Exploring human biology with N-of-1 clinical trials.

Studies on humans that exploit contemporary data-intensive, high-throughput 'omic' assay technologies, such as genomics, transcriptomics, proteomics and metabolomics, have unequivocally revealed that humans differ greatly at the molecular level. These differences, which are compounded by each individual's distinct behavioral and environmental exposures, impact individual responses to health interventions such as diet and drugs. Questions about the best way to tailor health interventions to individuals based on their nuanced genomic, physiologic, behavioral, etc. profiles have motivated the current emphasis on 'precision' medicine. This review's purpose is to describe how the design and execution of N-of-1 (or personalized) multivariate clinical trials can advance the field. Such trials focus on individual responses to health interventions from a whole-person perspective, leverage emerging health monitoring technologies, and can be used to address the most relevant questions in the precision medicine era. This includes how to validate biomarkers that may indicate appropriate activity of an intervention as well as how to identify likely beneficial interventions for an individual. We also argue that multivariate N-of-1 and aggregated N-of-1 trials are ideal vehicles for advancing biomedical and translational science in the precision medicine era since the insights gained from them can not only shed light on how to treat or prevent diseases generally, but also provide insight into how to provide real-time care to the very individuals who are seeking attention for their health concerns in the first place.

Agricultural Safety Education: Formative Assessment of a Curriculum Integration Strategy.

The purpose of this study was to assess an agricultural tractor and machinery safety curriculum for teacher training that focused on hands-on integration activities to assist with training youth in machinery safety skills. Teachers attended a single ten-hour summer training seminar hosted in Montana, South Dakota, or Utah during 2017. Teachers completed the National Tractor and Machinery Safe Operation (NSTMOP) exam to measure their existing knowledge prior to beginning the training. Upon seminar completion, teachers took an NSTMOP post-test to measure their knowledge gain of agricultural safety practices and hazard recognition associated with machinery and tractors. A total of 116 teachers completed the training. Fifty-three participants (45.7%) identified as female, and 63 (54.3%) identified as male. The average participant was 35 years old (SD = 11.3) and had 9.5 years of teaching experience (SD = 9.2). The average NSTMOP pre-test score was 35.2 out of 48 (SD = 3.3), and the average NSTMOP post-test score was 40.3 out of 48 (SD = 4.1). Participants' scores increased by ten percentage points. A paired-samples t-test was used to determine statistical significance. The difference between pre-test and post-test was significant (t(109) = 11.9, p < 0.001). Open responses indicated continuation of hands-on activities that focused on "how to teach" skills training that is relevant to the students. Teachers suggested developing new activities each year with a rotation of topics for upcoming seminars. Research is needed to determine the training's influence on the behaviors of young workers in agriculture.

Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

Background: Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. Material and methods: ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. Results: The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4-NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s): ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

A novel system for expansion and delivery of human keratinocytes for the treatment of severe cutaneous injuries using microcarriers and compressed collagen.

Cell therapy with autologous or allogeneic keratinocytes applied as a single-cell suspension is well established in clinical practice in the treatment of severe burn injuries to augment epithelial barrier restoration. Yet, the application of cell sprays can lead to significant cell loss owing to lack of adhesion of cell suspension to the wound bed. The development of a robust and controllable method of transplanting cells onto the wound bed is yet to be established. The ability to control adhesion and distribution of cells by using a cell carrier embedded in a biodegradable scaffold could significantly improve the treatment of cutaneous wounds with keratinocyte cell therapy. Several microcarrier-based systems for expanding keratinocytes already exist. A new method for expansion of human keratinocytes in a feeder-free, defined medium system on microcarriers has been developed. The cells retained their basal, proliferative phenotype after rapid expansion in a clinically relevant time-frame. The cell-laden microcarriers were further incorporated into collagen scaffolds fabricated by plastic compression. When cultured in vitro, cells continued to proliferate and migrate along the surface of the collagen scaffold. Using an in vitro wound bed model, cells were observed to form mostly single cell layers and in some areas multiple cell layers within 8 days, while retaining their basal, proliferative phenotype, indicating the suitability of this cell transplantation method to improve epithelial barrier restoration. This advanced cell expansion and delivery method for cutaneous cell therapy provides a flexible tool for use in clinical application. Copyright © 2017 John Wiley & Sons, Ltd.

Plasma levels of interleukin-6 and interleukin-18 after an acute physical exercise: relation with post-exercise energy intake in twins.

Increased interleukin-6 (IL-6) plasma levels have been described to occur during physical exercise. A relative reduction in energy intake after physical activity has also been reported after exercise, indicating a possible involvement of IL-6 as an anorexigenic factor. Given the possible effect of interleukins on appetite, we assessed whether a controlled physical activity bout is related with changes in IL-6, IL-6 soluble receptor (IL-6sR), gp130 and interleukin-18 (IL-18) plasma levels, as well as their relation with post-exercise energy intake. A co-twin intervention study was carried out with five young male monozygotic twin pairs. One co-twin performed 45 min of submaximal exercise on a treadmill near the anaerobic threshold ending with 7 min at 90 % VO(2) max, while his co-twin remained non-active. Ad libitum energy intake was tested through a carbohydrate-rich meal test. Venous blood samples were drawn at baseline, immediately after exercise and after the meal ingestion. Plasma concentrations of IL-6, IL-6sR, gp130 and IL-18 were measured via ELISA. IL-6 plasma levels increased after physical activity bout (2.6-fold change; p = 0.04). A less marked trend, although still significant, was observed for plasma levels of IL-6sR and gp130. Plasma levels of IL-18 did not significantly change during exercise. The twins who exercised exhibited significantly lower energy intake (181 versus 1,195 kcal; p = 0.04), compared to the co-twins who remained resting. The present study in monozygotic twins shows increased IL-6 plasma levels after acute physical exercise with a significant reduction in energy intake, supporting a linkage between IL-6 and acute post-exercise eating behaviour.

Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health.

Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductase.

Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies.

Immunomodulation of innate immune responses by vasoactive intestinal peptide (VIP): its therapeutic potential in inflammatory disease.

Since the late 1970s a number of laboratories have studied the role of vasoactive intestinal peptide (VIP) in inflammation and immunity. These studies have highlighted the dramatic effect of VIP on immune cell activation and function, and studies using animal models of disease have indicated that VIP has significant therapeutic and prophylactic potential. This review will focus on the effects of VIP on innate immune cell function and discuss the therapeutic potential for VIP in inflammatory diseases of humans.

Congenital disseminated neurofibromatosis type 1: a clinical and molecular case report.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition with a birth incidence of 1/3,500. Around 50% of cases are due to new mutations. The NF1 gene maps to 17q11.2 and encodes neurofibromin. NF1 is a "classical" tumor suppressor gene. Congenital disseminated NF1 is rare with just two cases previously reported. We present a deceased baby with congenital disseminated NF1 in whom we performed molecular studies. A germline mutation (R461X) in exon 10a of the NF1 gene was found. A 2 bp deletion (3508delCA) in codon 1170 of exon 21 was identified in DNA derived from some tumor tissue. Loss of heterozygosity in NF1 and TP53 was observed in other tumor samples. No microsatellite instability was observed in the tumor samples. This is the first report of molecular analysis of the NF1 locus in a patient with disseminated congenital neurofibromatosis. This case had a de novo germline mutation in NF1 and three documented somatic mutations in the NF1 and TP53 genes in tumor specimens.

Substrate changes associated with the chemistry of self-assembled monolayers on silicon.

Alkylsiloxane self-assembled monolayers (SAMs) are used in the semiconductor industry and, more recently, as proxies for organics adsorbed on airborne mineral dust and on buildings and construction materials. A number of methods have been used for removing the SAM from the substrate after reaction or use, particularly plasmas or piranha (H2SO4/H2O2) solution. However, when the substrates are reused to make new SAMs, the impact of the cleaning methods on the chemistry of subsequently formed SAMs on the surface is not known. Here we report atomic force microscopy, X-ray photoelectron spectroscopy, Auger electron spectroscopy, and Fourier transform infrared studies of changes in a silicon substrate upon repetitive deposition and removal of SAMs by these two methods. It is shown that a thicker layer of silicon oxide is formed, and the surface becomes irregular and roughened, particularly after the piranha treatment. This layer of silica impacts the structure of the SAMs attached to it and can serve as a reservoir for trace gases that adsorb on it, potentially contributing to the subsequent reactions of the SAM. The implications for the use of such surfaces as a proxy for reactions of organics on airborne dust particles and on structures in the boundary layer are discussed.

Transmission disequilibrium testing of dopamine-related candidate gene polymorphisms in ADHD: confirmation of association of ADHD with DRD4 and DRD5.

Attention-deficit hyperactivity disorder (ADHD) is one of the most common childhood behavioral disorders. Genetic factors contribute to the underlying liability to develop ADHD. Reports implicate variants of genes important for the synthesis, uptake, transport and receptor binding of dopamine in the etiology of ADHD, including DRD4, DAT1, DRD2, and DRD5. In the present study, we genotyped a large multiplex sample of ADHD affected children and their parents for polymorphisms in genes previously reported to be associated with ADHD. Associations were tested by the transmission disequilibrium test (TDT). The sample is sufficient to detect genotype relative risks (GRRs) for putative risk alleles. The DRD4 gene 120-bp insertion/deletion promoter polymorphism displayed a significant bias in transmission of the insertion (chi(2)=7.58, P=0.006) as suggested by an analysis of a subset of these families. The seven repeat allele of the DRD4 48-bp repeat polymorphism (DRD4.7) was not significantly associated with ADHD in the large sample in contrast to our earlier findings in a smaller subset. We replicate an association of a dinucleotide repeat polymorphism near the DRD5 gene with ADHD by showing biased nontransmission of the 146-bp allele (P=0.02) and a trend toward excess transmission of the 148-bp allele (P=0.053). No evidence for an association was found for polymorphisms in DRD2 or DAT1 in this sample. The DRD5 146-bp (DRD5.146) allele and the DRD4 240-bp (DRD4.240) allele of the promoter polymorphism emerge as the two DNA variants showing a significant association in this large sample of predominantly multiplex families with ADHD, with estimated GRRs of 1.7 and 1.37, respectively.

Genome-wide scan of reading ability in affected sibling pairs with attention-deficit/hyperactivity disorder: unique and shared genetic effects.

Attention-deficit/hyperactivity disorder (ADHD) and reading disability (RD) are common highly heritable disorders of childhood, which frequently co-occur. Data from twin and family studies suggest that this overlap is, in part, due to shared genetic underpinnings. Here, we report the first genome-wide linkage analysis of measures of reading ability in children with ADHD, using a sample of 233 affected sibling pairs who previously participated in a genome-wide scan for susceptibility loci in ADHD. Quantitative trait locus (QTL) analysis of a composite reading factor defined from three highly correlated reading measures identified suggestive linkage (multipoint maximum lod score, MLS>2.2) in four chromosomal regions. Two regions (16p, 17q) overlap those implicated by our previous genome-wide scan for ADHD in the same sample: one region (2p) provides replication for an RD susceptibility locus, and one region (10q) falls approximately 35 cM from a modestly highlighted region in an independent genome-wide scan of siblings with ADHD. Investigation of an individual reading measure of Reading Recognition supported linkage to putative RD susceptibility regions on chromosome 8p (MLS=2.4) and 15q (MLS=1.38). Thus, the data support the existence of genetic factors that have pleiotropic effects on ADHD and reading ability--as suggested by shared linkages on 16p, 17q and possibly 10q--but also those that appear to be unique to reading--as indicated by linkages on 2p, 8p and 15q that coincide with those previously found in studies of RD. Our study also suggests that reading measures may represent useful phenotypes in ADHD research. The eventual identification of genes underlying these unique and shared linkages may increase our understanding of ADHD, RD and the relationship between the two.

Biased paternal transmission of SNAP-25 risk alleles in attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is the most common childhood psychiatric disorder, affecting 5-10% of school-age children. Although the biological basis of this disorder is unknown, twin and family studies provide strong evidence that ADHD has a genetic basis involving multiple genes. A previous study found an association between ADHD and two polymorphisms in the 3' untranslated region (UTR) of SNAP-25, a gene encoding a synaptic vesicle docking protein known to play a role in the hyperactivity observed in the Coloboma mouse strain. In this paper, we test biased transmission of the 3' UTR SNAP-25 haplotype using a larger ADHD sample of 113 families with 207 affected children. Using the transmission disequilibrium test (TDT), we found a trend consistent with biased transmission of the TC haplotype of SNAP-25 in all transmissions and detected a significant distortion (P=0.027) when paternal transmissions were evaluated.

Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction.

BACKGROUND: Surgical resection of adenocarcinoma of the stomach is curative in less than 40 percent of cases. We investigated the effect of surgery plus postoperative (adjuvant) chemoradiotherapy on the survival of patients with resectable adenocarcinoma of the stomach or gastroesophageal junction. METHODS: A total of 556 patients with resected adenocarcinoma of the stomach or gastroesophageal junction were randomly assigned to surgery plus postoperative chemoradiotherapy or surgery alone. The adjuvant treatment consisted of 425 mg of fluorouracil per square meter of body-surface area per day, plus 20 mg of leucovorin per square meter per day, for five days, followed by 4500 cGy of radiation at 180 cGy per day, given five days per week for five weeks, with modified doses of fluorouracil and leucovorin on the first four and the last three days of radiotherapy. One month after the completion of radiotherapy, two five-day cycles of fluorouracil (425 mg per square meter per day) plus leucovorin (20 mg per square meter per day) were given one month apart. RESULTS: The median overall survival in the surgery-only group was 27 months, as compared with 36 months in the chemoradiotherapy group; the hazard ratio for death was 1.35 (95 percent confidence interval, 1.09 to 1.66; P=0.005). The hazard ratio for relapse was 1.52 (95 percent confidence interval, 1.23 to 1.86; P<0.001). Three patients (1 percent) died from toxic effects of the chemoradiotherapy; grade 3 toxic effects occurred in 41 percent of the patients in the chemoradiotherapy group, and grade 4 toxic effects occurred in 32 percent. CONCLUSIONS: Postoperative chemoradiotherapy should be considered for all patients at high risk for recurrence of adenocarcinoma of the stomach or gastroesophageal junction who have undergone curative resection.

Refining the ADHD phenotype using affected sibling pair families.

Attention deficit hyperactivity (ADHD) is a highly heritable behavioral disorder characterized by symptoms of inattention, hyperactivity, and/or impulsivity. Detection of susceptibility genes underlying ADHD may benefit from refinement of the ADHD phenotype into components that reflect more specific gene to behavior pathways and through the reduction of etiological heterogeneity. Using affected sibling pair (ASP) families, we are examining familial clustering of ADHD symptoms, neurocognitive task performance, and the occurrence of comorbid conditions to attempt to refine the phenotype and/or identify clinical features that may be used to stratify ADHD subjects to reduce etiological heterogeneity.

Susceptibility markers in colorectal cancer.

Many susceptibility factors contribute to an individual's risk of developing colorectal cancer. Family history of colorectal cancer (particularly with early age of onset), maleness and increasing age are all factors associated with increasing risk. About three quarters of colorectal cancers are thought to be due to somatic mutations, and both high- and low-penetrance predisposing genes contribute to the remaining quarter of cases. Many of the highly penetrant dominant genes are known, but others remain to be identified. Describing the contribution of individual genes is likely to be very complex, as some modify the impact of other genes and other environmental factors rather than incurring a direct, easily attributable effect. The two dominant predisposing syndromes are familial adenomatous polyposis and Lynch syndrome, the first due to a mutant tumour-suppressor gene APC, and the second due to mutations in a number of genes responsible for mismatch repair in DNA at cell division. Establishing genetic susceptibility for colorectal cancer will soon be possible, and could save lives by allowing targetting of screening and the encouragement of preventive behaviours. However, there will always be a risk of making healthy people "sick" through the identification of predisposing genes, and there are many potential ways by which a gene carrier may be stigmatized by society, insurance companies and employers.

Macrocephaly in autism and other pervasive developmental disorders.

To assess the prevalence of macrocephaly (head circumference > or = 1.88 standard deviations above normative data for age and sex or > 97th centile) in autism and other pervasive developmental disorders, 41 children with autism, and a comparison group of 21 children with tuberous sclerosis complex (TSC) or an unspecified seizure disorder were studied. Familiality of head circumference was also assessed from measurements of 133 first-degree relatives. Significantly higher rates of macrocephaly were found in probands with autism (12.2%) and their first-degree relatives (15.5%) when compared against a published normative sample. The incidence of macrocephaly in the comparison group of probands with TSC and seizure disorder (9.5%) and their first-degree relatives (8.3%) was higher than normative data as well, although the relation between macrocephaly and autism was more pronounced. Head circumference and extreme scores reflecting macrocephaly were moderately heritable in the present sample (H2 = 0.47). The increased prevalence of macrocephaly in relatives of children with autism compared with control children suggests that this characteristic may be a familial risk factor in the pathogenesis of autism.

Evidence for linkage of a tandem duplication polymorphism upstream of the dopamine D4 receptor gene (DRD4) with attention deficit hyperactivity disorder (ADHD).

Attention deficit hyperactivity disorder (ADHD) is a common childhood-onset neurodevelopmental disorder. Evidence from twin, adoption, and family studies provide support for a genetic contribution to the etiology of ADHD. Several candidate gene studies have identified an association between a 7-repeat variant in exon 3 of the dopamine 4 receptor gene (DRD4) and ADHD. However, in spite of the positive reports finding association of the exon 3 VNTR with ADHD, several other polymorphisms within DRD4 have been identified that conceivably could contribute to risk for ADHD. Recently, another common polymorphism of the DRD4 gene has been described involving a 120-bp repeat element upstream of the 5' transcription initiation site. In this report, we describe results of analysis of the DRD4 120-bp repeat promoter polymorphism in a sample of 371 children with ADHD and their parents, using the transmission disequilibrium test (TDT). Results showed a significant preferential transmission of the 240-bp (long) allele with ADHD. Exploratory analyses of the Inattentive phenotypic subtype of ADHD strengthened the evidence for linkage. These data add further support for the role of DRD4 variants conferring increased risk for ADHD, and imply that additional studies of DRD4 and other related genes are needed.