The National Lung Cancer Screening Trial (NLST) demonstrated the use of low dose helical computed tomography (LDCT) scans for lung cancer screening. However, the NLST was implemented in urban hospitals and prior to the Lung CT Screening Reporting and Data System (Lung-RADS). In this retrospective cohort study, 774 eligible patients received LDCT screening using Lung-RADS criteria. Eighty-four patients (10.9%) had subsequent testing performed compared to 24.2% in the NLST study. Of those with subsequent testing, 21.4% were diagnosed with lung cancer compared to only 4.6% in the NLST study. Lung-RADS significantly reduced unnecessary testing while identifying higher rates of lung cancer compared to the NLST. Lung cancer is the leading cause of cancer-related mortality in the USA, accounting for one in four cancer-related mortalities . KentuckWhile there have been tremendous strides in screening for other types of cancers, such as colon, prostate, and cervical, lung cancer screening still remains an area of controversy. The National Lung Cancer Screening Trial (NLST) was a large, multicenter, randomized controlled trial that compared annual low dose helical computed tomography (LDCT) to chest X-ray in high-risk individuals as a tool for lung cancer screening ,7. The NAdditionally, the NLST was conducted prior to the advent of Lung CT Screening Reporting and Data System (Lung-RADS) . Lung-RASt. Claire Healthcare (SCR) is a 159-bed community hospital in NE Kentucky and is registered with ACR to perform lung cancer screening. SCR provides healthcare services to 11 counties in northeastern Kentucky, all of which are rural counties, with a total population of 166,130 people served. Their lung cancer screening program commenced in 2015 and includes trained on-site radiologists who read all LDCTs and a data manager to ensure all data are entered into the ACR system. Annual reminders are sent out to patients by the radiology suite but shared decision-making and tobacco cessation must be performed and documented by the ordering provider, which is verified by radiology. All readings follow the ACR Lung-RADS reporting criteria and data are uploaded into the SCR ACR database for tracking and monitoring purposes.https://www.acr.org/Clinical-Resources/Reporting-and-Data-Systems/Lung-Rads. While the Lung-RADS criteria were not in effect during the NLST study, the retrospective analyses of the NLST study by Lung-RADS categories by Pinsky et al. were used to compare SCR results [et al. results, we also collapsed the Lung-RADS results from our study into the same categories. Some readings in the NLST had more than one category findings (example 3 and 4B). For these cases, we attributed their count to the category with the greatest probability of malignancy.SCR de-identified medical record and ACR data from August 2015 to August 2018 were used to determine the distribution of findings by Lung-RADS category. Details of the Lung-RADS categorization can be found on the ACR website at:  results . In thatWe further assessed the number of subsequent testing till diagnosis (if any), ranging from additional imaging to resections performed based on the findings of the LDCT, and calculated the percentage that resulted in a cancer diagnosis. SCR results were then compared to NLST baseline LDCT results and number of subsequent testing and cancer diagnoses. Subsequent testing in our study included follow-up imaging, biopsies, or any other procedures and the definition was mirrored as it was described in the NLST study. All study procedures were approved by the University of Kentucky and St. Claire Regional Medical Center Institutional Review Boards.There were a total of 774 SCR patients who received a baseline LDCT for lung cancer screening between August of 2015 and August of 2018 . The meaet al.’s retrospective analyses of the NLST trial [Among baseline SCR LDCT readings, 85.2% were categorized as Lung-RADS 1 or 2, 8.0% as Lung-RADS 3, and 6.7% as Lung-RADS 4A, 4B, or 4X . These rST trial .n = 84), 21.4% were diagnosed with cancer compared to 4.6% in the NLST study who had subsequent testing.In terms of subsequent testing and cancer diagnoses, we found 84 patients (10.9%) had further testing performed at SCR compared to 24.2% in the NLST study . MajoritControversy surrounded the announcement of the lung screening guideline when published by the United State Preventive Services Task Force . The conIn this study, we are able to demonstrate how a small rural community hospital successfully screens lung cancer in a region with one of the highest lung cancer rates in the USA. The utilization of Lung-RADS in the screening protocol demonstrated 13.3% fewer additional testing performed, when compared to the NLST. Among SCR patients who had additional testing performed, the percentage of patients identified with lung cancer was approximately five times higher compared to the NLST population. This demonstrates that the application of Lung-RADS in the screening procedures resulted in fewer subsequent testing procedures despite the high rate of lung cancer identified compared to the NLST. The rates of cancer identified at SCR can also be attributable to the fact that the prevalence of lung cancer is highest in Eastern Kentucky compared to the rest of the USA. This is an important factor when determining the value of LDCT screening in populations with high risk of developing lung cancer. Overall, lung cancers identified through lung cancer screening (LCS) in Eastern Kentucky were double (2.3%) compared to the NLST study (1.1%) . We alsoThe NLST was conducted across urban and metropolitan sites. The infrastructure, resources, and access are different in rural communities. A secondary aim of this study was to evaluate the feasibility of implementing LDCT screening guidelines in a rural community. We sought to assess whether subsequent testing rates would be higher compared to the NLST study. In fact, SCR conducted fewer subsequent tests compared to both the NLST study and VA system study ,10,14. SThere are limitations in our study that should be noted. While we compared SCR screening outcomes to a study that applied the Lung-RADS criteria post-hoc to the NLST study, there could be significant differences in false-positive rates and subsequent testing rates if the Lung-RADS was actually applied during the NLST a priori. Nonetheless, the intention of our study was to demonstrate the difference of subsequent testing rates with the application of Lung-RADS criteria at the time of LCS compared to the NLST study, which lacked such criteria. Another limitation is that we conducted the study in a region of the country with the highest rates of lung cancer . This imWe conclude that the application of the Lung-RADS has the potential to reduce unnecessary testing; and when testing is warranted, a higher rate of lung cancers can be identified. Further research is needed to assess whether these findings are replicable in other rural regions where lung cancer is comparable to national rates.


Drosophila immune cells was until recently limited to three major cell types, based on morphology, function and few molecular markers. Three recent single cell studies highlight the presence of several subgroups, revealing a large diversity in the molecular signature of the larval immune cells. Since these studies rely on somewhat different experimental and analytical approaches, we here compare the datasets and identify eight common, robust subgroups associated to distinct functions such as proliferation, immune response, phagocytosis or secretion. Similar comparative analyses with datasets from different stages and tissues disclose the presence of larval immune cells resembling embryonic hemocyte progenitors and the expression of specific properties in larval immune cells associated with peripheral tissues.The catalog of the  Immune cells are able to move and connect distant tissues and organs. This feature likely accounts for their pleiotropic role as sensors and regulators of the internal state in homeostatic, challenged and pathological conditions. While pleiotropy seems to arise from immune cell heterogeneity, the cause and nature of cell diversity is still poorly understood. How much does it depend on intrinsic differences dictated by cell autonomous cues vs. environmental conditions met by these cells during their life? To address the longstanding question on the impact of nature vs. nurture, of cell identity vs. cell state, we first need to characterize the different subtypes in depth.Drosophila shares with vertebrates several factors controlling the differentiation of the myeloid lineage , feeding 3rd instar larvae (48 h ALH) and wandering 3rd instar larvae (96 h ALH)  were dowPearson correlation were computed as follow. Pseudo-transcriptomes were generated for the hemocyte subgroups from the stage 6 embryos data , from thThe dot plot  was gene3. The source code was downloaded from the GitHub repository https://github.com/aertslab/pySCENIC.git. The https://resources-mirror.aertslab.org/cistarget/. The analysis was carried out on the wasp infested expression matrix from Cattenoz et al. deposited in the ArrayExpress database at EMBL-EBI4 under the accession number E-MTAB-8698.To identify the regulons enriched in the lamellocytes, we ran Single-Cell regulatory Network Inference and Clustering (SCENIC)  through 5 the regulatory elements within 5 kb upstream the TSS and the transcript introns6 were used for the analysis. The most significant regulons showing differential activity among clusters were determined with Mann-Whitney U-test -3xmcherry , rabbitThe following secondary antibodies were used at 1:500: FITC donkey anti-chicken IgG (Jackson ImmunoResearch Labs Cat# 703-095-155), FITC goat anti-mouse IgG (Jackson ImmunoResearch Labs Cat# 115-095-166), Cy3 donkey anti-mouse IgG (Jackson ImmunoResearch Labs Cat# 715-165-151), Cy3 goat anti-rat IgG (Jackson ImmunoResearch Labs Cat# 112-165-167), Cy3 donkey anti-rabbit IgG (Jackson ImmunoResearch Labs Cat# 711-165-152), Cy5 goat anti-mouse IgG (Jackson ImmunoResearch Labs Cat# 115-175-003) and Cy5 goat anti-rat IgG (Jackson ImmunoResearch Labs Cat# 112-175-167), Cy5 goat anti-rabbit IgG (Jackson ImmunoResearch Labs Cat# 111-175-144), Alexa Fluor 647 goat anti-mouse IgG (Jackson ImmunoResearch Labs Cat# 115-605-166).The slides were analyzed by confocal microscopy (Leica Spinning Disk and Leica SP8) with 20x, 40x, and 63x objectives, using hybrid detectors in photon counting mode. DAPI was excited at 350 nm, the emission filters 410–510 were used to collect the signal; FITC was excited at 488 nm, the emission filters 498–551 were used to collect the signal; Cy3 was excited at 568 nm, emission filters 648–701 were used to collect the signal, and Cy5 was excited at 633 nm; emission signal was collected at 729–800 nm. The images were analyzed with Fiji .Leptopilina boulardi as well as wounding triggers precocious lymph gland histolysis. These challenges also lead to the differentiation of lamellocytes that encapsulate the wasp eggs or participate to the wound closure . The progenitors undergo several rounds of division, differentiate into plasmatocytes and crystal cells and migrate along stereotyped routes to spread throughout the organism (PM hemocytes) . The sec closure  and the  closure .The larval PM hemocytes were recently analyzed in three scRNAseq studies , followip < 0.01 in The systematic comparison of the lists of markers from the three studies allows the identification of common subgroups . The marFor example, PM9 in Tattikota et al. dataset shares a significant number of markers with PL-0, PL-3, PL-vir1, PL-prolif, PL-Inos, and PL-robo2 in Cattenoz et al. dataset . The subThe marker comparison shows a large correlation across studies for crystal cells and lamellocytes: 53 crystal cell markers and 265 lamellocyte markers are commonly found by Tattikota et al. and Cattenoz et al. . All theThe markers involved in mitosis, including Cyclin B (CycB) , the phoTwo datasets contain subgroups of plasmatocytes enriched in transcripts involved in immune pathways: PL-AMP, PL-Rel and PL-vir1 in Cattenoz et al.; PM3, PM5, PM6, and PM7 in Tattikota et al. The comparison of the markers indicates a correlation between PL-Rel/PL-AMP and PM3/PM6/PM7 as well as between PL-vir1 and PM3/PM5 .PM6/7 and PL-AMP are highly enriched for AMP expression . Of note, the AMPs Mtk, DptB, and Drs are enriched at higher levels in the dataset from Tattikota et al. than in the one from Cattenoz et al. This is likely due to the induction of these AMPs by clean wounding, a condition specific to the dataset from Tattikota et al. All these subgroups also express high levels of Matrix metalloproteinase 1 (Mmp1), involved in wound healing . PM3 andPM5 and PL-vir1 present common expression profiles, distinctive from the other subgroups. They are not enriched in AMPs  and seemThe comparison of the markers consistently identifies a subgroup of cells representing less than 1% of the hemocyte population across the three studies: PL-ImpL2 , PM11 T, and theThe datasets from Fu et al. and from Cattenoz et al. identified phagocytic plasmatocytes, plasmatocytes secreting storage proteins and plasmatocytes secreting opsonins.et al.) are enriched for the phagocytic receptor NimC2  and PL-robo2  and PL-Lsp  display a highly distinctive expression pattern of proteins secreted in the hemolymph such as Larval serum protein 1 alpha , Larval serum protein 2 (Lsp2), Apolipophorin (Apolpp), and Odorant binding protein 99b (Obp99b) . All theAt last, the thanacytes  and PL-Pcd  express low levels of NimC1 and secrete the opsonins Thioester-containing protein 2 and 4 (Tep2 and Tep4) that promote the phagocytosis of bacteria and the activation of the Toll pathway . The thaThese three subgroups were not identified in Tattikota et al. dataset. Their markers are detected in some cells but these cells are not clustered together and are associated with the pool of unspecified plasmatocytes, with whom they share the majority of the markers.The datasets from Tattikota et al. and Cattenoz et al. include conditions promoting the differentiation of the lamellocytes and identify two subgroups. One of them expresses both plasmatocyte and lamellocyte markers and likely corresponds to plasmatocytes that are transdifferentiating into lamellocytes . The second subgroup lacks most of the plasmatocyte markers and expresses the lamellocyte markers strongly . For the sake of simplicity, we will call the first and the second subgroups intermediary and mature lamellocytes, respectively.To identify the molecular pathways activated during lamellocyte differentiation, we carried out a regulon analysis using SCENIC  on the dThe regulons CrebB, Foxo and REPTOR-BP are involved in the maintenance of energy metabolism upon food restriction or molecular stress . They maDrosophila, we have screened tissue specific or embryonic scRNAseq datasets containing hemocytes  and compared their molecular signatures to the dataset of Cattenoz et al.In mammals, macrophages are conditioned by their microenvironment, which leads to molecular signatures specific to the tissues in which they reside . To asseThe scRNAseq analyses on larval eye discs  reveal aNext, to determine if the eye disc associated hemocytes represent a subgroup that was not defined in Cattenoz et al. dataset, we compared their pseudo-transcriptome to the combined pseudo-transcriptome of all subgroups from Cattenoz et al. . This coOne of the main characteristics of the unspecified plasmatocytes is the expression of Extra Cellular Matrix (ECM) components such as Pxn, which is necessary for the assembly of the basement membrane . Pxn is Two scRNAseq datasets on the larval brain from 1st instar to 3rd instar larvae  report tThe single cell analyses suggest that the larval hemocytes originate from the proliferative subgroup. We therefore predicted that the prohemocytes present at the early embryonic stages may resemble this subgroup and compared the larval dataset from Cattenoz et al. with the one obtained from scRNAseq on embryos at stage 6 . At thisDrosophila larva. Molecular features consistently found across the studies allow the identification of robust plasmatocyte subgroups in addition to crystal cells and lamellocytes. The proliferative subgroup resembles the prohemocytes present in the early embryonic stages and the plasmatocytes associated with different larval tissues seem to present specific features.The comparative analysis of the scRNAseq datasets confirms the diversity of the immune cells present in the The present analysis identifies common subgroups with specific potentials  or lacking specific properties (unspecified plasmatocytes) . We cannot exclude that discrete subgroups were not found because of their low frequencies in the specific tissue. This may notably be the case of the hemocytes associated with the eye discs, which display markers of the large subgroup of unspecified plasmatocytes. In addition, the dissection procedure may remove the less tightly associated cells, rendering our interpretation incomplete. The development of The comparison of scRNAseq datasets brings robustness to the precise information on the cellular diversity gained with a single dataset. However, this approach is highly dependent on the analysis pipelines followed within each study. Since the number of studies using scRNAseq are currently booming and in parallel, new analytical tools are constantly generated, the comparability between datasets is not always straightforward . GeneratDrosophila lead to the differentiation of specific subgroups. Future lineage tracing assays will help assessing the relative impact of intrinsic and environmental cues on the presence of the different subgroups as well as their stability. The generation of subgroup specific drivers will make it possible to assess the role of the different subgroups in physiological and pathological conditions. Finally, bulk RNAseq of distinct subgroups (sorted according to the markers from the scRNAseq) will generate deeper molecular signatures. The combination of more refined experimental and analytical approaches will determine with higher precision the similarities and differences between subgroups and reveal the function of the subgroups. This will contribute to unravel the heterogeneity and the biology of the macrophage populations.In sum, the comparison of the three scRNAseq datasets allows the definition of common features and robust subgroups, an important asset to understand the biology of hemocyte heterogeneity. Some subgroups show a distinctive transcriptomic signature, while others present many shared markers, suggesting an origin according to nature and nurture, respectively. Increasing evidence highlight the influence of the microenvironment in governing the gene expression profile in mammalian tissue-specific macrophages, despite their common embryonic origin . We expewww.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-8698.The data was deposited in the ArrayExpress database at EMBLEBI (PC and AG: conceptualization, investigation, writing—original draft, funding acquisition, and supervision. PC, AG, AP, and SM: methodology and writing—review and editing. AG: resources. All authors contributed to the article and approved the submitted version.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.


The KEGG pathway and GO analysis have considerably augmented some of the miRNAs that play a key role in regulating basic biological processes. Our research offers valuable understanding into miRNA biology and aids to comprehend the purpose of miRNA in regulating major biological changes in animals.Sheep have an indispensable position in the world. Studies have shown that the reproduction of sheep is regulated by the photoperiod. However, sheep are short-lived animals that often rub in the fall. MicroRNAs are a class of non-coding RNAs of 21 to 25 nucleotides that play an important role in animal development. The expression and role of miRNAs in the estrus regulation of sheep are unclear. In this study, we present miRNA expression profiles in the sheep pituitary gland in the estrus and anestrus states. We detected a total of 199 miRNAs and 25 differentially expressed miRNAs in sheep pituitary gland during estrus and anestrus states. Six miRNAs were examined by reverse transcription quantitative-PCR and were significantly differentially (p < 0.05) expressed miRNAs, that are miR-143, miR-199a, miR-181a, miR-200a, miR-218, and miR-221 in both estrus and anestrus states. miRNAs containing estrus-related terms and pathways regulation are enriched using enrichment analysis from gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, we also envisioned a miRNA–mRNA interaction network to understand the function of miRNAs involved in the pituitary gland regulatory network. In conclusion, miRNA expression profiles in sheep pituitary gland in the anestrus and estrus deliver a theoretical basis for the study of pituitary gland biology in sheep.MicroRNAs (miRNAs) are a class of small non-coding RNAs, molecules of 21 to 25 nucleotides in length, that regulate gene expression by binding to their target mRNA and play a significant role in animal development. The expression and role of miRNAs in regulating sheep estrus, however, remain elusive. Transcriptome analysis is helpful to understand the biological roles of miRNAs in the pituitary gland of sheep. A sheep’s pituitary gland has a significant difference between estrus and anestrus states. Here, we investigate the expression profiles of sheep anterior pituitary microRNAs (miRNAs) in two states, estrus and anestrus, using Illumina HiSeq-technology. This study identified a total of 199 miRNAs and 25 differentially expressed miRNAs in the estrus and anestrus pituitary gland in sheep. Reverse transcription quantitative-PCR (RT-qPCR) analysis shows six differentially ( MicroRNAs are a class of small non-coding RNA molecules, of about 21–25 nucleotides in length that regulate gene expression by base pairing with their target mRNAs, primarily leading to protein inhibition. Initially, genes encoding miRNA in the nucleus are converted into primary transcripts by RNA polymerase II . Lin-4 wThe pituitary gland can be considered as the “master” endocrine organ of the body, acting as the central endocrine regulator of animal growth, bone metabolism, and the cell generation cycle . It takeIn recent years, miRNAs, which are essential for the development of the anterior pituitary gland, have been studied step by step. For example, in mice miR-26b regulates the expression of Pit-1 (pituitary-specific positive transcription factor 1) by inhibiting Lef-1 (lymphoid enhancer factor 1) expression. Lef-1 is a key transcription factor that is involved in many growth processes. It is a sequence-specific DNA-binding protein that is expressed in pre-B and T lymphocytes of adult mice . Lef-1 mPOMC (pro-opiomelanocortin) is a precursor of the pituitary melanocortin-related peptide and is regulated primarily by CRF (corticotropin-releasing factor). Previous studies revealed that miR-375 is greatly expressed in mice pituitary and that overexpression of miR-375 negatively regulates CRF-stimulated POMC expression and ACTH secretion . All of To the best of our knowledge, the role of miRNAs in sheep seasonal estrus regulation and mode of expression in the pituitary is still unclear. Studies have shown that sheep reproduction is regulated by the photoperiod . Estrus Ovine estrus regulations play an important role in growing meat and wool production in sheep commerce. According to reports, regular feeding causes estrus and therefore anestrus in sheep with low feeding status .Kazakh sheep in northern China are known to both breeders and consumers due to its quality meat and high adjustability. However, the anestrus period in these sheep is usually very long from April to July. Therefore, during anestrus season ewes are unable to mate and lambs are not available for quite a few months .In the past few years, the usage of Illu-HiSeq sequencing of the transcriptome has increased with the prospect of classifying differential expression and discovering new miRNAs. In order to fully explore and understand the role of miRNAs in pituitary regulation, we determined miRNA expression profiles in sheep in estrus (E) and anestrus (A) phases by Illu-HiSeq sequencing, assuming that there are differentially expressed miRNAs in these two physiological states. These data will help us to understand the role of miRNAs in the regulation of seasonal fertility, pituitary growth, and development in animals.All experiments involving animals were carried out under the regulations approved by the Animal Care and Use Committee of Shihezi University (SU-ACUC-08032).Healthy Kazakh ewes used in this study lived in the natural pasture of Shihezi, Xinjiang, China. Six ewes, about two years old, weighing 45 kg were selected. Three ewes were selected during the estrus season , and three ewes were selected during the anestrus season .One month before estrus, rams and ewes were kept together for no less than 10 min daily. Twice a day estrus was monitored according to the well-known estrus sign by experts.Once the back pressure test was carried out the sheep showed a rigid stationary state, the vulva turned red and extruded, and clear yellow mucus released from the vulva, which is a sign of estrus period; these sheep were part of the estrus pituitary group (PGE). During our first estrus observation on the first day of the estrus cycle, three sheep were selected for slaughter in order to collect the anterior pituitary gland. On the other hand, if there is no evidence of estrus after 34–36 days of second estrus cycles, it is called anestrus period . At thisAccording to the manufacturer’s instructions, the pituitary tissues of sheep estrus and anestrus were homogenized independently in liquid nitrogen, and total RNA was extracted using Trizol method .The quantity and quality of total RNAs were confirmed by the Bioanalyzer 2100 system and the RNA 6000 Nano kit . After testing the sample, a small RNA library and high-throughput sequencing, three samples of both PAG and PGE were sequenced independently by Beijing Nuowheyuan Bioinformatics Co., Ltd. Subsequently, a sequencing library was made using the NEBNext Super Direction RNA Illumina , following the manufacturer’s recommendations. Using the specific structure of 3’and 5’ end of sRNA, total RNA was used as an initial sample to be ligated at both ends of the sRNA and reverse transcribed into cDNA. Then, the target DNA fragment was isolated by gel electrophoresis and the gel was recovered for the cDNA library. After pooling, RNA-Seq sequencing was performed according to the requirements for effective concentration and target data. Statistical analysis of the length of sRNA was performed and sRNA shorter than 18 nucleotides were filtered for successive analysis. Types of sRNA are determined by the length of the distribution peak. Gene annotation and reference genome were downloaded from sheep reference genome . Using BTotal RNA was extracted from sheep pituitary using RNA Kit . cDNA was synthesized from the purified 500 ng total RNA using reverse transcription PCR (RT-PCR) Kit . RT-PCR was performed using specific primers for six miRNAs . RT-PCR amplification products were examined by electrophoresis and DNA sequencing to confirm the presence of miRNAs. Reverse transcription quantitative-PCR (RT-qPCR) was used to verify differential expression. All miRNAs identified according to the manufacturer’s protocol were subjected to RT-qPCR analysis using SYBR Green (Takara). RT-qPCR was performed using the following conditions: Initial denaturation at 94 °C for 10 min, subsequently 40 cycles at 55 °C for 30 s, 55 °C for 10 s, and 72 °C for 32 s, using a LightCycler 480 II system . U6 was used as a core reference to normalize miRNA expression. Three independent biological replicates were performed for all samples of qPCR. Expression levels of these genes were determined using SYBR Green Real-Time PCR. Next, the data were analyzed using the formula 2ΔΔCt. Primers are listed in MiRNAs bind to the 3-UTR (untranslated region) of the target mRNA, destabilize the mRNA, and perform translational silencing to suppress protein production . To bettp < 0.05 were considered noteworthy for enrichment analysis.The results were presented by cytoscape 3.7.2. Encyclopedia of Genes and Genomes (KEGG) enrichment analysis  and the To scientifically study the uniqueness and affluence of miRNAs, we scrutinized miRNA expression data from the pituitary gland in both estrus and anestrus states by Illumina HiSeq according to the flowchart a. Over 2Secondary structures of identified miRNAs were obtained by aligning the reference sequence with specific sequences in miRBase a. A totaTo understand the expression levels of both known and novel miRNAs in each sample, we used TPM to normalize expression levels and detect them by the density distribution of TPM a. A totaIn order to verify the accuracy of the differentially expressed miRNA by RNA-seq data analysis, six differentially expressed miRNAs were randomly selected for RT-qPCR analysis and the amount of the selected miRNA was normalized to the amount of U6 snRNA. Analyzing the results, we found that the overall trend of this data was the same as our RNA-seq data and the results of the two methods were highly consistent. Compared to the PGA library, the results demonstrated that oar-miR-143, oar-miR-199a-3p, oar-miR-181a, and oar-miR-218a were upregulated, but oar-miR-200a and oar-miR-221 were downregulated . The datp < 0.05) were expressively enriched in three GO analysis categories (p < 0.05)   and invo < 0.05) b. Data a < 0.05) c.Although transcription studies have been performed on sheep genomes, to our knowledge this is the first report on miRNA expression profiles in sheep pituitary gland in the estrus and anestrus states, enriching information on the miRNA database in this animal species.MiRNAs are a class of small non-coding RNAs. Different studies have revealed that miRNAs are closely linked to the growth and development of plants and animals. Carletti et al. found that miR-21 promotes the survival of the ovarian follicles during ovulation in mice . In addiGO analysis specified that most of the differentially expressed miRNAs involved 28 molecular functions, 33 cellular components, and 92 biological processes. Simultaneously, analysis of the KEGG pathway reveals that miRNAs are involved in signaling pathways and in metabolic regulation, disease, endocrine, and cellular signaling that are directly linked to the pituitary function. Similarly, MAPK (mitogen-activated protein kinase) signaling pathway involves a variety of signaling cascades, of which Ras-Raf-Mek-ERK1/2 (MAPK / ERK) is one of the most dysregulated in human cancers. It has an important regulatory role in keeping the balance between Sox2 + pituitary cell proliferation and differentiation . In addiIn conclusion, this study provides valuable information about miRNAs biology and aids us to comprehend miRNAs role in regulating basic biological developments like pituitary development. However, further experimentation is required to carefully evaluate the purpose of miRNAs and their predictive target analysis.


Understanding carriage and transmission potential of SARS-CoV-2 in children is of paramount importance to understand the spread of virus in school and community settings.We performed an updated rapid review to investigate the role of children in the transmission of SARS-CoV-2. We synthesized evidence for five categories and results are reported narratively.A total of 33 new studies were included for this review. We did not identify additional studies that reported documented cases of SARS-CoV-2 transmission by children. We identified 15 new studies that demonstrate children’s susceptibility and transmission risk of SARS-CoV-2 with evidence provided on the chance of being index or secondary cases, the potential of faecal-oral transmission, and the possibility of asymptomatic transmission. There is little data on the transmission of SARS-CoV-2 in schools. There were three studies reporting COVID-19 school outbreaks in France (Oise), Australia  and Israel. The remaining four studies found that all reported cases did not infect any other pupils or staff. With data from seven studies and governmental websites, the proportion of children among all confirmed COVID-19 patients was estimated for 29 countries, varying from 0.3% (lowest in Spain) up to 13.8% (highest in Argentina). Lastly, we identified seven studies reporting on PIMS-TS linked to COVID-19 among paediatric patients.There is somewhat limited evidence available for quantifying the extent to which children may contribute to overall transmission, but the balance of evidence so far suggests that children and schools play only a limited role in overall transmission. The outbreak of coronavirus disease 2019 (COVID-19), caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a global health, societal and economic threat. Understanding carriage and transmission potential of SARS-CoV-2 in children is of paramount importance to prevent the spread of the virus in school and community settings.Initial evidence suggests children may be less frequently infected than adults, and typically tend to have mild symptoms when infected. Emerging reports of a very rare Paediatric Inflammatory Multisystem Syndrome Temporally associated with SARS-CoV-2 (PIMS-TS) suggest a link to COVID-19 in paediatric patients [Our early rapid review of transmission in children identified several relevant studies and provided useful but limited information on susceptibility and transmission in children. We have now updated our rapid review and included large seroprevalence surveys, national or municipal population screening studies and tracing studies on school-based clusters of transmission. We aim to keep updating the rapid review to include new studies as they become available and to re-evaluate the conclusions given the rapid pace of ongoing research on COVID-19.theWe searched PubMed, medRxiv and the WHO COVID-19 database on 21 June 2020 with entry date limits from late 2019  and checked by a senior epidemiologist (ET). We synthesized evidence thematically and reported results narratively.The initial search retrieved 2034 titles of articles. After screening, 33 new studies were eligible for inclusion since our last search on 30 April 2020 (There is limited evidence detailing transmission of SARS-CoV-2 from infected children based on our previous review (covering the period up to 30 April 2020)  and two From this update, we identified 15 new studies that demonstrated the children’s susceptibility and transmission risk of SARS-CoV-2 with evidence provided on the chance of being index and secondary cases, the potential of faeco-oral transmission, and the possibility of asymptomatic transmission.Contact-tracing studies suggested that children were not likely to be the index case in households and children contacts were less likely to be secondary cases than adult contacts. A collection of international family clusters found that children were the index case in only 3 (10%) of 31 individual cluster studies . Data fret al conducted a retrospective multi-centre study, and followed up all children from 883 families with SARS-CoV-2 infected members in Zhejiang Province, China [Persistent shedding of SARS-CoV-2 in stools of infected children, which raised the concern of faeco-oral transmission, is supported by a new study. Hua e, China . They foThe proportion of children who are truly asymptomatic or pre-symptomatic remains unknown. Data from Italian emergency departments suggested that 21% of SARS-CoV-2 PCR-positive children were asymptomatic at the time of testing, but there was no follow-up to determine whether these children developed symptoms later (pre-symptomatic) . HoweverA hospital-based study in South Korea examined the viral load of 12 infected children and demonstrated that symptomatic children had higher initial viral load in nasopharyngeal swab specimens than asymptomatic children, but found no differences in faeces or saliva specimens . AdditioFour new studies on SARS-CoV-2 transmission in school settings were identified. A cross-sectional study in Belgium examined the transmission risk of SARS-CoV-2 in day-care settings. They randomly sampled 84 children who were attending day-care after the outbreak of COVID-19, and found no asymptomatic carriage of SARS-CoV-2 among young children . AnotherThe very large Spanish ENE-COVID seroprevalence survey (n = 61 075) conducted between 27/04/2020-11/05/2020 found lower seroprevalence in children (n = 6527) . SpecifiInternational research confirms that the percentage of children among the confirmed COVID-19 patients is low. A study analysed the global COVID-19 prevalence for 23 countries with data available for paediatric cases . It was A new paediatric multisystem inflammatory syndrome has been identified and temporally associated with COVID-19. Recent reports from Europe and North America have described a small number of children being admitted to intensive care units with PIMS-TS having some features similar to Kawasaki disease and toxic shock syndrome. This is rare but with common characteristics that have been observed during the COVID-19 pandemic reported from different cohorts -27. A reThis updated rapid evidence review summarises the most recently available evidence to understand the role of children in SARS-CoV-2 transmission. There is a lack of direct evidence on the dynamics of child transmission, however the evidence to date suggests that children are unlikely to be major transmitters of SARS-CoV-2.Direct evidence showing children as a source of transmission is scarce and largely based on small studies or studies investigating few paediatric cases. We did not identify more studies that reported documented cases of SARS-CoV-2 transmission by children. The original two studies included in our last rapid review  indicateBy synthesizing preliminary evidence in this update, our study shows that children have lower susceptibility for SARS-CoV-2 infection. A meta-analysis of eight studies reporting an OR for secondary attack ratios of children (vs adults) indicated that children were perhaps 50% less susceptible to infection than adults (odds ratio (OR) = 0.44, 95% CI: 0.29-0.69), with substantial heterogeneity . Of themThe persistent shedding of SARS-CoV-2 in stools of infected children were continually reported by new studies, which is in accordance with our findings from three studies in our last update, showing that SARS-CoV-2 may be present in the gastrointestinal tract for a longer duration than viral presence in the respiratory system -41. TherThere are very few data sets available on school specific transmission. Although a few outbreaks have been observed in schools, eg, in France (Oise), Australia  and Israel, it is not certain whether the source was from children or adults ,43  have implemented seroprevalence surveys. Data from these countries confirm that the percentage of children among the confirmed COVID-19 patients is small, varying from 1% in young children up to 6% in older children . In addiBeyond the seroprevalence, we also estimated the percentage of children among the confirmed COVID-19 patients based on data from governmental websites and publications. Data for 29 countries shows a substantial varying range (0.3%-13.8%) of proportion of paediatric patients. Although we have tried to find the most recent and comprehensive data for as many countries as possible, data for paediatric patients are not always available for most countries, or have not been recently updated on their governmental websites. We supplemented the governmental data by extracting relevant information from the publications, however, some of them are now out of date at the time of writing. We need to acknowledge that the reported figures may change along with progress of the pandemic in reported countries.It is widely acknowledged that children are less likely to suffer severe and critical COVID-19 disease than adults ; howeverThis rapid review has a number of limitations. Despite experienced reviewers undertaking searches, screening and data extraction, due to the tight deadline the literature review and data extraction were done by one person per article and so it is possible that some key articles may have been missed. We have not performed risk of bias assessment for the included studies and so this may have biased the results. Many of the included studies are pre-print publications or reports and therefore not peer-reviewed. The views expressed represent those of the authors and are not a substitute for professional medical advice.In conclusion, although there is limited evidence available for quantifying the extent to which children may contribute to overall (nationwide) transmission, given the lower susceptibility, relatively small proportion of infections and lower chance of being index cases, children are unlikely to have been significant drivers of the epidemic so far and there is not any evidence yet to indicate they are at higher risk of causing super-spreading events in the community or schools. The balance of evidence suggests that children play only a limited role in overall transmission, but it is noted that the relative contribution of children to SARS-CoV-2 transmission may change with reopening of society and schools.


Demand for the fabrication of high-performance, transparent electronic devices with improved electronic and mechanical properties is significantly increasing for various applications. In this context, it is essential to develop highly transparent and conductive electrodes for the realization of such devices. To this end, in this work, a chemical vapor deposition (CVD)-grown graphene was transferred to both glass and polyethylene terephthalate (PET) substrates that had been pre-coated with an indium tin oxide (ITO) layer and then subsequently patterned by using a laser-ablation method for a low-cost, simple, and high-throughput process. A comparison of the results of the laser ablation of such a graphene/ITO double layer with those of the ITO single-layered films reveals that a larger amount of effective thermal energy of the laser used is transferred in the lateral direction along the graphene upper layer in the graphene/ITO double-layered structure, attributable to the high thermal conductivity of graphene. The transferred thermal energy is expected to melt and evaporate the lower ITO layer at a relatively lower threshold energy of laser ablation. The transient analysis of the temperature profiles indicates that the graphene layers can act as both an effective thermal diffuser and converter for the planar heat transfer. Raman spectroscopy was used to investigate the graphite peak on the ITO layer where the graphene upper layer was selectively removed because of the incomplete heating and removal process for the ITO layer by the laterally transferred effective thermal energy of the laser beam. Our approach could have broad implications for designing highly transparent and conductive electrodes as well as a new way of nanoscale patterning for other optoelectronic-device applications using laser-ablation methods. For the fabrication of transparent electronic devices, such as touchscreen panels (TSPs) and flat-panel displays (FPDs), it is essential to develop transparent conductive electrodes (TCEs) with high transmittance and low resistivity. Although indium tin oxide (ITO) has been mainly applied as a typical material for TCEs in electronic devices because of its high figure of merit and good compatibility with conventional semiconductor-processing technology ,2,3, the2 electronic hybridization configuration [Graphene has been widely attempted to be used as one of the promising candidates for TCEs in various optical and electronic device applications owing to its high transmittance and high flexibility ,13,14. Tguration ,16,17. OIn this study, to realize a higher conductance of a TCE in an electronic device, a chemical vapor deposition (CVD)-grown graphene layer was transferred to indium tin oxide (ITO)-film-coated glass or polyethylene terephthalate (PET) substrates, and graphene on an ITO (graphene/ITO) double layer was suggested as a new TCE material. However, it is very difficult to produce fine electrode patterns because of the difference in etch selectivity between graphene and ITO layers. In the case of the wet etching process for the removal of the ITO layer, it is indeed challenging to maintain graphene-electrode patterns without damage. The electron-beam-lithography and plasma-etching techniques have been reported as some patterning methods for graphene ,19,20. O2) at a flow rate of 60 sccm, a few-layered graphene film was grown with methane (CH4) feedstock gas at a flow rate of 5 sccm. Subsequently, polymethyl methacrylate (PMMA) was spin-coated over the CVD-grown graphene layer on the Cu foil; then, the whole PMMA/graphene/Cu foil structure with Cu foil downside was carefully floated on ammonium persulfate 2S2O8, Sigma-Aldrich Korea Ltd., Seoul, Korea)) solution to etch off the Cu foil [A-few layered graphene films were produced in a CVD furnace and transferred to soda-lime-glass and PET substrates ,28,29, o Cu foil ,31, foll Cu foil , despite4) laser system  was used for the direct patterning of the prepared graphene/ITO-layered films  and 0.27637 , respectively. Those of the graphene/ITO/PET substrate were 0.18134  and 0.56833 , respectively. There is a tendency that in the same substrate, the lateral etch rate of graphene is higher than that of the ITO layer. This could be because of the difference in the thermal conductivity as well as film thickness between the two layers. For the same material, for example, the graphene layer with PET shows a higher value with increasing σ compared to that with a glass substrate. This result would be possibly due to the lower thermal conductivity [The difference in the laser-driven etched spot patterns between the graphene upper and ITO lower layers can be explained as a result of the higher thermal conductivity of graphene than that of the ITO layer . Therefouctivity ,40 as weuctivity . The theuctivity . The bou∂T∂z|ITO . κ is thComsol Multiphysics [The transient temperature distribution confirms the different characteristics of thermal-energy transport on graphene/ITO double and ITO single layers, enabled by the laser irradiation for 10 ns . The anaiphysics . First, iphysics  to 533 Kiphysics ) of the iphysics .−1. For the region 1, the three characteristic bands, D-, G-, and 2D-band, indicative of the existence of a graphene layer were found [−1) of weak intensity and both G- (~1580 cm−1) and 2D-band peaks (~2700 cm−1) on the graphene layer were observed. When the Raman spectra were acquired along the edge of a laser-etched spot, where the graphene upper layer was etched selectively but the ITO lower layer remained (region 2), very small traces of both the D- and G-band peaks were observed, but no 2D-band peak was detected, indicating that most of the graphene upper layer had been removed as a result of laser ablation. Compared to the Raman spectra of a center region of the laser-etched spot, on which no graphene-related peaks were observed (region 3), some of the residual amorphous carbon on the ITO lower layer was found, which is expected to have resulted from the incomplete heating and removal process for the ITO lower layer by the laterally transferred effective thermal energy of the laser beam.To confirm the removal of the graphene/ITO double layer after laser ablation, Raman spectroscopy was performed to examine a laser-etched spot on the graphene/ITO double layer a, where re found ,55,56,57In conclusion, we experimentally showed the laser-ablation-assisted patterning of graphene/ITO double layers for future TCE applications. To elucidate the underlying mechanism for the difference between ITO single and graphene/ITO double layers during the laser-ablation process, we also conducted a theoretical analysis of transient temperature spatial distribution, revealing thermal-energy transport on graphene/ITO double and ITO single layers. From the analysis, we demonstrated a role of effective thermal-energy transfer during laser ablation, indicating that the graphene layers can act as both an effective thermal diffuser and converter for the planar heat transfer. The analysis of Raman spectroscopy also supported this laser-ablation mechanism of the selective etching of graphene layers due to the effective thermal-energy transfer. Our study may provide insight into designing optimal electrode patterning using graphene layers, which would not only enable better electrode formation with high transparency and conductance for TCE applications but would also serve as a new way of nanoscale patterning for other optoelectronic-device applications using laser-ablation methods. Future work will be carried out towards the further reduction of the size of the pattern width as well as the development of complex shapes of pattern design.


Magnetocaloric materials based on field-induced first order transformations such as Ni-Mn-Ga-Co are promising for more environmentally friendly cooling. Due to the underlying martensitic transformation, a large hysteresis can occur, which in turn reduces the efficiency of a cooling cycle. Here, we analyse the influence of the film microstructure on the thermal hysteresis and focus especially on large angle grain boundaries. We control the microstructure and grain boundary density by depositing films with local epitaxy on different substrates: Single crystalline MgO(0 0 1), MgO(1 1 0) and Al Solid state cooling with magnetocaloric materials is a promising way for more efficient and environmentally friendly cooling techniques . The HeuHere, we use locally epitaxial films to understand how nucleation and phase growth depend on the film microstructure. For this, we compare the microstructure of Ni-Mn-Ga-Co films grown by DC magnetron sputter deposition on three types of substrates. The term “local epitaxy” describes that the film is not necessarily single crystalline, but may exhibit several, well defined orientation relations to the substrate. This results in large angle grain boundaries. We examine the impact of these grain boundaries on the martensitic transformation, specifically on the transition and hysteresis width. The hysteresis width Ni-Mn-Ga-Co films with a thickness of 800 nm were prepared by DC magnetron sputter deposition in a UHV chamber  (sample A), MgO(1 1 0) (sample B), and Alents see . Being ae before . The rouTo examine the influence of the different microstructures on the martensitic transition, we compare magnetization measurements in dependence of the temperature for the three films see . For eacn values . The marthe film . Not onlThe different transition behaviours of the films are well visible when plotting the volume fraction of martensite as a function of temperature. Since the magnetization measurements are not only influenced by the martensitic transition, but also by the magnetization change in vicinity to the Curie temperature of the austenite, it is difficult to analyse the fraction of transformed material from the magnetization curves. Therefore, we chose to analyse additional resistivity measurements see  to estimFor a discussion of the different transition behaviours of the films, we consider the following characteristic temperature spans: The transition temperature intervals anch see . This vaFrom the magnetization and resistivity measurements in Secondly, the hysteresis width undaries a,c, the undaries b. In par compare . While tIn the following, we describe how grain boundaries influence the martensitic transition in general and analyse why the different transition behaviours of sample B and C originate from the presence of large angle grain boundaries. The grain microstructure of the films is formed at high temperatures during deposition when the material is austenitic. This means that the martensitic microstructure has to adapt to the already existing grain boundaries. These large angle grain boundaries are generally incompatible with twin boundaries, a characteristic feature of the martensitic transition, which connect differently oriented martensitic variants. Therefore grain boundaries act as barriers to the formation of a compatible martensitic microstructure. To illustrate the impact of grain boundaries on the martensitic microstructure in films, the transition with grain boundaries (a) and without grain boundaries (b) is sketched in With this knowledge, we can explain the different transition behaviours of the three Ni-Mn-Ga-Co films: Sample A on MgO(0 0 1) is single crystalline, therefore the transition takes place in a small temperature interval with a uniform temperature dependence and without a two-step transition. In contrast, sample B on MgO(1 1 0) exhibits a large grain boundary density and a large variety of grain sizes ranging from 1 to 10 For an application in magnetocalorics, not only the hysteresis width but also the transition width for a complete transformation cycle should be as small as possible . TherefoA broad martensitic transition in Ni-Mn-based films with grain boundaries can be seen in previous publications ,22,23 anTo summarize, we presented results of the martensitic transition for epitaxial Ni-Mn-Ga-Co films grown on MgO(0 0 1), MgO(1 1 0) and Al


It is possible that the Coronavirus Disease 2019 (COVID-19) pandemic will also have long-term impacts on the cohort that was in utero during the pandemic, from exposure to maternal infection and/or the stress of the pandemic environment. We discuss how COVID-19 disease during pregnancy may affect fetal and postnatal development with adverse impacts on health and aging. Severe maternal infections are associated with an exaggerated inflammatory response, thromboembolic events, and placental vascular malperfusion. We also discuss how in utero exposure to the stress of the pandemic, without maternal infection, may impact health and aging. Several recently initiated birth cohort studies are tracking neonatal health following in utero severe acute respiratory syndrome virus 2 (SARS-CoV-2) exposure. We suggest these cohort studies develop plans for longer-term observations of physical, behavioral, and cognitive functions that are markers for accelerated aging, as well as methods to disentangle the effects of maternal infection from stresses of the pandemic environment. In utero exposure to COVID-19 disease could cause developmental difficulties and accelerated aging in the century ahead. This brief review summarizes elements of the developmental origins of health, disease, and ageing and discusses how the COVID-19 pandemic might exacerbate such effects. We conclude with a call for research on the long-term consequences of in utero exposure to maternal infection with COVID-19 and stresses of the pandemic environment.The 1918 Influenza pandemic had long-term impacts on the cohort exposed  We discuss how maternal infections and in utero exposure to the pandemic environment may affect postnatal development and lifetime cardiovascular, metabolic, and neurodevelopmental health. The effects of SARS-CoV-2 on pregnant women and neonates are compared to other coronaviruses, SARS (severe acute respiratory syndrome) and MERS-CoV (Middle East Respiratory Syndrome). We then suggest indicators of maternal, neonatal, and later life health that should be monitored following in utero exposure to SARS-CoV-2. Lastly, we consider how to disentangle the effects of maternal infection versus the effects of prenatal exposure to the stress of the pandemic environment, both positive and negative.By the end of 2020, about 300,000 infants may be born in the United States to mothers infected by the severe acute respiratory syndrome virus 2 (SARS-CoV-2) sometime during their pregnancy. Millions more will be born to mothers and families who have experienced tremendous stress and change in their daily lives and environments due to the pandemic. Postnatal effects of in utero during the pandemic.,in utero during the peak pandemic period, experienced more depression after age 50, diabetes and ischemic heart disease after age 60, and mortality after age 65, with some variation in estimated effects.–in utero in 1919 had more renal disease, circulatory and respiratory morbidities, and diabetes after age 60.The 1918 Influenza pandemic  had multiple health consequences for the cohort that was in utero exposure or indirectly by its multifarious stresses. Men in the 1918–1919 U.S. flu cohort were shorter at WWII enlistment than flanking cohorts,in utero married men with less education.,in utero during the 1918 epidemic was also confirmed in the parents of the Wisconsin Longitudinal Study participants,No data on birth weight are available for the 1918 Influenza cohort; however, there are many indications that early life physical health and neurodevelopment were directly affected either by in utero in 1918–1919 are based solely on birth dates and cannot identify individuals whose mothers actually experienced infections. Thus, the analysis is restricted to population data on timing of infections and birth dates related to outcomes many decades after the pandemic. Thus we cannot readily distinguish direct effects on long-term health and well-being due to exposure to the infection itself versus the stress effects due to exposure to the pandemic environment. These socio-environmental stressors include potentially increased levels of uncertainty, undernutrition, under- and unemployment, poverty, and loss of loved ones. Despite this, the findings from the 1918 flu pandemic of lasting consequences for those exposed prenatally warrants study of similar programming effects from the current pandemic. Unlike the earlier pandemic, the current pandemic provides opportunity to study individuals with known in utero exposure to SARS-CoV-2 throughout their life based on known maternal infection and socio-environmental stresses. We can add further critical comparisons to individuals exposed in utero to the pandemic environment but with no maternal infection, which potentially allows disentanglement of the effects of exposure to infection versus pandemic environment.These historical associations for the cohort ,Maternal viral infections can affect the fetus or neonate through multiple pathways. Maternal infections can directly infect the fetus as well as the placenta.in utero metabolism to cause intrauterine growth restriction (IUGR).Virus-related maternal inflammatory processes can impact the placenta and disturb We summarize the main points of the COVID-19 literature published thus far, bearing in mind that these findings are preliminary as they are based on small numbers, specific locations, and, often, more severely ill patients.,,Pregnant women do not appear to be at increased risk of contracting SARS-CoV-2, as their rates of infection seem to parallel the rates of infection in their surrounding communities. When pregnant women do contract the SARS-CoV-2 virus, the majority experience a similar clinical course as age-matched non-pregnant women; however, reports increasingly show need for more hospitalization, mechanical ventilation, and intensive care.As in the general population, a minority of pregnant patients develop severe and critical illness, with cardiopulmonary compromise, and even multi-organ failure. SARs-CoV-2 can cause an exaggerated inflammatory response and coagulation activation in some individuals, causing worse outcomes. Whether the immunologic changes of pregnancy affect the exaggerated inflammatory response is unknown. The limited data suggests that COVID-19 pregnancies have similar elevations of cytokines  and chemokines  as other COVID-19 patients.Little is known about the impact of the SARS-CoV-2 virus on the placenta and the impact of the associated inflammatory and thromboembolic responses on the placenta. There are some reports of neonatal infections from SARs-CoV-2 infection of the placenta and its transplacental transmission.,,Rates of preterm birth may be increased by maternal SARS-CoV-2 infections, with estimates ranging from 12% to 27%.Additionally, rates of C-section delivery are increased in pregnant women with SARS-CoV-2 infections. In a systematic review, C-sections occurred in 52%–85% of COVID-19 deliveries, which may also be overestimated.Stillbirths among women with COVID-19 so far have been rare and seem to have been related to very severe maternal illness. Infected mothers in one prospective study had significant several-fold increased rates of stillbirth.Data on effects of first and second trimester maternal infections is very limited, as is data for spontaneous abortions. There is a theoretical concern that fever in the first trimester may increase risk for miscarriage and congenital anomalies. Further studies of the outcomes of maternal infections, especially in the first and second trimesters are needed.In utero transmission needs further study to substantiate reports of transmission, based on polymerase chain reaction assay for SARs-CoV-2 in the neonate and/or placenta, or elevated IgM antibodies.in utero transmission are complicated by the initial lack of criteria defining vertical transmission. Horizontal spread of the disease to neonates from family members or other household contacts occurs and is generally mild.Most newborns born to mothers with COVID-19 disease have been healthy, and most commonly suffered from sequelae of prematurity rather than the effects of SARS-CoV-2. The rate of congenital infection from SARS-CoV-2 at this time is estimated at <3%, approximating the rates of other congenital infections.in utero. Such effects may span the lifecycle of these babies; during this time it may be possible to intervene to prevent later life problems. Cohort studies include the UCSF PRIORITY: Pregnancy Coronavirus Outcomes Registry and the American Academy of Pediatrics Society of Neonatal and Perinatal Medicine (AAP SoNPM) – National Perinatal COVID-19 (NPC-19) Registry, a collaboration of the AAP SoNPM with the Vermont Oxford Network (VON). Neither of these registries are currently supported for the long-term follow-up which is warranted by important scientific and clinical reasons.The COVID-19 cohort provides a natural experiment to examine the effects of maternal viral infection while infants were in utero for childhood development and adult health COVID-19 birth cohort studies consider immediate collection of data from the mother, fetus, neonate, and placenta. These initial data should be followed by analysis of child growth and development and lifelong study of health, behavioral patterns, and cognitive functioning. Table We suggest that to capture the consequences of viral exposure The child should be followed for indicators of growth, motor, and neurological development milestones. Throughout life, the standard blood-based indicators should be collected. Composite measures derived from clinical chemistry and physical exams can be used starting in childhood to categorize the “Pace of Aging.”in utero exposure to the pandemic environment and the effects of different regional and national policies to address viral spread. The COVID-19 pandemic has increased levels of stress, unemployment, food insecurity, and domestic violence, and diminished or disrupted prenatal care. These factors may have programming effects and/or direct effects on neonatal health. The pandemic-related lockdown policies may benefit some individuals; pregnant women staying home who may rest more, and have decreased exposure to other infections, and women with job stability, may benefit from less stress from work and commuting time. These benefits are evidenced by reports of decreased rates of preterm birth for the general population during the pandemic.The COVID-19 pandemic also provides an opportunity to study the effects of in utero exposure. We suggest the desirability of enrolling an additional child born either shortly before or shortly after the COVID-19 pandemic to the same mother. These study groups would allow comparison between infants who were exposed to maternal infection and those who were prenatally exposed to the pandemic without having experienced maternal infection. These infants can also be compared to those who were born before or conceived after the pandemic, providing further insight on the effects of the pandemic environment. The inclusion of information on social and economic stresses will allow comparisons between countries taking different measures to reduce spread of the virus, These types of comparisons may give us further insights beyond the effects of COVID, such as socioeconomic and social policies that may decrease risk of preterm birth, which has eluded the maternal-infant health community for decades. This should be balanced by further investigating reports of generally increased stillbirth and potential contributors.Studies would include mother and child dyads and a selection of mothers with symptomatic and nonsymptomatic COVID-19 diagnosis, as well as a group of mothers and babies with no in utero during this pandemic based on the 1918 influenza birth cohort. From comparisons of the 1918 Influenza with the current COVID-19 pandemic, we suggest studies of markers from birth through adulthood that are indicators of the altered development and accelerated aging that could be experienced in the century ahead.Maternal COVID-19 infections and exposure to the pandemic environment may have long-term effects on growth and aging for the cohort We need to evaluate whether the effects of COVID-19 can be generalized to other viruses. While the current pandemic is similar in some ways to the earlier, there are important differences. COVID-19 resembles the 1918 Flu in that many infections were relatively uncomplicated cases, but deadly for a small proportion of cases. Surprisingly, given the 100 year difference in time, the mortality during the two pandemics appears fairly similar in New York at the height of the pandemic; an increase over baseline of about 200 deaths per 100,000 population in the 1918 epidemic and an increase of about 150 per 100,000 in 2020.


The challenges with reconstruction of penile defects are plenty. In addition, no single and universally accepted reconstructive method exists for penile defect repair. Herein, we present the application of a circumferential penile shaft defect reconstruction using pull-up double-opposing keystone-designed perforator island flaps (KDPIFs) in one patient. This is the first case report of a circumferential penile shaft defect reconstruction using KDPIFs.A 43-year-old man who injected petroleum jelly into his penis 10 years ago presented with multiple firm nodular mass-like lesions adherent to the overlying skin along the penile shaft. Our urologic surgeon removed the foreign bodies and performed a primary closure with undermining. However, wound dehiscence developed, and skin necrosis was exacerbated 5 days postoperatively.We performed debridement, and the final post-debridement defect was circumferential (5.5 × 12 cm) from the base of the glans to the midpoint of the penile shaft.We covered the defect using pull-up double-opposing KDPIFs (10 × 13 cm each) based on the hot spots of the superficial external pudendal artery perforators on each side from the suprapubic area to the scrotum.The flaps survived perfectly, with no postoperative complications. The patient was satisfied with the final outcome and had no erectile dysfunction or shortening of penile length after a 6-month follow-up.We successfully reconstructed a circumferential penile defect with pull-up double-opposing KDPIFs both esthetically and functionally. Our technique can be a good alternative modality for extensive penile defect reconstruction. However, tissues having these properties are difficult to obtain. Thus, penile defect reconstruction is challenging. There are various reconstructive options including skin grafts, loco-regional flaps, and free flaps for the coverage of penile defects. Until recently, there has been no single and universally accepted reconstructive method for penile defect repairs. Reconstructive surgeons choose the proper method on a case-by-case basis because each technique has advantages and disadvantages. Furthermore, sometimes, the penile defect cannot be covered following previous reconstructive methods. Therefore, new or modified reconstructive techniques are warranted to cover the defect successfully and achieve better cosmetic and functional outcomes. The goals of penile reconstructive surgery are to provide reliable and durable coverage of the penis with minimal donor site morbidity both functionally and esthetically. Herein, we present and describe our modified keystone-designed perforator island flap (KDPIF) technique using pull-up double-opposing KDPIFs for the coverage of a circumferential penile shaft defect. To our knowledge, this report presents the first case of a reconstruction of a circumferential penile shaft defect using KDPIFs.Penile defects are an uncommon and difficult problem to address physically and psychologically.22.1A 43-year-old man visited the Department of Urology at our hospital for the removal of foreign bodies in his penis. The patient readily proffered the information that he injected petroleum jelly subcutaneously into the penis with the intention of increasing its girth about 10 years ago. Physical examination revealed multiple firm nodular mass-like lesions adherent to the overlying skin along the penile shaft. Our urological surgeon performed a circumferential incision, removed the foreign bodies and fibrotic tissues, and performed a primary closure with undermining. However, wound dehiscence developed 5 days postoperatively and skin necrosis was exacerbated circumferentially. Thus, the patient was referred to the Department of Plastic and Reconstructive Surgery for wound management and reconstruction.2.2We planned the debridement of necrotic tissues and flap coverage. The operation was performed with the patient in the lithotomy position under general anesthesia. We performed debridement using the Versajet II hydrosurgery system , and the final post-debridement defect was circumferential 5.5 × 12 cm) from the base of the glans to the midpoint of the penile shaft. We designed double-opposing KDPIFs (10 × 13 cm) based on the hot spots of the superficial external pudendal artery perforators on each side from the suprapubic area to the scrotum , and bilateral scrotal flap with or without V-Y incision.,5 The scrotal skin resembles the nature of the penile skin with regard to color and texture. Especially, the scrotal skin has high elasticity and pliability and is the most appropriate reconstructive option for penile shaft skin because its durability to withstand erection and friction can be acquired.,3,5,12 Thus, reconstruction with scrotal skin flap in penile defects can provide both aesthetic and functional advantages over other reconstructive options, such as skin grafts, other local flaps, and free flaps. Among various types of scrotal flaps, some techniques, such as the Cecil-Culp technique and bipedicled scrotal flap, require a 2-stage operation.,12 Other techniques, such as advancement musculocutaneous scrotal flap, bipedicled anterior scrotal flap, and bilateral scrotal flap, are performed as a one-stage operation.–3,5 In case of the extensive and circumferential penile defect coverage, both dorsal and ventral areas of the defects are covered with scrotal skins in these scrotal flap techniques. However, the dorsal areas of penile defects are relatively far from the scrotum and, therefore, can have different skin properties from the scrotal skin. Thus, we consider that it is more suitable, with respect to reconstructive principles, to cover the dorsal penile defects with the skins that are much closer. In our case, we covered the circumferential penile shaft defect with the pull-up double-opposing KDPIFs. Each keystone flap was positioned on each side with respect to the midline from the suprapubic area to the scrotum as our design. Consequently, each dorsal and ventral defect in our case could be covered with the closest skin.The scrotal flap is the most common local flap technique for penile defect coverage. Various types and modifications have been devised for extensive  penile defects, which include the Cecil-Culp technique ,,13 KDPIFs have a curvilinear-shaped trapezoidal design and are essentially comprised of two end-to-side V-Y flaps. They are traditionally classified into four types: type I (skin incision only), type II , type III (opposing keystone flaps designed to create a double-keystone flap), and type IV .,13 The KDPIF technique has the advantage of simple defect-adaptive design, easy reproducibility, safety, and short procedure time due to its inherent characteristics for minimal flap undermining and dissection. Several studies have described KDPIF reconstruction for perineal defects.,15 The perineum is a perforator-rich area, and the scrotum receives plentiful blood supplies from the perineal branch of the internal pudendal artery, the external pudendal branches of the femoral artery, and the cremasteric branch of the inferior epigastric artery.–3,7,12 In our case, we used double-opposing KDPIFs based on the hot spots of the superficial external pudendal artery perforators and performed much more undermining at the ring-shaped area of flap surrounding the base of the penile shaft in contrast with the minimal undermining concept of the original KDPIF technique. Nevertheless, all flaps fully survived with no complications. We considered that our surgical technique resulted in more reliable vascular perfusion due to the rich blood supply of the scrotum.The KDPIF, devised by Behan in 2003, has been popularly used to cover cutaneous defects at various anatomical locations. In our surgical method, we consider that four end-to-side V-Y flaps of double-opposing KDPIFs had considerable effects on securing a sufficient penile length by advancements in both dorsal and ventral sides. Our patient had no tightening during penile erection and was satisfied with the postoperative penile length. Meanwhile, a previous study demonstrated that KDPIF reconstruction could guarantee favorable subjective and objective outcomes, which can be achieved by considering relaxed-skin tension lines, skin creases, and esthetic subunits in the design of the flap. Another study described that any single large local flap could be insufficient and unsatisfactory for extensive perineal defects that exceed the midline of the body, and multiple perforator flaps can achieve better functional and esthetic results. In the present case, we used 2 (double-opposing) keystone flaps in each side of the defect and designed the flaps with consideration for relaxed-skin tension lines, skin creases, and anatomical divisions. Thus, the final result showed a favorable outcome with the skin creases mimicking the natural creases.A previous study described that a V-Y incision on symphysis pubis in addition to the bilateral scrotal flap was effective for preventing shortening of penile length.Although we performed a successful penile defect reconstruction with KDPIFs, the current report has some limitations that should be acknowledged. We included only one case and used a non-randomized retrospective design. Thus, selection biases and the presence of confounding factors are unavoidable. Further prospective large-scale studies with other comparison groups are required to confirm the consistency of the observed favorable outcomes. Moreover, further studies are needed to address the limitations of the technique, including perfusion physiology and flap survival.5To the best of our knowledge, there has been no report of KDPIF reconstruction for isolated penile defects, and the current report presents the first case of a successful reconstruction of circumferential penile shaft defect using pull-up double-opposing KDPIFs both esthetically and functionally. Our technique can be a good reconstructive option to consider when covering extensive  penile defects.www.editage.com) for English language editing and publication support.We would like to thank Editage (Conceptualization: Chi Sun Yoon, Kyu Nam Kim.Data curation: Hyun Gun Lee.Formal analysis: Hyun Gun Lee, Soo Yeon Lim.Investigation: Chi Sun Yoon, Kyu Nam Kim.Methodology: Chi Sun Yoon, Kyu Nam Kim.Writing – original draft: Hyun Gun Lee.Writing – review & editing: Soo Yeon Lim, Chi Sun Yoon, Kyu Nam Kim.


The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, we focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8–5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg). This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patients. The SARS-CoV-2 infection  is associated with the development of acute respiratory distress syndrome (ARDS). ARDS is present in approximately 75% of patients in need of intensive care and is recognized as the main cause of mortality [The scientific community firmly believes that thrombolytic action against fibrin depots in the lungs could be a powerful key to improving the ventilation–perfusion ratio of COVID-19 patients . Indeed,Plasminogen is a key component of the endogenous fibrinolytic system. It is a single chain plasma protein MW 92 kDa), and it is the inactive precursor (zymogen) of the serine protease plasmin (fibrinolytic enzyme). The conversion of plasminogen into plasmin occurs in nature under the action of the plasminogen activators, t-PA and u-PA. The formed plasmin cleaves the clots into soluble degradation products. Furthermore, plasminogen acts as a regulator in physiological processes such as wound healing 2 kDa, an and as aTherefore, the presence of plasminogen at pulmonary level could play a dual active role against the pathophysiology of ARDS, counteracting both the acute alveolar inflammation and lysing the clots resulting from pulmonary microcoagulopathy. Additionally, although patients at an early stage of COVID-19 infection present normal levels of plasminogen, patients progressing towards severe ARDS symptoms demonstrate lower plasminogen values due to its consumption of fibrinolysis activation .In the epidemiological emergency context generated by the SARS-CoV-2 infection, the administration of plasminogen in COVID-19 patients was subject to preliminary clinical evaluation, coordinated by the Talengen Institute of Life Sciences . The stuIndeed, the administration by inhalation route permits direct delivery of the drug to the site of action, thus favoring the rapid onset of therapeutic effects. Pulmonary administration allows for the achievement of high local drug dosages, limiting systemic toxicity and side effects, which may occur with parenteral administration . SimilarAdministration by inhalation is strictly related to the use of medical devices, such as nebulizers, pressured inhaler, etc. Among these, the simplest way to introduce a drug into clinical practice is to aerosolize it starting from its aqueous solution by using continuous nebulization. Presently, there are three main nebulization technologies, i.e., pneumatic (jet nebulization), ultrasonic, and using a microperforated vibrating membrane. Each device is designed to produce aerosols with controlled droplet size distributions to reach different segments of the respiratory tract. However, every nebulization technique causes shear stress and heat, which can affect drug physiochemical properties, thus precluding its therapeutic action . It is tSince different nebulization technologies could provide diverse harmful stresses and aerosol outputs, it is necessary to collect preclinical technological information on the appropriate choice of nebulizer as a function of drug product and therapy .In the present paper, jet and ultrasonic nebulization were evaluated for the pulmonary delivery of plasminogen by using a pharmaceutical grade homogeneous aqueous solution of the plasma derived protein. Presently, pharmaceutical grade plasma-derived plasminogen (PLG) is available as eye drops (Kedrion S.p.A.) and currently used as an orphan medicinal product (OMP), according to Italian law 648/96, for the treatment of ligneous conjunctivitis. The conceptualization of the research considers that the requirements of the European Pharmacopoeia (Ph. Eur.) 10th edition for inhaled solutions are similar to those for eye drops (solutions) and that the dosage form prepared according to GMP standards guarantees the safety of pharmaceutical quality for a ready translation to clinic.2O.The physicochemical properties of PLG-OMP and isotonic PLG-OMP solutions (iPLG-OMP) were evaluated and are shown in 99mTc) [Solution properties are known to affect both jet and ultrasonic nebulization . Concern99mTc) . Further99mTc) . TherefoBoth PLG-OMP and iPLG-OMP samples underwent nebulization procedures. Two commercial devices with different nebulization technologies were applied, namely the jet nebulizer OMRON A3 complete (flow setting within 2–15 L/min) and the ultrasonic device, PIC airproject plus. Despite having different operating principles, the two devices are meant to provide similar Mass Median Aerodynamic Diameter (MMAD) aerosols, as declared by manufacturers, particularly referring to the ampule position 3 of the jet nebulizer  and the PIC ultrasonic nebulizer (US). Concerning the ampule position 2 of the jet nebulizer , it generates aerosols with a higher MMAD and is more suitable for the treatment of tracheo-bronchial pathologies, as declared by manufacturer and in line with general correlation between MMAD and the deposition in the respiratory tract . DespiteAliquots of aerosolized PLG-OMP and iPLG-OMP were collected and submitted for biochemical characterization. The same analyses were performed on not-aerosolized control solutions (plain PLG-OMP and iPLG-OMP samples) as well as on the residual solutions collected from the device ampoules at the end of the nebulization. The stability of the aerosolized protein toward the nebulization processes, starting from either PLG-OMP or iPLG-OMP samples, was related to several parameters, including autoactivation of plasminogen to plasmin, the formation of Lys-plasminogen (I and II), the presence of protein fragments or aggregates and the maintenance of plasminogen-specific activity.Plasminogen is the zymogen of the plasmin enzyme. Under stressful conditions, the zymogen can activate itself and exert a catalytic function by activating other plasminogen molecules. Therefore, the presence of an activated enzyme is generally considered an important stability issue ,27. In oSDS-PAGE of aerosolized PLG-OMP and iPLG-OMP samples was performed in order to provide further evidence of the structural integrity of the protein in the nebulized samples, as well as detect the eventual formation of fragments. Data from three batches of nebulized products proved that over 90% were structurally intact plasminogen (in agreement with the specification of the finished PLG-OMP product), as shown in Glu-PLG is the native form of plasminogen. The prefix Glu is due to the presence of glutamic acid as the first amino acid of the chain. Lys-PLG is the most common truncated form of plasminogen, missing the first 77 amino acids and displaying lysine as the first amino acid of the chain. The properties of Lys-PLG are different from Glu-PLG, especially in the readiness of activation. Lys-PLG within the PLG-OMP product and iPLG-OMP solutions is considered an impurity or an unwanted pre-activation state; the presence of Lys-PLG in aerosolized samples would reveal the occurrence of aggressive stress. Under specific electrophoretic conditions, the Glu-PLG migrates as two separate bands corresponding to the two glycoforms: Glu I and Glu II. If present, the Lys-PLG is visualized as two separate bands deriving respectively from Glu I and Glu II and which are named Lys I and Lys II .In all the analyzed samples, the percentage of protein bands referable to Lys-Plasminogen were found to be 0%, regardless of the applied nebulization procedure. In all samples, it was possible to appreciate the protein bands attributable to Glu-PLG forms as indirect confirmation of the previously described results , Table 2Plasminogen-specific activity was measured in all aerosolized samples and compared with control PLG-OMP and iPLG-OMP solutions.Both PLG-OMP and iPLG-OMP solutions were analyzed using the Plasminogen potency evaluation, and the protein concentration was measured by the Bradford protein assay. Subsequently, the specific activity was calculated and compared with those of the sample Control .Enzymatic activity was found in the three batches of PLG Samples analyzed after nebulization, reflecting the fact that the protein, in addition to structural integrity, also maintained total/partial functional integrity. The nebulization of both PLG-OMP and iPLG-OMP solutions involves a modest loss of specific activity, compared with the control solutions. In particular, the specific activity of aerosol samples is higher for pneumatic nebulization and lower for ultrasonic nebulization, according to the J2 > J3 > US trend. The PLG-OMP samples nebulized with Omron Jet, in particular the ones using position 2, provided specific activity very similar to that of the Control samples. This evidence of the functional integrity of the product after nebulization would make this condition preferable.Despite maintaining specific activity in the range 4.5–5.0 IU/mg, the dilution carried out to prepare iPLG-OMP increased the susceptibility of plasminogen to nebulization.It should be noted that the part of the product that was not nebulized was identified as a residue. PLG-OMP and iPLG-OMP Residual samples maintained functional integrity. In fact, the specific activity of these samples was comparable to that of the controls, with the exception of the residue of the ultrasonic samples, where the specific activity showed a significant reduction, confirming a greater aggressiveness of the ultrasonic setting on protein stability. These data are in agreement with those observed using the SDS-PAGE method.To determine the droplet size distribution of the aerosols, laser diffraction measurements were performed. As displayed in However, it should be noted that the aerosol produced by US nebulization is not homogeneous over time, as observed by monitoring the Transmission value of detector 0 during the entire nebulisation period (data not reported). The results evidenced a heterogeneous polymodal size distribution, displaying droplets with median diameter values differing by two orders of magnitude. This behaviour is recorded for both the reference physiological solution and the samples, this being the limit of device performance. The wide polydispersion obtained with US nebulization is confirmed by the high SPAN value .(50), with about 60% and 25% of droplets sized below 10 μm and 5 μm, respectively. Better yield is obtained by the J3 setting, which provides higher shear stress and, consequently, lower-sized particles, resulting in more than 80% and more than 40% of droplets being sized below 10 μm and 5 μm, respectively.Concerning the jet nebulizations, the generated aerosols have a narrow particle size distribution, with small SPAN values and median diameters analogous to those obtained for the NaCl reference solution. Additionally, these parameters fall within the range indicated by the device manufacturer. It is also worth noting that the percentages of droplets below 10 μm and 5 μm are strictly dependent on nebulization settings and not on the nebulised solution sample, since both PLG-OMP and iPLG-OMP share similar physical properties with the NaCl reference solution. In particular, lower air flow position (J2) corresponds with bigger dvFrom a physical point of view, both types of samples  comply with the general specifications reported in FUXII for liquid preparations for nebulization. Both are homogeneous solutions, their pH (pH 6.5) falls within the required pH range 3.0–8.5 and, being sterile, they satisfy microbiological requirements. Furthermore, the data obtained do not significantly deviate from the 0.9% saline solution values, suggesting a good nebulization yield in agreement with the specifications of the nebulization devices. According to what is reported in the literature, the physical characteristics detected are in line with the data of the formulations used for treatment of the lower airways . The droFurthermore, since the nebulized solutions are homogenous and have physical properties analogous to those of NaCl 0.9%, the size distribution obtained by laser diffraction analysis could be assimilated into the aerodynamic distribution of the droplets ,30. ThisTo support the inherent feasibility of the nebulization of Plasminogen, it is important to underline that meticulous biochemical analysis highlighted the functional and structural integrity of the product. Moreover, no indicator of product degradation has been observed.In particular, the Protein composition (SDS-PAGE) did not show the presence of low molecular weight fragments or aggregates in any of the analyzed samples. The presence of Lys Plasminogen was also not found in any of the analyzed samples, reflecting the fact that the sample did not undergo any pre-activation. All tests carried out seem to be in tune and confirm each other’s results. However, a part of the product, called Residue because it was not nebulized, remained active and showed no signs of degradation. More sensitive chromatographic methods  as well as dynamic light scattering analysis (DLS) will be set for future corroboration of the presently acquired data. Such methodologies are frequently applied in parallel with biochemical investigation in order to assess protein stability during the optimization of inhalable formulations .Jet nebulization was less aggressive towards the nebulized plasminogen, particularly for the J2 mode, which is characterized by a more rapid nebulization. However, the J3 setting also allowed for good maintenance of plasminogen stability and, considering the higher percentage of aerosol respirable fraction, it is possible to conclude that this setting is adequate for the treatment of bronchopulmonary pathologies.Indeed, the collected data represent a starting point for deeper investigations, including the ability of aerosolized plasminogen to reach the low respiratory tract and eventually pass the alveolar capillary membrane . The goaA similar approach is presently under investigation in COVID-19 patients with nebulized heparin as an anticoagulant agent , which i®, Laboratorie Unither, Amiens, France), saline solution NaCl 0.9% , water for HPLC . The Plasminogen Kit (IL) consists of lyophilized chromogenic substrate S-2403, pyroGlu-Phe-Lys-pNA.HCl  and bulking agent; the streptokinase reagent , fibrinogen, and human serum albumin. HemosIL Calibration Plasma  and HemosIL Normal Control ASSAYED (Werfen), HemosIL Factor Diluent (Werfen); 4th international standard for Plasmin (NIBSC), MiniGel (NuPAGE); LDS Sample Buffer 4× (NuPAGE), Antioxidant (NuPAGE); MOPS SDS Running Buffer 20× (NuPAGE); Bis-Tris Precast Gels (NuPAGE); Simply Blue Safe Stain, Molecular Weight Standards ; Glu-Plasminogen Standard ; Lys-Plasminogen Standard (Sigma-Aldrich); Albumin Standard (Kedrion); water for HPLC (Sigma-Aldrich); Coomassie Protein Assay Reagent .Human plasminogen eye drops 1 mg/mL , Kedrion S.p.A.; excipients water and NaCl); saline solution NaCl 3% .Isotonic PLG-OMP solutions (iPLG-OMP) were prepared by the simple dilution of 6 parts of PLG-OMP with 1 part of LibernarAliquots of 5 mL (PLG-OMP and iPLG-OMP specimens) were atomized using either ultrasonic ultrasonic airproject plus (PIC) or pneumatic (Jet) OMRON A3 complete (OMRON). In the case of pneumatic nebulization, two delivery modes, position 2 and 3 of the bulb, were adopted. Nebulization was conducted until no cloud was produced. Atomized samples were collected by condensation into a glass bioaerosol impinger (0–4 °C) and stored at −20 °C until analysis . Both th2O), cinematic viscosity was determined by a Cannon-Fenske (50) viscosimeter under ASTM D-445 standard procedure, using water for HPLC use as a reference; liquid surface tension was measured using a Force Tensiometer . All measures were performed under controlled temperature (22 ± 1 °C).Both PLG-OMP and iPLG-OMP underwent extensive physical characterization, as described below. pH measurements were performed using pHmetro Orma electronic p200; solution density was evaluated using the hydrostatic method  calibrated with water for HPLC use; osmolality was measured by a Roebling osmometer  .Protein quantificationProtein concentration was determined spectrophotometrically according to the Bradford methods  with BioPlasminogen specific activityPlasminogen activity (PLG:Act) was quantified by a Hemosil Plasminogen test-kit (Instrumentation-Laboratory) according to manufacturer’s instructions. A Hemosil calibration plasma kit was used for calibration. Plasminogen specific activity (IU/mg) was calculated referring to the protein concentration determined for control and aerosolized specimens.Plasmin activity (PL:Act)Stress-induced conversion of plasminogen to plasmin was evaluated by measuring plasmin activity (PL:Act) in aerosolised samples. PL:Act was quantified by using a lyophilized chromogenic substrate  from a Hemosil Plasmin Inhibitor test-kit (Instrumentation-Laboratory) according to the manufacturer’s instructions. International Standard Plasmin Concentrate  was used as calibrator. The p-nitroaniline released from the chromogenic substrate was kinetically monitored at 405 nm and was directly proportional to the quantity present in the analyzed sample.Protein characterizationGlu-PLG. SDS-PAGE was carried out under denaturing conditions using 4–12% gel . Plasminogen and home-made PLG standard samples were prepared using an LDS sample buffer 4× (invitrogen) as a denaturing agent, followed by thermal denaturation at 95 °C for 5 min. 5 µg of protein PLG samples were loaded onto the gel, and the electrophoretic run lasted 55 min at 200 volts. In a second test, the gel was stained with Simply Blue Safe protein stain (Invitrogen) for 1 h and subsequently destained with water. The gel was acquired at Chemidoc MP using ImageLab software Lys-PLG. SDS-PAGE was carried out under denaturing conditions using 8% gel (Invitrogen). Plasminogen and Lys-PLG standard samples were prepared using an LDS sample buffer 4× (Invitrogen) as a denaturing agent, followed by thermal denaturation at 95 °C for 5 min. 1 µg of protein PLG samples were loaded onto the gel, and the electrophoretic run lasted 55 min at 200 volts. In a second test, the gel was stained, destained and acquired as previously described.Native glutamic acid-plasminogen (Glu-PLG) and Lysine-plasminogen (Lys-PLG) were determined by SDS-PAGE. The results were expressed as % of the band attributed to Glu-PLG or Lys-PLG, with respect to all bands present in the Line .Droplet size distribution measurements were conducted at Alfatestlab S.r.l  using Malvern Panalytical’s Spraytec laser diffraction system . 5 mL of each PLG-OMP and iPLG-OMP solution was nebulized by applying the mouth mask of each nebulization apparatus, located at 80 ± 2 mm from the laser cell. Measurements were performed according to ISO 13320:2009 standards at 23 ± 2 °C and a relative humidity of 50 ± 5%.t-test.PLG-OMP specimens were collected from three product batches, and the same batches were used for the preparation of iPLG-OMP samples. Each analytical test was conducted at least in triplicate on each batch sample. When possible, the data sets were statistically compared by applying a Student The study assesses the feasibility of delivering inhalable plasminogen starting from human plasminogen orphan medicine (PLG-OMP). The research has shown that, even if none of the tested conditions massively degraded plasminogen, the ultrasonic and jet nebulization of PLG-OMP were not interchangeable. It is also concluded that PLG-OMP can be nebulized as it is. Additionally, if it would be necessary to dilute PLG-OMP to iPLG-OMP within a clinical setting, it can still be nebulized, although a lower activity of the drug should be expected.Considering the recent approval of clinical trials based on the inhalation of fibrinolytic drugs such as t-PA , the pre