Mitochondrial dysfunction plays a key role in the pathophysiology of neurodegenerative disorders such as ataxia and Parkinson's disease. We describe an extended Belgian pedigree where seven individuals presented with adult‐onset cerebellar ataxia, axonal peripheral ataxic neuropathy, and tremor, in variable combination with parkinsonism, seizures, cognitive decline, and ophthalmoplegia. We sought to identify the underlying molecular etiology and characterize the mitochondrial pathophysiology of this neurological syndrome.Clinical, neurophysiological, and neuroradiological evaluations were conducted. Patient muscle and cultured fibroblasts underwent extensive analyses to assess mitochondrial function. Genetic studies including genome‐wide sequencing were conducted.c oxidase deficiency, and multiple mtDNA deletions. We identified a splice acceptor variant in POLG2, c.970‐1G>C, segregating with disease in this family and associated with a concomitant decrease in levels of POLG2 protein in patient cells.Hallmarks of mitochondrial dysfunction were present in patients’ tissues including ultrastructural anomalies of mitochondria, mosaic cytochrome POLG2 deficiency to include an overwhelming, adult‐onset neurological syndrome that includes cerebellar syndrome, peripheral neuropathy, tremor, and parkinsonism. We therefore suggest to include POLG2 sequencing in the evaluation of ataxia and sensory neuropathy in adults, especially when it is accompanied by tremor or parkinsonism with white matter disease. The demonstration that deletions of mtDNA resulting from autosomal‐dominant POLG2 variant lead to a monogenic neurodegenerative multicomponent syndrome provides further evidence for a major role of mitochondrial dysfunction in the pathomechanism of nonsyndromic forms of the component neurodegenerative disorders.This work extends the clinical spectrum of  POLG and a dimeric accessory subunit encoded by POLG2. Pathogenic POLG variants are a common cause of primary mitochondrial disorders typically resulting in adPEO, but can also lead to a phenotypic spectrum manifesting variably across brain, muscle, and/or liver .Mitochondrial dysfunction caused by disturbances of mtDNA integrity gives rise to a diverse array of neurological, neuromuscular, and multisystem disorders. The clonal expansion of multiple mtDNA deletions is typically associated with late‐onset primary mitochondrial disease characterized by autosomal‐dominant progressive external ophthalmoplegia (adPEO) as the predominant clinical feature.POLG2. The first reported case of POLG2 disease was documented in a patient with adult‐onset autosomal dominant PEO in whom multiple mtDNA deletions were detected.POLG2 disease was a patient who developed ptosis at age 30, but not ophthalmoplegia, and proximal and distal myopathy in her late forties. Muscle biopsy showed clonally expanded multiple mtDNA deletions.POLG2 variant and showed no signs of disease at age 57, indicating possible incomplete penetrance. Subsequently, three cases with pediatric‐onset autosomal dominant disease with varying phenotypes have been reported: a young adult with disease onset of adPEO in late teens and two unrelated infants presenting with hypotonia, seizures, and liver disease.POLG2‐related disorders display a spectrum of clinical phenotypes with a wide range of age of onset.Few families have been reported to have disease due to variants in POLG2 splice acceptor variant segregating with disease, leading to loss of POLG2 protein in patient cells. This work extends the clinical spectrum observed with POLG2 disease and lends further support for the role of the accumulation of mtDNA deletions in neurodegeneration.We describe a severe neurological syndrome in a large family with cerebellar ataxia, axonal sensory ataxic neuropathy, and tremor. Some subjects also presented with parkinsonism, seizures, cognitive decline, and ophthalmoplegia. Most died within 15 years of onset of clinical symptoms of disease. Genetic studies revealed a μm) were cut from transversely orientated muscle blocks and subjected to standard histological and histochemical analysis including COX, succinate dehydrogenase (SDH), and sequential COX‐SDH oxidative enzyme reactions. Large‐scale mtDNA rearrangements within the major arc were screened by long‐range PCR to amplify a ~13.0 kb product in wild‐type mtDNA . The level of deleted mtDNA in individual muscle fibers isolated by laser microcapture was determined by quantitative real‐time PCR using the ABI PRISM Step One real‐time PCR System  to confirm the presence of clonally expanded mtDNA deletions.Cryostat sections . All genetic alleles studied were annotated in reference to POLG2 NM_007215.Whole exome sequencing was performed using Illumina paired‐end precapture libraries constructed according to manufacturer's protocol. Sequence data were aligned, variants were called by GATK, and custom Perl scripts were used to annotate variants as previously described.7 cells following Graham & Rickwood method.Fibroblasts were grown in DMEM High Glucose with L‐Glutamine and Sodium pyruvate  with 15% FBS. Crude mitochondrial fraction was prepared from 1 × 10POLG2 oligonucleotides: forward 5′AAACCCTGTGGAACCTAGGAG3′ and reverse 5′AGCATGCCTCGGTCTAGGTC3′. Each sample was run in triplicate and was normalized to GAPDH.RNA extraction was performed using TRIzol and purified using PureLink RNA minikit (Life Technologies). First‐strand cDNA synthesis was done using SuperScript III First‐Strand Synthesis System for RT‐PCR (Life Technologies) and oligodT. Quantitative RT‐PCR experiments were performed using StepOne Plus RT‐PCR system (Applied Biosystems). μg/mL was added, and the cells were incubated for 30 min at 37°C. Membrane potential was determined by the ratio of red/green fluorescence in eight random 400× microscopic fields and expressed as mean ± standard deviation (SD).Fibroblasts were grown on glass chamber slides and treated with 30 ng/mL rotenone for 4 h at 37°C, or the same volume of solvent. 5,5′,6,6′‐tetraethylbenzimidazolyl‐carbocyanine iodide  of 5 Consent for the study was obtained from all subjects and a local ethics review board approved the study.Seven native Belgian individuals from two sibships belonging to the same extended family presented with a neurological disorder involving cerebellar and sensory ataxia with tremor Fig. . InitialPatient II‐1, presented at age 53 with gait disturbances. He experienced both cerebellar and sensory ataxia, progressing within 6 years to a loss of ambulation. Additionally, he experienced head tremor followed closely by an arm tremor that was both intentional and resting. Electrophysiological studies, comprised of recordings of nerve conduction velocities (NCV), electromyography (EMG), and somesthesic‐evoked potentials (SEPs) indicated an axonal sensorimotor neuropathy in the lower limbs, and a pure sensory neuropathy of upper limbs. SEPs indicated a near‐absent spinal response and a very low‐amplitude cortical response. Patients II‐8, II‐11, II‐12, II‐13, and II‐17 showed similar clinical signs and courses with details shown in Table One subject, II‐10, displayed a significantly different clinical presentation and clinical course compared to the rest of the kinship. Her disease presented at age 77 years, two decades later than most others in this family, when she received a diagnosis of asymmetrical Parkinson's disease based on the onset of a resting tremor of the left hand, mild left‐sided rigidity, generalized akinesia (right<left), hypomimia, head tremor, mild reduction in stride length, reduced arm swing, and mild dystonic movements of the left foot. While mild tandem ataxia was observed, notably, patient II‐10 had no sensory complaint at the onset of symptoms and NCV and brain MRI were normal. In a 5‐year follow‐up exam, only moderate reduction in pallesthesia of lower limbs was noted, whereas NCV and MRI were not repeated. This subject's parkinsonian symptoms improved with levodopa treatment.Two additional patients displayed parkinsonism in combination with ataxia and peripheral neuropathy as described above. Patients II‐13 and II‐17 had bilateral bradykinesia, reduced postural reflexes, and facilitory paratonia without resting tremor or cogwheel rigidity at the onset of their illness. In contrast to subject II‐10, these parkinsonian symptoms did not respond to levodopa.status epilepticus at the end of their seventh decade. Two females II‐8 and II‐17 had isolated episodes of generalized tonic‐clonic seizures a few years after the onset of their symptoms.Five patients  developed progressive cognitive impairment of the frontal‐subcortical subtype leading to profound dementia in two of them . The three males II‐1, II‐11, and II‐12 had a rapidly progressive cognitive decline followed by generalized tonic‐clonic seizures a few months before death in Two patients displayed ophthalmological signs. Patient II‐17 showed classic hallmarks of PEO with ptosis but no nystagmus, at age 59. By the age of 68, oculomotor examination revealed ophthalmoplegia with moderate limitation of amplitude and marked slowness of horizontal and vertical saccades and pursuit movements. Patient II‐13 had moderate ophthalmoparesis without ptosis at 72. It is noteworthy that the age of onset of this patient's ophthalmologic dysfunction was beyond the age of death of most affected subjects in this family.Affected individuals displayed cerebellar and brainstem abnormalities on T2‐Weighted and T2 FLAIR images with focal hyperintense lesions of middle and upper cerebellar peduncles, posterior area of the pons and mesencephalon , and the periaqueductal grey matter. They also exhibited various degrees of multifocal hyperintense hemispheric white matter lesions, cortical atrophy, and pontocerebellar atrophy Fig. . PatientSkeletal muscle biopsies from four affected individuals were examined for evidence of mitochondrial dysfunction. Electron microscopy ultrastructural studies revealed several anomalies including subsarcolemmal accumulation of abnormal mitochondria with paracrystalline inclusions Fig. A. HistopMolecular studies revealed that muscle from all four patients studied showed evidence of multiple mtDNA deletions Fig. C. SinglePOLG2 c.970‐1G>C, in affected individual II‐17. Variants in POLG2 are known to cause multiple mtDNA deletions which was one of the molecular phenotypes observed in muscle biopsy from our patients. The POLG2 c.970‐1 G>C variant has not been previously reported in the POLG2 disease literature and is not present in ExAC database. Sequencing of family members confirmed POLG2 c.970‐1G>C segregated with disease in this pedigree  and the father of the second sibship (I‐3) were siblings Fig. . LikewisPOLG2 splice acceptor variant, c.970‐1G>C, segregating with disease that caused diminished levels of POLG2 mRNA and protein in patient tissue. To our knowledge this is the first example of a POLG2 variant causing reduction in protein levels. The clinical signs in this family are distinct from previously reported cases of POLG2 deficiency and the syndrome described here extends the POLG2‐related phenotypic spectrum.We describe a neurological syndrome comprised of cerebellar ataxia, axonal sensory ataxic neuropathy, and tremor in combination with epileptic seizures, cognitive decline, parkinsonism, and ophthalmoplegia. Brain imaging studies indicated cerebellar and brainstem atrophy. Genetic studies identified a heterozygous POLG2 is embryonic lethal, POLG2 expression was associated with late‐onset ptosis and myopathy. Thus, the c.970‐1G>C splice acceptor variant in POLG2 we identified that causes reduction in POLG2 expression is consistent with previous POLG2 mutations causing mtDNA deletions and late‐onset mitochondrial disease.Recent microscopy reveals that POLG2 expression and subcellular localization is critical for mtDNA replication.POLG2 mutations, other genetic causes of primary multiple mtDNA deletion syndromes most often present as adPEO with myopathy, but also manifest a broad range of age of onset with variable phenotypes and expressivity. The neurological syndrome described here shares some similarities in clinical presentation with a POLG‐related syndrome, sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO).C10orf2, can lead to an ataxia‐neuropathy syndrome that includes dementia.POLG,C10orf2,OPA1,MPV17Like A thorough consideration of diseases included in the differential diagnosis is particularly relevant given the demonstrated variable expressivity of this variant and others leading to similar phenotypes. Among additional neurological syndromes that overlap with this disorder, but do not have a primary mitochondrial etiology, a special attention should be paid to Fragile X‐associated tremor/ataxia syndrome (FXTAS), as it includes a similar age of onset with progressive intention tremor, parkinsonism, cognitive decline, and generalized cortical atrophy and hypersignals in the middle cerebellar peduncles on MRI.POLG2 c.970‐1G>C, that likely results in reduced efficacy of the mtDNA polymerase. mtDNA deletions may also occur in the tissues of individuals without a primary mitochondrial disorder through DNA damage induced by ROS coupled with improper repairThe mtDNA deletions identified in the patients’ tissues in this study result from faulty replication of the mitochondrial genome due to the germline transmission of a genetic variant, Conception and design of the study: LVM and PEB. Acquisition and analyses of data: PEB, AB, VA, BDP, RVC, RWT, ELB, LH, KC, GS, MG, AR, GD, PL, PH, JJM, JB, MMH, and FGD. Drafting the manuscript or figures: PEB, LVM, WCC, JP, RWT, BDP, AB, and VA.The authors declare no competing interests.Table S1. Electrophysiological studies: conduction studies on sensory and motor nerves.Click here for additional data file.


Since alcohol use disorders are among the most prevalent and destructive mental disorders, it is critical to address factors contributing to their development and maintenance. Drinking motives are relevant driving factors for consumption. Identifying groups of drinkers with similar motivations may help to specialize intervention components and make treatment more effective and efficient. We aimed to identify and describe distinct motive types of drinkers in dependent males from two diverse cultures (Uganda and Germany) and to explore potential differences and similarities in addiction-related measures. Moreover, we investigated specific links between motive types and childhood maltreatment, traumatic experiences, and symptoms of comorbid psychopathologies.N = 75). Subsequently we compared the identified motive types concerning their alcohol consumption and alcohol-related symptoms (Alcohol Use Disorders Identification Test), history of childhood maltreatment (Childhood Trauma Questionnaire), trauma exposure , psychopathology  and deficits in emotion regulation .To determine distinct drinking motive types, we conducted latent class analyses concerning drinking motives  in samples of treatment-seeking alcohol-dependent men (r = 0.25 to r = 0.48). However, the motive types did not differ significantly on alcohol consumption or alcohol-related symptoms and traumatic experiences apart from childhood maltreatment.We found two congruent drinking motive types in both contexts. Reward-oriented drinking motives like the generation of positive feelings and enhancing performance were endorsed almost equally by both motive types, whereas high relief motive endorsement characterized one group, but not the other. The relief motive type drank to overcome aversive feelings, withdrawal, and daily hassles and was characterized by higher adversity in general. Emotional maltreatment in childhood and psychopathological symptoms were reported to a significantly greater extent by relief drinkers (effect sizes of comparisons ranging from The chronology of addiction development and patterns of drinking motivation seem to be similar across cultures, i.e., that motive targeting interventions might be applicable cross-culturally. Addressing comorbid symptomatology should be a key treatment component for relief drinkers, whereas finding alternatives for the creation of positive feelings and ways to counteract boredom and inactivity should be a general treatment element. Globally, alcohol abuse and dependency is one of the key causes of premature illness and death, loss of functionality and productivity, social decline, delinquency, and domestic and community violence , 2. AlcoOne such construct, the self-medication hypothesis, describes the use of drugs to alleviate or suppress suffering as one attempt to cope with stressors as well as psychopathological symptoms and the related negative affective and physiological states . When seA more recent model, the affective processing model , borrowsModels like these seem to be supported by high comorbidity rates between substance-related disorders and depressive, anxiety, and trauma and stressor-related disorders . FurAccording to Cox and Klinger , positivAnother concept closely related to drinking motives is drinking expectancy . ExpectaSince drinking typically starts in adolescence, an abundance of research has focused on drinking motivations in young samples of high-income countries, with the aim being to understand what makes them start and continue drinking .According to the global status report on alcohol and health , Uganda The current literature on drinking motivation implies that relief and reward drinking or enhancement, coping, social, and conformity drinking are correlated, suggesting that drinking motives are dynamic and can change intraindividually. In addition, individuals can follow several drinking motives concurrently even within specific situations , 65–68. We additionally expected that the individuals especially characterized by coping motives would start drinking earlier, consequently report higher chronicity of alcohol dependency, and show more symptoms of other mental health disorders. Accordingly, we assumed that early aversive experiences would predict class membership, i.e., relief drinkers should report more experiences of childhood maltreatment in their families of origin. From a clinical point of view, these aspects are of particular interest since they clarify whether patients with alcohol use disorders reporting specific motives or motive constellations differ on therapeutically relevant measures.n = 49) recruited from wards specialized in the treatment of substance-related and addictive disorders at two psychiatric hospitals. The Ugandan participants were alcohol-dependent men (n = 26) living in Gulu, who were interested in taking part in an inpatient treatment program that was announced via the radio and a community-based organization called Program for Prevention, Awareness, Counseling and Treatment of Alcoholism (PACTA). Alcohol-related diagnostic status was recorded according to the ICD-10 by the practitioners routinely charged with the task of diagnosing and treating dependent patients in the respective wards at treatment entry .The German sample consisted of alcohol-dependent men  from all subjects in accordance with the Declaration of Helsinki . NeitherEthical approval for the studies was provided by the Ethical Committee of Bielefeld University following the guidelines of the German Psychological Society. These guidelines are in agreement with the American Psychological Association’s code of ethics. In addition, all materials and procedures have been reviewed by the Lacor Hospital Institutional Research and Ethics Committee, Gulu, and approved by the Ugandan National Council for Science and Technology.Versions of the screening instruments in Luo, which is the local language in northern Uganda, have been previously created and utilized. The procedure of translation and adaptation is described elsewhere , 72.Aversive events experienced in the respondents’ families of origin were assessed with the Childhood Trauma Questionnaire (CTQ) in the German sample –75. The Trauma exposure beyond childhood maltreatment was only assessed in the Ugandan sample. The Violence, War and Abduction Exposure Scale is a 34-item checklist of potentially traumatic events that was developed specifically for use in the northern Ugandan context , 81. It Alcohol consumption and alcohol-related symptoms were measured using the 10-item interview version of the Alcohol Use Disorders Identification Test [AUDIT ]. Items We created a Drinking Motive Scale on the conceptual basis of Cox and Klinger’s  motivatiPTSD symptoms were assessed in the Ugandan sample only. The Posttraumatic Stress Diagnostic Scale  providesThe 15-item Depression-Section of the Hopkins Symptom Checklist [DHSCL ] was useTo measure the presence of psychopathological symptoms in the German sample, we used the Brief Symptom Inventory [BSI ]. The BSThe Difficulties in Emotion Regulation Scale [DERS ] was appSince our main interest was not grouping drinking motive items, as in variable-centered approaches like factor analysis, but grouping respondents based on the patterns of their answers concerning their drinking motives, we chose the person-centered analytic approach of LCA. Finite mixture models, like LCA, provide a framework for identifying subgroups of individuals that are not directly observable. To be able to compare response patterns of Ugandan and German alcohol dependents, we calculated two separate LCAs. The LCAs provided us with information on the probability of an individual in a certain motive class to endorse a certain drinking motive  and on the relative prevalence of a certain motive class (class probability). Sample sizes for LCAs were reduced by 5 individuals in the German and by 1 individual in the Ugandan sample, due to missing information of at least 1 of the 30 motive items.We compared models with differing numbers of classes on the values of common parsimony indices, the Akaike Information Criterion (AIC), and the Schwarz Bayesian Criterion (SBC). The AIC hinted at different solutions in the two samples, and the solution in the German sample was not parsimonious (suggesting five classes). In both samples, the SBC also referred to as Bayesian Information Criterion (BIC) produced lowest values for a two class solution. The model that produces the lowest values on parsimony indices can be judged the best model. In addition, simulation studies have indicated that commonly the BIC was the best information criterion for identifying the correct number of classes . Given tSubsequent comparisons of the resulting two motive types across the two diverse samples were possible for most variables, since they were assessed equally in both samples. However, psychopathology was assessed with different screening instruments; therefore, we created a composite measure of psychopathology consisting of the mean of z-transformed PTSD and depression scores in the Ugandan sample and the z-transformed global severity score of the BSI in the German sample. To test the robustness of the findings on psychopathology, we additionally compared the motive types on depression symptoms only. Since the BSI and the DHSCL have a shared history of instrument development the 6 items of the depression subscale of the BSI exactly match 6 of the 15 DHSCL items. The depression symptom score consequently combined the z-transformed scores on these six items in both samples.Data analyses were carried out with JMP version 13.1.0 .The samples of treatment-seeking, alcohol-addicted men from two diverse cultures and vastly divergent living contexts did not differ in current age, age at first consumption, or age at the beginning of dependency. Consequently, they did not differ concerning the chronicity of addiction. While the typical amount of consumption in standard drinks (containing 13 g of pure ethanol) also did not differ, the Ugandan sample was significantly more affected by alcohol-related symptoms (as indicated by the AUDIT score). Both samples were equally affected by emotional maltreatment in their families of origin. However, they differed in their histories of physical maltreatment, with the Ugandan sample being affected more heavily. Further characteristics, such as traumatic events beyond childhood maltreatment and different measures of psychopathological symptoms that were only assessed in one of the two samples, are displayed in Table The two separate LCAs yielded similar results. Two distinct motive types were found in both the German and Ugandan sample. One motive type was characterized by high endorsement of all items that are linked to negative reinforcement. Individuals in that group had a high relief motivation connected to alcohol. This became evident in the domain of social functioning, where overcoming insecurities in social interactions was especially relevant for the relief motive type. The same held true for overcoming aversive feelings, images, and memories and the suppression of current problems. Moreover, these same items were the ones that separated the distinct motive types in both samples . Age was about 6 years younger in the relief-motivated group. Psychopathological symptoms (non-composite scores) showed relevant differences in both samples with the relief motive type being consequently more severely affected. Depression symptoms in the Ugandan sample (as measured by the DHSCL), general psychopathology in the German sample (as measured by the BSI), and problems with emotion regulation (as assessed by the DERS in the German sample) presented with medium to large effect sizes. However, the medium effect for symptoms of depression did not reach significance in the Ugandan sample due to the small sample size. Differences in PTSD symptoms in the Ugandan sample did not yield significant results.Some indicators were assessed in one of the samples only. Traumatic events outside the family of origin including war events were assessed in the Ugandan sample only. Motive types did not differ concerning these events as indicated by small to negligible effect sizes. An exception was age at the time when the worst traumatic event had happened although this difference was non-significant ; therefore, we called this group after its unique feature “relief motive type,” whereas motives representing positive reinforcement (reward motives) were about equally endorsed by both motive types in both samples.p < 0.05]. It may additionally be intensified by the fact that shortages in alcohol availability due to access or financial constraints are forcing Ugandan addicts to bear withdrawal symptoms more often than German addicts. Access to alcohol, and therefore the potential to consume at or even before the first hints of craving or withdrawal, is likely to be a given in the German sample at any time. Consequently, German addicts may not pursue motives connected to counteracting withdrawal. The fact that excessive sweating is the only relief-oriented item that is not differentiating between the Ugandan motive types may be due to the fact that sweating may not be recognized as a sign of withdrawal because it is omnipresent in every day life in Uganda and might be attributed to physical labor, climate, or frequent illnesses like malaria.The only difference between the German and the Ugandan sample were motive items related to avoidance of withdrawal symptoms. Both motive types endorsed these items in about equally low rates in the German sample, whereas in the Ugandan sample, all items except drinking to counteract excessive sweating were strongly endorsed by the relief motive type, but not the non-relief motive type. This difference may be a correlate of the significantly higher severity of alcohol-related symptoms in the Ugandan versus the German sample and more specifically in the Ugandan relief drinkers versus the Ugandan non-relief drinkers [mean AUDIT scores M = 30.4 (SD = 5.0) versus M = 25.3 (SD = 6.4), In agreement with the results of this study, other researchers have compared up to 13 countries and found cross-cultural consistencies in drinking motives as well. However, these studies have looked at exclusively non-treatment-seeking Western youths using factor analytic approaches. A four-factor model of drinking motivation has been confirmed in all countries, with the same ranking of youths’ drinking motive endorsement. Social motives were most frequent, followed by enhancement, coping, and conformity motivations , 68, 90.In accordance with previous research findings, we did not find clear-cut coping, social and enhancement, or relief drinkers versus reward drinkers, as theoretical considerations according to the motivational model of alcohol use or the psychobiological model of craving for alcohol may imply , 34, 35.In agreement with these findings, Dvorak and colleagues  found thr = 0.13.We found an approximate 2:3 ratio of motive type classification in both countries. In the Ugandan sample, we found 60% relief drinkers and 40% non-relief drinkers, whereas prevalence rates were almost the exact opposite in the German sample, with more participants being non-relief drinkers (59%). The German and Ugandan samples did not differ in the levels of general psychopathology and depression symptoms as measured by the established composite scores, i.e., differences in symptomatology do not explain the reverse prevalence rates. Furthermore, it is rather unlikely that the higher load of war-related and other traumatic experiences outside the family of origin explains the higher prevalence of relief drinkers in the Ugandan sample, since within Ugandan dependent men, relief motive drinkers did not report more traumatic experiences than non-relief motive drinkers, although events of forced perpetration were reported slightly more by relief drinkers. However, the difference was non-significant with a small effect size of Relief drinkers in our samples started drinking heavily significantly earlier and thus became addicts at around age 26, which was more than 6 years earlier than non-relief drinkers. This was also reflected by the fact that the relief drinkers in our samples were, on average, 8 years younger than the non-relief drinkers. They additionally drank slightly more (about four standard drinks) on a typical drinking day and consequently reported being affected more by alcohol-related symptoms. However, in this study, these differences did not reach significance and were mainly driven by the Ugandan sample.Most of the literature examining drinking motives in dependent samples  or in yoLittlefield et al.  suggesteAlthough our study neither draws on prospective information nor has the power to build more complex models incorporating multiple predictors to affirm interpretations, it seems that timely, more proximal traumatic events are not as relevant as childhood maltreatment in the prediction of who will become a relief drinker later in life. As opposed to other types of familial , war-related, or more general traumatic experiences, emotional maltreatment in childhood was especially closely connected to later drinking to alleviate aversive feelings and memories, symptoms of  anxiety, and current stress on top of an enhancement motivation. Similar results have been found by Potthast et al.  in theirMost of the few studies investigating traumatic experiences, drinking motives, and drinking behavior simultaneously were again conducted in young, mostly college student samples. Cross-sectionally, both drinking to cope and drinking to enhance mediated the association between students’ childhood trauma exposure and drinking behavior , 112. HoConsequently, another correlate of being a relief drinker in our samples was suffering from more severe psychopathology. Differences between the relief motive type and the non-relief motive type were significant with medium to large effect sizes  development of mental and physical health, and, finally, to assess whether the effects of prevention or intervention strategies vary across the motive types. The latter would ultimately inform motive type-tailored intervention strategies that may lead to improved prevention and treatment success, as well as higher intervention efficiency.Wurdak et al.  evaluateEthical approval for the studies was provided by the Ethical Committee of Bielefeld University following the guidelines of the German Psychological Society. These guidelines are in agreement with the American Psychological Association’s code of ethics. Additionally, all materials and procedures have been reviewed by the Lacor Hospital Institutional Research and Ethics Committee, Gulu, and approved by the Ugandan National Council for Science and Technology. All subjects gave written informed consent in accordance with the Declaration of Helsinki.VE made substantial contributions to the conception and design of the work, the acquisition, analysis, and interpretation of the data; drafted, revised, and approved the manuscript; and ensures the accuracy and integrity of any part of the work. MP made substantial contributions to data acquisition, revised the manuscript critically for important intellectual content, approved the current version to be published, and agrees to be accountable for all aspects of the work to be accurate and integer. FN made substantial contributions to the design and interpretation of the data, revised the manuscript critically for important intellectual content, approved the current version to be published, and ensures the accuracy and integrity of any part of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zebrafish is a widely used model organism for studying heart development and cardiac-related pathogenesis. With the ability of surviving without a functional circulation at larval stages, strong genetic similarity between zebrafish and mammals, prolific reproduction and optically transparent embryos, zebrafish is powerful in modeling mammalian cardiac physiology and pathology as well as in large-scale high throughput screening. However, an economical and convenient tool for rapid evaluation of fish cardiac function is still in need. There have been several image analysis methods to assess cardiac functions in zebrafish embryos/larvae, but they are still improvable to reduce manual intervention in the entire process. This work developed a fully automatic method to calculate heart rate, an important parameter to analyze cardiac function, from videos. It contains several filters to identify the heart region, to reduce video noise and to calculate heart rates.The proposed method was evaluated with 32 zebrafish larval cardiac videos that were recording at three-day post-fertilization. The heart rate measured by the proposed method was comparable to that determined by manual counting. The experimental results show that the proposed method does not lose accuracy while largely reducing the labor cost and uncertainty of manual counting.With the proposed method, researchers do not have to manually select a region of interest before analyzing videos. Moreover, filters designed to reduce video noise can alleviate background fluctuations during the video recording stage (e.g. shifting), which makes recorders generate usable videos easily and therefore reduce manual efforts while recording.The online version of this article (10.1186/s12859-018-2166-6) contains supplementary material, which is available to authorized users. Congenital heart disease (CHD) is the most common congenital defect, which accounts for 3% of infant deaths . To bettTo date, there have been many methods that assess cardiac functions in zebrafish embryos/larvae –13. ThesAlthough the videos were obtained under different conditions, all of the aforementioned methods have to face two important issues after video acquisition: (a) identification of the heart area and (b) image processing to enhance heartbeat signals in the video. This work has developed a novel method for heart rate estimation. For the first issue, this work proposes a half-heart mask to identify the ROI. For the second issue, this work proposes that Empirical Mode Decomposition (EMD) could be used as a high-pass filter to reduce video noise. Another contribution of this work is the comparison strategy between the proposed method and manual counting. This work designed an experiment to quantify manual error, which was used as a reference to evaluate the computational error. The experimental results show that the proposed method achieves the same error level as human.This article is organized as follows. Section 2 describes the proposed method, which includes three major steps. Section 3 describes its performance, including the details of zebrafish larva preparation, video acquisition and an experiment to compare the calculated heartbeat rate with manual counting. Section 4 concludes this work and discusses possible extensions.The concept of the proposed heart rate calculation is based on brightness intensity of each video frame. Fig. Figure y. This work defines the variation level of brightness intensity of a pixel at  in an n-frame video as the variation of the n-1 brightness intensity differences of two consecutive frames at that pixel:yi,j,k is the brightness intensity of pixel  in the k-th video frame. The variation levels of all pixels for a video are normalized to  so that they can be rendered to a grayscale image as shown in Fig. This work developed a procedure for half-heart mask generation, which consists of four filters. Given a zebrafish larva video, the first filter is to calculate, for each pixel, the variation level of brightness intensity through the video. This filter first converts each video frame to a grayscale image. In this work, 12 color-to-grayscale methods analyzed in  have beeθ. The θ% pixels with the highest variation levels are converted to black (region of interest), otherwise are converted to white (region of non-interest). Since the target area of interest is heart, the ideal θ of a zebrafish larva video depends on its heart-to-video ratio: the area ratio of the heart region to the entire video. The heart-to-video ratios of the 32 testing videos collected in this work varied from 0.9% to 10.0%. This work used θ = 4 for all testing videos. The result of this filter is a binary mask as shown in Fig. The second filter is to find a region in which the brightness intensity varies drastically. This filter converts the grayscale image of variation levels into a binary mask by setting a percentage threshold, eps) and minimum number of points (minPts). For a black pixel, q, its eps-neighborhood contains pixels of which the distance to q is less than eps. If the eps-neighborhood has more than minPts pixels, q is defined as a core pixel. The scanning process of DBSCAN picks an arbitrary pixel p from the un-scanned black pixels to start. If p is a core pixel, p and its eps-neighborhood form a new island. All pixels in this island are then scanned and their eps-neighborhoods are added into the island. The newly added pixels are also scanned to see if more pixels can be added to the island. This scanning process is conducted recursively until no more pixels can be added to island. If p is not a core pixel, p is considered as a noise and assigned to no island. Then, DBSCAN moves to the next un-scanned black pixel and stops when all black pixels have been scanned. Figure eps = 20 and minPts = 300 for all testing videos, of which the video resolution was 1280 X 720. If the video resolution changes, minPts should be adjusted because the number of pixels in the same area changes. The eps should also be adjusted to maintain the pixel density (minPts/eps2). For example, if a video resolution changes from 1280 X 720 to 640 X 360 (i.e. area becomes 1/4), the minPts is suggested to be 300/4 = 75 and the eps is suggested to be 20/sqrt(4) = 10. The result of this filter is the largest island of DBSCAN as shown in Fig. The third filter is to further reduce the archipelago-like mask of Fig. n-frame video results in n centroids. The k-means algorithm [n centroids into the two clusters: one contains frames where most blood cells are in the atrial and the other contains frames where most blood cells are in the ventricular. The k-means algorithm partitions centroids based on their locations into k disjoint clusters. When clustering n data points into k cluster, the k-means algorithm minimizes the following equation:cj is the center of the i-th cluster, pj is the j-th data point, and wi,j equals to 1 if pj belongs to cj or 0 otherwise. This work sets k = 2 for atrial and ventricular. After k-means, the two cluster centers are connected to form a line segment. The perpendicular bisector of the line segment cuts the heart mask into two halves. The final mask of this step is the first half as shown in Fig. The fourth filter is to split the heart mask of Fig. lgorithm  is then y-value at position x is defined as the brightness intensity sum of all pixels in the x-th frame of that video. Peaks in an h-signal correspond to heartbeats  mixed with noise signals of frequency higher than the target signal . Moving s. Second, the local maxima of s are connected as an upper envelope; while the local minima of s are connected as a lower envelope. Third, the mean curve, m, between the upper and lower envelopes is interpolated. Fourth, the component, h, is calculated as the difference between s and m. If h is not an IMF, the above four operations are repeated with s = h to obtain a new component. The entire loop stops when some component fulfills the requirements of IMF. The final component is the output of the high-pass filter as well as the output of the h-signal extraction step. The subsection “Effects of half-heart mask generation” discusses the low frequency noise and the effects of the proposed EMD-based filter on it.In addition to high frequency noise, the smoothed h-signals of some zebrafish larva videos have low frequency noise such as the light curve in Fig. n peaks are detected in the considered time span, t. The conventional formula of heart rate is n/t. This formula assumes that the time span contains roughly n periods. In practice, however, the time from the span start to the first peak plus the time from the last peak to the span end is not guaranteed to be a complete period. To prevent such an uncertainty, this work proposed a heart rate formula that trims the time outside the first and last peaks:tn and t1 are the timestamps of the last and first peaks, respectively, in the considered time span.This step calculates heart rates based on peaks in h-signals. Common recording operations (e.g. setup and remove the camera) might introduce fluctuations at the start and end of a video. To preclude such interferences, only the middle 20% time of an h-signal is considered for heart rate calculation. If the duration of an h-signal is less than 15 s, the middle 3 s are considered. In this step, the input h-signal is decomposed into data points. Each data point is the brightness intensity sum of a video frame. A data point is detected as a peak if its value is greater than its two consecutive points. Suppose The subsection “Table The proposed method was applied to calculate the heartbeat rates of 32 zebrafish larval cardiac videos. Each video was also counted by 11 operators for manual counting results. The 11 operators came from a bioinformatics laboratory with basic training. In nature, the heart rates delivered by an operator have certain differences when counting the same video multiple times. To alleviate such manual instability, a video was counted 30 times by an operator. With 30 repeated counting results of 11 operators, this work defined two kinds of manual errors: (a) individual error, which measures the variation between repeated experiments of an operator (i.e. human instability) and (b) crowd error, which measures the variation between different operators. The answer of a video for computational results and for crowd error was the average of all manual counting results for that video. The answer of a video for individual error was different by operator, where the answer of an operator was the average of his 30 counting results.Table n in Eq. t1 in Eq. tn in Eq. t1) and the last (tn) counts leads to individual error. This work outperformed manual counting in heart rate calculation because of eliminating such instability. Finally, as in heartbeat detection, the half-heart mask and noise filters played important roles in heart rate calculation. The MAE, RMSE, rMAE and rRMSE of this work were 15.8%, 13.4%, 16.4% and 14.0% to those of this work (−). The gap was larger than that in heartbeat detection.For heart rate calculation, this work achieved the best performance in all evaluation indices. Note that the error of number of heartbeats and that of heart rate cannot be compared directly since they are in difference scales. The time span of consideration . The performance of manual counting depended on the operator (relatively large RMSE and rRMSE). The proposed half-mask mask and noise filters largely reduced the heart rate error . These results indicate that the proposed method can accurately and stably calculate heart rate from zebrafish larval cardiac videos.As described in the subsection “Table In summary, the large contribution of the varied region mask came from filtering out a large area. The heart mask yielded a relatively small contribution, but it is a necessary step for the half-heart mask. The large contribution of the half-heart mask came from accurately identifying pixels whose brightness intensities vary in a similar phase. This phase consistency was important to enhance stability.n is the number of peaks, pi is the i-th peak, li is the left trough of pi, while ri is the right trough of pi . Table The proposed method has been tested on two videos provided in , where oAlthough making a conclusion based on only two independent videos might be too arbitrary, the results reveal the potential of the proposed method on heart videos of other species.This work introduced a pipeline that calculate heart rate from zebrafish larval cardiac videos. The proposed method contains filters to identify the heart region automatically and can be performed without pre-selecting a region of heart manually. It also contains filters to withstand background fluctuations during the video recording stage, which makes recorders generate usable videos easily. The experimental results show that the proposed method does not lose accuracy while largely reducing the labor cost in long-period heartbeat counting. The automatic and accurate heart rate calculation method proposed in this work can be extended to calculate other cardiac functions like heartbeat regularity.https://merry.ee.ncku.edu.tw/zebrafish/ was setup to supplement the static figures in this manuscript. The source code of the proposed method is deposited in Github at https://github.com/mbilab/zebrafish.As the target of the proposed method is video, an online demo page at Additional file 1:Table S1. Performance of heart rate estimation with different color-to-grayscale methods. (DOCX 14 kb)


Research relating to alcohol use amongst university students primarily examines the effects of binge drinking. Researchers rarely focus on a range of drinking styles including light or non-drinking. This study was designed to gain an in-depth understanding of the lived experiences of female, first year UK undergraduates, who do not drink alcohol. Semi-structured interviews were conducted with eight participants. Narratives were analysed using interpretative phenomenological analysis (IPA; by Smith and Osborn (Sage 51-80,  Excessive alcohol consumption is a major health concern in the UK, with drinking playing a significant social role in British culture .Eight female, non-drinking undergraduates participated. The mean age of the participants was 21.5 years (SD = 5.29 range = 18–33 years). All participants had not drank alcohol for approximately 2 years or more. To protect anonymity, names of all participants have been replaced with pseudonyms  at the University of Lincoln . Informed consent was obtained from all participants included in the study prior to data collection. Data were collected through semi-structured interviews, conducted within a comfortable and private room on the university campus.In total, 15 open-ended interview questions were asked . Given the sensitive nature of the study, questions were tailored to be sensitive and respectful, whilst being clear and concise for the interviewee to easily understand. An interview schedule acted as a guide to structure the interview, to enable participants to direct the interview focus where possible. Interviews lasted on average 41 min. At the end of the interview, all participants were debriefed, thanked for their participation, and given the opportunity to ask questions.Data were analysed in accordance with the principles of interpretative phenomenological analysis .Many participants commented that being a non-drinker in an environment where drinking alcohol is engrained within its culture leads to difficulties when attempting to socialise with peers. All eight participants explained that not drinking in university was a lifestyle choice deemed abnormal or “some sort of disease”  I’ve actually been known to explain to people, (…) that I have medical reasons and that’s why I can’t drink because it’s just easier than trying to explain to somebody that you don’t want to drink. (…) The second you mention medical they go, oh right you know and they back off (…) they’re not so keen to pressurise you (…) it’s just easier than trying to say look I don’t wanna drink and I’m not gonna drink. Extract 1—Many expressed feelings of social isolation, feeling as though their peers do not want to socialise with them. Participants intentionally deceived their peers to gain acceptance. In social situations, participants lied about their drinking status, misleading their peers to believe that abstinence was only temporary, to alleviate the risk of peer scrutiny. In the following extract, Bethany employs deception. She uses an excuse as a method of escaping from a potentially negative social encounter:Bethany’s social experiences appear to be particularly strained, as she describes her attempt to alleviate scrutiny and peer pressure. She finds it easier to pretend that she cannot drink for medical reasons, as it is easier than explaining that “you don’t want to drink”. This infers that past conversations about her non-drinking lifestyle have received unfavourable reactions. Peers may not have been accepting of her being a non-drinker, leading Bethany to feel she has to adopt the identity of being disabled with pain. She goes to the length of feigning an injury to provide a socially acceptable justification for her non-drinking. Using crutches as a prop to increase the legitimacy of her excuse, Bethany prevents being pressurised to drink alcohol. She explains that providing a medical reason for not drinking, instead of saying that she simply does not want to, reduces the risk of confrontation from others and avoids revealing her true non-drinking identity.Danielle) and could be very persuasive. Excuses and tentative replies to offers of alcohol-related activities have been used as a means of managing these challenges. Jessica drew attention to the avoidance strategy she used:That’s how people are social. My flatmates would ask me (…) are you coming out tonight (…) when I say “no, I’ll give this one a miss”, (…) it makes me feel really antisocial. Every time I say no, it gives off the message that I don’t wanna be social and they’ll stop asking me. If they ask me in the morning (…) it’ll be “I’ll think about it”, then in the evening I’ll be like “I’ll have an early night”. I find it quite difficult ‘cause it’s me saying I don’t want to do this with you is being personal. Extract 2—Many participants expressed disapproval for parties and nightclubs, because alcohol consumption was the dominating feature of the event. Many felt uncomfortable in these environments, which led to attempting to avoid attending future events of this type. Participants explained that the beginning of the academic term is seen as an opportunity to make new friends and socialise with new housemates; therefore, offers to attend alcohol-related events were frequent. They expressed that trying to decline invitations from their peers and new housemates was challenging, as this needed to be performed strategically without causing offence. Many felt that it was difficult to decline invitations as drinking students were described as having “strong personalities”  I asked a second year (…) I said oh is there anything for non-drinkers but (…) they couldn’t really think of any or couldn’t remember any. I know they could do more. The only thing that wasn’t clubbing or entirely alcohol related was the quiz, which I went to (…) But that was the only thing and I mean one night out of a whole week which is built up and it’s Fresher’s Week, it’s meant to be a big thing, so I feel like I missed the boat a little bit there. Extract 3—In addition to difficulties in social management, all participants in this research highlight the impact that non-drinking has on their ability to form friendships and socialise comfortably with others. All participants explain difficulty in finding opportunities to socialise with their peers at university. As previously explained, Fresher’s Week is one of the main methods of meeting new people at the start of the academic year; participants in this research confirm this, highlighting its social significance. However, their experiences of Fresher’s Week were significantly restricted. Being a non-drinker resulted in exclusion from social opportunities, especially during the start of their university experience. When the researcher asked Megan about the activities available for non-drinking students in the local area, she acknowledged that opportunities to socialise without alcohol were scarce:Megan explained that she attended only one event during Fresher’s Week, as only one activity was not alcohol based. This was the view of all eight participants in this research; they all reported that the majority of the activities in the city revolve around heavy drinking.Megan sought to find events that she could attend; however, current students were unaware of any activities that she would be able to go to. This may be due to a lack of activities available or a lack of awareness from other students, as it is reasonable to assume that the university may not advertise activities that will not attract the majority of students. For those who are abstainers or light drinkers, the scarcity of non-drinking activities may act as a barrier to meeting new people. Megan also indicates feels she was unable to take advantage of the social opportunities that Fresher’s Week provided.In line with all other participants interviewed, Megan believes the university should cater for the needs of first year, non-drinking students by hosting and increasing the promotion of alternatives to alcohol-dominated events. These events need to be promoted during Fresher’s Week to ensure that the week is equally inclusive for both drinkers and non-drinkers and provides all first year students ample opportunity to socialise and bond with new people.Bethany). Many expressed that the behaviour and personality of their peers changed when intoxicated. This meant that when trying to form a bond, participants felt that the friendship was not genuine, because the drunken individual would not be able to remember former interactions or conversations that occurred between them. Danielle expressed the importance of feeling part of a friendship group, but has struggled with this as a result of being a non-drinker:I’ve definitely struggled to make friends and I still feel like I haven’t made a proper group of friends and I see everyone else with lots of friends (…) I don’t think alcohol is the blame for that but I think it would have been easier if I had been like that (…) I definitely think it’s harder to fit in. I feel everyone formed friendships now and it’s like too late to start going in to them, so I feel like I just don’t fit in with them. Extract 4—Being a non-drinking student has a considerable impact on the ability to make and maintain friendships. For all participants interviewed, making friends was a significant challenge. One student explained that she could count her number of friends “on one hand” . Danielle felt as though she does not “fit in with them”, acknowledging “them” to be a distinct social group of which she was not a part. This demonstrates a divide between drinking students and non-drinking students and the differentiation between their behaviours and personalities.This interview was conducted 3 months after the participants started university. Danielle implies that to join or establish a friendship group, she would have to do so quickly otherwise she will have missed her opportunity. Danielle emphasises the pressure to make friends, implying that if she cannot make friends within her first term, then she may not be able to for the remainder of her time at university. This highlights an additional concern expressed by Danielle and other interviewees; the risk of isolation. Another student expressed that she felt lonely and “isolated at times”  and conform to the typical student lifestyle. This deviation from the norm, led to the requirement of justification and explanation as to why they would not be drinking. One participant hypothesised that this was due to the fact that “not everybody understands the concept of not drinking” (Rachel). Jessica describes the extent to which, on one occasion, peer pressure led her to drinking alcohol.They’d be like, you really do want to though don’t you, they’d pour a drink out for me, sort of say oh just have a drink, (…) well the second time I went out I sort of caved into that. Extract 5—All eight participants interviewed expressed that interactions with drinking students were often unpleasant. As a result of university drinking culture, it was highlighted that, as a first year undergraduate, students were “expected to go out and get drunk”  Then I think she basically told someone else (…) “she’s quite boring isn’t she cause she doesn’t drink” and “who doesn’t drink at uni, drinking’s like the whole life of uni” (…) The thing is the girl who said I’m quite boring (…) she opened up (…) she was just like I didn’t really expect you to be like this (…) I kind of like thought you’re just someone who doesn’t drink so I just thought you didn’t really go out much so I just thought you were quite a boring person. Extract 6—In contrast, other participants expressed experience of social aggression in a different manner. All participants felt that they were perceived as unsociable or boring, with seven out of eight students subject to hurtful comments either to their face, or behind their backs. During a university society meeting, one participant, Laura, was asked whether she drank alcohol. When she said no, a member of the society meeting spoke about Laura negatively to another group member behind her back. At a later date, the girl who made the initial negative comments about Laura said she mistook her for a boring person, just because she was a non-drinker. Laura gives an account of her experience:Unlike other participants in this study, Laura felt confident admitting to being a non-drinker within a group. However, this act of confidence led to social aggression from her peers, and she was immediately subject to scrutiny, negative comments, and labelling. Drinkers made an immediate interpretation of Laura’s personality, linking non-drinking with being boring and unsociable. It is implied that Laura is in fact an outgoing, social person, and her personality defies the lingering non-drinker stereotype. It seems that alcohol played a central role in influencing alcohol-consuming peers to make an immediate judgement of Laura, instead of coming to a more rounded, reasonable understanding of Laura’s real self; her true personality and sociability are misunderstood and hidden as a consequence.I was really worried about coming to uni because I don’t drink. I didn’t want to get to uni and them to think I was boring for not drinking. I was really nervous about it. Extract 7—Laura also implies that her peers may be mocking her lifestyle choice. The drinking student classes alcohol as “the whole life” of university which suggests that they view Laura’s non-conformity as atypical. Laura explains that the individual who was judgemental towards her had an expectation about Laura’s personality, based purely on her teetotalism and deviation from a social norm. This extract highlights the judgement non-drinkers face whilst attending university, experiences that only occur as a result of non-conformity to a typical student lifestyle. It is this deviation from the typical university lifestyle and cultural norm that caused students in this study to feel uneasy starting university. Megan sheds lights on her feelings:It is implied here that before starting at university, Megan was aware of the predominating alcohol norms and expectations when attending university in the UK, which as a non-drinking individual caused her to experience apprehension about starting university. She was concerned about the existing social stigma of being a non-drinker and did not want to be classified or stereotyped as boring by her university peers. This may potentially be due to the fact that these existing perceptions of non-drinker characteristics could leave her automatically susceptible to feelings of alienation and social acceptance during the start of her university experience. It is possible that her peers may treat her differently for being a non-drinker, which caused Megan angst, and she may not be given adequate opportunity to behave in a way that represents her true self when meeting new people as a consequence of existing stereotypes.This study provides a valuable contribution to the understanding of the lived experiences of female, non-drinking students during their first year of undergraduate study attending a UK university. Through the use of IPA methodology, the research provides a rich understanding of the challenges experienced by this sample, adding to the small body of literature on the topic.It was evident from all interviews that non-drinkers acknowledge that alcohol consumption is a dominant norm amongst university students in the UK, which plays a significant role in social environments. The decision not to drink alcohol by these young women has led to them learning how to prepare for and respond to social pressures, people’s expectations, and the consequences of being a non-drinker in an excessive drinking culture. Many were unsure how to handle non-conformity to cultural norms, leading to the development of coping strategies to manage peer pressure to drink alcohol or to engage in the prevailing alcohol fuelled student life such as pre-drinking. This study highlights that despite being a female non-drinker, which despite literature indicating that in some situations is deemed as more tolerated, they too experienced considerable challenges with peer acceptance.Given that alcohol is a key tenet for social interaction within the undergraduate population Gill , being aFor this sample of female UK undergraduates, it seemed important to seek acceptance from their peers. As a result of identifying as and presenting themselves as a non-drinker caused concern for participants, and led to having to strategically manage social situations, as it was easier to mislead new acquaintances about their non-drinking status as an opposed to admitting to being a non-drinker. Through finding excuses or avoiding uncomfortable conversations about drinking, participants would not only avoid peer pressure and encouragement to drink, but would also reduce the risk of being excluded from social events with new acquaintances altogether. Our findings are similar to Conroy and de Visser  in whichThese findings also draw comparisons with previous research that highlighted difficulties for non-drinking students with regard to the social adversities and pressures that can occur when identifying as a non-drinking individual , across year groups, and in what way being a non-drinker has impacted them across the years. In addition to continuing qualitative research in this area, quantitative measures can aim to build on qualitative findings to develop a wider picture of the experiences of female non-drinking undergraduates on a larger scale. If the consensus is that a large proportion of students are experiencing social difficulties whilst at university, these findings can help UK universities to ensure that appropriate support is in place for students who are struggling to cope with the stressors of university, including being equipped with the skills to successfully manage challenging situations, including handling peer pressure.Building on a small body of literature, this study has presented an account of the lived experiences of eight non-drinking, female UK students, in their first year of undergraduate study. The use of interpretative phenomenological analysis allowed for an in-depth, idiographic exploration of this particular group of students. Findings have highlighted that subversion of the cultural alcohol norms within a university setting can result in female non-drinkers living and learning to live with challenges including social constraints, peer pressure, and criticisms from peers regarding leading a non-drinking lifestyle. Whilst these findings related to a small group of non-drinking students, this study has highlighted the importance of focusing research attention towards non-drinking students. Beneficially, this research provides an understanding of the potential challenges that may occur if an undergraduate decides to abstain from alcohol, which can help university to reconsider not only the support that should be available to those who are struggling to manage with their university journey, but also provide a range of non-alcohol dominated events to ensure all students feel socially inclusive.


The goal of the present study is to learn the activity of p21-saRNA-322 in colorectal cancer. Three human colorectal cancer cell lines, HCT-116, HCT-116 (p53–/−) and HT-29 were transfected with the p21-saRNA-322. The expression of P21 protein and p21 mRNA were measured using the Western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The effect of p21-saRNA-322 on cancer cells was evaluated in vitro; and furthermore, a xenograft colorectal tumor mode in mice was established to estimate the tumor suppressing ability of p21-saRNA-322 in vivo. The results showed that in all three colorectal cancer cell lines, the expression of p21 mRNA and P21 protein were dramatically elevated after p21-saRNA-322 transfection. Transfection of p21-saRNA-322 caused apoptosis and cell cycle arrest at the G0/G1. Furthermore, anti-proliferation effect, reduction of colonies formation and cell senescence were observed in p21-saRNA-322 treated cells. Animal studies showed that p21-saRNA-322 treatment significantly inhibited the HT-29 tumor growth and facilitated p21 activation in vivo. These results indicated that, p21-saRNA-322-induceded up-regulation of p21 might be a promising therapeutic option for the treatment of colorectal cancer.The double stranded small active RNA (saRNA)- p21-saRNA-322 inhibits tumor growth by stimulating the p21 gene expression. We focused our research of p21-saRNA-322 on colorectal cancer because 1) p21 down-regulation is a signature abnormality of the cancer, and 2) colorectal cancer might be a suitable target for  Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death worldwide . DespiteP21, a multifunctional cyclin-dependent kinases (CDK) inhibitor (CKI), is a downstream protein of P53. During the responses to DNA damage, P53 was induced and specifically bound its consensus binding sequences in the regulatory region of p21 (wild-type p53-activated fragment) to trans-activate the genes, causing prevention of cell cycle progression via arresting cells in the G1/s . In addiSmall RNAs (sRNAs) are an abundant class of small non-coding RNAs that could regulate diverse developmental and physiological processes via multiple mechanisms . In the in vitro and in vivo. However, to bring the p21-saRNA-322 into drug R&D, a proper drug delivery system has become a key issue. It appears that colorectal cancer could be a potential target for the p21-saRNA-322, as p21 down-regulation is a signature abnormality of the cancer and in rectal application might be a suitable approach for p21-saRNA-322 delivery.Since Li et al. synthesized p21-saRNA-322 , researcThe goal of the present study is to learn the activity of p21-saRNA-322 in colorectal cancer. We investigated the ability of p21-sa-RNA-332 to activate p21 expression and its effect on colorectal cancer cell lines, as well as in xenograft colorectal tumor mice. In what presented below we show that p21-saRNA-332 might be a promising alternative way in the treatment of colorectal cancer.Three well-characterized human colorectal carcinoma cell lines  and HT-29) were chosen to study the ability of p21-sa-RNA-332 to activate p21 expression. Before transfection, the levels of p21 and p53 in the three cell lines were determined at both the mRNA and protein levels. As shown in Figure The p21-saRNA-322 was used to activate p21 expression, in which HCT-116, HCT-116 (p53−/−) and HT-29 cells were transfected with 25 nM of p21-saRNA-322 using scramble RNA as well as the untreated as references. 24, 48 and 72 hrs later, the expression of p21 mRNA was tested by RT-PCR and P21 protein level was analyzed with Western blot. As shown in Figure Then, we investigated the effects of p21-saRNA-322 activated p21 expression on colorectal cancer cells.0/G1 phase was increased in the p21-saRNA-322 treated group  and 81.3% for HT29), as compared to that in the mock group  and 70.0% for HT29) and the scrambled RNA treated group . The transfection with p21-saRNA-322 also respectively caused decrease in the S phase cells  and 10.9% for HT29 in p21-saRNA-322 group, in the comparison to those in the mock group: 37.7, 36.2 and 25.7%, and the scrambled RNA treated group: 34.1, 35.2 and 25.7%, respectively in the three cell lines), suggesting a cell cycle arrest at the G0/G1 checkpoint. These results are in agreement with previous studies [In the present study, we investigate the influence of cell cycle distribution by p21 activation via p21-saRNA-322 in human colorectal cancer cells using flow cytometric analysis. As shown in Figure  studies .A large body of literature indicated that p21 was an important apoptosis protein in colorectal cancer –35. TherAccumulative research proved that, p21 induction is a determinant in the regulation of colorectal cancer cells proliferation , 36. In A group of evidence demonstrates that p21 is required for cellular senescence , 38–40. in vivo, HT-29-Red Fluc colorectal cancer cells were injected subcutaneously into nude mice. Bioluminescence signal of tumor growth was measured by testing photons/second following intraperitoneal administration of luciferase substrate. The HT-29-Red Fluc colorectal tumor-bearing mice were randomly divided into three groups, phosphate buffer saline (PBS), scramble RNA and p21-saRNA-322 groups. No statistical difference in signal intensity or body weight was detected among groups at time of randomization. Three days after tumor implantation, intratumoral injection  was applied every three days for 5 weeks. Bioluminescence signal was measured every week to monitor the growth of tumor mass. At the end of the study, all of the mice were sacrificed and tumors were surgically removed. Representative photographs of the host mice and the corresponding tumor masses are indicated in Figure 3, 0.72 cm3, and 0.13 cm3, respectively; the median weights were 0.61, 0.63 and 0.10 g, respectively. The results revealed that, p21-saRNA-322 treatment significantly reduced the tumor burden, whereas no significant difference was detected in the 2 reference groups. Tumor bioluminescence within each mouse was monitored longitudinally and plotted over 5 weeks for all treatment groups. As shown in Figure To determine whether the stimulation of p21 expression by p21-saRNA-322 suppresses tumor progression in vivo, which seemed to be the mechanism responsible for the tumor growth inhibition.Immuno-histochemistry (IHC) test revealed that, cells positive for p21 were very rare and randomly dispersed in the tumor tissue of the control groups, while the p21-saRNA-322-treated xenograft tissue were symbolized with intense nuclear staining of p21 Figure . The resIn the present work, we explored the therapeutic potential of p21-saRNA-322 on human colorectal cancer. Three well-characterized human colorectal carcinoma cell lines HCT-116, HCT-116 (p53−/−) and HT-29 were chosen based on their dissimilarity in p53 and p21 gene expression. Among the three cell lines, the lowest expression of p21 was observed in HT29 cell line and the p53 expression was deprived in HCT-116 (p53−/−) cells. Based on that, we investigated whether the introduction of p21-saRNA-322 could activate p21 gene expression in the colorectal cancer cells, and whether the activity of p21-saRNA-322 was influenced by intrinsic p21 expression and p53 integrity. We found that induction of p21 gene was quite robust to all of the three colorectal cancer cell lines, and importantly, for p53 negative ones. Moreover, the elevated p21 expression was detectable for over 10–15 days after p21-saRNA-322 transfection, longer than siRNA induced gene expression change –16. ThisThe major effect of p21-saRNA-322 is to directly up-regulate p21 expression. p21 is a multifunctional cyclin-dependent kinases (CDK) inhibitor (CKI), which suppressed tumorigenesis by inhibiting cell cycle progression –31, inhiin vitro and in vivo via arresting cell cycle inhibiting cell proliferation and colony formation, and inducing cell apoptosis as well as cellular senescence. Result for the activity of p21-saRNA-322 on CRC has not been reported yet.Recently, literatures reported a small active RNA (saRNA)-induced gene expression activation phenomenon known as RNA activation (RNAa). Distinguished from small interference RNA (siRNA), specific small double strand RNA  could directly activate target gene transcription through binding specific gene promoter regions , recruitin situ drug delivery might overcome those obstacles. The advantages of the in situ delivery are focused-delivery of RNA into target tumor tissue, decreased adverse events as well as reduced immune reactions. Several local delivery strategies for therapeutic RNA have been developed, including intracranial [in-situ administration route. In the present study, we have successfully verified the cancer suppressing effect of p21-saRNA-322 on xenograft animal model. Based on that, our next attempt is to develop a clinically relevant orthotopic animal model to evaluate the therapeutic effect of p21-saRNA-322 using rectal administration technique. We consider it an important step toward possible future application in human.saRNA induced gene activation represents a novel approach for the treatment of human cancer; however, RNAa-based clinical research has been restricted due to the susceptibility of RNA molecules to serum nucleases, renal clearance and non-specific bio-distribution. In addition, potential activation of innate immune system by nuclear acids could lead to unexpected toxicities and undesirable effects , 44. Stuacranial , intraocacranial  and intracranial . Naked sacranial . Kang etacranial . Our preacranial , 50. ThuThe saRNAs  were designed as described by Li et al  and chemCAMS. HT-29 was cultured in McCoy’s 5A (Modified), HEPES medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). HCT-116 and HCT-116 (p53−/−) were maintained in RPMI1640 supplemented with 10%FBS, 100 U/ml of penicillin, and 100 ug/ml of streptomycin. All cell lines were cultured in a humidified atmosphere containing 5% CO2 and maintained at 37°C.The human CRC cell line HCT-116 and HT-29 were abstained from China Infrastructure of Cell line Resources . HCT-116 (p53−/−) cells (chosen as deficiency of p53) was kindly gifted by Ph. D Zhan from cancer hospital 4 cells/well and cultured overnight to 10–20% confluence. For HT-29, the original density was 1–1.5×105 cells/well. Transfections of p21-saRNA-322 and scramble RNA were carried out at a concentration of 25 nM/ well using Lipofectamine 3000 reagent  according to the manufacturer’s protocol and lasted for 24, 48 and 72 hrs.The day before transfection, HCT-116, HCT-116 (p53−/−) cells were seeded into 12-well plates with antibiotics-free growth medium at a density of 4–5 × 10® Plus RNA Purification Kit . The concentration of RNA was measured by spectrophotometer ND2000 . 5ng of total RNA was used for quantitative RT-PCR, which was performed on the Applied Biosystems 7500 Fast Real-Time PCR System  using Power SYBR® Green RNA-to-CT™ 1-Step Kit . PCR amplification included an initial denaturation step (95°C for 10 min), 40 cycles of denaturation (95°C for 10 s), and annealing (60°C for 1 min). Values are showed as fold-differences compared to that of untreated reference group. Expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Cells were collected at 24, 48 and 72 hrs after treating with 25 nM p21-saRNA-322, scramble RNA treated as well as the untreated cells were used as references, rinsed twice with ice-cold PBS. Total RNA was extracted using TRIzolThe p21 PCR primer sequences: 5′-cttcga ctttgtcaccgaga-3′ (forward), 5′-ggtccacatggtcttcctct-3′ (reverse); GAPDH PCR primer sequences: 5′-AGAACA TCATCCCTGCCTCT-3′ (forward), 5′-CTGCTTCACCAC CTTCTTGA-3′ (reverse). All experiments were done in triplicate and independently validated three times.+ electrophoretic imaging system .Cells were collected at 24, 48 and 72 hrs after treatment, rinsed twice with ice-cold PBS, harvested, lysed in Pierce RIPA buffer . Cell lysates were clarified by centrifugation at 12000 × g for 30 min at 4°C and protein concentrations were determined using the BCA protein assay reagent . Cell lysates (protein concentration was adjusted by adding certain amount of RIPA buffer) were subjected to 12% sodium dodecyl sulfate-polyacrylamide electophoresis (SDS-PAGE) gels, separated by SDS-PAGE and electrophoretically transferred to Invitrolon polyvinylidene difluoride (PVDF) membranes . Membranes were blocked with 5% skim milk and then incubated overnight with the appropriate primary antibodies  followed by matching horseradish peroxidase-conjugated secondary antibodies. After washing each sample three times for 10 min with 15 ml TBST, immobilon Western Chemiluminescent HRP Substrate  was added and the result was tested with ChemiDoc XRS5, 1 × 105 and 2 × 105 cell/plate, respectively. After cultured for 24 hrs, cells were transfected with 25 nM p21-saRNA-322 using Lipofectamine 3000. Scramble RNA treated and untreated cells were used as reference. 48 hrs after treatment, cells were harvest, rinsed by PBS twice and fixed in (80%) cold ethanol at 4°C overnight. Fixed cells was collected by washing with PBS twice and centrifuged at 300 × g for 5 min. 500 μL of Prodium Iodide (PI) staining solution  were added into each tube, incubated for 30min at 37°C in the darkness. The stained cells were immediately analyzed at BD FACSCalibur . Distribution of cell cycle was showed as the percentage of cells in G0/G1, G2/M and S populations.Flow cytometry assay was used to analyze cell cycle. HCT-116, HCT-116 (−/−) and HT-29 cells were plated onto the 6-well plate at the density of 1 × 10Apoptosis of colorectal cancer cells were assessed by flow cytometry after co-staining of cells with Annexin V-FITC and PI in accordance with manufacturer’s recommendations . Annexin V FITC positive, PI negative cells and double-positive cells were considered to be in early and late stages of apoptosis, respectively .4 cells (5 × 104 cells for HT-29 cells) in 1 mL of complete cell culture medium per well for proliferation assay. Every 24 hrs for the following 6 days, a batch of cells were collected and the cell number was detected using TC 20TM Automated Cell Counter (Bio Rad). All experiments were performed in six duplicate.The cell count detection via TC 20TM Automated Cell Counter (Bio Rad) was performed to assess the effect of p21-saRNA-322 on cell proliferation. HCT-116, HCT-116 (p53−/−) and HT-29 Cells were transfected with 25 nM p21-saRNA-322 using Lipofectamine 3000 for 12 hrs, scramble RNA treated cells as well as the untreated cells were used as references. Following treatments, cells were plated in 12-well plates at a density of 1 × 103 cells per well. Culture medium was changed every 3 days. Colony formation was analyzed 12 days after treatment by staining cells with a 0.05% crystal violet solution for 1 hr.As described by wang et al with little modulation , HCT-1162, 5 mM each of potasium ferricynide and potassium ferrocynide in 40 mM citric acid/sodium phosphate buffer) for 2–8 hrs at 37°C in dark. After incubation, the cells were washed with PBS and images were captured using an Olympus  inverted light microscope. The experiment was conducted triplicate and counting was done on three randomly selected fields each plate.Described by Phalke et al , senesce5 Bioware Brite HT29-Red-Fluc  colorectal cancer cells suspended in 50 uL PBS was injected subcutaneously into the right posterior limb. Three days following tumor cell implantation, in vivo bioluminescence imaging of HT29-Red-Fluc cells was evaluated on a IVIS Spectrum Imaging System  following intraperitoneal administration of 200 uL D-luciferin (15 mg/kg) , Bioluminescence signal was measured using Living Image® 4.3.1. Regions of interest were defined to quantify signal intensity. The mice were randomized to three groups. Group 1 (five mice each) received intratumoral injection of Lipofectamine3000-encapsulated p21-saRNA-322 (30 ug/mouse) every three days for 5 weeks beginning at day 4. Group 2 and 3 (five mice each) were injected intratumorally with Lipofectamine3000-encapsulated scramble RNA (30ug/mouse) or Lipofectamine3000-mixed PBS every three days for 5 weeks beginning at day 4. Bioluminescence signals were monitored weekly. The study was terminated 5weeks following treatment. All mice were subsequently euthanized and tumors surgically removed, the volume of the tumor was calculated with the formula: V = (width2 × length0.5) and tumor weight was assessed. The tumor tissue was placed in 10% neutral buffered formalin for immune histochemistry (IHC). Results are expressed as means ± SD.Six- to eight-week-old male nude mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co. Ltd (Beijing. China). All animals in this study were housed under pathogen-free conditions and were maintained in accordance with the guidelines of the recommendations for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee of the Ethics of Animal Experiments of the Chinese Academy of Medical Sciences & Peking Union Medical College. 3 × 10t-test, using SPSS15.0 Sigma Plot software . The correlation between variables was analyzed using Pearson correlation coefficient and p < 0.05 was considered to be a statistically significant difference.Data are expressed as mean ± standard deviation (SD). Statistical analysis of data was done with Student’s in vivo, we extended the research into the nude mouse xenograft model, and found that the p21-saRNA-322 in situ administration significantly inhibited xenograft growth. These results indicate that the strategy of treating colorectal cancer via p21-saRNA-322 merits further investigation.The present study demonstrated that introduction of p21-saRNA-322 significantly increased p21 expression in colorectal cancer cells, which in turn generated negative biological effects on colorectal cancer cell growth. To confirm the role of p21-saRNA-322