ADRA2A rs553668, DRD2 rs1124491 and SLC6A4 rs6354 were identified to be associated with ADHD. A significant multiplicative and additive gene-environment interactions were observed between the PSE and the ADRA2A rs553668 in relation to ADHD and ADHD-ODD. The risk of the genetic variants in ADHD was increased significantly if the child had prenatal tobacco exposure. The genetic risk for ADHD could be influenced by the presence of environmental risks. The environmental and the genetic risks are not distinct to each other. More gene-environment interaction studies were needed to reveal the etiology of ADHD.The purpose of our study was to test the hypothesis that prenatal tobacco smoking exposure (PSE) could modulate the association of genetic variants with ADHD. A community based case-control study was conducted among Chinese children and 168 ADHD patients and 233 controls were recruited by using combination diagnosis of DSM-IV, SNAP-IV and semi-structured clinical interview. Logistic regression analysis was performed to estimate the effect of prenatal tobacco smoking exposure and genotype frequencies on ADHD susceptibility individually by adjustment for potential confounders. Multiplicative and additive interaction analysis were performed to evaluate the interactions between risk genes and PSE with regard to ADHD. Prenatal tobacco smoke exposure was a significant risk factor of ADHD even after adjusted for other potential confounders.  Previous studies reveal that individuals affected by ADHD frequently lead to various social functional impairment, such as social communication disorder, bad peer relationships, academic performance difficulties3, develop personality disorder, substance abuse and even criminality5.Attention-deficit/hyperactivity disorder (ADHD) is a chronic neurodevelopmental disorder characterized by developmentally inappropriate level of inattentiveness and hyperactivity. It is one of the most frequently diagnosed disorders among children and adolescents, with the pooled prevalence of 7.2%7. The genetic architecture of ADHD is complex and numerous single nucleotide polymorphisms (SNPs) of genes involved in the regulation of neurotransmitter systems are identified to be associated with ADHD10.Among them, genes involved in the dopaminergic, serotonergic and noradrenergic pathways are the most common candidate genes for ADHD studies13.As a complex multi-factorial disease, exact etiology of ADHD is still elusive. The biologic basis for ADHD is thought to be largely genetic14. Some prenatal and perinatal factors are reported to be associated with ADHD, such as younger maternal pregnant age15, caesarean, induced delivery, preterm birth, low birth weight16, and mother smoking during pregnancy. With regard to prenatal smoking, it is constantly identified to be a risk factor for offspring hyperactivity symptom and ADHD19. Evidence from epidemiological researches suggests that maternal cigarette smoking and environmental tobacco exposure during pregnancy are all in association with high risk of ADHD. Hyunjoo Joo20 and his colleagues have described that independent and combined exposure to pre- and postnatal secondhand smoke were associated with ADHD, particularly with the hyperactivity symptom domain. A large population-based birth cohort have provided that maternal smoking during pregnancy was associated with children hyperactivity symptom19. Although large numbers of studies demonstrated a relationship between maternal smoking exposure during pregnancy and ADHD, most of previous studies have investigated the effect of smoking exposure only and there were few studies on gene–environment interactions which were more likely to play the real role in the etiology of ADHD21.Adding to genetic influence, environmental factors are assumed to be co-involved in the etiology of ADHD21, but it may be required to uncover the genetic liability14. The studies about the G ene× Environment studies of gene and smoking exposure on ADHD are limited. A gene-environment interplay study reports that the co-exist group of prenatal smoking exposure and 10 R/10 R genotype of DAT gene showed a higher mean scores of hyperactivity-impulsivity and inattentiveness than other groups22. But the findings remain speculative since the participants are lack of a clinical diagnosis of ADHD, and the ADHD scores of the participants was derived from the Conners’ Parent Rating Scale which will lead to the overdiagnosis of the ADHD.Importantly, the environmental risk is not distinct to the genetic influence23. Therefore, gene and environment interaction research will be an inevitable way to explore the etiologic mechanisms of ADHD. However, studies about the joint effects of gene and environmental factors on ADHD is limited14. To fill the knowledge gap, we conducted the case-control study to elucidate the potential synergistic effect of the prenatal tobacco exposure during pregnancy and children genetic variants on children diagnosed ADHD and its symptom domains.Developing of novel technologies have made it more common in identifying genetic risk factors of ADHD, however, proved the genetic architecture of ADHD to be far more complex, since the genetic influences expressed only additional risks to disease susceptibilityp < 0.05). There were no significant difference between ADHD patients and controls in terms of family history of neural system diseases, parental marital status, preterm birth (pregnancy < 37weeks), Low birth weight(birth weight <2500 g) and delivery mode, levels of blood lead and postnatal tobacco exposure. Meanwhile, ADHD children scored higher of SNAP-IV scales  and of CBCL total scores than controls . Thus they were excluded from further analysis. Consequently, a total of 19 SNPs remained in the subsequent analysisA total of 22 SNPs were detected. The genotype distributions of ADRA2A rs553668, DRD2 rs1124491 and SLC6A4 rs6354 were nominally significant at the uncorrected 0.05 significance level with adjustment for age and gender. Among these SNPs, ADRA2A rs553668G allele was associated with an increased risk of ADHD in the additive model, and SLC6A4 rs6354 (GG + GT) was associated with an increased risk of ADHD, whereas DRD2 rs1124491 (AA) was associated with a decreased risk of ADHD. After FDR adjustment, SLC6A4 rs6354 remained statistically significant , but the SNP was not associated with ADHD-ODD. Additionally, individuals who carried DRD2 rs1124491 AG or GG genotype displayed a risk association with inattention symptom compared to AA genotype . Compared with those carrying TT genotype, the risk association with ADHD-ODD was increased significantly in individuals carrying the SLC6A4 rs6354 GT or GG genotype , but no association was observed in other ADHD symptom domains. The ORs and 95% CIs changed slightly in different adjustment model  showed a risk association with ADHD and all symptom domains, and slight change of the OR and 95% CI was observed in different adjustment model . The strong association was shown in ODD symptom domain  and in the hyperactivity symptom domain , and RERI was 1.88(95% CI = 0.06–4.12), Meanwhile, multiplicative interaction was displayed in this group (Pmul = 0.003). The multiplicative and additive interactions were found in all symptom domain of ADHD. However, no multiplicative and additive interactions were detected between DRD2 rs1124491 polymorphism and PSE, nor between SLC6A4 rs6354 polymorphism and PSE , compared with AA carrier without PSE. But the OR for the rs553668 GG or AG carriers with PSE, was significantly increased to 3.54 in children, especially a strong association with hyperactivity and ADHD-ODD, and the effect size did not diminished even after adjusted by potential environmental influencing covariates. In addition, the study found three SNPs of ADRA2A rs553668 GG or AG genotype were in concordance with the findings of several studies, and indicated that rs553668, being known as the DraI site in the 3′-untranslated region of ADRA2A gene, A-to-G variant increased the risk of ADHD25. Meanwhile, the findings showed the DRD2 rs1124491 in the significant association with inattention type of ADHD. DRD2 gene (Dopamine Receptor D2 gene), encodes the D2 subtype of the dopamine receptor. A population-based birth cohort have reported an association of DRD2 rs1124491 with ADHD in males and low persistence in female ADHD patients27. Moreover, a common variant SLC6A4 rs6354 was identified to be in association with ADHD-ODD. SLC6A4 rs6354 located in the untranslated(exon 2) 5′-UTR region of SLC6A4 gene—a potential module of exonic splicing enhancer or silencer by bioinformatics prediction— and may affect the expression level or exonic splicing of serotonin28. Evidences show that one repeat length polymorphism-STin2 located in the untranslated exon 2 of SLC6A4 gene may be significantly associated with major depressive disorder and suicide29.With regard to the findings in the genetic analysis, the results of risk association with ADHD in the children carrying 30 and his colleagues found maternal smoking during pregnancy was shown a significant association with offspring ADHD symptom scores in twins study after adjustment by genetic factors, although the main effect of cigarette exposure was small. One gene-environment interaction study evaluated maternal smoking and the DAT gene. In the cases with co-exist of maternal smoking during pregnancy and 10 R/10 R genotype of DAT gene, the mean score of hyperactivity-impulsivity and inattentiveness derived from the Conners’ Parent Rating Scale were higher than other groups22. The findings in the current study infer that the susceptibility of ADHD for the unfavorable ADHD genotypes carriers might increase when the children having the history of in-utero exposure to cigarette smoking.A limited number of G × E studies have shown that genetic factors interact with maternal smoking to heighten risk for ADHD. Anita Thapar31. The neurotoxic effects of nicotine exposure on human fetal brain development have not been studied directly. However, a large number of animal studies have found that nicotine has obvious neurotoxic effects on fetal brain development32. Animal models confident that the nicotine can readily cross the placental barrier into fetal circulation, and the blood-brain barrier, which protects the adult brain from toxic chemicals, is not fully formed until about six months after birth33. So, nicotine can cross the blood-brain barrier and specifically binds to nicotinic acetylcholine receptors (nAChRs) in the fetal brain34, and nAChRs have an important role in regulating synaptic plasticity and brain development. Concerning of animal models, chronic nicotine exposure to animal brain developmentally leads to long-term changes of decreasing presynaptic nAChRs function, which cause altering nicotine-stimulated release of those neurotransmitters and the altering continues for a long time even after nicotine exposure has been removed36. And neurotransmitters dysregulation of acetylcholine, dopamine and serotonin are assumed to be the reason of ADHD21. The decreased presynaptic nAChRs function may increase the risks of dysfunctions of dopaminergic, serotonergic and noradrenergic neurotransmission caused by genetic variants. That may explain the strong joint effects of prenatal tobacco exposure and genetic variants on all ADHD subtypes. But the exact reasons of the increasing risks require more extensive research.However, the molecular mechanisms responsible for the interaction remain unknown. Nicotine, the main psychoactive component of tobacco, can accumulated in human brain fast when they are smoking38. Moreover, evidences support that longer term low level exposure (<10 µg/dL) associated with intellectual impairment40. Unfortunately, the effects of lead exposure on ADHD are less clear. In the current study, no statistically significant association was found between low blood lead levels (<10 µg/dL) and diagnosed ADHD and all the symptoms. A growing body of evidence now support that low level exposure (<5 µg/dL) also associated with cognitive impairment, suggesting that there is no threshold for developmental neurotoxicity42. Stephani Kim5 and his colleagues examined the relationship between blood lead levels and ADHD in children, and found that the odds ratios for the risk of ADHD is 4.63 (95% CI: 1.36–15.72) in the “≥2 mg/dL” group and the odds ratios increased to 7.25 (1.66–31.67) in the “≥3 mg/dL” group. However, some studies reported that the association between blood lead levels and ADHD did not exhibit a dose-response relationship43.Lead is a heavy metal that exists in the environment in nature, and it is a most widely used neurotoxicants. Many studies have shown that high blood lead levels (≥10 µg/dL) are harmful to children neurodevelopment21, aiming to minimize the overdiagnosis or underdiagnosis. However, several limitations have to be considered during interpretation of the findings. The major limitation of this study is that the small sample size has decreased the power to detect the effects of the SNPs and the gene-environment interactions. One inherent methodologic problem in this case-control design is that the retrospective assessment of PSE might introduce systematic error in the calculation of the association between PSE and ADHD because of recall bias. In order to diminish the recall bias, we recruited the new diagnosed patients from the community survey. Although the regression model controlled for important confounding variables such as age, gender, mother and father education, and postnatal smoke exposure status, it did not take into account other possible environmental pollutants, such as manganese, mercury, arsenic and bisphenol A and phthalates45, which can be a focus of future research. Psychological and social environment are also the important for child psychological development, but those did not measured in our study. This is a weakness of the study. The participants in our study were enrolled from one city of China, which limited the results interpretation in general populations. Finally, its cross-sectional nature limits explication about the causal effects of G × E interactions. Future research may focus on a longitudinal study to confirm the causality.To the best of our knowledge, this paper is the first G × E interaction research about SNPs and prenatal tobacco smoke exposure in association to ADHD and all the subtypes. The major strength of this study is the standardized procedures of ADHD assessment in children and the precise and rigorous diagnosisThis study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, and all methods were performed in accordance with the approved guidelines and regulations. Informed consent was obtained from all participants and their parents.Children who were diagnosed for ADHD by pediatric psychiatrists were recruited as the cases in the Liuzhou Women and Children Healthcare Hospital, and 6–12-year-old healthy children from 2 elementary schools in Liuzhou City were also recruited as the controls. The exclusion criteria for both groups were: 1) a history of mental retardation, autism spectrum disorders, bipolar disorder, language disorders or learning disabilities, pervasive developmental disorder; 2) a history of brain disease, seizure disorder, or other neurological disorders; 3) intelligence quotient (IQ) < 70; and 4) the presence of any chronic physical disease; 5) a blood lead concentration ≥10(µg/dL).Permissions were given for the eligible patients and controls to participate in the study, and those children whose parents could not be contacted or denied consent for participation were excluded. As a result, a total of 168 cases and 233 controls (6–12 years of age) were recruited.46. The 4-point response is scored 0–3  on SNAP-IV scale. Subscale scores are calculated by summing the scores on the items in the specific symptom domains , in which average score 0–1 means no features, 1.1–1.5 means subclinical, 1.6–2.0 means significant, and >2.1 means very severe. We defined oppositional defiant disorder as the ODD symptom domain score ≥1.6. The Child Behavior Checklist (CBCL) scale was conducted to evaluate child potential behavior problems and the Wechsler Intelligence Scale for Children (WISC) was used to evaluate full-scale IQ of all participants. All instruments used in the current study are validated Chinese versions.The status of both the case and control subjects were confirmed with a semi-structured clinical interview with the parents and children that was conducted according to the DSM-IV ADHD Rating Scale by a pediatric psychiatrist. Additionally, diagnoses were made that included both in categories and severity ratings using the 26-item Chinese version of SNAP-IV scaleTo measure lead concentrations in blood, 2–3 mL of whole blood was drawn from each child and kept it in heparin-containing tubes. Blood lead level in whole blood was measured using graphite furnace atomic absorption spectrometry . The limit of detection for blood lead was 0.1 µg/dL. None of the blood samples showed below the limit of detection.Genomic DNA was extracted from peripheral blood leukocytes using the TIANamp® Blood DNA Kit DP318 .47, and included 7 genes . The screening procedure for the candidate single nucleotide polymorphism(SNP) was as follow: First, we searched the F-SNP database (http://compbio.cs.queensu.ca/F-SNP) to retrieve information about the predictive functions of the SNPs within these genes and those of potential effects on splicing, transcription, translation, and post translation processes were selected. Second, we filtered the SNPs by the MAF of CHB  in the database of 1000 genomes (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/) and selected SNPs with MAFs > 5% for the next step. Third, we tested the linkage disequilibrium (LD) among selected SNPs using SNAP Pairwise LD (http://www.broadinstitute.org/mpg/snap/ldsearchpw.php), and only one was reserved if several SNPs were in strong LD with each other (r2 ≥ 0.80). Consequently, a total of 16 SNPs were retained. High-quality genotyping was performed on the Sequenom MassARRAY platform  by BIO MIAO BIOLOGICAL TECHNNOLOGY  following the standard experimental procedures from the manufacturer. In addition, samples with a call rate <80% or Hardy-Weinberg equilibrium (HWE) < 0.05 in the control group were removed.The candidate genes within the dopamine, norepinephrine and serotonin neurotransmitter pathways were selected based on recent findingsTobacco exposure status was obtained from the children’s parents by a questionnaire. The questions were constructed in two parts: mother tobacco exposure and child secondhand smoke exposure. Firstly, we asked whether the mother smoked before pregnancy or not (>100 cigarettes) and whether she smoked during pregnancy. If yes, we then asked how many cigarette expensed per day. Only three of the mothers reported being an ex-smoker or having smoked during pregnancy. Besides, we inquired whether or not she was exposed to secondhand smoke during pregnancy at home or in the workplace and how long she stayed in the smoking circumstance per day/week. Secondly, we investigated whether any family members smoked at home after the child birth and how many cigarette the members expensed per day/week.A face-to-face questionnaire survey was administered for their parents by a trained interviewer to collect socio-demographic variables, prenatal and perinatal characteristics. Socio-demographic variables included age, gender, and parental marital status . Prenatal and perinatal characteristics included birth weight, pregnancy age of mother, preterm birth and the mode of delivery . The family history of neural system diseases and ADHD were asked additionally, but no participant declared of ADHD family history in their families.t test, when appropriate. Hardy-Weinberg equilibrium (HWE) for genotypes was calculated in controls by a goodness-of-fit chi-squared test. Logistic regression analysis was used to estimate the effect of PSE and genotype frequencies on ADHD susceptibility by computing odds ratios (ORs) and 95% confidence intervals (95% CIs). Four inheritance models  were estimated for each SNP. Meanwhile, the statistical power to detect the effects of the SNPs was calculated by Power v3.0.049, and for SNPs with MAF of 0.11(rs6354), 0.43(rs1124491), and 0.44(rs553668), for example, we calculated that the power for our sample size to detect an OR of 1.50 was 43.6, 74.1 and 74.8%, respectively.Differentials in the distribution of socio-demographic variables, prenatal and perinatal characteristics between patients and controls were compared by chi-squared test or https://sourceforge.net/projects/mdr/) was used to assess the gene-environment interactions between risk genes and the environmental risks. The details analytic protocol was described in the previous study51. Furthermore, multiplicative and additive interactions were computed. Multiplicative interaction was evaluated by the interaction term of logistic regression model. Synergy index (SI), attributable proportion due to interaction (AP), and relative excess risk due to interaction (RERI) and their corresponding 95% CIs were calculated by additive interaction analysis, as suggested by Tomas Andersson52. If there is no additive interaction, RERI and AP are equal to 0, or SI are equal to 1. The multiplicative and additive interaction analyses were adjusted for age of the child, gender, pregnancy age of mother, and postnatal smoking exposure. Since a higher proportion of males and younger ADHD children(<9 years old) was noted in our study.The Multifactor Dimensionality Reduction (MDR) 2.0 beta 8.1 program . All tests were two-tailed, and statistical significance was defined at supplementary table


Ocular trauma is a major cause of monocular blindness worldwide. Vitrectomy at correct timing can significantly improve the efficacy and prognosis, but the timing of vitrectomy has remained highly controversial for decades. Trauma cases are different from each other, thus, a flexible timing system based on the details of each individual case is recommended. Unfortunately, no such a timing system is available for clinical application up to now. To establish the vitrectomy timing individualization system for ocular trauma (VTISOT), we first identified 6 independent tPVR risk factors  by retrospective study. Then, the tPVR score was established by binary logistic regression analysis. Most importantly and critically, the vitrectomy timing individualization system for ocular trauma was established based on the identified tPVR risk factors and the tPVR score. The following evaluation of the VTISOT showed that the patients consistent with the VTISOT principles exhibited reduced tPVR incidence and better surgical results. In short, the VTISOT principles were established, which may provide a new approach to individualize the timing of vitrectomy and improve the prognosis after trauma. Among these factors, there is obviously no way of reducing the damage inflicted by the initial injury. Fortunately, with the introduction and development of vitrectomy, the quality of surgical reconstruction has substantially improved. However, unfortunately, the scarformation reaction of the body, described as traumatic proliferative vitreoretinopathy (tPVR) in the posterior segment, has remained a crucial limiting factor in the rehabilitation of the severely injured eye1.Ocular trauma is a major cause of monocular blindness worldwide. The prognosis for a serious eye injury is primarily determined by the initial injury, the quality and timing of surgical intervention, and the biological reaction of the body5. The main reason for this long-standing controversy is that trauma cases are different from each other, and it is impossible for us to provide a unified surgical timing for various eye injuries. Thus, a flexible approach based on the details of the individual case is recommended6. Unfortunately, no vitrectomy timing individualization system is available for clinical application up to now.Vitrectomy at the correct timing not only reduces the incidence of tPVR but also significantly improves the efficacy and prognosis. But the timing of vitrectomy has remained highly controversial for decadesIn this study, we established the vitrectomy timing individualization system for ocular trauma (VTISOT) on the basis of tPVR risk factors and the tPVR score, providing a new method to individualize the timing of vitrectomy and improve the prognosis for ocular trauma.This retrospective study was approved by the institutional review and ethics boards of the Hebei Provincial Eye Hospital, Hebei, China and followed the tenets of the Declaration of Helsinki. All research was performed in accordance with relevant guidelines and regulations. Informed written consent was obtained from all the parents.7, injured eye (OD or OS), preoperative best-corrected visual acuity (BCVA) and intraocular pressure (IOP), cornea (clear or injured), hyphema (grades 0~4)8, lens (injured or uninjured), length of laceration, zone of wound7, retinal condition, vitreous hemorrhage (VH), timing of surgery, intra-operative major hemorrhage, iatrogenic retinal injury, and postoperative tPVR, BCVA, retinal detachment (RD), infection, and globe survival .We enrolled consecutive patients who were evaluated at the ocular trauma department of the Hebei Provincial Eye Hospital between Jan 2014 and Nov 2017. Their medical records were reviewed for the following data: age, sex, time of injury, type of injury The inclusion criteria were as follows: (1) patients diagnosed with eye injury; (2) those who underwent pars plana vitrectomy. The exclusion criteria were as follows: (1) patients who were injured for more than 10 days at the initial presentation; (2) a history of eye injury; (3) those in which endophthalmitis or intraocular foreign body was diagnosed at the initial presentation; (4) those who underwent early endoscopic vitrectomy due to severe corneal edema and opacity; (5) those who underwent postponed pars plana vitrectomy due to severe corneal edema and opacity; (6) massive choroidal hemorrhage diagnosed by preoperative ultra B-scan; (7) those who underwent only anterior vitrectomy; (8) incomplete medical records; (9) those with a follow-up period <6 months after vitrectomy.The tPVR risk factors were identified by univariate and multivariate analyses. The tPVR score was established by binary logistic regression analysis according to the identified tPVR risk factors. Then, the vitrectomy timing individualization system for ocular trauma (VTISOT) was established based on the tPVR risk factors, the tPVR score and the vitrectomy-associated characteristics of the injured eyes.To verify the validity of the VTISOT principles, we divided the patients who underwent vitrectomy into two groups according to the consistency between the actual timing of vitrectomy and the individualized timing from VTISOT: Consistency Group and Inconsistency Group. Then, we compared the differences in tPVR, postoperative RD, globe survival, intra-operative major hemorrhage, iatrogenic retinal injury, postoperative infection, postoperative BCVA and BCVA improvement between the two groups.10. Thus, the tPVR in our study was defined as the cellular membranes within the vitreous cavity or on either retinal surfaces or obvious retinal shrinkage after trauma.According to Pastor JC, etc., PVR is defined as the growth and contraction of cellular membranes within the vitreous cavity and on both retinal surfaces, and in many cases a fibrotic process of the retina itself11.A red reflex is present with no retinal detail observed posterior to the equator or no red reflex or the same amount of blood was observed during vitrectomy. The hemorrhage is equivalent to grades 3 and 4 in T Göncü’s vitreous hemorrhage grading7.Open-globe injury: Zone 1: Isolated to cornea ; Zone 2: Corneoscleral limbus to a point 5 mm posterior into the sclera; Zone 3: Posterior to the anterior 5 mm of sclera. Closed-globe injury: Zone 1: external ; Zone 2: anterior segment ; Zone 3: posterior segment Retinal laceration appears but sclera does not have a full-thickness wound in zone 3 of the open-globe eye injury, for example, a corneal penetrating eye injury by a steel nail with retinal laceration in zone 3.A portion of or the entire retina cannot be recognized by B-ultrasonography. In other words, the vitreoretinal interface is seriously blurred.4.Based on F Kuhn’s review, the timing of vitrectomy in our research includes the following options: early (days 2–4), delayed (days 5–7), late (days 8–14), and very late (past 2 weeks). The timing refers to the interval of injury and vitrectomy measured in days7.Based on the grading method of DJ Pieramici, the VA was divided into the following 7 grades: no light perception (NLP), light perception (LP), hand motion (HM), counting finger (CF) ~ 4/200, 5/200~19/100, 20/100~20/50, and ≥20/4012.Grade 0: without blood; Grade 1, hyphema filling less than one-third of the anterior chamber (AC); grade 2, hyphema filling one-third to one-half of the AC; grade 3, hyphema filling more than half of the AC but less than the total; and grade 4, total hyphema with either red or black blood clotsP < 0.05.Differences in BCVA and BCVA improvement between groups were assessed using the Mann-Whitney U test. Differences in age and length of laceration between groups were assessed using Student’s t-test. Differences in other characteristics between groups were assessed by Fisher’s exact test or the Chi-Square test. Multivariable analyses for tPVR risk factors were performed by binary logistic regression analysis. To determine the cutoff value of the tPVR score, ROC curve analysis was applied. All analyses were performed using SPSS software ver. 21.0 . The level of significance was set at A total of 625 consecutive injured eyes that met the initial inclusion criteria were evaluated. Of these, 320 eyes were excluded because of exclusion criteria. A total of 305 eyes from 305 injured patients were included in our final analysis. Of the 305 included eyes, 137 (49.9%) eyes had no tPVR, and 168 (55.1%) eyes had tPVR after vitrectomy. The demographics and ocular characteristics of the 2 groups (no tPVR group and tPVR group) are summarized  was considerably reduced compared with other groups . This finding indicates that early vitrectomy may decrease the incidence of postoperative tPVR.To predict the incidence of postoperative tPVR before vitrectomy, we established the tPVR score using binary logistic regression analysis Table .Table 5VThe tPVR score = Logit (P) = Zone 3 Injury × 1.84 + Z3R Laceration × 2.721 + Massive VH × 0.785 + Retinal Disorder × 0.785 + Rupture × 0.914 + Contusion × 1.48 + Perforating × 2.247 − 2.583.ROC curves revealed that compared with any individual tPVR risk factors, tPVR score predicted tPVR better Fig. . The cutFour main tPVR risk factors and classification of high/low tPVR risk before vitrectomy1.1Four main tPVR risk factors: Zone 3 Injury, Z3R Laceration, Massive VH, and Retinal Disorder.1.2High tPVR risk case: A case that includes one or more of the above 4 main tPVR risk factors.1.3Low tPVR risk case: A case that includes none of the above 4 main tPVR risk factors.2.Timing of PPV surgery2.1High tPVR risk cases: Days 2–4 after injury.2.2Low tPVR risk cases: Days 8–14 after injury.3.Individualized adjustments of surgical timing (on the basis of not violating the above principles)3.1Adjust surgical timing by tPVR score3.1.1 There is a possibility of tPVR when the tPVR score is greater than 0.589. The greater the tPVR score, the higher the probability of tPVR.3.1.2 Early surgery may reduce the incidence of tPVR. Thus, the greater the tPVR score, the earlier the surgery.3.1.3 The surgical timing should be delayed properly when tPVR score is less than 0.589. The lower the tPVR score, the later the surgery.3.2Adjust surgical timing by characteristics of the injured eye3.2.14.Corneal edema: The surgical timing should be delayed properly when the cornea is not clear enough due to edema. Otherwise, endoscopic vitrectomy or temporary keratoprosthesis should be applied3.2.213. Suprachoroidal hemorrhage: The surgical timing should be delayed properly when suprachoroidal hemorrhage exits3.2.34. Unsutured wounds: Unsutured wounds (too posterior to reach) generally require some delay, and the IOP should be tightly controlled during vitrectomy3.2.44. Lens injury: Severe glaucoma second to lens injury requires the earlier vitrectomy3.2.54. Vitreous hemorrhage: The more severe the VH, the more urgent its removal3.2.64. Low IOP: The lower the IOP, the earlier the vitrectomy should be performed (to prevent secondary ciliary body damage and consequent phthisis)The timing of vitrectomy for ocular trauma is mainly based on the tPVR risk factors. The individualized adjustments are based on the tPVR score and the vitrectomy-associated characteristics of the injured eyes. The following principles apply to vitrectomy for non-endophthalmitis and non-IOFB.To evaluate the VTISOT, we divided the patients into 2 groups: inconsistency group 164, 53.8%) and consistency group . The consistency group exhibited a reduced incidence of tPVR 27.0%), postoperative RD (9.9%), and iatrogenic retinal injury (4.3%) and increased incidence of globe survival (98.6%) compared with the inconsistency group   were established based on the independent tPVR risk factors and the tPVR score.5. Numerous patients miss the best opportunity to regain their sight or even anatomically lose their eyeballs .Ocular trauma is a major cause of monocular blindness worldwide. Despite progress made in surgical techniques, pharmacological adjuncts, and preventive measurements, the clinical outcome remains unsatisfactory. Especially, The timing of vitrectomy for ocular trauma has remained highly controversial for decades4. Issues involving logistics and infrastructure must also be taken into consideration4. Thus, a flexible plan based on the above details is recommended6. Therefore, we tried to established the vitrectomy timing individualization system for ocular trauma (VTISOT).The main reason for this long-standing controversy in vitrectomy timing after trauma is that no two injuries are identical and no two surgeons are the same. An individual plan must be fashioned in each case based on the specifics of the injured eye and patient and the capabilities, experience and expertise of the surgeon15. Despite tremendous advancements in surgical and pharmaceutical approaches for management of tPVR, it continues to remain a clinical challenge. Moreover, there is no objective and systematic basis for clinical assessment of tPVR risk factors, and no method is available to predict the incidence of tPVR in a specific injured patient. The tPVR risk factors that can be targeted for clinical intervention may represent a breakthrough in treating tPVR and a key to the vitrectomy timing individualization, so it is important to identify it first.The tPVR is a one of the leading causes of blindness following ocular trauma13.In our study, 6 tPVR risk factors were identified by univariate and multivariate analysis. Among them, 5 factors, including Zone 3 Injury, Z3R Laceration, Massive VH, Retinal Disorder and Type of Injury, are immutable and objective after injury. Only vitrectomy timing can be intervened clinically. Our research also demonstrates that the incidence of tPVR is decreased by early vitrectomy, which is consistent with recent research16. The following reasons account for the reported differences. First, although there is a PVR grading system, the definitions of tPVR differ from each other. For example, Cardillo et al. defined tPVR as any intraocular cellular proliferation associated with any degree of detachment15. In their report, retinal detachment was a prerequisite, so the incidence of tPVR in their report was relatively low. In most other reports, retinal detachment was not necessary for the definition of tPVR. In our research, tPVR was defined as cellular membranes within the vitreous cavity or on either retinal surfaces or obvious retinal shrinkage after trauma, which covered a relatively wide range of conditions and resulted in relatively increased tPVR incidence. Second, the timings of vitrectomy differed. Wang’s research revealed a significant difference in tPVR incidence between the early surgery group  and late surgery group 13. Third, the severity of injuries in each reports might be different. Thus, the definition of tPVR should be standardized in future studies.The reported incidence of tPVR after vitrectomy varies widely (11~66.7%)To the best of our knowledge, a suitable method to predict the incidence of tPVR has not been reported prior to this study. Our study provides a good idea for solving this problem by a simple calculation. The predicted incidence of tPVR = exp(tPVR score)/(1 + exp(tPVR score)). The ROC curve reveals that the prediction accuracy is 78.3% (95% CI: 73.1~83.4%), and the best values of sensitivity and specificity obtained with this model are 61.9% and 81.3%, respectively.17, this system provides an objective foundation for surgeons to fashion individual plans and achieve patient cooperation.Although the values of sensitivity and specificity of these type of predictive models may be inadequate for a direct clinical use18. Nevertheless, the tPVR score is the first predictive model of tPVR based on systematic clinical analysis. Although we have not validated this model, it provides a good idea and method for predicting tPVR. The external validation of this model can be performed by our team or other interested teams in the future.Another problem that must be discussed is that this type of formulas must be validated externally. External validation aims to address the accuracy of a model in patients from a different but plausibly related population, which may be defined as a selected study population representing the underlying disease domain5. Kuhn et al. reports that primary comprehensive reconstruction with immediate vitrectomy at “zero hour” after injury is the best option for severe trauma with high PVR risk19. The advantages of this option are numerous: the risk of a subsequent development of endophthalmitis is eliminated; if an infection is present, it can be dealt with properly; all tissue pathologies that do not represent irreversible damage may be treated; the inflammatory reaction and thus the risks of PVR and secondary ciliary body destruction are reduced, etc4. However, in reality, this is not always possible. Various obstacles may exist. For example, the facility might not be properly prepared to offer the full armamentarium for complex vitrectomy, at least not outside “normal business hours”. Alternatively, the surgeon has no or only limited experience of vitreoretinal techniques; or the nurse is not familiar with the equipment. In addition, corneal opacity and the risk of expulsive choroidal hemorrhage may represent obstacles for immediate vitrectomy4. If comprehensive primary reconstruction is not possible, the next best option is vitrectomy performed within 100 hours or 2–4 days of the injury4. However, some others regard “days 7–14”21, “days 4–10”15 or “within 14 days”6 as best timing. Some even think that the 4th week is the best option in management of a gunshot perforating eye injury3. Hermsen’s early study showed that the best results were achieved when vitrectomy was performed between 15 and 30 days following injury22.The timing of vitrectomy for ocular trauma has remained highly controversial, with the exception of IOFB and endophthalmitis at initial presentationWith the advancement of vitrectomy, few ophthalmologists support the options post 2 weeks. The main controversy is concentrated on immediate (zero hour), early (within 100 h or days 2–4) or late (days 7–14) at present. To date, ophthalmologists are still confused about the best timing for traumatic vitrectomy.4.In this study, both univariate and multivariate analysis showed that the timing of vitrectomy was closely related to the tPVR, and the incidence of tPVR in the early surgery group  was considerably reduced compared with other groups . This finding indicates that early vitrectomy may decrease the incidence of postoperative tPVR, which is consistent with the latest studiesThe VTISOT we established integrated the tPVR risk factors, the tPVR score and the individual ocular features affecting surgical operation and efficacy, including corneal edema, suprachoroidal hemorrhage, unsutured wounds, lens injury, vitreous hemorrhage and low IOP. The evaluation results demonstrated that the patients consistent with the VTISOT principles exhibited reduced tPVR incidence and better surgical results, including reduced incidence of tPVR, postoperative RD and iatrogenic retinal injury; increased incidence of globe survival, and better postoperative BCVA and BCVA improvement.25. Rojas J, et al. developed three predictive models of PVR using genetic variables only. Although none of them has greater ability than that achieved by clinical predictive formulas24, genetic predictive variables could be added to any of the previously established clinical formulas, to improve their predictive capability and thus allow us to have a better tool in the prophylaxis of PVR25. So, the genetic predictive variables could also be added to the tPVR prediction and the VTISOT principles in future studies.Some researchers have recently described the potential contribution of genetic components to PVR, suggesting that it is a complex disease in which many environmental, clinical, and genetic variables may interactAlthough the VTISOT has included all clinical factors we can consider, the improvement is still possible in the future. For example, with the inclusion of more participants and genetic factors and the performance of external validation, risk factors will be more precise, and their weights in the equation will be more precise. However, currently, clinicians, especially inexperienced surgeons, can consider the VTISOT as a reference to select the timing of vitrectomy for trauma.In conclusion, we established the vitrectomy timing individualization system for ocular trauma (VTISOT) on the basis of tPVR risk factors and the tPVR score. The patients consistent with the VTISOT principles exhibited reduced tPVR incidence and better surgical results, providing a new idea to individualize the timing of vitrectomy after trauma. Further clinical studies will be useful to evaluate and improve the VTISOT.Data of Characteristics


The location of the high mountains of southern Europe has been crucial in the phylogeography of most European species, but how extrinsic (topography of sky islands) and intrinsic features  have interacted to shape the genetic structure in alpine restricted species is still poorly known. Here we investigated the mechanisms explaining the colonisation of Cantabrian sky islands in an endemic flightless grasshopper. We scrutinised the maternal genetic variability and haplotype structure, and we evaluated the fitting of two migration models to understand the extant genetic structure in these populations: Long-distance dispersal (LDD) and gradual distance dispersal (GDD). We found that GDD fits the real data better than the LDD model, with an onset of the expansion matching postglacial expansions after the retreat of the ice sheets. Our findings suggest a scenario with small carrying capacity, migration rates, and population growth rates, being compatible with a slow dispersal process. The gradual expansion process along the Cantabrian sky islands found here seems to be conditioned by the suitability of habitats and the presence of alpine corridors. Our findings shed light on our understanding about how organisms which have adapted to live in alpine habitats with limited dispersal abilities have faced new and suitable environmental conditions. Understanding the evolution of Pleistocene glacial periods in temperate regions has been instrumental in disentangling much of the biogeographic distributions and genetic structure of their biotas. The effect of Quaternary climatic oscillations on the extant genetic diversity in the European and North American biotas is well-documented ,2,3,4. TMountain chains are considered hot spots of biodiversity due to the high levels of unique taxa they host, which is a direct consequence of harbouring a rich range of habitats in a relatively small area ,13,14. BUnderstanding how flightless organisms cope with environmental changes can inform us about how heterogeneous landscapes shape the genetic structure of montane biotas over time. Ultimately, this information will shed light on functional connectivity of disjunct sky islands, providing robust guidelines for conservation managers. Under a recurrent scenario of glacial and interglacial periods, it is not evident how animals with reduced dispersal capabilities respond, although it must be also dependent on the speed of the environmental changes . It is pChorthippus cazurroi). This grasshopper shows a patchy distribution in the Cantabrian Mountains, occurring along a narrow range between 1400 and 2570 m above sea level. Such a narrow distribution appears ideal to make inferences about how landscape features shaped the demographic history and genetic variability of alpine taxa. To achieve our objectives, firstly, we scrutinised the maternal genetic structure from all Cantabrian sky islands where this taxon occurs. We studied mitochondrial data because when the range of a species is exhaustively sampled, it is possible to get robust inferences of its genetic structure due to the absence of recombination and fast coalescent time [With the present study we aimed at unravelling the mechanisms explaining the dispersal process and evolution of spatial genetic structure in an alpine flightless grasshopper  for females (n = 285) and 8.27 ± 0.74 mm for males (n = 156), meanwhile, C. yersini (a relative species capable of short flight and occurring in sympatry) shows longer wing length: 22.07 ± 1.46 mm and 17.88 ± 1.42 mm for females (n = 189) and males (n = 134), respectively. However, both species show closer total body length (19.01 ± 1.83 mm versus 27.20 ± 1.46 mm) for C. cazurroi and C. yersini females, respectively; and 13.53 ± 1.63 mm versus 20.34 ± 1.56 mm for males, respectively. The species is found on grasslands habitats over 1400 m above sea level (a.s.l.) on limestone massifs. This grasshopper species shows the typical univoltine life cycle with nymphs emerging after snow is melted (June–July). After four moults, the nymphs mature to adult stage, which appears to be especially abundant in August, when most reproduction occurs. In controlled laboratory conditions, with food provided ad libitum, females survive on average 21 ± 1 Standard Error days, during which they can lay up to 11 egg pods, each containing on average 8 ± 3 SE eggs [ountains . The verles n = 1, respectChorthippus cazurroi in the Cantabrian Mountains. In particular, the species was found in 22  using the primers LCO1490 and HCO2198 [TM nucleic acid gel stain . We performed sequencing reactions using the Perkin Elmer BigDye v. 3.1  terminator reaction mix in a volume of 10 µL, following the procedures and conditions described in Pato et al. [® 3130xl Genetic Analyzer . Sequences were edited and aligned by hand using BioEdit v. 7.2.5 [We extracted DNA from the hind femur or head using the Qiagen DNeasy kit  and following the manufacturer’s protocol. To determine genetic variability of  HCO2198 . Polymer HCO2198 ,39. PCR v. 7.2.5 . Unique eµ) [We calculated genetic diversity statistics   per locaeµ) . We infeeµ) .We evaluated how two migration models, LDD and GDD  of 0.1 kmeach see  that covWe grouped the 22 localities into seven regions according their spatial distribution . SummaryAs indicated above, we generated 30,000 simulated datasets for each of the two migration models. In addition, we simulated 100 PODS for each model to evaluate the accuracy of the selection method. Considering that the accuracy of the estimation for a particular real dataset may differ among ABC techniques ,55, we aWe estimated the population genetic parameters with the Rrej, Rreg, and Rnn ABC approaches . We usedChorthippus cazurroi’s whole distribution range included 22 localities along the sky islands of the Cantabrian Mountains  for distinguishing between the investigated models (LDD versus GDD) under all the applied ABC approaches . The ana-06 (5.15 × 10−7–9.43 × 10−6). Here we present the parameter values estimated with the highest probability (mode of the posterior distribution), and the variance in the precision of these estimates  for the interpretation of these results.We found that the ABC approaches provided a reasonable power to estimate the population genetics parameters under the GDD model, after using the 100 PODS, although, as expected, the accuracy of this estimation slightly varied among the studied parameters and the applied ABC approaches . The popOur inferred haplotype network suggests a scenario of recent colonisation of the Cantabrian Mountains sky islands, led by a starting population of more than 500 individuals. Our findings support a colonisation by a gradual dispersal process rather than the presence of LDD events such as those caused by upland winds or biotic factors. Such a contemporary expansion is well-matched with the limited genetic structure found, which explains the pattern of incomplete lineage sorting revealed by the haplotype network analysis, and presenting haplotypes shared between massifs. This genetic structure is compatible with a scenario of incomplete lineage sorting, which is common in species with recent differentiation processes ,60.Chorthippus cazurroi started a process of slow and gradual range expansion along the Cantabrian sky islands using, most likely, narrow alpine corridors to achieve its current distribution  surrounding the sky islands. This finding is relevant in order to predict future movements of this species and assess ecological connectivity among disjunct sky islands distributed throughout heterogeneous and unsuitable landscapes. Undoubtedly, this information will be also critical in future management actions with the goal of preserving genetic diversity and ecosystem function. Importantly, our results confirm that this flightless species do not participate in LDD movements or, if they did, such circumstance did not shape its current genetic structure. Long distance dispersal constitutes a recognised strategy performed by diverse organisms with the aim of colonising areas away from the source, rapidly, and without loss of genetic diversity ,29,70. IOur approach provides a framework to improve our understanding, and ecological meaningfulness, of colonisation and diversification processes of non-vagile organisms throughout the sky islands of Cantabrian Mountains. Nonetheless, we are aware that our biologically informed hypotheses hinge upon required assumptions, which can be challenging. Our models assume an ancestral population from which the species spread along the sky islands. We chose such a source population according to our knowledge on the evolution of the last glacial period in the Cantabrian Mountains, the potential habitats where the ancestral population could have occurred, and the high values of genetic diversity found. However, the clear pattern of migration shown by our analyses (GDD) is unlikely to be affected by the origin of the expansion . AnotherChorthippus cazurroi populations established in the Cantabrian Mountains present a simple pattern of progressive and slow gradual migration. We also found that these populations evolved with a slow increase of their population sizes over time, which could be explained by the patchy distribution of suitable habitats and hard environmental conditions present in the region. In conclusion, our findings have shed light on the processes involved in the formation of biodiversity in alpine flightless animals occurring on southern temperate mountains and, importantly, have provided an ideal framework to integrate genetic divergence at the population level with ecological information related to animal differentiation such as morphological and acoustic traits.Overall, we showed that


PIK3CA mutation and was sensitive to the PI3K inhibitor, alpelisib (BYL719). We review the available literature on metastatic MPTT, detail the patient’s course, and present a whole genome analysis of this rare tumor.Proliferating tricholemmal tumors (PTTs) are rare benign neoplasms that arise from the outer sheath of a hair follicle. Occasionally, these PTTs undergo malignant transformation to become malignant proliferating tricholemmal tumors (MPTTs). Little is known about the molecular alterations, malignant progression, and management of MPTTs. Here, we describe the case of a 58-year-old female that had a widely metastatic MPTT that harbored an activating  While MPTT has the potential for local recurrence and metastasis, fewer than 30 cases of metastatic malignant proliferating tricholemmal tumor  have been detailed in the literature  are benign neoplasms of the external hair sheath.The patient’s course is outlined in Fig. A 58-year-old, previously healthy, white female presented to her primary care provider (PCP) with the desire to remove a right posterior scalp cyst for cosmesis. This non-inflamed, non-draining, painless, 1–2 cm cyst had been present for close to 10 years without change in size or fluctuance. The cyst was initially drained by the PCP, but, when it recurred 6 months later, the PCP excised the cyst and sent the specimen for routine pathology. The initial read of the tissue sample was high-grade invasive carcinoma with squamous features and arising in association with a PTT. Based on the pathology, the PCP referred the patient to a plastic surgeon for a more definitive excision of the lesion and repair of the defect. The lesion was excised with negative margins and pathology read as invasive high-grade squamous cell carcinoma (SCC). Eight months post resection, the lesion recurred locally along with a palpable right posterior cervical lymph node (LN). A positron emission tomography (PET) scan at that time demonstrated hypermetabolic activity in the posterior occiput and in a posterior neck LN. Fine needle aspiration (FNA) of both the primary scalp lesion and LN were completed, and pathology was reported as SCC, similar to the primary lesion.29 The patient tolerated the adjuvant chemoradiotherapy with expected toxicities including nausea and fatigue.With a working diagnosis of locally advanced SCC, the patient’s care was referred to a tertiary care center. There, a dermatopathologist re-evaluated the previous biopsy specimens and altered the diagnosis from SCC to MPTT Fig. . Subsequ30) identified a PIK3CA c.G3140A (p.H1047R) mutation. Given the paucity of evidence for this mutation in this cancer, the patient was treated with a standard regimen for metastatic SCC (consisting of docetaxel and cisplatin every three weeks).31 At the same time, the patient was put on the waitlist for NCT01219699, a phase I study of oral BYL719  in adult patients with advanced solid malignancies, whose tumors have an alteration of the PIK3CA gene.32Fifteen months after her neck dissection (1 year after completing chemoradiotherapy), FNA of a suspicious right paraspinal LN at C5 documented disease recurrence. The patient underwent a revision neck dissection  with final pathology specimens consistent with metastatic MPTT. A subsequent PET scan demonstrated hypermetabolic activity in a right supraclavicular LN, multiple mediastinal LNs, and in a 0.8 × 1.1 cm right lower lung lobe parenchymal nodule. An endobronchial ultrasound (EBUS) and transbronchial needle aspiration (TBNA) showed involvement of level 4 and 7 mediastinal LN by carcinoma. Molecular profiling of the tissue using a multiplexed PCR assay  scan (she was asymptomatic at this time). The patient then enrolled in the trial of alpelisib at 450 mg daily. Her only suspected adverse effects related to the study drug were nausea and weight loss. After 3 months of treatment, she demonstrated a partial response per RECIST Fig.  and S1.3ing Fig. .32). Two months after stopping alpelisib, a surveillance CT scan demonstrated peritoneal carcinomatosis. Within days of this CT scan, the patient was admitted to the hospital with increased work of breathing requiring 4 L/min oxygen. The patient opted for no aggressive measures and was symptomatically treated until her death a week later.Four months after starting the study drug, the patient developed a community acquired pneumonia (CAP), and the treatment was suspended. Although the CAP resolved both clinically and radiographically, the patient was started on 2 L/min oxygen due to a persistent cough that was not responsive to therapy. It was unclear if the patient had progressive disease or a drug-related pneumonitis at this time . Second, we performed WGS on the primary tumor and compared it to normal liver tissue from the patient. The WGS coverage was 29.3× for the normal and 29.0× for the tumor samples. Using a robust analysis pipeline34  of the tumor. First, we verified that the numerous cancerous lesions found at autopsy were all metastases from the same cancer and not secondary cancers. Ten of the lesions that were sampled during autopsy were molecularly profiled with the SNaPshot assay,e34 Fig. , we idene34 Fig. .Fig. 2Ge35 database 37 tool—ANKRD20A1 c.G599A (p.R200Q) and DCAF12L1 c.G1366T (p.G456W). As a validation of our methods, PIK3CA H1047R was identified by our WGS pipeline and predicted to be cancer-causing per FATHMM. Targeted dideoxy sequencing was performed on 1/3 of the mutations to corroborate the SNV calls, and each of the mutations was validated exclusively in tumor tissue  were detected, including 6139 germline variants already reported in the dbSNP (v137)se Table . Most ofse Table . Among tsue Fig. .DNAH17 and PGS1, which were each amplified to 8 copies. We also identified 59 SVs spanning 662 unique genes , and the rest were equally divided amongst inter-chromosomal translocations (n = 11), insertions (n = 12), and deletions (n = 13). None of the SVs discovered in our WGS samples appear to be relevant for MPTT oncogenesis or metastasis. However, further examination of gene or protein expression could help determine if a tumor relationship exists.Our analysis also uncovered a variety of somatic copy number variations (CNVs) and structural variants (SVs). Specifically, we identified 238 CNVs covering 1,495 unique genes Fig. . At the nes Fig. . Most of1) or with those altered in cutaneous SCC  . Moreover, the altered genes in metastatic MPTT did not significantly overlap with genes associated with the outer root sheath/bulb of hair (the putative origin of MPTTy) Table . Finally12 MPTT is difficult to distinguish from SCC due to its shared behavior (invasion), common location , and overlapping histology—including the potential presence of spindle cells.38 However, SCC is classically heralded by actinic keratoses, and histologic evidence of keratin production suggests SCC. Meanwhile evidence of tricholemmal keratinization, a lobular pattern , as these are known to occur on the scalp but not to metastasize.1 Our sequencing data hint that cutaneous SCC and MPTT are genetically distinct and could potentially be differentiated with a gene signature. Moreover, due to the low prevalence of PIK3CA mutations in cutaneous SCC,43 the malignancy has the potential to be differentiated with a simple gene (PIK3CA) test. However, more data are needed to confirm this hypothesis. For now, consultation with a board-certified dermatopathologist is recommended if a diagnosis of unusual squamous cell carcinoma or MPTT is considered.MPTT is a rare entity, with just a few hundred cases described in the literature. However, the true prevalence of the cancer may be obscured by its common misdiagnosis as SCC and by its abundance of alternative names.ern Fig. , and rea7 However, it is unclear how this process unfolds and/or how often MPTT metastasizes. Categorization of the process has been attempted based on histomorphology;2 however, cases with little or no cytologic and architectural atypia may exhibit aggressive behavior and vice-versa.44 It is surprising that MPTT does not metastasize more often given the immune privilege of cells in the hair shaft; one would expect that hair shaft tumors (secondary to their low level of major histocompatibility molecules) would more readily metastasize.45 More studies are needed to understand the biology and clinical course of MPTT, especially with regards to biomarkers that may predict disease severity.We performed an extensive literature review and uncovered all known cases of metastatic MPTT Table . While M46 Later studies concentrated on the elevated proliferation rate of these malignancies as demonstrated by Ki67 immunostaining.47 To date, only two studies have examined the genetics of these tumors, and both focused on TP53 mutations.48 Genetic analysis of this patient’s MPTT did not reveal any alterations in TP53.There have been just a handful of investigations into the molecular alterations of MPTT. Early studies identified and focused on the high percentage of aneuploid cells found in MPTT.51 Interestingly, the amount of structural alterations, including focal and chromosome-arm-level CNVs, is well above the average of most tumors types.52 While the structural alterations did not reveal any obvious fusion, amplification, or driving event, analysis of the mutations found in MPTT uncovered two potentially cancer causing mutations—ANKRD20A1 R200Q and DCAF12L1 G456W. ANKRD20A1, a poorly studied ankyrin family member, is thought to be involved in mediating protein-protein interactions but has only been studied as a biomarker in Behçet’s disease.53 DCAF12L1 is involved in germ cell development and is linked to extracellular signal-regulated kinase signaling.54 Interestingly, both of these genes’ products are normally expressed only in ciliated tissues per the human protein atlas.55 As with any genetic study, more laboratory work is needed to validate any potential functional significance of these mutations.Our WGS of a metastatic MPTT instead identified numerous exonic and structural variants Fig. –4 and PI1 Interestingly, the hair shaft is driven by Wnt signaling, and alterations in this pathway lead to a distinct type of tumor: pilomatricoma.56 No genes linked with Wnt signaling were altered in MPTT. Likewise, the similar cutaneous SCC often arises from alterations in Notch signaling43 and none of these mediators were altered in MPTT. The difference in genetics between MPTT, cutaneous SCC, and the normal hair shaft suggests the existence of an alternate mechanism of oncogenesis. This finding also challenges the dogma that MPTT arises from the outer root of the hair shaft.1 More MPTTs should undergo genomic profiling to uncover this pathway and help treat patients.Given that the outer root sheath is a proliferative compartment, it is surprising that not many of these genes were altered or deregulated. In contrast to similar proliferative compartments such as the gut or bone marrow, the hair follicle appears to have evolved a striking resistance to oncogenic stimuli, either because of a relatively protected anatomic location, unknown biologic factors, or combination of the two.2 Mohs surgery and excision with frozen section margin assessment is an intriguing emerging option as it has the benefit of decreasing the rate of recurrence while simultaneously sparing tissue in larger lesions.57 However, as this case highlights, early involvement of a head and neck surgeon may be beneficial in cases of biopsy-proven MPTT due to their tendency for locoregional spread and cervical LN metastasis.Due to its poorly understood biological behavior, local excision with wide margins (≥2 cm) is the most common primary treatment for MPTT in the literature. However, given the relatively high local recurrence rate (4–7%) with a median literature follow-up of 15 months, other surgical treatment modalities are being investigated.61 The few studies that do exist have shown: that ethanol injections may be useful for local recurrence; that chemotherapy regimens containing cisplatin, doxorubicin, and vinca alkaloids can be beneficial for metastatic disease; and that radiotherapy can be effective in preventing primary lesion recurrence. Here, we show that metastatic MPTT can be responsive to a variety of chemotherapy regimens and a small molecule inhibitor of PI3K, in the setting of a somatic PIK3CA activating mutation –approved protocols. Specifically, tissue was collected for study through the Head and Neck Cancer Tissue Repository and Clinical Database (IRB #030062). Written informed consent was obtained from the patient. All samples were de-identified, protected health information reviewed according to the Health Insurance Portability and Accountability Act (HIPAA) guidelines, and studies conducted in accordance with the Declaration of Helsinki. The patient consented to be treated with BYL719  under NCT01219699, a phase I study of oral alpelisib (a PI3Kα inhibitor) in adult patients with advanced solid malignancies (Vanderbilt CTSR #PHI-1065). The patient’s spouse consented for a full hospital autopsy without limits, allowing for any tissue, organs, or fluids that are removed from the body to be studied, disposed of, or used for teaching and research. All witnessed and signed consent forms are on file.Immunohistochemistry (IHC) of patient samples was performed at the Vanderbilt Translational Pathology Shared Resource core laboratory. Formalin-fixed paraffin-embedded tissue was used for all immunohistochemistry. For hematoxylin and eosin (H&E) staining, slides were prepared using an automated H&E stainer (Gemini) using the manufacturer’s protocol and customary dyes . Ki67 staining was prepared using an automated IHC slide stainer  with slight deviations from the manufacturer’s protocol: antigen retrieval was performed at a pH of 9 for 20 minutes; slides were incubated with Ready-To-Use anti-Ki67  for 60 minutes; and BOND Polymer Refine detection system (Leica) was used for visualization. Both H&E and IHC slides were digitized using a light microscope and camera (Nikon).Quantification of Ki67 positive nuclei was performed by a trained pathologist. Briefly, 10x digitized images  containing at least 100 nuclei were manually counted for Ki67 positivity. This was repeated three times (using different sections of tumor) for both pre- and post- PI3K treatment. Data were plotted with Graphpad Prism 7 and significance of difference determined using an unpaired t-test.Samples were processed per the Vanderbilt Head and Neck Cancer Tissue Repository and Clinical Database standards operating procedures. Surgical specimens were flash frozen in liquid nitrogen. Following H&E staining for tumor content, the freshly frozen tissue then was macro-dissected to enrich for ≥70% tumor content by a trained histotechnologist. Ten 30 µM slices of enriched tumor tissue were subsequently transferred to a microcentrifuge tube. Genomic DNA was extracted from this tube using standard proteinase K digestion and phenol extraction. Briefly, tumor tissue was incubated in DNA extraction buffer  and proteinase K overnight at 56 °C. Lysates were then treated with RNase A for 1 h at 37 °C, phenol:choloroform extracted, and DNA precipitated using isopropanol. The resulting gDNA was run on an agarose gel.DNA library preparation and whole genome sequencing was performed by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core laboratory. Briefly, 200 ng of intact genomic DNA was fragmented to an average size of 300 bp on a LE220 focused ultrasonicator (Covaris). Fragmented DNA was used to generate sequencing-ready libraries with indexed adaptors (Illumina). Library quality was assessed using the 2100 Bioanalyzer (Agilent) and libraries were quantitated using KAPA Library Quantification Kits (KAPA Biosystems). Pooled libraries were subjected to 100 bp paired-end sequencing on a HiSeq2500 (Illumina) per the manufacturer’s protocol. CASAVA v1.8 (Illumina) was used to generate de-multiplexed FASTQ files.64 to check for sequence quality  and indels) unique to the tumor tissue using VarScan v2.3.5,70 MuTect v1.1.3,71 and Strelka v1.0.672 using default parameters. A mutation was preserved if <2 reads supported the variant allele in the normal sample, if its average Phred quality score was >30, if it was not a strand bias artifact, and if it was not included in dbSNP v137.35 Tumor specific copy number variants were detected using Control-FREEC73 in its default settings. Somatic structural variants were characterized from the analysis-ready alignment files using tool CREST v1.174 with default parameters. Variant annotation was performed using ANNOVAR v2013-5-9.75For an overview of the analysis pipeline, see Figure ity Fig. . Reads wAs detailed above, DNA was extracted from freshly frozen tissue sections using the DNeasy Blood & Tissue Kit (Qiagen). PCR was performed with HotStarTaq Master Mix (Qiagen) using 50 ng DNA and the primers in Table 79 and, if necessary, converted to human homologs using biomaRt.80 Similarly, genes known to be mutated in cutaneous squamous cell carcinomas were compiled from previous studies.43 These sets of genes  AND . Studies from all available dates were included in the search, but language was limited to English language studies only. The database search yielded 930 results linked to the search query. Titles and abstracts were screened for further study based on their description of metastatic MPTT, including its molecular alterations, malignant progression, and management. Full text review of pertinent articles contributed the data for Tables Further information on research design is available in the Supplementary AppendixReporting Summary


Hepatitis A and B disease burden persists in the US. We assessed hepatitis A and hepatitis B vaccination series completion rates among 350,240 commercial/Medicare and 12,599 Medicaid enrollees aged ≥19 years. A vaccination series was considered as completed provided that the minimum interval between doses, as defined by the CDC, and the minimum number of doses were reached. We stratified completion rates by vaccine type  at initial vaccination for each cohort. In the commercial/Medicare cohort, the series completion rate was 32.0% for hepatitis A and 39.6% for hepatitis B among those who initiated with a monovalent vaccine, and it was 36.2% for hepatitis A and 48.9% for hepatitis B among those who initiated with a bivalent vaccine. In the Medicaid cohort, the series completion rate was 21.0% for hepatitis A and 24.0% for hepatitis B among those who initiated with a monovalent vaccine, and it was 19.0% for hepatitis A and 24.6% for hepatitis B among those who initiated with a bivalent vaccine. In conclusion, hepatitis A and B vaccination series completion rates were low, and appeared to be lower among Medicaid than among commercial/Medicare enrollees. Commercial/Medicare enrollees who initiated with a bivalent vaccine had higher series completion rates than those who initiated with monovalent vaccines – an observation that was not made among Medicaid enrollees. What is the context?Hepatitis A and B are vaccine-preventable liver diseases. Although the reported incidence of hepatitis A and hepatitis B infections have dropped significantly since the introduction of hepatitis vaccines, surveillance reports indicate that hepatitis A and B infections persist in adults.There is limited information on hepatitis A and hepatitis B vaccine series completion.What is new?In our large cohort study we found that commercial/Medicare and Medicaid enrollees had low rates of vaccination series completion for both hepatitis A and hepatitis B vaccines.What is the impact?These results demonstrate the need for interventions to increase hepatitis A and B vaccine series completion.1,2 Since the Centers for Disease Control and Prevention's (CDC) introduction of hepatitis A vaccination recommendations for infants in high-risk communities in 1996,3 followed by a recommendation for routine pediatric vaccination nationwide in 2006,4 and of routine hepatitis B vaccination recommendations for children in 1991,5 the reported incidence of hepatitis A has dropped by more than 90% and that of acute hepatitis B by more than 80% in the United States (US).6Hepatitis A and B are vaccine-preventable liver diseases caused by hepatitis A (HAV) and hepatitis B viruses (HBV), respectively.7. In 2015, the estimated number of new HAV infections in the US was 2,800 , while that for new HBV infections was 21,900 , representing increases over the previous year of 12% and 14%, respectively.8,9Despite this decrease, the disease burden of hepatitis A and B persists. Consistently, the US National Viral Hepatitis Action Plan 2017–2020 states that hepatitis B is still a serious threat to the health of the US population despite a ‘coordinated national response’ initiated in 20114 and hepatitis B vaccinations.10 In the US, the available types of hepatitis vaccines for adults are the monovalent hepatitis A (HepA) vaccines, the monovalent hepatitis B (HepB) vaccines, or a combined bivalent hepatitis A/hepatitis B (HepA-HepB) vaccine.11,12 The monovalent HepA vaccines are inactivated whole-virus vaccines, and the monovalent HepB vaccines are recombinant vaccines. The bivalent HepA-HepB vaccine contains one pediatric dose of monovalent HepA vaccine and one adult dose of monovalent HepB vaccine.11,12 Potential reasons for the persisting vaccine-preventable hepatitis disease burden may be suboptimal vaccination rates and poor vaccination coverage among adults, especially among adults at a high-risk for hepatitis A or B, compared to infants and children.7,13 In 2015, for adults aged ≥19 years, reported HepA vaccination coverage (≥2 doses) and HepB vaccination coverage (≥3 doses) was 9.0% and 24.6%, respectively.13For adults at high risk for infection, morbidity, or mortality, the US Advisory Committee on Immunization Practices (ACIP) issued specific recommendations for hepatitis A14 determined that HepA and HepB series completion rates in the Vaccine Safety Datalink population ranged between 40% to 65% among those who received a first dose, depending on the hepatitis vaccine (HepA or HepB), with higher completion rates in those aged <18 years. However, this study was done prior to the US Food and Drug Administration approval of bivalent vaccine in 2001.15 Bivalent vaccine introduction was expected to increase adherence to vaccination schedule, by offering fewer medical visits and by reducing the number of injections required by the separate administration of each monovalent vaccine.16 Other studies assessing HepA, HepB or HepA-HepB series completion were either based on a select patient population, such as travelers,17 or were conducted outside the US.17,18Literature on adherence to the CDC guidelines for HepA, HepB, and HepA-HepB vaccine series completion schedules among adults in the US is limited. One studyIn this context, we conducted a retrospective longitudinal cohort study using recent insurance claims data to assess HepA and HepB vaccination series completion rates among US adult enrollees of commercial/Medicare and Medicaid insurances, separately. We further stratified the results by type of vaccine, monovalent or bivalent, that was received when the vaccination series was initiated.The eligible population consisted of 350,240 commercial/Medicare patients and 12,599 Medicaid patients. As shown in Among Medicaid patients , full se19Assessing HepA and HepB vaccination series completion rates in a large sample from a US insurance claims dataset, we found that 32–36% of commercial/Medicare insurance enrollees received the full HepA vaccination series, and 40–49% received the full HepB vaccination series. The rate varied depending on whether they initiated on a monovalent or bivalent vaccine. Between 65% and 76% of patients initiating HepB vaccination received ≥2 doses. Although this analysis was not designed to compare completion rates between commercial/Medicare and Medicaid enrollees, the respective ranges for Medicaid enrollees were observed to be lower: 19–21% and 24–25% completed a full HepA and HepB vaccine series, respectively, and 52–54% received ≥2 HepB doses. In general, Medicaid enrollees face challenges in terms of gaps in access to certain providers because of provider shortages in low-income communities, lack of transportation, and lower physician fees and participation in Medicaid compared to commercial insurance.14 It was found that within one year of the first dose in adults, completion rates ranged 25–44% for HepA and 41–62% for HepB according to the age group .14 Their results also corroborate an observation of lower completion rates among Medicaid enrollees in the current study. The latest findings presented at 2017 congresses were derived from the same database as in our study.20,21 They showed that 27%, 51%20 and 40%21 of the patients received ≥2 doses of HepA, HepB, or HepA-HepB vaccines, respectively. No differentiation, however, was made between Medicaid and commercially insured individuals20 who have different socioeconomic, disease and disability status that cannot be controlled for in this database22; nor did the definition of series completion account for switching between the bivalent and monovalent hepatitis vaccines.21This study updates previously published material on HepA and HepB vaccination series completion rates among adults in the US, which are limited to only one full publication and two announcements in 2017 conferences. Nelson et al. report the results of a retrospective population-based study of patient vaccination data recorded from 1996 to 2004 in the administrative databases of seven health care organizations participating in the Vaccine Safety Datalink Project.13,27 used the National Health Interview Surveys (NHIS) to assess hepatitis vaccination coverage but none examined the series completion rates neither the type of vaccine  administered.Finally, a number of studies17 In another study, medical visits prior to or after starting HepA or HepB vaccination series was found to facilitate series completion.14,28Potential barriers to vaccination series completion may include administrative or financial barriers, limited access to vaccination services, or lack of awareness of recommended schedules among patients. For example, travelers who were partially vaccinated with HAV and HBV, believed that they had received the recommended number of HepA and HepB vaccine doses.29 Previous investigations have shown that electronic medical record (EMR) reminders significantly improved the rates of influenza and pneumococcal vaccination in a population of patients over 65 years of age.30 More recent analyses31,32 compared 3-dose HepB vaccine completion rates in patients with diabetes aged 19–59 years between two large US managed care organizations, Kaiser Permanente Southern California in which an electronic alert and a reminder were implemented in the EMR and Kaiser Permanente Northern California in which no such reminder was used.31 The site with EMR reminders increased vaccination coverage rapidly from 7.6% in 2012 to 29.4% in 2015, while no increase was seen at the site with no EMR reminder.31,32 EMR are increasingly being adopted by health systems,31 and might therefore be a valuable tool for improving vaccination completion.Our results highlight the need for future research to determine the effectiveness of interventions to increase vaccination completion rates. Electronic health records with complete information on patient's immunization history are among the Standards for Adult Immunization Practice recommended by the National Vaccine Advisory Committee.Selection of patients with a HepA, HepB, or HepA-HepB vaccine was based on CPT codes, and coding inaccuracies may lead to misclassification bias and misidentification of patients with a hepatitis vaccine. In addition, CPT codes cannot distinguish between brands of hepatitis vaccines and thus cannot differentiate between vaccination schedules for the two HepA vaccines. Completion windows were adjusted to enable completion of the longest time window within each vaccine series. Finally, our population was covered by employer-sponsored health care with supplemental Medicare plans, or Medicaid, and may not be generalizable to an uninsured population.We used the Truven Health Analytics MarketScan claims database, which is a large US administrative claims database. Data retrieved included Q1 2007-Q3 2015 data from the Commercial Claims and Encounters database and the Medicare Supplemental and Coordination of Benefits database  and Q1 2007-Q4 2014 data from the Medicaid Multi-State database (“Medicaid” cohort). The Truven MarketScan database is de-identified and complies with the Health Insurance Portability and Accountability Act (HIPAA) to preserve patient anonymity and confidentiality.The date of the first Current Procedural Terminology (CPT) code for a HepA, HepB, or HepA-HepB vaccine was defined as the ‘index date’. More precisely, patients who initiated a series for HepA and another series for HepB vaccine with monovalent vaccines had distinct index dates that corresponded to the first dose of each vaccine series. However, once a patient had initiated a HepA or HepB series, their index date for each series was locked. Thus, patients who initiated with a monovalent vaccine could not have a later bivalent index date, and patients who initiated with a bivalent vaccine could not have a later monovalent index date. Identification of subsequent doses was also based on CPT codes. Study periods consisted of a ‘baseline period’ that was the period from the date of health plan enrollment up to and including the index date, and an ‘observation period’ that was the period from the index date to the end of insurance coverage or data cut-off .10.1080Patients were included in the study if they had ≥1 claim with a CPT code for HepA, HepB, or HepA-HepB vaccine, were ≥19 years of age at index date, and had continuous insurance enrollment for ≥12 months before the index date (“baseline period”) and ≥18 months after the index date (“observation period”). This was to allow for the evaluation of vaccination series completion, because the minimum required time for the completion of a full series vaccination against HepA is 6 months and against HepB is 4 months . Patient11,12 (HepA and HepB vaccine series completion was assessed separately for HepA and HepB and was defined as a series of either monovalent HepA or HepB vaccines, bivalent vaccines, or a combination of monovalent HepA or HepB vaccines with bivalent HepA-HepB vaccine, as shown in 11,12 . The proAll analyses were stratified by commercial/Medicare and Medicaid patient cohorts. Descriptive statistics were generated to summarize patient baseline characteristics and series completion. Additionally, the number and proportion of patients completing ≥2 doses of HepB and the full HepA and HepB series were assessed and analyses were stratified according to whether the initial dose was a monovalent or bivalent vaccine.HepA and HepB vaccination series completion rates were low among US adults, and appeared to be generally lower among Medicaid than among commercial/Medicare enrollees. Adult commercial/Medicare patients starting hepatitis vaccination series with the bivalent vaccine had higher rates of completion than those starting with monovalent vaccines – an observation that was not made among Medicaid enrollees. Low rates of HepA and HepB vaccination series completion rates among adults in the US, demonstrate the need for interventions that increase series completion, thereby offering optimal protection for individual patients, and contributing to public health.Not applicable.nd Annual International Meeting; May 20–24, 2017; Boston, MA, USA.ISPOR 22


Tenebrio molitor larvae meal showed the highest apparent digestibility coefficient, being attested as a potential alternative feed for Nile tilapia fingerlings. Those findings may contribute to sustainable development of the tilapia production around the world.Insects can be a source of nutrients for aquatic organisms, replacing scarce or unsustainable foods. The diversity of insect species contributes to their variable nutritional composition, which fish may digest differently. Nile tilapia is a very important fish for aquaculture, which requires great quantities of quality protein and other dietary nutrients on its initial rearing phase. Therefore, it is important to better understand the technical feasibility of using insect meal as a nutrient and energy source for Nile tilapia fingerlings. In this study, Nauphoeta cinerea meal (NCM) (Blattodea), Zophobas morio larvae meal (ZMM) (Coleptera), Gromphadorhina portentosa meal (GPM) (Blattodea), Gryllus assimilis meal (GAM) (Orthoptera) and Tenebrio molitor larvae meal (TMM) (Coleptera)) and three replicates (tanks), each containing 50 fish. The control diet had no insect meal included and the other five treatments comprised 80% commercial diet and 20% test ingredient with 0.1% chromic oxide as an inert marker. TMM presented a higher ADC for dry matter, protein, corrected protein and chitin than to other treatments (p < 0.01). GPM presented the highest ADC for lipids (p < 0.01). In general, the TMM presented better ADC of nutrients and energy and all the insect meals evaluated are potential feed for Nile tilapia fingerlings. Insects are a valuable source of nutrients for fish, but little is known about their nutritional value for Nile tilapia fingerlings. To evaluate the nutritional value and energy apparent digestibility coefficient (ADC) of five insects for Nile Tilapia male fingerlings, 900 fish were distributed in 18 fiberglass conic tanks, in a completely randomized design, with six dietary treatments (control,  The use of alternative ingredients in aquaculture feeds is intended to minimize the dependence on scarce, expensive or unsustainable feedstuff. The main ingredients traditionally used in fish feed are commodities whose price is set by the inconstant global supply and demand. Soybean meal is one of the most used vegetal protein sources in aquafeed formulation due to its high protein content, and amino acid profile as well as its price . HoweverInsect meal has stood out as an alternative ingredient to be included in animal feeds . InsectsClarias gariepinus [Oncorhynchus mykiss [Cyprinus carpio [Pelteobagrus fulvidraco [Oreochromis sp. [Oreochromis niloticus [Nauphoeta cinerea meal (NCM). The authors concluded that cinerea cockroach meal may be used as feed for Nile tilapia fingerlings. Jabir et al. (2012) [Zophobas morio larvae meal (SMM) and fishmeal (FM), the main dietary protein sources for juvenile red tilapia. Protein and lipid ADC in the SWM based diet was lower  than in the FM diet . The authors concluded that the ADC of SWM would need to be improved for it to be a reasonable alternative for a partial or complete replacement of FM in the red tilapia diet. Studies in recent years have evaluated the use of insects as feed for freshwater fish, such as African catfish riepinus ,11, rains mykiss , Jian cas carpio , yellow lvidraco , red tilomis sp.  and Nileiloticus . Frecciailoticus  did not . (2012)  comparedNile tilapia is the fourth most farmed fish species around the world, representing 8% of total global inland production  and 36% Nauphoeta cinerea and Gromphadorhina portentosa), one cricket species (Gryllus assimilis) and two species of tenebrio (Zophobas morio and Tenebrio molitor) for Nile tilapia fingerlings. Therefore, the aim of this study was to assess the nutrient and energy apparent digestibility coefficient from five insect meals; two cockroach species . The committee only assess studies involving animals from the phylum Chordata, subphylum Vertebrata, which is why the procedures with involving insects are not involved in the mentioned protocol. Oreochromis niloticus (3.0 ± 0.2 g of mean weight). The trial was set up in a recirculating system adapted to the Guelph system, equipped with a fiber mesh pad filter, a submerged biological filter and an ultraviolet filter. The system was supplied with constant aeration, and thermoregulation (26 ± 1 °C). Oxygen saturation in the water was 85% ± 2%; the ammonia and nitrite concentration amounted to 0.2 ± 0.1 and 0.3 ± 0.1 mg/L, respectively. A 12-h light/dark cycle was adopted. An in vivo digestibility trial was performed to assess the apparent digestibility coefficients (ADC) of the dietary nutrients and energy. The feeding trial was carried out at the Experimental Aquaculture Station of the Animal Science Department at the Federal University of Lavras in a completely randomized design with six dietary treatments  and three replicates (fiberglass cylindro-conic tanks with 250 L capacity) each containing 50 reversed male Nile Tilapia fingerlings Nauphoeta cinerea, Gromphadorhina portentosa and Gryllus assimilis were harvested as adults while Zophobas morio and Tenebrio molitor were harvested as larvae.The insect meals were obtained from the Laboratory of Entomoculture of at the Institute of Agricultural Sciences of the Federal University of Minas Gerais . All the insects were reared on a vegetal diet , killed by immersion in boiling water, dried in a forced air oven (50 °C for 48 h) and milled in an electric screw meat grinder . 2O3) as an inert marker. The control diet was used as the reference diet, and the data collected from this group was used to calculate the test ingredient’s ADCs, as described below. After 10 days of adaptation to laboratory conditions, the fish were fed three times daily to the point of apparent satiation for 15 days with the experimental diets, with an average of 18 g of feed per feeding per tank. After the last daily feeding, tanks were cleaned and plastic tubes connected at the bottom of each tank for feces collection overnight, for 15 days. The next morning, the tubes were removed and feces dried in a forced-air drying oven (60 °C) for 48 h and stored in a freezer (−20 °C) for laboratory analysis.The fish diets comprised 20% test ingredient  and 80% control diet  with 0.1% chromium oxide   for dry The ADC of nutrients and energy from the experimental diets was calculated following the equations Bureau and Hua  proposed(1)ADC is the apparent digestibility coefficient;Id is chromic oxide’s concentration in the diet (%); If is chromic oxide’s concentration in the feces (%); Nd is the nutrient’s concentration in the diet (%); Nf is the nutrient’s concentration in the feces (%);(2)ADCi is the nutrient’s apparent digestibility coefficient in the test ingredient;ADCdt is the nutrient’s apparent digestibility coefficient in the test diet; ADCref is the nutrient’s apparent digestibility coefficient in the reference diet; r is the reference diet’s proportion in the test diet (0.6994); i is the test ingredient’s proportion in the test diet (0.3); Nref is the nutrient’s concentration in the reference diet (% as fed); Ni is the nutrient’s concentration in the ingredient (% as fed).ADC (1) and ADCi (2) were calculated according to the following equations:Data is expressed as mean and standard error of the means (SEM). Normality and homogeneity of variances were tested using the Shapiro–Wilk and Levene tests, respectively. Data were analyzed by one-way ANOVA followed by Tukey’s multiple range test at a 1% probability level. All statistical analyses were performed using SPSS 22.0 software package .p < 0.01). Tenebrio molitor meal presented a higher ADC for dry matter, protein, corrected protein and chitin than the other tested ingredients (p < 0.01). The cricket Gryllusassimilis meal had the lowest digestibility for protein and corrected protein (p < 0.01). For energy, Zophobas morio and T molitor meals presented higher ADC’s than the other treatments (p < 0.01), and G. assimilis and the cockroaches, Nauphoetacinerea and Gromphadorhinaportentosa, did not differ. The lipid ADC of G. portentosa meal was the highest among the treatments (p < 0.01). The highest chitin ADC was found in T. molitor meal and the lowest in N. cinerea meal (p < 0.01). The ADC of the insect meal’s dry matter, protein, corrected protein, energy, lipids and chitin are presented in Insects have been widely studied as fish feed in the last few years, but only a few studies have focused on the nutrient and energy digestibility. The variation in nutrient and energy digestibility is based on the insect species, life stage, and the fish species , and mayT. molitor meal. A reduction in nutrient digestibility due to the presence of chitin was expected ,12, as PN. cinerea, G. portentosa and G. assimilis, ranging from 42.6% to 61.7%. This decrease in dry matter ADC can be explained by the feed’s bromatological composition, in which higher chitin values were found. Shiau and Yu [T. molitor and Z. morio the dry matter ADC was higher than the other diets’, which corroborates Shiau and Yu’s [T. molitor and Z. morio had the lowest chitin percentage in their composition.Low values of dry matter ADC are found for u and Yu , using dand Yu’s  researchN. cinerea, G. portentosa and G. assimilis meals, in the present study, presented lower protein ADC, which can be connected to this feed’s protein quality, that is, their amino acid composition or even because of their higher chitin content. However, tenebrios, T. molitor, and Z. morio meals showed dry matter ADC close to the reported values for fishmeal and soybean meal [Tenebrio molitor meal was also close to that of those feeds [We have shown in the results the corrected protein parameter because chitin is a nitrogen-compound, so Kjeldahl’s method may overestimate the insect meal’s protein content . Neverthean meal  for Nilese feeds .Gammarus kischineffensis and Astacus leptodactylus) for Nile tilapia and reported chitin ADC of 71.5% and 69.3%, respectively. For cobia Rachycentron canadum, chitin ADC from brown shrimp (Penaeus aztecus) and crab meals (Brachyura) were 78.2% and 66.8% respectively [Moreover, the type of chitin matrix in insects may negatively influence chitinase activity and thus protein digestibility . Those eectively . The chiG. portentosa meal presented the highest chitin content (28.94%) and the highest lipid ADC (98.8%) among treatments. Additionally, N. cinerea and G. assimilis showed 91.6% and 87.9%, respectively, and this reduction was not a reflex of chitin levels, as Shiau and Yu [According to Tanaka et al. , chitin u and Yu  demonstrBombyx mori) have high amounts of digestible fatty acids and were as effective as sardine oil in providing energy to induce fast growth in common carp (Cyprinus carpio) fed 3%, 6% and 9% silkworm meal or sardine oil. Those authors also reported that fat digestibility increased with an increasing inclusion level of non-defatted insect meal. Lipid digestibility among fish is known to be quite variable and dependent on many factors, such as the inclusion level and fat source [Nandeesha et al.  reportedt source . In genet source . G. portentosa meal presented the highest percentage of lipid digestibility (98.8%) and low protein and corrected protein digestibility (61.6% and 58.3% respectively). Nandeesha et al. [The a et al.  reportedTenebrio molitor larvae meal is the most suitable feed for Nile tilapia fingerlings among the insect meals evaluated. Further investigation is needed to understand the nutritional value and optimal inclusion level of various insect meals for fish.In general, all the insect meals studied are potential alternative ingredients for aquafeed formulation.


A compact optical nanofiber sensor enables detection of 100 nm particles in the field. This device improves our ability to track air quality with high spatiotemporal resolution.1 and costing US$225 billion globally in 2013 according to the most recent data from the World Health Organization (WHO) and World Bank, respectively2. Despite this impact, air pollution levels continue to increase, with 92% of the world’s population living in environments in which WHO air quality guidelines are not met3. In the United States, the Environmental Protection Agency (EPA) regulates six criteria air pollutants, one of which is particulate matter (PM)4. While portable sensors for particles under 2.5 μm in diameter (known as PM2.5) exist, compact sensor development for ultrafine particles, which are <100 nm in diameter, has lagged. Ultrafine particles are unregulated by the EPA, and due to their small size, they can directly enter the lungs or the brain and have been linked to cancer and Alzheimer’s, among other diseases5. The main source of ultrafine particles is exhaust from motor vehicles, combustion, and industrial processes6. Despite local variations in air pollution levels7 for both fine and ultrafine particles, air monitoring systems are few and far between, numbering approximately 2–3 stations for cities of 1,000,000 people or more in the United States8. Furthermore, such monitoring systems are large , Xiao’s sensor can also provide highly precise size information, which can help to better establish correlations between pollution composition and health hazards and can inform future environmental regulations.The serpentine nano-optical fiber sensor measures particulate concentrations by monitoring step-wise drops in the optical power transmitted through the fiber as particles land. These changes in power can be related to the size of the particle that has landed on the fiber using Rayleigh–Gans scattering theory12, Xiao’s sensor does not require a tunable laser, which makes this system cheaper and more portable.The sensor was used for particulate matter monitoring in Beijing over a period of a month, and the data agree well with official data from the Beijing Municipal Environmental Monitoring Center, demonstrating that the sensor can be used for real-world, long-term monitoring. In these experiments, measurements were taken every few hours. Unlike other precise particle sensing systemsThe future of air pollution monitoring technology is low cost, chemically specific, sensitive, weather protected, mobile sensor arrays capable of being worn or attached to existing infrastructure Fig. . When us