The persistence of bovine tuberculosis (bTB) in certain cattle herds is a major concern in countries pursuing disease eradication worldwide. The chronic nature of the disease, the lack of performance of diagnostic tools, and the presence of wildlife reservoirs may lead infected herds to require longer periods to achieve the officially tuberculosis-free (OTF) status. Here, we evaluated the impact of farm and breakdown characteristics on the probability of disease persistence in infected farms in Castilla y Leon, a bTB-endemic region of Spain, using survival and logistic regression models. Data from bTB breakdowns occurring in 3,550 bTB-positive herds detected in 2010–2017 were analyzed. A multivariable Cox proportional hazards model was fitted using time to recover OTF status as the response variable, and a multivariable logistic regression model using the chronic status (yes/no) for herds experiencing particularly long breakdowns as the outcome variable was also used. Both analyses revealed that county-level bTB herd prevalence, herd size, number of incoming animals in the previous 3 years, number of skin test reactors in the disclosing test, and number of days between the disclosing and follow-up tests were associated with increased breakdown duration. Production type was not consistently associated with chronic infection, suggesting that once infected, it is not a significant predictor of outbreak duration beyond the initial stages of the breakdown. Province-level location and number of animals that are bacteriology-positive also affected significantly the expected herd breakdown duration, but their effect became less significant over time. Risk factors identified in this study may help to identify herds more prone to suffer chronic bTB infection that may require additional control measures early on in a breakdown. Mycobacterium tuberculosis complex, mainly Mycobacterium bovis and Mycobacterium caprae, which has a major impact in the economy of affected countries due to its effect on trade. Despite being eradicated in several countries (Bovine tuberculosis (bTB) is a zoonotic disease affecting cattle caused by members of the ountries , 2, bTB ountries . In Spaiountries .M. bovis in the environment and the aggregation at communal water and food sources that can promote closer contacts between cattle and may increase the likelihood of contact with infected wildlife reservoirs  and the predominance of extensively managed bullfighting and beef herds , 16. TheAlthough several studies have been conducted on herd-level risk factors for bTB in Spain , 19, to In accordance with EU Directive 64/432/EEC , the SpaResults), these were grouped into two categories for simplicity . Additionally, data about the number of movements (contacts) and animals received by each farm during 2007–2016 were also available. Finally, information on the date and type of bTB test , number of animals tested, and number of reactors found in the SIT and/or IFN-γ assay for herd tests performed in the frame of the bTB eradication program during 2007–2018 was collected. For herds subjected to whole-herd depopulation, information on the date of depopulation was also obtained. The total number of cattle older than 6 weeks tested per herd in routine and follow-up tests was used to calculate herd size.The primary population was comprised of all cattle herds in Castilla y Leon subjected to bTB testing during 2010–2017. Information on herd characteristics, cattle movements, and bTB status of farms was collected through the SITRAN Information System . DemograResults). A bTB breakdown was formed by all related bTB-positive herd tests. The bTB breakdown duration was defined as the period elapsed from the first bTB-positive herd test until the first negative herd test following the last related bTB-positive herd test , herd type, median herd size in the year the bTB breakdown started, relative change in herd size prior to the disclosing bTB-positive herd test (percentage difference between the herd size in the first positive herd test of the bTB breakdown and the median herd size the previous year), number of herds sending animals to the bTB breakdown herd (in-degree) and number of incoming animals in the previous 3 years (for breakdowns occurring in 2010–2017), number of days between the disclosing and follow-up tests, and number and proportion of positive animals to the SIT and bacteriology in the disclosing test. For herds in which bTB was disclosed through pre-movement tests or abattoir inspection, the number of animals tested in the previous routine test was used to calculate the relative change in herd size at the disclosing test. Models considering certain risk factors  did not include breakdowns starting before 2010/2011 as no information for those factors was available. Herd size was categorized into three categories based on terciles , relative change in herd size at the disclosing bTB-positive herd test, number and proportion of SIT reactors in the disclosing test, number and proportion of bacteriology-confirmed animals in the disclosing test, in-degree in the 3 years prior to the start of the bTB breakdown, and number of days between the disclosing and follow-up tests were categorized into quartiles, while county-level herd prevalence was categorized into quintiles.The association between each available predictor variable and the outcome variable “duration of the bTB breakdown” was explored in a survival analysis. The survival distributions for the categories of each of the considered variables were plotted and compared using the log-rank test. These analyses accounted for left truncation  and right censoring  as seen elsewhere . Here, tZ1…Zk, so that:Univariable Cox proportional hazards models were fitted to compare the hazard ratios of resolving an outbreak for herds depending on the covariates t represents the survival time, λ0(t) is the baseline hazard function, and βi is the regression coefficient of covariate Zi  scores. The choice of variable to include in the model for correlated variables  was based on the AIC (i(t) using a parametric continuous function to construct time-dependent covariates . A control was any herd with a bTB breakdown duration less than or equal to the median bTB breakdown duration in the whole study population, i.e., irrespective of the number of positive herd tests  as previously performed (A case control study to identify risk factors associated with herds with an increased duration of bTB breakdowns (“chronic herds”) was also carried out. For this, we defined a case as any herd with a bTB breakdown duration equal to or greater than 784 days. This 784-day threshold was selected as our aim was to identify bTB breakdowns with an unusually long duration for Castilla y Leon, and this included only the top 25% longer resolved bTB breakdown of herds with ≥3 bTB-positive herd tests  to ensure a mean VIF of <5 among the variables (p ≥ 0.05). Results in the model were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). The Hosmer–Lemeshow statistic was used to test the goodness of fit of the model  as the outcome variable. Risk factors that were significant in the univariable model at a liberal ariables  before bariables . The finhe model .Statistical analyses were performed using R version 3.5.0 . Survivan = 1,452/3,550, 40.9%), followed by Avila , Leon , and Segovia , whereas the rest of the provinces accounted for the remaining 23.9% (n = 848) of the total number of bTB-positive herds in Castilla y Leon  experienced only one bTB-positive herd test, 18.1% (n = 642) two, and 32.4%  of the herds were positive in three or more herd tests. Four hundred nine herds tested negative at least once between bTB-positive tests within the same breakdown. The median bTB breakdown duration in herds with ≥3 bTB-positive herd tests after the exclusion of 49 herds that were subjected to whole-herd depopulation during the bTB breakdown was 511 days [interquartile range (IQR) = 284–784].According to the definition used in this study, there were 3,550 (20%) bTB-positive herds out of 17,793 tested herds in Castilla y Leon at some point during the period 2010–2017. Beef was the predominant production type among the bTB-positive herds , followed by dairy , mixed (beef and dairy) , bullfighting , fattening , and raising heifer herds . Throughout the study period, the majority of bTB-positive herds was found in the province of Salamanca  out of the remaining 3,454 bTB-positive herds detected in the period 2010–2017 did not clear the infection during the study period or were depopulated and were therefore considered censored observations in the analysis. In addition, 247  of the bTB breakdowns had started before 2010 and were therefore left-truncated.p <0.001) associated with an increasing time to recover OTF status  and in-degree. The final Cox model was fitted with both time-fixed (production type and number of SIT reactors in the disclosing test) and time-varying coefficients , and obtained coefficients did not differ substantially (<19% change) compared with those estimated in the univariable models except for production type, province, and number of SIT reactors in the disclosing test , 2. TimeA total of 347 herds with a bTB breakdown duration equal to or greater than 784 days (cases) and 1,443 herds suffering a bTB breakdown with a duration ≤ 133 days (controls) were considered in this analysis. No herd had more than one chronic bTB breakdown. Spatial distribution of these chronic and non-chronic herds in Castilla y Leon from 2010 to 2017 is represented in p < 0.001, Mann–Whitney test) larger for cases  compared to controls .The majority (>80%) of both case and control herds were beef/fattening . Overallp < 0.001), and the latter was selected for the multivariable model based on better AIC. According to the final multivariable model including 1,657 herds with complete information on all covariates considered, herd size, number of incoming animals in the 3 years before the outbreak, county-level prevalence before the outbreak, the province where the herd was located, number of SIT-positive and bacteriology-confirmed animals in the disclosing test, and an increasing number of days between the disclosing and follow-up tests were all associated with being a chronically infected bTB herd .The proportion of herds with missing information for any given variable was always <8%; out of the 1,790 herds considered in the case-control study, no information for number of incoming contacts  in the 3 years prior to the start of the bTB breakdown and county-level herd prevalence was available for breakdowns starting before 2010 (51 herds) and 2011 (130 herds), respectively, and were subsequently removed from models considering these variables . MoreovebTB herd , while pbTB herd . There wThe persistence of bTB in certain cattle herds in terms of either herd recurrence or prolonged periods of restriction is a major problem in countries pursuing disease eradication worldwide. Among the factors that may substantially extend the time to recover OTF status and thus hamper eradication programs are the chronic nature of the disease, the presence of wildlife reservoirs, and the lack of performance of diagnostic tools. Several studies have conducted risk factor analyses for chronic bTB herd breakdowns in Europe , 35–38, In this study, we defined two positive bTB herd tests as potentially related if they were separated by no more than 18 months, the median duration of bTB outbreaks in herds with ≥3 bTB-positive herd tests, which represented 32.4% of the total bTB-positive herds during 2010–2017. Herds with ≤ 2 bTB-positive herd tests were not considered to select the threshold in order to focus on problematic herds. The median bTB breakdown for all bTB-positive herds in Castilla y Leon duration was 133 days, which is in agreement with values reported in Northern Ireland .The univariable survival analyses revealed that production type was associated with longer bTB breakdown durations: unsurprisingly, outbreaks in bullfighting herds were significantly longer compared to beef/fattening and dairy/mixed herds in agreement with previous studies conducted in a different region in Spain . Some ten = 777) included herds in which IFN-γ was being used , what indicates that even in the absence of SIT reactors, there was conclusive evidence of the presence of disease, since the IFN-γ test is only used on units confirmed by bacteriology or based on epidemiological grounds  , suggestive of active circulation of the disease in the herd, was associated with increased duration/risk of being a case herd compared with the reference category (one reactor). However, herds in which no SIT reactors had been found in the disclosing test were also at higher risk , 3 compae herds) . During e herds) , 44, rese herds) . AdditioWe also found evidence of time-varying effects for the number of bacteriology-positive animals in the disclosing test, province, and number of days between the disclosing and follow-up tests in the survival analyses, as the mean estimates did not distribute evenly throughout the study time. The interval of time between the disclosing test and the follow-up test was significantly associated with both outbreak duration and the odds of a chronic infection, with herds tested at increased intervals  in order to focus on outlier herds in the Castilla y Leon context. This threshold is well above the 1-year threshold used to differentiate chronic infections arbitrarily selected in previous studies in Ireland  and NortThe case-control study further revealed the effect of farm-level management and outbreak characteristics with bTB persistence in infected herds: an increased number of bacteriology-positive animals in the disclosing test were significantly associated with increased odds of experiencing long-duration breakdowns. These findings are consistent with a higher risk of chronic infection in herds with multiple SIT reactors, as these herds are more likely to have a higher proportion of animals confirmed through bacteriology. This result may also be related to a higher bacterial excretion in animals with an advanced disease stage and thus leading to a higher risk of bTB persistence due to the increase of the time of contact with susceptible animals .Even though chronic case herds were widespread throughout Castilla y Leon , infecteThe association between an increased herd size and the risk of experiencing a chronic outbreak could be explained with the increased odds of finding reactors  in larger herds , 48, sinThe number of incoming animals in the 3 years preceding the start of the bTB breakdown was retained in the case-control logistic model based on AIC and in agreement with findings in the survival analysis, even though no significant differences between the variable categories were observed. Still, several studies have consistently evidenced that cattle movements may be important in bTB transmission , 50, andM. bovis , since no information on them was available for these outbreaks. These outbreaks had been considered to estimate the overall median duration of outbreaks in the region and the threshold for selection of chronic herds. Nevertheless, if these are excluded for these calculations, no major differences were observed , suggesting that they are not significantly affecting the study definitions.The inclusion of these variables  in the final models forced the elimination of outbreaks starting before 2011 , and whole-genome sequencing (WGS)], but this information was not available for a large proportion of the herds evaluated here.Other factors not explicitly included in the study that could be related to increased duration of outbreaks include wildlife variables or the presence of concurrent infections compromising the diagnostic sensitivity. The effect of paratuberculosis in bTB diagnostic tests has been described in the past in Spain , 55, 56 Time-varying effect emerges when the proportional hazards assumption is not fulfilled , and forOur findings should be interpreted with caution, as associations found here may not reflect causation. Still, our results demonstrate that certain farm and breakdown characteristics (available at the beginning of the breakdown) can help to predict the probability of experiencing a chronic bTB infection among those already infected, what may help to implement specific measures aimed at preventing reinfection  or decrease residual infection . Further research is needed to better understand the mechanisms behind persistence of bTB in the region, including the risk factors associated with bTB recurrence and the role of wildlife reservoirs in bTB reinfection and/or maintenance in the herd.The data analyzed in this study is subject to the following licenses/restrictions: datasets included in this study are owned by the Official Veterinary Services in Castilla y Leon and include information that cannot be made publicly available. Reasonable data requests can be submitted to the authors of the study and will considered individually. Summary data is provided in the article. Requests to access these datasets should be directed to mingonol@jcyl.es.Ethical review and approval was not required for the animal study because data from animals used in this study was generated through the normal functioning of the tuberculosis eradication campaigns in Spain and therefore no additional procedures were performed as a result of this study.PP and JA designed the study with the help of AG, JN, and OM, who also provided the data. PP analyzed the data with the help of JA, BR, JB, and JLS in data interpretation. PP and JA wrote the initial manuscript and received substantial feedback from all other authors.PP and BR were employed by MAEVA SERVET SL. The remaining 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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new coronavirus that was recently discovered in 2019. While the world is working hard to overcome and control the coronavirus disease 2019 (COVID-19) pandemic, it is also crucial to be prepared for the great impacts of this outbreak on the development of antimicrobial resistance (AMR). It is predicted that inappropriate and too much use of antibiotics, biocides, and disinfectants during this pandemic may raise disastrous effects on antibiotic stewardship programs and AMR control all around the world. Furthermore, the use of certain antibiotics alone or in combination with antiviral agents or other medications for the treatment of secondary bacterial infections among COVID-19 patients may be regarded as a major factor that negatively affects host immune response by disrupting mitochondrial function and activity. Herein, we suggest that the current management strategies to control AMR and prioritize antibiotic stewardship schemes should be extremely highlighted in relation to the COVID-19 outbreak. The rising concerns about excessive use of antimicrobials and biocides and taking too much hygiene also need to be addressed during this pandemic due to their impacts on AMR, public health, and the environment. Globally, coronavirus disease 2019 (COVID-19) has created a health catastrophe that has had an immense impact on social care systems, economy, and financial resources. The importance of infection control and prevention measures such as social distancing, hand hygiene, and self-isolation has now been advised at a communal level . The COVThe World Health Organization (WHO) is worried that the trend will later be fueled by the wrong consumption of antibiotics during the COVID-19 pandemic. Indeed, when physicians are not aware or do not have access to the laboratory diagnostic findings related to the etiology of the infected patients, they tend to drive higher administration of different antimicrobials . Recent The uncontrolled use of biocide-based products could lead to the unusual release of such antimicrobials into environmental resources and cause the selective survival of bacterial strains carrying resistance genes during the COVID-19 catastrophe . FurtherStaphylococcus aureus (MRSA) in the hospital environment . Based on their approach, empirical antifungal therapy does not seem to be beneficial, and about 21% of patients who received empirical antifungals such as voriconazole, isavuconazole, and caspofungin had adverse effects without providing a survival benefit. Therefore, incorporation of strategic algorithms for accurate diagnosis and effective treatment of invasive fungal disease is vital in critically ill patients with COVID-19 and subsequently may result in a definitive diagnosis and successful antifungal therapy throughout the period of severe respiratory distress.Antimicrobial resistance has been emphasized as the most considerable threat to global health and economy in recent years, but it can hide behind COVID-19 for a while. It is predicted that the current emergence of the COVID-19 pandemic can seriously facilitate the growth rate of AMR worldwide; thus, the continuing and preventive measures against AMR should not be neglected during this global disaster . Unfortuironment . Until nironment . In anotironment , 11% of ironment , putativIstituto Superiore di Sanità in Italy, 84% of the expired SARS coronavirus 2 (SARS-CoV-2)-positive patients used antibiotic therapy, whereas 54% received antiviral therapy and 31% received steroid therapy during hospitalization  and microecological niches where various bacterial species are present is critical. Generally, it is supposed that high concentrations of such microbicidal agents could completely kill most bacterial species especially those that provide beneficial services to the ecosystem and to other living organisms while inhibiting the emergence of drug-resistant microorganisms . On the n health .qac genes in Staphylococcus, Enterococcus, and Escherichia coli (It should be noted that understanding the transferability of disinfectant resistance genes can considerably help to limit the outspread of such mobile genetic elements among microorganisms . Albeit hia coli . Taken tSince there is currently no specific and effective therapeutic agent, medication, or vaccines available to treat COVID-19 patients, the human immune system is considered as the solely active weapon against the virus. Unfortunately, off-target sites of majority of the approved antibiotics include mitochondria, which are evolutionary linked to prokaryote cells . MitochoBefore the emergence of the SARS-CoV-2 pandemic, the world was already demanding immediate, coordinated, and ambitious actions to avert the potentially disastrous AMR crisis and its related economic and health consequences. Now that we are in the midst of a global crisis, which is disrupting normal life for everyone, causing hundreds of thousands of people sick or hospitalized, and killing some of the most high-risk individuals, a better understanding of the pathophysiology of COVID-19 and advocating of potential effective therapeutics for the treatment of secondary bacterial co-infections seem to be critical. Also, prospective monitoring of the co-infections in patients with COVID-19 could help apprehend whether such co-infections affect disease progression and may interfere with antiviral treatment. Furthermore, current management practices and strategies to control AMR and prioritize antibiotic stewardship schemes should be extremely highlighted in relation to the COVID-19 outbreak. The concerns about consumption of “too many antimicrobials and biocides” and taking “too much hygiene” also need to be addressed during this pandemic due to their impacts on AMR, public health, and the environment.SR and AY contributed significantly to the literature review and wrote the draft of the manuscript. AY worked on concept and design of the study and interpreted the collected information. BH, HA, and MZ provided clinical advice and guidance for the improvement of the manuscript. AY critically revised the final version of the manuscript. All authors approved the final version of the manuscript and the authorship list.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.


This Viewpoint calls for a greater understanding of the role that water plays in the transmission of anti-microbial resistance and covid-19 in protracted urban armed conflict, in order to develop a ‘pathogen-safe’ practice. It argues that dealing with the twin threats is difficult enough in the best of circumstances, and is so little understood in war zones that surgeons and water engineers now question if their practice does more harm than good. Experience suggests that the known transmission routes are complicated by a great number of factors, including the entry of heavy metals through bullets in patients’ wounds, hospital over-crowding, mutation in treated water or wastewater, and other threats which endure long after the bombing has stopped. The skeleton research agenda proposes greater sewage surveillance, testing of phages and monitoring of treatment designed to dispel or substantiate these assertions. If covid-19 is indeed accelerating the perils of anti-microbial resistance (AMR) in the US and UK, [The rampant virus and the mutating bacteria must force a change to the traditional public health response of agencies that are active in humanitarian contexts. This obliges no less than a re-conception of practice, and can come only after development of the knowledge and policy base.The ‘antibiotics apocalypse’ has already claimed hundreds of thousands especially in low and middle-income countries . ResponsWe lose pan-drug resistant patients every day when the fighting is most intense, and every week between the bouts of violence, as these continue to be shaped by ongoing tensions and economic sanctions. Consider the eight-year-old girl from Aleppo who came in February 2020 with bilateral above-knee amputations and burns, and who died of infections despite the tetracycline and colistin we gave her. Or the hundreds of teenagers shot in the knees who overwhelmed hospitals in Gaza in March 2018 . Many ofmore antibiotics are dispensed to covid-19 patients [Covid-19 amplifies the manslaughter when the family that cannot be kept out of the wards returns to care for their injured, and even patients .One Health WASH in Health Care Facilities and similar initiatives stand out amidst this field, and have suggested numerous transmission route diagrams, as in Figure 1 [131415Figure 2 [192021We know that standard drinking water and wastewater treatment may get the virus , but notFigure 1 13141516. 1 131415. But theFigure 2 19202122.In the disastrous ‘ecology of war’ which characterizes protracted conflicts, the impact of sanctions goes well beyond the blast zone, and endures long after the dust settles 24. We ha2528Blind of insight, we double-down on scrubbing-up, over-chlorinate the water, treat the wastewater, rebuild antibiotic stewardship programs and blend them with covid-safe practice, scream at families to social-distance, track-and-trace, hope and pray. To little apparent avail. The patients keep coming and our options keep narrowing.Figure 2 are the most relevant to covid-19 and AMR. We must determine if the direct link between the conduct of hostilities and diarrhea multiplies or (somehow) reduces AMR [Of course, better disinfection and more beds will help, but only a knowledge base will help us get ahead in the game. We first need to establish which of the transmission routes proposed in uces AMR , for exauces AMR 34.We must then determine the best ways to prevent or interrupt the pathways. What is the scope for bacteriophages , for exaThe answers can lead to policy that guides ‘pathogen-safe’ practice in war zones, and improves the quality of life of hundreds of millions . Until t


This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development. Keratan sulfate (KS) is a widely distributed glycosaminoglycan (GAG) component of proteoglycans (PGs) in the extracellular matrix (ECM) and on cell surfaces of tensional and weight-bearing connective tissues such as ligaments, tendons, articular cartilage, and intervertebral discs (IVD), in addition to corneas and the central and peripheral nervous systems (CNS/PNS) ,2,3. HisMAb BKS-1 identifies KS in early tissue development in KS-PGs containing low sulfation KS glycoforms as well as highly sulfated KS in mature tissues. In corneal tissues, MAb 5-D-4 and BKS-1 detect similar corneal KS-PG populations by Western blotting  and, in A recent study with corneal KS also showed that this GAG interacted with a diverse range of morphogens, growth factors, nerve growth factor receptors, and members of the Robo, Slit, Ephrin, Ephrin receptor, and Semaphorin families of neuroregulatory proteins . The strAll materials and supplier details were as specified earlier .Human cartilage was donated by total knee replacement patients using the North Sydney Area Health Authority Ethics protocol 0404-103M (Oct 2004–2008): J Melrose, C Little, D Sonnabend—“Pathobiology of the small leucine rich repeat proteoglycans in cartilage, intervertebral disc and tendon degeneration” which covers the use of human tissues discarded at surgery. Late fetal (embryonic days 19–20) and adult (4 month) spinal tissues from white Wistar rats were used in this study as described previously . These wThe LUM-1 and KER-1 core protein antibodies used in this study  have beeEscherichia coli and HEK 293T cells were purchased from OriGene  and used as positive control standards in Western blots. A rabbit polyclonal antibody to keratocan  raised to C-terminal amino acids 227-257 was also obtained from abcam . A rabbit monoclonal Lumican antibody  was obtained from Santa Cruz Biotechnology . A rabbit polyclonal lumican antibody (PR-353) to the C-terminal amino acid sequence LRVANEVTLN was a gift from Prof Peter Roughley, McGill University, Canada. The KTN, B-9, and PR-353 antibodies were used in Western blotting with human knee cartilage extract or recombinant lumican and keratocan proteins to confirm that KER-1 and LUM-1 identified keratocan and lumican core proteins as shown previously in a number of studies -propanesulfonic acid) pH 7.0 SDS electrophoresis buffer. Gels were electroblotted to nitrocellulose membranes (0.22 μm) in NuPAGE transfer buffer containing 10% methanol at 30 V constant voltage for 1 h. SeeBlue-2 pre-stained protein molecular weight standards  were also electrophoresed. Blots were blocked for 3 h in 5% bovine serum albumin in 50 mM Tris–HCl 0.15 M NaCl  and anti-lumican, anti-keratocan, or anti-BKS-1 antibodies (1 μg/mL) added overnight in 2% bovine serum albumin in TBS. Goat anti-rabbit or anti-mouse IgG alkaline phosphatase conjugated secondary antibodies (1/5000 dilution) were added for 1 h. The blots were then washed in TBS and developed with NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3’-indolyphosphate) substrates in 0.1 M Tris–HCl pH 9.5 containing 5 mM MgClThe schematic shown in Western blotting of extracts of SC and IVD from adult (4 month) old rat spinal tissues showed that both tissues contained lumican and keratocan and BKS-1(+) positive KS-PGs a. LumicaAt 4 months the rat vertebral column was skeletally mature and its associated connective tissues had fully formed. Alcian blue staining for sulfated GAG a and IHCImmunolocalization studies of late fetal rat spinal tissues with BKS-1(+) showed strong ECM labeling of keratanase-generated KS neoepitopes within the cartilaginous inner AF of the developing IVD a. LabeliLabeling of BKS-1(+) was highly prominent within the NP and AF of the IVD; however, it was not detected in the CEPs or adjacent GPs a. LumicaKeratocan and lumican both have documented roles in the regulation of nerve migration during the development of the CNS/PNS. vβ3- integrin mediated cell binding and has been isolated as a minor, leucine and aspartic acid-rich KS-PG from mineralized tissues [vβ3- integrin and has been immunolocalized to pre-dentin during tooth formation [BKS-1(+) members of the small leucine rich proteoglycan (SLRP) family, including lumican and keratocan, have previously been reported in the development of the annulus fibrosus of the rat IVD . In the  tissues . Osteoadormation .The present study indicated that lumican and keratocan were prominent matrix components of the adult rat SC. These SLRPs were originally identified as bone and cartilage matrix PGs that bind and regulate growth factors such as TGF-β1  to modulKeratocan also has neuro-directory, growth factor, and morphogen interactivities supportive of SC developmental processes ,16,63. TShh acts as a differential regulatory switch in the transformation of motor neurons to oligodendrocytes during SC development and re-myelination during axonal repair [In the present study, Western blotting with the KER-1 and LUM-1 antibodies alongside commercial keratocan and lumican antibodies validated both KER-1 and LUM-1 and confirmed that they detect 37 kDa keratocan and 51 kDa lumican core proteins in rat spinal tissues and human articular cartilage. BKS-1(+) also identified these core protein species. Higher power images of BKS-1(+) immunolocalizations in 4 month rat SC highlighted the subtlety in expression of KS on different cell types: BKS-1(+) identified positively-stained microglia throughout the grey and white matter whereas positively stained neurons were confined to the grey matter. The midline of the developing brain and SC is a place where axonal guidance decisions effects the patterning of sensory and motor systems. A variety of glial structures develop along the midline crucially directing axons to the left or right side of the CNS during spinal development ,78,79. Kl repair  supportiBKS-1 immunolocalizations are accurate quantitative measures of KS since one BKS-1 epitope is labeled per KS chain in all KS glycoforms irrespective of their charge status whereas MAb 5-D-4 binding is multivalent and identifies highly charged hexasaccharide KS epitopes. While this improves the sensitivity of detection of KS by MAb 5-D-4, it imparts a bias towards detection of highly sulfated KS chains. BKS-1 is therefore a useful new KS antibody with significant utility in identifying KS in early tissue development in KS-PGs containing low sulfation KS glycoforms as well as highly sulfated KS in mature tissues. During fetal development, BKS-1(+) detects a yet to be identified KS-substituted PG other than keratocan and lumican within the pre-ossification centers of the vertebral bodies. MAb BKS-1 also strongly delineates KS-PGs within the inner annulus of the developing IVD. In the adult rat, BKS-1(+) expression patterns correlate closely with KS SLRPs in the SC and IVD tissue. In fetal SC and vertebral spinal tissues a close correlation is observed between BKS-1(+) and lumican, but not keratocan expression which is absent from the IVD and only weakly present in GP. The vertebral growth plate of the rat spine also appears to contain a non-glycanated form of lumican which awaits further characterization.


Kaposi’s Sarcoma (KS) is a reticuloendothelial system tumor, that may involve the skin, mucosa and viscera . It can Whit regard to the clinical presentation, each recognized variant has different manifestations and different visceral involvement. It has been estimated that KS confined to the penis is uncommon and is more often observed in patients with AIDS , represeThe aim of this study is to describe an uncommon clinical presentation of genital KS in HIV-seronegative man and to perform a narrative literature review of the cases described to date.KS, first described by Moritz Kaposi in 1,872, is a rare neoplasm that origins from the endovascular cells in a multifocal way-This enigmatic infrequent malignant disease has since received much resonance after the AIDS epidemic in the early 1980s, with an incidence of classic KS ranged from 0.01 per 100.000 personyears for the UK and 0.2 per 100.000 personyears for the USA. However, currently, the incidence of KS is reported to be 200-fold higher in recipients of solidorgan transplants, known as iatrogenic KS form, rather than in the general population . The incKSHV is a large double-stranded DNA herpesvirus with a protein covering by an icosahedral capsid, surrounded by tegument and enclosed in a lipid envelope derived in part from the cell membrane. Different glycoproteins in the viral envelope interact with celltypespecific cellular entry receptors such as integrins , the cystine-glutamate transporter xCT, heparan sulfate and the tyrosine protein kinase receptor EPHA2). KSHV can infect several different cell types, including endothelial cells, B cells, epithelial cells, dendritic cells, monocytes and fibroblasts. Once inside the cell and after uncoating the virus genome enter in the nucleus where enter in lantecy phaseas episome and undergoes sporadic bouts of lytic reactivation . Virus cThe behavior of the disease varies from a singular lesion localized in the skin, to a fleeting extensive respiratory and gastrointestinal visceral involvement. All variants of KS cutaneous lesions usually present as multiple, pigmented, raised or flat, painless that do not blanch. Classic variant  is typically confined to lower limbs with few lesions. Visceral and mucosal disease is rare and usually occurs in the gastrointestinal tract. Endemic is a typical manifestation of African children often present with multiple lymph nodes with lymphoedema and a very aggressive natural history of the disease, including visceral disease. AIDS-related is characterized by multiple cutaneous lesions on the limbs, trunk and face. Mucosal lesions, such as oral lesion, are common (identified in 20% of patients) and visceral involvement is seen in 15% of patients. Related with Iatrogenic immunodeficiency, such as in organ transplantation. Often presents as cutaneous KS lesions but both mucosal and, rarely, visceral disease can occur. Finally, in MSM patients the clinical manifestations included lesions that can occur at any skin sites, usually few. Visceral and mucosal disease is rare . RegardiA 71-year-old heterosexual, Caucasian man, referred to our department for the presence of penile neoformation appeared from at least 6 months. At the clinical examination, a 0.6mm x 0.6mm x 0.3mm red painless radish nodule hemangioma-like was found on the gland near the frenulum  and 1B. An English-language literature research was conducted, focusing on the cases of penile KS in HIV positive and negative patients . Two difNew-onset apparently benign lesions of penis in immunocompetent patients, even in absence of risk factors for sexually transmitted diseases, should be always investigated, because it could represent the first manifestation of primary KS in which penis could be the only isolated clinical presentation. The surgical management could represent a good therapeutic option, leading to disease clinical resolution with no further recurrence, thus providing histological diagnosis.


We argue that the COVID-19 pandemic will result in increased antimicrobial resistance (AMR). Broad-spectrum antibiotic use is common among hospitalized COVID-19 patients and in excess of reported secondary infection rates, suggesting unnecessary prescribing. Selection pressure is likely to be particularly intense in COVID-19 epicentres and within non-epicentre hospital units dedicated to COVID-19 care. Risk factors that increase the likelihood of hospitalization or poor outcomes among COVID-19 patients, such as advanced age, nursing home residence, debilitation, diabetes and cardiopulmonary or other underlying systemic diseases, also predispose to AMR infections. Worry for AMR emergence is heightened since first-wave COVID-19 epicentres were also AMR epicentres. Disruptive direct and indirect effects of COVID-19 globally on economic systems, governance and public health expenditure and infrastructure may fuel AMR spread. We anticipate that the impact of COVID-19 on AMR will vary between epicentres and non-epicentres, by geographic region, hospital to hospital within regions and within specific hospital units. In reports from China, organisms included pandrug-resistant (PDR) Clearly, no conclusions about relationships between COVID-19 and AMR can be drawn from published reports. There are several reasons, however, that we expect AMR to emerge during the pandemic.Candida. This population may drive high rates of broad-spectrum antibiotic use, especially since empiric therapy upon hospital admission will likely be directed against healthcare-associated pathogens.Broad-spectrum antibiotic use has been widespread among hospitalized COVID-19 patients, and in excess of reported secondary infection rates. Therefore, much of the use to date has been unnecessary. Antibiotic therapy will likely remain common owing to difficulties in conclusively excluding secondary pneumonia or nosocomial superinfections in many cases. A large percentage of patients admitted to hospitals with COVID-19 are from congregated living facilities such as nursing homes, prisons and psychiatric hospitals.COVID-19 strained hospital capacity to breaking point in hard-hit epicentres globally. As such, overall antibiotic use in these locations and at facilities in other locations that cared for large numbers of COVID-19 patients was likely up sharply, even if non-emergency hospital services were suspended or regional antibiotic trends were down. For many hospitals outside of epicentres, antibiotic use within ICUs or wings that converted into COVID-19 units was likely increased, particularly if normalized to BDOC Figure. TherefoA. baumannii and MBL-producing Enterobacteriaceae in Asia and ESBL-producing Enterobacteriaceae in the USA.,Worry for emergence of AMR is heightened by the fact that first-wave COVID-19 epicentres were also AMR epicentres. As of 31 May 2020, countries with the largest numbers of diagnosed COVID-19 cases were the USA, Brazil, Russia, the UK, Spain and Italy,Escherichia coli with colistin resistance due to plasmid-borne mcr1 arose in pigs in China, which led to worldwide human dissemination in 2015 and horizontal gene transfer to other Enterobacteriaceae.,While antibiotic exposure is undeniably a major risk factor for AMR,,The volume of human antibiotic consumption has an inconsistent impact on AMR rates.We anticipate that the impact of COVID-19 on AMR will be uneven, varying between epicentres and non-epicentres, by geographic region, hospital-to-hospital within regions and within specific hospital units. Initially, local AMR patterns are likely to be exacerbated. As travel resumes, the risk of dissemination of novel resistance determinants will increase. AMR screening programmes should be implemented for asymptomatic hosts and livestock following antibiotic exposure. Research protocols can evaluate the impact of antibiotic use on microbiota and resistomes, as part of more sensitive surveillance.None to declare.


Cellulose is one of the most abundantly available natural polymers. Carboxymethyl cellulose (CMC) belongs to the cellulose family and has different degrees of substitution. Current research comprises the fabrication and characterization of CMC nanofibers using polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) as capping agents and carriers for sustainable food packaging applications. Recently authors successfully fabricated smooth and uniform nanofibers of stated polymers and optimized the ratios of three polymers for continuous production. However, in this research, it was further characterized for mechanical properties, surface properties, structural properties, air permeability, and chemical properties to confirm the suitability and scope of tri-component nanofibrous mats in food packaging applications. Different fruits and vegetables were packed in a plastic container and closed by nanofiber mats and by a plastic lid. All samples were observed after a specific period of time (fruits were kept for 40 days while vegetables were kept for 10 days in the controlled environment). It was observed in the results that fruits and vegetables closed by nanofiber based webs exhibited better freshness and lower accumulation of moisture as compared to that of containers with plastic lids. From the results of performed tests, it was observed that nanofiber mats possess enough mechanical, structural, and morphological properties to be used as food packaging. Sustainability is one of the key considerations while developing materials, methods, or systems. Materials from sustainable sources are always preferred over unsustainable materials. Natural polymers have a vast range of applications in every area of science ,3,4,5,6.CMC is easily soluble in water and a hydrophilic polymer. CMC is soluble in cold water and does not form a gel. There are unsubstituted sites present on the backbone of CMC, which lowers the solubility of CMC in cold water. In cold water the unsubstituted sites of CMC do not work properly but as the water temperature goes up gel formation rate increases because of unsubstituted cellulose sites along the backbone chain as temporary cross-linkers between the chains. Viscosity of CMC and gel formation rate is directly proportional to each other. Increase in viscosity makes the CMC difficult to electrospin. PVA is well renowned polymer due to its easy spinnability, biocompatibility, and good thermo mechanical properties ,42. Hydr−1), polyvinyl alcohol (PVA) , and sodium carboxymethyl cellulose (Na-CMC)  were procured from Sigma-Aldrich . Polymers were not further modified physically or chemically before using.Polyvinylpyrrolidone (PVP) . Added in each solution 30 min before electrospinning was 2% glutaraldehyde (25% dilution) (PVA concentration in spinning solution was fixed at 6% −1. Prepared nanofibers’ morphology was examined using the scanning electron microscope (SEM) . Acceleration energy was kept at 10 kV for all samples. Diameters of nanofibers were calculated by Image J. software and taking average of 50 random readings from different nanofibers from each image. The degree of hydrophilicity of prepared nanofibrous mats was confirmed by measuring the water contact angle (WCA) of each nanofibrous sample. WCA was measured by using the contact angle analyzer . Degradation behavior of nanofibers under high temperatures was analyzed using the thermogravimetric analyzer . The thermal degradation test was carried out in ambient atmosphere (in ambient air) and static mode while the incremental rate of heating was 10 °C/min in temperature range of 25–500 °C. Sample weight for all specimen was 10 mg each. Crystalline structure of CMC based composite nanofibers was observed by the X-ray diffraction (XRD) spectra of each sample. XRD spectra were recorded at room temperature (25 ± 3 °C) using Rotaflex RT300 mA, Rigaku, Osaka, Japan, an angle (2θ) ranging from 5 ≤ 2θ ≤ 70°. Mechanical properties of prepared composite nanofibers were assessed by the universal testing machine (UTM), Tensilon RTC 250A; A&D Company Ltd., Osaka, Japan. ISO 13634 testing standard was followed to prepare and characterize all specimens (note that n = 5 for each type of nanofibers), crosshead speed was 5 mm/min and the test was performed at room temperature. Air permeability of CMC based electrospun nanofibers was performed using Air Permeability Tester Kato Tech Co., Ltd., Osaka, Japan. Average resistance (R) was calculated by measuring the AP of 5 specimens for each type of nanofibers. Suitability of electrospun nanofibers for food packaging applications was assessed by comparing nanofibers and plastic packaging for different fruits and vegetables in controlled environment (relative humidity 75% and temperature 2 °C (275 K)).Possibility of any chemical interactions among functional sides of PVA, PVP, and CMC, PVP was examined using Fourier transform infrared spectroscopy (FTIR) with an attenuated total reflection (ATR) prestige-21 . FTIR-ATR spectra were taken from wave number ranging from 600 to 4000 cm−1 a broader peak, which was associated with the hydroxyl group in PVA and CMC main chains, was observed in the ATR spectra. However, the stated peak was becoming more flatter in the case of CMC based composite nanofibers. It was concluded in the results of WCA that CMC based composite nanofibrous mats presented a bigger contact angle (˃90°). Characteristic peaks of PVA were observed at 3200–3450 cm−1, which indicates the presence of the hydroxyl group (–OH stretching) in PVA chains (however limited due to crosslinking but still available). No hydroxyl peak was found in the case of a composite nanofiber, which indicates completely restricted –OH functionality in composite nanofibers. In the PVA spectrum, the symmetric and asymmetric peak (–CH2–) was observed at 2900 cm−1 [−1, and peaks from 2850 to 2980 cm−1 were associated with CH stretching. Peaks at 1430 cm−1 and 1370 cm−1 were associated with the CH deformation band  not a single nanofiber was observed. Nanofibers of PVA/PVP composite were also smooth as it can be shown in w/w) of its carrier PVA. So, the authors recommended that PVA:PVP:CMC = 6:12:3 concentration is most suitable for smoother and continuous nanofibers production without beads formation.Surface structure and diameter of nanofibers were examined by scanning electron microscope (SEM). It is already stated that it is very difficult to electrospin CMC due to its high viscosity and poor conductivity. However, PVA can be easily electrospun even at a different concentration but a concentration from 6% to 10% is the most appropriate to get smooth nanofibers. PVP possesses low viscosity at lower concentrations, which makes it difficult to electrospin. PVP helps to decrease the viscosity of CMC and imparts conductivity in it. CMC and PVP together give ideal viscosity and conductivity, which show results in the form of good nanofibers. In our recent research the concentration of PVP and CMC was optimized to obtain uniform nanofibers. SEM images of CMC based electrospun nanofibrous mats and neat PVA nanofibers are shown in Measuring diameters of nanofibers is one of the important tools to assess uniformity of mats. Diameters of the CMC based composite, PVA/PVP, and PVA nanofibers were calculated by taking 50 readings of random nanofibers using Image J. software. Nanofiber based food packaging have an advantage over paper based or film based food packaging because of the unique porous structure of nanofiber mats. Air permeability (AP) allows food items especially fruits and vegetables to breathe , which keeps them fresh for a longer period of time. The hydrophilic or hydrophobic nature of polymeric nanofibers plays an important role while targeting final applications . Food paPVA and PVP show thermal stability above 300 ℃ while CMC is thermally unstable above 280 °C. Crystalline structure of PVA nanofibers was compared with that of tricomponent composite nanofibers of PVA/PVP/CMC by the XRD spectra. It can be observed  that PVAMechanical properties are important for any material, which is designed for commercial applications such as packaging. Tensile strength was measured for PVA, PVA/PVP, PVA/CMC, and tricomponent PVA/PVP/CMC nanofibers, and the effect of loading of CMC on the mechanical properties of the final product was compared with that of pure PVA or PVA/PVP nanofibers. An experimental study was carried out to investigate suitability potential of nanofibers based food packaging by comparing with plastic based packaging. Fruits and vegetables of a similar type and freshness were taken and packed in different containers. One container for each type was packed by the plastic lid and the other was packed by nanofiber mats . All samples were placed in a controlled environment in the same refrigerator. Vegetables were examined after 10 days while fruits were placed for 40 days. CMC was selected as a target material for electrospun nanofiber based food packaging due to biocompatibility, biodegradability, and sustainability. From the results of morphological characterization, it was concluded that CMC could be electrospun along with PVA and PVP, and possess uniform morphology. Diameters were also found to be in uniform range. The water contact angle was also measured to confirm the stability of nanofibrous mats in outdoor use. It was concluded that all nanofibers were found to be hydrophobic. It was because of crosslinking of PVA, PVP, and CMC. In general all these polymers are hydrophilic and water soluble in the uncrosslinked form, however, these were transformed to being water insoluble and hydrophobic due to crosslinking. Tensile properties were also found to be conformed to the target application as all nanofibers of the tricomponent blend had tensile strengths well above 10 MPa. Air permeability is one of the important factors that were evaluated for nanofibrous mats and it was concluded that all nanofibrous mats possess excellent air permeability, which will keep fruits and vegetable fresh for a longer time. It was also observed in the results that fruits and vegetables closed by nanofibers based webs exhibited better freshness and lower accumulation of moisture in the containers. Considering all results it is suggested that PVA/PVP/CMC nanofibrous mats can be used for food packaging.


Prokineticin 1 (PROK1) is a key regulator of embryo implantation and placentation, and its dysregulation is associated with pregnancy complications, such as pre‐eclampsia and foetal growth restriction. We have previously shown that insulin strongly enhances the expression of PROK1 in human decidualizing stromal cells. Here, we demonstrate that dihydrotestosterone (DHT), but not testosterone, potentiates insulin to up‐regulate PROK1 in these cells. However, the androgens alone do not influence the expression of PROK1. Our findings suggest that insulin and androgens both are involved in the regulation of PROK1 that could have implications for normal and pathological pregnancies. Prokineticin 1 (PROK1) is highly expressed in female reproductive organs, such as ovaries, placenta and endometrium, and has emerged as a crucial negative regulator of extravillous trophoblast invasion.Recently, we showed that insulin highly up‐regulates PROK1 in decidualizing human stromal cells.2Endometrial biopsies were collected from nine regularly cycling, healthy volunteers between age 18‐35 and body mass index 19‐28 on cycle day 5‐9 as previously described.6, 2'‐O‐dibutyryladenosine cAMP and 1 μmol/L medroxyprogesterone‐17‐acetate were carried out as previously described.Isolation of endometrial stromal cells and in vitro decidualization using 0.5 mmol/L NT method.Total RNA was extracted, and cDNA was synthesized as described previously.Secreted PROK1 protein was measured from undiluted conditioned media using ELISA  according to the manufacturer's instruction. The limit of detection (LOD) was 16 pg/mL. Values below the LOD were replaced by half of LOD.All data were log‐transformed, and normal distribution was challenged with the Kolmogorov–Smirnov test. Two‐way ANOVA with Sidak's multiple comparison test was used to determine the effect of insulin and/or androgen exposure.3P < .001) and protein expression of PROK1 (P < .001) in human decidualized stromal cells, while neither DHT nor testosterone had a significant effect alone on PROK1 expression when compared to treatment with decidualization agents (MPA and db‐cAMP) alone. Insulin+DHT or insulin+testosterone also significantly increased both gene and protein expression of PROK1 compared with treatment with only decidualization agents . Insulin+DHT or insulin+testosterone significantly increased PROK1 gene  and protein expression  compared with DHT or testosterone treatment. Insulin + DHT enhanced both gene (P < .01) and protein expression of PROK1 (P < .05) even more in comparison with insulin treatment (Figures There was a main effect of insulin exposure in decidualized stromal cells with increased PROK1 at mRNA and protein level. We could not detect main effect of DHT/testosterone exposure. There was an interaction effect between insulin and DHT at both mRNA and protein level, but no interaction between insulin and testosterone. Insulin enhanced both gene ( Figures .4Here, we demonstrated that DHT or testosterone exposure alone has no effect on PROK1 in decidualizing stromal cells, however, DHT, but not testosterone, potentiates insulin to increase PROK1 at mRNA and protein level. The difference between the effect of testosterone and DHT might be partially explained by the twofold higher affinity and fivefold decreased dissociation rate of DHT to the androgen receptor compared with testosterone.Experimental evidence suggests that PROK1 promotes proliferation, but impairs migration and invasion of trophoblast cells and their differentiation in order to remodel the maternal spiral arteries.In conclusion, our results suggest that insulin and androgens both are involved in the regulation of PROK1 in human decidualizing stromal cells. This could be of relevance for implantation, placentation and the development of placenta‐related pregnancy complications.The authors declare no conflicts of interest.DU and ALH designed the study; ALH enrolled the participants and took the endometrial biopsies; DU and CGB performed the experiments; DU and CGB analysed and interpreted the data; DU visualized the data; DU wrote the initial draft of the manuscript; ALH had overall responsibility for the study; all authors contributed to writing the manuscript, have seen, reviewed and approved the final version of the manuscript.


The crystal structures of the PATZ1 BTB domains from mammals and fish contain homodimers. The core dimer conformation of these BTB proteins is dynamically stable, despite the presence of highly flexible regions in the dimerization interface. CD8 gene in lymphocyte development and interacts with the p53 protein to control genes that are important in proliferation and in the DNA-damage response. PATZ1 exerts its activity through an N-terminal BTB domain that mediates dimerization and co-repressor interactions and a C-terminal zinc-finger motif-containing domain that mediates DNA binding. Here, the crystal structures of the murine and zebrafish PATZ1 BTB domains are reported at 2.3 and 1.8 Å resolution, respectively. The structures revealed that the PATZ1 BTB domain forms a stable homodimer with a lateral surface groove, as in other ZBTB structures. Analysis of the lateral groove revealed a large acidic patch in this region, which contrasts with the previously resolved basic co-repressor binding interface of BCL6. A large 30-amino-acid glycine- and alanine-rich central loop, which is unique to mammalian PATZ1 amongst all ZBTB proteins, could not be resolved, probably owing to its flexibility. Molecular-dynamics simulations suggest a contribution of this loop to modulation of the mammalian BTB dimerization interface.PATZ1 is a ubiquitously expressed transcriptional repressor belonging to the ZBTB family that is functionally expressed in T lymphocytes. PATZ1 targets the  It was first discovered in a yeast two-hybrid (Y2H) experiment, in which it associated through its BTB  domain with the BTB domain of the transcription factor BACH2 , another BTB domain-containing transcription factor , also known as ZBTB19, is a transcription factor that is present in all vertebrates Fig. 1a. It waet al., 2011b), all of which contain an N-terminal BTB domain and a variable number of ZF motifs at their C-terminus. Two members of this family contain an additional motif in the form of an AT hook (PATZ1 and PATZ2/ZBTB24).Structurally, PATZ1 belongs to the POZ (pox virus and zinc finger) or ZBTB (zinc finger and BTB) family of transcription factors  proteins creates a charged groove that binds nuclear receptor co-repressors such as NCOR1, NCOR2 (SMRT) and BCOR : LRF/Pokémon (ZBTB7A), PLZF (ZBTB16), MIZ1 (ZBTB17), BCL6 (ZBTB27), MYNN (ZBTB31), FAZF (ZBTB32), KAISO (ZBTB33) and HKR3/TZAP (ZBTB48). In order to obtain biological insights into the binding of PATZ1 to co-repressors, we determined the atomic structure of the PATZ1 (ZBTB19) BTB domain. To probe the role of the unique A2/B3 central loop, we obtained crystal structures of both the mouse PATZ1 BTB domain and its zebrafish orthologue.2.2.1.LALIGN; Gasteiger et al., 2003et al., 2015Escherichia coli Rosetta 2(DE3) strain and grown at 310 K by shaking at 180 rev min−1 in Terrific/Turbo Broth (TB) medium supplemented with 50 µg ml−1 kanamycin and 33 µg ml−1 chloramphenicol until the absorbance at 600 nm reached a value of 0.6. Expression of the fusion protein was induced by the addition of 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and growth was continued for 16 h at 291 K. The zebrafish (Danio rerio) PATZ1 BTB protein-coding sequence (1–135) was PCR-amplified from zebrafish genomic DNA (a kind gift from Dr S. H. Fuss), cloned and expressed in the same bacterial expression plasmid as described above.The human isoform 3 (Q9HBE1-3) and mouse (Q9JMG9) PATZ1 BTB sequences are 98.9% identical  and disrupted by sonication on ice. The lysate was clarified by centrifugation at 26 700g for 45 min at 277 K. The supernatant was applied onto a HisPur Cobalt Resin column (Thermo Fisher) previously equilibrated with wash buffer . Following a 10 min incubation at 227 K and the application of wash buffer, the protein was then eluted by the addition of elution buffer . The collected eluate was concentrated to 2.6 mg ml−1 using a Sartorius Vivaspin 20 column (10K molecular-weight cutoff) for additional purification by size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 75 prep-grade column  in gel-filtration buffer  at 277 K. Fractions were analyzed on a 14% SDS–PAGE gel by electrophoresis and those containing PATZ1 BTB were pooled and concentrated to 9 mg ml−1. The zebrafish PATZ1 BTB domain (69% sequence identity to mouse BTB) was expressed and purified as described above for the mouse BTB domain. The final concentration of the protein was 8.5 mg ml−1.The cells were harvested by centrifugation, resuspended in 25 ml lysis buffer , 25%(w/v) PEG 1500. Crystals of the zebrafish PATZ1 BTB domain appeared after 72 h in 40%(v/v) PEG 500 MME, 20%(w/v) PEG 20K, 0.1 M Tris pH 8.5, 0.06 M magnesium chloride hexahydrate, 0.06 M calcium chloride dihydrate.All crystallization experiments were performed at 291 K using the sitting-drop vapour-diffusion method. The initial screening of 768 conditions was performed at the University of Sussex crystallization facility using a Crystal Phoenix dispensing robot to pipette 0.1 µl protein solution and 0.1 µl precipitant solution into single drops in 96-well plates. Crystals of the mouse PATZ1 BTB domain appeared after 72 h in 0.1 2.3.xia2 , with one molecule in the asymmetric unit.For data collection, single crystals were briefly immersed in mother liquor supplemented with 20% glycerol prior to flash-cooling in liquid nitrogen. For mouse PATZ1 BTB, data were collected to 2.29 Å resolution on beamline I04 at Diamond Light Source, Didcot, UK. The diffraction data were indexed, integrated, scaled and reduced with xia2 and XDS , with one molecule in the asymmetric unit.For zebrafish PATZ1 BTB, data were collected to 1.8 Å resolution on beamline I04 at Diamond Light Source. The diffraction data were indexed, integrated, scaled and reduced with Detailed X-ray data-collection and refinement statistics are given in Table 12.4.Phaser  as the search template. The identified solution was then subjected to rounds of manual rebuilding with Coot and refinement with Phenix to give final Rwork and Rfree factors of 21.5% and 22.4%, respectively. The final structure was validated with MolProbity and deposited together with the structure factors in the Protein Data Bank as entry 6guw.The structure of zebrafish PATZ1 BTB was solved by molecular replacement with Detailed X-ray data-refinement statistics are given in Table 12.5.et al., 2007Homo sapiens (NP_114440.1) and Mus musculus (NP_001240620.1) for mammals, one organism only was chosen for each group of different species of vertebrates: Danio rerio (XP_009300883.1) for fish, Xenopus laevis (XP_018117120.1) for amphibians, Thamnophis sirtalis (XP_013922905.1) for reptiles and Parus major (XP_015499085.1) for birds. Sequence limits were determined based on the annotations of the BTB domain in the UniProt database . ZBTB4 was excluded from this alignment because of its N-terminal serine-rich repetitive insertions, which are beyond the scope of this description. Shading was added according to the percentage of similarity for every position in the alignment visualized in Jalview 2  in PyMOL . The coordinates of the loop model were added to the crystal structure and fragments were joined using the VMD AutoPSF plugin. SymmDock  was modelled as a monomer using the NAMD  simulations in M-ZDOCK  and the stability of the modelled structure was assessed by MD as before.In one of the MD simulations of human PATZ1 BTB, the flexible loop region in one of the monomers formed an extra β-strand structure. The model was further refined by once more duplicating this monomer into the dimeric form. This refinement was processed by taking a frame from the run in which the root-mean-square deviation (r.m.s.d.) with the initial structure, excluding the loop, was minimal (3.04 Å) and by mirroring the information from the monomer with the formed secondary structures to the other monomer with 3.3.1.Supplementary Fig. S1). The murine PATZ1 BTB domain protein was expressed from a construct encoding amino acids 12–166 preceded at the N-terminus by 20 amino acids comprising a His tag and an HRV-3C protease digestion site . The last ten of these amino acids (ALEVLFQGPG) are visible in the structure and fold into a β-strand (β0) antiparallel to the first N-terminal PATZ1 β-strand (β1) . Superposition of this structure with that of BCL6 BTB in complex with co-repressor peptides , although their amino-acid sequence is not conserved. Attempts to crystallize the mouse PATZ1 BTB construct following cleavage of the His tag failed, suggesting that the extra N-terminal amino acids are likely to aid the crystallization process in this case.Here, we report the crystal structures of the BTB domains of murine and zebrafish PATZ1 Fig. 2. In a sia and 1b). This large loop replaces a shorter amino-acid stretch that forms a β-strand (B3) in other ZBTB family proteins, as described in detail by Stogios et al.  protein is predicted to contain a 31-amino-acid A2/B3 loop (residues 75–105) that is partially conserved in cKrox (ZBTB15), which sets PATZ1 apart from the other ZBTB family members . This crystallization artefact presumably aids crystal packing, as the ‘B3 strand mimic’ appears to stabilize the β-sheet formed by B1 and B2. To test this hypothesis, we crystallized a mouse PATZ1 BTB construct lacking the last seven C-terminal amino acids. The crystals of this protein diffracted poorly and to lower resolution (3.4 Å), showing the same homodimeric BTB domains in a different crystal packing (data not shown). This suggests that these seven amino acids were in fact important for stabilizing the B1–B2 β-sheet and for crystal packing (hence the better diffraction), yet their absence did not encourage the folding of the A2/B3 loop.Interestingly, in the mouse PATZ1 BTB structure a seven-amino-acid stretch from the C-terminus of an adjacent molecule in the crystal unit cell extends into the region normally occupied by the B3 β-strand in other ZBTB proteins (a). The mammalian A2/B3 loop could be an evolutionarily acquired insertion sequence encoding an intrinsically disordered loop . The zebrafish PATZ1 BTB domain was expressed from a construct encoding amino acids 1–135 preceded at the N-terminus by 20 amino acids comprising a His tag and an HRV-3C protease digestion site. The 20 amino acids at the N-terminus and the first ten residues of the zebrafish PATZ1 BTB domain are not visible in the electron-density map. In addition, seven amino acids belonging to the A2/B3 loop are also missing from the electron density. When excluding the central loop, the murine and zebrafish sequences share 83.7% identity, whilst the structures can be superimposed with an r.m.s.d. of 0.62 Å . The zebrafish BTB domain also has the same quaternary structure as the mouse BTB domain: a strand-exchange homodimer. Owing to the absence of the long disordered A2/B3 loop, the zebrafish PATZ1 BTB domain is structurally more similar to other ZBTB proteins than the mammalian PATZ1 BTB domain. The zebrafish PATZ1 BTB domain also has a seven-amino-acid sequence between A2 and B3 for which no discernible electron density could be found. This loop was modelled using ModLoop .Sequence alignment shows that while the length of the A2/B3 loop is conserved in all PATZ1 orthologues, it is conspicuously absent in those from fish and amphibians Fig. 1a. The m Å Fig. 2e. The zf and 3c) and that at least ten amino acids within the modelled loop contribute to the homodimerization interface . Interestingly, the β-strand that is generated in the simulations contains the threonine residue that is the only amino acid that differs between the human and mouse BTB domains. Other BTB domains such as LRF and MIZ1 also contain flexible loops in this region  we found no evidence for such oligomerization in mouse or zebrafish PATZ1.The human and mouse PATZ1 BTB domains have a single amino-acid difference (T91A). To detail the possible structure and dynamic behaviour of the A2/B3 loop in human PATZ1 BTB, we performed molecular modelling and molecular-dynamics simulations (MD). MD simulations lasting 200 ns indicated that while the overall dimer forms a stable structure, the A2/B3 loop region is uniquely flexible. The simulations also suggest that a new β-strand could form within this loop . The interfaces retrieved from PDBePISA . Using the VMD tools, we found a dramatic increase in the number of charged residues (mostly negative) that participate in interface contacts (marked with asterisks in Table 2c and 3d). During the simulation, the A2/B3 loop region significantly contributes to the interface in both models, resulting in flexibility of the salt bridges that form between a single charged amino acid from one monomer and multiple opposite charged amino acids from the opposite monomer. While Arg47 of mouse PATZ1 BTB is only engaged with Glu75 in the crystal structure, MD show that it can contact a broader number of charged residues, including those from the flexible loop . We also find that two of the three degron residues  that are predicted to play a role in BTB heterodimer degradation participate in the interaction interface of both BTB homodimer structures.In order to understand the dynamics of these interfaces, we assessed the number of contact-forming residues in the energy-minimized modelled BTB domains . To examine the structural similarity between the PATZ1 and BCL6 BTB domains, we calculated the r.m.s.d. (1.56 Å) between individual monomers. The structural similarity between BCL6 and PATZ1 was more evident when the flexible PATZ1 loop was excluded (r.m.s.d. of 1.23 Å). Comparison of the surface charge distributions of the two proteins indicates major differences . Specifically, the BCL6 lateral groove contains a high density of positively charged amino acids that interact with the co-repressors . Surprisingly, the surface of PATZ1 corresponding to the BCL6 lateral groove did not contain as many basic residues . In fact, this region of mouse and zebrafish PATZ1 is highly conserved  and contains more acidic amino acids.Even though BCL6 and PATZ1 are structurally very similar, their corresponding co-repressor binding interface sequences are not conserved Fig. 4d. To exet al., 2007Supplementary Fig. S5) is part of the charged pocket that is conserved between BCL6, LRF and PLZF  may indicate altered binding modalities and/or affinities for co-repressors. In fact, a structurally conserved charged pocket has previously been hypothesized to be involved in ligand interaction .Another feature that is common to PATZ1 and BCL6 is their BTB domain-mediated localization to nuclear speckles (Huynh et al., 2013The MD simulation shows that alternative contacts are possible for several charged residues at the homodimer interface Table 2. Becauseet al., 2017BTB domains are attractive targets for anticancer compounds. Compounds that prevent homodimerization or result in the degradation of BCL6 (Kerres mouse PATZ1 BTB domain, 6guvPDB reference: zebrafish PATZ1 BTB domain, 6guwPDB reference: 10.1107/S2059798320005355/ni5006sup1.pdfSupplementary Figures. DOI:


In utero patency of the ductus is maintained by low oxygen tension, high levels of prostaglandins, nitric oxide and carbon monoxide. After birth, ductal closure occurs first by functional closure, followed by anatomical remodeling. High oxygen tension and decreased prostaglandin levels mediated by numerous factors including potassium channels, endothelin-1, isoprostanes lead to the contraction of the ductus. Bradykinin and corticosteroids also induce ductal constriction by attenuating the sensitivity of the ductus to PGE2. Smooth muscle cells of the ductus can sense oxygen through a mitochondrial network by the role of Rho-kinase pathway which ends up with increased intracellular calcium levels and contraction of myosin light chains. Anatomical closure of the ductus is also complex with various mechanisms such as migration and proliferation of smooth muscle cells, extracellular matrix production, endothelial cell proliferation which mediate cushion formation with the interaction of blood cells. Regulation of vessel walls is affected by retinoic acid, TGF-β1, notch signaling, hyaluronan, fibronectin, chondroitin sulfate, elastin, and vascular endothelial cell growth factor (VEGF). Formation of the platelet plug facilitates luminal remodeling by the obstruction of the constricted ductal lumen. Vasa vasorum are more pronounced in the term ductus but are less active in the preterm ductus. More than 100 genes are effective in the prostaglandin pathway or in vascular smooth muscle development and structure may affect the patency of ductus. Hemodynamic changes after birth including fluid load and flow characteristics as well as shear forces within the ductus also stimulate closure. Current pharmacological treatment for the closure of a patent ductus is based on the blockage of the prostaglandin pathway mainly through COX or POX inhibition, albeit with some limitations and side effects. Further research for new agents aiming ductal closure should focus on a clear understanding of vascular biology of the ductus.Failure of ductus arteriosus closure after preterm birth is associated with significant morbidities. Ductal closure requires and is regulated by a complex interplay of molecular and mechanical mechanisms with underlying genetic factors.  Fetal circulation is a unique event, functionally different from pediatric and adult circulation. Ductus arteriosus (DA) is a small, simple vessel with just a few milimeters length and width, but has a profound impact on the survival of the fetus and newborn and plays an essential role in normal postnatal adaptation after birth. It functions as a bridge between two large vessels in fetal life, but sometimes it becomes the “bridge between life and death” in preterm infants. It has baffled scientists since ancient times, and beginning from Galen and Ibn-al Nafis, Vesalius, Leonardo Botallo and William Harvey have commented on its structure and function .During fetal development, cardiovascular septation occurs during days 22–28 of gestation and looping of ventricles completes. At this stage, bilateral aortic arches and bilateral DA form but over the next week, the right aortic arch and ductus involutes. Ductus arteriosus arises from the left sixth embryonic arch; leaves the pulmonary artery very close to the bifurcation point and inserts at the transition zone between the aortic arch and descending aorta, distal to the origin of the left subclavian artery. Usually it has a tubular shape but may be funnel-shaped with a narrow end on the pulmonary side and a wide end on the aortic side  and the anatomical step (remodeling). Interestingly, the closure of DA resembles the process after vascular injury or atherosclerosis in the adult arteries . UnderstSmooth muscle tone is regulated by phosphorylation/dephosphorylation of myosin light chain (MLC). MLC is phosphorylated by Calcium/calmodulin-dependent MLC-kinase (MLCK) and dephosphorylated by calcium independent MLC-phosphate (MLCP). Increased cytosolic calcium activates MLCK, which leads to MLC phosphorylation and vasoconstriction  Figure . PatencyProstaglandins are synthesized from arachnidonic acid by cyclooxygenases COX1 and COX2 within the ductal muscle. Circulating prostaglandins, originated from the placenta contribute to the patency of DA . Prostag2) is the major arachnidonic acid product of the ductus but although it is more prevalent, it is less potent than prostaglandin E2 (PGE2), which is the most important prostaglandin to regulate the patency of DA (2 interacts mainly with its receptor EP4. EP4 activates voltage-gated potassium channel (Kv) and outward K+ current increases, thereby inducing membrane hyperpolarization, which inhibits Ca2+ influx via the voltage-gated L-type calcium channels (CaL) and decreases intracellular calcium , which inturn activates protein kinase (PKA), which inhibits myosin light chain creatine kinase (MLCK). Inhibition of MLCK blocks phosphorylation of myosin light chains, which results in inhibition of vasoconstriction, hence vasodilation occurs , by PKG leads to uptake of Ca2+ in sarcoplasmic reticulum, lowering intracellular calcium  is produced by endothelial nitric oxide synthase in the luminal endothelium and endothelium of vasa vasorum and maintains the patency of DA by acting through cGMP. After its production, it diffuses into the SMC and binds with soluble guanylyl cyclase, producing cyclic guanosine monophosphate (cGMP). cGMP activates cGMP-dependent protein kinase (PKG), which induces vasodilation  Figure . NO is ather one . This mi2 sensing cytochrome P450, thus interrupting endothelin-1 signaling (Hem oxygenase 1 and hem oxygenase 2 which produce carbon monoxide (CO) are found in the endothelial and smooth muscle cells of the DA. CO dilates the ductus by inhibition of Oignaling , 20  inhibitors such as milrinone and amrinone have positive inotrope and vasodilator effects. Inhibition of PDE3 also induces dilation of the fetal and postnatal ductus arteriosus in humans. This effect is mediated by an increase in cAMP and cGMP in vascular smooth muscle  is expressed heavily in ductal SMC and acts as a calcium channel which increases intracellular Cain utero .in utero are summarized in Factors that maintain ductal patency Functional closure of the ductus occurs by 8 h in 44% of normal term infants and almost 100% are closed by 72 h. . The ratConstriction of the ductus is maintained via3 factors:4 receptors on the ductal wall.a) Decreased concentration of prostaglandins by the loss of placental source and increased removal by the lungs, and decreased number of EPb) Increased arterial oxygen pressure.c) Decreased pulmonary vascular resistance resulting in decreased blood pressure within the lumen of the DA.2 concentrations drop rapidly due to removal of placental production and increased metabolism in the lungs   . The act15-PGDH) . 15-keto to PGE2 . On the  to PGE2 . Bradykitriction .2 is higher than that in the term infant, which is attributed to increased binding to EP2, EP3, and EP4 receptors  and should be considered one of the body's major systems such as cardiovascular, nervous or endocrine systems .2  especially H2O2, which changes the cellular redox potential and alters the function of redox-sensitive genes, second messenger systems and oxygen-sensitive potassium channels, which control SMC membrane potential. Production of adenosine triphosphate (ATP) through oxidative phosphorylation increases with rising oxygen levels and inhibits KATP channels  oxidase. Proximal electron transport chain is the major oxygen sensor in the ductal SMC. This is reflected in the observation that electron transport chain inhibitors  mimic hypoxia and constrict the ductus, as well as activating the carotid body. When POchannels . These cchannels .2induced gene expression of ion channels  and T type (CaT) calcium channels, and opening of inositol triposphate (IP3) sensitive sarcoplasmic reticulum calcium stores which release calcium  expression, which promotes phosphorylation of myosin phosphatase inhibiting MLCP. Activation of the Rho-kinase pathway induces calcium sensitization, which sustains ductal constriction through positive feedback mechanism . Stimulation of these receptors induces IP3 production by phospholipase c, which leads to an increase in intracellular Ca2+ (Oxygen stimulates the release and synthesis of endothelin-1 (ET-1), acting on type A receptors . This is not profound enough to cause cell death but interferes with the ability of the ductus to close .Glutamate promotes ductal constriction by glutamate inotropic receptor subunit 1 (GluR1)-mediated noradrenalin production in animal models . It may +2 from endoplasmic reticulum, and inhibits the production and activity of prostaglandins. It is used frequently in extremely low birth weight infants for the prevention and treatment of apnea and bronchopulmonary dysplasia. However, it does not affect the contractile response of the ductus at therapeutic concentrations  plays an important role in regulating the body fluid volume and blood pressure. It is secreted from the ventricles in response to increased cardiac stress and increases intracellular cGMP, inducing vasodilation. In PDA, the volume of left-to-right shunting is associated with the BNP level and higher levels predict a hemodynamically significant PDA and a poor response to indomethacinin preterm infants , 64. BraProgesterone regulates prostaglandin synthesis and prostaglandin sensitivity of smooth muscle cells. Progesterone withdrawal in late pregnancy coincides with the increasing sensitivity of ductus arteriosus to constriction by indomethacin . This ef2 levels after birth, which impairs ductal vasodilation and a concomitant rise in oxygen which leads to vasoconstriction. Intracellular calcium increases, secondary to inhibition of Kv channels and KATP induced membrane depolarization. Increased calcium within the cell activates Ca2+–Calmodulin dependent MLCK activation, which phosphorylates MLC , notch signaling, Vascular Endothelial Growth Factor (VEGF) and fibronectin coupled with hyaluronan. The proliferation of SMCs is mediated by retinoic acid and notch signaling. Mounds are formed by expansion of the neointima by hyaluronan, which creates a suitable space for migrating SMC from the muscle into the intima and proliferating endothelial cells. The process begins with the accumulation of hyaluronan beneath the endothelial cells, accompanied by loss of laminin and type IV collagen and their separation from the internal elastic lamina. Influx of water into the hyaluronan widens the subendothelial space. Hyaluronan potentiates the migration of SMCs to the subendothelial space through hyaluronan binding protein, synthesized by SMCs  pathway in the ductal wall  has many pro-inflammatory effects, but also inhibits SMC proliferation and hyaluronan accumulation during the remodeling process of the ductus. The up-regulation of IL-15 in the ductus is mediated through the prostaglandin pathway. IL-15 upregulates the expression of EPal cells . In lateal cells .Notch receptors are critical for cell differentiation and there are 4 Notch receptors in humans. Notch 2 and Notch3 receptors are abundant in smooth muscle and they regulate proliferation, migration and angiogenesis in the vasculature. A decrease in the number of Notch receptors in SMCs is associated with downregulation of gene expressions related to contractile elements . Notch s2, IL-15, and oxygen , which stimulates hyaluronan production in the SMCs  is necessary for recruitment when physiologic shear forces are present and this interaction is only possible when the flow is very slow. Therefore, leukocytes can adhere to the ductal wall only after vasoconstriction and loss of ductal flow has occurred , 100 MonDuring the initial process of vasoconstriction, detachment of endothelial cells triggers the adhesion of platelets to the “injured” site, forming a platelet plug, which further blocks the lumen. Mice with disrupted platelet function or administered antibody against platelets have impaired ductus remodeling . The effAlthough they originate from the same neural crest lineage, there are major transcriptional differences between the DA and aorta. Several studies highlight the effect of genetic predisposition to the patency of DA. These include either the prostaglandin pathway , or vascular smooth muscle development or structure. Most of these information is obtained through mouse models. Overall, there are more than 100 genes are effective in the development of DA , 109.2 synthase to prostaglandin H2, which in turn is metabolized by PGE synthase to prostaglandin E2 (PGE2). Prostaglandin endoperoxide synthase 1 (Ptgs1 or COX1) and (Ptgs2 or COX2) is the rate limiting enzyme in the production of prostaglandins from arachidonic acid. Mice deficient in Ptgs1 have normal survival after birth whereas 35% of mice deficient in Ptgs2 die shortly after birth because of PDA (2 binds to the cell through its receptor EP4 (Ptger4) and mediates vascular smooth muscle relaxation and intimal thickening. Mice lacking these receptors die due to PDA (Hpgd gene encodes hydroxyprostaglandin dehydrogenase 15-(NAD) which is responsible for PGE2 metabolism. The deletion of this gene leads to elevated levels of PGE, leaving the DA open and resulting in death (Arachidonic acid is metabolized by PGHe of PDA . If bothe of PDA . Prostage to PDA . Furtherin death . Mice dein death .Pde5a mediates vasoconstriction in SMC of DA and its expression decreases in 6 h after birth (The effects of prostaglandins are mediated through cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). Phosphodiesterases degrade these compounds and several genes have been proposed for phosphodiesterases. er birth .The genes upregulated during ductal closure are phosphodiesterase 4B (PDE4B), desmin pyruvate dehydrogenase kinase isozyme 4 (PDK4), and insulin-like growth factor binding protein 3 (IGFBP3). Downregulated genes include cyclin-dependent kinase inhibitor 1C (CDKN1C) and alpha-2-glycoprotein1 (AZGP1). PDE4B limits the accumulation of cAMP, minimizing the vasodilator effect of endogenous prostaglandins. Therefore, increasing PDE4B would facilitate ductal constriction. Desmin is a component of the cytoskeleton and its increase indicates the maturation of SMCs. PDK4 and IGFBP3 regulate cell proliferation and apoptosis, which are operative in ductal closure. Increased transforming growth factor β-receptor II regulates PDK4 also , 116. WiMyh11) have delayed closure of DA  and its effector Rock2 is upregulated in the newborn DA and inhibition of this system may result in the patency of DA  and a subunit of L-type calcium channels (Cacna2d1) are highly expressed in the DA (Kcnk3 and Abcc9) are also upregulated in the DA (BKca) are abundant in vascular smooth muscle and they are expressed significantly higher in DA . There are 2 membrane receptors for ET-1, ET infants , 126.rs2234693, prostaglandin I2 synthase gene rs493694)( and another polymorphism in the interferon gamma gene rs2430561 is associated with a decreased risk of PDA and bronchopulmonary dysplasia (rs1056567) gene, angiotensin II type 1 receptor AGTR1 gene and TFAP2β (rs987237) gene, are associated with increased risk of PDA (Agtr2) is upregulated in late preterm infants also , and fibronectin (Fn1) genes which may draw attention in the future  and the pressure gradient between both ends , and inversely proportional to the blood viscosity and length of the tube . Moreover, increased venous return from the lungs to the left ventricle will cause an increase in the left ventricular end-diastolic pressure, which will increase atrial natriuretic peptide (ANP), with evolution of pulmonary venous hypertension and pulmonary hemorrhage. On the other hand, left to right shunting at the ductus will decrease the systemic perfusion  leading to systemic hypoxia and organ dysfunction. Shorter diastolic time and increased myocardial oxygen demand may result in endocardial ischemia .There is scarce information on the effects of dilation mediated by the mechanical force of the flow, or through shear forces and shear stress. Although there is no clear evidence, it may be speculated that there are some mechanosensors in the ductal wall, which can sense the biomechanical changes in the lumen and regulate ductal closure. The effect of laminar flow vs. turbulent flow across the ductus is also understudied. In case of laminar flow, endothelial cells are less likely to proliferate and nitric oxide synthase and prostacyclin are upregulated , 147. HoA sharp decrease in pulmonary artery pressure, as it happens after surfactant replacement therapy may increase the left-to-right shunt through the ductus, leading to pulmonary overflow and hemorrhage. In preterm infants, increased pulmonary fluid and protein load is eliminated by an increased lung lymph flow, named as “edema safety factor.” Therefore, ductal closure within the first 24 h of birth do not have any significant effect on the course of respiratory distress syndrome. However, if ductus remains open for several days, this compensation cannot be maintained and alteration in the pulmonary mechanics and pulmonary edema ensue. The impact of patent ductus arteriosus on patient outcomes seems to be a function of magnitude of shunt, rather than its mere presence. Closure of the ductus improves lung mechanics in these infants . Early d2. Phospholipase A2 stimulates the release of arachnidonic acid precursors needed for prostaglandin synthesis; thus in response to steroids, prostaglandin production is inhibited  or the peroxidase pathway (acetaminophen). The infant's gestational age, as well as the infant's race, ethnicity, growth, and exposure to antenatal corticosteroids modify the effects of prostaglandins and determine the rate of drug-induced closure , 169.2 receptors are down-regulated after birth. However, in the preterm infant, the production of receptors increases, rendering the ductus susceptible to vasodilation and resistant to indomethacin or ibuprofen. COX1 and COX2 expression increases with advancing maturity in the fetus  inhibits POX enzyme which catalyzes the conversion of PG2 to PGH2, without any peripheral vasoconstriction .Some authors have suggested to use a combination therapy of ibuprofen and paracetamol as a first line therapy for ductal closure. However, preliminary reports suggest that the effectiveness of combination therapy does not differ compared to monotherapy of either drug . In anotOther non-steroidal anti-inflammatory drugs such as aspirin, metamizol, nimesulid, and diclophenac have constrictor effects on the ductus –203.Glucocorticoids have also vasoconstrictive effects on the ductus, as well as synergistic effects with non-steroidal anti-infective drugs (NSAIDs), as a result of decreasing the sensitivity of the ductal SMCs to prostaglandins  Figure . In the There are many substances and foods with anti-inflammatory and anti-oxidant properties, such as those rich in polyphenols or flavonoids. For example, 30–40% of solid extract of green tea composes of polyphenols, mainly catechins. They exert their anti-inflammatory effects through the inhibition of endogenous prostaglandins. Similar effects have been reported with black tea, resveratrol, mate tea, orange juice and dark chocolate . ExcessiATP channel inhibitor, used for treating diabetes. It is shown that DA constricts in response to glibenclamide, and constriction is inhibited by diazoxide, a KATP channel activator (ATP channel genes, have PDA, often resistant to medical treatment, requiring surgical closure most of the time (ATP channels may modulate DA tone.Genetic therapies may include endothelin-signaling genes or blockage of potassium channels. Channel isoforms have distinct pharmacological and physiological characteristics, making them ideal targets for ductal-selective therapies . Glibencctivator . Childrethe time . Agonist4 inhibition (Novel treatment strategies for PDA include combined use of ibuprofen and paracetamol , EP4 inhhibition , NOS inhhibition , enhancihibition , glutamahibition  especial1 is the main drug used for vasodilatatory effects of the ductus. PGE1 binds to the EP4receptor which increases intracellular cAMP. Milrinon is a phosphodiesterase 3 inhibitor, which can also increase cAMP levels and dilate the ductus (PGEe ductus .Non-selective endothelin receptor antagonist TAK-044 and inhibition of Notch pathway may have vasodilating effects on the ductus , 91. Nat2 breakdown (Antenatal corticosteroids are used for the maturation of preterm lung, but their effects on the development of patent ductus arteriosus, intracranial hemorrhage, and necrotizing enterocolitis are also well-studied . Howeverreakdown .4 which is a calcium channel blocker may result in delayed ductal closure in infants (4 exposure is not associated with an increased risk of hemodynamically significant PDA; in fact, it may be associated with a decreased likelihood of hemodynamically significant PDA (ATP channel activator may induce ductal opening (2 production, thus dilating the ductus in animals but its effect is limited in humans (Antenatal MgSO infants . Howevercant PDA . Diazoxi opening . Furosemn humans .Ductus arteriosus is an intriguing and complex vessel with many distinct features. Understanding the precise mechanisms of regulation of the ductus is essential in order to improve individual pharmacological treatments. The debate about PDA should not be “treat all” or “treat none,” but rather, “who to treat.” This approach necessitates individualized treatment. Treatments targeting endogenous ductus regulation pathways other than prostaglandins, such as cell cycle regulators, transcription factors, and epigenomic regulators should be developed. Since vascular constriction and ductal wall remodeling are required for complete closure, treatments that favor vasoconstriction and remodeling would also be ideal for infants with persistent PDA. Novel therapies for ductal closure in preterm infants should also focus on a clear understanding of genes which are operative on ductal patency, as new genome-wide studies uncover genes associated with PDA. Evolving pediatric clinical pharmacology heralds next generation solutions for unresolved issues of today, such as drugs with selective DA effects, without any side effects.FO is responsible for the design, research, writing, and editing of the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.