Ficedula albicollis), a passerine with complex song. We also aimed to reveal among‐individual differences in behavioral responses by testing whether individual differences in plasticity were repeatable. The presence of general plasticity across traits and/or contexts was also tested. To assess plasticity, we documented (1) short‐scale temporal changes in song traits in different social contexts , and (2) changes concerning the height from where the bird sang (singing position), used as a proxy of predation risk and acoustic transmission conditions. We found population‐level relationships between singing position and both song length (SL) and complexity, as well as social context‐dependent temporal changes in SL and maximum frequency (MF). We found among‐individual differences in plasticity of SL and MF along both the temporal and positional gradients. These among‐individual differences in plasticity were repeatable. Some of the plastic responses correlated across different song traits and environmental gradients. Overall, our results show that the plasticity of bird song (1) depends on the social context, (2) exists along different environmental gradients, and (3) there is evidence for trade‐offs between the responses of different traits to different environmental variables. Our results highlight the need to consider individual differences and to investigate multiple traits along multiple environmental axes when studying behavioral plasticity.Individual animals can react to the changes in their environment by exhibiting behaviors in an individual‐specific way leading to individual differences in phenotypic plasticity. However, the effect of multiple environmental factors on multiple traits is rarely tested. Such a complex approach is necessary to assess the generality of plasticity and to understand how among‐individual differences in the ability to adapt to changing environments evolve. This study examined whether individuals adjust different song traits to varying environmental conditions in the collared flycatcher , whether individuals adjust different song traits to varying environmental conditions. We recorded a population‐level relationship between singing position and song length (SL), and repeatable among‐individual differences in plasticity of SL and maximum frequency along both the temporal (order) and positional (singing position) gradients. Our results show that the plasticity of bird song exists along different environmental gradients and that it may potentially evolve.This study examined in the collared flycatcher ( We evaluated whether plasticity covaries between traits and between environmental gradients at the individual level. We also examined if individual differences in song plasticity were repeatable. To assess song plasticity, we documented short‐term variations in different song traits associated with changes in both social and nonsocial environments using the reaction norm approach . The study plot contains approximately 800 artificial nest boxes.The collared flycatcher is a hole‐nesting, long‐distance migratory passerine. Most males arrive earlier (around the middle of April) than females to the breeding grounds and occupy territories that consist of a small area around a natural tree cavity or a nest box. Male collared flycatchers start singing after arriving and usually stop singing after pairing , separated from each other by a few sec intervals Gelter, . Collare2.2Data were collected during the courtship period of the collared flycatcher (within the period from 11 April to 7 May) between 2008 and 2019.First, we captured males and females to be used as stimulus birds to elicit songs from the focal males  a male decoy as a social stimulus, mimicking natural situations associated with territorial intrusion or (2) a female stimulus, mimicking nest box inspection by females during mate‐sampling. The displayed stimulus was chosen based on field constraints (such as the availability of stimulus birds and the capacity of recording experimenters). During the assays, stimulus females were placed on the top of the nest box, while stimulus males were positioned 1.5–2.0 m away from the nest box. The different positions were necessary to represent better the different natural situations mimicked with the stimuli. The focal males usually stopped singing and fled away when we approached the nest box. They were exposed to the stimuli for 5 min after returning to their territory. This time period has been reported to be sufficient for the focal male to interpret the situation as a visit from a prospecting female or a territorial intrusion . We determined the age of males  based on their plumage . A total of 98 recordings  were made for 85 males, after presenting male stimuli, 46 recordings (1092 songs) were made for 45 males after presenting female stimuli and 73 recordings (2046 songs) were recorded for 66 males without a stimulus.Three independent song traits with biological relevance in the study population were used during the statistical analysis. Specifically, we considered for each song the song length (SL), maximum frequency (MF), and short‐term complexity (hereafter complexity), which probably play a role in inter‐ or intrasexual communication in the study species  software. We chose only songs for which the spectrograms were clearly distinguishable from the background noise. Syllables were manually selected and from the syllable segments we automatically extracted five spectrographic features with the Ficedula Toolbox , applying the reaction norm approach  and date of the recording (as control variables), the order of songs within the recording in interaction with context  and singing position. The random part of the model contained year, focal bird identity, a random slope over the order of songs and over the singing positions for focal bird identity with correlations between random intercepts and slopes. The whole random structure for individual identity  was estimated separately for the three contexts in which the recordings were made, as we expected context‐based patterns based on our previous research  in song traits within which we had found among‐individual variability in the random slopes. In the repeated measures scenario, we had 77 recordings from 37 birds from different days or years . We only used data from birds where the repeated measurements were recorded in the same context (24 out of 37). Repeatability was calculated with a two steps procedure using univariate LMMs including only individuals with repeated song recordings. We used a two‐step procedure instead of more complex models due to our modest sample size in terms of individuals with repeated data. First, we built two models similar to those described above for each song trait–context combination where we had found variation in random slopes, one for the first and one for the second recordings of the same birds. Age, date, the order of songs, and singing position were the fixed effects, while year and identity  were included as random effects. All posterior samples for the slope estimates for each individual were extracted. Second, we built models using individual slope estimates  as the response variable, order of recording as a fixed factor, and individual identity as a random effect. Based on the posteriors of the latter models, we calculated repeatability , and the 95% credible intervals.2.4.3− or p+), which is the proportion of posterior samples of the median sign   and singing position. The response of SL to the order of songs was significantly different in the female and no stimulus context compared to that in the male context. Specifically, males did not change their song in the male context, but sang longer songs gradually after removing the female stimulus and over time in the no stimulus context Table . Also, t3.2Regarding SL, we found among‐individual differences in plasticity for the order of songs in the male stimulus and no stimulus contexts, and for singing position in the male stimulus context Table . RegardiWe also compared the random intercept and slope variance estimates among contexts and traits Table . We foun3.3p− = 99.7% and −0.711  p− = 99.5%, respectively, showing negative relationships between the plastic responses of the same or different traits to different environmental factors.In the recordings of males after they were exposed to male stimuli, we found significant covariance 1) between the random slopes for the order of songs and singing position in SL, and (2) between random slopes for singing position in SL and for the order of songs in MF  at both the population and individual levels. Among‐individual differences in plasticity of SL and MF in response to the order of songs or to singing position seemed repeatable in certain contexts, although the estimates had wide credible intervals. In contrast, there was no evidence for consistent variation across recordings for song complexity. Among‐individual variance in plastic responses of song in social contexts may have implications for reproductive success , for changes in the song types used throughout the breeding season  and zebra finches (Taeniopygia guttata), for example, sang shorter songs to females  for minimum frequency ; Investigation ; Methodology (lead); Visualization (lead); Writing – original draft (lead). David Canal: Investigation ; Writing – review & editing . Gergely Hegyi: Investigation ; Writing – review & editing . Katalin Krenhardt: Investigation ; Writing – review & editing . Miklós Laczi: Investigation ; Writing – review & editing . Gábor Markó: Investigation ; Writing – review & editing . Gergely Nagy: Investigation ; Writing – review & editing . Balázs Rosivall: Investigation ; Writing – review & editing . Eszter Szász: Investigation ; Writing – review & editing . Sándor Zsebők: Data curation ; Investigation ; Resources ; Writing – review & editing . László Zsolt Garamszegi: Conceptualization (lead); Funding acquisition (lead); Investigation ; Supervision (lead); Writing – original draft (supporting); Writing – review & editing .Supinfo S1Click here for additional data file.


A transmural esophageal tear complicated by large size and unusual location was diagnosed in a 59‐year‐old man after undergoing pneumatic balloon dilation for achalasia. It was closed with six endo‐clips. The patient recovered and was discharged with ordinary diet 8 days later, after receiving supportive care for a week. Complicated esophageal perforations could also be managed with endoscopic intervention. As this depends on timely diagnosis, our key clinical message is that a careful endoscopic evaluation of the area and physical examination after PBD could obviate the need for later open surgery, reducing the morbidity associated with post‐PBD esophageal perforation. Being followed by a significant decrease in LES pressure, PBD has shown to be associated with a remarkable resolution of achalasia symptoms. In <5% of cases, however, PBD leads to esophageal perforation—a complication linked to severe morbidity.2In April 2018, a 59‐year‐old man presented to the gastroenterology clinic of the Alzahra University Hospital complaining of a progressive dysphagia to liquid and solid diet. The onset of his symptoms dated back to 2 years before presentation, when he started to experience a gradually worsening sensation of chest pain and retrosternal fullness when consuming solid food. Ever since, he mentioned that he had to eat very slowly and consume a lot of fluids with his meals. As his symptoms progressed, he experienced the same sensations while consuming liquid diet as well.Upon presentation, physical examination and laboratory tests were suggestive of a mild anemic state and otherwise unremarkable. Initial endoscopic evaluation revealed evidence of esophageal candidiasis along with a severe constriction in the LES region. His esophageal candidiasis resolved 7 days later after treatment with oral fluconazole . He was eventually diagnosed with achalasia based on typical findings in his history of illness, follow‐up upper gastrointestinal endoscopy, and upper gastrointestinal X‐ray series  and propofol . His vital signs were being monitored throughout the procedure by the team of anesthesiologists and gastroenterologists who were performing it. Under direct endoscopic vision, the scope was passed to the stomach and second portion of duodenum, facilitating the placement of a guide wire in the gastric antrum. A 30 mm achalasia balloon dilator (Boston Scientific) was thereafter, passed into the esophagus on the guide wire. After being placed in the proper site under direct endoscopic vision, the balloon dilator was inflated for 60 s with 10 PSI pressure per protocol. It was then successfully deflated and withdrawn. Immediate endoscopic inspection after dilation revealed what appeared to be a 3 cm linear, deep mucosal tearing at the left lateral side of the lower esophagus. Closer inspection revealed that it was a complete transmural tear of the esophageal wall. Transmural esophageal perforation was immediately diagnosed and confirmed, after the patient developed subcutaneous emphysema at the anterolateral sides of his chest and neck. Although large, the tear was linear and seemed amenable to endoscopic repair; hence, the team decided to attempt for an endoscopic closure of the tear using endo‐clips. First, the distal margin of the tear was closed to prevent any further widening, and then, it was closed in an upward direction. A total of six clips were used to close the entire tear , and was prescribed with intravenous (IV) fluids and antibiotics. There, he had subcutaneous emphysema in his chest, neck, and face. A subsequent chest radiograph 4 h after the procedure revealed left pneumothorax, which was managed with a chest tube placed in the fifth intercostal space. He was then put on mechanical ventilation while being closely observed in the ICU. 24 h later, he regained spontaneous ventilation. He was therefore, extubated, and did not show any sign of respiratory distress thereafter. After 6 days of parenteral diet upper gastrointestinal X‐ray series and computer tomography studies were performed, revealing no leakage. Hence, oral liquid diet was initiated, which was well tolerated. On day seven post‐PBD, regular diet was resumed, which was well tolerated as well without any complications. On day eight post‐PBD, after a follow‐up chest radiograph confirmed complete expansion of the left lung, the chest tube was removed and replaced with a petroleum sterile gauze dressing, and the patient was discharged. In a follow‐up visit in July 2018 (3 months post‐PBD), the patient reported complete resolution of his achalasia symptoms, without any further sequelae.3We reported a case of a PBD‐associated transmural esophageal tear with a large size and in an unusual location causing pneumothorax and subcutaneous emphysema. The tear was managed with endoscopic clipping and supportive care and the patient recovered without sequalae in a week. This case could be regarded as a replicative highlight of the effectiveness of endoscopic intervention for management of esophageal perforationFurthermore, we reviewed all of the reported cases of post‐PBD transmural esophageal perforations in MEDLINE and Google Scholar using the keywords “Pneumatic balloon dilation,” “achalasia,” “transmural tear,” and “esophageal perforation”—with proper Boolean operators and specific vocabulary considerations. Conservative management of esophageal perforation with IV fluids and antibiotics has been effective with minimal recovery times in contained perforations.4We reported our experience in managing a complicated PBD‐associated esophageal perforation case with endoscopic clipping and supportive care. Compared with other PBD‐associated esophageal perforation cases reported in the literature which were managed with endoscopic interventions, our case was unique in terms of its associated complications, that is, the location and size of the tear, and subsequent development of pneumothorax and subcutaneous emphysema. In addition to surgery—and subject to confirmation by stronger studies—endoscopic intervention could be regarded as a safe and effective option for the uncomplicated PBD‐associated esophageal perforations, as well as the cases with the mentioned complications. As the usage of this method depends on prompt diagnosis, the key clinical message in this case is that an exhaustive endoscopic inspection of the area along with physical examination after PBD could obviate the need for later open surgery, reducing the morbidity associated with post‐PBD esophageal perforation.MM has managed the case and gathered the relevant case information, validated the findings, and drafted the initial draft. NS has searched databases for relevant data, reviewed the relevant literature, and reviewed and edited the manuscript. Both of the authors have reviewed the final manuscript and approved it for publication.In accordance with national guidelines, a written informed consent was obtained from the reported patient for publishing his anonymized case along with any unidentifiable images.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS‐CoV‐2 can spread irrespective of a patient's course of disease, these institutions’ continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS‐CoV‐2 screening facilities with a built‐in biosafety level (BSL)‐2 laboratory were set up to allow the testing offer to be brought close to the subject group's workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build‐in laboratory with two similar stations commencing operation until June 2020. During the 15‐month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios. BSLbiosafety levelHAhigh availabilityHIShealth information systemLIMSlaboratory information management systemPCRpolymerase chain reactionQRquick responseRKIRobert Koch‐InstituteRNAribonucleic acidRT‐qPCRreverse transcription‐quantitative PCRSIDspecimen identifierSOPstandard operating procedureVMvirtual machineVPNvirtual private network1The first year of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic was characterized by serious long‐term impacts on society and the economy. The rapid spread of SARS‐CoV‐2 is intensified by asymptomatic carriers and pre‐symptomatic transmission . MedicalPopulation screening has to provide sufficient accessibility and throughput capabilities, resulting in a high test frequency  with fast sample‐to‐answer times in order to represent effective measure for the assessment and control of virus spread . VariousMany university research laboratories provided SARS‐CoV‐2 molecular diagnostic testing in response to the pandemic and test capacity shortage , 11–13. The project described in this article was intended to create additional testing capacity using university facilities, staff members, and their expertise. The project was conducted at a university without a medical school, but was supported by individual institutions of a medical university in planning, organization, and medical guidance. At the outset of the project, a sufficient number of biosafety level (BSL)‐2 laboratories for the ribonucleic acid (RNA) isolation upfront of a PCR analysis were not available on campus. Mobile screening facilities for both specimen collection close to the subjects’ workplace and RNA isolation in an integrated BSL‐2 laboratory were developed to address this issue. The pre‐processed, noninfectious samples were distributed among the university's labs for PCR analysis. A custom health information system (HIS) and laboratory information management system (LIMS) were developed to support the process flow of collecting subject data and tracking and reporting samples as well as analysis results to local public health departments and subjects. The utilized screening facilities, diagnostic methods, HIS, and LIMS underwent constant development toward a more automated workflow during the project. This development includes the addition of test centers for antibody screening indicating a subject's previous SARS‑CoV‑2 infection and its immune response to infection and vaccination. The implementation of serological testing was undertaken to provide insights into the seroconversion of subjects and record the number of subclinical cases of SARS‐CoV‐2 infections. All proceedings were subject to extensive compliance with regulatory directives, mainly including certification of the diagnostic laboratory and fulfillment of health‐related data policies.2In order to ensure timely implementation and start of operation of the screening facilities and diagnostic laboratory, the various tasks were divided among specialized working groups and university institutions. Figure FIGURE 1Processes displayed by material and data workflow. (A): Representation of material and data flow. Prepared test‐kits for PCR and antibody testing (a) were provided to screening facilities, where subject data was entered and samples  were collected (b). Swabs were handed to a BSL‐2 laboratory for RNA isolation (c), followed by PCR analysis (d). Serum samples were used for antibody detection analysis (e). Lab processes were controlled by the LIMS, which also stored raw analysis data (f). Raw analysis data was evaluated and confirmed results (g) were sent to the HIS, upon which sample result and subject data were merged (h). Positive PCR test results were submitted to the health department and a medical staff member informed the subject, whereas all other test results were sent to the subject via e‐mail (i). (B): Implementation of the laboratory and digital infrastructure processes. Subject and sample admissions started with manually entering or recalling a subject's personal information  from a previously handed‐out QR code. Subsequently, the acquisition of voluntarily given answers to medical study questionnaires in the HIS data entry software occurred. Subjects were then given printed handouts with their QR code for future visits and a consent form to be signed before taking either an oropharyngeal swab or a blood sample. The collected sample was pseudonymized with an identifier label from a pre‐assembled test kit, and the completed dataset was stored in the HIS database. Depending on the type of specimen, samples were then handed to the appropriate laboratory for analysis. Upon retrieval, the arrival of samples was documented. For PCR and antibody analysis, plate and rack information were created by scanning and sorting samples. Analysis results were evaluated and confirmed by lab managers. If no valid result was achieved and a reserve sample was available, analysis was performed again. Confirmed results (including the need for sample recollection) for each pseudonymized sample were sent to the HIS database for report generation and informing subjects. In case of a positive PCR test result, a medical staff member received the subject and sample information to contact the subject directly. At the same time, the report was sent to the public health departmentPopulation screening for SARS‐CoV‐2 presents one possible means to control infection spreading. PCR tests remain the gold standard for the detection of an acute SARS‐CoV‐2 infection. University‐based diagnostic laboratories can provide much required PCR testing capacities due to their expertise and equipment. This article summarizes and shares how screening facilities and diagnostic laboratories can be set up and operated under the difficult conditions  of a pandemic. An important feature of the screening facilities described is their mobility, which allows them to be set up flexibly close to groups of test subjects. The described integration of a BSL‐2 laboratory into a container‐based screening facility enables the expansion of laboratory capacities. The modular description of the laboratory methods used and the setup of the  infrastructure enables adaptation based on existing expertise and equipment. The software and documentation developed as part of this work are publicly available online to assist others in setting up further studies .2.1Main challenges—especially during the initial phase of the SARS‐CoV‐2 pandemic—included the supply and provisioning of consumables and safety equipment. These tasks were therefore implemented and maintained in‐house. This mainly included the in‐house production of test kits and disinfectants after a change in the corresponding legal basis . TranspoTo uniquely identify specimens, custom adhesive labels were provisioned. Each label contains a unique specimen identifier (SID) in both a human‐readable and a machine‐readable quick response code (QR code) format for automatic data entry using a handheld scanner. The SIDs are defined as unsigned integers encoded using a character set based on the RFC3548 specification . Only unTest kits with prelabeled consumables specific to the performed analysis were prepared in‐house. PCR test kits consisted of labeled reaction tubes and RNA columns placed in clear zip bags; test kits for antibody detection only contained labeled reaction tubes. Each test kit also included additional adhesive labels for uniquely identifying swab and serum gel tubes directly after sample collection. Swab tubes and serum gel tubes were not included in the prepackaged test kits and instead provided in bulk packages at the testing location to account for repeated sample collection in case of initial failure. The correct assembly of test kits was recorded in a standard operating procedure (SOP) and each assembled kit was digitally verified by scanning its content. To ensure RNase‐free assembly, all work areas used were cleaned and disinfected prior to test kit packaging, including RNaseZap .2.2The targeted population groups for PCR screening were selected regarding an increased infection risk based on professional activity and continuity of the provided medical care structure. In this regard, employees of medical workplaces , educational institutions, and companies of systemic importance were offered participation in the PCR test offer. Invitations were specially extended to subjects meeting the following guidelines recommended by the Robert Koch‐Institute (RKI): being over 50 years of age, having comorbidities , or taking immunosuppressants  . IndividIn times of high SARS‐CoV‐2 incidence, subjects were invited for weekly participation in the PCR test offer, with biweekly tests recommended during phases of low incidence. A pharyngeal smear was performed for PCR testing by swabbing the region between the anterior and posterior palatal arches on the subject. Most throat swabs were performed at a mobile screening facility by trained medical personnel. At nursing and retirement homes, swabs for PCR testing were taken in collaboration with physicians and senior staff members and were collected on‐site and picked up for further processing. Teachers at three schools provided self‐collected pharyngeal swabs after receiving appropriate in‐person and video supported training. Subject participation in antibody detection tests was offered twice in the autumn of 2020 and spring of 2021. Blood samples for detecting SARS‐CoV‐2‐specific antibodies were taken from the cubital vein by certified medical personnel. After informed consent, samples, subjects’ contact information, and clinical parameters were collected.Furthermore, subjects were questioned on each day of testing whether COVID‐19 symptoms or risks regarding SARS‐CoV‐2 infection were or had been present. Later, vaccination status of subjects was also inquired. These additional parameters were answered voluntarily at each appointment to support epidemiological research. During the first time being tested, subjects received a printout featuring an encrypted QR code containing the provided contact information to accelerate data entry during subsequent tests. After sample collection, the labeled sample tube  and its respective test kit were then passed to the appropriate laboratory for analysis. By physically separating subject information and the SID at this point, only pseudonymized samples were given to the laboratory.2.3To ensure a high acceptance and participation of the target population groups, mobile container‐based screening facilities were developed to be placed as close as possible to the targeted population while also providing the required BSL‐2 laboratories on‐premise. Each screening facility consisted of two intermodal (“shipping”) containers connected along their longitudinal side. Figure One major challenge for container‐based laboratory setups is compliance with the appropriate BSL and ISO requirements . Following BSL‐2 specifications, all interior surfaces, including the selected technical appliances such as computers, keyboards, and handheld scanners used for sample tracking, were made out of easily sterilizable materials. To further adhere to a BSL‐2 containment level, waste was stored in designated containers placed in each section which were regularly cleared and decontaminated prior to disposal.One container comprised the main staff entrance leading into a hygiene lock, featuring two medical and laboratory staff changing room areas. This container also stored consumables  and a secured server rack  according to the manufacturer's instruction. The swabs obtained by an oropharyngeal smear were immersed in lysis buffer. 50% of the resulting suspension of the analysis material was subjected to RNA isolation according to Chomczynski and Sacchi, leaving a reserve sample for retesting . All steTo increase throughput, semi‐automatic RNA isolation using Qiagen's QIAcube  was tested during the further course of the project since the underlying RNA isolation method remained unchanged. Only slight modifications of the established SOP were required, and simultaneous isolation of 12 specimens was possible.Later on, a predominantly automatic workflow for RNA isolation was implemented using the MagMAX Viral and Pathogen Nucleic Acid Isolation Kit by Thermo Fisher  alongside the automated Auto‐Pure 96 Nucleic Acid Purification System by Allsheng . This workflow only requires the swab to be placed in RNA lysis buffer in the container‐based BSL‐2 laboratory. This step inactivates present RNases and stabilizes intact viral and human RNA. After this step, the specimen is no longer considered infectious and can be transferred to a BSL‐1 laboratory for automatic RNA isolation. Reformatting of the prepared lysate from reaction tubes to 96‐deepwell‐plates was performed with an epMotion 5073 Liquid Handling System by Eppendorf , followed by the automatic RNA isolation using the Allsheng purification system. For PCR analysis, the isolated RNA was transferred into reaction tubes using an epMotion or an rLINE Robotic Liquid Handling Dispenser Module by Sartorius .Direct detection of SARS‐CoV‐2 RNA by real‐time reverse transcription‐quantitative PCR (RT‐qPCR) is considered the gold standard in diagnostics. Following the protocol described by Corman et al. and recommended by the WHO, RT‐qPCR was performed using TaqMan Fast Virus 1‐Step Master Mix by Applied Biosystems  , 25. SAR2.5To detect SARS‐CoV‐2‐specific antibodies in serum, two different qualitative and complementary detection methods were used: the electro‐chemiluminescence immunoassay Elecsys Anti‐SARS‐CoV‐2 by Roche Diagnostics  and the microarray‐based immunoassay SARS‐CoV‐2 ViraChip IgG Test Kit by ViraMed Biotech AG . Elecsys was used to detect IgM and IgG antibodies against the virus’ N antigen. The second test was performed utilizing the ViraChip test kit detecting IgG antibodies against S1, S2, and N virus antigens. Seropositivity was declared when at least one of these antibody types was present.Elecsys Anti‐SARS‐CoV‐2 was performed with a cobas e411 analyzer by Roche Diagnostics  using manufacturer's materials and reagents according to manufacturer's instructions directly after serum preparation via centrifugation in serum gel tubes. Apart from sample preparation, analysis using the cobas e411 was fully automated. For the SARS‐CoV‐2 ViraChip IgG test performance, samples were manually transferred into reaction tubes and then diluted by a pipetting robot  before manual washing. To increase throughput, the washing of samples was later automated using a Hydroflex Washer by Tecan . Test readout was performed with ViraChip Scanner and analyzed with ViraChip Software . Antibody detection was set up approximately 4 months after the project started. Sample tracking and analysis report was implemented based on the previously established LIMS and HIS. The detailed procedures for detecting SARS‐CoV‐2‐specific antibodies are described by Jonczyk et al. , 28.2.6To account for the specific setup of laboratory and analysis methods, it became necessary to develop a custom LIMS rather than licensing an established LIMS at the project's start. LIMS and HIS development were intentionally split into discrete, stand‐alone software applications. This separation allowed for independent scaling of each application while also facilitating the prospective replacement or integration of custom applications with established LIMS or HIS products.The LIMS is designed to track every sample, well position, plate, and raw RT‐qPCR and antibody analysis data. The LIMS comprises a document‐based database to store all information. Laboratory routines were aided by software defined processes and direct integration and control of laboratory devices into the LIMS. Users were guided through the necessary manual steps defined by the respective SOP of each analysis by a laboratory user software connected to the database. They were required to confirm steps taken before continuing.Upon arrival in a laboratory, samples were registered before further processing. The SIDs on labeled specimen tubes are scanned using a handheld scanner and linked to well positions on a plate to accession samples. The LIMS generates a plate definition file containing the SID for each sample loaded into the analysis software that drives the qPCR or ViraChip scanner. Following laboratory analysis, raw data files obtained from laboratory devices are ingested into the LIMS, and each sample was evaluated, resulting in a positive, negative, inconclusive, or invalid result. Following review by senior laboratory scientists, results are confirmed for further processing by the HIS.2.7A custom HIS was developed due to the limited time available and desired flexibility of the data acquisition required to enter subject information and link subjects to their respective specimens. The development of the custom HIS was initially focused on utility software used in test kit production and a graphical data entry system deployed at the mobile screening facilities. Printing of the customized adhesive labels was centralized to ensure uniqueness and therefore unambiguousness of provisioned SIDs without the need for sample collection workstations to maintain a constant connection to a centralized server or configuration overhead with the implementation of a local SID provisioning service. This feature allowed subsequent collaborating external laboratories to report lab results in a consistent namespace. Central printing of labels also reduced operating costs instead of printing labels on‐site.The data entry front‐end system facilitated the acquisition of subject‐specific information such as personal data and state of health and linked the collected specimen to a single subject. Subjects received printouts of the recorded personal data, SID, and legal information during their test appointment. These printouts contained a subject‐specific and encrypted QR code to allow quick recall of subject information during subsequent appointments without requiring a connection to the database system. Acquisition of personal data was initially determined by RKI requirements regarding correct reporting of subjects who tested positive for SARS‐CoV‐2 via PCR . ThroughThe related subject information and clinical data were stored in text‐based files comprising key‐value pairs for the properties of each specimen. This approach ensured compatibility with future additions or alterations to the required data collection. During this initial stage of data entry, the related files were stored locally on encrypted drives and manually backed up. The data entry software ensured complete and conflict‐free storage of files. Files were manually collected from workstation clients for further processing. Upon completion of the laboratory analysis, the result files provided by the LIMS were matched with the respective HIS file in a spreadsheet application and checked twice, followed by manual test result notification.For PCR tests, subjects received an electronic mail reporting non‐positive results. In case of inconclusive or invalid results, subjects were invited to provide an additional sample. Positive PCR test results were disclosed to medical team members, which then communicated positive results to the subject via phone, emphasizing fast compliance with the required steps mandated by health authorities . In Germany, a secure digital interface for reporting positive test results was not provided, which required the transmission of positive PCR test results to local health authorities to be performed manually by telefax. A written result was sent electronically to each subject for reporting antibody screening results. Antibody screening results were not required to be disclosed to the authorities.Advancements towards an automatic workflow of processing lab result reports were made by first automating file transfer from workstation clients to data storage servers with redundant storage and automated backups. Both HIS files and lab results directly obtained from LIMS were periodically parsed into a SQL database. Scripted queries to the database facilitated automation of test result reporting to subjects. Automated reporting of positive PCR test results to local health authorities was set up using a secure online faxing server. The initial manual processes remained valid operation modes throughout the project course to provide fallback methods, for example, in case of connectivity loss or required maintenance. Given collaborations with external laboratories, an import module for structured data was added to the HIS to allow for the import of result files.2.8Similar to the provisioning of consumables and safety equipment, the acquisition of technical equipment proved challenging during the initial stage of the SARS‐CoV‐2 pandemic. The project's digital infrastructure relied on stand‐alone encrypted clients and manual data backup and transfer tasks in the first few weeks. Once the availability of hardware components improved, the digital infrastructure was scaled to a reliable server setup. Figure LIMS and HIS databases were deployed using virtual machines (VM) on centralized servers. A set of three hypervisor servers with shared storage was set up to provide a high availability (HA) environment featuring live migration of VMs to ensure uninterrupted operation even during server maintenance. Additional servers were deployed for regular backups. Servers were distributed among different buildings and backed by uninterruptible power supplies (UPS) to eliminate any single point of failure or data loss in case of power outages. Physical access to servers was limited to system administrators. Staff members only received access to the deployed clients upon completion of a technical briefing and data regulations and safety training. Authentication of users was governed by domain‐based user administration.An HA firewall protected internal networks that allowed granular access from external authenticated devices and users using virtual private network (VPN) connections. Unidirectional flow of subject‐related data from remote locations  was established. All client‐to‐server communication was encrypted. Container‐based screening facilities included a locally placed encrypted server with redundant storage for temporary caching of files and mirroring of domain information. This ensured stable operation in the cases of poor internet availability on‐site.2.9The laboratory operation was subject to extensive quality management and compliance with applicable policies and guidelines to establish and maintain the required quality, safety, and performance in the use of diagnostics, and to ensure the reliability of the results obtained with them. Certification of the laboratory operation was achieved in order to be able to report the obtained results to subjects and local health authorities. For this purpose, a quality management system in accordance with the “RiLiBÄK” directive  was established. This directive describes basic requirements  for quality assurance.The safety concept and the prerequisites for operation were precisely documented in SOPs, risk assessments and hygiene plans, and checked and confirmed by the responsible occupational safety department  and the trade supervisory office. Operating instructions were prepared in accordance with the RiLiBÄK specifications for devices, analysis systems, and procedures and released after external review. The instructions were regularly updated and were accessible both digitally and at the workplace for authorized personnel. Before using the respective laboratory methods, especially after method modification, they were verified in at least three independent test runs with samples whose test results were known. The verification of RNA isolation and PCR analysis was performed using 30 samples, the test procedures of the antibody module were each verified with 260 samples of different origin. To assess the quality of the PCR analysis, laboratory operations were tested by an external quality assurance service program for the detection of the virus genom .To ensure reliable operation over time, the guidelines provide for regular maintenance and calibration of the equipment in accordance with the manufacturer's specifications. Quality control for PCR analysis included an extraction control in the form of detecting human RNA for each specimen. Positive and negative controls were added to each plate and verified during PCR analysis. For quality control of the antibody detection using the Roche automated electrochemiluminescence immunoassay, control samples provided by the manufacturer were analyzed twice daily. For the antibody detection using the microarray‐based immunoassay SARS‐CoV‐2 ViraChip IgG Test Kit, control samples from the manufacturer with known results were integrated on each analysis plate. In addition, internal controls  are located in each well by default to perform a functional check for each analyzed sample.A separate data protection concept for handling the collected subject data was drafted and submitted to the appropriate data protection officer for review. It complies with Article 5 of the General Data Protection Regulation (GDPR) and the data protection principles and other data protection requirements set out therein. An ethics vote of the responsible medical association  and Institutional Review Board approved the evaluation of the collected subject data in a pseudonymized form in the context of medical studies .3Less than 4 weeks after the first imposed lockdown in Germany, and despite many issues regarding the provision of required materials , a first mobile container‐based testing station commenced operation in April 2020 . In addi2. A three‐phase power supply was required to operate the test station, which a mobile generator can also provide. The water needed for the operation was provided through water tanks. Subject and sample data was able to be transmitted to a central server via encrypted mobile storage media if no active internet connection was available on‐site. These design considerations allow for a flexible setup even in rural areas. However, the correct and safe implementation of PCR and antibody detection tests is limited to trained personnel, which might become challenging in some areas. Here, the decentralized collection of samples and subsequent transport to a central laboratory presented a common approach that can reduce the demand for biotechnology professionals. The presented setups were used for decentralized sample collection feeding into the container‐based BSL‐2 laboratories for RNA isolation and subsequent transport to campus laboratories for PCR analysis. The lack of centralized BSL‐2 laboratories still created a high need for personnel to operate each laboratory station.The container‐based setup with a built‐in BSL‐2 laboratory enabled high throughput of sample acquisition and processing with minimum requirements posed to the installation site. The shipping containers allowed easy relocation and required a footprint of 33 mThe setup was constantly improved during the course of the project and, in particular, extended to include automation steps regarding laboratory analysis and reporting of results. Due to the forward compatibility considered from the beginning and the iterative improvement of processes, this approach allowed the setup to be launched quickly and scaled at a fast pace. Over time, subjects also became familiar with the PCR testing procedure and the collection of personal data and clinical parameters was significantly accelerated. Here, the personal QR codes, in particular, played a major role, which reduced the initial average of 8 min for capturing subject data and taking the swab to approximately 4 min. With these improvements, at a single mobile container‐based screening facility over 220 tests were collected in 8 h. Swabs taken before noon in a container‐based screening facility close to the university's BSL‐1 laboratories used for PCR analysis could be evaluated the same day. Most other samples could be analyzed within 36 h.The devised screening facilities and diagnostic laboratory were successfully used in the context of two medical studies. Table Figure More than 33,000 PCR tests and close to 7000 antibody detection tests were performed. Over 7200 subjects participated in the PCR test offer, and more than 4000 subjects participated in antibody detection tests. Subjects accepted the offer for PCR testing with varying frequencies. In Cohort 1, the number of PCR tests performed ranged widely between one and 46 tests per subject, with a median of three tests. About 31% of the subjects underwent PCR testing only once during the several months of the project period. For population screening using PCR tests to be an effective measure, it must be coupled with strict precautionary measures  and a high participation rate of subjects at risk . CompareOverall, 53 subjects with acute SARS‐CoV‐2 infection were detected by PCR analysis . After a confirmed positive PCR test in one nursing home, thanks to the quick deployment of a mobile test station, residents and staff members were tested repeatedly, documenting a localized outbreak in late October of 2020  showed a low infection rate among these subjects , the total cost per PCR test within this project was approximately 60 €, divided equally between sample collection (including transport) and lab costs. Despite the added overhead of sample collection on‐site, performing a PCR test within this project was only slightly more expensive than the billable cost  of preventive PCR testing of hospitalized subjects in Germany . Due to Within this project's scope, setups and operating procedures for outdoor and indoor screening facilities were developed. These mobile setups, suitable for a flexible choice of location, made it possible to bring the test offer close to the target populations’ workplace or place of living. Furthermore, the container‐based screening facilities included BSL‐2 laboratories providing additional capacities to process the potentially infectious samples. Table Much of the testing shortfall, especially early in the pandemic, can be traced to a shortage in critical supply chains, personnel required, expertise, or instrumentation necessary to collect samples and perform PCR analysis. University‐based research laboratories can provide much‐needed assistance to increase PCR testing capacity. However, adjusting legal frameworks  can become necessary to allow operation under the difficult conditions imposed by a pandemic. Improving the digital infrastructure in public health can lead to the removal of barriers to enable simplified connection of university‐based research laboratories to the health care system in times of crisis. In addition, government funding often becomes necessary for university‐based research laboratories to account for the extensive costs.4Population screening regardless of the presence of COVID‐19 related symptoms using PCR tests represents a valuable method to limit the spread of SARS‐CoV‐2. Targeted screening of subjects in medical, educational, or system‐critical professions and subjects at high risk of severe disease progression can help to maintain critical infrastructure and reduce mortality rates.This report describes the implementation, continuous optimization, and operation of mobile screening facilities and diagnostic laboratories by non‐medical university research institutions during the first 15 months of the SARS‐CoV‐2 pandemic.In line with implementations and shared experiences from other groups, it is clear that university‐based research laboratories represent an important resource when facing a pandemic scenario. The authors recommend government entities to financially support universities with the capability of adding to the testing capacity and provide the required infrastructure, legal framework, and health policy guidelines to enable the university‐based operation of diagnostic laboratories to effectively combat pandemic scenarios. This report can assist others in quickly establishing similar setups valuable for pandemic response.The authors have declared no conflicts of interest.An ethics vote of the responsible medical association  and Institutional Review Board approved the evaluation of the collected subject data in a pseudonymized form in the context of medical studies .Informed consent was obtained as part of the digital registration process at the mobile testing sites.SUPPORTING MATERIALClick here for additional data file.


Dear Editor‐in‐ChiefWe thank the readers for their valuable and interesting feedback on our recent article “Electrical flash burns due to switchboard explosion”.They point out that modified moist occlusive burn therapy (MMOBT) may be an alternative treatment for electrical flash burns. Although moist occlusive burn therapy (MOBT) and moist exposure burn therapy (MEBT) had been widely used in burn units, the optimal treatment remains controversial. In 2012, Mabrouk et al. found that moist occlusive dressing (Aquacel[®] Ag) significantly improves the management and healing rate of partial thickness facial burns with better long‐term outcome compared with moist open dressing (MEBO[®]). Scar quality was improved in the occlusive group. The frequency of dressing changes, pain and patient discomfort were also reduced in the occlusive group.However, according to the readers' perspective, there are still three shortcomings as follows.First, the number of cases in our study was relatively small, which could reduce the credibility. Actually, due to a low morbidity of electrical flash burns, it is really hard to collect sufficient samples within a short time. Undoubtedly, we promise to incessantly collect as many cases as possible in future studies. It is hoped that we can achieve more substantive results and share more practical clinical experiences.The second shortcoming is the lack of comparison of the safety and efficacy with other topical agents. In general, surgical debridement and covering with burn cream is the primary choice for emergency management of burn injuries. As shown in our previous study, MMOBT is conducted by the combination application of chitosan‐based biogel and a layer of sterile polyethylene film.Electrical injury may result from contact with faulty electrical appliances or machinery or contact with open household wiring or electrical power lines. The severity of electrical injury depends on the voltage, type of current and duration of contact with the source. High voltage always causes severe injuries. Higher than 1000 V is considered as a high voltage level, while lower than 1000 V is considered as a low voltage level.Low‐voltage injuries tend to cause small, deep contact burns at the exit and entry sites. However, high‐voltage injuries can be further divided into true high‐tension injuries and electrical flash injuries.True high‐tension burns are caused by high‐voltage current passing through the body, while electrical flash burns are caused by tangential exposure to a high‐voltage current arc where no current actually flows through the body Figure . ObviousFinally, the third shortcoming could be summarised as three points: (1) conceptual distinction between electrical injury and electrical flash burn; (2) difference between the consequences of high‐voltage current and low‐voltage current; (3) prerequisite condition for the occurrence of electrical flash burns. To illustrate the above issues, our reply is as follows:In summary, we concluded that MMOBT is a promising therapy for the treatment of cervicofacial burns. However, to confirm the superiority of MMOBT, further study is still needed.This study was supported by the Innovative Research Foundation of Changzhi People's Hospital (202005).The authors declare no conflicts of interest.


Successful parenting requires constant inferring of affective states. Especially vital is the correct identification of facial affect. Previous studies have shown that infant faces are processed preferentially compared to adult faces both on the behavioural and the neural level. This study specifically investigates the child‐evoked neural responses to affective faces and their modulation by motherhood and attention to affect. To do so, we used a paradigm to measure neural responses during both explicit and implicit facial affect recognition (FAR) in mothers and non‐mothers using child and adult faces. Increased activation to child compared to adult faces was found for mothers and non‐mothers in face processing areas  and areas associated with social understanding  when pooling implicit and explicit affect recognition. Furthermore, this child‐evoked activation was modulated by motherhood with an increase in mothers compared to non‐mothers in the left precuneus. Additionally, explicitly recognising the affect increased child‐evoked activation in the medial superior frontal gyrus in both mothers and non‐mothers. These results suggest preferential treatment of affective child over adult faces, modulated by motherhood and attention to affect. Increased activation to child compared to adult faces was found for mothers and non‐mothers in face processing areas  and areas associated with social understanding  during implicit and explicit affect recognition. Furthermore, this child‐evoked activation was modulated by motherhood with an increase in mothers compared to non‐mothers in the left precuneus. Additionally, explicitly recognising the affect increased child‐evoked activation in the medial superior frontal gyrus in both mothers and non‐mothers. It is, therefore, not surprising that infants and children are treated differently than adults  combines several processes: one needs to process the visual information, extract relevant features connected to the portrayed information and infer the affective state based on these features. Additionally, affective faces can also induce an emotional reaction in the perceiver. Therefore, FAR combines affective and cognitive aspects of social understanding Kanske, . In GobbWhen parents encounter the face of their own child as opposed to the face of an unfamiliar child, they show increased neural responses in areas of the extended system, including the insula, amygdala, anterior cingulate cortex and precuneus . Additionally, we hypothesised stronger child‐evoked activation in the explicit than in the implicit FAR task due to the increased attention to the affective content of the faces (Hypothesis 3: EXPchild>adult > IMPchild>adult). Lastly, we expected the modulation of child‐evoked activation by motherhood to be decreased in the explicit compared to the implicit affect recognition task (Hypothesis 4: interaction motherhood * attention).This functional magnetic resonance imaging (fMRI) study aimed to investigate neural responses to affective child versus adult faces, henceforth referred to as child‐evoked activation, and potential modulations of this child‐evoked activation. Specifically, we were interested in the influence of motherhood and attention to affect. We adapted an established paradigm . However, ROIs and neuroimaging analyses as well as all Hypotheses 3 and 4 have not been preregistered.The study design, sample size, data collection procedures, behavioural analyses and Hypotheses 1 and 2 have been preregistered prior to data collection  and a grey blank screen was presented between blocks for 9.5 s on average .The FAR task was presented in three runs of 36 blocks each. Each block started with a sentence of either the IMP or the EXP condition, followed by four trials of either adult or child faces . Next, we collected field maps  which were followed by T2*‐weighted echo‐planar imaging (EPI). The EPI sequence measuring brain activation during the FAR task consisted of three runs .All neuroimaging data were collected at the Berlin Center for Advanced Neuroimaging using a 3T scanner . We acquired structural images with a T1‐weighted magnetically prepared rapid acquisition gradient echo . We also included the runs, conditions of no interest and the response times as covariates of no interest. The response time was added to control for differences in response time between conditions. Since we were interested in child‐evoked activation, we created three differential contrasts for each participant: one measuring child‐evoked activation in the EXP task (EXPchild>adult), one measuring child‐evoked activation in the IMP task (IMPchild>adult) and one measuring child‐evoked activation pooled over both tasks (EXP + IMP: child > adult). The pooled differential contrast was used on the second level in a one‐sample t‐test over both mothers and non‐mothers to evaluate Hypothesis 1. The task‐specific differential contrasts were entered in a flexible factorial on the second level, including the factors subjects, motherhood (yes or no), attention to affect (EXP and IMP) and the interaction between motherhood and attention. To evaluate Hypothesis 2, we pooled over tasks and compared ‘motherchild>adult’ with ‘non‐motherchild>adult’ and for Hypothesis 3, we pooled over groups and compared ‘EXPchild>adult’ and ‘IMPchild>adult’. Lastly, we computed the interaction between motherhood and attention to affect in the flexible factorial to evaluate hypothesis 4. We performed a ROI analysis using one single mask to perform a small‐volume correction for all contrasts evaluating our hypotheses. This mask included the following regions : insulae, amygdalae, fusiform gyri, medial superior frontal gyri, precunei and posterior cingulate cortices. The mask was created using MARINA .All analysis of the functional neuroimaging data were conducted based on the general linear model (GLM) in SPM12. First, we specified and estimated a GLM for each participant with four covariates of interest .Generally, the samples of mothers and non‐mothers collected in this study were comparable. Computed Bayesian Mann–Whitney‐3.2BF10 = 3.19e + 20), making it more than twice as likely as the second‐best model, additionally including the factor motherhood , very strong evidence for the protagonist (BFincl = 69.74) and anecdotal evidence for the interaction of task and protagonist (BFincl = 2.39). On the other hand, evidence was revealed against the predictor motherhood , the interaction between task and motherhood , the interaction between protagonist and motherhood (moderate: BFincl = 0.28) and the three‐way interaction . This indicates that even though motherhood did not influence response time, task and protagonist as well as their interaction did, which strengthens the decision to include response time in the first level GLM of the neuroimaging analysis.Response times did not differ between mothers and non‐mothers. However, there were differences due to the task, protagonist and the interaction between task and protagonist, with the IMP task leading to longer response times than the EXP task and children leading to longer response times than adults , medial superior frontal gyrus  as well as the bilateral insulae , therefore supporting hypothesis 1 (child>adult). This child‐evoked activation was significantly stronger in mothers in the left precuneus compared to child‐evoked activation in non‐mothers pooled over both tasks . This partly supports Hypothesis 2 by showing increased child‐evoked activation in mothers in the extended system. However, there was no difference in neural response in areas associated with affective social understanding. Additionally, child‐evoked activation as a response to the EXP task was stronger than in response to the IMP task in the left medial superior frontal gyrus for mothers and non‐mothers combined , partly supporting Hypothesis 3 (EXPchild>adult > IMPchild>adult). Again, modulations affected an area associated with cognitive social understanding but not areas associated with affective social understanding. There were no significant differences in activation in any of the ROIs in the contrasts ‘non‐motherschild>adult > motherschild>adult’, ‘IMPchild>adult > EXPchild>adult’ or in the interaction between child‐evoked activation, motherhood and attention to affect; therefore, Hypothesis 4 was not supported.Both the hypotheses‐guided ROI and the explorative whole‐brain analyses revealed child‐evoked activation in both mothers and non‐mothers, which was further modulated by motherhood and attention to affect  and areas associated with social understanding . This child‐evoked activation was modulated by motherhood and attention to affect: First, mothers exhibited an increase of child‐evoked activation in the left precuneus. Second, child‐evoked activation was increased in the left medial superior frontal gyrus when participants were explicitly asked to recognise the displayed emotion instead of assessing an unrelated physical feature. There was no interaction of both modulating factors on child‐evoked activation. All results were independent of any behavioural differences between mothers and non‐mothers. However, response times in the implicit condition were more prolonged than in the explicit condition as well as longer in response to child compared to adult faces. In conclusion, affective child faces were processed preferentially compared to adult faces, and this child‐evoked activation was modulated by motherhood and increased by directing the attention to the affective content of the faces.This study shows bilaterally stronger activation in both the core and the extended face processing system in response to affective child compared to adult faces in both mothers and non‐mothers, supporting Hypothesis 1. Concerning the extended system, there were effects both in areas associated with cognitive  and affective (insulae) social understanding , with mothers showing increased activation in the left precuneus of the extended system. The precuneus is a vital node in the default mode network (Fransson & Marrelec, both motherhood and child‐evoked activation. For example, non‐mothers may have used hormonal contraception more often and for a more extended period of their life. Studies indicate that hormonal contraception could alter the processing of affective faces (Hamstra, De Rover, De Rijk, & Van der Does, It is impossible to make claims about the direction of the modulatory effect of motherhood on child‐evoked activation based on the here presented data: while it may be the case that motherhood influences child‐evoked activation, it may as well be the case that having a brain system wired for preferential child processing more likely leads to motherhood. There are, however, several arguments to be made in support of the former statement, that is, a possible influence of motherhood on child‐evoked activation. First, longitudinal studies show structural and functional brain changes due to pregnancy and motherhood (Carmona et al., Although mothers and non‐mothers were comparable in most aspects tested in our sample, mothers were more likely to be in a relationship than non‐mothers. In combination with mothers and non‐mothers rating the importance of having children similarly, this could indicate that being in a relationship influenced the likelihood of an existing wish for children to be realised. Additionally, there may be other differences between mothers and non‐mothers that were not measured in this study. Some possible factors include sexual orientation and fertility. Wanting a child but not being able to have one may significantly influence how child faces are processed. Future studies should attempt to find subgroups of non‐mothers to specify whether the found differences generalise to all non‐mothers.Concerning Hypothesis 3 on the modulation of child‐evoked activation by task, both ROI and whole‐brain analyses revealed stronger activation of the medial superior frontal gyrus in response to the explicit relative to the implicit task condition. The medial superior frontal gyrus is part of the extended face processing system and is associated with cognitive social understanding (see Figure All observed differences in neural processing between mothers and non‐mothers emerged despite no differences on the behavioural level. This suggests that mothers and non‐mothers did not differ in their performances as measured by response times in the explicit and implicit affect recognition task. There was no evidence for an interaction between motherhood and attention to affect or protagonist of the stimuli on the behavioural level. An explanation for missing behavioural effects may be that the task was chosen to increase detection efficiency for neural responses and not for sensitivity on the behavioural level. It is, therefore, possible that the task was not sensitive enough to detect behavioural differences as reported in other studies (Plank, Christiansen, et al., 5This study investigated child‐evoked activation during processing of facial affect and its modulation by motherhood and attention to affect. Affective child compared to adult faces led to increased activation in the core and the extended system of FAR in mothers and non‐mothers both during implicit and explicit affect recognition. Importantly, this child‐evoked activation was modulated by motherhood with stronger child‐evoked activation in the left precuneus in mothers compared to non‐mothers. This could indicate that mothers draw more heavily on the interoceptive investigation of their own emotions and personal memories when processing affective child faces. Additionally, the child‐evoked activation was increased in the medial superior frontal gyrus when participants were asked to recognise the displayed emotion compared to when they were asked to assess unrelated physical features. This suggests that differences between processing affective child and adult faces are increased when directing attention to the displayed emotion. There was no interaction between the modulatory effects of motherhood and attention to affect. This study is an essential contribution to studies showing differences in social understanding between mothers and non‐mothers, especially regarding the social understanding of children.The authors declare no potential conflict of interest.Irene Sophia Plank, Catherine Hindi Attar, Isabel Dziobek and Felix Bermpohl conceptualised and designed the study. Irene Sophia Plank took the lead on acquisition and analysis with vital contributions of Isabel Dziobek and Felix Bermpohl. Irene Sophia Plank wrote the draft. All authors contributed to the interpretation of the data and the revision of the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript.Appendix S1: Supporting InformationClick here for additional data file.


Traditional PET maps patterns of absolute tracer uptake but has demonstrated shortcomings in disorders such as brain neoplasm or focal epilepsy in the ability to resolve normally from pathological tissue. In this review, we describe an alternative process of metabolic mapping, dynamic PET. This new technology quantifies the dynamics of tracer uptake and decays with the goal of improving the functional mapping of the desired metabolic activity in the target organ. We discuss technical implementation and findings of initial pilot studies in brain tumor treatment and epilepsy surgery.Positron emission tomography with fluorine‐18 fluorodeoxyglucose ( These dThe longer scan times and the need to calibrate concentration‐time curves require procedures that differ from static PET. The process used in our ongoing studies is described belowPET is preceded by MRI for purposes of co‐registration. While the patient lays in the scanner with active scanning, intravenous FDG (dose = 10 mCi) is injected; images are collected over 60 min in list‐mode format.An unsedated patient will move over the course of 60 min. Automatic motion correction starts with an initial frame of images which is used to transform subsequent images.The stabilized images are not only co‐registered with MRI but segmented into anatomic regions of interest with the use of published MRI atlases.Calibration of radiation counts (providing the upper and lower limits of radiation uptake) occurs with calculation of the model‐corrected blood input function (MCIF). Details are provided below.kinetic mapping is applied across all voxels covering the whole brain PET volume. The computational burden is high enough to require parallel computing techniques for practical use.The calculation of tracer kinetics requires capturing reference values of blood tracer concentration, the blood input function. The input function describes the maximal level of radiotracer within the blood that is available for tissues to use. The ideal blood input function would be continuous intra‐arterial blood sampling over the whole period of PET acquisition obtained from a major artery. While dynamic imaging with animal models can be done with invasive intraarterial monitoring, practical human imaging requires less invasive approaches.One method of sampling that avoids arterial access, but still requires intravenous access, is heated vein blood sampling.A noninvasive approach is to acquire the blood input function through sampling of the image itself. The image‐derived blood input function, as developed by us and othersSuch large pools of blood, however, are not accessible in human brain imaging since these organs lie outside the limited field of view of PET scanners.PETIDIF) during the acquisition, mimicking the ideal continuous sampling of intraarrterial monitoring. A model IDIF that also corrects for spillover contamination consists of:STb = spillover contamination from the tissue to the blood at late time points, rb = blood recovery coefficient, tb and te = beginning and end of a time frame. CT(t), the model tissue, was obtained by solving FDG‐transport differential equations from blood to tissue spaces as described. Ca(t) is 7‐parameter model blood for FDG‐transport as described. The above model IDIF is then optimized using the following objective functions:Practical dynamic PET imaging, therefore, requires methods to accurately record the blood input function from the image of an unsedated human subject. We have developed a model‐corrected blood input function (MCIF),IDIF) (Equation O(p) used non‐linear regression analysis yielding the estimate of MCIF. Computation of MCIF is a semi‐automated process with a priori determination of the lower and upper bounds and the initial guess values of the parameters, an approach recently validated with arterial blood sampling in rodent total body dynamic PET imaging (see details below).ModelPeak is computed from the model equations for the IDIF ,18F‐fluoroethyl‐l‐tyrosine (18F‐FET).Glioblastoma multiforme (GBM) is a malignant, infiltrating, primary brain tumor that has an annual incidence of 3.19 cases per 100,000 person‐years and poor prognosis with a 5‐year survival rate of 4%–5%.l‐amino acid analogs to statistically model survival in patients with recurrent malignant gliomas. They found that kinetic parameters obtained early after the start of treatment  provided sufficient information to predict the outcome with reasonable confidence using regression techniques.In this setting, dynamic PET remains an important option to consider. For example, recent work by Wardak et al1‐k3 and Ki) and total blood volume (TBV) for each tumor for each patient  or, when available, the combination of follow‐up imaging with surgical pathology (n = 7). The lesion‐related kinetic rate constants provided the independent variables of predictive input values. After creating the model with total of 34 lesions  across 25 patients, the resulting model predicted lesion type from a test set. The model accurately predicted 95% of tumors and 75% of necrotic lesions. This pilot study suggests dynamic FDG‐PET imaging could improve the diagnostic accuracy of brain tumor treatment.To provide objective distinctions between tumor and normal tissue, we created a statistical model with the dependent outcome of lesion status, coded as tumor progression or tumor necrosis, for any given patient based on gold standards of expert interpretation of MR imaging obtained in follow up  and dynamic FDG‐PET. Four had unilateral mesial temporal/hippocampal regions of hypometabolism on dynamic FDG‐PET concordant with an anticipated surgical target; one underwent laser interstitial thermal therapy of the ipsilateral hippocampus at the site of dynamic FDG‐PET hypometabolism and was seizure‐free for 30 months at last follow‐up. These patients represent the potential of improved sensitivity of determination of surgical targets.A patient who had previous unsuccessful right frontal lobectomy had right mesial temporal hypometabolism on dynamic FDG‐PET. Subsequent intracranial monitoring disclosed multifocal seizure foci. This patient represents the potential ability of dynamic PET to provide a prognosis against further surgical intervention.Finally, three patients had dFDG‐PET findings indicating unilateral mesial temporal hypometabolism in the context of bilateral mesial foci proposed by the Epilepsy Surgery Committee review. Each declined intracranial monitoring. These two patients represent the potential of dynamic PET to guide invasive evaluations to bring more patients to future surgery.Overall, the pilot study suggests that dynamic FDG‐PET may indicate focal regions of hypometabolism in epilepsy surgery subjects whose standard static FDG‐PET was unhelpful.From a technical standpoint, the second study offers several innovations that could improve subsequent work. Our use of a semi‐automated, objective procedure of model‐corrected blood input function offers practical and theoretical advantages over manually selected initial images of the internal carotid artery. This innovation turns what in the past was an operator‐dependent and manually‐calculated process into a rigorous, semi‐automated method that has not only informed our preliminary work but has been rigorously validated in a prior animal‐model work with arterial blood sampling.In summary, this review has provided an overview of the biological and technical underpinnings on how dynamic PET can better capture functional differences in normal versus pathological brain regions. Recent technical innovations may facilitate easier and more widespread use of dynamic PET. Current work has centered on brain neoplasm and focal epilepsy; future work may yield promising results in degenerative disease, such as dementia, traumatic brain injury, or neurovascular disorders.The authors report no conflicts of interest.Dr Quigg conceptualized and undertook the human research, wrote and edited the manuscript.Dr Kundu developed and undertook engineering, human, and animal research, and wrote and edited the manuscript.


KRAS mutation analysis suggested implantation of sigmoid colon cancer to the post‐endoscopic submucosal dissection site of intramucosal rectal cancer.A 70‐year‐old woman was diagnosed with intramucosal rectal cancer and advanced sigmoid colon cancer at the same time. First, the intramucosal rectal cancer was curatively resected by endoscopic submucosal dissection, and surgery was subsequently performed for sigmoid colon cancer. After 20 months, a follow‐up colonoscopy revealed a tumor growth at the ulcer scar of the endoscopic submucosal dissection. Histological findings and  In the surgical field, implantation of exfoliated cancer cells has been recognized as one of the mechanisms contributing to tumor recurrence at the suture site after surgery for colorectal cancer.KRAS mutation in the recurrent lesion.Here, we report a case of cancer recurrence due to the implantation of sigmoid cancer into the post‐ESD ulcer of intramucosal rectal cancer. Implantation of sigmoid cancer was strongly supported by an identical A 70‐year‐old woman was referred to our hospital for the treatment of a rectal tumor located 10 cm from the anal verge and advanced sigmoid colon cancer located 20 cm from the anal verge. The rectal tumor was endoscopically diagnosed as intramucosal rectal cancer (0–IIa+IIc) and an en bloc resection was performed by ESD in advance Figure . The resTwenty months after surgery, a computed tomography scan showed a thickening of the rectal wall, and a colonoscopy revealed a submucosal tumor‐like lesion at the post‐ESD scar Figure . Local rKRAS mutation analysis indicated that the intramucosal rectal cancer was positive for p.G13D, and both the primary sigmoid cancer and recurrent cancer at the ESD scar were positive for p.G12V. These results strongly suggested that the recurrent lesion was a cancer cell implantation from sigmoid colon cancer.KRAS p.G12V mutation in both the primary sigmoid cancer and the recurrent lesion confirmed the cancer cell implantation.We reported a case of implantation of sigmoid colon cancer into the post‐ESD site of intramucosal rectal cancer. An identical Umpleby et al. previously demonstrated the presence of viable exfoliated cancer cells in the lumen of patients with advanced colorectal cancer.KRAS gene mutation in the recurrent lesion was consistent with sigmoid colon cancer but differed from rectal cancer. These findings strongly suggested cancer cell implantation from the sigmoid colon cancer to the ESD site.There are a few reports of tumor cell implantation associated with endoscopic resection. Tajika et al. reported a case of cancer cell implantation from rectosigmoid colon cancer into the endoscopic mucosal resection (EMR) site of a synchronous rectal carcinoid.Various factors may influence the establishment of tumor cell implantation into the endoscopically resected site. Maeda et al. have proposed two possible factors involved in cancer cell implantation: the presentation of viable cancer cells and the susceptibility of damaged tissue to colonization and proliferation of cancer cells.KRAS p.G12V mutation in both sigmoid cancer and recurrent lesion. KRAS mutation in codon 12 has been reported to confer increased oncogenic potential of cancer cells when compared to the codon 13 mutation,KRAS p.Q61R mutation in both the primary and recurrent lesions of possible cancer cell implantation,KRAS mutation analysis may be a good biomarker.The malignant potential of cancer cells may be one of the risk factors for cancer cell implantation but there are few reports on this matter. In this study, we detected a KRAS mutation may be helpful for the diagnosis of cancer cell implantation.In conclusion, endoscopists need to pay attention to the risk of tumor cell implantation when performing endoscopic resection of a colorectal tumor in the presence of advanced colorectal cancer. Analysis of the None.


In the era of biomedical HIV prevention and treatment technologies, such as treatment as prevention (TasP) and pre‐exposure prophylaxis (PrEP), there is momentum to develop and rigorously evaluate interventions focused on PrEP among those at risk for HIV acquisition and antiretroviral therapy (ART) adherence among people living with HIV. While HIV status‐specific interventions focused on PrEP or ART provide valuable information, status‐segregated interventions can create, perpetuate, and even increase HIV stigma among transgender women of colour and other marginalized communities in the United States (US).Due largely to community advocacy, discourses that support status‐neutral approaches have emerged in the scientific literature. Although US‐based funding mechanisms have typically designated awards focused on a specific HIV status, intervention developers and implementing agencies find creative ways to design and implement status‐neutral programmes despite such restrictions. We present our experience with intervention research in New York, Detroit, New Orleans, Puerto Rico and the San Francisco Bay Area, all Ending the HIV Epidemic (EHE) priority jurisdictions. Kickin it with the Gurlz’ was developed to be status‐neutral through two grants due to community demands for a unifying approach. The Transgender Women Engagement and Entry to (TWEET) Care Project was designed to improve HIV care engagement for transgender women living with HIV, but developers realized the importance of including participants of any HIV status. Healthy Divas was designed for transgender women living with HIV but subsequent implementing agencies prioritized adapting it to be status‐neutral. These examples support the urgency of designing, implementing and evaluating status‐neutral interventions.Community‐based organizations strive for inclusivity in their programming and are rightly often reluctant to segregate services based on the HIV status of their clients. As researchers, we have an ethical imperative to work to reduce HIV stigma and respond to the needs of those most impacted by HIV, including transgender women of colour. As such, we call upon funders to develop mechanisms that support the development and testing of HIV status‐neutral interventions to reduce HIV stigma and support community building, thereby increasing the possibility of fully realizing the benefits of biomedical HIV prevention and treatment technologies for all. In tIn the United States (US), transgender women of colour have expressed the urgency of grounding HIV prevention and care programming in their lived experiences, noting that oppression rooted in ethno‐racism and cisgenderism and resultant social determinants of health  take precedence over HIV , 11, 12.Evidence also suggests that HIV status‐neutral interventions are preferred in community‐based settings , 15, as 2Even though US federal funding mechanisms often designate awards to focus on people of a specific HIV status, intervention developers and implementing agencies can and do find creative ways to design and implement status‐neutral programmes despite such restrictions. For example, the Health Resources and Services Administration (HRSA) funded the Enhancing Engagement and Retention in Quality HIV Care for Transgender Women of Colour Initiative in 2012. This Special Projects of National Significance (SPNS) initiative supported nine demonstration projects to develop and evaluate innovative interventions to reduce HIV health inequities among transgender women of colour living with HIV . As partchose to share their HIV status over the course of the project, which fostered self‐efficacy in communicating about HIV, allowed group members to form deeper connections with each other and provided an opportunity to collectively challenge prevailing HIV stigma. In Detroit, TWEET has continued to be implemented as a peer‐delivered status‐neutral intervention even after the end of the funding to meet the community demand for such a unifying and supportive group led by transgender women of colour.Three organizations replicating TWEET in HRSA's E2i Initiative   also adon = 11) and focus groups (n = 12) to adapt the intervention content, participants expressed a desire for status‐neutral groups citing the importance of “breaking down stigma in community.” While only possible through two separate funding mechanisms, our team is in a position to examine the feasibility and acceptability of a status‐neutral intervention with transgender women of colour designed to address both HIV prevention and care continua outcomes at the outset.Beyond TWEET, status‐neutral interventions that simultaneously address trauma are urgently needed in Detroit. As Michigan's HIV epicentre , patternIn San Francisco, the UCSF Center of Excellence for Transgender Health (UCSF CoE) has spent the better part of a decade developing and testing a peer‐led, gender‐affirming intervention for transgender women living with HIV, called Healthy Divas. Significant funding and resources have been invested in pilot testing and conducting a randomized controlled trial of the intervention (R01MH106373). Based on the urgent need for such a programme, Healthy Divas was selected for national dissemination by HRSA's E2i Initiative . FurtherThe UCSF CoE has also been conducting PrEP research with Latina transgender women at La Clínica de la Raza, a community‐based clinic in a predominantly Latinx neighbourhood of Oakland, California. Triunfo was designed as a peer‐led, PrEP education and community mobilization project to encourage PrEP uptake and adherence among Latina transgender women at risk for HIV acquisition. As a result of the focus on those not living with HIV, anyone in the community who was not able to participate in Triunfo was outed as living with HIV, which resulted in inadvertent reinforcement of HIV stigma and a sense of the programme being unnecessarily exclusionary and divisive. Our university‐academic partnership recently received funding to expand the programme at La Clinica to include Latina transgender women living with HIV and study the implementation of a status‐neutral version of the intervention designed to improve both HIV prevention and HIV treatment.3With evidence and advocacy to support HIV status‐neutral approaches as a means to eradicate HIV stigma globally , 24, 25,Social support, community connection and social capital are important mechanisms that reduce the deleterious impact of HIV stigma on HIV care continua outcomes, including engagement in care and viral suppression , 27, 28.HIV status‐neutral interventions have tremendous potential to reduce HIV stigma through building solidarity and information exchange among trusted peers, as well as addressing pressing needs that undermine successful engagement across both the HIV prevention and care continua . HoweverCommunity‐based organizations strive for inclusivity in their programming and are reluctant to segregate their services based on HIV status. We have an ethical imperative to respond to the needs of those most impacted by HIV, specifically transgender women of colour. Our HIV intervention efforts must be designed to decrease or eliminate HIV stigma among marginalized communities who experience multiple and interlocking systems of oppression. Funding priorities focused exclusively on status‐segregated intervention research create an ethical dilemma for HIV researchers wishing to collaborate effectively with communities most impacted by HIV. As such, we call upon funders to develop funding mechanisms that support the development and testing of HIV status‐neutral intervention research to reduce HIV stigma, support community building and fully realize the benefits of biomedical HIV prevention and treatment technologies for all.The authors declare no competing interests.KEG, GR and JMS wrote the first draft of the manuscript. BMM, LJC, LAR and MC contributed to the refinement and presentation of the interventions and programmes presented. All authors contributed to the writing of the manuscript, and all approved the final draft.


Computational tools such as those estimating the dissimilarity of candidate antigens to the human proteome or molecular modelling tools predicting the likelihood of a given TCR to interact with distinct antigens are promising but are still in their infancy.In the NeoScreen study, only CD8 T cell responses were reported, although neoepitope‐specific CD4 TILs may also be enriched. CD4 help was shown to be key to promote and support anti‐tumour CD8 responses during successful immunotherapy.Of interest, B cell engineering may be further improved to potentiate the stimulation of tumour antigen‐specific clonotypes. Via TCR discovery engines such as NeoScreen and also via alternative strategies, including the screening of the most frequent clonotypes in patients’ baseline tumours, In the context of ACT, it is well established that cell state in infusion products is of paramount importance.Next‐generation ACT could be further improved using treatment combinations. Of interest, TIL therapy may be combined with standard‐of‐care cancer treatments such as chemotherapy and radiotherapy which can release tumour antigens, thus recruiting cellular immunity.


Startup plants include seedlings and basal and epicormic resprouts. It has long been held that startups have different strategies from adult plants, but theory for what trait differences to expect is limited and not yet quantitatively tested. Three applicable concepts are analogous to human startup firms, R‐shift, and trait‐growth theory. All three suggest startups should be built with lower construction costs than established plants. This appears to be almost always true in terms of leaf mass per area (LMA), though many comparisons are complicated by the startups growing in lower light. Trait‐growth theory predicts LMA should increase progressively with height or total leaf area, driven by higher conductive‐pathway costs associated with each unit leaf area, and by greater reward from slowing leaf turnover. Basal resprouts often have somewhat higher LMA than seedlings, but possibly this is simply because they are larger. A number of eminently testable questions are identified. Prospects are good for a theoretically cogent and field‐tested body of knowledge about plant startups. Startup businesses are a recognized category, different in several ways from established enterprises Box . SimilarBox 1Startup firms are a recognized category within human economics, business management and entrepreneurship. Features that they share with startup plants include the following Cohan, . (1) LarMarkets and profitability are very uncertain for startup firms. This is because their products are typically novel. So‐called ‘angel’ investors accordingly spread their investment across many startups, accepting the high risk of failure in exchange for high returns from the minority of successes. Seedlings similarly experience much uncertainty, not because they are a novel product but due to the chances of finding themselves in different microsites. The genes shared by seedlings from an individual mother are analogous to an angel investor inasmuch as their success can be thought of as an aggregate across all the mother’s seed production.et al., The unpredictable microsites faced by individual seedlings (but not so much by resprouts) favour plastic adjustment of traits  make predictions. Other traits are discussed briefly in Supporting Information Notes Startup firms are a recognized category in business. The analogy of startup plants to startup firms , which account in order for photosynthesis, respiration, and tissue turnover losses. The values of M,r and k refer to mass, maintenance respiration rate, and turnover rate for different tissues . Dividing by leaf mass per area (LMA) converts leaf mass Ml into leaf area, p¯E is the assimilation rate of CO2 per unit leaf area, and α adjusts gross primary production for growth respiration and other factors affecting the conversion of fixed carbon fixed into biomass.The right‐hand side of Eqn The second key equation expresses the importance of allocation. For example, the rate of leaf area growth can be expressed asB/dt aredAldMa the rate of deployment of new leaf area per mass invested, accounting for LMA and also for support costs elsewhere in the plant. Deployment is potentially increased by economizing on construction costs.dMadB the fraction of biomass produced that is allocated to vegetative growth vs to reproduction. Growth rate is increased by reducing investment in reproduction.The two terms on the right‐hand side in addition to det al., B/dt, these two effects will be equal and opposite  should be directed to the Click here for additional data file.