The continuing evolution of SARS-CoV-2 variants not only causes a long-term global health concerns but also encounters the vaccine/drug effectiveness. The degree of virus infectivity and its clinical outcomes often depend on various biological parameters  of an individual along with multiple environmental factors . Thus, despite the extensive search for and use of several vaccine/drug candidates, the combinative influence of these various extrinsic and intrinsic risk factors involved in the SARS-CoV-2 virus infectivity has yet to be explored. Previous studies have reported that environment temperature is negatively associated with virus infectivity for SARS-CoV-2. This study elaborates on our previous findings, investigating the link between environmental temperature and other metabolic parameters, such as average total cholesterol and obesity, with the increase in COVID-19 cases. Statistical analysis conducted on a per country basis not only supports the existence of a significant negative correlation between environmental temperature and SARS-CoV-2 infections but also found a strong positive correlation between COVID-19 cases and these metabolic parameters. In addition, a multiphase growth curve model (GCM) was built to predict the contribution of these covariates in SARS-CoV-2 infectivity. These findings, for first time, support the idea that there might be a combinatorial impact of environmental temperature, average total cholesterol, and obesity in the inflation of the SARS-CoV-2 infectivity. This novel virus spread rapidly across the globe and has been declared as a pandemic by the World Health Organization in March, 2020. As of July, 2021 (data considered in our study), ~ 200 million cases have been confirmed, with over 4 million deaths worldwide. In order to curb the spread of this novel virus, multiple comprehensive measures will have to be devised and thus, it compelled us to elucidate additional factors which might affect the transmission of this virus. Indeed, various nanotechnology based approaches as preventive measures and detection of SARS-CoV-2 have been suggested to combat COVID-19 pandemic3. Previous studies have demonstrated that environmental temperature affects the spread of other respiratory viruses including rhinovirus, influenza A and B, and other coronaviruses4. These data suggested that environmental temperature might also play a role in the transmission of SARS-CoV-2. We recently published an article demonstrating a strong negative correlation between environmental temperature and SARS-CoV-2 infection of a country, worldwide5. This conclusion was made at the beginning of COVID-19 pandemic  when the data was too much dynamic and fluid. Thus, to further validate our previous findings, we have analysed the link between monthly average environmental temperature (MAET) and COVID-19 cases of a country by using data from March, 2020 to July, 2021.An international public health crisis emanated from Wuhan (Hubei province), China with the emergence of a new coronavirus strain later identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)7. Additionally, a study conducted in Wuhan observed that a number of obesity-associated conditions, including diabetes, hypertension, and coronary artery disease, were common comorbidities in hospitalised patients8. Hence, obesity and BMI were the other chosen factors for this study. Statistical analysis identified a strong negative correlation between MAET and the number of COVID-19 cases in a country. Additionally, we identified a positive correlation between the previously mentioned metabolic parameters and the number of COVID-19 cases in the country. This data implicates additional variables, particularly environmental and metabolic factors, collaborates to support the rapid spread of the SARS-CoV-2 infection.Because of the inconsistent results between studies on the role of temperature in SARS-CoV-2 infectivity, we looked into other factors which might also be contributing to the spread of the virus. In this present study, we probed the correlation between average total cholesterol, obesity, and BMI all associated with metabolic processes with confirmed SARS-CoV-2 cases. Apart from being the predominant component of lipid microdomains, cholesterol has also been investigated in the efficient binding of the SARS-CoV virus to the ACE-2 receptor, even before the current COVID-19 pandemicTo investigate whether the geographical location has any impact on SARS-CoV-2 infection, we first identified the top 75 countries for COVID-19 infection  with COVID-19 cases per million are shown in Table 5. Thus, to examine the interdependence of cholesterol levels and total COVID-19 cases, we conducted a univariate analysis between average total cholesterol and the month-wise COVID-19 cases per million by both Pearson and Spearman’s correlation methods. A strong positive correlation was observed between cholesterol and total COVID-19 cases per million of a country  approach to capture the growth of COVID-19 cases for considered time periods. Furthermore, the multiphase GCM elected to study the intra-individual and inter-individual changes in trajectories for considered countries. In the absence and presence of various covariates, we assigned the ranks to different fitted multiphase models as per their performances. It was observed that model-8 had the lowest AIC and BIC values and large enough TLI value Table , and so ) unit change in slope , 1.0465  and −1.1209  unit changes in slopes  illustrate an indirect effect on the outcome variable through latent variables From Table , it can 15. Reports in the literature suggest that temperature might be an important factor accounting for the transmission of other coronaviruses, like SARS-CoV16 and MERS-CoV17 because of (i) increased virus half-life at lower temperatures, (ii) greater stability in nasal passages when the epithelial surface is cold, and (iii) greater stability in lower humidity as compared to intermediate humidity17. Considering these facts, we conducted a study to establish a link between environmental temperature and COVID-19 cases. We observed that countries with more COVID-19 cases were mostly located north of the latitude of Wuhan, China where the pandemic started in December, 2019. Thus, we performed a detailed country-wise statistical analysis which established a significant negative correlation between COVID-19 cases and MAET of a country5. However, this initial finding was limited to the COVID-19 data from March and April, 2020. While there are a few studies whose findings are broadly congruous with our own regarding temperature and SARS-CoV-2 caseload18, others have found no correlation between temperature and infection rate13. In this study, we have validated the relationship between MAET and COVID -19 cases per million from March, 2020 to July, 2021 of a country. Univariate analysis by both Pearson and Spearman’s methods indicated a negative correlation between temperature and COVID-19 cases  E4 genotype have an increased risk of severe COVID‐19 infection. Though increased cholesterol levels promote ACE2 and furin trafficking inside host cells21, cholesterol plasma levels are found to be decreased in patients post-infection. In brief, high cholesterol present in host cell membrane, virus particles, and human blood may augment the virus entry processing in the host cells23. Our data depicted that geographic locations above 23.5oN latitude and towards the poles had a higher prevalence of high average total cholesterol levels, often overlapping the areas with the highest COVID-19 cases  compared to patients with normal weight, even at a younger age26. A cohort study showed that obesity is an important factor in disease severity of SARS‐CoV‐2, having the highest impact on patients with a BMI ≥ 3527. Moreover, in vitro experiments have shown that ACE2 and TMPRSS2, two essential entry components for SARS-CoV-2 infection, are highly upregulated in the lung epithelial cells of obese patients28. Therefore, we explored the relationship between BMI and obesity and COVID-19 caseload. Upon identifying the geographical locations of countries with a higher prevalence of BMI and obesity, significant overlap was observed with those with high numbers of COVID-19 cases  in winter months may further potentiate virus infectivity since the virus may persist for longer times in a relatively lower humid atmosphere47.However, other intrinsic factors like hypertension, cardiovascular diseases, renal diseases, and cancer, as well as extrinsic factors like relative humidity and indoor temperature, have not been included in these analyses. Specifically, indoor temperature may contribute to virus infectivity. Actually, cold temperatures and low relative humidity (RH) adversely increase the half-lives of the virusIn brief as a conclusive remark, such multiphase growth curve models may be used to depict the contribution of various covariates with COVID-19 cases. Moreover, the individuals with metabolic disorders such as high cholesterol and obesity may have additional risk for this virus infectivity especially in winter months or while living in colder environments. Thus, this study further recommends that a nationwide policy is to be framed in order to combat COVID-19 pandemic and its clinical outcomes for taking care of vulnerable individuals with such metabolic diseases of a cold country. However, further study is required to know whether the infectivity rate of mutant variants of SARS-CoV-2 depends on these factors.48. We considered the total cases per million as the variable of interest for the COVID-19 case dataset for each month. The environmental temperature data for different countries was collected from climatestotravel.com, as described in our recent publication5. The latest data for cholesterol and obesity were acquired from the WHO website and BMI from ghdx.healthdata.org. These datasets are publically available. All data collection was performed in accordance with the relevant guidelines and regulations.COVID-19 case data was collected for every month from March, 2020 to July, 2021 from2021 from ourworldindata.org/coronavirus9. This online server is freely available. The top 75 countries for SARS-CoV-2 infections have been marked in the world map. The top 75 countries for highest average total cholesterol, BMI, and obesity levels in their population have also been marked on the world map.The geographic location of different countries has been marked in the world map with the help of world map marker (worldmapmaker.com), explained previously 51. Statistical analysis was considered to be significant for Univariate analysis was performed using the Spearman and Pearson methods to determine whether to accept the null hypothesis between the two variables, as described previouslyFor this work, we applied the multiphase latent GCM to study the variable of interest (cases per million) over the defined time period by incorporating time invariant covariates (TICs) and time varying covariates (TVCs). Here, total cases per million over the months was taken as variable of interest while average total cholesterol and BMI were considered as TICs and temperature as TVC. Further, TICs and TVC were examined to see whether the changes in cases per million over months may be explained by the presence of these predictors. R software was used for this analysis. For the modelling purposes, “lavaan” package in the CRAN directory was utilized.In model fitting, a latent GCM approach was used for COVID-19 cases per million in the presence of different associated covariates: temperature, cholesterol and BMI. The latent GCM captures the growth trajectory of the variable of interest over the defined time period. TICs and TVCs were considered to see whether the changes in cases per million over months may be explained by the presence of these predictors. Here, cholesterol and BMI were treated as TICs, and temperature as TVC. The general structure for the GCM in presence of TIC and TVC can be given as below:52. As observed from the trajectory plot, the COVID-19 case growth rates were different in the considered months. Therefore, it was more suitable to fit multiple splines than just one line for the defined time slot to capture the COVID-19 cases for different countries. Based on this proposition, we divided the total COVID-19 cases per million over the months into four phases for modelling purposes. Four vectors The multiphase GCM is based on two or more regression lines which allows modelling of multiple processes that are responsible for the intra-individual changes over the time periodsThe parameter Some well-known fitting criteria such as AIC, BIC and TLI were used for choosing the best model. The lower values of AIC and BIC with sufficiently large value of TLI directs to choose a better model. So, a model having lowest values of AIC and BIC with large enough TLI was preferred to others.


The onset of the COVID-19 pandemic necessitated the rapid transition of many types of substance use disorder (SUD) treatments to telehealth formats, despite limited information about what makes treatment effective in this novel format.This study aims to examine the feasibility and effectiveness of virtual intensive outpatient programming (IOP) treatment for SUD in the context of a global pandemic, while considering the unique challenges posed to data collection during an unprecedented public health crisis.The study is based on a longitudinal study with a baseline sample of 3642 patients who enrolled in intensive outpatient addiction treatment  from January 2020 to March 2021 at a large substance use treatment center in the United States. The analytical sample consisted of patients who completed the 3-month postdischarge outcome survey as part of routine outcome monitoring .χ22=0.4, P=.81), overall quality of life , financial well-being , psychological well-being , and confidence in one’s ability to stay sober . Individuals in hybrid programming were more likely to report a higher level of general health than those in virtual IOP .No significant differences were detected by delivery format in continuous abstinence (Virtual outpatient care for the treatment of SUD is a feasible alternative to in-person-only programming, leading to similar self-reported outcomes at 3 months postdischarge. Given the many obstacles presented throughout data collection during a pandemic, further research is needed to better understand under what conditions telehealth is an acceptable alternative to in-person care. Substance use disorder (SUD) is a chronic relapsing disease associated with numerous psychosocial harms and health sequalae. Addiction was a leading global cause of disability and death prior to the COVID-19 pandemic -4. IsolaPrior to the COVID-19 pandemic, virtual services showed promise but were slow to develop. Early applications have shown promise as a means of preventing premature dropout from SUD treatment . The panThe onset of the pandemic became a catalyst for addiction treatment programs to quickly pivot to provision of services through telehealth formats despite limited data to guide their delivery. Change was facilitated by paradigm shifts in federal, state, and local policies and in organizational and provider practices ,17. AlthThere is still a significant need for research related to the application and assessment of telehealth for SUD. Unfortunately, best practices for patient outcome collection for SUD treatment in mixed settings have yet to be established. In this paper, we describe how the COVID-19 pandemic presented a novel opportunity to bridge the gap and assess the effectiveness of a virtual intensive outpatient programming (IOP) for substance use treatment through the examination of short-term postprogram outcomes of adults who received IOP services through different delivery formats at the largest SUD treatment provider in the United States.The Hazelden Betty Ford Foundation (HBFF) is the largest national provider of addiction services in the United States. The HBFF utilizes evidence-based practices through a multidisciplinary and integrated approach to addiction treatment across varying levels of care. In 2019, the HBFF piloted a single virtual intensive outpatient group with planned expansion of virtual services in 2020, as informed by routine outcome monitoring (ROM) data. The HBFF has an established infrastructure and process for collection of ROM data that has been used and refined since 1974. ROM data provide an understanding of real-world conditions, offering applied generalizability to community-based treatment settings where the majority of care is provided ,22. ThesThis study presents 3-month findings  from a 12-month longitudinal assessment of patients, 18 years and older, who were discharged from IOP between January 2020 and March 2021 (N=3642). Patients were separated by 3 distinct treatment delivery settings in response to pandemic changes: (1) in-person care only ; (2) hybrid, in-person, and virtual care ; and (3) virtual care only .The study was reviewed and approved by Emory University’s Institutional Review Board (STUDY00001822) and was determined to have met the human research exemption since all data were collected within the context of the HBFF’s standard ROM practices.Trained research data collection specialists (RDCS) utilized a systematic and manualized process for data collection. Web-based surveys were automatically assigned to IOP patients at 1, 3, 6, 9, and 12 months postdischarge, with survey completion windows open for 30 days. Survey links were emailed to patients, with reminder prompts every 3 days for up to 2 weeks. Patients were contacted by an RDCS every 4-7 days to complete the survey over the phone or to encourage patients to complete it online if not initially completed. Patients were still prompted to provide responses if admitted to a different level of care.In response to a notable decline in response rates at the beginning of the pandemic, RDCS spent over 1200 hours attempting to contact patients to complete the 3-month survey throughout the course of the virtual IOP study. The timing of data collection was also impacted. Virtual IOP began in March 2020, while data collection began in May 2020, largely due to reallocation of resources to facilitate the transition of direct care to virtual services. To retrospectively capture an in-person IOP comparison group, all patients discharged from an in-person IOP on or after January 1, 2020, were opted into receiving IOP outcome surveys. However, due to the 30-day survey windows, the majority of in-person and hybrid patients were excluded from completing the baseline survey at admission and the 1-month postdischarge survey, impacting response rates for those time points .Demographic information was collected from patient electronic medical records. The majority of the full sample of patients were White  and male , with a mean age of 39.1 (SD 13.5) years.abstinent since discharge vs relapsed) during the follow-up period was assessed using a question from a modified Form 90 Alcohol Questionnaire (Form 90-AQ)  vs 1014/2503 [40.5%]) and to get discharged against staff/medical advice (107/1060 [10.1%] vs 531/2503 [21.2%]). Completers also showed a longer length of stay in IOP care (mean 58.10 [SD 36.16] days) than noncompleters (mean 49.57 [SD 38.73] days). There was no difference between those who stepped down into IOP from a higher level of programming within the HBFF .overall perceived quality of life or in the total number of poor physical and mental health days at 3-month follow-up. Finally, there were no differences detected between IOP setting and the individuals’ confidence in their ability to stay sober, financial well-being, or psychological well-being.A few differences emerged between IOP settings across multiple domains of functioning . There wgeneral health, where those in the hybrid group (mean 4.08 [SD 0.75]) were more likely than those in the virtual group (mean 3.89 [SD 0.83]) to report a higher level of general health.The only significant difference by IOP setting that emerged was in the overall quality of one’s This study is the first of its kind to assess telehealth for SUD in the IOP setting in a large cohort of patients (N=1000+). No meaningful differences in outcome measures were identified between delivery settings at 3-month follow-up, with individuals reporting similar levels of continuous abstinence, quality of life, and social/emotional well-being. Our findings in regard to continuous abstinence were consistent with previous studies following patients at 3-6 months postdischarge from IOP  . These rHistorically, peer-based connections and the therapeutic milieu have been integral parts of addiction treatment. Concern has been expressed by the addiction treatment community regarding the shift to virtual services and its impact on group engagement and patient-centered outcomes . These pOur findings aid in establishing a platform for future evaluation of data collection processes that inform the effective development of standardized protocols for routine outcomes data practices, including frequency of contact, method of outreach, and training of staff. Standardized protocols must consider the context for accurate interpretation of collected data. For example, differences in response rates emerged in this study based on the timing of data collection in relation to the global pandemic and due to unanticipated staff burden and should be interpreted in this context. At 3-month follow-up, response rates were lowest for those in in-person IOP . These rates are likely attributable to these being completed by patients between May and August 2020, timing that coincided with major city- and statewide lockdowns and great uncertainty about the unknowns presented by the pandemic. Furthermore, because of the large opt-in of the in-person cohort at the beginning of data collection (May 2020), many participants at 3-month follow-up were given access to the survey well into the 30-day response window, reducing the likelihood that they would have adequate time to complete it prior to survey close. In contrast, response rates for those in virtual IOP  were most impacted by a higher-than-projected admission rate of individuals into IOP throughout the study period, resulting in a larger sample size than originally anticipated. As a result, there were proportionally lower staffing levels than would be typically allocated for the final sample size, which may explain the lower response rates for the virtual group.A key strength of this study is the breadth of data collected from such a large number of patients receiving SUD treatment during a period of extensive change. Although these results are mainly descriptive, these analyses are necessary to carefully evaluate the impacts of a global shift in treatment approach. A number of limitations should be considered when interpreting results. Similar to existing research used with ROM data -39, respFinally, although our sample was representative of HBFF program participants, it differs from the general population in a few specific ways that are important to acknowledge when considering the generalizability of the findings. First, patients in the sample were primarily White and non-Latinx. As a result, there may have been too few non-White/Latinx participants to detect differences. However, this sample directly compares to findings from the 2020 National Survey on Drug Use and Health (NSDUH)  in termsResults from this study suggest that virtual outpatient care for the treatment of SUDs is a feasible alternative to in-person care, leading to similar rates of self-reported continuous abstinence, health, and well-being in patients at 3-month follow-up. This study should serve as a baseline for the assessment and refinement of the role of virtual services in the field of addiction treatment in order to better understand under what circumstances telehealth can function as an effective alternative to the established in-person standard of care.


Fungi are well equipped to cope with oxidative stress and the reactive oxygen species that are, in the case of phytopathogens, produced mainly by the plant host for defence purposes. Peroxidases represent the major line of evolution for rapid decomposition of harmful peroxides in all aerobically metabolising organisms. In all the sequenced fungal genomes, many divergent genes coding for various peroxidases have been discovered, and Hybrid B heme peroxidases represent a distinctive mode of fungal-gene evolution within a large peroxidase–catalase superfamily that ranges from bacteria to plants.hyBpox genes, mainly within the genomes of Sclerotiniaceae , which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium-crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum, although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1-specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae. We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.In this study, we focus on a detailed bioinformatics analysis of  During their unique evolution in various environments, fungi were adapted mainly for aerobic metabolism and are mostly well equipped with efficient antioxidants , the mosSclerotium cepivorum Berk is a typical phytopathogenic representative and, more specifically, it is a plant necrotroph [Allium species, particularly onions, leeks, and garlic by producing a characteristic strong colour changes [The filamentous ascomycetous fungus crotroph . It caus changes . This ca changes . Efficie changes .Sclerotium cepivorum was released previously, [The complete genome of viously,  showing Sclerotium cepivorum, Berkeley CBS 276.93 was obtained from Fungal Biodiversity Centre . Only this strain was used throughout all experiments. Fungal culture was routinely grown at 25 °C in potato-dextrose-broth medium  containing 4 g potato infusion and 20 g dextrose, with the addition of fresh, sterilised onion extract, pH 5.1 for 1 L final volume. This fungus was grown either on solid agar medium (with the addition of 15 g/L agar) or in the liquid medium that was used for cultivation in 500 mL Schott flasks. In this case, the two cultures were grown in a final volume of 100 mL, without shaking, at 25 °C for 7 days, as optimised previously [2O2 was added to the first culture at a final concentration of 1 mM, and the second culture was grown in parallel without the addition of peroxide. Growth was continued for 30 min. at the same temperature with agitation of 130 rpm on a rotary shaker. Obtained samples were used for the isolation of DNA or RNA . Total RNA of this fungus from the frozen mycelium of samples with and without the addition of 1 mM H2O2 was purified with RNeasy Plus Mini Kit . From the obtained RNA libraries, corresponding cDNA was prepared with First Strand cDNA synthesis kit  by using the random hexamers approach. Resulting cDNAs were amplified and then sequenced with the hyBpox-specific primers described in Fungal genomic DNA from 100 mg of frozen S. cepivorum CBS 276.93 was investigated from the liquid PDB cultures obtained under conditions described in 2 transformation of the relative protein abundances, Student’s t-tests were calculated and the results were visualised as a volcano plot.The fungal protein secretome of www.psc.edu/biomed/genedoc accessed on 2 February 2022). Putative signal sequences of analysed peroxidases with the detection of Sec/SPI cleavage sites were predicted with SignalP 6.0 server [Complete protein sequences of 170 peroxidases belonging to the peroxidase–catalase superfamily . The minimum probe radius was set to 0.7 to detect even the narrower tunnels present in the ScepHyBpox1 model. The remaining parameters were kept at their default settings. The coordinates of the starting point of the tunnel search were calculated using the Arg69 and His73 residues together with the heme cofactor. The AutoDock Vina plugin [Tunnels in the protein were identified using the Caver 3.0.3  plugin ia plugin  was thenScephyBpox1-mRNA was found in both fungal mRNA libraries, either induced with the addition of 1 mM hydrogen peroxide  or also in the control sample without this addition. After performing cDNA synthesis within both independent mRNA libraries, the same ScephyBpox1 product was obtained from the fungal strain CBS 276.93, with a total length of 1614 base pairs for the open reading frame. The presence of 1 spliced intron with a length of 54 nucleotides was detected by direct comparison of genomic and RT-PCR fragments  and also shared with Family III earlier . A diversification had occurred already within the early steps in the evolution of the distinct HyBpox subfamily; the basal clade of still-putative Hybrid B heme peroxidases is positioned among Chytridiomycetous fungi with high bootstrap support. Based on investigated microfossils, it was reported  that zoo genomes ,35. In sxist cf. , which pAllium species, high bootstrap support is achieved. This particular sequence of Hybrid B peroxidase from Sclerotium cepivorum can thus be considered a typical model protein for all closely related heme peroxidases from the mostly phytopathogenic ascomycetous class of Leotiomycetes; however, the corresponding native peroxidases, other than the newly discovered ScepHyBpox1, still remain unknown at the protein level.By further following the unique history of this subfamily in the robust evolutionary tree presented, the ascomycetous descendants that could have emerged through a lateral gene transfer event from some Basidiomycota predecessor serving as gene donor can be observed in the next clades. This is supported by a high bootstrap value . HoweverNext, we analysed the conserved and unique features of the newly discovered subfamily named Hybrid B heme peroxidases and compared them with already well-known sequence features in corresponding amino-acid positions within the peroxidase–catalase superfamily. The complete multiple sequence alignments of selected Hybrid B heme peroxidases is presented in For an easy overview, only 14 representative protein sequences from all resolved HyBpox subfamily clades of the evolutionary tree shown in R-X-X-F-H-D consensus sequence for all Family II and Family III heme peroxidases [S. sclerotiorum and S. trifoliorum HyBpox as Y-H-D.K-T-A- The catalytic histidine responsible for the correct orientation and heterolytic cleavage of incoming peroxide is strictly conserved on the distal side. Additionally, the importance of the preceding arginine for the heterolytic cleavage of hydrogen peroxide was verified experimentally in distantly related CcP [In the first two panels, the N-terminal heme peroxidase domain is presented, whereas in the last two panels, the C-terminal sugar-binding domain (CBM) that is typical among all known heme peroxidases solely for HyBpox proteins is shown. The highly conserved motifs on the distal a and prooxidases  that shaated CcP . Althougated CcP  and appeL/M-V-A-C-G-H-T/V for all Hybrid B peroxidases and is also located in the α-helix. However, there are some differences also, including a deletion in Family-II and Family-III representatives . To verify the relevance of this motif, we applied PSI-Blast (position-specific iterated) search in a non-redundant protein-sequence database and found that it is frequently present in numerous WSC-containing proteins (water soluble carbohydrate-binding domain) [L/M/V-T-I/V-T/S-A-A-V-R-A-D-R-I that has large differences only in the sequence of basal chytridiomycetous HyBpox and is completely absent in the sequences of Family II and III heme peroxidases. We again used PSI-Blast to reveal the relationships. The output again demonstrated its presence among numerous WSC-containing proteins and many of them without a preceding domain.In contrast to all manganese peroxidases, versatile, and lignin peroxidases, both known Hybrid B peroxidases, possess a unique CBM-containing domain. Details can be found in the panel of  domain) . Some ofS. cepivorum was modelled together with structures of its selected evolutionary ancestors in order to determine whether the typical heme-containing fold allowing peroxidase activity was preserved during the evolution of this subfamily, and how the second, non-heme domain is spatially related with the larger peroxidase domain. For this purpose we used the methodology of AlphaFold 2 [The spatial structure of yet completely unknown Hybrid B heme peroxidase from the phytopathogen haFold 2  because haFold 2 . AlthoughaFold 2  and theyThe larger peroxidase domain that is mainly α-helical, as in the majority of other heme peroxidases from the whole peroxidase., Refs. ,44,45 ca., Refs. , and theThe smaller CBM domain occurs in contrast with a much lower portion of α-helical and more β-strand structural content that can be attributed mostly as β-sandwich. This typical double-domain structure of Hybrid B heme peroxidases represents a strictly conserved fold, observed also for the six reconstructed ancestral sequences in important evolutionary nodes cf. , which aThe details of the reconstructed active centres of all the AlphaFold models were inspected in the presented phytopathogenic peroxidase and in its six ancestors in order to follow the fine details of the specific spatial orientation of the conserved amino acids allowing peroxidase reactivity. The output, again presented in the structural overlay, is shown in In principle, all above presented structural figures clearly show that the N-terminal heme-binding peroxidase domain was highly conserved during its entire evolutionary history, thus allowing essential and efficient peroxidase activity. On the other hand, there is additional variability, which is not involved in peroxidation cycles, in the additional CBM-binding domain on the C-terminus of Hybrid B peroxidases.Finally, two potential substrates were docked in the active centres of all of the models presented here. Hydrogen peroxide is the first and most common substrate reduced in the active centres of all peroxidases. The second potential substrate—guaiacol—is used by a large group of evolutionary-related peroxidases as an electron acceptor for reactive enzyme intermediates. Results are presented in In We demonstrated that in a typical secreted heme-peroxidase representative from a phytopathogenic fungus, namely ScepHyBpox1 and its ancestors, there exists an important novel and still rather undiscovered heme peroxidase subfamily that may have an important function for pathogenic fungal cells in their defence against oxidative bursts caused by the host plant. Corresponding mRNA is produced rather constitutively and is translated into a unique two-domain protein representing the sole secreted peroxidase of this filamentous fungus. This fact underlines its importance for the survival of the described phytopathogen and probably also for related pathogenic fungi from the whole class of Leotiomycetes during infection cycles and when attacking plant tissues. Numerous Hybrid B heme peroxidase sequences obtained from different fungal families of both Ascomycota and Basidiomycota were systematically collected for phylogenetic purposes. Even the presence of corresponding Hybrid B heme peroxidase sequences that are supposed to be an early diverging lineage of the whole fungal kingdom were detected among Chytridiomycota. All representatives of the newly discovered peroxidase subfamily presented here reveal surprisingly high overall sequence similarity which is mainly localised in the larger heme-peroxidase domain on the N-terminus of the protein, irrespective of their trophic levels in various ecosystems. The C-terminal domain that is directly fused with the larger α-helical peroxidase domain is quite unusual for all previously described heme peroxidases, but it is also present in all of the known deep ancestors of this subfamily in nearly the same orientation where the CBM-containing domain is located near the region of the proximal side of heme. The proposed “hybrid“ character of the proteins analysed herien is concealed in this unique double-domain architecture and apparently has deep evolutionary origins among early-diverging aquatic fungal lineages. This observation is in noticeable in constrast to, e.g., lignin peroxidases among early-diverging fungi that have not yet been described. In contrast, the presence of these flexible enzymes among Chytridiomycota before their presence among Dikarya indicates that, in addition to their basic protective function against reactive oxygen species, they may have been involved in the ancient extraction of useful nutrients from early land-colonising plants and their algal relatives . In thisIn this study, a new type of Hybrid B heme peroxidase was detected and investigated on the proteomic level as the sole peroxidase in the secretome of an important phytopathogenic fungus. The reconstruction of its broad phylogenetic relationships in corresponding peroxidase (sub)families reveals its importance as a model protein representing numerous, related heme peroxidases. Its unique two-domain architecture was modelled, and a typical tunnel leading to the distal heme cavity was resolved by the advanced method of structural modelling methodology represented by AlphaFold and CAVER. The performed docking of two potential substrates must be verified in a highly purified sample of this or a related Hybrid B heme peroxidase. The observed Hybrid B heme and CBM-fusion proteins are now attractive targets for future functional and engineering studies.


A metallic nano-trench is a unique optical structure capable of ultrasensitive detection of molecules, active modulation as well as potential electrochemical applications. Recently, wet-etching the dielectrics of metal–insulator–metal structures has emerged as a reliable method of creating optically active metallic nano-trenches with a gap width of 10 nm or less, opening a new venue for studying the dynamics of nanoconfined molecules. Yet, the high surface tension of water in the process of drying leaves the nano-trenches vulnerable to collapsing, limiting the achievable width to no less than 5 nm. In this work, we overcome the technical limit and realize metallic nano-trenches with widths as small as 1.5 nm. The critical point drying technique significantly alleviates the stress applied to the gap in the drying process, keeping the ultra-narrow gap from collapsing. Terahertz spectroscopy of the trenches clearly reveals the signature of successful wet etching of the dielectrics without apparent damage to the gap. We expect that our work will enable various optical and electrochemical studies at a few-molecules-thick level. A pair of parallel metallic plates create a uniform electric field between them when a voltage difference is applied, provided that the separation between them is sufficiently small compared to the size of the plates themselves. They have not only become the central building block of electrochemical studies, but also greatly benefitted the field of nanophotonics, as the distribution of electromagnetic fields near a pair of parallel metallic plates can be expressed analytically and is exactly known. Therefore, metallic gap structures have provided an ideal testbed for quantitative analysis on light–matter interactions, as well as enhancing the interactions via strong electromagnetic field enhancements ,2,3,4,5.In such a context, the invention of atomic layer lithography made a huge breakthrough by making possible the experimental realization of wafer-scale arrays of horizontally aligned metal–dielectric–metal structures with a dielectric gap as small as 1 nm . Selecti2) and vaporizing the CO2 at its critical point to minimize the force applied to a system in the drying process  technique, whose working principle relies on replacing liquid water with liquid carbon dioxide  time-domain spectroscopy. The spectroscopic method can non-destructively evaluate a gap structure without having to make an electronic contact and, therefore, prevents unwanted damage to the sample in the measuring process. In detail, we created THz pulses with 800-nm optical pulses from a Ti:sapphire oscillator and a DC-biased gallium arsenide photoconductive antenna. The THz pulses passed through a 1 × 1-mm rectangular aperture and the sample, subsequently, was incident on zinc telluride crystal for electro-optic detection. The detected field amplitude was transformed into a frequency domain spectrum via fast Fourier transform. All the measured spectra were normalized with respect to transmission spectra of the aperture plus bare silicon substrate, so that performance of the gap could solely be determined.The final structure of our nano-trenches was an array of 1.5-nm-wide coaxial apertures with a rectangular perimeter of 10 × 40 μm, with separation of 10 μm from both sides, as depicted in −4, and therefore, the field enhancement at the gap reaches over 2000 at resonance, finding great potential in nonlinear optical studies or sensing applications.The measured THz transmission spectra of the nano-trench samples before and after etching are summarized in When the gap undergoes the normal wet etching and blow dry process, we observe a significant decrease in transmission or equivalently decreased near-field enhancement in the nano-trenches (red line). As metallic nanogaps incorporate field enhancement via capacitive accumulation of charges , the decWe also observed that the performance of the fabricated sample depends strongly on the etching time, i.e., the amount of time the sample stays in the wet etchant. This may be inferred from the fact that the alumina spacer exists not only between the metallic layers but also underneath one of the layers, which leads to a possible collapse of the nano-trenches upon complete etching of the underlying alumina layer a. Such a2 inside the CPD chamber. Therefore, proper design of the sample holder and careful handling of the samples can further improve the yield of our process. Furthermore, as 1.5 nm is the smallest gap width achievable with our facilities, and since a smaller gap is much more prone to capillary collapses, we expect even higher success rates for nano-trenches with larger gap sizes. We briefly mentioned here that we also successfully applied our method on 5-nm-wide nano-trenches (data not shown), and the result was consistent with a previous report where 5-nm-wide nano-trenches were fabricated without the CPD process.The overall yield of our etching and CPD process is 90 percent for the 1.5-nm gap width and is limited by occasional damage to the sample caused by the turbulent flow of liquid COIt is worth discussing the relatively short etching time of ~20 s for the nano-trench sample, considering that the gap width is only 1.5 nm wide and water or etchant molecules are expected to enter the gap very inefficiently. This can be understood in two aspects. First, the chemical reaction between KOH and alumina is extremely favored, which can be inferred from the reported etch rate of >2500 nm/min . TherefoOur scheme could also be improved by further optimization of the fabrication parameters. For instance, using a negative photoresist instead of image-reverse resist will simplify the procedure and may lead to better control over the geometry of the metallic layers. Furthermore, one could use electroplating instead of lift-off to achieve a steeper sidewall angle of the metallic layer, which can improve the optical properties and mechanical stability of the fabricated nano-trench structure. Such improvements may lead to realization of nano-trenches with even smaller gap widths and stronger field enhancement, which could open a new venue towards strong interaction of terahertz waves with a single-molecule-thick layer.In summary, we developed a method for fabricating high-aspect-ratio metallic gaps, or nano-trenches, as narrow as 1.5 nm. This was achieved by fabrication of a metal–dielectric–metal gap structure and subsequent wet etching of the gap-filling dielectric. We found that the CPD technique efficiently eliminates the surface tension of water in the drying process, which, if present, leads to collapsing of the nanostructure. Successful etching or collapsing of the nano-trenches could be non-destructively determined with THz spectroscopy, as the optical properties of the gap structure are very sensitive to the formation of conducting channels. Unlike other spacer-based metallic gap structures, the nano-trenches have no filling material within the gap, which means that the optical hot spot is freely accessible to various types of materials. Therefore, the nano-trenches could find numerous potential applications in ultrasensitive molecular detection, nonlinear optics, and electrochemical studies.


The coronavirus disease (COVID-19) pandemic has led to an unprecedented worldwide burden of disease. However, little is known of the longer-term implications and consequences of COVID-19. One of these may be a COVID-19 associated coagulopathy that can present as a venous thromboembolism (VTE) and further, as multiple paradoxical cerebral emboli.A 51 year old man presented to the emergency department with multiple simultaneous embolic cerebral infarctions 11 months after mild COVID-19. In the subacute phase of the COVID-19 illness the patient developed increasing shortness of breath and was found to have an elevated D-dimer and multiple bilateral segmental pulmonary emboli. He was subsequently treated with 3 months of anticoagulation for a provoked VTE. The patient then presented 11 months after the initial COVID-19 diagnosis with multiple simultaneous cerebral infarctions where no traditional underlying stroke etiology was determined. A patent foramen ovale (PFO) and an elevated D-dimer were found suggesting a paradoxical thromboembolic event due to an underlying coagulopathy.This case report highlights the one of the potentially more serious complications of long-term COVID-19 where VTE due to a persistent coagulopathy is seen almost a year after the initial illness. Due to the highly prevalent nature of PFO in the general population, VTE due to COVID-19 associated coagulopathy could lead to ischemic stroke. This case report highlights the possibility for an underlying COVID-19 associated coagulopathy which may persist for many months and beyond the initial illness. The longer-term implications and consequences of COVID-19 remain largely unmapped. One of the previously identified longer-term complications is a COVID-19 associated coagulopathy. This associated coagulopathy can present as a venous thromboembolism (VTE) and, as yet unreported, multiple paradoxical cerebral emboli. Furthermore, the temporal correlation of the emergence of a COVID-19 associated coagulopathy to the primary illness remains unknown. In cases of milder COVID-19 symptoms, an associated coagulopathy can remain undiagnosed and result in thromboembolic events after the initial illness.A 51 year old man presented to the Emergency Department (ED) with acute onset left sided peripheral facial palsy and a complete right sided homonymous hemianopsia.The patient was a Caucasian male of normal BMI with no prior history of hypertension, hypercholesterolemia, diabetes, smoking and or heart disease. He had been in good health until March 2020 when he returned from a group holiday to Austria. The patient started feeling unwell with a fever and was diagnosed with coronavirus disease (COVID-19) along with several members of the group that he had travelled with. A nasopharyngeal PCR swab confirmed the presence of SARS-CoV-2 RNA on the 22nd of March 2020. The patient was admitted the hospital on the 29th of March 2020 after increasing shortness of breath and onset of diarrhea and vomiting. The patient was febrile but had no oxygen requirement and was therefore diagnosed with mild COVID-19. He was admitted for observation, managed conservatively, and was discharged home 3 days later on the 1st of April 2020 after showing clinical improvement.The patient was readmitted 2 days later on the 3rd of April 2020 with increasing shortness of breath. Clinically, pulmonary embolism (PE) was suspected with no signs of hemodynamic compromise. The diagnosis was confirmed by a CT Pulmonary Angiography (CTPA) Fig.  which deOn the 26th of February 2021, approximately 11 months after being diagnosed with COVID-19, the patient contacted the emergency medical services due to acute onset visual field disturbances. The ambulance arrived on scene minutes after the onset of symptoms. A preliminary examination by the paramedics revealed as right sided visual field defect and a left sided facial palsy. The patient had a heart rate of 70 beats per minute and blood pressure of 147/89 mm Hg. The prehospital oxygen saturation was 98 % whilst breathing room air. Based on their initial findings, the paramedics suspected a stroke, and the patient was blue lighted to the ED.On admission to the ED the patient was taken directly to the CT lab. The patient had a temperature of 35.5 ℃, ECG showing normal sinus rhythm, blood glucose of 5.8 mmol/L, a blood pressure of 157/99 mmHg and an oxygen saturation of 99 % on room air. A rapid neurological examination revealed a left sided peripheral facial palsy, right sided homonymous upper quadrantopia. A National Institutes of Health Stroke Scale (NIHSS) exam revealed a score of 3.A CT scan was performed including a pre- and intra- cerebral angiography. A subsequent cerebral perfusion scan was also performed. The CT examination was performed using the Siemens® Somatom Definition Flash using GE Healthcare® Omnipaque intravenous contrast and standardized protocols for angiography and perfusion.The plain CT showed no signs of hemorrhage and based on the clinical suspicion of stroke the decision to treat with intravenous thrombolysis was made. Alteplase was administered at a dose of 0.9 mg/kg as per guidelines. The CT angiography three occluded intracerebral vessels, occlusions of the P2 and P3 segments of the left posterior cerebral artery Fig. .Fig. 3CThe patient was admitted to the stroke unit and showed neurological improvement after Alteplase administration. The following day the patient had a NIHSS of 0.During hospital admission examinations were performed to determine ischemic stroke etiology. Ultrasound of the pre- and intra- cerebral vessels showed no significant stenoses or occlusions. Five day in-hospital cardiac rhythm monitoring showed sinus rhythm without any signs of atrial fibrillation and or other arrythmia. The patient underwent a Trans- Thoracic Echocardiography (TTE) which was normal and a Trans- Esophageal Echocardiography (TEE) where a patent foramen ovale was detected (PFO). The right to left shunting was visualized on color doppler and whilst using contrast Fig. . A bloodThe patient was discharged with a NIHSS of 0 following an Embolic Stroke of Unknown Source (ESUS). The patient is currently being treated with 20 mg Rivaroxaban OD, and atranscatheter closure of his PFO has been scheduled.Patients with COVID-19 are frequently found to have underlying coagulopathy. This is demonstrated by evidence of microthrombosis/venous thrombus, elevated D-dimer and fibrinogen, activated complement system and von Willebrand Factor (vWF), increased inflammatory cytokines  and antiphospholipid antibodies, as well as macrophage/endothelial cell dysfunction [Our case presented with VTE in the form of PE during the sub-acute phase of COVID-19 infection, then later, arterial thromboembolism in the form of acute ischemic stroke 11 months after COVID-19.Large arterial vessel stroke can be a presenting feature of COVID-19 in young patients. This was demonstrated in early case series during the pandemic, of which the majority 4 of 5) had elevated levels of D-dimer at diagnosis . There a of 5 hadOf interest PFO was also found, which has previously been reported to complicate PE in the context of COVID-19 infection in a young female . UnderlyVenous thromboprophylaxis is now routinely considered for patients admitted with COVID-19 infection. There is little evidence to suggest that COVID-19 infection in the absence of clinically evident VTE warrants the use of therapeutic anticoagulation to address an underlying coagulopathy. There are similar short-term risks of both thrombosis and bleeding in these patients . In the This case reports highlights the more serious, longer term complications of COVID-19 where multiple embolic infarctions presented almost 1 year after the acute illness. The COVID-19 associated coagulopathy potentially persisting many months after diagnosis in patients with a milder clinical course of illness.The long-term consequences of COVID-19 infection are only just beginning to surface potentially impacting a great number of individuals. More studies and reports are needed to map out the potential long-term consequences of COVID-19 associated coagulopathy.


Introduction. Conduct disorder is a psychiatric diagnosis characterized by repetitive and persistent norm-breaking behavior. This study aimed to compare the risk of conduct disorder between first- and second-generation immigrant children and adolescents and their native controls. Methods. In this nationwide, open-cohort study from Sweden, participants were born 1987–2010, aged 4–16 years at baseline, and were living in the country for at least one year during the follow-up period between 2001 and 2015. The sample included 1,902,526 and 805,450 children-adolescents with native and immigrant backgrounds, respectively. Data on the conduct disorder diagnoses were retrieved through the National Patient Register. We estimated the incidence of conduct disorder and calculated adjusted Hazard Ratios. Results. Overall, the adjusted risk of conduct disorder was lower among first-generation immigrants and most second-generation immigrant groups compared with natives . However, second-generation immigrants with a Swedish-born mother and a foreign-born father had a higher risk of conduct disorder than natives. Similar results were found for sub-diagnoses of conduct disorder. Conclusions. The higher risk of conduct disorder among second-generation immigrants with a Swedish-born mother and the lower risk among most of the other immigrant groups warrants special attention and an investigation of potential underlying mechanisms. According to United Nation (UN) estimates from the last decade, an increasing number of people have left their home countries to seek a safer and better life elsewhere . War, coImmigrants experience substantial stress before, during, and after immigration. Upon arriving in the host country, issues like struggles with adapting to a new culture and securing jobs and housing can affect immigrants’ own as well as their children’s mental health . MissingConduct disorder is a psychiatric disorder in children and adolescents that is characterized by repetitive and persistent norm-breaking behavior and violation of other people’s rights . AffectiSweden has received many refugees and non-refugee immigrants over the past few decades. By 2018, approximately one-third of the Swedish population were first- or second-generation immigrants . In the This was a population-based retrospective open cohort study. Pseudonymized data from several nationwide registers were provided to us by Statistics Sweden (SCB)  and The Eligible participants were born 1987–2010, and were registered as residents of Sweden during at least one year during the study period between 1 January 2001 and 31 December 2015. The enrollment included individuals aged 4–16 years at baseline, which could occur between 1 January 2001 and 31 December 2013. In total 2,707,976 children-adolescents were included in the study population that consisted of 1,902,526 (70.3%) natives (defined as Swedish-born with both parents born in Sweden), 228,889 (8.4%) first-generation immigrants (defined as born abroad with both parents born abroad), 160,839 (5.9%) second-generation immigrants with a foreign-born father (and a native mother), 133,217 (4.9%) second-generation immigrants with a foreign-born mother (and a native father), and 282,505 (10.4%) second-generation immigrants with two foreign-born parents. All second-generation immigrant groups were defined as born in Sweden.The follow-up continued until participants turned 18 years, emigrated, died, or at the end of the study on 31 December 2015, whichever came first. Approximately 90% of the participants from each of the native/immigrant groups were followed until the end of the study or the age of 18 years. In total, we accrued a total of 20,181,775 person-years of observations from participants including the following: natives , first-generation immigrants with two foreign-born parents , second-generation immigrants with two foreign-born parents , second-generation immigrants with a foreign-born mother , and second-generation immigrants with a foreign-born father .We used the following nationwide registers: The Register of the Total Population (RTB), the Multi-Generation Register, the Medical Birth Register, the Cause of Death register, the Migration Register, the Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA), and The National Patient Register (NPR). Data on conduct disorder diagnoses were obtained from the National Patient Register (NPR). The NPR had full national coverage for inpatient and outpatient hospitalization records during the entire study period and has been validated for research purposes .We used maternal income as a surrogate of socioeconomic status. We obtained data on maternal income from the LISA database at Statistics Sweden. Statistics Sweden calculates disposable income based on total income from all potential sources, including wages, welfare benefits, other social subsidies, and pensions. We obtained annual disposable income for all females 18–65 years living in Sweden during the observation period. To account for inflation, we categorized income into quintiles relative to all other females assigned a disposable income in that year. We then created year-specific income quintiles and used the maternal income quintile for the year of start of follow-up for each participant. Few individuals lacked information on income in the total study population. As a higher proportion of foreign-born parents lacked information on income, we did not impute missing data on income since we judged that this information was not missing at random between subgroups. However, we included those with missing income values in the lowest income quintile.In the NPR all psychiatric diagnoses for the study period were registered using the 10th version of the International Classification of Disease (ICD 10) . We searn = 23,392; 10.2%); a majority of these did not have their biological mother in Sweden . We assigned the lowest maternal income value (first income quintile) to those with a missing maternal income. We evaluated the proportional hazards assumptions for the Cox regression models using log-log survival plots and did not find any meaningful deviations from the assumption indicating that there was no effect modification from time in the models. We used Stata (version 15.1) for data management and statistical analysis [We estimated non-adjusted incidence rates of conduct disorder per 100,000 person-years and report 95% confidence intervals (CI) for the estimates. Using Cox regression, we ran sex-specific models and estimated hazard ratios (HRs) of conduct disorder with 95% CI for the immigrant groups compared with natives in two steps. In Model 1, we controlled for birth year and age at the start of the follow up. In Model 2, we additionally controlled for maternal income. Maternal income (see below) could not be attained for some participants including some of those from the first-generation immigrant group , perinatal , familial , and neighborhood risk factors  may contribute to the risk of developing conduct disorder . DespiteAmong all immigrant groups, only those second-generation immigrants with a foreign-born father and a Swedish-born mother had markedly higher risks of conduct disorder compared with natives while first-generation immigrant children and adolescents as well as second-generation immigrants with two foreign-born parents had lower risks. All residents in Sweden are entitled to free healthcare until they turn 18 years old. Moreover, schools are actively engaged in maintaining the mental health of their students, but the use of these services may be hampered because some immigrant populations have different patterns of using healthcare services for their children’s psychiatric problems ,34. PoorMost known non-genetic risk factors of conduct disorder are caused or associated with parental stress and conflict in the family. Marriage or partnership between a native and an immigrant person may result more frequently in divorce or break-up due to cultural differences and conflicts in parenting . A studyThe context of the host country and cultural stress may also play major roles in the integration of immigrants and in maintaining their mental health. In a study from the USA, bicultural stress was associated with changes in aggressive and rule-breaking behavior among newly arrived immigrant Hispanic high school adolescents . This poThe results of this study must be interpreted in the light of some limitations. We identified participants with a conduct disorder diagnosis through their inpatient and outpatient hospital referrals in the patient register. While trained doctors with psychiatric expertise have made most of these diagnoses, some inconsistencies in diagnostic procedures over time and across hospitals could not be ruled out. Besides, cultural differences between natives and some immigrants may affect immigrant children’s likelihood of obtaining a conduct disorder diagnosis. Although we accounted for several potential confounders, our analysis did not include all potential confounders . Future research should attempt to account for residual confounding in their analyses. Despite these limitations, however, this study is the first national cohort study comparing the burden of conduct disorder between immigrant children-adolescents and natives using high quality data. For example, we had no loss to follow up as each person could be tracked for emigration or death because of the unique identification number assigned to each person with a residence permit in Sweden.Our results indicated that first-generation immigrants and second-generation immigrants with two foreign-born parents had a lower risk of conduct disorder. In contrast, a markedly higher risk of conduct disorder among male and female second-generation immigrants with a foreign-born father and a Swedish-born mother was found. Future studies should investigate the underlying mechanisms behind these findings including potential inequities in the access to or the utilization of mental health services across natives and different immigrant groups.


Surprisingly, our analysis of the big image data shows the anomalous presence of excessive platelet aggregates in nearly 90% of all COVID-19 patients. Furthermore, results indicate strong links between the concentration of platelet aggregates and the severity, mortality, respiratory condition, and vascular endothelial dysfunction level of COVID-19 patients.A characteristic clinical feature of COVID-19 is the frequent incidence of microvascular thrombosis. In fact, COVID-19 autopsy reports have shown widespread thrombotic microangiopathy characterized by extensive diffuse microthrombi within peripheral capillaries and arterioles in lungs, hearts, and other organs, resulting in multiorgan failure. However, the underlying process of COVID-19-associated microvascular thrombosis remains elusive due to the lack of tools to statistically examine platelet aggregation  in detail. Here we report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring of the blood of COVID-19 patients ( The authors report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring to elucidate the underlying process of COVID-19-assocaited microvascular thrombosis. In fact, earlier autopsy reports on patients who died with COVID-19 have shown widespread thrombotic microangiopathy (TMA) characterized by extensive diffuse microthrombi present within peripheral capillaries and arterioles in lungs, hearts, and other organs, resulting in multiorgan failure11. This is aligned with respiratory failure due to severe diffuse alveolar damage being the primary cause of death in COVID-1910. It is also consistent with our current understanding of the pathophysiological mechanism of COVID-19 in which SARS-CoV-2’s entry into host cells is mediated by the angiotensin-converting enzyme 2 (ACE2) receptor, which stimulates the renin-angiotensin-aldosterone system and causes vascular endothelial damage followed by vasculitis , resulting in the formation of microthrombi6. In response to a number of reports that anticoagulant therapy with heparin leads to better prognosis in COVID-19 patients12, both domestic and international organizations have issued clinical practice guidelines recommending that all hospitalized COVID-19 patients should receive thromboprophylaxis (mainly heparin treatment) even without clear symptoms of thrombotic complications and understanding their efficacy14. Moreover, post-COVID-19 syndrome manifested by persistent and prolonged aftereffects has been reported to be linked to COVID-19-associated microvascular thrombosis16.With the increasing number of global case reports since the beginning of the COVID-19 pandemic, it has become clear that COVID-19-associated microvascular thrombosis is one of the key factors for the severity and mortality of COVID-1920 in vivo. For example, optical microscopy21 can directly probe platelet aggregation with high spatial resolution and has, in fact, visualized platelet hyperactivity in COVID-19 by identifying the presence of platelet aggregates  including macrothrombocytes22. However, visual inspection under the optical microscope is too slow and labor-intensive to analyze platelet aggregates in a statistically meaningful manner and can only provide qualitative results. Flow cytometry, on the other hand, enables statistical analysis of large populations of cells by measuring their physical and chemical properties via impedance, scattering, or fluorescence measurements in flow27 and has been used to identify leukocyte hyperactivity in COVID-19 via fluorescent probes30. However, the lack of spatial resolution in flow cytometry prohibits accurate differentiation between single platelets and platelet aggregates32 and hence cannot accurately characterize platelet activity. Likewise, light transmission aggregometry33, although widely used in clinical laboratory testing, only evaluates the average agonist-induced aggregation properties of many platelets in vitro through ensemble measurements and cannot provide details of platelet aggregation such as the size distribution or single-to-aggregate population ratio. Moreover, D-dimer testing, another widely used diagnostic, is conventionally used to estimate the presence of thrombi by measuring the cross-linked fibrin monomers  produced when thrombi are degraded by fibrinolysis34. D-dimer testing has been reported as useful for assessing the severity of COVID-19, as an elevated level of D-dimers (>1 µg/mL) at admission is associated with an increased risk of both required mechanical ventilation and death due to complications36. However, D-dimer testing does not probe platelet activity in vivo and is not sensitive to microvascular thrombosis including TMA (unless it becomes severe)37. Medical imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and ultrasonography can directly identify thrombi in the body, but do not have sufficient spatial resolution to recognize circulating platelet aggregates and microthrombi38. Finally, postmortem examination can directly identify microthrombi but is only applicable to dead patients. To date, statistical morphometric understanding of platelet aggregation has been inaccessible and hence overlooked, as optical microscopy (a high-content but low-throughput tool) has been the main method used to examine platelet aggregation in detail thus far32.Unfortunately, the underlying process of the incidence of COVID-19-associated microvascular thrombosis remains elusive. This is due to the lack of tools to statistically examine the detailed characteristics of platelet activity, or more specifically platelet aggregation n = 25,000) of single platelets and platelet aggregates (including platelet-platelet aggregates and platelet-leukocyte aggregates39) in blood samples, by optical frequency-division-multiplexed (FDM) microscopy32 on a hydrodynamic-focusing microfluidic chip41 . Importantly, this big image database of platelet aggregates enabled the previously unfeasible application of advanced computational tools44 which provided unique and insightful analytical results, as we demonstrated here.In this Article, to better comprehend the transient process of microthrombus formation in COVID-19, we obtained the landscape of circulating platelet aggregates via massive single-cell image-based profiling and temporal monitoring of the blood of COVID-19 patients. As shown in Fig. n = 110) who were clinically diagnosed with COVID-19 based on their reverse transcription-polymerase chain reaction (RT-PCR) test results  in each sample. Negative control data  on 67 different dates  than in female patients (n = 37) although their statistical significance is not very high, which is aligned with earlier reports that male patients are more prone to intensive treatment unit admission and death than female patients48. In addition, the ability of our method to highly resolve platelet aggregates and apply morphometric analysis to their images enabled identifying the presence of leukocytes. Fig. 35. Fig. Moreover, we investigated the influence of sex differences and leukocytes. Specifically, Fig. 2 (oxygen saturation level) level is reasonable since oxygen administration required to maintain the SpO2 level in good condition reflects the disease state of patients. These strong correlations between the concentration of platelet aggregates and the WBC, FVIII, VWF:RCo, and TM levels indicate that the high concentration of platelet aggregates is linked with systemic microthrombus formation and fibrinolysis, which are expressed by the high D-dimer level, and with vascular endothelial damage , which is expressed by the high FVIII, VWF:RCo, and TM levels. These links are consistent with earlier reports on severe vascular endothelial damage in the lungs and widespread microthrombi in the alveolar capillaries of COVID-19 patients49.We compared the landscape of circulating platelet aggregates with conventional clinical laboratory and physical findings  as potential explanatory factors (Supplementary Data p = 0.009) and FVIII (p = 0.016) as the most significant predictors of the concentration of platelet aggregates on the highest concentration day.To further investigate significant explanatory factors for the concentration of platelet aggregates, we performed a multivariate regression analysis  using the vascular endothelial disorder marker group , the coagulation/fibrinolysis marker group , and the group of other markers having strong correlations with the concentration of platelet aggregates on the highest concentration day . As shown in Fig. 34 and is known to be insensitive to microvascular thrombosis, including TMA37, whereas our method characterizes the initiation of microthrombus formation and the degradation of microthrombi20. The latter method is, therefore, more sensitive with high resolution to platelet hyperactivity, which is a suggested mechanism of widespread microthrombus formation45. In addition, the fact that only one severe case with a low D-dimer level was identified as shown in Fig. 50. In other words, the correlation between the D-dimer level and the concentration of platelet aggregates in Fig. Next, since earlier reports show the onset of COVID-19-associated thrombosis even with low D-dimer levelsOur temporal monitoring of the concentration of platelet aggregates in the blood of COVID-19 patients enabled us to probe the pathological conditions of COVID-19 patients, 76% of whom received anticoagulant therapy with heparin. Figure 6. Since VWF is a factor that mediates platelet adhesion and aggregation at the site of vascular endothelial injury, it is released into the blood in large amounts during the inflammation-mediated endothelial injury. Our findings  for the mild and moderate patients. In fact, the average numbers of measurements per patient were 1.9 (mild), 2.6 (moderate), and 7.3 (severe) times. The fact that the high AUC in the ROC curve of the concentration of platelet aggregates on the first measurement day is as large as 0.9  need to be carried out. Moreover, a prospective study of prediagnosed patients with COVID-19 needs to be conducted to evaluate the diagnostic potential of our method. Third, a prospective study is needed to determine whether the concentration of platelet aggregates can predict worsening respiratory status and the development of microthrombi as well as evaluate the efficacy of therapeutics. With these additional studies, our method may hold promise for predicting respiratory failure at an early stage and for developing treatment strategies for preventing the transition of patients to ventilatory management.There are a few limitations to this retrospective observational study and potential solutions to them. First, all the subjects were hospitalized patients at the University of Tokyo Hospital alone, which may have introduced a selection bias. Further multi-hospital studies are needed to draw more general conclusions. Second, our method is not directly applicable to COVID-19 diagnosis because the identification of circulating platelet aggregates is not the direct evidence of microvascular thrombosis while extensive diffuse microthrombi are associated with multiorgan failure2 level, body temperature, respiratory severity) and laboratory test data were obtained from the electronic medical records of the patients using a standardized data collection form. Informed consent for participation in the study was obtained from the patients using an opt-out process on the webpage of the University of Tokyo Hospital. Patients who refused participation in our study were excluded. Written informed consent was obtained from the healthy subjects as well. The demographics, clinical characteristics, and laboratory findings of patients with COVID-19 are shown in Supplementary Data This study was conducted with the approval of the Institutional Ethics Committee in the School of Medicine at the University of Tokyo  in compliance with the relevant guidelines and regulations. The subjects used in this study were patients who were clinically diagnosed with COVID-19 based on their reverse transcription-polymerase chain reaction (RT-PCR) test results , fibrin monomer complex level (FMC), antithrombin level (AT), thrombin antithrombin complex level (TAT), von Willebrand factor activity level (VWF:RCo), fibrinogen/fibrin degradation product level (FDP), thrombomodulin level (TM), and coagulation factor VIII activity level (FVIII) .Blood samples from the patients were used for routine laboratory tests such as the leukocyte count (WBC), red cell count (RBC), platelet count (PLT), prothrombin time international normalized ratio (PT-INR), activated partial thromboplastin time (APTT), D-dimer level, fibrinogen level (Fbg), C-reactive protein concentration level (CRP), alanine transaminase concentration level (ALT), lactate dehydrogenase concentration level (LD), and creatinine kinase level (CK). The cryopreserved plasma was used for additional tests such as plasma plasmin-αII) Fig. . All theThe FDM microscope is a high-speed, blur-free, bright-field imaging system based on a spatially distributed optical frequency comb as the optical source and a single-pixel photodetector as the image sensor. Since the optical frequency comb is composed of multiple beams which are spatially distributed, it is capable of simultaneously interrogating the one-dimensional spatial profile of a target object . In addition, since each discrete beam of the optical frequency comb is tagged by a different modulation frequency, a spatial-profile-encoded image can be retrieved by performing Fourier transformation on the time-domain waveform detected by the single-pixel detector. As shown in Supplementary Figure 2 were considered as the images of platelet aggregates in all the patient and healthy subject (control) samples. The concentration of platelet aggregates was defined by the ratio of the number of acquired images containing platelet aggregates to the total number of acquired images  in each sample. In all 110 patient datasets, 106 of them have 25,000 images while only 4 of them  have 20,000 images due to a data-recording error, but this should not influence the statistical accuracy of our data analysis since the number of acquired images is significantly large. The presence of leukocytes in platelet aggregates was also identified by analyzing the morphology of the platelet aggregates by their images using custom codes in Python 3. For confirmation, the fluorescence signal of anti-CD61-PE and anti-CD45-PC7 was also used. CD61-PE/CD45-PC7 double-positive events were counted as platelet aggregates containing leukocytes. CD61-PE-positive and CD45-PC7-negative events that had CD61-PE signal intensity greater than a threshold value were counted as platelet aggregates excluding leukocytes. The presence of excessive platelet aggregates was determined by calculating the mean and standard deviation of the distribution of the concentration of platelet aggregates in the control samples and evaluating if the concentration of platelet aggregates in a patient sample exceeded the threshold (mean + standard deviation). If a tighter threshold (mean + two standard deviations) was used to calculate the presence of excessive platelet aggregates, then the ratio of the number of patients with excessive platelet aggregates to the number of all patients is 75.5%, while the ratio of the number of patients with excessive platelet aggregates and low D-dimer levels (≤1 µg/mL) to the number of all patients with low D-dimer levels (≤1 µg/mL) is 62.8%. Statistical analysis was performed using the rank-sum test including the Kruskal-Wallis test, Mann–Whitney U test, and Wilcoxon rank-sum test with significance levels at 0.05. Each statistical correlation value was obtained by calculating Spearman’s rank correlation coefficient between the concentration of platelet aggregates on the highest concentration date and the corresponding clinical parameters. The line fitting function of the Origin 2021b software was used to draw linear fits to show the correlation between the concentration of platelet aggregates and each clinical parameter. A 95% confidence interval calculated from the standard error of measured y values is shown as a gray area in each panel  in bright-field images were segmented in MATLAB R2020a for calculating the concentration of platelet aggregates in each sample. First, a 10x interpolation by the interp2 function (MATLAB R2020a) was applied to each image for achieving segmentation results. Then, the outlines of the object regions were detected by using the edge detection function with the Canny method in MATLAB R2020a. Morphological operations like dilate, fill, and erode were applied to fill and refine the object regions for obtaining their masks as well as for eliminating the background noise. After the segmentation, the size of the object  in each image was calculated by multiplying the pixel size of the segmented region, which was extracted by the regionprops function, and the pixel resolution after interpolation (80 nm x 80 nm/pixel). All the images with objective areas larger than 48 µmges n = 2,000 in eFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Nephrotic syndrome is a unique clinical disorder, which provides interesting teaching opportunities that connect physiological and pathological aspects to clinical practice. During the current COVID-19 outbreak, in-person teaching in our institution was not permitted, thus creating a unique challenge for clinical skills teaching. A case-based electronic learning (e-learning) activity was designed to replace the traditional in-person teaching of renal semiology. e-Learning activities have been shown to be effective for knowledge retention and increasing novice learners’ performance. However, major knowledge gaps exist concerning the satisfaction of learners with e-learning activities as the sole form of teaching, specifically for undergraduate clinical skills education.Our study aimed to prospectively assess undergraduate medical students’ perceptions of and satisfaction with an e-learning activity teaching renal semiology.All second-year medical students (novice learners) from the medical faculty of the University of Geneva, Switzerland, undertook the e-learning activity and were invited to participate in a nonmandatory, validated web-based survey, comprising questions answered using a 10-point Likert scale and one qualitative open-ended question. For comparison and to provide further insights, 17 fourth- to sixth-year students (advanced learners) were prospectively recruited to participate in both the e-learning activity and the evaluation. A mixed methods analysis was performed.P<.001), clarity of objectives , and attainability of objectives . Both groups showed high interest in the inclusion of the activity as part of a blended learning approach; however, there was low interest in the activity being the sole means of teaching.A total of 88 (63%) out of 141 novice learners and all advanced learners responded to the evaluation survey. Advanced learners reported significantly higher satisfaction with the e-learning activity (mean 8.7, SD 1.0 vs mean 7.3, SD 1.8; Case-based e-learning activities might be better suited for advanced learners and could increase learners’ satisfaction within a blended teaching instructional design. More research on students’ satisfaction with e-learning activities in the field of clinical skills education should be done. In addition, more effort should be put into finding alternative teaching tools for clinical skills education in light of the ongoing COVID-19 pandemic and future health crises. Nephrotic syndrome, a potentially life-threatening clinical disorder, occurs due to the increased permeability of the basal membrane of the renal glomerulus, which could be caused by different etiologies. It is more common among children; however, it can occur at any age . Due to In the medical faculty of the University of Geneva, Switzerland, a 2-hour session on teaching mentioned elements of nephrotic syndrome is typically held in a small group (10-12 students) and uses problem-based learning (PBL) based on a clinical vignette. Due to the ongoing COVID-19 pandemic, in-person teaching activities at our institution have been canceled. Videoconference-based lessons did not yield student engagement during the sessions. Therefore, our team sought to design an alternative learning activity to replace the traditional way of teaching nephrological semiology.Electronic learning (e-learning) modules have been shown to be as effective as traditional teaching  and to iRedesigning our traditional activity as an e-learning module could therefore provide several advantages both in the context of the current pandemic as well as for future teaching activities. Students will have a didactic and validated source of information that meets the expectations of their educators. With the ubiquity of smartphones and other internet-connected devices -10, studWhile several recent systematic reviews and meta-analyses provide evidence on the potentially beneficial nature of e-learning modules compared to traditional learning in terms of knowledge acquisition and clinical performance , importaIn our paper, we aimed to prospectively study second-year (out of six) medical students’ satisfaction and perceptions of an e-learning module designed to replace the traditional teaching of nephrological semiology and compare the results to those of more advanced learners undertaking the same activity and evaluation. Our main hypotheses were that students would consider e-learning engaging due to the use of rich media and flexible since they would be able to perform it asynchronously, and that the proposed instructional design would be preferred to videoconference-based lessons.As the study was based on the analysis of anonymous data, no ethics committee approval from our institution was required.In the medical faculty of the University of Geneva, Switzerland, clinical skills teaching begins in the second year of medical school and continues in a transversal fashion throughout the whole 6-year curricula . TeachinWe prospectively included all second-year medical students (novice learners) from our institution. It should be noted that due to the COVID-19 pandemic, these students have never experienced in-person PBL classes. As data were anonymous, we do not have precise baseline characteristics; however, the students’ ages ranged from 20 to 27 years.For comparison and to provide further insights, 17 fourth- to sixth-year medical students (advanced learners) were invited to undertake the same e-learning activity and evaluation. Of importance, all advanced learners previously participated in the same activity in its traditional in-person PBL form during their second year of medical school.The e-learning activity was designed using the latest evidence-based teaching methods. Instructional design was based on self-directed learning using a case-based learning activity of a patient presenting symptoms suspected of nephrotic syndrome. The learning objectives, which are listed in Students were provided with reading material to review before completing the e-learning module. The activity concentrated on history taking, clinical examination, differential diagnosis, laboratory examination, and treatment plans for a patient presenting with symptoms suspected of nephrotic syndrome.The estimated time to complete both the readings and the e-learning module was approximately 2 to 3 hours long in a self-paced manner. Students were given 2 weeks to complete the module.The e-learning module was created using Rise Articulate 360  . The actAt the end of the module, students were provided with self-assessment multiple-choice questions based on knowledge acquired through the module.All students were given the opportunity to complete a 13-question survey. Survey questions were based on a validated tool   and weret test for means. Stata, version 16 (StataCorp LLC), was used for all statistical analysis [P<.05 was used to indicate significance. Qualitative data were analyzed using a thematic analysis approach.A mixed methods analysis was performed. Quantitative data were presented as mean (SD). We compared data between the 2 student groups using the analysis . A two-sIn total, 141 second-year medical students (novice learners) and 17 advanced medical students (advanced learners) undertook the e-learning activity. Among the novice learners, 88 (62%) students responded to the web-based survey, of whom 81 (92%) said they had completed the activity using their personal computer, 9 (10%) used a tablet, and 1 (1%) used a smartphone. Among advanced learners, all 17 students participated in the evaluation, of whom 12 (71%) used their personal computer and 5 (29%) used a smartphone to complete the activity.P<.001; user-friendliness: mean 9.0, SD 1.1 vs mean 7.6, SD 1.8, P<.001). Similarly, clarity and attainability of learning objectives were rated significantly higher among advanced versus novice learners . Both groups showed moderate to low preference for e-learning activities compared to videoconference-based activities, though e-learning preference was significantly higher among the advanced students . Both groups showed a low and similar preference for in-person traditional PBL learning over the proposed e-learning module . However, while significantly higher among advanced learners, both groups saw a high to extremely high need for the e-learning module to coexist and be complementary to traditional learning activities . Both groups appreciated the integration of rich media in the e-learning module, with significantly higher satisfaction among the advanced learners . Both groups highly and similarly appreciated the possibility to undertake the learning activity in an asynchronous fashion  and moderately appreciated the lack of need to physically attend the class .Both novice and advanced learners’ survey results are summarized in A total of 22 (25%) novice learners and 11 (65%) advanced learners answered the open-ended comments section. Most comments from novice learners concentrated on three main themes. Students’ reflections are summarized in During the postactivity Zoom session, the main themes discussed in the open-ended comments section were repeated by novice learners. Objective appreciation by the teachers leading the activity, however, indicated good attainability of the learning objectives, as manifested by the pertinent questions raised by students about the material and a good level of group discussion throughout the session.Our study examined the satisfaction and perceptions of novice and advanced learners from an e-learning module designed to teach nephrological semiology as part of a mandatory clinical skills seminar targeted at second-year medical students in the medical faculty of the University of Geneva, Switzerland, during the COVID-19 pandemic. We provide several important insights. First, advanced learners showed significantly greater satisfaction with the e-learning activity and seemed to prefer this format over videoconference-based activities. Second, both novice and advanced learners seemed highly opposed to the idea of replacing in-person PBL activities with e-learning as the sole means of teaching but presented high interest in having the e-learning module as part of a blended learning approach. Finally, the integration of rich media seems to improve satisfaction and engagement for both novice and advanced learners in the presented e-learning module.Several studies have already examined undergraduate students’ satisfaction and acceptability of e-learning–based activities ,22 and hIn fact, the current outbreak caused a major disruption within higher education and forced many institutions to revisit and reinvent teaching activities with the upscaling of online learning, as in-person teaching was not allowed for sanitary reasons . ClinicaIn our study, novice learners showed only moderate satisfaction and attested to only moderate clarity and attainability of learning objectives after following the e-learning activity for nephrological semiology. Advanced learners, however, attested to a significantly higher level of satisfaction, as well as clarity and attainability of objectives, from the same activity. This could be explained in several ways. First, our instructional design was based on self-directed learning, which was shown to be more challenging and less accepted by beginners  as they Interestingly, both groups similarly agreed that online-based learning should not replace traditional in-person learning activities, and that blended learning, with the e-learning module as a complement to traditional teaching, could present an important learning benefit. This may be more evidence of medical students’ preference for in-person learning, especially in clinical skills education, as well as for the possible benefits of e-learning modules for life-long learning since this type of activity was highly valued by the students in our study who had already undertaken the activity in its traditional form and might have seen the module as an efficient repetition of already-acquired knowledge. Lastly, both student groups seemed to find asynchronous e-learning to be significantly advantageous in terms of individual time constraints, which could provide insights into the acceptability of this form of learning among medical students and as a means for increasing engagement in certain activities and earlier introduction of self-directed learning in the curricula.Our study has several limitations. The sample size of both groups was small, and the observational nature of this monocentric study could decrease the level of confidence in the results. Moreover, we did not directly compare traditional and online activities in different groups due to the pandemic situation. Finally, we did not assess knowledge retention using a standardized test as this was out of the scope of this study. However, the fact that both the activity and the evaluation were prospectively designed and were based on current evidence-based teaching methods and validated evaluation tools highlights important strengths of our work. The high survey response rate in both groups was another strength of the evaluation and may be indicative of undergraduate students’ motivation to actively participate in this curricular design. Lastly, the evaluation was made on an actual, ongoing teaching activity, which could provide real-world and important insights concerning learners’ satisfaction with e-learning–based activities.In the context of the current pandemic, novice medical students expressed only moderate satisfaction from an e-learning module intended as the sole means of nephrological semiology teaching and showed a clear preference for in-person, problem-based teaching complemented by e-learning for blended learning activities in the future. In addition, case-based e-learning activities might be better fitted for more advanced learners. Additional and larger studies should be performed to assess medical students’ satisfaction with online-based versus traditional learning activities in order to adapt the instructional design of alternative clinical skills teaching among health professionals and to improve preparation and clinical training in the context of future pandemics.