes taking measurements with a sample rate of one hour. Different procedures for probe cleaning were used during and after the field experiment.The effectiveness of the tested techniques was evaluated by three different metrics: the monitoring results during the field test, visual inspections of the probes during and after the experiment, and the relative signal loss of the calibrations before and after the test.The chlorine production technique outperformed the other ones, producing reliable measurements along the experiment, and the results show effective protection against micro and macro-biofouling. The results for the copper biocide were not as good as for the chlorine but also demonstrated to be an effective technique. On the opposite, the PDMS, epoxy, PLA, and ABS did not yield satisfactory results for biofouling protection.Future work should now focus on the chlorine production technique. For this experiment, the chlorine probe was designed to produce seawater electrolysis using 1050 µW of electric power, but similar results might be achieved with lower power. Since power efficiency is a concern for long-term monitoring, the optimal power to protect against biofouling must be addressed. In addition, the copper technique should not be discarded, and other structural housings based on copper materials can be tested.Biofouling is still a problem that affects the performance of optical instruments and has been obstructing long-term monitoring studies with fully automated systems, without the need for pauses in the tests for cleaning, new calibrations, or other maintenance purposes. The scientific community has been focusing attention on this problem and new and innovative technologies have been emerging. However, most of these technologies are still confined to the laboratories and have not been truly applied in real sensors and tested in the field. We believe that experiments like the one we presented must be replicated to fully understand the potential of these emerging technologies, which can only be accomplished when applied to devices measuring physical or biological water parameters and tested in the field.


Varicella zoster virus (VZV) and simian varicella virus (SVV) cause varicella (chickenpox) in children and nonhuman primates, respectively. After resolution of acute disease, the viruses establish latent infection in neural ganglia, after which they may reactivate to cause a secondary disease, such as herpes zoster. SVV infection of nonhuman primates provides a model to investigate VZV pathogenesis and antiviral strategies. The VZV and SVV genomes are similar in size and structure and share 70–75% DNA homology. SVV and VZV DNAs are co-linear in gene arrangement with the exception of the left end of the viral genomes. Viral gene expression is regulated into immediate early, early, and late transcription during in vitro and in vivo infection. During viral latency, VZV and SVV gene expression is limited to transcription of a viral latency-associated transcript (VLT). VZV and SVV are closely related alphaherpesviruses that likely arose from an ancestral varicella virus that evolved through cospeciation into species-specific viruses. Human herpesvirus 3) produces varicella (chickenpox) in children, characterized by vesicular skin lesions over the entire body . Th. Th17]. The SVV genome encodes 73 identified open reading frames (ORFs), of which three  are duplicated within the IRS and TRS . VZV DNAThe major differences in genetic content of SVV and VZV DNA occur at the left ends of the viral genomes . SVV DNAThe SVV left end includes an ORF A that is not found within the VZV genome . The SVVThe SVV ORF B, as originally identified on the SVV genome, is now known to be a homolog of the VZV ORF 0 (or ORF S/L), sharing 35% amino acid identity . The VZVThe SVV ORF C is a more recently identified gene located at the extreme left terminus of the SVV genome . The SVVThe SVV ORF C, SVV ORF 0, and VZV ORF 0 are orthologs of the herpes simplex virus (HSV) unique long ORF 56 (UL56) gene family, which is conserved among alphaherpesviruses . These UInitial transcript mapping of the SVV and VZV genomes during lytic infection employed Northern blot analysis and labeled restriction endonuclease DNA fragment probes ,29. ThesMost recently, RNA sequencing approaches  have provided comprehensive analyses of the SVV and VZV transcriptomes ,33. The Similar to other herpesviruses, the SVV and VZV genomes are coordinately expressed into kinetic stages. While the cell-associated nature of SVV and VZV complicates the generation of high-titer viral stocks needed for synchronous in vitro infection, studies confirm that the viruses express IE, early, and late gene products.IE gene expression does not require prior viral protein synthesis and generates regulatory proteins that transactivate the promoters of viral genes. SVV and VZV DNAs have been predicted to express four IE genes, ORFs 4, 61, 62, and 63, based upon homology to the HSV-1 IE genes. The IE62, encoded by ORFs 62 and 71, is considered as the major viral transactivator of IE, early, and late gene promoters . The SVVThe SVV and VZV ORF 61 encode a homolog to the HSV-1 ICP0 transactivator protein. The SVV IE61 transactivates its own promoter as well as IE, early, and late gene promoters . The proStudies of the SVV and VZV ORF 63 gene products have shown differential effects on the regulation of viral promoters. While the SVV 63 protein did not transactivate the ORF 21 early gene promoter by itself, the SVV 63 upregulated SVV 62 transactivation of the ORF 21 promoter in neuronal cells . In contThe SVV ORF 4 protein only weakly transactivated an early promoter but stimulated IE62 transactivation of the same promoter . As indiHerpesvirus early gene products include enzymes involved in DNA synthesis. Several SVV and VZV early genes have been characterized including the deoxyuridine nucleotidohydrolase , uracil glycosylase (ORF 59), and thymidine kinase (ORF 36), which express enzymes involved in viral DNA replication and repair ,44,45,46Herpesvirus late genes encode structural proteins including the viral capsid and envelope glycoproteins. Several VZV ORFs encode proteins involved in the structure and assembly process of viral capsid formation . While SThe development of RNA-Seq and dRNA-Seq methodology has permitted low titer, cell-free virus synchronous infection to analyze the kinetics of VZV transcription and gene expression . VZV-infFollowing primary varicella infection, SVV and VZV establish life-long latency within cells of neural ganglia ,51. InduSVV gene expression in neural ganglia of latently infected monkeys is restricted to a viral latency-associated transcript (VLT) . This trSubsequently, RNA-Seq was employed to reveal a VZV VLT in human trigeminal ganglia . The VZVRNA-Seq analysis has also provided a detailed characterization of the SVV VLT . SimilarExpression of the SVV and VZV VLT oriented antisense to the ORF 61 and HSV-1 ICP0 homolog is consistent with gene expression during latency of other alphaherpesviruses . HSV-1, The role of the SVV and VZV VLT in viral latency and reactivation is not understood. A SVV ORF 61 deletion mutant, with a corresponding disrupted VLT, produced clinical varicella in rhesus macaques and established latent infection in neural ganglia . In addiHerpesviridae family an estimated 400 million years ago (mya) [Alphaherpesvirinae assembled based upon alignment of amino acid sequences for six conserved genes among alpha-, beta-, and gammaherpesviruses [simplexvirus , and simian agent 8 (SA8)), and varicellovirus  genera [A primordial herpesvirus infecting ancestral vertebrates gave rise to the go (mya) ,59. Figu) genera .An ancestral varicella virus may have arisen in an early African primate around 70 mya . Fossil VZV evolution continues as evident by the current existence of five major phylogeographical clades, which are differentiated by single-nucleotide polymorphisms (SNP) . Clades The SVV deltaherpesvirus (DHV) is currently the only SVV isolate that has been completely sequenced and annotated . DHV was


Immune checkpoint inhibitors (ICIs) are changing all aspects of malignant tumour therapy as an immunotherapy subverter in oncology. However, the current ICIs might induce systemic immune activation in other tissues and organs since they are not tumour-specific, causing the immune system to attack some normal tissues and organs of the human body. The toxicity can also amplify greatly although combined immunotherapy for cancer has increased the curative efficacy. The LC4 peptide was modified to improve its tumour-targeting ability and reduce peripheral immune system activation, which was obtained through phage display peptide library screening and could block the CTLA-4/CD80 interaction. The LC4 peptide as a result, like other ICIs, exerts anti-tumour effects by refreshing T cell function, and also activates the peripheral immune system. We used the PLGLAG peptide as a linker at the C-terminal of LC4 to connect with a tumour-targeting peptide RGD to increase the tumour tissue targeting ability, and obtain LC4-PLG-RGD. Further experiments demonstrated that the anti-tumour LC4-PLG-RGD activity was better than LC4 in vivo, and the ability to activate the peripheral immune system was weakened. In conclusion, LC4-PLG-RGD can increase the ICIs tumour-targeting and reduce excessive peripheral tissue immune activation, thereby reducing the side effects of ICIs, while increasing their anti-tumour efficacy. This study confirmed that enhanced ICI tumour targeting can effectively reduce immune-related adverse reaction occurrence.The online version contains supplementary material available at 10.1007/s12672-022-00562-6. Immune checkpoint inhibitors (ICIs) have become a central cancer treatment pillar, with almost half of all patients with metastatic cancer in economically developed countries eligible for ICIs treatment . ICIs arImmune checkpoint therapy can inhibit immune checkpoint activity, release immune brakes in the tumour microenvironment, and reactivate T cell immune tumour response, thus achieving anti-tumour effect. Therefore, increasing ICI enrichment in the tumour microenvironment and decreasing peripheral immune system activation is an effective method to realise this idea. It was reported that dual-targeted antibodies improve T lymphocyte infiltration in tumour tissues and thus achieve a stronger anti-tumour effect than single targeted antibodies , while t+ T cells and CD4+T cells, and constitutively expressed on Treg cells [CTLA-4 plays a negative regulatory role in the initial T cell activation stage, and is mainly expressed on activated CD8eg cells . Blockineg cells . The CTLIntegrin is an important cell adhesion receptor, which is highly expressed in tumour vascular endothelial cells and some tumour cells . RGD pepWe first obtained CTLA-4 affinity peptide LC4 by phage display peptide library screening in this study. LC4 peptide has been demonstrated to effectively block CTLA-4 /B7 protein interactions and has good anti-tumour effect in vitro and in vivo. PLGLAG was used to connect LC4 with RGD, a tumour-targeting peptide sequence with high affinity for integrin αvβ3, and obtain a modified peptide LC4-PLG-RGD. LC4-PLG-RGD as a result has better anti-tumour effect, could reduce peripheral immune system activation, and reduce irAEs produced in CTLA-4 treatment.Phage display peptide library technology was used to screen affinity peptides that bind to CTLA-4. Bio-panning five rounds on the hCTLA-4 protein were performed to obtain the hCTLA-4 binding peptides. Only eight peptides were identified and the amino acid sequences with the enriched phage clones were shown in Table The microscale thermophoresis (MST) binding curves of hCTLA-4 binding peptides to hCTLA-4 were shown in Fig. We constructed a CHO-K1 / hCTLA-4 cell line with high CTLA-4 expression to determine whether the affinity peptide specifically binds to CTLA-4 molecules on the cell membrane. CTLA-4 affinity peptides with biotin label were synthesised and co-incubated with CHO-K1/hCTLA-4 cell line. LC4, LC7 and LC8 had affinity with CHO-K1/hCTLA-4 cell line compared with the GA peptide group, which proved that these three peptides could specifically bind with hCTLA-4 at the cellular membrane Fig. .Fig. 2ThTargeting CTLA-4 to treat tumours requires blocking the interaction between CTLA-4 and its CD80/CD86 ligand. The affinity between CTLA-4 and CD80 is higher than that between CTLA-4 and CD86 ), and on+ T cells and CD4+T cells upon activation  for 14 days to identify the in vivo LC4 effect. The results showed that LC4 could significantly inhibit CT26 tumour growth in a dose-dependent manner, but had no significant effect on the weight of mice Fig. A, B.Fig.+ T cell and CD4+ T cell infiltration were detected in tumours. As a result, CD8+ T cell and CD4+T cell infiltration in the LC4 group (4 mg/kg/day) significantly increased compared with the control group , LC4-PLG-RGD or RGD-PLG-LC4 (6.2 mg/kg/day) by injected in tail vein for 14 days Fig. A–D. The +T cells, CD4+T cells, and FOXP3+Tregs were detected in tumours. As a result, the infiltration of CD8+T cells and CD4+T cells significantly increased both in the LC4 and LC4-PLG-RGD groups, while the CD4+T cells percentage in the LC4-PLG-RGD group was significantly higher than that in the LC4 group , perforin, and IFN-γ. CD4+T cells can produce direct anti-tumour effects by secreting IFN-γ, We next detected the T cell ability in the spleen and draining lymph nodes to proliferate and secrete cytokines by flow cytometry to figure out whether LC4-PLG-RGD activates peripheral immunity .CD8The RGD modified LC4-PLG-RGD ability to stimulate proliferation and T cell secreting cytokines in the peripheral immune organs was weakened compared with LC4, which proved that RGD modification successfully reduced the systemic effect LC4 administration on peripheral immune organs and achieved specific targeting on tumour tissues.While ICI therapy has improved melanoma patient outcomes, it has also resulted in unique immune-related adverse events (irAEs) rise , 16. The+T cell percentage in tumour tissue and activate the infiltrating T cell function in tumour tissue in vivo, which shows that LC4 peptide has ICIs activity. The spleen and draining lymph nodes are important peripheral immune organs. On the one hand, their activation is conducive to body anti-tumour, while on the other hand, systemic immunity generation may cause immune-related adverse reactions [+IFN-γ+T cell and CD4+IFN-γ+T cell percentage in the spleen and drain lymph nodes, which is consistent with the reported ICIs (antibody) effect [+T cell chemotaxis, which means its mechanism needs to be further studied.Peptide drugs has the advantage to conveniently synthesise compared with antibody drugs, lower immunogenic response, and transform easily. In this study, the peptide LC4 with specific affinity to the human CTLA-4 protein by phage display was identified. LC4 peptide could effectively block the CTLA-4/B7 protein interactions, activate peripheral immune organ activity while activating T cell activity in tumour tissues. Both LC4 peptide and ipilimumab can effectively block CTLA-4/B7 binding to activate the immune response compared with ipilimumab alone. LC4 could increase CD8eactions , 20. Bot) effect . Pan Zhe) effect , 22. How+ T cells [+T cells (Th1) secreting IFN-γ are related to the autoimmune response occurrence [+T cells and CD4+T cells secreting IFN-γ ratio in the spleen and draining lymph nodes in CT26 xenograft mouse models significantly increased after treatment with LC4 peptide. This suggested that although no obvious pathological changes were observed by H&E staining, the CD4+T cell secreting IFN-γ increase proved that CTLA-4/B7 interaction blocking by LC4 still resulted in irAE increase. At the same time, CD8+T cells secreting IFN-γ increase suggested that the peripheral immune organs were indeed activated, which may be accomplished with the assistance of activated CD4+T cells. Based on the above results, we next improved LC4 targeting to enable LC4 to carry out local immunotherapy at the tumour tissue in order to reduce peripheral immune system activation.CT26 colon tumour model was used to verify the anti-tumour activity of LC4 in vivo, which has the highest immunogenicity, and is the model most responsive to CTLA-4 inhibitor treatment. We took out the CT26 tumour-bearing mouse model’s heart, liver, lung, and kidney, and observed the tissue sections under microscope after H&E staining. No obvious pathological changes were found in the organs of mice after treatment with LC4 (data not shown). Cha E et al. has shown that patients who produce irAEs after treatment with CTLA-4 antibody are characterised by the proliferation of autoreactive CD4 T cells . Dardalhcurrence . In our + T cells and CD4+ T cells in tissues, reduce the Treg cell proportion infiltrated by tumour, and weaken the activation ability of T cells in peripheral immune organs. The limitations of this study was only observed the functional change of T lymphocytes in different organizations and the stimulus, according to the data to speculate the immune toxicity reaction, but not to look at the actual toxic effects such as colitis, to directly prove the increase tumor targeting can improve the antitumor activity of the immune checkpoint inhibitors and reduce its peripheral toxicity., and this part needs to be further improved..We coupled RGD and LC4 by use PLGLAG, RGD can bring LC4 into tumour tissue and cut the linker PLGLAG to expose LC4 peptide under the action of matrix metalloproteinases, which has a high expression in tumour cells. Our studies demonstrated that the LC4-PLG-RGD anti-tumour activity is better than LC4 in vivo, which can infiltrate more CD8In conclusion, a novel peptide LC4 targeting CTLA-4 was identified by phage display bio-panning, which has specific affinity with CTLA-4 and blocks the interaction between CTLA-4 and its CD80 ligand, which inhibits tumour growth. We modified LC4 to obtain LC4-PLG-RGD peptide since it can activate T cells in tumour tissues, as well as T cells in peripheral immune organs, whose ability to stimulate the T cell activation in peripheral immune organs was weakened, and could target tumour tissue while inhibiting tumour growth. Its anti-tumour activity is better than that of LC4. The results of this study suggested that enhancing checkpoint inhibitor tumour targeting can not only enhance their anti-tumour effect, but also effectively reduce their side effects, which is a new strategy in cancer immunotherapy.Female 6-week-old BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . The animals had free access to food and water and were maintained in a specific pathogen-free facility (24 °C ± 1 °C). Animal welfare and experimental procedures were carried out and approved in accordance with the Ethical Regulations on the Care and Use of Laboratory Animals of Zhengzhou University .5 syngeneic CT26 cells to establish colorectal cancer xenograft model. Tumour sizes were measured using a digital caliper, and tumour volumes were calculated as Eq. , 0.1 M NaHCO3 pH8.6 blocking buffer containing 5 mg/mL BSA  was added for 3 h incubation at 4 °C. After washing with TBST, 1013 pfu phages were added for 1 h incubation at 25 °C with gentle shaking. Then, the unbound phages were removed. Gly-HCl 200 μL 0.2 M (pH2.2 with 0.1% BSA) was used to harvest the binding phages. Bio-panning 84 phage clones were selected randomly and sequenced after five rounds.The Ph.D.™ -12 Phage Display Peptide Library Kit was purchased from New England Biolabs . The panning experiment was conducted according to the manufacturer’s instructions. HCTLA-4 protein 100 μg/ml was coated onto a 96-well plate overnight at 4 °C with gentle agitation in a humidified container in 0.1 M NaHCOGGGK-biotin at carboxyl terminal for flow cytometry (FCM) binding assay. Peptide sequences were as follows: GAGAAGGAGGGGGGGK-biotin ; SWHWHTHVRHQMGGGK-biotin (LC3-biotin); WGHSHFSHWKGRG- GGK-biotin (LC4-biotin); LDRPSSLAHLASGGGK–biotin (LC7-biotin); RHHSSNP-RDTAPGGGK-biotin (LC8-biotin).Peptides were synthesised in our lab by using standard solid phase Fmoc synthesis strategy and purified to more than 95% purity by RP-HPLC. Their molecular weights were assessed by mass spectrometry. Peptides were added with 2 under fully humidified conditions.Murine colorectal cancer cell lines CT26 were cultured in DMEM medium . DMEM and RPMI 1640 medium were supplemented with 10% FBS , 100 μ/mL penicillin  and 100 mg/mL streptomycin (Solarbio) at 37 °C with 5% COMST measurements were performed using the Monolith NT.115 system  to assess the CTLA-4 affinity binding peptides to Human CD80-Fc protein. The CTLA-4 binding peptides were first diluted to 400 μmol/L with MST buffer, and twofold serial dilutions were carried out to obtain 16 concentration gradients subsequently. Equal Red-NHS647 dye-labelled protein and peptide volumes were incubated at room temperature for 5 min, the mixture was loaded onto standard capillaries (NanoTemper Technologies GmhH), and immediately placed in an MST instrument for detection. The dissociation constant (Kd) was determined using the NanoTemper analysis software MO. Affinity Analysis v2.2.4.5 CHO-K1 cells transfected with pLVX-Puro/hCTLA-4 and pLVX-Puro/mCTLA-4 were used. Cell suspension was incubated with rat serum to block Fc receptors for all flow cytometry assay, and stained with corresponding antibody or biotin-peptides at 4 °C for 30 min, and then analysed by flow cytometry after washing twice.In brief, 5 × 105 CHO-K1 cells with further incubation at 4 °C for 30 min after incubation for 30 min at 4 °C. Subsequently, the anti-human IgG Fc-PE (eBioscience) was added and incubated at 4 °C for 30 min, and the cells were washed and analysed by a FACS Calibur flow cytometry (BD Bioscience). The reaction system without peptide was used as a positive control, and the system in which cells only reacted with the flow anti-Fc-PE antibody served as a negative control.CHO-K1 cells expressing hCTLA-4 and mCTLA-4 were used for cell-based blocking assay. Briefly, the assay was carried out in PBS pH 7.2, each 50 µL reaction system contained a final CTLA-4 binding peptides 200 µmol/L concentration and 10 ng of Human CD80-Fc protein. The mixture was added to 3 × 105 cells/well) were cultured in 48-well flat-bottomed plates with 500 μL IMDM containing 10% FBS. The PBMCs were stimulated by 1 μg/mLanti-CD3 (eBioscience) and 0.5 μg/mL anti-CD28 antibodies (eBioscience). The activated PBMCs were incubated with 200 μmol/L peptides. Protein transport inhibitor cocktail  was added to each well and incubated over 4 h. Cells were collected and stained with surface markers antibodies anti-human CD4-PerCP-Cy5.5 , anti-human CD8-APC  prior to fixation and permeabilisation. Permeabilized cells were then stained with anti-human anti-Human IFN-γ PE , and analysed by FACS Calibur flow cytometry (BD Bioscience).PBMCs from healthy donors were separated by density gradient centrifugation means . PBMCs . Briefly, ELISA plate was IFN-γ capture antibody coated with 100 μL/well overnight at 4 °C, aspirated and washed three times with washing buffer (PBS pH 7.2 with 0.05% Tween-20), and then blocked with ELISA/ELISPOT buffer for 1 h at room temperature. The diluted supernatant samples or IFN-γ standard were added into wells and incubated for 2 h at room temperature. Bound biotinylated IFN-γ were detected using avidin-HRP and 1 × TMB solution. Finally, a stop solution of 50 μL/well was added, and the plate optical density was measured at 450 nm using an EMax PlusMicroplate Reader .+T cells and CD8+ T cells.Tumour-bearing mice were sacrificed on the last treatment day. Tumour tissues were minced and digested in collagenase IV  and Dnase I (Sigmae-Aldrich) for 30 min at 37 °C, and passed through a 70-mm cell strainer. Part of the cells were collected and stained with surface markers antibodies after centrifuging, anti-mouse CD45 FITC , anti-mouse CD3 PerCP-eFluor 710 , and anti-mouse CD8 APC  for 30 min at 4 °C to detect CD4+ Tregs. The cells were washed and analysed by a FACS Calibur flow cytometry.Another cell part was stained with surface markers antibodies anti-mouse CD45 FITC , anti-mouse CD4 APC , and anti-mouse CD25 PE  prior to fixation and permeabilisation. Permeabilized cells were then stained with anti-mouse FOXP3PE-Cy7  for 30 min at 4 °C to detect FOXP3Single cell mouse spleen and draining lymph node suspension were prepared by gentle mechanical disruption. Tumour-infiltrating lymphocytes were isolated from tumour tissues. Cells from CT26 xenograft model were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate  and 1 μmol/L ionomycin (Sigma eAldrich) for 4 h. Cells were then stained with surface markers antibodies anti-mouse CD3 PerCP-eFluor710 , anti-mouse CD8 APC , or anti-mouse CD4 APC  prior to fixation and permeabilisation. Permeabilized cells were then stained with anti-mouse IFN-γ PE , analysed by FACS Calibur flow cytometry.p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant differences.Data are shown as mean ± SD and statistical significance between groups was analyzed using Excel according to the 1-tailed Student's test between different groups. *Additional file1 (PPTX 946 KB)


Fluid overload and venous congestion are associated with morbi-mortality in the ICU (intensive care unit). Administration of diuretics to correct the fluid balance is common, although there is no strong relationship between the consequent fluid loss and clinical improvement. The aim of the study was to evaluate the ability of the portal pulsatility index, the renal venous impedance index, and the VEXUS score (venous ultrasound congestion score) to predict appropriate diuretic-induced fluid depletion.The study had a prospective, observational, single-center observational design and was conducted in a university-affiliated medico-surgical ICU. Adult patients for whom the clinician decided to introduce loop diuretic treatment were included. Hemodynamic and ultrasound measurements  were performed at inclusion and 2 hours after the initiation of the diuretics. The patients’ characteristics were noted at inclusion, 24 h later, and at ICU discharge. The appropriate diuretic-induced fluid depletion was defined by a congestive score lower than 3 after diuretic fluid depletion. The congestive score included clinical and biological parameters of congestion.95%:0.70–0.92, p = 0.001), followed by the renal venous impedance index . The baseline VEXUS score  was poorly predictive of appropriate response to diuretic-induced fluid depletion.Eighty-one patients were included, and 43 (53%) patients presented with clinically significant congestion score at inclusion. Thirty-four patients (42%) had an appropriate response to diuretic-induced fluid depletion. None of the left- and right-sided echocardiographic parameters differed between the two groups. The baseline portal pulsatility index was the best predictor of appropriate response to diuretic-induced fluid depletion (AUC = 0.80, CIThe portal pulsatility index and the renal venous impedance index were predictive of the appropriate response to diuretic-induced fluid depletion in ICU patients. The portal pulsatility index should be evaluated in future randomized studies.The online version contains supplementary material available at 10.1186/s13054-022-04180-0. In recent years, there has been a growing interest in the diagnosis and treatment of fluid overload and venous congestion , 2. The Physicians empirically use diuretics, mostly loop diuretics , to corrRecently, several ultrasound indices of venous congestion have been studied in the context of heart failure and/or cardio-renal syndrome , 5, 10. The primary objective was to evaluate the ability of the portal pulsatility index to detect appropriate response to diuretic-induced fluid depletion. The secondary objectives were to evaluate the value of the renal venous impedance index and the VEXUS score to detect appropriate response to diuretic-induced fluid depletion.We performed a prospective, observational, single-center study in a cardiovascular medico-surgical ICU of a tertiary university medical center  between 2019 and 2020. This study was approved by the French Comité de Protection des Personnes (2018/7148). All patients received a written informed letter and gave consent to participate. The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.We consecutively included all patients with age > 18 years, presence of clinical signs of fluid overload, absence of fluid responsiveness assessed by an increase in stroke volume following passing leg raising; and for whom the clinician decided to introduce loop diuretic treatment for several days. The decision to initiate loop diuretic treatment was left to the treating physician and was noted from medical data. The main non-inclusion criteria were prior diuretic treatment during the ICU stays, permanent atrial fibrillation, veno-venous hemofiltration or dialysis, and patients with unstable shock (variation of blood pressure > 10% despite hemodynamic treatment and/or need to increase hemodynamic support).2) was calculated as π × LVOT2/4. The VTIAo  was measured by PW (pulsed wave) Doppler and a five-chamber apical view. SV  was calculated as VTIAo × AVA, and CO  as SV × HR (heart rate). In patients with TR (tricuspid regurgitation), peak TR velocity was measured by CW (continuous wave) Doppler, and the right ventricle–right atrium pressure gradient was calculated. The right ventricular systolic function was assessed by measuring TAPSE (tricuspid annular plane systolic excursion) and RVFAC . The hepatic venous flow was recorded from the subcostal window.Transthoracic echocardiography was performed by an experienced physician (advanced level training) using a Philips Envisor ultrasound system , according to current guidelines , 18. TheThe HV-S (supra-hepatic vein systolic) and HV-D (supra-hepatic vein diastolic) velocities were measured, and the S/D (systolic/diastolic) ratio was calculated. With the patient in the supine position, the diameter of the inferior vena cava was measured in the subcostal view at 1 cm from its junction with the right atrium. The maximum and minimum diameters of the inferior vena cava were measured, and the percentage of change in diameter was calculated.Vmax − Vmin)/(Vmax). We calculated intra- and inter-observer reproducibility. Intra and inter-observer reproducibility was 10% (4–16) and 15% (11–28), respectively.The PI  was assessed by pulsed-wave Doppler evaluation of the portal vein in the liver hilum . The VmaRRI  and RVI  were measured using a transparietal 5 MHz pulsed-wave Doppler probe. Doppler measurements were performed in the interlobar arteries/veins of the upper, median, and lower segments of each kidney , 19. For−1 [Based on the literature , 22, we −1 , 22. The−1 , 22. TheThe VEXUS score was calculated as previously described . The perPatients were followed during all their ICU stay. Based on an expert opinion statement  that sugClinical examination, lung ultrasound, urine output, diuretic, vasoactive treatments, NT-proBNP, and blood creatinine were recorded at inclusion, 24 h later, and at ICU discharge. Cumulative fluid balance and cumulative percentage of fluid overload were calculated at inclusion, 24 h later, and at ICU discharge. The VEXUS score and the congestion score were calculated at inclusion and 2 h later.The primary endpoint was the appropriate diuretic-induced fluid depletion on the last day of ICU .p value of 0.10  were then included in a multivariate logistic model with a backward selection procedure. All reported probability values were two-tailed, and a p value ≤ 0.05 was considered statistically significant. All analyses were performed using R software version 3.4.4 .We calculated that a sample size of 74 patients would be sufficient to demonstrate that the pulsatility portal index can predict the response to diuretic with an AUC (area under the curve) between 0.7 and 0.8, a power of 90%, an alpha risk of 0.05 and a beta risk of 0.1. We included up to 81 patients to take into account the risk of incomplete data. Two groups of patients were compared: patients with and without positive clinical response to diuretic-induced fluid depletion. Normality was assessed using the Shapiro–Wilk test. Accordingly, quantitative data are presented as medians (interquartile range) or as mean (standard deviation). Qualitative data are presented as frequency and percentage. Quantitative and qualitative variables were analyzed by using the Mann–Whitney test, the Student test, the paired Student test, or the Wilcoxon test, as appropriate. Bonferroni post hoc corrections were used to assess statistical significance, as appropriate. To assess the relationship between echographic parameters and the positive clinical response to diuretic-induced fluid depletion, we performed a ROC (Receiver Operating Characteristic) curve with calculating the AUC. The method described by DeLong et al. was used to compare the areas under the ROC curve associated with the variables . The cutn = 51 (63%) and the median SAPS II (Simplified Acute Physiology Score II) was 46 ± 18 (Table n = 25), positive fluid balance (n = 63) and congestion (n = 18). Of the 81 patients, the median score was 3 (2–4), and 43 patients (53%) had a congestive score over 3 , the total daily dose (70 mg (35–134) versus 60 mg (40–120), p = 0.311), and the total fluid balance at day one (− 505 ml (− 1465–385) versus − 770 ml (− 1451–155), p = 0.458) did not differ between appropriate diuretic-induced fluid depletion group and control group.The diuretic bolus dose (40 mg (23–48) versus 40 mg (40–80), p = 0.137, and 91 mmol l−1 (33) versus 93 mmol l−1 (36), p = 0.87. The VEXUS score was higher for patients with diuretic-induced fluid depletion than for the control group (2 (0–3) vs 0 (0–2), p = 0.03).Hemodynamic and echocardiographic parameters at baseline and at 2 hours for both groups of patients are given in Table p = 0.001). The venous impedance pattern was significantly worse in patients with appropriate diuretic-induced fluid depletion (2 (1–2) vs 3 (2–3), p = 0.011). The VEXUS score was higher in patients with appropriate diuretic-induced fluid depletion (2 (0–3) vs 1 (0–2), p = 0.002). At 2 h, the portal pulsatility index was higher in patients with appropriate diuretic-induced fluid depletion (34% (23–54) vs 24% (12–31), p = 0.001). None of the other echocardiographic parameters differed between the two groups, even right-sided echocardiographic parameters and the IVC diameter  vs 28% (22–35), p < 0.05). None of the other echocardiographic parameters differed between the two groups.The venous impedance pattern was significantly worse in patients with appropriate diuretic-induced fluid depletion (95% 2.8–158.9), p = 0.003), cardiac index (OR = 6.9 (CI95 17–28.5), and the impedance venous pattern (OR = 6.3 (CI95% 2.2–18.2) were associated with the diuretic-induced fluid depletion response. The AUC of the portal pulsatility index was 0.80    .At baseline, the portal pulsatility index and venous impedance pattern were associated with the response to diuretic treatment. When analyzed using multivariate logistic regression, portal pulsatility . The AUC of the impedance venous pattern was 0.77 . The VEXUS score was poorly predictive of appropriate diuretic-induced fluid depletion with an AUC of 0.67 .At 2 h, the portal pulsatility index and venous impedance pattern predicted with high specificity appropriate diuretic-induced fluid depletion. The AUC of the portal pulsatility index was 0.72 (CIThis is an exploratory pragmatic study regarding the prediction of response to fluid depletion in ICU patients. Our results may be summarized as follows: (1) patients with an appropriate response to diuretic-induced fluid depletion had high pulsatility portal index and worst renal venous impedance. (2) Right- and left-sided echocardiographic parameters were not associated with an appropriate response to diuretic-induced fluid depletion. (3) Pulsatility portal index and renal venous impedance were the best predictors of appropriate diuretic-induced fluid depletion response to diuretic. The changes in pulsatility portal index and venous renal index predicted the appropriate decongestion with fluid depletion but not response per se to diuretic therapy (amount of fluid). Pulsatility portal index and renal venous impedance are two parameters that may indicate that volume can be mobilized in a congestive state.Interestingly, in our cohort with patients considered as being in fluid overload, less than half of the cohort had appropriate decongestion, and might truly have needed fluid depletion. The renal response to diuretic (quantified by diuresis and natriuresis) was the same in both groups of patients (responders vs non-responders) and indifferently of appropriate decongestion. This underlines the need to assess the venous congestion more than fluid overload. That also explains the growing interest in the assessment of congestion with different parameters  or scores that are associated with clinical outcomes, and that may also predict response to treatment , 4, 25. These results can be explained. Congestion is a complex clinical and hemodynamic syndrome that is the consequence of several cardiac and non-cardiac phenomena. In ICU, underlying disease , positive pressure ventilation, and therapeutics  can further alter cardiovascular homeostasis and increase the prevalence of congestion. Of the factors involved in congestion, volemia is the most difficult parameter to assess in ICU. Parameters usually used to assess volemia evaluate a preload state, and they can be altered by cardiovascular disease, thus they are not good indices of volemia. In the present cohort, patients were fluid unresponsive ), and they had frequently right and left-sided echocardiographic parameters of heart failure . But none of these parameters were associated with an appropriate diuretic-induced fluid depletion response. Interestingly, our findings support a recent consensus paper that proposes to analyze signs of venous stasis to determine fluid status .The inferior vena cava diameter and the supra-hepatic venous flow are two parameters that may better reflect the severity of the underlying cardiovascular homeostasis than patient volemia . The infThe pulsatility portal index is an indicator of congestion in relation to volemia may be because of the reservoir role of the splanchnic circulation. A recent study on a healthy subject has demonstrated that fluid expansion increased portal blood flow and its pulsatility only in the patient who did not increase their cardiac output . In thisOn the contrary, the inferior vena cava and the hepatic veins may be more sensitive to heart condition and thoraco-abdominal interactions . Thus, aMost patients’ characteristics were in accordance with guidelines on diuretic-induced fluid depletion. Despite these points, and as previously demonstrated the response to diuretic-induced fluid depletion fluid is highly variable and poorly predictive . We did The study has limitations. This is an exploratory, prospective monocentric observational study that includes usual limitations of such designs. Because of inclusion/non-inclusion criteria, we have selected a specific cohort with a high prevalence of cardiovascular comorbidities, with marked signs of left-sided heart failure, mostly fluid unresponsive, and who were included later after admittance to ICU. These points reflect a selected cohort of cardiovascular patients for whom fluid depletion may be necessary, such as in patients with pulmonary weaning failure . IndicatIn conclusion, our study demonstrates that the Doppler study of the portal flow provides information on hemodynamic congestion and fluid overload. The pulsatility portal index and venous renal index predict the appropriate response to diuretic-induced fluid depletion in ICU patients. Further studies integrating these indices may confirm these observations and clarify the usefulness of these indices.Additional file 1: Table S1. Hemodynamic and echocardiographic measurements according to congestive response to diuretic treatment (Appropriate fluid depletion vs Control). $p < 0.05 comparison between two groups of patient. *p < 0.05 comparison with baseline.


While a 3-dose mRNA-1273 primary series is recommended for moderately or severely immunocompromised (IC) individuals in the U.S., some IC individuals do not complete the 3-dose series. We conducted a matched cohort study to evaluate the relative vaccine effectiveness (rVE) of the 3-dose mRNA-1273 primary series vs. 2 doses of mRNA-1273 in preventing SARS-CoV-2 infection and severe COVID-19 disease in IC individuals.nd dose date. Third doses were accrued from 08/12/2021 to 12/31/2021, with follow-up through 1/31/2022, spanning the delta and omicron periods. Outcomes were SARS-CoV-2 infection (positive molecular test or diagnosis code), COVID-19 hospitalization, and COVID-19 hospital death. Adjusted hazard ratios (aHR) with confidence intervals (CI) were estimated by Cox proportional hazards models. Adjusted rVE (%) was calculated as (1-aHR) x 100.IC individuals aged ≥18 years with ≥12 months of Kaiser Permanente Southern California membership who received 3 doses of mRNA-1273 ≥24 days apart were 1:1 matched with randomly selected IC 2-dose recipients on age, sex, race/ethnicity, and 2rd dose (Table 3).Our study included 21,942 3-dose and 21,942 2-dose mRNA-1273 IC recipients. Adjusted rVE of 3 doses compared to 2 doses of mRNA-1273 against SARS-CoV-2 infection, COVID-19 hospitalization, and COVID-19 hospital death were 55.0% (95% CI: 50.8–58.9%), 83.0% (75.4–88.3%), and 87.1% (30.6–97.6%), respectively (Table 1). Adjusted rVE against SARS-CoV-2 infection ranged from 43.0% to 59.1% across subgroups of age, sex, race/ethnicity, history of SARS-CoV-2 infection, pregnancy, and comorbidities (Table 2). Point estimates of the 3-dose rVE were higher against COVID-19 infection and hospitalization in the first 3 months, compared to 3–6 months after the 3rd primary series dose for adequate protection of IC individuals.Three doses of mRNA-1273 provide additional protection against SARS-CoV-2 infection and severe outcomes for IC individuals, compared to 2 doses, highlighting the importance of completing 3-doses for IC populations. However, possible waning of protection against SARS-CoV-2 infection and severe outcomes after 3 months supports the ACIP recommendation of a booster dose at least 3 months after the 3Jennifer H. Ku, PhD MPH, GSK: Grant/Research Support|Moderna: Grant/Research Support Lina S. Sy, MPH, Dynavax: Grant/Research Support|Glaxosmithkline: Grant/Research Support|Moderna: Grant/Research Support|Seqirus: Grant/Research Support Lei Qian, PhD, Dynavax: Grant/Research Support|Glaxosmithkline: Grant/Research Support|Moderna: Grant/Research Support Bradley Ackerson, MD, Dynavax: Grant/Research Support|Glaxosmithkline: Grant/Research Support|Moderna: Grant/Research Support|Pfizer: Grant/Research Support|Seqirus: Grant/Research Support Yi Luo, PhD, Glaxosmithkline: Grant/Research Support|Moderna: Grant/Research Support|Pfizer: Grant/Research Support|Seqirus: Grant/Research Support Julia Tubert, MPH, Moderna: Grant/Research Support|Pfizer: Grant/Research Support Gina Lee, MPH, Glaxosmithkline: Grant/Research Support|Moderna: Grant/Research Support Ana Florea, PhD MPH, Gilead: Grant/Research Support|GSK: Grant/Research Support|Moderna: Grant/Research Support|Pfizer: Grant/Research Support Carla Talarico, PhD, Moderna: Employee of and a shareholder in Moderna Inc. Sijia Qiu, MS, Dynavax: Grant/Research Support|Moderna: Grant/Research Support Yun Tian, MS, Glaxosmithkline: Grant/Research Support|Moderna: Grant/Research Support Hung Fu Tseng, PhD MPH, GSK: Grant/Research Support|Janssen: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Seqirus: Grant/Research Support.