There has been no study in Japan on the predictors of risk for acquiring SARS-CoV-2 infection based on people’s behaviour during the COVID-19 pandemic. The aim of this study was to document changes in risk behaviour during the New Year’s holiday season in 2021 and to identify factors associated with high-risk behaviour for infection using a quantitative assessment tool.A longitudinal survey.Multiphasic health check-ups for the general population in Iwate Prefecture.Serial cross-sectional data were obtained using rapid online surveys of residents in Iwate Prefecture from 4 to 7 December 2020 (baseline survey) and from 5 to 7 February 2021 (follow-up survey). The data in those two surveys were available for a total of 9741 participants.We estimated each individual’s risk of acquiring SARS-CoV-2 infection based on the microCOVID calculator. We defined four trajectories of individual risk behaviours based on the probabilities of remaining at low risk, increasing to high risk, improving to low risk and persistence of high risk.Among people in the low-risk group in the first survey, 3.6% increased to high risk, while high risk persisted in 80.0% of people who were in the high-risk group at baseline. While healthcare workers were significantly more likely to be represented in both the increasing risk and persistently high-risk group, workers in the education setting were also associated with persistence of high risk .In determining countermeasures against COVID-19 (as well as future outbreaks), health officials should take into account population changes in behaviour during large-scale public events. Risk behaviours associated with contracting COVID-19 were assessed in a large sample of the general population living in one Japanese prefecture.Longitudinal assessments were conducted at two time points, straddling the 2021 holiday season.The brief online survey approach allowed for rapid, real-time tracking of population changes in behaviour.A limitation of the study was the low response rate to the baseline survey (25%) as well as the follow-up survey (40%), although the direction of the bias is likely to be conservative.Cases of COVID-19 spread worldwide soon after the first case was identified in China in 2019. The worldwide death toll from COVID-19 as of 19 November 2021 is 5 139 910.In December 2020, Japan experienced a third wave which was much larger than the second wave, peaking at 8045 new cases on 8 January 2021 (compared with 1575 cases at the peak of the second wave on 31 July 2020).3One of the reasons for the increase in the number of COVID-19 cases in Japan was the movement of people during the traditional New Year holiday. New Year’s Day is the most important holiday in Japan, when many people return to their hometowns to spend time with their family, relatives or local friends. The typical number of days that people take off during the New Year’s holiday is about 1 week (7 days) from 28 December to 3 January. Although there is a decrease in the movement of people because schools, businesses and workplaces are closed for several days, large crowds of people gather to pray at local shrines or temples on New Year’s Day .4The Japanese government opted to not restrict the movement of people during the period before and after New Year’s Day. Some people also ignored the government’s request to refrain from unnecessary outings while other people continued to take preventive measures against infection. However, the number of people who ignored government directives is not known. Studies have shown that there are variations in the tendency to adopt protective measures such as social distancing based on people’s age, gender, socioeconomic characteristics  and political ideology.The aim of our study was to determine different groups of individuals based on their trajectories of risk behaviour during the New Year holiday season and to determine factors associated with behaviour with a high risk for COVID-19 using a quantitative risk assessment tool, ‘microCOVID’.Iwate Prefecture is located in the north-eastern part of Japan (about 500 km from Tokyo) with a population of about 1.2 million . The tot910.1136/bmjopen-2021-054770.supp1Supplementary dataRapid online surveys of residents in Iwate Prefecture were conducted by the Office of Medical Policy in the Department of Health and Welfare in Iwate Prefecture Government using a popular social network platform called LINE . By using the LINE app, Iwate Prefecture has been providing information about COVID-19 every day since the beginning of the pandemic, including information on the number of new cases and characteristics of patients. A series of cross-sectional surveys were started in December 2020 to investigate people’s behaviour in order to prevent infection and promote health consciousness. The surveys have been conducted every 2 months. An online questionnaire was administered to a total of 100 958 people who had registered by the time of the baseline survey. We sent out notification about the survey via the LINE platform on 4 December 2020, and 25 411 individuals answered from 4 to 7 December . We conducted a second survey of registered people from 5 to 7 February 2021. We analysed the results of these two surveys. After excluding individuals who were lost to follow-up in the second survey (15055), those who were missing data needed for the calculation of microCOVID scores either in first or second survey (n=446), those who live outside Iwate (n=34) and those who had other missing values for analyses (n=135), data were available for a total of 9741 participants .We applied a weighting system to calculate the level of behavioural risk for acquiring COVID-19 infection, called microCOVID.10.1136/bmjopen-2021-054770.supp2Supplementary dataActivity risk indicates the chance that an activity will transmit COVID-19 from a person who currently is infected with COVID-19. According to microCOVID, the transmission risk is estimated to be ‘about 9% per hour’ from spending more than 10 min indoors or in close proximity with an unmasked person who is COVID-19 positive. Coefficients of risk are assigned to other types of interaction which differ from that reference value. The coefficients were calculated based on the following factors: duration of interaction, mask wearing (respondents and other persons), indoor/outdoor environment, distance from each other, volume of conversation and frequency (times a week) . With reAge, sex, municipality, occupation, frequency of eating out compared with the past year, returning home during the New Year holiday and visits to the local shrine/temple were included as independent variables in the analysis. Participants were divided into three age categories: young (people less than 39 years of age), middle age (people of 40–59 years of age) and elderly (people aged 60 years or older). Residential areas were classified into inland versus coastal/mountainous based on the geography of Iwate Prefecture. Occupations were assessed by asking ‘what is your current job?’ Participants were divided into five groups: healthcare workers, workers in service industries , education sector (teachers or students), government workers and all others . The frequency of eating out compared with the past year was classified into three groups: ‘decreased by 80% or more’, ‘decreased by 50%–70%’ and ‘decreased by 40% or less’. With regard to the first shrine visit of the year, participants were divided into three categories: ‘never observe the ceremony’, ‘visited’ and ‘did not visit this year as a preventive measure’. We asked respondents about the extent of preventive measures adopted during the second state of emergency compared with that in the first one. The participants were divided into three groups: lower (lower degree or no preventive measure), same (same as the first one) and higher (higher degree).2 test. Logistic regression analyses were conducted to identify the characteristics related to high-risk behaviour. We conducted analyses separated by low risk and high risk in the first survey to determine the predictors of each type of trajectory. We imputed missing covariate data by multiple imputation using Markov chain Monte Carlo method, creating five imputed data sets. We used the Statistical Package for Social Sciences (SPSS) software program V.25.0 (IBM) for all analyses. All statistical tests described were two sided, and analysis items with p values <0.05 were considered statistically significant.For analysis of the trajectories of behaviour risks, we calculated the risk scores for each individual in the first survey and that in the second survey. Baseline characteristics were compared for the low-risk and high-risk microCOVID groups in the second survey using the χNo funding was available for patient or public involvement in this project. No patients were involved in setting the research question or the outcome measures. Patients were not invited in the design, or conduct, or reporting, or dissemination plans of our research.We evaluated the predictors of high-risk behaviour for SARS-CoV-2 infection during the Japanese New Year’s holiday season in one prefecture. Overall, only a small percentage (<4%) of individuals transitioned from low risk (before the holidays) to high risk. However, the majority of individuals (80%) who were in the high-risk behaviour group at baseline remained high risk at follow-up.The characteristics of individuals transitioning to high-risk behaviour included younger age, people working in the healthcare sector, as well as individuals who admitted to practising a lower degree of preventive measures during the state of emergency. Risk factors for persistently high risk were mainly based on occupation, viz working in the healthcare sector and education sector.et al examined the associations between preventive behaviours for COVID-19 and health belief factors, fatalistic beliefs and demographic factors among 750 Iranian adults during the period from 11 to 16 March 2020. They showed that the adoption of preventive behaviour was higher in women than men and higher in urban dwellers than rural dwellers.et al examined the associations of internet use, risk awareness and demographic characteristics with engagement in preventive behaviours in the USA (n=979) from 10 to 14 April 2020.et al investigated the characteristics related to positive and negative attitudes towards social distancing as a preventive measure in the USA (n=1074) in May 2020.Previous studies have examined factors associated with risk behaviours during COVID-19.et al showed that the hazard ratio of the risk for COVID-19 was 3.40 (95% CI 3.37 to 3.43) among front-line healthcare workers.et al reported that the risk ratio for testing positive for COVID-19 or death caused by COVID-19 was 7.43 (95% CI 5.52 to 10.0) among healthcare workers.Identifying high-risk occupational settings for acquiring COVID-19 infection remains an important goal for policy, for example, prioritising vaccinations. Healthcare work was recognised as a high-risk occupation during the 2009 influenza A (H1N1) pandemic.et al also reported that social and education workers had a significantly higher risk for COVID-19 than did non-essential workers in the UK and the USA.22Interestingly, people in the education sector had substantially higher ORs for high-risk persistence even though they did not gather in schools for 3 weeks during the winter vacation. After our investigation period, which included 2 weeks after the second survey during the period from 8 December 2020 to 21 February 2021, about 14% of the total number of patients with COVID-19 was reported to be among teachers and school-age children in Iwate. A meta-analysis showed that children and adolescents have lower susceptibility to SARS-CoV-2; the pooled OR of being an infected contact in children compared with that in adults was 0.56 (95% CI 0.37 to 0.85).Initially, we hypothesised that people who made visits to shrines during the holidays  would have high risk for SARS-CoV-2 infection. In fact, our study did not suggest increased risk, possibly because of near universal mask wearing, and the fact that praying at shrines and temples occurs outdoors. On the other hand, although there are some other risky behaviours that typically occur during the New Year holiday season in Japan, we could not collect detailed information on these events, for example, ‘Bonen-kai’ (end-of-year parties in workplaces),The present study had several limitations. First, we did not ask questions correlated with healthy behaviours such as educational attainment or household income. We also did not ask questions about job titles, which might influence the risk of exposure to potentially high-risk environments. Second, there may have been selection bias since the respondents were limited to people registered in a health information programme about COVID-19 maintained by the Iwate Prefecture Government. We compared the characteristics of the participants at baseline (December 2020) with the characteristics of the whole population in Iwate in 2020 . While tHealthcare workers and people in the education sector (teachers and pupils) were found to have the highest behavioural risk during the New Year holiday season in Japan. Our results offer some clues for who should be prioritised for COVID-19 vaccination as the programme is rolled out in Japan.


RB1 mutation. The identification of low-penetrance mutations in the RB1 gene and the study of their inheritance in pedigrees is contributing to understanding the mechanisms underlying the development of retinoblastoma with low penetrance. It is important both for further expansion of knowledge in the field of molecular genetics of retinoblastoma, and for competent genetic counseling and subsequent clinical management of families with this form of the disease. Our results support an assumption that parental origin of an RB1 mutation influences the likelihood of developing retinoblastoma. We also revealed a relatively high frequency of asymptomatic carriage of the RB1 mutations among the parents of retinoblastoma patients, highlighting the utmost necessity for molecular analysis among the probands’ relatives irrespective of their clinical status and family history of retinoblastoma.Some families with hereditary retinoblastoma exhibit mild phenotype with low penetrance and variable expressivity, including complete absence of clinical signs of the disease in some carriers of the germline RB1 alterations causing hereditary low penetrance retinoblastoma and to evaluate how the parental origin of an RB1 mutation affects its phenotypic expression. By NGS and MLPA, RB1 mutations were found in 191 from 332 unrelated retinoblastoma patients. Among patients with identified RB1 mutations but without clinical family history of retinoblastoma, 7% (12/175) were found to have hereditary disease with one of the parents being an asymptomatic carrier of an RB1 mutation. Additionally, in two families with retinoblastoma history, mutations were inherited by probands from unaffected parents. Overall, nine probands inherited RB1 mutations from clinically unaffected fathers and five, from mothers. Yet, we gained explanations of maternal “unaffectedness” in most cases, either as somatic mosaicism or as clinical presentation of retinomas in involution, rendering the proportion of paternal to maternal truly asymptomatic mutation carriers as 9:1 (p = 0.005). This observation supports an assumption that parental origin of an RB1 mutation influences the likelihood of developing retinoblastoma. Additionally, our study revealed a relatively high frequency of asymptomatic carriage of the RB1 mutations among the parents of retinoblastoma patients, highlighting the utmost necessity of molecular analysis among the probands’ relatives irrespective of their clinical status and family history of retinoblastoma.Our aim was to identify  Retinoblastoma is the most common cancer affecting the retina in children, with an incidence of 1:16,000–1:18,000, accounting for 3% of all pediatric cancers . A tumorRetinoblastoma is usually diagnosed in the first two years of a child’s life. The main clinical symptoms of retinoblastoma are leucocoria, strabismus, poor vision, redness of the eye with pain in it, and proptosis. Ophthalmoscopy reveals unifocal or multifocal intraretinal transparent tumor nodes. As the tumor grows, which can occur in a three-dimensional plane, the nodes become opaque, dull white, and vascularized. Additionally, with retinoblastoma, diffuse retinal detachment and tumor seedings into the vitreous body are revealed ,4. In apRetinoma is diagnosed in both childhood and adulthood. Most patients are diagnosed with retinoma at their visit to an ophthalmologist for an eye examination because of retinoblastoma in their family members, especially first-degree relatives. The diagnosis can also be made in the case of complaints of decreased vision or strabismus. Ophthalmoscopic features of retinoma are translucent gray nodes in the retina, calcification, changes in the pigment epithelium of the retina, and chorioretinal atrophy ,7. RetinHowever, the differential diagnosis of retinoblastoma and retinoma may be challenging. When making a differential diagnosis, the age of the patient is of great importance. In addition, the absence of tumor progression without any treatment speaks in favor of retinoma.RB1 tumor suppressor gene [Molecularly development of retinoblastoma is based on biallelic inactivation of the sor gene ,9. Both sor gene .RB1 alleles predisposes to tumor development and determines familial transmission, while a somatic mutation of the other allele is acquired in utero or in early childhood and triggers tumorigenesis. Familial retinoblastoma manifests itself at an earlier age compared with the sporadic form (the mean age at diagnosis is 12 months) and is multifocal and bilateral in the majority of cases. Carriers of a germline mutation are additionally at higher risk of developing subsequent non-ocular primary cancers [Hereditary retinoblastoma is diagnosed in 40% of cases. In this form of the disease, a germline mutation of one of the  cancers . The dis cancers .Thus, it is of immense importance to understand the mechanisms that underlie hereditary retinoblastoma with low penetrance in order to improve our knowledge of the molecular genetics of this disease and to provide better genetic counseling and subsequent clinical management.RB1 mutation [RB1 mutation [The hereditary retinoblastoma phenotype is thought to depend on the functional type of the underlying mutation . In turnmutation ,14.Peripheral blood samples were obtained from 332 unrelated retinoblastoma patients, including 226 with the unilateral and 106 with the bilateral form. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the Research Centre for Medical Genetics. Written informed consent was obtained from each participant involved in the study. In Russia, conservative treatment of retinoblastoma is widely available, thus design of the present study was based on molecular testing of blood samples only.RB1 mutations were found in blood cells either by sequencing or MLPA, both parents agreed to give their blood samples and were tested for the carriage of the mutation found in the proband. In clinically familial cases, extra relatives underwent molecular testing provided that they were available and agreed to give their blood samples .A family history of retinoblastoma was known for 16 (4.8%) patients, 2 of which had unilateral and 14, bilateral retinoblastoma. In all pedigrees where Genomic DNA was isolated from peripheral blood lymphocytes by standard phenol–chloroform extraction.RB1 gene was performed by high-throughput parallel sequencing using the Ion Torrent platform . The panel of primer pairs for library preparation was designed using AmpliSeq Designer software . Target regions include all coding sequences of the RB1 gene, adjacent intron regions, and untranslated regions (5′-UTR & 3′-UTR). For library preparation, Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) was used. The reaction was carried out according to the standard protocol recommended by the manufacturer. Aliquots from the prepared libraries were subjected to clonal emulsion amplification on the Ion OneTouch instruments using the Ion OneTouch 200 Template Kit . Sequencing was performed on the Ion Torrent PGM genomic sequencer using an Ion PGM 200 Sequencing Kit . The results were analysed with Torrent Suite software consisting of Base Caller, Torrent Mapping Alignment Program TMAP, and Torrent Variant Caller. Genetic variants were annotated with ANNOVAR software  [Screening for point mutations and small insertions/deletions of the PA, USA) . Visual PA, USA) .Once a candidate genetic variant was detected in a proband, DNA samples of the proband’s parents and sibs were tested for this variant by Sanger sequencing of the DNA fragments amplified by PCR from the flanking primers. The direct sequencing of individual PCR products was performed on the automatic genetic analyzer ABI PRISM 3500 (Thermo Fisher Scientific) according to the manufacturer’s protocols.RB1 gene was performed by MLPA. The MLPA reaction was carried out with a SALSA MLPA P047-D1 RB1 kit according to the standard protocol recommended by the manufacturer  (accessed on 2 August 2021). Capillary electrophoresis of MLPA products was performed on an ABI PRISM 3500 genetic analyzer in accordance with the manufacturer’s instructions. The data obtained were processed using Coffalyser.NET software provided by MRC Holland.Screening for large deletions within or containing the Paternal to maternal truly asymptomatic mutation carrier groups were compared using Fisher’s exact test.RB1 gene either by DNA sequencing or by multiplex ligation-dependent probe amplification (MLPA)  with low penetrance and/or variable expressivity of the disease . In all families with retinoblastoma history, we identified molecular genetic alterations of the n (MLPA) .RB1 gene were identified in 175 cases. The latter underwent familial segregation analysis, and 7% (12/175) were found to have hereditary disease with one of the parents being an asymptomatic carrier of an RB1 mutation  were found to have retinomas at involution by fundoscopy .Thus, after in-depth molecular and clinical evaluation, we gained explanations of maternal “unaffectedness” in most cases, either as somatic mosaicism or as clinical presentation of retinomas in involution, rendering the proportion of paternal to maternal truly asymptomatic mutation carriers as 9:1 . This diRB1 mutation from their fathers who were asymptomatic carriers or had a milder disease form  in 11 families and from the mothers who were asymptomatic carriers in five families. Additional clinical and molecular genetic tests revealed retinoma at the involution stage in the asymptomatic mothers in three families and mosaicism for the mutation with a low proportion of cells carrying the mutant allele in the mother in one family. Thus, in all cases with unexplained incomplete penetrance, it is paternal inheritance of the mutation that was observed in the majority of families in our cohort (11 families vs. 1 family where the mutation was inherited from the mother).A total of 15 inherited low penetrance mutations were observed in our cohort of retinoblastoma patients. The mutations led to the disease with incomplete penetrance in 16 families . The probands with retinoblastoma inherited the RB1 gene mutations in 191 (58%) of them. Such efficacy of RB1 germline mutation screening in an overall cohort of retinoblastoma patients is consistent with previous reports [RB1 mutation screening in blood samples depends on the clinical form of retinoblastoma  and on the family history (inherited or sporadic disease). In our cohort, causative mutations were found in all families with retinoblastoma history. In a group of 316 sporadic retinoblastoma patients without a family history  mutations in the RB1 gene in peripheral blood DNA were detected in 55% (175/316) of cases. Among them, 98% (91/93) of patients with bilateral form of the disease demonstrated RB1 mutations. Such frequency of RB1 mutations in the bilateral form of the disease is consistent with the results previously reported by other authors [In this work, molecular genetic assessment of DNA samples from peripheral blood lymphocytes of 332 unrelated retinoblastoma patients resulted in identification of causative  reports ,21. Effi authors ,21. AmonRB1 mutations in retinoblastoma patients is possible in about 95% of cases, irrespective of clinical form of the disease and family history, only if the tumor material is available for molecular genetic testing. We reported a 95% efficacy of RB1 mutation screening in our early studies, when enucleation was an inevitable treatment option and tumor samples were available for each proband under study [RB1 gene mutations in families with low penetrance hereditary retinoblastoma.Identification of er study . Advanceer study , thus deRB1 alleles [RB1 mutations are classified into three categories depending on the pRB functional activity. One category includes the mutations that lead to the absence of the RB1 protein product from the cell and, therefore, loss of its function (nonsense and frameshift mutations). These mutations cause a premature transcription termination and subsequent nonsense-mediated degradation of the defective mRNAs [The hereditary retinoblastoma phenotype is thought to depend on the type of the germline (first) mutation affecting one of the  alleles ,25. RB1 ve mRNAs . Patientve mRNAs . Mutatiove mRNAs . The thive mRNAs ,24.RB1 gene. We suggest that it is the location in the first exon, abolishing the expression of a longer, naturally weakly expressed transcript and retaining the expression of the shorter one, that provides a possibility for such loss of function mutations to manifest as low penetrant. Modulation of penetrance of the disease caused by frameshift mutations may also be achieved by internal translation initiation. Sanchez et al. (2007) reported a family with a low penetrance RB1 mutation comprising a 23-basepair duplication in the first exon of RB1 (c.43_65dup) producing a frameshift in exon 1 and premature chain termination in exon 2. The authors demonstrated that this mutation did not cause appreciable NMD, and transcript expression in tissue culture cells and translation in vitro revealed that alternative in-frame translation start sites involving Met113 and possibly Met233 were used to generate truncated RB1 products (pRB94 and pRB80), known and suspected to exhibit tumor suppressor activity [Of the ten low-penetrance mutations that the retinoblastoma patients inherited from their fathers, five are splice site mutations, three are missense mutations, and two are frameshift deletions . Thus, eactivity .RB1 mutation is currently thought to provide a molecular mechanism that underlies the variation in phenotypic expression of the same mutation in different members of a family with hereditary retinoblastoma [An effect of the parental origin of the blastoma ,14,20.RB1 gene is known to harbor a 1.2-kb imprinted region presented by a CpG island (CpG 85) in intron 2 that shows differential methylation depending on the parental origin of the allele; i.e., the region is methylated in the maternal chromosome and nonmethylated in the paternal one. Two other CpG islands, CpG 106, and CpG 42, reside in the RB1 gene. The island CpG 106 includes the promoter and exon 1 and is characterized by biallelic lack of methylation, thus allowing expression of the major pRB-coding transcript from both RB1 alleles. The island CpG 42 is in intron 2, is methylated in both chromosomes, and lacks regulatory activity [The activity ,20.PPP1R26P1 protein-coding gene, which is in chromosome 9 and is integrated in RB1 in the inverse orientation. CpG 85 acts as a promoter for an alternative RB1 transcript, which is expressed only from the non-methylated paternal chromosome. In addition, although the total expression level of mRNA transcripts synthesized from the paternal allele may be expected to be higher than from the maternal one, expression from the paternal allele is actually two to three times lower because transcriptional interference arises when both regular and alternative transcripts are expressed [There is evidence that CpG 85 is part of a 5′-truncated processed pseudogene that originates from the xpressed ,20.RB1 alleles because the expression profile of the maternal allele becomes similar to that of the paternal one [Demethylation of CpG 85 in lymphoblast cell lines treated with the demethylating agent 5-aza-2’-deoxycytidine has been observed to result in equal levels of mRNA expression from the two rnal one .RB1 expression from the paternal and maternal alleles. The mouse RB1 gene lacks a CpG island homologous to human CpG 85. The observation indicates that differentially methylated CpG 85 is responsible for biased allelic expression of human RB1 [Mice have not been observed to have imbalanced levels of uman RB1 .RB1 expression from the paternal allele compared with the maternal one. Transcriptional interference is a mechanism where transcription of one gene suppresses transcription of another [RB1 transcript, blocks the progress of the transcription complex that synthesizes the major transcript from the same allele, thus decreasing the total expression level from the paternal chromosome. Thus, the above data demonstrate that RB1 expression is epigenetically regulated depending on the parental origin of the allele [It is currently thought that transcriptional interference is responsible for lower  another . A transe allele ,20.RB1 mutation correlates with disease penetrance [RB1 mutations inherited from the father more often cause retinoblastoma and determine its more severe form than the same mutations inherited from the mother. Tumor suppressor activity of pRB is thought to be high enough to prevent retinoblastoma when a low-penetrance mutation is inherited maternally. In contrast, when a mutant allele is transmitted from the father, low residual activity of the protein results from its lower expression and mimics a loss-of-function mutation, leading to retinoblastoma [Other studies have confirmed that the sex of the transmitter of an netrance ,28,29. ARB1 mutation penetrance may be explained by this effect, or whether the search has to be undertaken for other molecular mechanisms, such as, for example, impact of genetic variants outside the RB1 gene (“genomic environment”). One way to address this question is to accumulate observations of different RB1 low penetrance mutations in well characterized trios. Observation that males constitute an overwhelming number of truly asymptomatic RB1 mutation carriers may be interpreted as evidence that a parent-of-origin effect is a principal, if not a sole explanation for the incomplete penetrance of the RB1 gene mutations. Our present study is one that fully supports this conclusion. It is of note however, that the numbers of non-penetrant transmitting parents reported here are small, but this is a consequence of the low frequency of asymptomatic carriage of retinoblastoma in the population. The data of course should not be over interpreted: more substantiated conclusions may be made at least in the meta-analyses based on studies like ours, when the number of such increases.Although several reports have already shown evidence of a parent-of-origin effect in retinoblastoma presentation, an intriguing question is whether all cases of incomplete RB1 gene that should be classified as low penetrance mutations, that were not previously reported, and support low penetrance for three previously reported ones, c.607+1G > T [RB1 mutations among the parents of retinoblastoma patients, highlighting the utmost necessity for molecular analysis among the probands’ relatives irrespective of their clinical status and family history of retinoblastoma. Knowing the relatives RB1 status does not affect treatment and further clinical management of the proband. However, the identification of asymptomatic carriers of the RB1 gene mutation in a family is important for genetic counseling of a family in terms of further childbirth, since carriers of RB1 mutations with low penetrance have an increased risk of having a child with retinoblastoma compared to the general population. In addition, carriage of a low penetrance mutation in the RB1 gene increases the risk of developing other malignant neoplasms in various locations throughout life [In this study of low penetrance hereditary retinoblastoma, we described a significant group of patients without family history of retinoblastoma, that is, all patients were admitted as cases with sporadic disease. In the previously published articles on low penetrance hereditary retinoblastoma, in most cases, familial cases were described. Additionally, we demonstrated new mutations in the 7+1G > T ,22,28, c7+1G > T , and c.97+1G > T . Our stuout life ,29.


At a regional and local level, the COVID-19 pandemic has not spread out uniformly and some German municipalities have been particularly affected. The seroepidemiological data from these areas helps estimate the proportion of the population that has been infected with SARS-CoV-2 , as well as the number of undetected infections and asymptomatic cases.In four municipalities which were especially affected, 2,000 participants will be tested for an active SARS-CoV-2 infection  or a past infection (blood specimen IgG antibody test). Participants will also be asked to fill out a short written questionnaire at study centres and complete a follow-up questionnaire either online or by telephone, including information on issues such as possible exposure, susceptability, symptoms and medical history.The CORONA-MONITORING lokal study will allow to determine the proportion of the population with SARS-CoV-2 antibodies in four particularly affected locations. This study will increase the accuracy of estimates regarding the scope of the epidemic, help determine risk and protective factors for an infection and therefore also identify especially exposed groups and, as such, it will be crucial towards planning of prevention measures. In December 2019, the first cases of a lung disease caused by a new coronavirus were described in Wuhan, China. Since then, SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) infections and cases of COVID-19 (Coronavirus Disease 2019) have spread worldwide, causing a pandemic with over 12 million confirmed diagnoses and 560,000 deaths. Germany has so far registered 195,000 SARS-CoV-2 infections, causing 9,064 fatalities either of or related to the development of COVID-19 (as at 13 July 2020) . InternaThe available data on the spread of SARS-CoV-2 infections in Germany has so far been based on confirmed SARS-CoV-2 case numbers reported to the local health authorities in line with Germany’s Protection against Infection Act (IfSG). These cases are diagnosed by polymerase chain reaction (PCR) tests. Based on current scientific data, however, an unknown proportion of SARS-CoV-2 infections can be assumed to take an asymptomatic or mild course, which means that many subclinical or mild infection courses are not diagnosed as SARS-CoV-2 infections. Furthermore, in line with the recommendations from the Robert Koch Institute (RKI), the European Centre for Disease Prevention and Control (ECDC) and the World Health Organization (WHO), PCR tests are conducted only with symptomatic patients likely to test positive for SARS-CoV-2. So currently, reported case numbers do not provide a reliable estimate of the actual prevalence of past SARS-CoV-2 infections in the population. In the COVID-19 Case-Cluster-Study, which was conducted in the Gangelt municipality  between March and April 2020, researchers reported an IgG seroprevalence of 15.5% and a factor of five for undetected infections (with regard to the number of registered SARS-CoV-2 cases) . HoweverDue to the high number of cases, seroepidemiological data from particularly affected locations facilitates a rather accurate estimate of past infections and provides a good indication of the number of undetected SARS-CoV-2 infections. Furthermore, research into the risk and protective factors for an infection help identify high risk groups, and this is crucial in terms of putting prevention measures in place. Conclusions related to the burden of disease, the number of asymptomatic infections, as well as the dynamic of infections can be drawn and, in part, transferred to locations not as impacted by the epidemic.In the context of the CORONA-MONITORING lokal study, antibody prevalence and the proportion of active infections was determined in four municipalities particularly affected by SARS-CoV-2 and a cumulative incidence of over 500 registered cases per 100,000 inhabitants over a timespan of one month.At each survey location the study aims to:Determine seroprevalence (proportion of the population that has been in contact with the virus) for each survey location by age group and sex,Calculate the proportion of undetected infections,Identify the proportion of asymptomatic infections.Due to the limited number of cases, some analyses cannot be conducted for each location; but will be conducted with aggregated data of all four sample points:Sensitivity analyses of infection rates and the proportion of undetected infections by taking into account the data of non-responders, as well as the data on reported and/or deceased cases.Presenting infection rates differentiated by exposure contexts  and use of public transport).Calculation of the proportion of undetected cases relative to the number of reported cases differentiated by risk group status  and age group, sex and education.Differentiation of symptomatic infections by sex and/or age group, as well as by exposure contexts  and use of public transport).Identification of risk and protective factors for a SARS-CoV-2 infection and the extent to which it is embedded in the respondent’s living, family and occupational situation.Calculation of infection mortality .Furthermore, the study aims to create a basis for longitudinal studies that will potentially facilitate follow-ups on the possible sequelae of people who have experienced a SARS-CoV-2 infection.The CORONA-MONITORING lokal study is a population-based, seroepidemiological observation study that will conduct cross-sectional examinations at four locations in Germany which were particularly affected by the COVID-19 epidemic. An ongoing acute transmission of SARS-CoV-2 would call for a repetition of cross-sectional surveys or supplementing the results with longitudinal examinations with serial serological testing and interviews with a selected group of participants.The study will be conducted at four municipalities particularly affected by the COVID-19 epidemic  among 2,000 individuals for each study. Municipalities are selected with regard to the epidemiological developments shortly before the local start of the study. The selection criteria for municipalities are a past or ongoing transmission as well as the willingness of local authorities to contribute towards the study.A random sample from population registries is provided to the RKI. This includes adults aged 18 years and older with no upper age limit who are registered residents in one of the surveyed municipalities. Proportional sampling is applied, i.e. population registries are asked to provide 4,000 to 5,000 randomly drawn addresses not stratified by sex and/or age group. Based on an expected response rate of between 60% and 70%, at first 2,900 randomly selected individuals are then contacted in writing. If the response rate is lower than expected, further random samples will be drawn within sex and/or age groups underrepresented among participants from the first sample. When further survey waves are conducted, this step is repeated. The municipalities that have been selected so far are Kupferzell (Baden-Wuerttemberg), Bad Feilnbach (Bavaria) and Straubing (Bavaria).Individuals will be included▶ if they are 18 years or older,▶ are registered residents of one of the four study municipalities,▶ can provide written consent to participate in the study, or, where necessary, written consent is provided by a legal representative,▶ are able to take part in the interviews (where necessary with the help of relatives) and the examinations at the study centres or during home visits.People who lack the necessary German language skills or where it is unclear whether they understand the study information leaflets or the consent forms are excluded from participating. Currently, we are considering translating the survey material into English and further locally relevant languages and seeking the services of interpreters.The individuals selected randomly from the population registries of the corresponding municipalities are first invited in writing to take part in the study. They receive an invitation by mail with materials informing them about the study . Those who are willing to participate can make an appointment at the study centres choosing from the days scheduled for the study either via an online calendar or by phone at the study hotline . DependiPeople in the age group 60 years and older, who have been defined as a high risk group for a COVID-19 infection, receive an adapted invitation letter that explicitly offers the option of a home visit . A telepPeople who contact survey staff and refuse to take part are asked to state the reasons for their decision. People who do not react to the letter and with whom no contact is therefore established will be chased-down after the field work has been completed and will be asked to fill out a non-responder questionnaire to estimate the representativeness of the study sample.Per municipality, one to two temporary study centres will be established. Centres will consist of a bus for examinations and a rented space. The latter will count with one to two receptions and two examination rooms as well as a laboratory (with a centrifuge and a laptop). The bus for examinations provides two further examination rooms.The study staff includes physicians, staff who have completed training in a medical profession , nutritionists and administrative employees. All in all, the study teams consist of around 25 individuals.In preparation for the CORONA-MONITORING lokal study the following training sessions were organised:▶ overview of the study objectives▶ overview of SARS-CoV-2 and COVID-19▶ measures of infection protection (including disinfecting measures)▶ working with personal protective equipment▶ participant reception and informed consent▶ conducting home visits▶ taking blood specimens and oropharyngeal swabs▶ specimen processing, storage and transport▶ break periodsAll the staff involved in the study took part in these training sessions .Participants then fill out a short questionnaire, provide a blood sample and an oropharyngeal swab. Oropharyngeal swabs are taken out of the opened mouth using the Copan Group  Copan Liquid Amies Elution Swab transport system. For venous blood collection, the Vacutainer System 8.5ml tube BD Vacutainer SST II Advance  with separating gel and clotting activator is used.Blood samples and oropharyngeal swabs are immediately taken to the laboratory. The swabs are stored in a fridge and blood samples centrifuged for 12 minutes at 4.400 RPM (corresponding to 3000 x g) 30 to 45 minutes after being taken in an Eppendorf model 5702 lab bench centrifuge  and filled into two serum tubes  and then also stored in the fridge. Consent forms and written questionnaires are sent with the specimens collected and stored at 4°C in actively cooled boxes  with a daily shuttle to the RKI for further processing.Home visits are conducted by teams of two people: one person to take specimens and one person as a driver. Procedures during home visits match those at the study centres. After the home visit, the driver takes the consent form, the questionnaire and the specimens to the study centre for further processing.Around one to two weeks after the examination, a follow-up interview via an online questionnaire is scheduled. If a participant prefers, this interview can also be conducted as a telephone interview .At the study centres and during home visits, participants are asked to wear facemasks and are, if necessary, provided with one. Participants are also asked to maintain physical distance. During specimen collection, the examination staff wear personal protective equipment and particle filtering FFP2 masks. Examination rooms are regularly cleaned, aired and surfaces disinfected after every examination. Participants with cold symptoms are asked not to come to the study centre and whenever possible an appointment for a home visit is made.Participants receive their PCR laboratory and antibody test results in the form of a pseudonymised result report. The following values are tabulated with their corresponding reference intervals:▶ PCR test result for SARS-CoV-2 virus material on the oropharyngeal swab (positive/negative).▶ ELISA (enzyme-linked immunosorbent assay) for SARS-CoV-2 IgG antibodies in the blood .Before communicating the results, a trained study physician conducts a plausibility check for the test results. The administrative survey staff send out the pseudonymised result report accompanied by a personalised letter. Notifiable laboratory results are forwarded to the responsible health office (in writing) within 24 hours and the participant is informed about the result by the medical study staff (by telephone and in writing). In addition, infected participants receive the RKI’s information leaflet on self-isolation.Quality assurance measures during the study procedures are organised through an internal QA taskforce, which has previously been established at the RKI for other studies.Sensitivity, specificity and cross-reactivitySensitivity indicates how well a test can correctly identify a person with SARS-CoV-2 specific antibodies.Specificity indicates how well a test can correctly identify a person without SARS-CoV-2 antibodies.Cross-reactivity describes the capacity of antibodies to bind to antigens with similar docking sites. With regard to SARS-CoV-2, this means that an antibody does not bind only to SARS-CoV-2 but potentially also to other coronaviruses spread in Germany .At all stages of data collection, Standard Operating Procedures (SOP) have been defined. The study team receives training based on a fixed training protocol before data collection begins. Furthermore, before actual data collection, a pretest was run to test and where necessary adapt all steps. Continuous supervision by the leading field staff and the QA team during data collection is supplemented by regular further training provided to the study team; these training sessions are firmly anchored within the schedule. Where necessary, additional individual training sessions can be carried out.All the steps during laboratory examinations are documented  to accomplish QA and to make sure that samples are not mixed up.Two in-house PCR tests are used to test for the SARS-CoV-2 genome. Test 1 detects the E-gene adapted to  and is mTo detect IgG antibodies against the new coronavirus, the study uses Euroimmun’s  commercial laboratory test ‘Anti-SARS-CoV-2-ELISA (IgG)’. The test has been validated by a number of laboratories . With a sensitivity of 93.8% and a specificity of 99.6%, the test is of high quality and of low cross-reactivity . The useAs in all test procedures with a specificity of <100% – in particular when seroprevalence in the population is low – a certain proportion of false positives is inevitable. A neutralisation test was therefore performed on all ELISA reactive samples at the National Consultant Laboratory for Coronaviruses at the Charité-Universitätsmedizin Berlin. The result of this test was used for scientific evaluation. The exact method of this plaque reduction neutralisation test is described here .IgG antibody tests indicate whether someone has in the past had contact with the new coronavirus. Whether, and if yes, for how long an individual is then immune to the new coronavirus, cannot yet be safely said. Even people with a positive antibody test should therefore keep to the recommended hygiene and behaviour rules in their private and professional contexts. The same in any case applies to people without a positive antibody test.Laboratory examinations to detect an acute or past SARS-CoV-2 infection are supplemented by interviews with study participants to gain further information on a set of questions such as potential exposure, susceptibility, symptoms and medical history. Interviews consist of two parts: a short questionnaire that participants fill out either at the study centre or during home visits in the form of a written questionnaire, as well as a more detailed questionnaire that participants are asked to fill out one to two weeks after the appointment. Preferably, the administration is web-based, which means that participants receive access to an online questionnaire which they are asked to complete. Participants that cannot or do not want to fill out this questionnaire online can complete it via a computer-assisted telephone interview (CATI). We now present the expected results and the statistical procedures involved in data analysis.Data analysis involved applying a weighting variable to better reflect the structure of the general population in the selected municipalities regarding age and sex. In addition, weighting is applied regarding education by sex and age group. The marginal distribution is approximated referring to the administrative district based on micro census data 2017 .Weighting allows true but unknown parameters in the population to be better reflected although it is also related to an increase in the variance of estimators gained in the study. Based on experiences made in previous studies and assuming a relatively high willingness to participate and low selectivity, we set this factorto estimate case numbers at 1.43 (=1/(70%), where 70% describes the ‘effectiveness’ of the weighting variable, a measure for the spread of weights). For younger adults (18-to 34-years-old) and the older age group (65 years and older) based on previous studies effectiveness is set slightly lower  , 23.Based on these assumptions, with a net sample of n=2,ooo individuals we can achieve the accuracies given in If the requirement for estimate precision defines that the variation coefficient of the prevalence estimate is to be less than one sixth , then all the expected confidence intervals are in this sense acceptable for estimates regarding the general population. For the estimates stratified by age group, this requirement is not met if seroprevalence is only 3%, or only 3% or 5% for the oldest age group. However, it is possible to increase the precision of the estimators for individual age groups, if we group estimators for several particularly affected municipalities. Moreover, 3% tends to be the lower limit for the seroprevalences that can be expected in particularly affected locations.Descriptive analyses are first conducted for the following parameters:▶ description of response and non-response,▶ sociodemographic characteristics ,▶ prevalence of SARS-CoV-2 IgG antibodies in blood samples, and/or positive oropharyngeal swab test in the corresponding population ,▶ prevalence of symptomatic and asymptomatic cases (based on test history),▶ undetected case rate of antibody prevalence relative to the number of reported cases,▶ antibody prevalence relative to local events visited.Further analyses look at the following questions:▶ Understanding SARS-CoV-2 risk and protective factors, as well as the extent to which they are embedded in the participants’ living, family and occupational situation.▶ Sensitivity analysis of infection rates and the proportion of undetected cases that take into account non-responder data as well as data on reported cases and fatalities.▶ Estimating the proportion of undetected cases for reported cases stratified by risk group yes/no  and age group, sex, and education.▶ Influencing factors and social contacts, mental health and everyday life.▶ Asymptomatic infections stratified by sex and/or age group, as well as by exposure contexts  and use of public transport).▶ Infection fatality stratified by age and sex.Studies in locations particularly affected by SARS-CoV-2 can contribute towards a better understanding of the spread of the infection. The proportion of people who have actually had an infection can help to estimate the number of undetected cases relative to the number of reported cases. This will provide a better idea of the local extent of the epidemic as well as the proportion of asymptomatic infections, and, by identifying active infections, the current infection dynamics.Currently, a number of seroepidemiological studies on SARS-CoV-2 are being conducted in municipalities particularly affected , 25. In The focused analysis of locations particularly affected with high rates of infection is an important contribution to understanding infection dynamics and the developments of population immunity while taking into account a diverse range of transmission dynamics. For example, the study can provide information on the likelihood of transmission, as well as on risk factors for a severe course of the disease, which are relevant not only to the local development of infections. Additional longitudinal studies with repeated serological testing of population subsamples can, due to the expected greater number of cases, provide information on the development of the epidemic and immunity of the population overtime. Furthermore, we can also keep track of people who have tested positive for SARS-CoV-2 antibodies, which should provide findings on the long-term effects of COVID-19. However, as these studies are focused on local hotspots and population structures, the results cannot be transferred to the general population and studies in particularly affected municipalities therefore cannot indicate the prevalence of SARS-CoV-2 antibodies in the general population in Germany. Possible limitations of the CORONA-MONITORING lokal study are a potential selection bias (only German speakers) which hopefully can be countered in the future by providing materials in several languages and interpreters. Recall bias is also a potential issue because many questions refer to the long time period since the beginning of the COVID-19 epidemic in Germany. A further point of discussion relates to how long SARS-CoV-2 antibodies remain detectable, as some studies indicate a decrease of antibody titers overtime . Still, In the context of the CORONA-MONITORING lokal study, SARS-CoV-2 seroprevalence is determined for four particularly affected locations. Findings can contribute to a better understanding of the local infection dynamics, but also allow comparisons of the local situations. A broader analysis of the data will allow us to determine risk and protective factors for an infection and a severe disease course and therefore identify high risk and exposure groups, which is essential for the planning of prevention measures.The spread of SARS-CoV-2 infections differs widely across Germany.Local infection hotspots can apparently often be traced to certain events or situations where pathogen transmission is increased.As part of the study, 2,000 participants per location are examined in a temporary study centre or during home visits for an active SARS-CoV-2 infection as well as SARS-CoV-2 IgG antibodies.Seroprevalence figures allow an estimate of the number of undetected and asymptomatic SARS-CoV-2 infections.The CORONA-MONITORING lokal study surveys the population antibody status at four locations in Germany that have been particularly affected by the SARS-CoV-2 pandemic, and this will enable an estimate of the local population seroprevalence.


We find that as Mg2+ concentration increases both actin and actin-microtubule networks undergo bulk contraction, which we measure as the shrinking width of each network. However, surprisingly, lowering the Mg2+concentration back to 2 mM does not stop or reverse the contraction but rather causes both networks to contract further. Further, actin networks begin to contract at lower Mg2+ concentrations and shorter times than actin-microtubule networks. In fact, actin-microtubule networks only undergo substantial contraction once the Mg2+ concentration begins to lower from 20 mM back to 2 mM. Our intriguing findings shed new light on how varying environmental conditions can dynamically tune the morphology of cytoskeleton networks and trigger active contraction without the use of motor proteins.The dynamic morphology and mechanics of the cytoskeleton is determined by interacting networks of semiflexible actin filaments and rigid microtubules. Active rearrangement of networks of actin and microtubules can not only be driven by motor proteins but by changes to ionic conditions. For example, high concentrations of multivalent ions can induce bundling and crosslinking of both filaments. Yet, how cytoskeleton networks respond in real-time to changing ion concentrations, and how actin-microtubule interactions impact network response to these changing conditions remains unknown. Here, we use microfluidic perfusion chambers and two-color confocal fluorescence microscopy to show that increasing magnesium ions trigger contraction of both actin and actin-microtubule networks. Specifically, we use microfluidics to vary the Mg Porcine brain tubulin and rhodamine-labeled tubulin were purchased from Cytoskeleton  and stored at −80°C in PEM-100 (100 mM PIPES (pH 6.8), 2 mM MgCl2, and 2 mM EGTA). Both actin and actin-microtubule networks were formed at a fixed concentration of 5.8 μM total protein concentration and included a fraction of labeled protein to image networks  were included. For both network types, the final solution was mixed and pipetted into the central channel of the microfluidic sample chamber  and Thermofisher (A12374) and stored at −80°C in Ca buffer [2 mM Tris (pH 8.0), 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaClnetworks . Actin ntructure . However chamber  and incuThe construction of the microfluidic device shown in The flow cell was filled with a 50:1 mixture of polyethylene glycodiacrylate (PEG-DA) and photoinitiator, 2-hydroxy-2-methylpropiophenone, diluted to 10% in 10 mM Tris-HCl, then exposed to UV through a custom photomask to form two semipermeable membranes of crosslinked PEG-DA. The flow cell was immediately flushed with DI to remove the un-linked PEG-DA solution. This process results in three channels separated by two semipermeable membranes: one central channel for holding the sample and two side channels to enable buffer exchange via diffusion. The flow cell was flushed with 5% Tween (in PEM-100) followed by PEM-100.μM fluorescein salt in the original polymerization buffer as a proxy to measure ion concentration. To enable buffer exchange, the outlet Tygon tubes were connected to a digitally controlled syringe pump and the inlet Tygon tubing was inserted into the desired buffer reservoir. When the syringe pump is turned on, buffer is pulled into the side channels from the reservoir at a flow rate of 3 μl/min to enable buffer exchange within the sample chamber via passive diffusion through the semipermeable membranes.The sample was pipetted into the central channel and the side channels were filled with the original polymerization buffer consisting of PEM-100, 2 mM ATP and 2 mM GTP (only for actin-microtubule networks). The flanking side channels were then connected to capillary tubing  at both ends. The sides of the device were sealed with epoxy to completely enclose the sample within the central chamber and the capillary tubing in the side channels. The capillary tubes were connected to separate Tygon tubing  before sealing all tubing with epoxy. The Tygon tubing on the inlet side of the microfluidic device was prefilled with the original polymerization buffer. We included 2 2, 2 mM ATP and 2 mM GTP (only for actin-microtubule networks). This buffer did not contain fluorescein. We pumped this buffer through for 90 min before switching back to the fluorescein-polymerization buffer and pumping for 50 min.The microfluidic experiment took place over the course of 3 h. In the first 10 min, the pump remained off in order to equilibrate the system. We then pumped in the fluorescein-polymerization buffer for 30 min to achieve simultaneous flow at an equal flow rate. After 30 min of pumping the fluorescein-polymerization buffer we switched the reservoir to the buffer containing PEM-100, 20 mM MgCl2+ concentration. The microscope is outfitted with 488 nm and 561 nm lasers and simultaneously records separate images for each laser channel. As such, we are able to separately visualize the cytoskeleton network in the sample chamber (568 nm laser channel) and the intensity of fluorescein dye in the side channels and sample chamber (488 nm laser channel). Time-series of 512 × 512 images (6.215 μm/pixel) in each channel were recorded for 3 h with each image taken every minute. Time-series from the network channel and fluorescein channel were analyzed separately using custom written Matlab code to determine network width and corresponding Mg2+ concentration.A Nikon A1R laser scanning confocal microscope with a 4× objective and QImaging QICAM CCD camera was used to collect time-series of labeled cytoskeletal networks and buffer channels. The low magnification objective enabled us to visualize the entire network to determine bulk morphological changes triggered by buffer exchange. It also allowed us to view the entire device in a single field of view to characterize the rate of buffer exchange and to correlate the network activity with Mg2+ concentration . This phase serves as a control to ensure that we achieve simultaneous flow with equal flow rate through both side channels. After 30 min we change the buffer to include 20 mM Mg2+ (Phase II). After 90 min of 20 mM Mg2+ buffer flow (t = 120 min), we reintroduce the original 2 mM Mg2+ polymerization buffer (Phase III).Once networks are formed in microfluidic chambers, each 170 min experiment proceeds as follows  both networks continue to contract even as the Mg2+ concentration is lowered back to the original concentration; and 3) the onset of contraction is delayed for actin-microtubule networks in comparison to actin networks.2+-driven contraction. The regions with larger standard deviation, at the beginning and end of the experiments, represent variations in the initial and final widths of the different samples, likely arising from small differences in the semipermeable membranes of each microfluidic chamber , and the filaments repel each other due to the cloud of positive ions surrounding them (and repelling each other). Squeezing the two filaments together would lower the entropy of the “trapped” ions and is thus avoided. This effect is similar to an osmotic pressure difference that drives water between the filaments to try to balance the concentration of cations near and far from filament surfaces [We can also understand this contraction process by considering the entropic cost of trapping the Mgilaments , 39. At surfaces , 40, 41.2+ concentration by reintroducing the original 2 mM Mg2+ polymerization buffer (Phase III). While Phase II contraction is largely driven by the free energy minimization of the cations, as we describe above, we interpret the Phase III contraction as arising from the free energy minimization of the filamentous network. Namely, as filaments are pulled toward each other and linked together by cation crossbridges, the initial configuration is not the most entropically favorable in terms of their configuration. There are likely mechanical stresses on the filaments that were pulled together by the increasing ion concentration. Thus, the filaments will rearrange and reorient to relieve this stress and increase their configurational entropy, even when the cation concentration is reduced. Because the relaxation timescales of entangled actin networks such as these can be as long as minutes to hours, [2+ concentration are quite strong. Further, there is no obvious driving force, analogous to the entropic force that drives the filaments together, that would force the filaments apart. Finally, there are theoretical predictions that suggest that thermal Casimir forces may play a role in electrolyte solutions confined by surfaces (which in this case are the filaments) and in biopolymer networks [The more counterintuitive and surprising result is that contraction continues when we lower the Mgo hours, , 42–44 tnetworks , 46. Thinetworks . This ef2+ cations (~20 mM Mg2+) for the onset of contraction than actin networks, and the most pronounced contraction occurs only after the ion concentration starts to drop  and/or a stronger entropic force (dictated by the cation concentration difference between the bulk and in between filaments) would be required for neighboring filaments to be driven together by the osmotic-like depletion of water between the filaments. As such, a higher Mg2+ concentration and longer time would be needed to induce contraction, just as we see in 2+ concentration and more time for thermal fluctuations to move neighboring filaments close enough to each other to allow for depletion-driven contraction, once the filaments reach this point they can move together more rapidly because there are fewer steric constraints (entanglements) restricting their motion. Namely, the empty voids in the mesh that filaments can freely move through are larger than in actin networks. While actin filaments are more flexible and can thus more readily respond to depletion forces, their smaller network mesh size  prevents the filaments from moving as quickly together as in the actin-microtubule composites.The longer relaxation times could explain the delay in contraction triggered by increasing Mgμm/min) to slower contraction of ~3 μm/min (2+ concentration) that rapid contraction of actin-microtubule networks would still occur at the same experimental time, governed by the intrinsic relaxation timescale of the network. However, we expect that the shift to slower contraction that occurs in Phase III would occur proportionally later, as it is determined by a shift to configurational entropy maximization that can only occur after the network has been in the presence of low ion concentration for a long enough time. In future work we will more fully explore these hypotheses by varying the times over which the networks are exposed to high and low cation concentrations as well as the maximum Phase II cation concentration.Finally, the shift that actin-microtubule composites exhibit at ~140 min (in Phase III) from relatively fast contraction (~13 3 μm/min  may indi2+ due to its importance in physiological processes such as actin and microtubule polymerization and myosin-driven actin contraction [2+, would produce similar results for polyelectrolytes with similar charge densities as actin and microtubules [2+ promotes actin polymerization and stabilizes filamentous actin, Ca2+ can destabilize actin filaments and promote depolymerization, which could negatively impact contractile behavior [We chose to focus our study on Mgtraction , 33, 48.otubules . Howeverotubules , 38. Fur2+-triggered contraction on this ratio. We expect our results to smoothly vary between the two cases we present here as we vary this ratio. While the networks we have studied do not have any crosslinking proteins present, we plan to incorporate actin and microtubule crosslinkers and determine their impact on the results. Based on our previous microrheology measurements on crosslinked actin-microtubule composites [We have previously shown that actin-microtubule composites exhibit increasing stiffness, mesh size and heterogeneity as the ratio of microtubules to actin is increased . In futumposites , 50, we 2+ concentration. The contraction dynamics are highly dependent on the network type, with actin-microtubule networks exhibiting a substantially delayed response relative to actin networks. We also show that both networks continue to contract during subsequent lowering of Mg2+ concentration, which we attribute to slow network rearrangement to maximize the configurational entropy of the filaments. Our results demonstrate unexpected ways in which the cytoskeleton can dynamically morph and contract in response to environmental stimuli, and how the varying mechanical and structural properties of actin and microtubules can tune the characteristics of this dynamic response.We have presented an experimental approach that combines diffusion-controlled microfluidics with two-color fluorescence confocal microscopy to measure bulk morphological changes to cytoskeletal networks triggered by increasing and decreasing concentrations of magnesium ions. We show that both actin networks as well as actin-microtubule networks undergo bulk contraction triggered by increasing Mg


This cohort study investigates differences in cognitive functioning by sex and genetic variant status among Colombian children the autosomal dominant Alzheimer disease presenilin 1 E280A variant. Do children from the Colombian kindred with autosomal dominant Alzheimer disease (ADAD) differ in general cognitive functioning based on sex or genetic status?In this cohort study of 1354 Colombian children (including 265 children with the presenilin 1 E280A variant), those with and without the variant did not differ on cognitive abilities. However, boys with the variant exhibited statistically significantly worse working memory performance compared with girls with the variant, as well as boys and girls without the variant.This study found that boys with the ADAD variant had increased risk of worse cognitive performance compared with girls with and without the variant, as well as boys without the variant. PSEN1) E280A variant had early life plasma biomarker findings consistent with amyloid β overproduction. However, the cognitive functioning of children with this variant has not been characterized vs those without the variant.We previously reported that children with the autosomal dominant Alzheimer disease (ADAD) presenilin 1  and sex.To test whether cognitive functioning of children with and without the Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV) to measure general cognitive functioning. Data were analyzed from July through November 2020.This cohort study was conducted among 1354 children (including 265 children with the variant) aged 6 to 16 years recruited from the Alzheimer Prevention Initiative Colombia Registry. Participants from the city of Medellín and surrounding suburban areas traveled to the University of Antioquia to undergo all procedures. Participants were administered a Spanish version of the WISC-IV cognitive performance by genetic status, sex, and the interaction of genetic status with sex. Urbanity, socioeconomic status, and education were entered as covariates.Univariate general linear models were used to characterize differences on WISC-IV indices. Irrespective of genetic status, boys had statistically significantly decreased mean scores on indices for working memory , perceptual reasoning , and verbal comprehension  compared with girls. In the interaction between sex and genetic status, boys with the variant had worse mean working memory index performance  than girls with the variant , as well as boys  and girls  without the variant.Among 1354 children with ADAD , there were 265 children with the variant (19.6%) and 1089 children without the variant (80.4%). Children with and without the variant did not differ by demographic variables or performance on PSEN1 variant had decreased working memory abilities compared with girls with the variant and boys and girls without the variant, suggesting a sex-specific genetic risk in early life cognitive performance among individuals with the PSEN1 variant. This increased risk of future cognitive difficulties among boys with the variant may have important downstream implications for learning and academic achievement and could be associated with sex differences seen in adulthood on episodic memory measures.This study found that boys with the  To our knowledge, however, no study has investigated the cognitive performance of children with ADAD variants compared with those without variants, nor the possible sex differences in cognitive performance in childhood in this population.Individuals with autosomal dominant Alzheimer Disease (ADAD) variants develop early onset Alzheimer disease (AD) with nearly 100% penetrance, providing a unique opportunity to characterize the earliest biological and cognitive changes associated with the disease.Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV),8 to assess cognitive functioning among children with the PSEN1 E280A variant and age-matched family members without the variant from the Colombian ADAD kindred. The literature on sex differences in cognition among children without developmental disorders is variable, with boys and girls showing distinct patterns of working memory strengths.9 However, previous research on the cognitive functioning of adult members from this PSEN1 kindred10 and findings from the literature about cognitive performance in children with the apolipoprotein ε4 (APOE ε4) variant,13 a known genetic risk factor associated with the more common sporadic form of late-onset AD, suggest that there may be differences in cognitive performance among children from this kindred. We hypothesized that children with an ADAD variant would perform worse on tests of verbal intellectual abilities compared with children from the same family without a variant and that boys with a variant would perform worse than girls with a variant across all WISC-IV indices.4In this study, we used a neuropsychological measure, a Spanish version of the STROBE) reporting guideline.This cohort study was conducted under guidelines approved by local institutional review boards. Ethics approval was obtained from the University of Antioquia Ethics Committee in Colombia. Parents or guardians of the participants provided written informed consent, and children assented before enrollment in the study and completion of the procedures. All data were acquired by investigators who were masked to the participants’ genetic status. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  for MCI and 49 years  for dementia.2 Given that PSEN1 E280A is an autosomal dominant variant, all children with the variant were heterozygous for PSEN1 E280A and had a biological parent who also had the variant.1 A blood sample was taken to confirm genetic status.A total of 1499 children were recruited from the Alzheimer Prevention Initiative Colombia Registry, which currently includes more than 6000 living members of the All children in the recruited sample lived within a 100-mile radius of Medellín, Colombia. Participants were asked to identity their sex assigned at birth, using a binary definition of sex . Potential participants were screened and excluded in advance for the presence of neurological disorders  and chronic psychiatric disorders .WISC-IV, a measure of cognitive functioning in children.8 Of 1499 children in the database, 145 participants with a Full Scale Intelligence Quotient (FSIQ) score lower than 70 on the WISC-IV (including 16 children with the variant) were excluded from the study, given that an FSIQ less than 70 is suggestive of an intellectual disability.8 We included 1354 children in the analysis, including 265 individuals (19.6%) with the PSEN1 E280A variant.Participants were administered a Spanish version of the PSEN1 E280A variant. There were 457 participants  who had a biological parent with the variant, whereas 897 children without the variant had an extended nonparent relative with the variant . The 192 children without the variant who had a biological parent with the variant were considered in a post hoc subsample analysis. This was done to determine if performance comparisons on the WISC-IV differed when comparing children with the variant against individuals without the variant who had a parent with the variant vs when comparing against the entire larger sample of children without the variant.Participants in this study were aged 6 to 16 years  and belonged to the same extended families who descended from a shared ancestor with the WISC-IV is a neuropsychological assessment commonly used to measure cognitive abilities in children aged 6 to 16 years.8 The WISC-IV has 15 subtests, although the first 10 subtests are considered the core subtests and yield an FSIQ and the scores on the following 4 indices: verbal comprehension index (VCI), perceptual reasoning index (PRI), working memory index (WMI), and processing speed index (PSI).8 The VCI consists of 3 subtests: vocabulary, similarities, and comprehension. The VCI is used to measure a child’s language-based intellectual abilities. The PRI measures nonverbal fluid reasoning and is assessed with the following subtests: matrix reasoning, picture concepts, and block design. The WMI evaluates attention and concentration and consists of 2 subtests: digit span and letter-number sequencing. The PSI measures the speed of mental processing14 and contains 2 timed subtests: coding and symbol search. Scores on the WISC-IV in this study were normed relative to a sample of Spanish-speaking children and adolescents from Mexico, given that the WISC-IV had not been normed in a Colombian population at the time of this report. This Spanish WISC-IV version is referred to as the Escala Wechsler de Inteligencia para Niños-IV.8 Raw WISC-IV data from the subtests were normed using the participant’s age and transformed into scaled scores, which have a mean (SD) of 10 (3). The WISC-IV technical manual8 explains in detail how the subtests are converted into the 4 index scores, which have a mean (SD) of 100 (15).Data were acquired at the University of Antioquia in Medellín, Colombia. Cognitive tests were administered in Spanish by psychologists trained in neuropsychological assessment (DR and NL). The t tests. We used χ2 tests to examine potential differences between children with and without the variant in sex ratio, current living zone , and socioeconomic status .Potential differences in age and education between children with and without the variant (as well as between boys and girls with the variant) were examined using Welch 2 independent sample WISC-IV index score), we examined if there were differences by genetic status (model 1), sex (model 2), and the interaction of genetic status with sex (model 3). Education, SES, and urbanity were entered as covariates in all models, and Bonferroni corrections were applied in all models. Violin plots were produced in R statistical software version 4.0.0  using ggplot to provide a visualization of differences in WISC-IV index scores by sex and genetic status.Univariate general linear models were used to characterize the association between genetic status and performance on the VCI, WMI, PRI, and PSI; these models were run on SPSS statistical software version 26 (IBM). For each dependent variable  and 1089 individuals without the variant (80.4%). Children with and without the variant did not differ by mean (SD) age , mean (SD) years of education , urban living status , SES , or proportion by sex in each group  , there were 265 individuals with the  P =.50) .WISC-IV indices differed between the groups when examining only participants who had at least 1 immediate parent with the variant. This subsample differed on urbanity, with more children with the variant living in urban areas than children without the variant, but not on other demographic variables (eTable 1 in the P < .001) and subsample  .For the WISC-IV indices by PSEN1 E280A status after controlling for sex, urbanity, SES, and education in the full sample. For example, the mean scores for children with the variant vs those without the variant were 90.09  vs 89.41  (P = .40) for the VCI and 91.26  vs 92.00  (P = .32) for the WMI (P = .19) for the VCI and 91.20  vs 92.81  (P = .10) for the WMI , PRI , and VCI  (eTable 4 in the P < .001) and PRI  , boys without the variant , and girls without the variant   and girls without the variant  but not boys without the variant  than girls with the variant  . The FigPSEN1 E280A ADAD in childhood. The PSEN1 E280A variant is associated with the development of the AD clinical syndrome in midlife with virtually 100% certainty.1 Compared with children without this variant, children with the variant from this kindred (aged 6-16 years) were previously found to have increased gray matter volume in temporal and parietal brain regions, as well as decreases in deactivation in the parietal regions during an episodic memory task.3 In adults with the variant from this cohort, sex differences in verbal memory performances favored women,4 warranting the study of sex differences in cognition in childhood. Our study is novel, given that it is the first report, to our knowledge, on cognitive functioning among children with an ADAD variant. The homogeneity of the clinical course of this single-variant PSEN1 E280A ADAD cohort allowed us to characterize the cognitive performance of children with the variant decades before the median age of onset of MCI for this variant (EYO −38 to −28 years for this sample).2In this cohort study, we used cognitive data from a large sample to examine the cognitive functioning of WISC-IV indices by PSEN1 E280A variant status after controlling for urbanity, SES, and education. We found no differences in the entire sample, which included children without the variant who had an extended family member  with the ADAD variant, as well as in a subsample that included only participants who had a biological parent who also had the PSEN1 E280A variant. When controlling for the same covariates, irrespective of genetic status, boys performed worse than girls in the full sample on the WMI, PRI, and VCI; in the subsample, boys performed worse than girls on the WMI and PRI. Additionally, we found a significant interaction between sex and genetic status in the full sample, wherein boys with the variant had worse WMI performance than girls with the variant, boys without the variant, and girls without the variant; in the subsample, boys with the variant performed worse on this index compared with girls with and without the variant. This partially supports our hypothesis that boys with the variant would exhibit weaker cognitive performance than girls with and without the variant, although we had anticipated to see significantly worse performances among boys with the variant compared with girls across all WISC-IV indices. The magnitude of the differences in WMI scores between boys with the variant and other groups was statistically significant but small from a clinical perspective. The largest difference of the adjusted means on this index between boys and girls corresponded to an approximately 5-point standard score difference .Contrary to our hypotheses, we found no differences in cognitive performance on the 4 from our group that found that women with the variant had better verbal memory performance than men with the variant. In prototypical aging and sporadic AD, healthy older women have been found to have higher scores on verbal episodic memory tests compared with healthy older men.5 The literature from children without developmental impairments suggests that there may be differential brain networks recruited during working memory tasks by boys and girls.15 Additionally, the type of working memory task itself may be associated with sex differences seen in children, with boys who do not have developmental impairments performing better on tests of mental arithmetic and girls without developmental impairments performing better on tests of digit span.9 In this study, working memory performance on the WISC-IV was evaluated with 2 auditory tests ,8 and we found that girls, irrespective of genetic status, had better WMI scores, aligning with prior meta-analytic research.9 Boys with the variant, however, appeared to be driving the at-large sex difference in this study, given that there were no significant differences in WISC-IV performance when examining boys and girls without the variant.Although we found a clinically small difference, our findings are consistent with a 2020 study16 It is possible that early life differences in working memory are associated with sex differences seen in adulthood on episodic memory measures in this cohort.4 Auditory working memory was also found to be an important factor associated with reading comprehension,18 and worse working memory performance in childhood was associated with an increased likelihood of dropping out of secondary school.19 Decreased working memory performance in boys with variants needs to be further studied, as this increased risk could lead to downstream learning difficulties that may impact early life educational attainment. Future investigations should explore how this ADAD variant is specifically associated with acquiring academic skills  and educational attainment , as well as the association of various sociodemographic factors with cognition among children with an ADAD variant. In this study, children with and without a variant did not differ on years of educational attainment, SES, or urbanity, but irrespective of genetic status, boys in this study attained approximately half a year less of formal education than their age-matched female peers. Enhancing early life educational experiences and quality has been suggested as a modifiable risk factor associated with dementia due to sporadic causes20 and is an important avenue of primary prevention research in ADAD. One study21 found that parental educational attainment can be associated with educational outcomes for children. Therefore, we need to explore possible interactions between parent education and cognitive and academic performance in individuals with and without the PSEN1 variant, as well as the association of SES, educational quality, and growing up in urban vs rural environments with cognitive outcomes.23There are potential cognitive and academic implications of worse working memory performances compared with their peers among boys with ADAD variants. In children without developmental impairments, auditory working memory performance has been associated with verbal episodic memory performance.PSEN1 E280A variant. The registry for this study, however, enrolls individuals without the variant who can trace their lineage to the extended PSEN1 E280A cohort but who do not necessarily have a parent with the variant. This likely contributed, at least in part, to bias in the sample. Another limitation of this study is that the environmental stress associated with caring for a parent with cognitive decline due to ADAD may not be an experience shared equally by children with and without the variant. Some children without the variant in the large sample did not have a biological parent with the variant but did have an extended relative with the variant. Owing to the bias of the large sample and the possible outcomes associated with caring for a parent who is progressing to the AD clinical syndrome, we chose to also run our analyses in a subsample consisting of children with and without the variant who also had at least 1 parent with the variant, and this analysis yielded similar results to those of the entire sample.This study has several limitations, including uncertainty regarding generalizability to children with other ADAD variants. The risk of bias in the unbalanced number of children with and without the variant in the sample must also be noted, given that theoretically the ratio should be closer to 1:1 given the autosomal dominant nature of the PSEN1 E280A variant. Some previous work has suggested that the early onset of menarche may be negatively associated with intelligence in the general population.24 Longitudinal studies are needed to identify the mechanism associated with these cognitive differences between boys and girls who have the variant and to determine the approximate age at which these differences emerge. Additionally, although participants were compared using the same WISC-IV norms from Mexico, WISC-IV norms for Colombia need to be developed given that children without the variant had lower index level performances than the expected mean value of 100 standard score seen in a reference sample.Another limitation of this study is its cross-sectional nature, whereas childhood is an extended period of physical and cognitive development. As the onset of adolescence is variable, our future work will consider the value of collecting data on the age of puberty onset and examine its association with sex differences in cognitive performance among individuals with the PSEN1 E280A variant did not differ from children without the variant in performance across a wide set of cognitive domains. Boys with the variant, however, had decreased working memory performance compared with girls with and without the variant. These results may provide a foundation to investigate the earliest cognitive signs of ADAD and underscore the need for primary prevention research aimed at enhancing early life cognitive and educational experiences of ADAD cohort members.We characterized the cognitive performance of children with an ADAD variant compared with children without the variant from the same kindred cohort. Overall, children with the