Maoto (ma-huang-tang) was developed in ancient China and is used to alleviate flu symptoms. Currently, no meta-analyses have evaluated the efficacy and safety of maoto for alleviating flu symptoms.Influenza is a common viral infection worldwide. Ichushi), two Chinese databases , and two Korean databases  for studies published in or before October 2017. Clinical studies that compared maoto plus neuraminidase inhibitors (NAIs) vs. NAIs alone, or maoto alone vs. NAIs alone, were included in the present analysis. The primary outcome measure (efficacy) was the length of time from the start of medication to resolution of influenza symptoms  and virus isolation. The secondary outcome measures (safety) were as follows: (1) side effects and adverse reactions, such as nausea, abnormal behaviour, or discontinuation of symptomatic treatment; (2) morbidity (complications caused by influenza infection) or mortality; and (3) hospitalisation for any reason.In the present study, we searched MEDLINE/PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, a Japanese database (N = 60) and ten non-randomised studies . We found that maoto plus NAIs was superior to NAIs alone in terms of the duration of fever in one RCT  and four NRSs . The duration of symptoms or virus isolation did not differ between maoto and NAIs. No severe side effects or adverse reactions were reported related to maoto or NAIs.Twelve relevant studies were identified, including two randomised controlled trials  contains supplementary material, which is available to authorized users. Influenza is a common viral infection worldwide, occurring in seasonal epidemics. According to the World Health Organization (WHO), 5–15% of the population is affected by annual epidemics of influenza infection in the upper respiratory tract .Three types of antiviral drugs have been approved by the Japanese national health insurance system for the symptomatic treatment of influenza infection, namely neuraminidase inhibitors (NAIs), adamantanes, and cap-dependent endonuclease inhibitors. Four NAIs—oral oseltamivir, inhaled zanamivir, inhaled laninamivir, and intravenous peramivir—are available and widely prescribed in Japan because they act against both types of the influenza virus, A and B. In contrast, amantadine and rimantadine (known as adamantanes) are less widely prescribed because they only act against the influenza A virus, which shows marked resistance to adamantanes . BaloxavA recent systematic review of oseltamivir revealed that it can shorten the duration of symptoms by 16.8 h in adults but does not reduce hospital admissions due to complications from influenza. The review also pointed out that the drug conferred an increased risk of nausea and vomiting in adults . The WHOGanmao capsules were more effective than amantadine at shortening the duration of influenza symptoms [maxingshigan-yinqiaosan, alone or in combination with oseltamivir, reduced the duration of fever in patients infected with the 2009 H1N1 influenza virus [Recently, the efficacies of several traditional Chinese medicine formulas as symptomatic treatments for influenza have been evaluated. For instance, a systematic review of the Cochrane library reported that symptoms . A randoza virus .Maoto (Ma-Huang-Tang in Chinese) has been widely prescribed as a symptomatic treatment for the common cold and flu according to claims in the Japanese national health insurance system. It can be prescribed to both children and adults. Traditionally, the symptoms that indicate maoto are headache, chill, fever, arthralgia, and cough, without sweating. Maoto can also be applied for rheumatoid arthritis, bronchial asthma, infant nasal obstruction, and difficulties in sucking milk.Maoto is a multicomponent formulation, originally extracted from four crude drugs, as follows: 5 g of ephedra herb, 5 g of apricot kernel, 4 g of cinnamon bark, and 1.5 g of glycyrrhiza root. It is currently prepared for prescription use in Japan as granules  or powder  through the process of decoction, concentration, drying, and the addition of an excipient. The maoto preparation is orally administered, usually after dissolution in warm water.maoto and its component ingredients are active. For instance, ephedra herb and its tannins inhibit endosome acidification and influenza A virus fusion to the cell membrane [maoto acts against influenza A in vitro, while laninamivir and amantadine do not. In addition, using multiple subtypes of the influenza virus , the authors found that maoto reduced the intracellular virus titre, as well as the levels of matrix protein 2 and nucleoprotein present in the experimental system. Nagai et al. [maoto (0.8 g/kg/day and 1.3 g/kg/day) had an antipyretic effect in the early phase of influenza A infection in mice and that the levels of anti-influenza immunoglobulin M, immunoglobulin A, and immunoglobulin G1 antibodies increased in nasal fluid, bronchoalveolar lavage, and serum. Thus, maoto, as a multicomponent formulation, has multiple effects on the life cycle of the influenza virus and broad influence on host metabolism [Some studies have demonstrated that membrane , 10. Glymembrane . Cinnamamembrane . Masui ei et al.  showed ttabolism .Maoto is a prescription drug that has been covered by the Japanese national health insurance system for over 40 years. The cost of maoto is much less than that of NAIs. Specifically, the officially set drug cost of maoto is 150 JPY (1.4 USD) per person, whereas the standard prescription of oseltamivir and acetaminophen costs 3260 JPY (29.6 USD). We previously calculated that, if half a prescription of oseltamivir was replaced with maoto, the annual saving in medical costs in Japan would be 9 billion yen (82 million USD) [maoto is also available in the pharmacy as an over-the-counter drug, although its concentration is half that of the prescribed medication.maoto in alleviating flu symptoms have been evaluated in clinical studies. These studies compared maoto with NAIs, or maoto plus NAIs with NAIs alone. However, the results have been inconsistent, and no meta-analysis has yet analysed the efficacy and safety of maoto in alleviating flu symptoms.The efficacy and safety of maoto in alleviating flu symptoms.Thus, the objective of this review and meta-analysis was to evaluate the efficacy and safety of maoto extract. The NRSs included quasi-RCTs, non-RCTs, prospective or retrospective cohort studies, historically controlled trials, and (nested) case-control studies [We included both RCTs and comparative non-randomised studies (NRSs) of prescription  studies .We included studies that enrolled patients who had uncomplicated influenza diagnosed using a rapid antigen detection test (RADT) and/or genetic detection with polymerase chain reaction. Studies were included regardless of the patients’ age.maoto. The possible comparisons were as follows: (1) maoto vs. placebo, (2) combination of maoto plus NAIs vs. NAIs alone, and (3) maoto vs. NAIs. Co-interventions were allowed if they were offered to both arms of the study. We excluded studies that included other herbal formulas.We included studies assessing the efficacy of The primary outcome measure (efficacy) was the length of time from the start of medication to resolution of influenza symptoms  and virus isolation.The secondary outcome measures (safety) were as follows: (1) side effects and adverse reactions, such as nausea, abnormal behaviour, or discontinuation of symptomatic treatment; (2) morbidity (complications caused by influenza infection) or mortality; and (3) hospitalisation for any reason.Ichushi), two Chinese databases , and two Korean databases . In this search, we used the key words ‘maoto/mao-to/ma-huang-tang’, and ‘flu/influenza’. Our search strategies are supplied in Additional file We searched the following databases for studies published in or before October 2017: MEDLINE/PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, a Japanese database  scanned the titles, abstracts, and keywords of every record retrieved. We located the full articles for further assessment when the information given suggested that the study (1) included patients with uncomplicated influenza and (2) assessed the efficacy of Two authors (TY and RA) independently extracted, checked, and made a review table of the articles. The extracted data comprised the (1) authors and title of the study, (2) year of publication, (3) study size, (4) age and sex of participants, (5) detailed methodological information, (6) dose and duration of the intervention, (7) details of control interventions, (8) outcomes, and (9) side effects or adverse reactions. TY entered these data into the Review Manager software.We assessed the reporting quality of each study, basing the assessment largely on the quality criteria specified by the Cochrane Handbook for Systematic Reviews of Interventions  for RCTsmaoto. To evaluate symptom duration, we ignored differences in the definition of symptom, as well as differences in the viral type (A or B) or surface antigen (e.g. H1N1 or H3N2). The unit of analysis was the individual participants. We tried to obtain any relevant missing data from the study authors by e-mail or telephone.We performed a quantitative meta-analysis when data for a given outcome were available in more than two of the included studies, regardless of the daily dosage or duration of https://community.cochrane.org/help/tools-and-software/revman-5).The present meta-analysis was performed using Review Manager (RevMan) Ver. 5.3 for Mac . This analysis quantified inconsistency across studies and allowed us to assess the effects of study heterogeneity on the meta-analysis. To combine studies, we used the fixed-effects model when the studies in a given subgroup were significantly similar . We included RCTs and NRSs in this meta-analysis, and RCTs and NRSs were always analysed separately. The overall effect was tested using the Z score, with P < 0.05 being considered statistically significant.We examined heterogeneity through visual inspections of forest plots, a standard χ2 test and I2 statistic, we used a random-effects model to analyse whether heterogeneity may have led to the different effects among studies. In this model, data should be continuous, thus we used the weighted mean difference and 95% confidence intervals to analyse data that had been measured using the same scale, and then combined the weighted mean differences (e.g. duration of fever). We attempted to determine the potential causes of heterogeneity by examining the individual study and subgroup characteristics.When heterogeneity was suggested by the χIchushi), 11 in MEDLINE, 11 in EMBASE, 11 in CENTRAL, 6 in VIP, 7 in CNKI, and one by a Google hand search. We did not find any relevant studies in KMbase or KAMJE. After removing 24 duplicates, another 239 records were removed because they were (1) not relevant comparative clinical studies of maoto, (2) basic research, (3) reviews, or (4) case reports. Subsequently, we excluded 12 full-text articles of clinical studies that (1) lacked relevant outcomes (N = 5), (2) involved different interventions , or (3) duplicated participants .In the present review, 241 studies were found in the Japanese database  were used as an intervention. The reason might be that the influenza infection is clearly indicated only for maoto produced by Tsumura Co. in Japan. No self decoctions from crude drugs were used in any of the studies.In most of the included studies, Among the 12 studies, there were two RCTs , 29 and maoto plus NAIs and those administered NAIs alone according to an NRS by Tsuji et al. [The duration of overall symptoms did not differ between patients administered i et al. .maoto plus NAIs was superior to NAIs alone in terms of the duration of fever in one RCT  and four NRSs  , and we chose a fixed-effects model for this outcome.The combination of P = 0.018; according to their analysis) in the maoto plus NAIs group.One NRS by Fukutomi et al.  reportedWe did not identify any articles mentioning the dosing times of acetaminophen or the duration of virus isolation after symptomatic treatment.maoto and those administered NAIs in either one RCT or three NRSs , and we chose a fixed-effects model for this outcome. Our search for studies comparing maoto vs. NAIs identified four reports from two first authors, Kawamura [The duration of overall symptoms did not differ between patients administered RSs Fig. . The hetKawamura , 23 and Kawamura . HoweverKawamura ) for symmaoto and NAIs in either the RCTs or NRSs. In an RCT by Nabeshima et al. [maoto group than in the oseltamivir group. However, this trial also included participants who were administered zanamivir, and the difference in fever duration was no longer significant when the participants who were administered zanamivir were combined with participants who were administered oseltamivir as the NAIs group. Eight NRSs were included in the meta-analysis of fever duration. The heterogeneity was significant , and we chose a random-effects model for this outcome . The tendency was rather opposite in children , and this discrepancy might be caused by two NRSs [maoto, this may have also contributed to the discrepant results in children. Regarding the daily dosage, although we noticed that the daily dosage of maoto used by children was generally less than that used in adults, we did not observe a relationship between the dosage and treatment efficacy in paediatric participants. We did not perform further subgroup analyses, such as age ≤ 10 years vs. > 10 years, viral type A vs. B, or the time from onset to consultation ≤24 h vs. > 24 h, since data could not be obtained for a sufficient number of cases.The duration of fever did not differ between a et al. , the autome Fig. . As a sutwo NRSs , 27 thatmaoto group than in the NAIs group on days 1, 2, and 3. They also reported that the severity of myalgia/arthralgia was significantly better in the maoto group than in the NAIs group at night on day 1.We could not perform meta-analyses on other symptoms like malaise or headache because most articles only reported the duration of overall symptoms and fever. An NRS by Nabeshima et al. in 2010  reportedmaoto group compared to in the oseltamivir group .This NRS by Nabeshima et al. in 2010  also repOne RCT and one NRS reported on virus isolation by RADT after drug administration on days 3 and 5 , 29. We maoto or NAI administration. Only one study [As shown in Table ne study  reportedne study  recordedmaoto may shorten the duration of fever when used alone or in combination with NAIs and that maoto may be a well-tolerated approach for alleviating flu symptoms. However, it should be noted that the quality of evidence for this conclusion was low. In the future, better-designed trials will be required to elucidate the efficacy of maoto.Although we could not reach a definitive conclusion because the included studies had a high risk of bias, the findings of this meta-analysis suggest that maoto because it has a strong odour and taste [maoto and NAIs, where no significant difference was found according to the present analysis. To fully understand whether maoto is as effective as NAIs, an ‘equal efficacy’ or ‘non-inferiority’ study should be performed. It is worth performing such studies because maoto is much less expensive than are NAIs (approximately 20-fold in Japan) [We rated the quality of the evidence from the included studies as very low to low for a variety of reasons. First, only two RCTs were included, and the participants in the RCTs were assigned to each arm using open allocation. Second, we could not obtain enough information regarding the participants’ demographic backgrounds, including possible confounders such as vaccination history. Third, no blinded studies were identified via the database search conducted in the present review, although it would be difficult to perform a double-blinded study of nd taste . Indeed,nd taste . Finallyn Japan) .maoto is consumed for several days, and we believe that the safety of this drug is high. However, it does contain ephedra, and clinicians should be cautious when prescribing it to patients who have thyroid, cardiovascular, or prostate dysfunction, or to elderly patients [In the present study, we did not note any severe side effects or adverse reactions. Usually, patients . We acknpatients .maoto should ideally be used according to the traditional diagnosis of influenza, as mentioned in the introduction, and the inclusion or exclusion criteria should contain traditional concepts or diagnoses. However, what constitutes a traditional diagnosis may differ among researchers and clinicians, and it is difficult to utilise strictly defined criteria in clinical trials. In our previous study, none of the symptoms, such as chill and absence of sweating, were themselves obligate indicators for maoto in the alleviation of influenza symptoms, suggesting that maoto can be used to treat probable influenza infection regardless of whether traditional concepts or diagnoses are used [In future studies, are used .maoto. However, the Cochrane collaboration did perform a systematic review of studies using traditional Chinese medicines, reporting that traditional Chinese medicines have clinical efficacy in alleviating influenza infection symptoms, similar to NAIs [maoto, a Japanese traditional medicine, not a traditional Chinese medicine.Our search did not reveal any other reviews on  to NAIs . The sys to NAIs  that wasmaoto may lower the fever duration when it is used alone or in combination with NAIs and is likely a well-tolerated treatment. However, the current evidence is too weak to draw a definitive conclusion. An RCT  must be conducted to determine the efficacy and safety of maoto. The present review and meta-analysis may be beneficial in estimating the effect size and calculating the sample size in future clinical trials.Even though a definitive conclusion could not be established owing to the small sample sizes and high risk of bias in the analysed studies, our findings imply that Additional file 1:Search Strategies and Results. (DOCX 29 kb)Additional file 2:Table S1. Basic characteristics of the included studies. (DOCX 27 kb)Additional file 3:Figure S1. Funnel plot of fever duration after drug administration: maoto plus neuraminidase inhibitors vs. neuraminidase inhibitors alone. SE, size of effect; MD, mean difference. (PDF 35 kb)Additional file 4:Figure S2. Funnel plot of overall symptom duration after drug administration: maoto vs. neuraminidase inhibitors. SE, size of effect; MD, mean difference. (PDF 38 kb)Additional file 5:Figure S3. Funnel plot of fever duration after drug administration: maoto vs. neuraminidase inhibitors. SE, size of effect; MD, mean difference. (PDF 74 kb)


Anopheles cruzii, Aedes fluviatilis, Aedes scapularis, Psorophora ferox, Culex quinquefasciatus, Culex mollis, and Culex intrincatus, together with specimens representing four subgenera and one genus of Culicidae  had their blood meals identified. Their diverse host use was evidenced by the identification of 19 species of vertebrate host, namely two amphibians, three mammals and 14 birds. Birds were the most commonly identified host in blood meals. These results not only show the diversity of mosquito hosts, but also underscore the challenges involved in monitoring arboviruses of public health importance, given potential combinations of host use for each mosquito species.The study of host associations of mosquitoes  provides valuable information to assist in our understanding of a variety of related issues, from their life-history to the entomological surveillance of pathogens. In this study, we identified and characterized mosquito blood meals from both urban and forested areas in the city of Paranaguá, state of Paraná, Brazil, by analyzing the amplification of host DNA ingested by mosquitoes under different storage conditions and digestion levels. Host DNA preservation was evaluated in fresh blood meals according to storage duration (30 to 180 days) and temperature (-20°C / -80°C) and, in digested blood, according the degree of digestion classified on the Sella scale. Molecular analysis of blood meals was based on DNA extraction and amplification of a fragment of the mitochondrial COI gene. We determined that, up to180 days of storage, the evaluated temperatures did not influence the preservation of fresh blood meals DNA, whereas the amplification success was increasingly reduced over the course of the digestion process. The species  Hematophagy is shared by females of most mosquito species , which uThe contact with different hosts can allow for the use of novel vertebrate species in the transmission of pathogens . A less Several approaches have been used to identify the blood meals of mosquitoes, such as serological and molecular methods. Assays such as precipitin tests and ELISA (Enzyme Linked Immunosorbent Assay), which were common prior to PCR-based blood meal analysis, tended to show low specificity, given that identification was based on tests that only indicated groups of vertebrates as potential hosts –20. CurrMolecular methods require maximum integrity of the DNA molecule for the optimal detection and amplification of the target fragment . In arthGiven that mosquitoes collected in the field show different degrees of digestion in their blood meal, it is thus important to assess the ideal storage conditions of mosquito blood meals after field collection. In addition, although storage at -80°C is the ideal way to preserve blood DNA, it is necessary to determine the temperature limits and duration of storage for mosquitoes used in studies seeking to identify their host choice . Thus, tThe protocol for the use of animals in the laboratory was approved by the Commission on Ethics for the Use of Animals of the Institute of Biological Sciences of the Universidade Federal do Paraná (CEUA/BIO-URPF) (Protocol number 719). All procedures for manipulation and animal care were carried out in strict conformity with the recommendations by the training manual of that commission. The collection of mosquito specimens in the field was carried out in a private urban area, which was authorized by the respective owners. In the forested area, in a public property, the collection permit was issued by the Instituto Ambiental do Paraná. In both areas, the collection of specimens did not involve threatened or protected species and did not entail losses for any vertebrate animal, nor risks to human health.Aedes aegypti belonging to a Rockefeller strain. These females were approximately 30 days old and were reared and kept in the Laboratório de Morfologia e Fisiologia de Culicidae e Chironomidae of the Universidade Federal do Paraná under temperatures between 25–29° C, 70–80% relative humidity, and 12:12 h (light:dark) photoperiod. Daily, these mosquito females had a 10% sucrose solution available for feeding. For the blood meal, anesthetized mice (Mus musculus) became available for approximately 40 minutes, following a protocol approved by the committee on ethics for animal experimentation. The assessment of the effect of the degree of blood digestion on DNA amplification, as well as the identification of vertebrate hosts, involved females collected in the field.In order to assess the effects of temperature and storage duration on the amplification of blood DNA, we used females of 2 and a population of approximately 26,000 inhabitants (in 2010) , all analyzed with respect to their blood source  had their host identified through blood meal analysis. As the molecular identification of mosquitoes has been shown to be efficient, mainly of cryptic species , throughd source . These nd source . These sd source .Cx. quinquefasciatus (n = 27), Cx. intrincatus (n = 1), Ae. scapularis (n = 1), which fed on avian (68.96%) and mammal blood (31.04%). Most blood meals of Cx. quinquefasciatus were identified as originating from chicken , followed by dogs (Canis lupus familiaris), humans (Homo sapiens), and passerine birds . The blood meal of Cx. intrincatus led to the identification of the heron Nyctanassa violacea, whereas the blood meal of Ae. scapularis was identified as coming from a domestic dog , Ae. fluviatilis (n = 1), Ae. scapularis (n = 2), Ps. ferox (n = 2), Cx. quinquefasciatus (n = 9), Cx. mollis (n = 1), Aedes (Ochlerotatus) sp. (n = 2), Culex (Culex) sp. (n = 1), Culex (Melanoconion) sp. (n = 1), Culex (Microculex) sp. (n = 3), and Limatus sp. (n = 1). Fourteen species of vertebrates were used as host, including wild birds , mammals , and amphibians . The genus Culex only had wild birds as their blood source, except for the subgenus Microculex which included a bird and two amphibians . The blood meals of Ae. scapularis were identified as blood from birds and horse, and another specimen of the subgenus Ochlerotatus and a female of Limatus fed on avian blood. All blood meals of An. cruzii, Ae. fluviatilis, and Ps. ferox were identified as human blood  had a minor impact on the amplification of host DNA. However, the degree of digestion had a strong negative influence on the amplification and identification of host DNA. Over the past few decades, it has been shown that DNA detection and amplification by PCR are more efficient for engorged mosquitoes kept at temperatures equal or below -70°C . In the analyses . On the analyses .For completely engorged mosquitoes with little or no interference in their digestion, especially engorged with fresh blood, the storage at -20°C for up to 180 days was sufficient to achieve good results in terms of PCR amplification. Tests with only fresh blood meals have limited the establishment of temperatures and storage periods more appropriate for blood already influenced by the digestion process, which can reduce the breadth of these factors. For field-collected specimens, DNA degradation due to the digestion process is unavoidable and, together with temperature, might reduce the viability and positive amplification of blood meals . HoweverIn the present study, amplification success decreased according to the degree of the digestion. Laboratory experiments that assessed blood digestion over the course of hours ,26,27 anCulex  were analyzed for the identified blood source. This analysis resulted in the identification of 19 vertebrate species as hosts . Given that the use of broad-spectrum molecular markers may not be sufficient to identify some of the species present in areas of high fauna richness such as PEP, success rates and specificity of identifications may be limited. However, the resulting identifications in this study allowed us to understand the host use of Culicidae species studied here and, through the identification of large groups of vertebrates, to infer about the potential risks of transmission of pathogens.Despite the relative efficacy for identifying mosquito species , moleculCulex . In addiCx. quinquefasciatus and Ae. scapularis) and one of vertebrate (Homo sapiens) were commonly recorded in both areas. As expected, the diversity of mosquitoes and hosts identified was higher in the forest area than in the urban area, since the PEP certainly has higher species richness [Only two species of mosquitoes , which were considered as opportunistic for choosing avian and mammalian hosts, including humans [The identification of avian blood in a single specimen of g humans . The lowAn. cruzii and Ae. fluviatilis) [Ps. ferox) [An. cruzii and humans and the record of non-human primates in the park [Ps. ferox raises concerns about the transmission risks of the Venezuelan Equine Encephalitis Virus [In PEP, opportunistic species (iatilis) ,69 or sp. ferox)  had thei. ferox) ,70,71. Tthe park  might inthe park . Likewisthe park  in associs Virus .The knowledge of the host use patterns of Culicidae can provide answers to issues that are still poorly understood for some species . Limits of temperature and storage time and better digestion conditions of blood meals investigated in the present study can contribute to the design of entomological surveillance efforts. Our results suggest that most investigated species selected hosts according to their availability in the environment, which is an important contribution given the scarcity of information on host preferences of most mosquito species, either in urban or wild habitats. Given the environmental disturbances and the possibility of emergence (or reemergence) of human pathogens, this type of effort would be instrumental to continually monitor host use of hematophagous insects.


About 1.25 million people worldwide die every year because of road accidents. Risk is higher when drivers use mobile phones, whereas seat belts help to prevent crash-related injury. We aimed to evaluate the prevalence, associated factors, and temporal trend of the use of seat belts and mobile phones among drivers and passengers in Florence, Italy (2005–2015).Use of seat belts and mobile phones use was monitored via direct observation in four areas in the province of Florence. We fitted Poisson regression models with robust variance to investigate the factors associated with the use of seat belts and mobile phones use by the drivers and to explore long-term trends and seasonal patterns in the two time-series.We observed a total of an overall 134,775 vehicles: seat belts were worn by 71.8% of drivers and front-seat passengers and 27.6% of back-seat passengers, while mobile phones were being used by 4.8% of drivers. Drivers were more likely to wear seat belt when transporting passengers  and while driving in the afternoon , and less likely when the front-seat passenger was not wearing seat belts . After an initial increase, seat belts use by the driver decreased over time (-0.5% each year during 2010–2015), with significant peaks and troughs in July and January, respectively. Mobile phone use by the driver was inversely associated with wearing seat belts  and carrying passengers . The proportion of drivers using mobile phones did not vary over time nor showed any clear seasonality.Drivers’ risky behaviours (not wearing a seat belt and using a mobile phone) are associated, showing a global misperception of risk among a subset of drivers. The number of passengers and their behaviour is also associated with the driver’s attitude. The effectiveness of primary enforcement laws has declined in Italy in recent years; therefore, other strategies should be devised and implemented. One of the most relevant priorities in Public Health is road accidents. Worldwide, about 1.25 million people die each year as a result of road traffic accidents, which have become one of the leading causes of death among people aged 15–29 years  plan andIn Europe, almost 85,000 people died of road traffic injuries in 2013 . CompareThe risk of road accidents is higher when the driver is using mobile phones (MP) in handheld mode, especially if the driver is texting, locating the phone, or dialling . The 201SB is one of the most cost-effective preventive measures to reduce injuries, disabilities, and mortality caused by road accidents . The preThe adoption of a specific legislation is a key factor in lowering the health consequences of road accidents. In Italy, the use of SB has been mandatory for front-seat passengers (FSP) of cars since 1989, and for all occupants since 1992 . Moreover, the use of MP in hand-held mode is not allowed while driving . the new points-based driving license, introduced by the new Highway Code in 2003 , is descThe purpose of our study is to evaluate the prevalence, associated factors and temporal trend of SB use  and of MP use among drivers in Florence, central Italy, from 2005 to 2015.The former Local Health Authority (LHA) of Florence has monitored the use of car safety devices and MP usage 16] sinc sinc16] Firstly, descriptive analysis was performed, using the chi square test to compare proportions.Secondly, two binomial multivariate Poisson regression models with robust variance were fitted to estimate the association between covariates and the use of SB (first model) or MP (second model) by the driver in terms of adjusted prevalence ratios. In the first model, we included the year, the season, the time of the day, the number of passengers, the use of SB by the FSP, the use of MP by the driver, and the site of observation as covariates. In the second model, we included the use of SB by the driver as the covariate and we dichotomized the year variable, since we did not find any trend in the descriptive analysis and there was no clear difference among the years.Thirdly, we analysed monthly trends of SB and MP usage prevalence. Since the number of observations changed over months, we obtained the percentage of SB non-use and MP use per month and then plotted the average proportion of drivers wearing SB and using the MP while driving for each month  during the study period.seesection below), and more stationary in the following years, we decided to only use data from 2010 to 2015 for the study of seasonality.We fitted additive Fourier terms  into Poisson regression models with a robust variance estimator to more accurately investigate the presence of seasonal patterns in the two time-series. Fourier terms were introduced in the model, which looked for cyclical patterns with 12-month, 6-month, and 3-month periods; smaller periods were not considered based on the visual inspection of the time-series. Since both time-series appeared more disturbed in the earlier years of our study period  was set at 0.05. Data were analysed with IBM SPSS Statistic 24 and Stata 14 statistical software.The overall number of observations during 2005–2015 was 134,775, with a maximum  observations per year in 2005 and a minimum  in 2009. On average, we observed 347.3 vehicles per day of observation (CI 95% 324.6–370.1) and around 3 days of observation per month. Overall, data on 134,775 drivers, 29,408 FSP and 2,872 BSP were collected. In As shown in Multivariate association between covariates and SB use while driving and multivariate association between covariates and MP use while driving are shown in MP use while driving did not change significantly over the years , but showed a lower use in spring  and summer  when compared to autumn. The highest use was in the south-eastern area, with a PR of 1.06  compared to the centre of Florence. On the other hand, the best habit was found in north-eastern area, with a PR of 0.28  compared to the Centre. Moreover, MP use was more frequent when the driver was alone in the car .The visual inspection of Considering the period between 2010 and 2015, Poisson analysis with Fourier terms for SB use by drivers shows two main components . Firstly) also shows a linear trend and a periodic annual component.Poisson analysis with Fourier terms for SB use by the FSP see  also shoBased on these results, we can make relevant considerations about the behaviour of drivers and passengers in Florence. We found a lower use of SB (71.8%) compared to other western European countries where their use is mandated by law, according to the WHO European status report on road safety . On the Our findings show that driver’s and passenger’s behaviours tend to be associated. This suggests that strategies to enhance proper behaviours should focus not only on drivers, but also on passengers. For example, educating children at primary and secondary school could have positive effects on their parents . AccordiBy focusing on MP use prevalence while driving, we see no trend over the years, despite the introduction of innovative devices and technologies such as Bluetooth, wireless headphones, and voice recognition software, which can reduce the need for texting and hand-held calling while driving.We found a decreasing trend in SB use by drivers, by 0.5% per year during 2010–2015. This might be partly explained by the rebound effect of the penalty points driving in the license system, introduced in 2003 , which hWe found that there was high seasonal variation in SB use by drivers. In winter, drivers tended not to fasten their SB, possibly due to cumbersome clothes which make it less comfortable to wear SB. This result is somehow counterintuitive, since driving conditions in winter could be perceived as riskier because of climatic conditions  . We alsoOur study has some limitations. First, since the aim of the study design is to minimize misclassifications of SB and MP use, observation sites and periods were chosen using a convenience-based sampling to improve visibility and safety of observers. Sites were chosen on the basis of two pilot surveys, conducted in 2003 and 2004, to test the feasibility. The chosen roads may not be fully representative of the study area, since they had to be large and straight enough to allow the observation. Drivers could be more or less prone to using SB or MP in these streets, thus introducing a bias. Periods were instead chosen according to technicians’ workload and weather conditions, which may have introduced some bias as well. However, the number of observations in each month of the year did not substantially vary across years, and observers were instructed in order to select vehicles in a systematic way, which should minimize the risk of sampling bias affecting our findings.2). This does not allow us to distinguish well among different road types and it could generate a sort of cluster effect since the same people tend to travel along the same road, especially in smaller municipalities. However, we consider it improbable that many vehicles were sampled more than once, given the different time periods of observation and the number of inhabitants of the municipalities.The second limitation is the small number of observation points . Anyway, direct observation reduced the overestimation of proper behaviour, typical of surveys using self-reported information ; over-reWe assessed the prevalence of SB and MP use among car drivers in Florence, Italy, during 2005–2015, and found that drivers’ risky behaviours were often combined, highlighting a general misperception of risk in a subset of drivers. In Italy, primary enforcement laws are already in force, allowing the police to stop motorists for not wearing SB. However, our data suggest that their effectiveness has declined in recent years, suggesting that alternative strategies should be devised and implemented to improve road safety. We believe it critical to adopt a public health perspective and rely on evidence-based prevention (EBP) strategies when planning interventions aiming to improve road safety. Under this regard, the Cochrane collaboration is currently assessing the effectiveness of several interventions aiming to promote the use of SB , such asS1 Fig(TIF)Click here for additional data file.S1 FileLorini C, Pellegrino E, Mannocci F, Allodi G, Indiani L, Mersi A, et al. Uso delle cinture di sicurezza e del cellulare alla guida a Firenze: trend dal 2005 al 2009. Use of seat belts and mobile phone while driving in Florence: trend from 2005 to 2009. Epidemiol Prev 2012; 36 (1): 34–40.(PDF)Click here for additional data file.S2 FileThe variable names are a) date = date of observation; b) zona = area of observation: Central (1), North-western (2), South-eastern (3), North-eastern (4)); c) orario = time of observation: 7-10(1), 10-12(2), 12-15(3), 15-18(4), 18-20(5); 4) P1 to P5 = use of SB by the driver, front seat passenger and back-seat passengers: yes(1), no(0); 5) cell = use of mobile phone by the driver: yes(1), no(0).(7Z)Click here for additional data file.


Preeclampsia is a condition associated with high rates of maternal-fetal morbidity and mortality. It usually occurs in 3–10% of nulliparous women and 18% of previously affected women. Different lines of evidence have demonstrated the role of the father in the onset of preeclampsia. The placenta is the cornerstone of preeclampsia and poses important paternal genetic determinants; in fact, the existence of a “paternal antigen” has been proposed. Nulliparity is a well-known risk factor. Change of partner to a woman without history of preeclampsia increases the risk; however, this change decreases in women with history of the condition. High interval between pregnancies, short sexual intercourse before pregnancy, and conception by intracytoplasmic sperm injection suggest a limited exposure to the so-called paternal antigen. A man who was born from a mother with preeclampsia also increases the risk to his partner. Not only maternal but also paternal obesity is a risk factor for preeclampsia. Fetal HLA-G variants from the father increased the immune incompatibility with the mother and are also significantly associated with preeclampsia in multigravida pregnancies. An analysis of a group of Swedish pregnant women showed that the risk for preeclampsia is attributable to paternal factors in 13% of cases, which could be related to genetic interactions with maternal genetic factors. This review aimed to evaluate the evidences of the father’s contribution to the onset of preeclampsia and determine the importance of including them in future studies. In 2013Preeclampsia is related to deficient placental implantation, with multisystem consequences  in the mother . Its incPreeclampsia is classified as mild and severe and can complicate to hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome and eclampsia .The pathophysiology of preeclampsia is characterized by placental hypoxia and/or ischemia leading to overexpression of hypoxia-inducible factor 1 (HIF1), which in turn increases the expression of the soluble isoform of vascular endothelial growth factor (sFlt) . HypoxicRisk factors for disease development include nulliparity, pregnancy with multiple products, previous history of preeclampsia, vascular and connective tissue diseases, maternal age of >35 years, Afro-American ethnicity, preexistent renal disease, arterial hypertension, type 2 diabetes, and obesity .Previous studies on preeclampsia have considered the mother’s participation almost exclusively. However, the placenta is a transient biparental organ with maternal and paternal contributions. In this way, changes on the expression profile of genes involved in the metabolism and transport in the placenta depend on various maternal and paternal genetic profiles .Increasing evidence has demonstrated the father’s role in the onset of preeclampsia. In The risk of preeclampsia is genetically attributable to the mother (35%), fetus (20%), and couple (13%) . Women aPaternal genetic material plays an important role for the onset of preeclampsia. A triploid (69 XXX) partial mole, with paternal isodisomy in the placenta and a disomic fetus, increases preeclampsia-like symptoms at 19 weeks of gestation . AlthougIn this review, we aimed to describe different lines of evidence regarding the paternal contribution on preeclampsia.p < 0.01)  . Ikedife < 0.01) . The Gua < 0.01) . The sam < 0.01) .When evaluating paternal vs. maternal half-sisters with preeclampsia, The same results were obtained by A woman pregnant by a partner who previously fathered a woman with preeclampsia was highly at risk to develop the disease. In 2004, Cnatinggius et al. evaluated three different scenarios: (1) mothers without preeclampsia and no partner change (control group); (2) mothers with previous preeclampsia, current preeclampsia, and change of spouse who was not fathered by a previous couple with preeclampsia; and (3) mothers with preeclampsia and change of couple who fathered another woman with preeclampsia. The maternal effect was 45%, meanwhile the paternal one was 10% . Conversp = 0.0003), suggesting a protective role. The authors report lower amounts of soluble HLA A and HLA B in the seminal fluid, in partners of preeclamptic women  and mother (OR = 3.3) are products of preeclampsia-complicated pregnancies has been observed; however, this study did not consider the changes of paternity . In men This observed risk increases when severe or early preeclampsia is considered  . Lie (20p = 0.05) higher frequency of preeclampsia in women conceiving by a sperm donor compared to partners in spermatozoa insemination. The type of spermatozoa and number of previous insemination cycles were the variables that influenced the risk of preeclampsia (p = 0.012); in fact, the authors observed that the fewer the number of the insemination cycles, the higher the risk of preeclampsia (in vitro fertilization (IVF), compared to those with primary sperm donation (IUI or IVF) followed by egg donation. A higher frequency of preeclampsia in the latter was observed; therefore, the authors conclude that double gamete donation is associated with increased risk for preeclampsia  must be recognized to avoid rejection . TrophobThe immune nature of preeclampsia was observed by p = 0.002) among primiparous women; further analyses suggested that the differences between pre-eclamptic cases and controls were primarily accomplished by a different transmission from the father of a 14 bp deletion/insertion polymorphism in the 3′UTR region (14 bp del/in) (p = 0.006) , and the frequency of double deletion in both the father and baby was lower in severe early-onset preeclampsia (p = 0.024). The analysis of couples did not reveal significant differences between cases and controls (= 0.006) . In Tan controls . A recencontrols .Semen is not sterile, and its microbes have also been considered as a potential cause of preeclampsia. Repeated exposure to semen seems to create a memory protecting women from preeclampsia and the same could be possible in the case of semen microbes. Therefore, common elements of preeclampsia and infections such as Galectin13, Toll-like receptors, and antiphospholipid syndrome are found .The genetic nature of preeclampsia has also been evaluated through (1) familial cases, (2) twin studies, (3) consanguinity studies, (4) candidate gene evaluation, and (5) linkage analysis. Genes are involved in different pathophysiological mechanisms involved in preeclampsia. Genetic studies that included mother/father/children triads, which allow to determine the sole or joint contribution for preeclampsia, were limited.(MTHFR) and FVL in 92 mother/father/child triads revealed an increased risk only in mothers carrying two mutated copies of MTHFR and one mutated allele in FVL. Therefore, the risk of preeclampsia is not increased in the presence of fetal MTHFR or FVL mutations  and Glu298Asp (eNOS) in control vs. preeclamptic groups in mothers, fathers, and child. The -93 polymorphism (LDL) was higher in preeclampsia only in mothers, but not in fathers or children. The transmission disequilibrium test revealed no differences in the rate of transmission of the studied common vs. mutated alleles  of ≥25 kg/m2 in their partners and studied children. EDN1 gene in mothers with preeclampsia and their partners, showing a significant negative association with the disease in case of fathers , which was strengthened after adjusting the paternal protective factors. The evaluation of two polymorphisms in SOD1 (+35A/C) and SOD2  genes in 698 mother/father/infant triads revealed a significant association with preeclampsia in fathers with Ala16Val (TT genotype) . This study revealed essentially the same risk for preeclampsia in both combined TT genotypes in mothers and fathers  and mother/father/infant triads  (MTHFR were evaluated in women, fetus, and fathers as risk factors for pregnancy-associated complications, including preeclampsia. The authors found significant differences between cases and controls in maternal Factor V and fetal MTHFR, but no differences on any of the polymorphisms analyzed in the father’s group (In < 0.001] . Polymor’s group .Epidemiological, clinical, immunological, and genetic evidences supported the contribution of fathers in the onset of preeclampsia. Despite this, few studies were intended to evaluate the spouses’ role. In this way, the evaluation of paternal-derived immune and genetic materials was performed in order to identify the risk and prognostic markers of preeclampsia. Recent advances in placental epigenetics, along with the use of OMICS tools, ensure the identification of molecular markers associated with the role of fathers in the development of preeclampsia.CG-H gathered and analyzed the data, and wrote the manuscript. MS-M gathered and organized the data and references. ET organized the data. MS-M, JG-O, and ET critically lectured the manuscript. BL-R gathered the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lead, mercury, and cadmium are common environmental pollutants in industrialized countries, but their combined impact on hypercholesterolemia (HC) is poorly understood. The aim of this study was to compare the performance of various machine learning (ML) models to predict the prevalence of HC associated with exposure to lead, mercury, and cadmium. A total of 10,089 participants of the Korea National Health and Nutrition Examination Surveys 2008–2013 were selected and their demographic characteristics, blood concentration of metals, and total cholesterol levels were collected for analysis. For prediction, five ML models, including logistic regression (LR), k-nearest neighbors, decision trees, random forests, and support vector machines (SVM) were constructed and their predictive performances were compared. Of the five ML models, the SVM model was the most accurate and the LR model had the highest area under receiver operating characteristic (ROC) curve of 0.718 (95% CI: 0.688–0.748). This study shows the potential of various ML methods to predict HC associated with exposure to metals using population-based survey data. People can be exposed to high levels of toxic metals from numerous sources, including contaminated air, water, soil, and food . Among tExposure to lead, mercury, or cadmium is known to cause various toxic effects and diseases, including disturbance of lipid metabolism or dyslipidemia . There aRecently, machine learning (ML) prediction models, such as logistic regression (LR) models, k-nearest neighbor (KNN), decision trees (DT), random forests (RF), and support vector machines (SVM) have been developed in many areas of health care research ,19,20. MThis study was based on data from KNHANES 2008–2013, provided by the Korea Centers for Disease Control and Prevention. Using data from the KNHANES 2008–2013 databases, 10,089 subjects aged 12 years and older who had no missing responses on the questionnaire were included in this study. The KNHANES program was approved by the KNHANES Institutional Review Board (IRB) and was conducted in accordance with the Ethical Principles for Medical Research Involving Human Subjects, as defined by the Helsinki Declaration . All study participants provided informed written consent.The KNHANES included well-established questions to determine the demographic characteristics and health status of the participants. This included questions on age, sex, income, physical exercise, smoking and drinking habits, and food intake. Daily energy intakes were assessed using 24 h recall and food intake frequency methods. The participants’ heights, weights and waist circumferences were measured. Then, the body mass index (BMI) was calculated as weight (in kilograms) divided by the square of height (in meters). Blood samples were collected by venipuncture after 10–12 h of fasting. Then, the total cholesterol was measured by enzymatic methods using commercially available kits  within 2 h of blood sampling. The criterion for HC was a total cholesterol level of 240 mg/dL or higher or the use of lipid-lowering medications . FurtherAll features were extracted from the original dataset and were transformed by scaling each feature to a range between zero and one using the ‘MinMaxScalar’ class from the pre-processing module of the Python scikit-learn library. After data processing, the input features were sex, age, income, BMI, waist circumference, exercise, smoking habits, alcohol drinking, and energy intake, as well as the blood concentrations of lead, mercury, and cadmium. Then, the entire data set was split into training and test sets at a ratio of approximately 7:3. Therefore, 8795 subjects were placed into the training set, while 1294 subjects were placed in the test set. The overall accuracy of each model was evaluated using k-fold cross-validation with k = 10. We used the default hyperparameter configurations unless otherwise specified. The LR model used L2 regularization with a primal formulation. The primal formulation was used because there are more samples than features. The regularization strength was set to 0.1, and the model was trained for 100 iterations before convergence. We used the KNN classifier ‘KNeighborsClassifier’ from sklearn.neighbors and the number of neighbor points was set to 3. In this study, the DT model used Gini impurity to measure the quality of split. The minimum number of samples required to split a node was set to two, and the minimum samples per leaf was set to one. The RF model used ten separate DT estimators and the SVM model used a linear type as its kernel with the shrinking heuristic enabled. The model used a C value of one and probability estimates were enabled to plot a receiving operating characteristic (ROC) curve for the model. All of the models were implemented using the scikit-learn library (version 0.20.1).t-tests. The statistical significance of categorical variables between the HC and control groups were determined using the Mantel–Haenszel chi-square test. The prevalence of HC was compared among tertiles of blood metal concentration using multivariate logistic regression after adjusting for the other metals. As a measurement of the performance of the prediction model, the accuracy was calculated for all data, the training set, and the test set. The ROC curve was also used as a metric to measure the prediction model performance. All statistical analyses were carried out using SAS v. 9.4 .We calculated the frequency and, where appropriate, the percentage or mean and standard deviation of the demographic characteristics to describe the sample population with respect to the HC categories. We compared the values of continuous variables between the HC and control (non-HC) groups using p < 0.01). The non-HC group had significantly higher mean monthly incomes, percentage of regular exercise, and mean daily energy intakes than the HC group (p < 0.01).p for trend = 0.001), mercury (p for trend = 0.020), and cadmium (p for trend < 0.001).p = 0.174). However, the p values between the SVM or LR model, and all other models were less than 0.003.The accuracies of all of the models are shown in Similarly, following the training of each model with the training dataset, the SVM and LR models had the best accuracy when analyzing the test dataset. On the other hand, the DT model has the lowest accuracy compared to other models .The area under ROC curve (AUC) for all of the prediction models is summarized in Accumulating epidemiological and experimental studies have provided strong evidence that lead, mercury, or cadmium exposure can affect lipid metabolism, including the disturbance of total cholesterol levels ,25,26. RIn the current study, we presented an empirical comparison of five different techniques for estimating the risk of HC using KNHANES data on 10,089 participants. The LR, RF, and SVM outperformed the DT and KNN techniques, achieving overall accuracies of >0.86 and area under the AUC of >0.60. The results of our study show that complex ML models, such as SVM, tend to outperform simpler models such as DT when predicting HC associated with exposure to heavy metals. As KNN is a non-linear classifier, it tends to perform better with many data points. Therefore, effective feature selection may improve the performance of the KNN classifier .The strength of this study is that it is based on a large dataset, reflected by the number of participants and the number of important confounding variables, including cigarette smoking, alcohol drinking, physical exercise, and energy intake. Limitations of our study include the lack of more features and lack of further parameter optimization to avoid overfitting. In addition, clinical data, such as history of medication and prevalence of diseases affecting the lipid metabolism, were not included in the models, which may decrease the prediction accuracy of some of the algorithms. Nevertheless, the results of this study suggest that the performance of ML models can be very sensitive to the values of the hyperparameters and selected features. Therefore, the hyperparameters and features used to build ML models should be carefully explored and tuned to achieve the best predictive accuracy.Prediction models using ML algorithms have shown solid prediction capabilities in various application domains including public health and environmental science. In this study, we presented a development and comparison of five popular ML models on predicating HC using a population-based database from the KNHANES. The results of this study showed that prevalence of HC among subjects exposed to lead, mercury, and cadmium could be predicted using different ML approaches with high accuracy. The results also show that various ML techniques can significantly vary in terms of their performance (accuracy and AUC) and that the SVM and LR models were the better models for predicting the risk of HC in the general population following exposure to lead, mercury, and cadmium. These findings suggest that ML approaches could be used as alternative methods in the prediction of the risk of HC due to exposure to metals.


HIV-associated encephalitis (HIVE) is one of the common complications of HIV infection, and the pathogenesis of HIVE remains unclear, while lncRNA might be involved it. In this study, we made re-annotation on the expression profiling from the HIVE study in the public database, identified the lncRNA that might be involved in HIVE, and explored the possible mechanism.GSE35864) of three regions of brain tissues  was chosen, updated annotation was performed to construct the non-cording-RNA (ncRNA) microarray data. Morpheus was used to identify the differential expressed ncRNA, and Genbank of NCBI was used to identify lncRNAs. The StarBase, PITA and miRDB databases were used to predict the target miRNAs of lncRNA. The TargetScan, PicTar and MiRanda databases were used to predict the target genes of miRNAs. The GO and KEGG pathway analysis were used to make function analysis on the targets genes.In the GEO public database, the microarray expression profile (Seventeen differentially expressed lncRNAs were observed in the white matter of brain tissues, for which 352 target miRNAs and 6,659 target genes were predicted. The GO function analysis indicated that the lncRNAs were mainly involved in the nuclear transcription and translation processes. The KEGG pathway analysis showed that the target genes were significantly enriched in 33 signal pathways, of which 11 were clearly related to the nervous system function.The brand-new and different microarray results can be obtained through the updated annotation of the chips, and it is feasible to identify lncRNAs from ordinary chips. The results suggest that lncRNA may be involved in the occurrence and development of HIVE, which provides a new direction for further research on the diagnosis and treatment of HIVE. Cognitive impairment is one of the challenges that HIV patients may face . HIV-1 eIn the human genome, more than 70% of the genes are transcribed into RNAs, but less than 2% of them are protein coding genes, and most of them are noncoding RNAs . Non-codIn recent years, expression microarray technology has played an important role in the research on the disease occurrence and development, and many research results can be reviewed and downloaded in the public database. Because updated annotation of microarray results has always been continuing, new results and novel revelation can be accomplished through the re-annotation and analysis of published studies on microarray results. In this study, we retrieved the HIVE study related microarray data from the GEO database, re-annotated and analyzed these data, identified the lncRNAs that might be involved in the pathogenesis of HIVE, and performed the correlation analysis (the work flow was shown in http://www.ncbi.nlm.nih.gov/geo), curated by the National Center for Biotechnology Information (NCBI), is a public functional genomics database, and the data could be downloaded for free. In the GEO database, the microarray expression profile (GSE35864) in three regions of the brain, basal ganglia, white matter, and frontal cortex, in normal, HIV infected, HIV infected with neurocognitive impairment, and HIV infected with both neurocognitive impairment and encephalitis patients was chosen. Twenty-four human subjects in four groups were examined: Group A (n = 6) HIV-1 uninfected with no neuropathological abnormalities at autopsy; Group B (n = 6) HIV-1-infected (HIV+) neuropsychologically normal with no neuropathology; Group C (n = 7) HIV+ with substantial HIV-associated neurocognitive impairment (HAND) as defined below, with no encephalitis (HIVE) or substantial neuropathological defect; Group D (n = 5) HIV+ with HAND and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix Human Genome U133 Plus 2.0 Array platform.The Gene Expression Omnibus . Morpheus (https://software.broadinstitute.org/morpheus/) was used to analyze online and identify the differential expressed “NR_” between the groups  of each region tissue . This analysis was based on the t-test and adjusted according to the characteristic that the noise of microarray data was correlated with the peak value of expression data. We considered that p-value <0.01 was statistically significant. The number of upregulation and downregulation was 100). The Wayne map was drawn based on the intersection of the above results to select differential “NR_” participating in the occurrence and development of HIVE. The pseudogenes, rRNAs, microRNAs and other short RNAs  were filtered out through Genbank from NCBI database. The final remainder was the differential expressed lncRNAs.First, the latest annotation files of HG-U133_Plus_2 Annotations, CSV format, Release 36 (7/12/16) of the Affymetrix Human Genome U133 Plus 2.0 Array were downloaded from the website https://www.ncbi.nlm.nih.gov/nucleotide), which were then input into StarBase (http://starbase.sysu.edu.cn/), PITA (https://genie.weizmann.ac.il/pubs/mir07/mir07_data.html) and miRDB (http://www.mirdb.org/), to predict the target miRNAs of the lncRNAs. The miRNAs were input into TargetScan (http://www.targetscan.org/vert_71/), PicTar (http://pictar.mdc-berlin.de/) and MiRanda (http://www.microrna.org/microrna/home.do) to predict the corresponding target genes.The sequences of lncRNAs were retrieved through NBCI Nucleotide (http://david.abcc.ncifcrf.gov/) is an online program that provides a comprehensive set of functional annotation tools for researchers to understand biological meaning behind many genes. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the identified DEGs using the DAVID database. After GO functional enrichment analysis, we considered the biology process terms with p-value <0.05 was statistically significant. For KEGG analysis, we considered the subpathway with p-value <0.05 was statistically significant.The Database for Annotation, Visualization and Integrated Discovery  of each region tissue . As shown in We re-annotated the The target micRNAs of the lncRNAs were identified from the database, and a total of 352 target miRNAs were predicted in 17 differentially expressed lncRNAs . The 6,6The 6,659 target genes were uploaded to the DAVID software, and the significant GO classification and KEGG pathway were selected. GO cell component (CC) analysis showed that the target genes were obviously clustered in the nucleus, cytoplasm, Golgi apparatus, lysosome, plasma membrane, etc. , Table 2p < 0.05), which were obtained through KEGG analysis, including mucin type O-Glycan biosynthesis, proteoglycans in cancer, pathways in cancer, glutamatergic synapse, long-term depression and other relevant pathways (pathways . Eleven There are two main highlights in the research. (1) The differentially expressed lncRNAs were identified through the re-annotation of published microarray results. (2) The target miRNAs of the lncRNAs and target genes were predicted using a bioinformatics method, and GO function and KEGG pathway analyses were performed to learn about the possible mechanisms of lncRNA involved in the occurrence and development of HIVE.β signaling pathway was also involved in the pathophysiology of the nervous system, which can limit inflammation and reduce neurological damage in the nervous system infection process (β signaling pathway was also related with the tolerance of dendritic cells (β signaling pathway can inhibit the progression of autoimMune encephalopathy (In recent years, hundreds of lncRNAs have been discovered, and the changes in the expression of lncRNAs have been associated with the occurrence and development of many diseases. Plenty of evidence has shown that lncRNA is involved in the replication process of the virus  and that process . The TGFic cells . Togethealopathy . For thealopathy . The actalopathy , and thealopathy . Moreovealopathy . TherefoThe brand-new microarray results from a different perspective can be constructed through the updated annotation of the precious and published microarray data. In this study, the lncRNA results were obtained through the re-annotation of microarray data, which provides the foundation for further research on the role of lncRNA in the occurrence and development of HIVE. From the point of view of the lncRNA-miRNA-target genes, cluster analysis was performed by various bioinformatic methods to explore the role of lncRNA. GO analysis showed that lncRNA may be involved in the occurrence and development of HIVE via its participation in the nuclear transcription and translation. The KEGG pathway analysis showed that most of the KEGG pathways with statistical significance were associated with the function of the nervous system. Therefore, we can speculate that lncRNA is indeed involved in the occurrence and development of HIVE, which is of great significance for future research on lncRNA on HIVE. It is also proved that it is feasible to identify lncRNAs from public database.10.7717/peerj.5721/supp-1Data S1Click here for additional data file.


Global trade agreements have shaped the food system in ways that alter the availability, accessibility, affordability, and desirability of ready-to-eat foods. We assessed the time trends of ultra-processed foods purchases in Mexican households from 1984 to 2016. Cross-sectional data from 15 rounds of the National Income and Expenditure Survey  were analyzed. Food and beverage purchases collected in a daily record instrument (over seven days) were classified according to their degree of processing according to the NOVA food framework: (1) Unprocessed or minimally processed foods; (2) processed culinary ingredients; (3) processed foods; and (4) ultra-processed foods. From 1984 to 2016, the total daily energy purchased decreased from 2428.8 to 1875.4 kcal/Adult Equivalent/day, there was a decrease of unprocessed or minimally processed foods  and processed culinary ingredients , and an increase of processed foods  and ultra-processed foods . Given that ultra-processed foods purchases have doubled in the last three decades and unprocessed or minimally processed foods purchased have gradually declined, future strategies should promote the consumption of unprocessed or minimally processed foods, and discourage ultra-processed foods availability and accessibility in Mexico. Since the 1980s, the food industry has dominated the food system with marketing strategies encouraging consumers to eat more ready-to-eat products in their diet . ConsequNationally representative dietary studies conducted with the NOVA food framework have indicated that a high dietary energy contribution of ultra-processed foods is associated with a lower dietary quality, which is characterized by a higher intake of free sugar and saturated fat, and by a lower intake of protein and dietary fiber ,4,7,10. In Mexico, ultra-processed food retail sales grew 29.2% (from 164.3 to 212.2 kg per capita/year) between 2000 and 2013 . As a coThis study used 15 rounds of the National Income and Expenditure Survey ) conducted by the National Institute of Statistics and Geography of Mexico ): 1984, 1989, 1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014 and 2016. All ENIGH rounds were collected between August and November . The ENIGH is a probabilistic based survey with a two-stage stratified clustered sampling design, representative at the national level, and urban and rural strata. It collects data on household expenditures in food and beverages, and sociodemographic characteristics such as household composition, income, head of household educational level, and place of residence . Detailed information about data collection is available elsewhere [n = 25,241) and with incomplete sociodemographic information (n = 1427). We also excluded households that reported a total energy purchased ≥10,000 kcal/Adult Equivalent (AE)/day (~percentile 99.5) because this quantity was considered implausible for individual consumption (n = 1797). Thus, we analyzed the daily food and beverage purchases from a total sample of 277,509 Mexican households.In each round, the number of households varied . In our analyses, we excluded households that did not report food and beverage expenditures , he/she was also asked to record all those food and beverages obtained as a gift, through subsidized prices, or produced by the family (non-monetary expenses). Both types of expenses were deflected by the national consumer price index of November 2016 to enable comparability over time. In this instrument, the quantity (kg or lt) of both types of expenses were recorded for each food item reported. For beverages, lt were converted to kg by using their density. The list of food items in the instrument were: (1) Single foods  or (2) group of foods .n = 14 food subgroups); (2) processed culinary ingredients (n = 3 food subgroups); (3) processed foods (n = 4 food subgroups); and (4) ultra-processed foods (n = 10 food subgroups). When the food or beverage item was composed by a food group, we classified it into one NOVA group based on the classification of the majority of the food or beverages included in the item.We classified the food and beverage items according to the NOVA framework into: (1) Unprocessed or minimally processed foods  Percentage of consumers (we considered the household as a consumer if it reported any expenditure for that NOVA food group); (2) percentage of expenditure; (3) percentage of volume; and (4) percentage of energy purchased. We also estimated the total expenditure, volume, and energy purchased per day by AE. Energy content of purchased items was estimated using the food composition database compiled by the National Council for the Evaluation of Social Development Policy ) and the AE was estimated by dividing the recommended dietary allowance (RDA) for energy of each household member (according to age and sex) by the energy RDA for an average adult  .Household size was defined as the sum of all the AE within the household. Household composition was classified in four types: (1) Adults only, (2) adults and children only, (3) adults and adolescents only, and (4) adults, adolescents and children. Head of household educational level was classified according to the maximum degree of studies as: Low (from 0 to 6 years of study), medium (from 7 to 12 years of study) and high (≥13 years of study). Household women’s occupation was identified based on the type of employment that they reported and was classified as: Work inside home or outside home. Household income was deflated using November 2016 as a base and divided by the household AE; additionally, we stratified households in each survey into quintiles according to their income/AE. We only present quintiles I, III and V . Place of residence was classified as rural (locations with <2500 inhabitants) and urban (locations with ≥2500 inhabitants). The country was divided into four regions with common sociodemographic characteristics: North region ; Central region ; Mexico City; and South region .We estimated means and proportions of sociodemographic characteristics to describe the households across the years.We estimated the total expenditure and volume of purchases per day per AE, and for each NOVA food group the % consumers, % expenditure, and % volume in each survey. We performed linear regression models with a continuous variable for the years to estimate the linear trend . In this analysis, we present the linear regression coefficient (crude and adjusted) associated with a 10-year change.We also estimated total energy purchased, and for each NOVA food group and subgroup the % energy purchased in each survey years. We performed the similar linear regression models previously described. In this analysis, we also present the linear regression coefficient (crude and adjusted) associated with a 10-year change.Additionally, we estimated the mean of daily energy purchased  of the NOVA food groups, and ultra-processed food subgroups across the years. For this estimation, to ease interpretation, we regrouped the 10 ultra-processed food subgroups into five: (1) Cookies, pastries, sweet bread, and breakfast cereals; (2) ultra-processed tortilla, bread and meats; (3) sugar-sweetened beverages ; (4) sweet and salty snacks; and (5) other ultra-processed foods.Finally, we assessed the trends in the % energy purchased of ultra-processed foods by household composition, head of household educational level, household women’s occupation, household socioeconomic status, residence area, and region. We performed multiple linear regression models  with an interaction term between the year (dummy variables) and each sociodemographic characteristic to estimate the adjusted mean of % energy purchased of ultra-processed foods for each year and sociodemographic characteristic strata. We assessed if the % energy purchased of ultra-processed foods in 2016, and the change from 1984 to 2016 had a statistically significant difference between sociodemographic characteristics.p value < 0.05 to test statistical significance.All the analyses were performed using Stata version 12.0  with the survey command to account for the design effect of complex surveys and weighting factors to obtain national representation. For some analyses, although they were conducted including all years, we only present the results of the ENIGH 1984, 1992, 2000, 2008 and 2016 to ease interpretation. We considered a In p < 0.05). Total expenditure and volume of purchases decreased over time. The % consumers and the contribution to volume and expenditure of unprocessed or minimally processed foods, and processed culinary ingredients decreased over time; whereas processed food and ultra-processed foods increased. However, the magnitude of the change differed. Processed foods’ indicators showed the smallest change across the years. The largest change in % consumers was observed for processed culinary ingredients, with a reduction of 8.32 percentage points (pp) for every 10 years; followed by ultra-processed foods with an increase of 3.91 pp for every 10 years. On the other hand, the largest changes in the % expenditure and in the % volume were observed for ultra-processed foods . Moreover, both indicators of ultra-processed foods had the greatest relative changes, the % expenditure increased by 88.05% and the % volume increased by 125.70% between 1984 and 2016. When we adjusted these indicators for sociodemographic characteristics, we observed similar trends but with a smaller magnitude in general. For instance, the % expenditure in unprocessed or minimally processed foods changed from −3.08 to −2.07 pp for every 10 years; whereas in ultra-processed foods changed from 3.80 to 2.71 pp for every 10 years.In p < 0.05), the total energy purchased decreased 169.01 kcal/AE/day , the relative change was −22.78%. For NOVA food groups, for every 10 years, there was a decrease of 3.04 pp in % kcal of unprocessed or minimally processed foods  and 1.79 pp in % kcal of processed culinary ingredients ; while there was an increase of 0.42 pp in % kcal of processed foods  and 4.42 pp in % kcal of ultra-processed foods . Some of the subgroups with the largest absolute change (for every 10 years) in each NOVA food group were corn (−3.07 pp), oils and fats (−1.16 pp), cheeses (0.31 pp) and carbonated sugar-sweetened beverages (0.73 pp). Additionally, some of the food subgroups with the largest relative change (from 1984 to 2016), in each NOVA food group, were poultry and game (+147.83%), sweeteners (+34.00%), processed meat (+83.33%) and salty snacks (+900.00%). After adjustment for sociodemographic characteristics, we observed similar trends in NOVA food groups and subgroups but with a smaller magnitude; although for some of them the magnitude was larger.In p < 0.05), and the change from 1984 to 2016 was larger (p < 0.05) in: Households in which the head of the family had a high educational level compared to those in which the head of the family had a low and medium educational level; households where the women work outside the home compared to those in where the women work inside the home; households with medium and very high socioeconomic status compared to those with low socioeconomic status; households from urban areas compared to those from rural areas; and households from the central and north region compared to those from the south region. On the other hand, there were no differences in the change from 1984 to 2016 among the households with different composition. However, we observed that the energy purchased contribution of ultra-processed foods was higher (p < 0.05) in 2016 in household with adults and adolescents, adults, adolescents and children, and adults and children compared to those with adults only.We studied the trends of food purchases over a one-week period from nationally representative samples of Mexican households. From 1984 to 2016, there was a decrease of unprocessed or minimally processed foods and processed culinary ingredients; however, in processed foods, there was a slight increase. On the other hand, for ultra-processed foods there was an important increase. The increase of ultra-processed foods was very sharp in earlier years, it increased from 10.5% kcal in 1984 to 22.3% kcal in 2006; whereas, from 2006 onwards, there were small increments and decrements with the highest point being 2012 with 23.7% kcal.Ultra-processed foods are expanding in the food system across the globe. We found in Mexico, that the energy contribution of ultra-processed foods to household’s purchases increased from 10.5% kcal in 1984 to 23.1% kcal in 2016. Similar trends, but in higher magnitude, have been documented in previous studies of household food expenditure from Brazil  , Canada Popkin et al. propose that income growth, policy liberalization, infrastructure improvement, urbanization and the rise of rural nonfarm employment have promoted changes in the food system and diet . MoreoveWe observed that the household income in Mexico rose from $10,514.5 in 1984 to $18,194.9 MXN/AE/quarterly in 2016. Parallel to the increase in income, the energy contribution of ultra-processed foods to total purchases maintained, in general, an upward trend across the years; some of the few exceptions were in 1996 and 2008, where a slight decrease from the previous survey was observed. It is possible that these decreases were related to the economic crisis at the end of 1994 and mid-2008 .At the beginning of the 1980s, Mexico faced a severe economic crisis characterized by the falling of international oil prices, the decline in the agricultural sector and the inability to pay international loans on time. To respond to this crisis, in 1986, Mexico joined the General Agreement on Tariffs and Trade (GATT) that facilitated the entrance of foreign food corporations . NeverthAs urbanization increases, supermarkets, convenience stores, and fast-food restaurants expand and catch a greater number of consumers . In paraPrevious studies have found that ultra-processed foods consumption differs among strata of sociodemographic characteristics ,5,6,8. IInterestingly, after we adjusted trends in the energy contribution of NOVA food groups and subgroups by sociodemographic characteristics  changes over time decreased in magnitude slightly but were still present in most food groups. This means that other factors, most likely environmental, macroeconomic or even cultural are also driving these changes. Moreover, there were some food subgroups such as corn tortilla, and milk, in which the adjusted trend was larger than the crude trend. This means that if sociodemographic variables, such as income and education, had not improved over time, the expenditure on corn tortilla should have increased more (because higher income and education are negatively associated with corn tortilla), and the expenditure on milk should have decreased more (because higher income and education are positively associated with milk).We found that among unprocessed or minimally processed foods, from 1984 to 2016, there was an increase in the contribution of corn tortilla , and a decrease in corn . Although corn tortilla is unprocessed or minimally processed, it resembles ultra-processed foods because it is convenient and does not require any culinary preparation to be consumed. Whereas corn is used in traditional culinary preparations . From a public health stand, it might be hard to limit the increased popularity of ready-to-eat products, but the creation and promotion of ready-to-eat products within the unprocessed or minimally processed foods could be an appealing alternative.In our study, we found that the purchases of ultra-processed foods increased, not only in terms of energy contribution , but also in terms of absolute energy . Moreover, this was the case even though the total kilocalories purchased decreased from 2428.8 to 1875.4 kcal/AE/day. The finding that total energy purchased decreased might be due to an increase in thepurchases away-from-home. Unfortunately, the type of food consumed away-from-home is not collected in this survey. Given the high number of food chain establishments in Mexico ,31, futuIn the last two surveys (2014 and 2016) we observed a decrease in the energy contribution of ultra-processed foods, especially from carbonated sugar-sweetened beverages and salty snacks. This could be due to the launch of the National Strategy for the Prevention and Control of Overweight, Obesity and Diabetes in 2014 , which iStrengths of our study include the use of national representative and systematically collected information. This enables the analysis of a large time frame trend of food purchases in Mexico. On the other hand, by using the NOVA food framework, we could identify how ultra-processed foods are becoming part of the dietary patterns of Mexican households.There are also limitations of our study. First, household’s food and beverage expenditure records do not consider waste and foods consumed away-from-home. Therefore, they do not accurately reflect dietary intake. For instance, in a previous analysis with 24-h recalls, from the 2012 National Health and Nutrition Survey, we found that the contribution of unprocessed or minimally processed foods, processed culinary ingredients, processed foods, and ultra-processed foods was 54.0%, 10.2%, 6.0%, and 29.8% kcal, respectively [Another limitation of our analysis is that breakfast cereals were not collected from 1984 to 2000, affecting the estimation of the ultra-processed foods purchases’ trend. Next, processed foods and ultra-processed foods tend to be reformulated by food industries. Since in our study we used only one nutritional composition table across all surveys, it is possible that the energy purchased contribution of some of these products might be under-estimated or over-estimated in some time periods. Finally, since our study focused only on energy, further studies are needed to evaluate how the trend of ultra-processed foods is associated with the trends in macro and micronutrients.Our study indicates that ultra-processed foods purchases in Mexico have doubled in the last three decades, paralleling a gradual decrease in unprocessed or minimally processed foods, and processed culinary ingredients purchases. Moreover, we found that this trend is only partially explained by sociodemographic changes in household size and composition, education level, income, women’s occupations, urbanization, and geographical region. Hence, other macroeconomic, environmental, and sociocultural factors must be important drivers of these trends. In order to improve the dietary patterns of Mexico, future food environment, food system, behavior-change communication and education strategies should promote the consumption of unprocessed or minimally processed foods such as fruits and vegetables, whole grains, nuts, and legumes; and discourage ultra-processed foods availability and accessibility.