COVID-19 vaccination is a key public health measure to mitigate the crippling effects of the pandemic. COVID-19 vaccination began in adults and targeted adolescents at a later stage. However, medical decision-making on its uptake among the latter was unknown, which could be affected by their literacy and concerns about the vaccine. The study aimed to elucidate the awareness, concerns and barriers of adolescents toward the COVID-19 vaccine.A cross-sectional online survey using a self-developed questionnaire was conducted between June to November 2021. The adolescent participants were students from institutes of post-secondary education who were recruited via convenience sampling. The data were collated from an officially approved electronic platform, audited and analyzed. Continuous and categorical variables were expressed as mean (standard deviation) and percentages, respectively.A total of 460 adolescents participated in the study . Most of them (91.5%) were aware of the COVID-19 vaccines. The main barriers to vaccination were uncertainty about long-term health risks (38.6%) and vaccine efficacy (37.3%). Regarding the potential vaccine side effects, they were concerned about: anaphylactic shock (73.2%), low blood pressure (58.3%) and fast heartbeat (58.0%). Only 58% expressed willingness for repeated COVID-19 vaccination.Despite high COVID-19 vaccine awareness, the adolescents were concerned about the potentially severe but rare side-effects. The study identified apprehension regarding vaccine efficacy and the potential long-term health impact as the main barriers to vaccination. Future studies should address these concerns to scale vaccination programs among adolescents. Since the outbreak of the COVID-19 pandemic and the emergence of various strains, vaccinations have emerged as a key solution to curb its spread. COVID-19 vaccinations have been proven to reduce both the incidence and severity of infections . The CDCVaccine hesitancy is defined as the delay in acceptance or vaccination refusal despite its availability . Rates oth May 2021, the Pfizer® COVID-19 vaccination received Food and Drug Agency (FDA) approval for use in individuals aged 12 years and older . Junior colleges are the equivalent of high schools, whereas polytechnics provide more practice-based learning experiences culminating in a diploma. ITE provides technical and vocational education .The survey consisted of 6 domains: demographic, history of severe allergic reaction to vaccines or medications, knowledge and attitude on the vaccine, concerns about the vaccination, factors influencing their vaccine uptake and willingness to proceed with COVID-19 vaccinations. Participants filled in their gender, ethnicity, year of birth and sites of Institute of Higher Learning but no participant identifier was collected. The questionnaire allowed them to respond by selecting either binary (Yes/No) or multiple allowable options. The questionnaire underwent face validation by a group of polytechnic students in an unpublished study.Awareness of common facts about the COVID-19 vaccine was assessed via questions regarding its efficacy, potential side-effects and cost.Concerns about the COVID-19 vaccine side-effects were determined by asking participants about their concerns regarding specific side-effects. The side-effects ranged from common but mild symptoms such as tiredness to more severe side-effects such as anaphylactic shock.Barriers to COVID-19 vaccination were assessed via questions encompassing vaccine accessibility, vaccine administration and COVID-19 vaccine-specific factors including efficacy and potential long-term side-effects.st June to 30th November 2021. The study team formulated a preliminary set of survey questions using Google Forms. After the finalization of the survey questions on Google Forms, the final survey was then created using the form builder tool called “FormSG,” which is a national survey platform to create digital government forms developed by Singapore's GovTech's Data Science & Artificial Intelligence Capability Centre. After the form is built-in FormSG, it is identified via a unique URL weblink. With the URL weblink, the corresponding QR code is created as well. The QR code will facilitate signing in to complete the survey simply just by scanning it via the application on mobile phones. Otherwise, the participants were able to access the survey via the weblink. The link and QR code were generated from FormSG and distributed to the students through snowball convenience sampling via Whatsapp, Telegram, emails and word of mouth. The submissions of the individual responses were collated via a dedicated email as pre-set in the FormSG. At the end of the study, all the individual email files were exported from Outlook as.pst file format. The.pst file format was next imported into the Data Collation tool in FormSG where it was converted into Excel. The data was next exported for analysis and visualization.The e-survey was conducted from 1Descriptive statistics were done to describe the sample and associations performed to consider relationships between gender and ethnicity with concerns and willingness to get COVID-19 booster jabs. Continuous and categorical variables were expressed as mean (standard deviation) and percentages, respectively.The demographic characteristics are shown in Overall, 265 (58%) adolescents were willing to repeat the vaccination, 33 (7%) were reluctant and the remaining 162 (35%) were ambivalent.The surveyed adolescents displayed high literacy of COVID-19 vaccines, such as vaccine efficacy, its side-effects (including non-interference with DNA), cost and implementation for local residents. In contrast, a 2020 survey by the Nanyang Technical University of Singapore showed one in four Singaporeans falsely believed that the COVID-19 vaccine alters DNA . UnfortuVaccine hesitancy was related to its perceived efficacy (37.4%) and effect on long-term health (38.7%). The vaccine has been proven to be efficacious with current available data not suggesting any known long-term detrimental effects to one's health . More adMost adolescents reported obtaining information from family (80.0%) and mainstream media (78.2%). In contrast, around 25% reported obtaining information from teachers and school briefings. With adolescents spending extended periods of their day in school, the education professionals should not miss the opportunity to provide them with the requisite knowledge and opportunity to clarify any doubts regarding the COVID-19 vaccine.th February 2022, 96% of adolescents aged 12 to 19 years of age received their primary doses of COVID-19 vaccines  public health program  approval for use in this population  by the Research Department in SingHealth Polyclinics. The publication cost was supported by the SingHealth Polyclinics – Centre Grant.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The provision of kangaroo mother care (KMC) involving continuous skin-to-skin care (SSC) is an important intervention in neonatal care, which is recommended even when women are infected with severe acute respiratory syndrome coronavirus (SARS-CoV-2). We report on a nosocomial outbreak of SARS-CoV-2 infections in a KMC ward.Contact tracing was conducted following the diagnosis of SARS-CoV-2 in a mother lodging in the KMC ward. All mother-newborn dyads in the KMC and healthcare workers (HCW) were tested for SARS-CoV-2 within 24–72 h of diagnosing the index case. Nasopharyngeal swab samples were obtained and tested from contacts, with a nucleic acid amplification test (NAAT) assay. Next-generation sequencing was done on positive samples. The secondary attack rate (SAR) was calculated assuming that the mother who presented with symptoms was the source of infection.Acinetobacter baumannii sepsis, and his post-mortem lung histopathology showed features compatible with SARS-CoV-2 pneumonia. The sequencing of 13 specimens, which included 1 mother-newborn dyad, indicated clustering to the same phylogenetic lineage with identical mutations. In assessing for factors contributing to this outbreak, it was found that spaces between beds were less than 1 m and mothers had their meals around the same table at the same time.Twelve (70.6%) of 17 mothers and 8 (42.1%) of 19 neonates who were in the KMC ward with the index case were found to be positive with SARS-CoV-2. Seven (87.5%) of the 8 neonates who tested positive had mothers who also tested positive. Seventy-five percent (9/12) of the mothers and 62.5% (5/8) of the neonates who tested positive were asymptomatic. Eight (27.6%) of 29 HCW were found to be positive and were all asymptomatic. One neonate died from We report on a nosocomial outbreak of SARS-CoV-2 in a KMC ward, affecting a high number of mothers and neonates, and to a lesser extent HCWs. Although it is difficult to point to the index case as the source of this outbreak, as asymptomatic individuals can spread infection, the inadequate adherence to non-pharmaceutical interventions was assessed to have contributed to the spread of infection. This highlights the need for awareness and adherence to mitigation strategies to avoid SARS-CoV-2 outbreaks. Respiratory droplets and aerosols are regarded as part of a continuum and represent the primary mode of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Smaller aerosol particles (<5 microns) may travel greater distances than the larger particles (>5 microns) and have been the mode of transmission in confined close contact spaces. A number of reports have shown a higher concentration of the SARS-CoV-2 closest to the infected or the presymptomatic index cases . FomitesIn a meta-analysis by Haley et al., the hospital-acquired secondary attack rate (SAR) varied from 0 to 17.1% with a pooled estimate of 3.6% (95% CI 1.0–6.9) and no difference between the patient population and the heathcare workers (HCWs). Household contacts had higher SAR than hospital-acquired infections. The SAR was highest in pre-symptomatic and symptomatic cases than in asymptomatic cases .via vertical transmission in utero  ward in a public, tertiary hospital in a low- and middle-income setting.An investigation for SARS-CoV-2 infection of all mother-newborn dyads lodging in a 24-h KMC at Chris Hani Baragwanath Academic Hospital (CHBAH), and healthcare workers, was conducted after one of the women tested positive for SARS-CoV-2 infection following investigation for upper respiratory symptoms. CHBAH is a tertiary public hospital in Johannesburg, South Africa. It has a total of 3,200 beds, including a 185-bed neonatal unit consisting of 18 intensive care, 48 high care, 100 standard care, and 19 KMC beds. The daily bed occupancy is generally more than 80%.The KMC ward is a facility for otherwise well preterm-born neonates who are awaiting weight gain, and their mothers are encouraged to lodge in the ward to offer skin-to-skin care (SSC) to their babies throughout the day. Neonates admitted to the KMC include those weighing between 1,000 and 1,700 g, not requiring supplemental oxygen or intravenous fluids, and whose mothers are available to room-in with the baby. Mothers provide SSC continuously on their beds except when bathing or having meals, during which the babies are placed in the bassinettes next to the mothers’ bed. The ward accommodates up to 19 mother-neonate dyads.The KMC ward is adjacent to another ward which is a standard care nursery (SCN) accommodating up to 20 neonatal admissions. Neonates admitted to SCN and KMC wards are assessed and managed by the same nursing and medical staff. Because of limited lodging facilities, women of neonates requiring hospital admission  not in the KMC ward were discharged home and could visit daily.At the onset of the COVID-19 outbreak in South Africa, mothers were screened daily for symptoms suggestive of COVID-19 while staying in the KMC ward. On 8 June 2020, one of the mothers manifested a fever and cough and was diagnosed with COVID-19 following testing positive for SARS-CoV-2 from nasopharyngeal swabs, which was tested using a nucleic acid amplification assay test (NAAT). This mother was considered the index case for SARS-CoV-2 infection in this ward. She had been lodging in the KMC ward since 29 May 2020. She was counseled on self-monitoring and sent home to self-isolate.Nasopharyngeal swabs were performed on all the other mothers and their babies who were residing in the KMC and investigated for SARS-CoV-2 using the NAAT assay, within 24 and 72 h after identifying the index case, respectively. The secondary attack rate (SAR) was calculated based on the probability of onward infection from an index case among a defined group of close contacts . NAAT was performed using the U.S. Centers for Diseases Control and Prevention’s emergency use authorization assays to detect SARS-CoV-2 . SamplesMothers and neonates who were tested SARS-CoV-2 PCR-positive underwent viral sequencing using Superscript IV with random hexamers  to generate cDNA. The Swift Biosciences’ Normalase Amplicon and the SARS-CoV-2 amplicon panel  were use1 were used to generate paired-end fastq reads  were filtered out of the pipeline which contains several python scripts that manage the analysis workflow. It performs alignment of genotypes in MAFFT, phylogenetic tree inference in IQ-Tree20, ancestral state construction, and annotation.3The Genome Detective 1.126 and the Coronavirus Typing Tooltq reads . The bcfThe medical records of all the mothers and neonates who were tested and had NAAT results available were reviewed for clinical characteristics and outcomes at hospital discharge. The proportion of mothers and neonates with positive SARS-CoV-2 results, including the concordance of SARS-CoV-2 results between neonates and their mothers, and between twins were assessed.The clinical and laboratory data were collected prospectively and information on outcomes was extracted from the medical records. Descriptive stats were represented as numbers and proportions.Permission to report on this outbreak was acquired from the hospital management, and ethics approval was obtained from the University of the Witwatersrand Human Ethics Research Committee (Protocol Number – M200505).There were 17 mothers and 20 babies (including 3 sets of twins) admitted in the KMC ward between 8 June 2020, the day the index case was investigated, and 10 June 2020 when her SARS-CoV-2 result was received. Thirteen (76%) mothers had been admitted to the ward since 29 May 2020 and four (24%) were admitted between 8 and 10 June 2020. The bed location of mother-newborn dyads who were tested following confirmation of COVID-19 in the index case is presented in Twelve (70.6%) of 17 mothers and 8 (40%) of 20 neonates (including neonates of the index case) tested positive for SARS-CoV-2 infection . One neoAll the three mothers who had twins were positive for SARS-CoV-2, but only one of two sets tested positive, and both neonates of the third set of twins were negative .Among the 12 women who tested positive and confirmed to be infected by SARS-CoV-2, nine (75%) were asymptomatic, and three had mild upper respiratory tract symptoms. All the women infected with SARS-CoV-2 were discharged upon diagnosis for self-isolation at home, and one was sent to a government isolation facility.Acinetobacter baumannii in blood. Post-mortem histopathological findings (13 days after the SARS-CoV-2 positive result), obtained by targeted minimal invasive tissue sampling, revealed hyaline membrane, type II pneumocyte proliferation, interstitial inflammation, markedly reactive bronchial cells, and intravascular thrombi, which could be consistent with COVID-19. The immediate cause of death was assigned to be attributed to A. baumannii nosocomial sepsis with the antecedent cause related to nosocomial SARS-CoV-2 and acute respiratory distress syndrome due to SARS-CoV-2 (Three (37.5%) of the neonates who had a reactive NAAT test were symptomatic. There was a single death in one of the neonates with COVID-19 who developed nosocomial pneumonia and warranted mechanical ventilation and cultured multi-drug resistant RS-CoV-2 .There were 13 babies in the adjacent SCN ward who were also tested, all of whom had a non-reactive NAAT for SARS-CoV-2. One of these neonates was tested positive on a subsequent visit to the pediatric casualty ward 10 days after having been discharged on 15 June 2020, and this neonate was declared to be deceased upon arrival at the hospital. The verbal autopsy revealed that the neonate had difficulty with breathing on the evening of 24 June 2020 and was taken to the family physician who sent the neonate home on unspecified treatment. The baby was noted to be unresponsive in the early hours of the morning of 25 June 2020 and brought to the hospital.The next-generation sequencing was successfully performed in samples from 10 (77%) of 13 women and 3 (38%) of 8 neonates, with the remaining samples not sequenced due to low viral loads (cycle threshold >32). The sequenced samples included one mother-newborn dyad. All of the genomes were of the C.35 PANGO lineage classification; furthermore, all of the genomes clustered very closely together during the phylogenetic analysis with identical mutations at D614G and P314L positions . A singlTwenty-nine healthcare workers  who worked in the KMC ward were also tested for SARS-CoV-2 as part of the outbreak investigation; 27.6%  had a reactive SARS-CoV-2 NAAT.On reviewing the activities in the ward, it was identified that the mothers shared the same table for meals without observing social distancing. The mothers had been wearing cloth face masks during their stay in the ward. During the assessment of spacing between beds, the distance between the edges of the beds for mothers was recorded as 75 cm and between baby and mother was recorded as 25 cm.We report on a nosocomial outbreak of SARS-CoV-2 in a KMC ward, affecting mothers and their infants. Forty percent of neonates and 70% of mothers in the KMC ward were positive during this outbreak and 27.6% of HCWs were found to be positive. Other than the index cases, most mothers and infants who tested positive were asymptomatic. Vertical transmission was considered to be unlikely as the babies in the KMC ward were chronologically older, thus, more likely to be a horizontal transmission.A number of studies have reported on the mother-to-infant transmission of SARS-CoV-2 but most of them were around the time of birth or soon after birth, and they reported low transmission rates of less than 3.2% . A more A. baumannii. In reviewing this death, the histopathological changes observed were not in keeping with A. baumannii sepsis. The second neonate who died was in the SCN during the time of this outbreak. Though he was negative at discharge, it is possible that he acquired the infection during this outbreak as he presented within 14 days of the outbreak, but the acquisition of infection from the home or community cannot be excluded.Many studies have reported low or no mortality in neonates infected by SARS-CoV-2. In this study, we report that one (12.5%) of the neonates who were positive subsequent to this outbreak died with features suggestive of SARS-CoV-2, though the immediate cause was assessed to be healthcare-associated infection due to All the specimens sent for next-generation sequencing had the same lineage classification, and the genome of each specimen was clustered closely together on the phylogenetic tree. Thus, there was a common source for the outbreak in the KMC ward. These were representative of the common SARS-CoV-2 PANGO lineages circulating during the pandemic . HealthcThe response to the outbreak was to increase the space between beds from 75 to 150 cm. Mothers were offered surgical masks to wear at all times and were divided into two groups when going for meals to allow a space of 150 cm between mothers during meals. Mothers who came from home were screened for symptoms and tested for SARS-CoV-2, and only those who tested negative were admitted in the 24-h KMC ward. The positive mothers were only accepted into the KMC ward to perform 24-h KMC after they had completed 10 days of isolation at home or in a government isolation facility. There have been no other cases of COVID-19 from the KMC ward since the implementation of the above measures.Limitations of the study are that we did not swab the environment, the beds, bed rails, bedside lockers, or any of the ablution facilities or the staff common areas. Environmental swabs and airborne samples were not taken to investigate for SARS-CoV-2. The HCW nasopharyngeal swabs were not sent for NGS testing as these were tested by a different laboratory and we had no access to these samples. Strengths are that it reports on an outbreak in a KMC ward and the importance of NPI measures and screening.Our recommendations are that all mothers coming for labor and delivery should be screened and/or tested for SARS-CoV-2. Mothers who tested positive should be isolated, and those whose babies are well, to room-in with their babies and provide intermittent SSC during breastfeeding, keeping the 1-m distance between baby and mother in between feeds, mothers should wear surgical masks, especially during breastfeeding and practice hand hygiene. The mothers who test positive and are to remain in hospital to provide continuous SSC in the KMC ward with other negative mothers, these positive mothers to only be admitted to the KMC ward after 10 days of isolation. For mothers whose infants might have been too sick or too small for them to lodge while waiting for their infants and therefore were sent home, they must be screened for symptoms and tested for SARS-CoV-2 before readmission to the lodger facility or KMC ward. Mothers who are symptomatic and/or test positive should not be admitted to KMC; they should be referred to their family doctor or physician depending on the severity of symptoms and to be in isolation or quarantine. They should be readmitted once they have been discharged by their physicians or are de-isolated. Mothers should be encouraged to express breastmilk and ask the family members to bring the breastmilk to the hospital for the baby.This study reports on a SARS-CoV-2 outbreak in a KMC ward with mothers, neonates, and HCWs infected. The majority of the infected neonates had a good outcome in terms of respiratory morbidity. There was a low mortality rate. The outbreak highlights the importance of adhering to NPI in mitigating the spread of SARS-CoV-2 in a hospital setting with such close and prolonged contact. As we aim for zero separation of mother–newborn dyads and promote rooming-in and breastfeeding, even in mothers who are infected with SARS-CoV-2, we must impress on strategies to curtail the spread of these infections.EPI_ISL_12690551, EPI_ISL_12690550, EPI_ISL_12690542, EP I_ISL_12690553, EPI_ISL_12690541, EPI_ISL_12690552, EPI _ISL_12690544, EPI_ISL_12690555, EPI_ISL_12690543, EPI_I SL_12690554, EPI_ISL_12690546, EPI_ISL_12690557, EPI_IS L_12690545, EPI_ISL_12690556, EPI_ISL_12690548, EPI_ISL _12690547, and EPI_ISL_12690549.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The data presented in the study are deposited in the GISAID EpiCoV repository and the accession numbers are: The studies involving human participants were reviewed and approved by the University of the Witwatersrand Human Research Ethics Committee. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.FN and SV conceptualized and analyzed the data of the study and wrote the first draft. FN, RT, AK, NN, SK, KS, NK, DK, TNM, CO-E, TM, and NC collected and analyzed data. VB, SJ, and SM analyzed and interpreted the NGS data. All authors analyzed the drafts and are accountable for the content.


The segmentation of 3D cell nuclei is essential in many tasks, such as targeted molecular radiotherapies (MRT) for metastatic tumours, toxicity screening, and the observation of proliferating cells. In recent years, one popular method for automatic segmentation of nuclei has been deep learning enhanced marker-controlled watershed transform. In this method, convolutional neural networks (CNNs) have been used to create nuclei masks and markers, and the watershed algorithm for the instance segmentation. We studied whether this method could be improved for the segmentation of densely cultivated 3D nuclei via developing multiple system configurations in which we studied the effect of edge emphasizing CNNs, and optimized H-minima transform for mask and marker generation, respectively.The dataset used for training and evaluation consisted of twelve in vitro cultivated densely packed 3D human carcinoma cell spheroids imaged using a confocal microscope. With this dataset, the evaluation was performed using a cross-validation scheme. In addition, four independent datasets were used for evaluation. The datasets were resampled near isotropic for our experiments. The baseline deep learning enhanced marker-controlled watershed obtained an average of 0.69 Panoptic Quality (PQ) and 0.66 Aggregated Jaccard Index (AJI) over the twelve spheroids. Using a system configuration, which was otherwise the same but used 3D-based edge emphasizing CNNs and optimized H-minima transform, the scores increased to 0.76 and 0.77, respectively. When using the independent datasets for evaluation, the best performing system configuration was shown to outperform or equal the baseline and a set of well-known cell segmentation approaches.The use of edge emphasizing U-Nets and optimized H-minima transform can improve the marker-controlled watershed transform for segmentation of densely cultivated 3D cell nuclei. A novel dataset of twelve spheroids was introduced to the public.The online version contains supplementary material available at 10.1186/s12859-022-04827-3. Confocal fluorescent imaging has become a leading imaging method to study cell samples and imaging in 3D is essential to fully understand the cell morphology and changes in it. The segmentation of cells or nuclei is a critical requirement for quantitative analysis. Segmentation is a prerequisite for extracting morphological shape features like volume, surface area, elongation and roundness or sphericity of individual nuclei. While analyses in 2D have been the standard for a long time, in recent years the interest in 3D modelling has been growing rapidly. 3D cell models have several advantages over 2D cell cultures. In the 3D model, cells start to express their natural organisational structures and extracellular matrices . The comTargeted molecular radiotherapies (MRT) allows targeting of high absorbed radiation doses selectively at a cell-level . The impWhile many approaches have been proposed for cell segmentation –14, one In recent years, approaches exploiting deep learning in the form of convolutional neural networks (CNNs) have also been used to generate markers and cell masks with state-of-the-art results –28. The In this study, we devised several system configurations where marker-controlled watershed was combined with CNNs and optimized H-minima transform for 3D nuclei segmentation. Namely, we first constructed a baseline method where two 3D U-Nets  created The experiments were performed using densely packed twelve HepG2 nuclei spheroids for training and evaluation in a cross-validation scheme and also four other datasets for evaluation. All samples of the datasets were expanded near isotropic. In addition to the baseline, the system configurations were compared to other H-minima-based marker-controlled approaches where nuclei mask were generated with traditional methods and also to multiple well-known reference methods or software.First, the original slices are downsampled or upsampled, depending on the size of input slices, to the size of input space and the interpolated expanded image stacks define the expanded space. The expansion is performed using odd integer factors, usually with a factor 3, instead of resampling the voxels precisely isotropic. In this way, the original slices are included in the expanded stack, which makes the transformation back to the input space straightforward. In the expansion, expandImageFilter of the Insight Toolkit with linear interpolation mode is used . Keras framework [Masking with U-Nets.In the training of a given ramework   , Jaccardionally,  presenteJaccard index, like SQ, is defined as the average of IoUs of matched objects. However, in the implementation provided by Piccinini et al.  the IoU In addition to PQ, JI and AJI, we defined nuclei number difference percentage (NNDP) asSystem configurations were compared to the ground truths using the defined evaluation metrics. With the twelve spheroids we used all metrics whereas with the independent datasets we computed PQ and JI. Before computing the evaluation metrics, the segmentations were always transformed from expanded space to the size of the original ground truths. This was performed by choosing matching slices and via 2D nearest-neighbourhood interpolation or extrapolation on the x-y-plane when the dimensions of this plane differed from The segmentations obtained using the configurations discussed in Section h-value. In preliminary experiments, the score, named as the optimal score, was used to determine the highest h-value of the estimation range discussed in Section Estimation of optimal h-value using the h-value was found to be 5.0 with the eleventh spheroid.Using the twelve spheroids, we made an experiment where the roundness score Watershed methods and performed masking by Otsu’s thresholding method [In addition to the deep learning baselines, we constructed four different H-minima transform-based marker-controlled watershed approaches for results comparisons: WS, aifWS, nlWS and blWS. The baselines were used to segment the twelve spheroids. All the baselines used the watershed method B discussed in Section g method . aifWS, g method  implemeng method , bilaterg method , and patg method . MoreoveWhile we tested various system configurations on all datasets in the independent datasets, we used the Embryo and Neurosphere datasets to perform results comparisons with the software reported by Piccinini et al. . Most ofTable Utilisation of the edge emphasizing U-Net models, especially the 3D ones, improved the results with all system configurations. The importance of the use of H-minima transform was slightly higher since the scores of h-value was quantified by comparing to an approach where the roundness score The performance of the estimation of In Table In this study, we implemented several novel system configurations which utilized U-Nets, optimized H-minima transform and marker-controlled watershed for 3D nuclei segmentation. The basis of the configurations was the the deep learning enhanced marker-controlled watershed method (U-Nets+SWS), similar to QCANet by Tokuoka et al. . In thisAll U-Nets were trained using standard architectures and a loss function which thus were not factors in the performance difference of the system configurations. Consequently, all results improvements were either due to the use of deep edge emphasis, optimized H-minima transform or both. While these methods have been discussed previously in the literature , 34, 36,In our experimental setup, all nuclei samples were expanded near isotropic using linear interpolation. The motivation for the expansion rose from our preliminary experiments in which we observed that the performance of the watershed algorithm was greatly improved when the input image was transformed isotropic. A similar situation occurred also with 3D CellProfiler as depicted in Table C method-based systems. Consequently, the creation of seeds was not a necessity.The system configurations were tested on the dataset of twelve spheroids and the independent datasets. The results on the twelve spheroids in Table U-Net+SWS, U-Net-Cell and all system configurations outperformed the traditional baselines. All traditional baselines, except CeP-based systems, used watershed method B but performed masking via filtering and Otsu’s thresholding. The parameters of these baselines were chosen optimally in an unrealistic setting. However, any system configuration using watershed method B significantly outperformed them. This result highlighted the importance of the generation of nuclei masks using U-Nets.C. Consequently, the results implied that relying solely on 2D U-Nets instead of 3D U-Nets would not be advisable, but that the former could be used for nuclei masking if markers were created from seeds via H-minima transform-based watershed.To the best of our knowledge, we are the first to compare the performance of 2D U-Nets to 3D U-Nets in a 3D nuclei segmentation task. The configuration h-value would have been chosen to maximise score h-value was almost as effective. Even if more sophisticated methods for finding the best h-value, locally or globally, do exist [The scores in  brackets in Table do exist , 17, 18,The three best system configurations, based on the cross-validation using the twelve spheroids, as well as the deep learning baselines, were also evaluated using the independent datasets. The nuclei in these datasets were not highly clumped but their imaging protocols and nuclei types, excluding the Liver dataset, differed from the twelve spheroids. For this reason, the main purpose of the evaluation on the datasets was to test the ability of system configurations to generalize to data dissimilar to the training data. Each system configuration and the baseline U-Net+SWS* included twelve different U-Net settings. We reported the average scores and their standard deviations of these settings in Table Another highlight of this study was the comparison of 3D version of U-Net-Cell , 35 agaiA limitation of machine learning based methods is the uncertainty of generalizability to other types of data sets or to datasets acquired at other sites. In our tests, the independent datasets included three datasets in which the imaging protocols and the cell cultures were different than in our training sets. We achieved satisfactory results on these datasets. However, only datasets having visually similar image contrast and image features were chosen for this study. It is likely that if applied to datasets that differ substantially from the data used in training, the models will fail. Another limitation is the tedious work to create manually segmented ground truths, especially in the case of 3D volumetric data. The use of synthetic data  and sparh-value locally instead of globally and the replacement of the roundness score with some other metric or a set of metrics. The training and evaluation sets should be enlarged to ensure the functionality of the system configurations with more diverse imaging protocols and cell cultures.Our study provided evidence that marker-controlled watershed for 3D nuclei segmentation could be improved via using edge emphasizing U-Nets for mask and optimized H-minima transform for marker generation. The methods of deep edge emphasis and the use of H-minima transform are not tied to any specific neural network architecture or training procedure and can be exploited alongside virtually any deep learning-based nuclei segmentation approach in the future. The software and thirteen cell nuclei spheroids with manually segmented ground truths were made publicly available. Interesting future work directions could be the estimation of Additional file 1: Detailed information on the datasets used in the study is given and optimization process of the baseline algorithms is described.


Charge scaling as an effective solution to the experiment–computation disagreement in molecular modelling of ionic liquids (ILs) could bring the computational results close to the experimental reference for various thermodynamic properties. According to the large-scale benchmark calculations of mass density, solvation, and water-ILs transfer-free energies in our series of papers, the charge-scaling factor of 0.8 serves as a near-optimal option generally applicable to most ILs, although a system-dependent parameter adjustment could be attempted for further improved performance. However, there are situations in which such a charge-scaling treatment would fail. Namely, charge scaling cannot really affect the simulation outcome, or minimally perturbs the results that are still far from the experimental value. In such situations, the vdW radius as an additional adjustable parameter is commonly tuned to minimize the experiment–calculation deviation. In the current work, considering two ILs from the quinuclidinium family, we investigate the impacts of this vdW-scaling treatment on the mass density and the solvation/partition thermodynamics in a fashion similar to our previous charge-scaling works, i.e., scanning the vdW-scaling factor and computing physical properties under these parameter sets. It is observed that the mass density exhibits a linear response to the vdW-scaling factor with slopes close to −1.8 g/mL. By further investigating a set of physiochemically relevant temperatures between 288 K and 348 K, we confirm the robustness of the vdW-scaling treatment in the estimation of bulk properties. The best vdW-scaling parameter for mass density would worsen the computation of solvation/partition thermodynamics, and a marginal decrease in the vdW-scaling factor is considered as an intermediate option balancing the reproductions of bulk properties and solvation thermodynamics. These observations could be understood in a way similar to the charge-scaling situation. i.e., overfitting some properties  would degrade the accuracy of the other properties . Following this principle, the general guideline for applying this vdW-tuning protocol is by using values between the density-derived choice and the solvation/partition-derived solution. The charge and current vdW scaling treatments cover commonly encountered ILs, completing the protocol for accurate modelling of ILs with fixed-charge force fields. Ionic liquids (ILs) derivatives as greener non-flammable alternatives to traditional volatile organic solvents are becoming increasingly popular in various areas of modern chemical research, e.g., gas adsorption, refrigeration, adhesive, and electrochemical devices ,4,5,6,7.Molecular simulation as a powerful tool to probe the microscopic motions of different components in complex systems has been widely applied in various physical, chemical, and biological situations ,38,39,40Observables commonly extracted from atomistic simulations of ILs derivatives are bulk properties, e.g., mass density, viscosity, and diffusion ,56,57,58Quinuclidinium and its derivatives as a family of bicyclic heterocycles with interesting electrochemical and pharmacological properties are crucial components in the ILs’ universe ,69,70,71We first check the quality of atomic charges for ILs investigated in the current work as a pre-simulation parameter validation. By reproducing the ab initio ESP of individual molecules, the RESP charge scheme ensures the accurate calculation of inter-molecular electrostatic interactions with the Coulomb equation. Therefore, the quality of atomic charges is evaluated with the ESP reproduction. The ESP relative root-mean-squared error (RRMSE) is often computed with reference to the target of the regularized charge fitting, i.e., the HF/6-31G* ESP in the current case. It is often argued that this ab initio level would produce low-quality (inaccurate) results and shifting to higher-level Hamiltonians is preferred ,77,78,79We then perform molecular simulations and check the bulk property widely used as the criterion for parameter adjustment, the mass density. We first follow the normal charge-scaling protocol by scanning the mass density as a function of the scaling factor of atomic charges, the results of which are presented in 2 ~0.999) with slopes −1.82 g/mL and −1.73 g/mL, respectively. The closeness of the two ~−1.8 g/mL slopes hints about the similar mechanisms of the vdW-scaling impacts on the bulk arrangement of quinuclidinium-based ILs. If we further transform the slopes into the relative variation by dividing them by the experimental mass densities, we reach the two almost identical values of −1.34 and −1.33 for the two ILs.Further insights could be obtained by replotting the density profile along the vdW-scaling parameter, i.e., following the charge-scaling method. As the charge-scaling factor 0.8 is observed to produce satisfactory descriptions of bulk property and solvation thermodynamics and is considered as a near-optimal solution in our previous works ,60,62 anWith a properly tuned force-field parameter set at 298 K, a further interesting investigation of the bulk density concerns its performance in the physiochemically relevant temperature range. To this aim, we extract further experimental values between 293 K and 343 K, and perform further unbiased simulations in the targeted temperature range. To make the temperature space more regular and include the existing 298 K data in this scan, the newly simulated temperatures consist of 288 K, 308 K, 318 K, 328 K, 338 K, and 348 K. Namely, the whole temperature scan is performed between 288 K and 348 K with a 10 K interval. The density profiles under the normal 100% GAFF2, 1.03-scaled, and 1.05-scaled parameter sets are presented in Finally, it is worth comparing the current simulation outcomes with predictions provided by other techniques. A machine-learning predictor trained with more than 2000 experimental points was recently reported to achieve accurate predictions for mass density . The preThe check of mass density has starkly demonstrated the effectiveness of vdW-radius scaling in the molecular modelling of ILs. In this section, we investigate the impact of such a treatment on solvation and partition thermodynamics through large-scale nonequilibrium alchemical free-energy calculations. The atomic charges of ILs are scaled with the near-optimal solution 0.8 following our previous works ,60,62, aThe computational results and the experimental values ,100,101 To evaluate the closeness of the absolute values of thermodynamic observables, we compute the mean absolute error (MAE), the mean signed error (MSE), and the root-mean-squared error (RMSE), the values of which are also provided in r. From the numerical data in To evaluate the consistency of the ranking information from simulation and experiment, we compute Kendall’s rank correlation coefficient (τ)  and PearOverall, the 100% GAFF2 parameter set achieves the best accuracy for solvation and partition thermodynamics. The 103% σ parameter set produces good solvation thermodynamics in both ILs, but the partition data have slightly larger errors. The 105% σ parameter set predicts solvation/partition thermodynamics with large errors and thus should not be used. Thus, the solvation- and partition-derived vdW-scaling factor is confirmed to be 100%. Combining the analysis of bulk property in the previous section with the current solvation/partition results, based on our previous experience in charge scaling, we recommend to use vdW-scaling factors between the density-derived choice 103% and the solvation/partition-derived solution 100%, as a balanced choice that simultaneously reproduces the bulk properties and solvation/partition thermodynamics with small errors. According to our previous charge-scaling experience, often scaling factors leading to slightly large mass densities are preferred. Therefore, in the current case, the balanced solution should be slightly smaller than 103%. Considering the sensitivity of thermodynamic observables to the variation of the vdW-scaling factor, values such as 102% should be the optimal choice for the current quinuclidinium-based ILs. Although it is preferable to derive some general scaling-factor applicable to most ILs, as the GAFF2 parameter set is pre-fitted without system-specific tuning such as the ab initio calculation-involved charge generation, the summarization of such a generally applicable vdW-scaling factor does not seem possible. However, the general guideline that the balanced vdW-scaling parameter should produce slightly larger mass densities than the experiment remains workable, which could guide ILs-specific vdW tuning in practical situations.The construction of the ILs system follows the previously accumulated guidelines in our previous works ,60,62. TThe 3D chemical structures of all molecules except [QUIN8] were taken from the PubChem database, and that of [QUIN8] was obtained by modifying the aliphatic chain of [QUIN6] due to the absence of its structure in the database. The atomic charges were obtained with the RESP  charge sFor each pure ionic solvent box, we performed 5000 steps of geometry minimization, 10 ns NVT equilibration, and 20 ns NPT equilibration to reach the ambient condition , after which a 30 ns NPT production run was initiated for density estimation. After that, the last snapshot was extracted as the starting configuration of the bulk ILs for alchemical free-energy calculation.The alchemical method constructs an artificial pathway connecting physical end states to sample the transformation indirectly ,113,114.Asolvation = − ln<exp(-Wgrowth)> with growthW being the dimensionless microscopic work accumulated during the solute-creation transformation. To avoid underestimation of uncertainty due to the intrinsic behavior of the analytical formula [For post-simulation analysis, the microscopic works can be related to equilibrium thermodynamics with appropriate estimators, e.g., the Jarzynski’s scheme or the Crooks’ scheme ,126,127. formula , we perfIn all simulations , the velocity rescaling algorithm  with a 0Molecular modelling is becoming a popular tool in the chemical research of green solvents. Fixed-charge additive force fields are commonly employed due to the time and length requirements of investigated processes. The force field selection of RESP+GAFF2 is found to be quite robust in modelling both the bulk properties and the interactions between ILs and external agents (solutes). As the full RESP charges do not properly take the polarization and charge transfer effects into consideration, it is common to scale down atomic charges via some property-matching procedure to minimize the experiment–calculation deviation. The bulk property widely selected as the matching target is the mass density. As the bulk-property matching procedure only considers the solvent–solvent interaction, often the best density-derived solution degrades the reproduction of the other component in the solution mixture, i.e., the solute–solvent interaction. Through large-scaling free-energy calculations for a spectrum of solute–solvent pairs and a detailed investigation of component-specific responses, we discover and elucidate the non-monotonic responding behavior of solvation and partition thermodynamics with respect to charge scaling and identify the scaling factor 0.8 as a solution generally applicable to typical ILs, producing a balanced description of solute–solvent and solvent–solvent interactions and thus simultaneously reproducing the experimental bulk property and solvation/partition thermodynamics. Although charge scaling as an effective treatment could handle the disagreement issue in many cases, there are situations when charge scaling seems insufficient to eliminate the systematic bias. In such cases, vdW parameters are often considered as another set of tunable parameters, which are varied to improve the experiment–calculation consistency.The current work based on two ILs from the quinuclidinium family accumulates extensive numerical experiences in fine-tuning the vdW parameters (the radius parameter σ to be specific). The charge-scaling treatment cannot really perturb the mass density, and vdW-scaling effectively brings the computational results close to the experimental reference. Further, the best vdW-scaling factor obtained at 298 K is found to be effective in the physiochemically relevant temperature range between 288 K and 348 K, confirming the robustness of the vdW-scaling treatment. From the computation of solvation and partition thermodynamics under these vdW-scaled parameter sets, we notice that the vdW-scaling treatment has an impact similar to charge scaling. The improvement of mass density is often accompanied by the deterioration of the other non-targeted observables . Based on this observation, similar to the charge scaling case, we recommend a similar procedure of selecting the vdW-scaling factor, i.e., choosing a vdW-scaling factor between the density-derived choice and the solvation/partition-derived solution, in order to achieve a balanced description of different interactions involved in the complex solution system. The current vdW-scaling along with the previous charge-scaling treatment covers almost all commonly encountered ILs species, thus completing the general guidelines for accurate modelling with fixed-charge force fields.


We observed a significant decrease in the mean fluorescence lifetime in COVID-19 non-survivors (p < 0.001), in bacterial pneumonia patients without COVID-19 (p < 0.001), and in patients diagnosed with idiopathic pulmonary fibrosis (p < 0.001), relative to healthy subjects. Furthermore, these results suggest that the development of pulmonary fibrosis may be a real and serious problem in former COVID-19 patients in the future. A reduction in the mean fluorescence lifetime of plasma was observed in many patients 6 months after discharge. On the basis of these data, it can be concluded that a decrease in the mean fluorescence lifetime of plasma at 450 nm may be a risk factor for mortality, and probably also for pulmonary fibrosis in hospitalized COVID-19 patients.A method of rapidly pointing out the risk of developing persistent pulmonary fibrosis from a sample of blood is extraordinarily needed for diagnosis, prediction of death, and post-infection prognosis assessment. Collagen scar formation has been found to play an important role in the lung remodeling following SARS-CoV-2 infection. For this reason, the concentration of collagen degradation products in plasma may reflect the process of lung remodeling and determine the extent of fibrosis. According to our previously published results of an in vitro study, an increase in the concentration of type III collagen degradation products in plasma resulted in a decrease in the fluorescence lifetime of plasma at a wavelength of 450 nm. The aim of this study was to use time-resolved fluorescence spectroscopy to assess pulmonary fibrosis, and to find out if the lifetime of plasma fluorescence is shortened in patients with COVID-19. The presented study is thus far the only one to explore the fluorescence lifetime of plasma in patients with COVID-19 and pulmonary fibrosis. The time-resolved spectrometer Life Spec II with the sub-nanosecond pulsed 360 nm EPLED® diode was used in order to measure the fluorescence lifetime of plasma. The survival analysis showed that COVID-19 mortality was associated with a decreased mean fluorescence lifetime of plasma. The AUC of mean fluorescence lifetime in predicting death was 0.853 (95% CI 0.735–0.972,  An outbreak of the novel coronavirus nCoV-19 SARS-CoV-2), responsible for the coronavirus disease-19 (COVID-19), was first reported in Hubei province, China, on 31 December 2019. It has rapidly spread globally, with approximately 3 million confirmed infections and 200,000 deaths within the first four months , respons,5. VarioSince 7.2–31% of COVID-19 patients suffer from virus-induced ARDS . Indeed,As progressive pulmonary fibrosis is one of the possible consequences of COVID-19 pneumonia, there is a great need for a new blood biomarker to detect it. Pulmonary fibrosis is characterized by excessive deposition and degradation of collagen in the tissue. Thus, most markers of pulmonary fibrosis are related to the search for proteolytically cleaved matrix degradation fragments, or neoepitopes, which are released into the blood circulation. Enzyme-linked immunosorbent assays (ELISA) are often used for the detection of various types of collagens. Some collagen-degrading matrix metalloproteinases (MMPs) are also considered markers of fibrosis ,21,22. TThere is still a great need for easy diagnostic methods that reveal strong predictors of mortality, and those that allow the assessment of the patient’s condition and post-infection fibrosis. Fluorescence spectroscopy of whole blood or its components has been a very convenient tool in the diagnoses of many diseases . The metp < 0.001) between plasma fluorescence lifetime and brain natriuretic peptide that is secreted by the left ventricle in response to mechanical wall stress, and is involved in cardiac remodeling. To our knowledge, there are no reports of the use of time-resolved fluorescence spectroscopy in COVID-19 patients and, in general, in patients with pulmonary fibrosis. The formation of excessive scar tissue in the lungs in COVID-19 may be associated with a higher risk of mortality. Meanwhile, an increase in the concentration of collagen degradation products in blood circulation during the process of lung remodeling in the early phase of wound healing may determine the extent of pulmonary fibrosis. The results of our previous study revealed that an increase in the concentration of hydrolysed collagen added to plasma reduced the mean fluorescence lifetime at 450 nm . MoreoveIn this study, we included patients  hospitalAll patients had undergone basic laboratory tests assessing the advancement of inflammation, the function of their liver and kidneys, and the parameters of their coagulation system. The mean percentage of lung involvement as assessed by the application of HRCT pneumonia analysis was 28%. The study group of COVID-19 patients was invited back for us to take another round of blood samples after 6 months. The reference group was 10 patients hospitalized in the pulmonological department from the same centre who were not treated for COVID-19. These patients were admitted urgently for bacterial pneumonia, after excluding SARS-CoV-2 infection. The next reference group was 9 patients with previously diagnosed idiopathic pulmonary fibrosis (IPF) that was identified in accordance with the 2018 recommendation of the scientific societies . The conA 4-millilitre blood sample was taken for examination from each subject included in the study. For each COVID-19 patient, the sample was drawn 2–4 days upon admission to hospital department. All samples were processed within 2 h of collection. Blood samples were added to standard sterile polystyrene tubes containing EDTA, and then centrifuged at 3500 rpm at 4 °C for 5 min to obtain plasma. The plasma fraction was collected and stored at −80 °C until measurement. Multiple freeze–thaw cycles were avoided. Measurements were taken within 1 h of defrosting the sample.® diode emitting light of a 360-nanometre wavelength was used in order to measure the fluorescence lifetime of plasma. Plasma samples were not diluted. The exposure time of the samples was 5 min. Fluorescence measurements of the plasma were made at wavelengths of 450 nm. The samples were brought to room temperature prior to performing the assay. The measurements were carried out with the use of quartz 3.5 × 10 mm cuvettes. A time-resolved spectrofluorometer Life Spec II  with a sub-nanosecond pulsed EPLED2) statistical analysis and the visual assessment of residual plots.The fluorescence lifetimes were obtained by the deconvolution analysis of the data using the multiexponential model of fluorescence decay, and the instrument response function was taken into account. Then, the mean fluorescence lifetime value (mFLT) was calculated as the weighted average of fluorescence lifetimes obtained from the three-exponential model of fluorescence decay. As averaging weights, the contributions of individual components (areas under decay curves) to the total fluorescence were taken. The appropriate number of exponents was determined on the basis of chi-square . In case of the non-normally distributed variables, differences were compared with the Mann–Whitney U test and the Wilcoxon signed-rank test, respectively, and dependencies were determined with Spearman’s rank correlation coefficients. The probability of surviving to day 30 in COVID-19 patients was performed by using Kaplan–Meier curves, and tested with the log-rank test (Mantel–Haenszel). Differences were considered significant at p < 0.05. As a class predictor for the receiver operating characteristic (ROC) curve for a given parameter (mFLT), the value of this parameter was used.The preliminary step of the statistical analysis was the Shapiro–Wilk test of the normality of the distribution of the measured parameters. If the variables were normally distributed, the differences between independent and dependent samples were compared with the Student’s As shown in The raw data obtained from the TCSPC method have a fairly large signal intensity spread. The same data after zero-max normalization clearly show faster fluorescence decay in the group of patients who did not survive COVID-19, compared to the patients who recovered (see the main plot in p < 0.001), in bacterial pneumonia patients without COVID-19 (p < 0.001), and in patients diagnosed with idiopathic pulmonary fibrosis (p < 0.001), relative to healthy subjects. Subgroup analyses revealed that the non-survivor group had a shorter fluorescence lifetime with a high level of significance (p = 0.001), relative to survivor patients. The 6-month post-hospitalized COVID-19 patients showed also a shorter mFLT compared to healthy subjects (p < 0.05).In this study, the authors analysed only values of mFLT weighted by the fractional contribution of each component calculated from three-exponential models. p < 0.001; cut-off points = 7 ns; 62% sensitivity and 100% specificity) differentiated the groups very accurately. The ROC curve analysis is shown in p = 0.001). There was no culmination of deaths within 30 days. There was also no correlation between mFLT and death time.The Kaplan–Meier survival curves  showed sp < 0.05) was observed only for some parameters: WBC (r = 0.28), neutrophils count (r = 0.34), and platelets count (r = 0.39). The mFLT was not dependent on the age and gender of patients. Moreover, prior medical conditions did not appear to affect mFLT. Smoking cigarettes was also not associated with the mFLT. There was also no correlation between the radiological score of computed tomography (HRCT) and the mFLT.In the presented study, no correlation of mFLT with most clinical parameters was observed. A slight correlation  was associated with a mortality rate of 27%, while no deaths were recorded at the higher levels.Understanding the course of COVID-19 among hospitalized patients is important for clinicians who are seeking to improve health outcomes in this high-risk population . EstimatIt is still premature to know whether lung changes occur as a temporary response to COVID-19 infection, and if they will spontaneously resolve over time, or if they represent an irreversible pathological feature caused by the viral infection that will persist in surviving patients. Surely, the data collected until now suggest that pulmonary fibrosis persists for many months after recovery from COVID-19 in some patients. Finally, it is still unknown whether the pulmonary fibrosis developed following COVID-19 infection is stable or progressive, especially considering possible genetics, aging, and metabolic risk factors; further investigations are needed ,52,53. IThis study has some limitations: small sample size in each group, a single-centre donation, and variable time interval of each patient from hospital department admission (up to two days difference) to collection of blood. The purpose of the study was to estimate the mortality of patients admitted to the hospital in a mildly severe to moderate state, not demanding invasive mechanical ventilation. For this reason, patients in very severe clinical conditions were excluded from this study.An unequivocal statement on whether the obtained results reflect actual pulmonary fibrosis requires confirmation, e.g., using lung biopsies and blood collagen degradation products concentration measurements. It is important to point out that this experiment was preliminary, and further investigation in this field of research is crucial. It should also be carefully examined whether other components related to COVID-19 mortality risk can influence the fluorescence lifetime at 450 nm.It was demonstrated that the method of detecting collagen degradation products based on time-resolved fluorescence spectroscopy may have significant diagnostic value in the assessment of pulmonary fibrosis. The presented results indicate that a shorter mFLT of plasma samples upon admission to hospital may predict a more severe course of the disease, leading to lung damage, and hence a poorer prognosis. Moreover, the method can be used to identify individuals with post-COVID-19 syndrome who are at high risk of developing pulmonary fibrosis for further diagnoses using highly specific diagnostic tests. Although this hypothesis requires further testing, the numerous benefits of using the fluorescence lifetime measurement encourage consideration of its application in clinical practice. The fluorescence lifetime detection method may also be suitable for clinical screening tests because this method is non-invasive, not time-consuming, and requires only simple pre-treatment of samples without using additional reagents.


SARS-COV-2 can contribute to long term consequences associated with sepsis and circulatory dysfunction. In this insightful paper, we highlight the emerging pathophysiology utilizing two case examples. Both systemic and organ specific features are discussed. In addition, a novel laboratory assay is presented that identified SARS-COV-2 in the circulation using conserved SARS ion channels rather than the spike protein. The presentation is linked to the pathophysiology with the emphasis for early recognition and continued research. This paper will serve as a catalyst for continued discovery. The two SARS-COV-2 cases shared here demonstrate the multiple organ involvement that can occur with this disease. Growing evidence points to the role of viral sepsis as a key component in severe SARS-COV-2 and even newer research reveals an assay that can identify the virus in the bloodstream. This new evidence creates the opportunity to improve patient outcomes by better understanding of its replication and transport in its human host. This paper will summarize two cases with multi-organ involvement and discuss the impact on those organ systems. In addition, it will summarize research of a new assay which identified live SARS-COV2 virus in the bloodstream of patients. The goal of this paper is to put forth thoughtful evaluation of the disease and promote development of new tools that will assist in identifying virus in the circulation as well as quantifying risk for disease severity to improve patient outcomes.COVID-19 is spread primarily when a person infected with SARS-CoV-2 exhales and another individual inhales the virus. Indoors, droplets from people speaking can linger in the air for approximately 8 to 14 minutes and this is likely a factor for increased transmission of COVID-19 in enclosed spaces . These sIon channels in SARS-COV-2 are conserved from SARS and allow rapid replication of the virus . VariantOnce the alveolus is exposed to the SARS-COV2 virus, the spike protein binds to the ACE2 receptor on the alveolar type II epithelial cell and begins the process of making the cell a host for replication of SARS-COV2 virus . The lun79year-old female with ascending aortic aneurysm, mild coronary artery disease, and hypertension. Patient was a neurologically intact female with sharp wit and excellent self-management of her health care who developed severe delusional symptoms during her SARS-COV-2 hospital stay and was declared incompetent to manage her affairs. Following recovery, she requested full psychiatric evaluation to re-evaluate her incompetency and was declared competent. This raises concerns regarding active SARS-COV-2 virus circulating and crossing the BBB.79 year-old female diagnosed at local hospital with SARS-COV-2. She was hospitalized early 2021 and treated for SARS-COV-2 pneumonia, hypoxia, and altered mental status. She did not require intubation. Her altered mental status persisted through her hospital stay and her medical power of attorney oversaw her care when she was discharged from the local hospital with improving pneumonia on early 2021. She was home for one day before shortness of breath and worsening mental status resulted in her transfer to a larger facility for care early 2021. There she recovered fully and requested psychiatric evaluation to reinstate her competence and care for herself. Patient returned home and has effectively managed her care since that time with a return to her physical and mental baseline.57 year-old male with mild sensory neuropathy secondary to disk space narrowing lumbosacral spine with surgical back history, Sick Sinus Syndrome, mild cardiomyopathy with pacemaker in 2011 followed by Automatic Implantable Cardioverter Defibrillator in 2015 after a seven-minute run of Ventricular Fibrillation. Cardiology check-up fall 2020 was unchanged from baseline with review of 2019 ECHO at 57% EF, MUGA 59% EF and Lexiscan with no ischemia or infarction.Fall 2020 patient was diagnosed at local urgent care with SARS-COV-2 by rapid antigen. By winter 2020 patient declined and was transferred by ambulance to a local hospital for shortness of breath. There, his SARS-COV-2 was confirmed, and he was diagnosed with hypoxia and acute respiratory alkalosis. By 6pm patient was intubated, and chest x-ray revealed bilateral infiltrates interstitial and alveolar. The patient was diagnosed with acute respiratory failure with hypoxia and flown to larger hospital to facilitate his more aggressive treatment needs. During flight his saturations were 85-91% on 20 of positive end expiratory pressure 55% O2. The following day the patient given continuous propanol for sedation due to the requirement of intubated ventilation and pronation. Patient treatment regimen required ventilation and pronation for 20 days when he was extubated. Neurology consult revealed severe chronic sensory motor polyneuropathy due to (CIDP) Chronic Inflammatory Demyelinating Polyneuropathy.Patient remained in hospital for another month before being transferred to rehab facility due to inability to walk and overall weakness with final diagnoses at discharge of Acute Hypoxic/Hypercapnic Respiratory Failure due to SARS-COV-2 Pneumonia, ARDS due to SARS-COV-2, Severe Sensory Motor Polyneuropathy due to CIDP, and Acute Metabolic Encephalopathy. Patient remained in therapy facility for 2 months when he was discharged home and monitored by home health. Following discharge patient developed shortness of breath and was seen at the local hospital where CTA revealed nine of ten segments of right lung with embolus and 1 embolus left lung. Patient was again transferred to a larger facility for management. It was five months from his diagnosis before he was walking again and eight months before he regained 90% of his baseline.The cardiac impact of Covid has been significant. Patients with heart disease often had more severe disease, worse progression, and poorer outcomes . In addiHigh expression of the ACEII receptor in myocardium represents a high risk for viral impact on cardiac tissue which can counter even the most aggressive efforts of ventilation . A 2022 Although many of the traditional manifestations of SARS-CoV-2 were recognized early in the pandemic that began in 2019-such as cough, fever, diarrhea, and fatigue-neurological manifestations of the disease were quickly determined to be a frequent and severe complication . It was The exact mechanism by which SARS-CoV-2 causes encephalopathy is uncertain; it is likely multifactorial and discussed in further depth below. However, there is evidence that it is mediated by endothelial cell dysfunction and subsequent blood brain barrier disruption, a dysregulated immune response from excessive proinflammatory cytokines, molecular mimicry, and direct invasion of neural tissues -30. CurrOverall, encephalopathy is associated with worse outcomes , and theChronic Inflammatory Demyelinating Polyneuropathy (CIDP) is an acquired inflammatory disorder of the peripheral nervous system that commonly presents as a predominantly symmetric motor weakness affecting both the proximal and distal muscles lasting longer than eight weeks . SensoryThere have been proposed associations with viral and bacterial infections as well as well as autoimmune disorders, including rheumatoid arthritis, myasthenia gravis, and multiple sclerosis . Severe The nature of SARS-CoV-2 provides valuable insight into its neurological manifestations, and through examination of the pathophysiology of SARS-CoV-2, its neurological sequalae will be explored. Severe SARS-CoV-2 infection may induce cytokine storming, which is a dysfunctional immune response caused by excessive proinflammatory cytokines release ,43. ThisHowever, the presence of encephalopathy in patients with severe illness may indicate additional mechanisms. Anosmia is a known feature of SARS-CoV-2 infection, and it has been proposed that SARS-CoV-2 may enter the Central Nervous System (CNS) through retrograde transport through the olfactory bulb ,29. Oncevia a synapse-connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways. Considering the high similarity between SARS-CoV and SARS-CoV2, it remains to make clear whether the potential invasion of SARS-CoV2 is partially responsible for the acute respiratory failure of patients with COVID-19. Awareness of this may have a guiding significance for the prevention and treatment of the SARS-CoV-2-induced respiratory failure [Additionally, the viral infection of endothelial cells then results in disruption of the blood brain barrier that triggers acute inflammation, resulting in neuron damage, disruption of neurogenesis, and encephalopathy . Further failure . This di failure .via molecular mimicry of NMDA receptors [via virus in the vascular system. Research as early as 2020 was showing the impact of both SARS-CoV-2 inflammatory response and the virus itself not only on organs but also on the vascular system [Neurological manifestations may also present after the acute phase of SARS-CoV-2 infection. There is evidence that SARS-CoV-2 may cause subsequent autoimmune encephalitis eceptors ,30,50. Teceptors -33. Liker system . This rer system . Now, inr system  we are pvia virus in the circulation is one that has potential to create new knowledge for better patient outcomes. Recognition of the complexity of damage that occurs within the vasculature as well as the organs it serves is a primary next step in our understanding. Tools such as the ion channel assay to evaluate for SARS-CoV2 in the blood may prove a novel way to evaluate for risk of severity and assist with prognosis and treatment in a global pandemic experiencing variation and diversity of disease. It’s mechanism of evaluation is on ion channels that are conserved within the different variants rather than on the spike protein which often has mutation. As vaccination numbers increase and treatments emerge it is easy to forget the significant impact this illness can have on the human multi-organ system and the vasculature system that serves it. However, longer data periods status post COVID-19 are demonstrating that we have not seen all the damage yet. These case studies are a reminder of that severity and the need to press forward with tools to identify risk for severe disease early. Testing that can determine if the virus is prevalent in the blood may be a tool to help identify patients at higher risk for severe illness. The assay developed in the Yu lab uses a methodology of fluorescence.6 This method detects not only positive from negative disease, but also offers the possibility for using the fluorescence to quantify virus amount in the circulation which may correlate with severity of disease. The possibility generates thoughtful consideration for future research.Evidence for severity of symptoms correlates well with sepsis and the presence of virus in the bloodstream. More data is required to evaluate if virus in the circulation correlates with more severe illness. As we are beginning to have data that lets research begin to establish prospective studies based on the path behind us, this evaluation of systemic injury


The aim of this study was to identify the occupational risk for a SARS-CoV-2 infection in a nationwide sample of German workers during the first wave of the COVID-19 pandemic (1 February–31 August 2020).Klassifikation der Berufe 2010). PCR-confirmed SARS-CoV-2 infections were assessed from self-reports. Incidence rates (IR) and incidence rate ratios (IRR) were estimated using robust Poisson regression, adjusted for person-time at risk, age, sex, migration background, study center, working hours, and employment relationship.We used the data of 108 960 workers who participated in a COVID follow-up survey of the German National Cohort (NAKO). Occupational characteristics were derived from the German Classification of Occupations 2010  3.3–4.1]. IR differed by occupational sector, with the highest rates observed in personal  and business administration  services and the lowest rates in occupations related to the production of goods . Infections were more frequent among essential workers compared with workers in non-essential occupations  and among highly skilled compared with skilled professions .The results emphasize higher infection risks in essential occupations and personal-related services, especially in the healthcare sector. Additionally, we found evidence that infections were more common in higher occupational status positions at the beginning of the pandemic. By 2022, the coronavirus disease COVID-19) pandemic had led to over 320 million infections and 5.5 million deaths worldwide 9 pandemi. HoweverGenerally, workers in essential occupations are supposed to be at highest risk for a SARS-CoV-2 infection . EssentiOverall, occupational infection risks have been studied only in few countries yet. However, the interplay of contextual aspects of the country  and the applied policy measures  is likely to cause country-specific differences. Therefore, further studies in different countries are necessary. We aimed to address this research gap for Germany, where currently available empirical evidence is limited to sick leave data of a small number of insurance funds .In addition to previous studies, we analyze occupational differences with both a focus on major groups  but also according to occupational status positions . The horizontal dimension classifies workers according to performed tasks and duties and creates occupational groups that have high in-group similarity  . HorizonTaken together, in this study, we aimed to add knowledge on occupational differences in SARS-CoV-2 infection risks by providing first analyses for Germany based on a nationwide sample of 108 960 workers. We present a comprehensive analysis of occupational infection risks and study different horizontal and vertical occupational characteristics.We used data from the German National Cohort (NAKO), which is the largest population-based cohort study in Germany and has therefore high potential for investigating health-related consequences of the pandemic . BetweenBetween 30 April–12 May 2020, all 197 834 participants who gave their consent to be contacted again were invited by letter or e-mail to participate in a COVID-19 survey that thematically focused on health-related consequences of the pandemic. From 30 April–20 November, 161 892 people completed the questionnaire (response 81.8%). For the purpose of this study, we linked data of this COVID-19 questionnaire with information about participants’ occupation obtained previously during the baseline interview. We hereby assumed that most of the participants’ occupation did not change between the baseline and COVID-19 questionnaire.The sample used for the following analyses was restricted to participants who completed the COVID-19 questionnaire and were currently employed or self-employed. Classification of persons in employment was based on labor force status assessed with the COVID-19 questionnaire and based on the concept of the International Labor Organization . As we wInfection with SARS-CoV-2. Infection with SARS-CoV-2 was assessed by two questions. The first question was: “Have you been tested for the corona virus once or several times in a doctor’s practice, in a test center or in a hospital since 1 February 2020?” Notably, performed tests were PCR tests as no antigen tests were available during this time frame. If participants replied with “yes”, a second question was posed, asking “Was at least one of the test results positive?” An infection with SARS-CoV-2 was defined as responding with “yes” to both questions. For those with a positive or negative test, a further question was posed asking for the date of the first test (if tested positive) or the date of the last test (if ever tested negative). This information was used to calculate the person-time at risk (see next paragraph). As different SARS-CoV-2 test rates may influence the likelihood for detecting infections, we report the number of conducted tests for each indicator of occupational grouping in supplementary e-Person-time at risk. Since individuals were observed for different lengths of time (some completed the questionnaire earlier than others), the individual person-time at risk was calculated and taken into account in the multivariable analyses. The person-time at risk was the number of days between the 1 February 2020 and the date the COVID-19 questionnaire was filled in (for those not tested or tested negative) or the date of the SARS-CoV-2 test (in case a person was tested positive).Occupation. Participants’ job title at the time of the baseline examination was ascertained by an open-ended question. A semi-manual coding procedure was applied by trained staff to convert responses into the five-digit occupational coding scheme German Classification of Occupations (Klassifikation der Berufe or KldB 2010) developed by the German Federal Employment Agency in cooperation with the German Federal Statistical Office . As the large number of 37 main groups is not practicable for analytical purposes, we aggregated them in five occupational sectors and 14 occupational segments  or non-essential. Essential workers, ie, workers in essential occupations in times of the pandemic, were defined based on the first three digits of the KldB-2010 in accordance with previous studies that relied on the List of the Berlin Senate Administration from 17 March 2020  unskilled or semiskilled occupations not requiring any formal training, (ii) skilled activities requiring vocational training, (iii) complex activities requiring further vocational training or a bachelor’s degree, (iv) and highly complex activities requiring an advanced tertiary degree. Supervisory and managerial responsibility was determined by the fourth digit of the KldB 2010 in combination with the denoted skill level. We differentiated between workers with (i) no supervisory or managerial tasks, (ii) supervisors and (iii) managers. Compared to supervisors, managers additionally have personnel and budget planning functions .Control variables. To control for possible confounding and to compare occupations with different socio-structural compositions, we controlled for age, sex, migration background, study center, weekly working hours, and the employment relationship (employee or self-employed) in multivariable analyses. We defined migration background if the interviewee reported that he or she or at least one of his or her parents was not born in Germany. To avoid over-adjustment bias, we have refrained from including health variables as smoking status or overweight, which are not considered as confounders but rather mediators in the relationship between occupation and infection . We used Pearson’s chi-squared test for categorical variables and two-sided t-test for continuous variables to investigate whether socio-demographic and employment-related characteristics significantly varied by test status .Third, we used modified Poisson regression analysis with robust variance estimation to compare incidence rates (IR) of SARS-CoV-2 infections by occupation . To handResults of regression analyses were presented as IR for horizontal indicators and as incidence rate ratios (IRR) for vertical indicators. IRs for horizontal indicators were used as they do not require the definition of a reference category, which is somewhat arbitrary in a nominal categorical variable as occupation. Therefore, we converted regression estimates by a post-estimation command into adjusted predictions at the means (APM) along with respective 95% confidence intervals (CI), indicating the IR of SARS-CoV-2 infections per 1000 workers for average values of covariates . RecalcuMissing values in variables of interest (0.2–6.4%) were imputed by a predictive mean matching procedure. Tables showing the pattern of missing information (e-table 2) as well as a comparison of the original versus imputed data (e-table 3) can be found in the supplementary material. All analyses were performed using Stata 16.1 MP .Among the 108 960 workers interviewed February–August 2020, 6062 (5.6%) reported to have been tested for SARS-CoV-2 at least once. Among them, 404 persons had a positive test result, resulting in a cumulative incidence in the sample of 0.37%. The mean person-time at risk was 109.5 days.The mean age of participants was 48.3 years [standard deviation (SD) 10.8], 51.2% were women, and 29.1% had a migration background. As shown in Additionally, we plotted IRR using non-essential workers as the reference category . When coWe estimated infection rates by ISCO major groups as an alternative occupational coding scheme (see supplementary e-table 4). We found the highest IR among technicians and associate professionals , followed by managers , and the lowest rates in craftsmen and related trades workers , as well as among machine operators and assemblers . As different SARS-CoV-2 test rates may influence the likelihood for detecting infections, we show the frequency of infections and tests conducted in each occupational group . Thus, reported infection rates in occupations that were affected by the lockdown do not truly reflect the risk of infection. A further point is that observed associations should not be generalized to subsequent phases of the pandemic, as several conditions have changed later on, including the occurrence of new virus variants, the implementation of workplace safety measures and the roll-out of vaccines. Another point is that it is unclear what share of infections in our population was transmitted during work, as we have no information where an infection originated. Furthermore, test rates varied by occupation and were considerably higher in medical occupations. Thus, we could underestimate infection risks in non-medical occupations. Moreover, our analysis is based on the occupation held at time of the baseline examination. Although studies indicate a low degree of occupational mobility over time (only around 4% of German workers change their occupation per year) , this miApart from these limitations, this study has several strengths. For the first time it was possible to link individual data on SARS-CoV-2 infection risks with occupation-related information based on a population-based study in 18 study centers in 13 federal states of Germany. The large number of cases in the NAKO allowed a comparatively robust estimation even of a rare event such as SARS-CoV-2 infection during the first wave. The extensive processing of occupational information allowed a large number of different occupational characteristics to be examined. Furthermore, by taking into account many socio-demographic and occupational covariates, important confounders could be controlled for. As occupational differences in infection rates remained robust even after adjustment for a wide range of confounder, this speaks in favor of robustness of the main findings.This study yields important insights into occupational SARS-CoV-2 infection risks in Germany for the first pandemic wave. Our results reinforce previous empirical evidence emphasizing higher infection risks among workers in essential occupations and personal-related services, especially in the healthcare sector. Additionally, we found evidence that infections were more common in higher occupational class positions at the beginning of the pandemic.The Federal Institute for Occupational Safety and Health  financially supported the scientific realization of this analysis (Grant BAuA-F2515). The analysis was conducted with data from the German National Cohort (GNC). The GNC is funded by the Federal Ministry of Education and Research (BMBF) [project funding reference numbers: 01ER1301A/B/C and 01ER1511D], the federal states and the Helmholtz Association with additional financial support by the participating universities and the institutes of the Helmholtz Association and of the Leibniz Association. We thank all participants who took part in the GNC study and the staff in this research program.The authors declared no conflict of interest concerning the research, authorship, and publication of this article.Ethikkodex) has been developed and the study is under steady surveillance by the ethical committees of the regional study centers (Written informed consent was obtained from all participants included in the study. An external ethics advisory board has been established that accompanies NAKO over the full study period. A ‘Code of Ethics’ of NAKO ( centers .The datasets analyzed during the current study are not publicly available due to privacy concerns but are available from the corresponding author on reasonable request.


Human protein kinases, the key players in phosphoryl signal transduction, have been actively investigated as drug targets for complex diseases such as cancer, immune disorders, and Alzheimer’s disease, with more than 60 successful drugs developed in the past 30 years. However, many of these single-kinase inhibitors show low efficacy and drug resistance has become an issue. Owing to the occurrence of highly conserved catalytic sites and shared signaling pathways within a kinase family, multi-target kinase inhibitors have attracted attention.To design and identify such pan-kinase family inhibitors (PKFIs), we proposed PKFI sets for eight families using 200,000 experimental bioactivity data points and applied a graph convolutional network (GCN) to build classification models. Furthermore, we identified and extracted family-sensitive (only present in a family) pre-moieties (parts of complete moieties) by utilizing a visualized explanation  method for deep learning, gradient-weighted class activation mapping (Grad-CAM).This study is the first to propose the PKFI sets, and our results point out and validate the power of GCN models in understanding the pre-moieties of PKFIs within and across different kinase families. Moreover, we highlight the discoverability of family-sensitive pre-moieties in PKFI identification and drug design. Over 300 protein kinases share a common biological function as ATP-dependent phosphorylation enzymes , with a To overcome these issues of drug resistance, various studies have suggested drug combinations or multi-targeting drugs to be an effective approach for complex diseases , 13–18. Graph convolutional network (GCN) is a recIn this study, we aimed to develop GCN models to identify PKFIs and to highlight the chemical pre-moieties. First, we collected PKFIs from the ChEMBL database , 29 and An overview of our method and models for identifying PKFIs is presented in Fig. To assess the PKFI differences between each kinase family, we applied GCN models on eight families in four kinase groups Table  individuTo evaluate the performance of each model, three metrics were applied: 1) accuracy (ACC) for general precision, (2) Matthew correlation coefficient (MCC) for measuring the quality of classification according to class-wise distributions, and (3) AUROC for measuring the composite index of sensitivity and specificity. The evaluation metrics of the test results of the eight PKFI sets are presented in Table  accuracyExplanation and visualization were generated using the Grad-CAM method ))7)). As After confirming the rationality of the explanations of Grad-CAM, we further compared the rationality of the explanations of inhibitors from different families see Fig. B. The EGIn addition to the intra-TK-grouped comparison, we examined the inhibitors of the PIM family in the CAMK group to further assess the inter-grouped differentiation on the explanation of PKFIs. Aligned by the position of the hinge region as the center, LGH-447 and AZD-1208 of the PThrough the comparison of inhibitors across three families, family-sensitive pre-moieties and environments were demonstrated, along with the actual kinase-binding inhibitor structures.While revealing the family-sensitive pre-moieties  by focusing along the decision process of the model, statistical significance must also be considered. Owing to the unfixed structure of pre-moieties, we utilized moiety-based fingerprint checkmol  to estabThe inconsistency between Grad-CAM-based family-sensitive pre-moieties and moiety-based checkmol fingerprint mapping might indicate that the information and rules of kinase-inhibitor interactions are hidden within the compound structures and compositions to a greater extent than we assumed. The GCN architecture is not only able to discover the specified local environments within inhibitors from each family but also shows the limitations of the current moiety-based describing methods. Furthermore, in this study, the GCN architecture has the potential to broaden the recognizable chemical moiety spaces and thus facilitate the rapid identification of potential inhibitors from large chemical spaces.In this study for exploring kinase multi-targeting, we formulated pan-kinase-family inhibitor sets for the first time and used GCN architecture to identify the hidden information in the PKFI sets of EGFR, JAK, and PIM families. We further applied Grad-CAM to visualize the effects of chemical environments on inhibitors that were considered by the models to make decisions. Validated by the kinase-inhibitor complexes, we discovered that the family-sensitive pre-moieties contain information on kinase-inhibitor interactions and are associated with different modes of inhibition of kinase activity. By comparing our discovered pre-moieties and the checkmol moiety-based fingerprints, we demonstrated the insufficiency of current moiety-based descriptors, which can be overcome by the GCN architecture for recognizing family-specific chemical environments. In summary, the GCN technique has the power to identify PKFIs and learn the undefined pre-moieties in the field of potential drug design and optimization.To access the information on kinase-inhibitor reactivity, we collected 195,802 sets of kinase-chemical activity data from ChEMBL and kinase profiling, which contain about 60,122 compounds belonging to 384 kinases grouped into 103 kinase families.50 bioactivity, (2) exclusion of relation of “ ~ ” and “ >  > ” for a certain activity, and (3) use of “Binding” assay type, “SINGLE PROTEIN” target type and confidence score 9 for retaining the direct experimental kinase-inhibitor interaction data. The resulting set contained 95,462 data points for 58,846 compounds and 382 kinases with IC50 < 500 nM as the activity cutoff.The ChEMBL data was obtained from the ChEMBL version 25 March 2019), which contains 15 million data points of 1.8 million chemicals and 12.5 thousand targets. We filtered our kinase-inhibitor set according to the following criteria: (1) selection of targets of 518 kinases [9, which To understand the complete test results between compounds and kinases, we collected kinase profiling data  published by Metz  and furtAfter collecting these two sources of data, we merged them with “80% voting” for duplicates, which meant that the final active/inactive labels for the duplicated data points were in agreement with 80%-consistent answers among its duplicates, and those with maximum consistency below 80% were excluded. The final kinase bioactivity dataset contained 195,802 test points, with 60,122 compounds and 384 kinases.i and family j must be no less than half of the total membership of family j with testing data on at least two kinase members. The final label of each PKFI is given as an answer when all the test points are consistently active or inactive.To establish the PKFI sets from kinase-inhibitor data, we further investigated the criteria for any compound Ci where To facilitate the classification of PKFIs with GCN frameworks, an attributed graph escribed , 31  was then applied after each convolution output to eliminate overfitting upon the pseudo-atomic order stipulated just for utilizing the GCN framework. Then, the final softmax binary classifier was applied to obtain a concatenation of three pooled feature maps to predict PKFIs Fig. .c of compound i, and the weight vector k features is calculated by the summation of the back-propagated gradients along the atom order dimension.Grad-CAM was originally designed and applied for convolutional neural networks (CNNs) , is an ak of compound i, we computed the Grad-CAM feature map of layer By defining the weights for each feature