Cardiovascular disease (CVD) is the leading cause of death worldwide, causing ~31% of all deaths .Hypertension, the principal cause of CVD mortality, is common, with an estimated 1.4 billion people living with hypertension globally. Rates of uncontrolled hypertension, defined by blood pressure systolic ≥140 mmHg or diastolic ≥90 mmHg, are high across low- and middle-income countries (LMICs), with LMICs suffering from disproportionate rates of premature mortality . HyperteHypertension programs in LMICs are often underfunded. While hypertension leads to significantly more deaths in LMICs than other conditions, ranking of funding priorities does not match the disease burden . Since 2Sodium intake may be the single most important modifiable factor for preventing elevated blood pressure in LMICs . EvidencThese interventions can be not only cost-effective, but also low-cost from the government perspective. A study from Argentina demonstrated that setting salt limits on bread and conducting mass media campaigns directed at lowering salt intake were both cost-effective at $50 USD/DALY averted and $179 USD/DALY averted, respectively, and affordable, with annual costs to the government for implementing each intervention in all of Buenos Aires of just $28,690 and $207,974 USD, respectively .In addition to ramping up prevention, there are significant efficiencies to be gained in care and treatment of hypertension. Diagnosis and treatment of hypertension have been shown to be cost-effective in LMICs . To acceThere is significant room for price reductions for both antihypertensive medicines and blood pressure measurement devices. In many LMICs, the public sector price for generic versions of common antihypertensives is higher, often two to fourteen times more expensive than international reference prices and more expensive than public procurement prices obtained in upper-middle-income countries . ObtainiNormative guidance supporting the use of blood pressure monitoring devices that meet WHO-recommended standards and the use of drug- and dose-specific hypertension treatment protocols enables countries to adopt and harmonize guidelines, increasing volumes for requisite medicines and devices. In HIV, guideline harmonization has led to a 70–80% price reductions for first-line medicines . In 2018Beyond focusing on improving the affordability of a limited number of preferred products, a number of market-based approaches may help to reduce medication and blood pressure device prices. Given the high prevalence of hypertension in LMICs, the scale of need is large, affording significant purchasing leverage. Pooling volumes through centralized procurement within one country or regionally between countries, as for example the PAHO strategic fund does for the PAHO region, can lead to price decreases of up to 90% across a range of medications, including cardiovascular treatments .Increasing price transparency through collecting and disseminating market information may also facilitate price reductions for essential medical products. Price transparency allows buyers to compare among various suppliers in a market, facilitating competition and possibly accelerating price decreases . HoweverFinally, using generic FDC of hypertension medications as opposed to combinations of single agent pills can reduce medication costs due to manufacturing or other efficiencies. A study of antihypertensives sold in the private sector in India demonstrated that the price of FDCs could be lower than their component single agent pills . SimilarHypertension care in many LMICs is centralized, with patients being seen at higher-level facilities and requiring frequent follow-up appointments . This moThe use of decentralized and differentiated service delivery (DSD) models can help save costs both from the health system and patient perspective. Decentralization refers to provision of care or specific interventions at lower-level primary health care facilities. Decentralization alone significantly reduces out-of-pocket costs for chronic diseases in LMICs . DSD incEfficiencies may be realized by ensuring quality care for those on treatment. Improvements in health care quality are an important condition for accomplishing the objectives of increased access to care . HyperteIn health programs with limited resources, approaches for short-term cost-savings must be weighed against potential negative impact on health outcomes and loss of long-term efficiencies. For example, as countries face a growing demand for NCD services, LMICs may employ user fees that patients to pay for services out-of-pocket to meet costs. These approaches need to be carefully calibrated to balance the need for cost containment with the need for optimal health care. In LMICs, where out-of-pocket spending constitutes a major share of health expenditure, user fees are associated with worse health outcomes and may lead to catastrophic costs for individuals . Other aScaling up effective hypertension programs is critical for controlling the growing burden of CVD, but implementing and sustaining these programs at a national scale will require additional investments by countries. Despite the clear evidence that hypertension prevention and treatment is cost-effective and saves lives, current funding constraints mean that presently programs do not provide adequate coverage to meet population health needs. However, programs can be made more efficient and less expensive per individual attaining control—potentially significantly less expensive—and countries and other health care payers can stretch their money by exploiting the efficiencies reviewed in this paper and ensure maximum benefit for the populations they care for. In many countries, robust hypertension control programs do not yet exist, and program start-up may require up-front financial investment, aggressive policy change, and adoption of new models of care. The medium- and long-term payoff from this up-front investment will lead to more affordable programs while expanding coverage, improving control, and reducing cardiovascular morbidity and mortality.The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.


Leishmania parasites undergoe post-translational modification as key regulators in several cellular processes, which represents an entire new field for drug target elucidation, once this is poorly explored. This perspective review describes the advances on antileishmanial metallodrugs and the elucidation of drug targets based on post-translational modifications, highlighting the limitations on the drug discovery/development process and suggesting standardisations focused on products addressed to who need it most.Despite the increasing number of manuscripts describing potential alternative antileishmanial compounds, little is advancing on translating these knowledges to new products to treat leishmaniasis. This is in part due to the lack of standardisations during pre-clinical drug discovery stage and also depends on the alignment of goals among universities/research centers, government and pharmaceutical industry. Inspired or not by drug repurposing, metal-based antileishmanial drugs represent a class that deserves more attention on its use for leishmaniasis chemotherapy. Together with new chemical entities, progresses have been made on the knowledge of parasite-specific drug targets specially after using CRISPR/Cas system for functional studies. In this regard,  Historical aspects and recent advances on antileishmanial metallodrugs,V) is the active principle of the main antileishmanial drugs currently in clinical use to treat all forms of leishmaniases since 1940s; reviewed by Haldar et al.,,The growing interest on developing bioactive compounds containing metals in their structure is due to the successful use of cisplatin in cancer chemotherapy,Leishmania spp. like L. major, L. amazonensis and L. infantum, including Sb-resistant strains.,in vivo efficacy assay; reviewed by Ong et al.L. amazonensis.Although classified as a heavy metal, bismuth (Bi) shares several physicochemical properties with Sb and arsenic (As), both latter considered metalloids. Being the most nontoxic heavy element to humans, together with no evidence of resistant parasites, Bi became an attractive alternative.,,,,in vitro activity against different Leishmania spp. at low micromolar range; reviewed by Rosa et al.,in vivo efficacy evaluation using experimental murine model, showing promising results with parasite burden reduction varying from 68 to 100% upon oral or IP  administration of 12.5 to 20 mg/kg/day of gold-based compound.,in vivo efficacy studies are presented in Inspired by the repurposing potential of the gold-based antiarthritic drug auranofin , severalL. amazonensis) comparable to amphotericin B in vitro.L. mexicana and DNA affinity studies suggested they can tightly bind DNA.Classified in the periodic table below gold (Au), silver (Ag) also displays good antimicrobial properties. When complexed with 1,10-phenanthroline-based ligands, Ag(I) complexes presented antileishmanial activity  murine model with low toxicity.L. major in vitro and prevented cutaneous leishmaniasis lesion caused by L. major in BALB/c mice at 5 mg/kg/day.L. donovani were achieved upon exposure to a quercetin conjugated gold nanoparticle.L. infantum, being more effective than Sb, leading to antiproliferative effect.2AgNps) combined with visible light exposure are promising alternatives to treat both VL and CL (Cutaneous Leishmaniasis).L. major being able to silent cysteine protease b (cpb) and gp63 coding genes.Like As and Sb, selenium (Se) is considered a metalloid and its antileishmanial activity has been explored L. major promastigotes, Ni(II) presented moderate activity,N,N,N’-trisubstituted acylthiourea presented high activity with an IC50 value of 0.45 µM against L. major.Like Au compounds, Ni(II) and Co(III) are transition metals that depending on its coordination and geometry diversity could also target TR leading to redox imbalance. When tested against in vivo efficacy in CL murine model due to L. amazonensis in BALB/c mice, upon intralesional administration of 75 µg/kg twice a week during 4 weeks.VOSalphen, a vanadium (V) complex with stilbene derivative, presented an impressive Leishmania spp., dose, therapeutical scheme, toxicological and drug combination approaches are limiting factors that impact study comparison and the advance in the field. Indeed, despite the increasing number of reports highlighting the importance of antileishmanial metallodrugs in vitro, very little goes further in the drug development pipeline.The aforementioned studies highlight the advances on the searching for metallodrugs against leishmaniasis. However, the lack of protocol standardisation on the use of animal models, administration route, experimental leishmaniasis-causing Challenges on drug discovery/development pipeline for antileishmanial metallodrugsInitiative (DNDi), a not-for-profit organisation is trying to reduce this gap in order to provide treatments to who need it most. But to achieve this aim, collaborative work and standardisation of protocols are required. DNDi have proposed the term target candidate profile (TCP) in order to standardise the minimum requirements for lead optimisation and drug development to treat leishmaniasis . In this context, we suggest additional standardisations in the pre-clinical scenario of antileishmanial drug discovery. The following points should be carefully addressed when developing antileishmanial metallodrugs. A compilation of these steps can be found in Target identification/validation would be the ideal beginning of the drug discovery workflow. However, several academic-based studies start from empirical approaches guided by a trial and error basis. Drug discovery and development are essentially by origin a multidisciplinary area were chemists, pharmacists, physicists, biologist, computer scientists, and so on, must work together even from the beginning delineating the rationale that will guide the whole process. Academic-focused projects within a context of limited resources where research groups are evaluated by the number of published papers and note necessary quality, will hardly pursue through the next steps. The growing number of manuscripts showing new chemical entities with potential antileishmanial property does not follow the emergence of new products to treat leishmaniasis, and the reason for that is a huge gap between academic research and the industry interest, passing through the economical politics from the State to save people from suffering of neglected tropical diseases, commonly associated to poverty. In this regard, the Drugs for Neglected Diseases Synthesis rationale - A great amount of data highlighting antileishmanial compounds started by collaborations where the chemist designed a series of chemical products based on different reasons  but rationale must be based on pharmacophore studies. Target-based or ligand-based approaches can be considered previous to a new synthesis. It can also reduce time and costs of further molecule optimisation during drug development steps. Rationale-guided synthesis would save time, money and increases success rate.The yield of an antileishmanial active compound and the complexity to obtain it must be included in this filtering equation. Sometimes this trial-and-error approach leads to an interesting compound, but there is no chance of enough quantity to pursue in vivo or more mechanistic assays. The group can meet the criteria for publishing a paper, but the contributions will be limited and the cycle tends to restart, generating “substrates” that cannot be translated into “products”.Computer-aid predictions - This is a very powerful tool, following the advances of artificial intelligence and machine learning predictors. Although it can drive very good predictions for organic compounds, it could not be the same when considering organometallic molecules, since molecular docking and molecular dynamic predictions for metal atoms can be an additional challenge.High throughput drug screening (HTS) - Several universities and research centers are able to collaborate with the pharmaceutical industry and perform HTS, mining drug candidates by fishing them based on phenotypic screenings. The limitation here is the filters required such as: solubility, one testing concentration, one target driven, stringent selectivity and potency. In this way, good candidates are filtered out. Sometimes, these rules are even incorporated in low or medium throughput screenings. There are several examples where selectivity index (SI) or IC50 did not discouraged an in vivo pilot PK (Pharmacokinetics) or efficacy studies.Culturing conditions - To perform phenotypic screening against Leishmania parasites and to test cytotoxicity in vitro on mammalian cells, it is essential to pay attention on culture medium composition, since metallic compounds could interact with thiols or albumin, interfering with drug availability. Metallic compounds could also react with the milieu constituents, transforming into new species.Parasite evolutionary stage - Despite the advantages of using promastigotes or axenic amastigotes for phenotypic screening, it is fundamental to include intracellular amastigotes, since metal-based compounds could act as prodrug where the presence of the host cell is essential for the mechanism of action. Drug-resistant parasites should also be included.Drug resistance prediction - It is strongly encouraged to evaluate the potential for selecting drug-resistant parasites. Stepwise drug resistance selection can provide clues on predicting parasite’s adaptations towards drug tolerance or developing specific drug resistance mechanisms.Drug combination - Is also encouraged and justified by reducing the chances of selecting resistant parasites and to improve therapeutical schemes, reducing dose and treatment time.in vivo, guided or not by previous in vitro isobologram tests.In vivo pharmacokinetic, toxicological and efficacy studies - Pilot assays could save time and energy before continuing drug discovery pipelines. Minimal in vivo requirements should be achieved before systematically investigate safety and efficacy. Efficacy studies must consider the disease establishment prior to the treatment.Animal model and administration route - The preferable administration route is oral and the in vivo VL model using golden hamsters is suggested as the first choice, since it provides more sensitive approach with systemic drug action.Antileishmanial metallodrugs: product market fiti when proposing the target product profile for antileishmanial drugs (https://dndi.org/diseases/cutaneous-leishmaniasis/target-product-profile/). Although the drug discovery process starts mostly at universities and research centers  eraLeishmania. However, after the advent of an efficient CRISPR/Cas system in Leishmania,,Leishmania species, such as L. braziliensis, the number of mutated genes raised from about 209 genes (before the CRISPR/Cas9 establishment) to about 400 genes nowadays.Leishmania, taking into account all parasite species  regulation, was released and will help us to advance in the development of antileishmanial drugs focused in this mechanism.The evidence used to validate drug targets can arise from genetic manipulations of a pathogen, such as by trying to obtain knockout/knockdown mutants of specific genes, which will give a clue about the gene essentiality/centrality and consequently would suggest the one as potential candidate for further studies as drug target. The process of discovering new targets with antileishmanial potential has undergone several changes in the last few decades with the establishment of new genetic engineering tools. During many years, the only tool available for gene replacement consisted of transfection of long linear constructs carrying a 0.5-1 kb long homology region, which significantly limited the success in obtaining gene deletion mutants in  species . Conside species . These nDrug target validation and the prospection of new antileishmanial candidates based on post-translational regulatory machinery,,,,,Leishmania parasite shifts between its invertebrate and vertebrate hosts. The regulation of PTMs levels is made possible by the action of a group of enzymes with activities against specific amino acid residues in particular proteins. The advent of highly sensitive proteomic approaches revealed the presence of a myriad of PTMs present in thousands of proteins involved in key cellular processes in trypanosomatids.,,,Leishmania.Environmental adaptation is regulated at different levels, from gene expression to enzymatic strategies. Reversible PTMs represent a relatively fast method to control protein activity, enabling more responsive cellular actions. Multiple PTMs, including phosphorylation, ubiquitination, methylation, crotonylation, acetylationProtein phosphorylation - Phosphorylation is arguably the most pervasive and best-studied PTM, and intimately associated in almost every cellular process. It plays key roles in modulating the activities of numerous enzymes by regulating protein-protein interactions or by changing protein conformation. This is achieved through the covalent attachment of phosphate groups to serine, threonine, and tyrosine residues by protein kinases, and their removal by protein phosphatases.,Leishmania parasite stages, confirming the importance of this modification during parasite adaptation throughout their life cycle,,,Leishmania kinome has 195 eukaryotic protein kinases (ePKs) that are classified according to catalytic domain conservation in six groups . Additionally, atypical kinases, which lack a conserved ePK structure, but are catalytically active, are present in Leishmania.,,L. mexicana kinome at key developmental stages of the parasite, revealing 44 protein kinases (PKs) essential for procyclic stage, 15 PKs important for procyclic to metacyclic differentiation in the vector and 29 PKs that are crucial for the parasite development in the mammalian host.Leishmania.,Protein acetylation - Protein acetylation occurs by the addition of an acetyl group in the Nε-amino group of the lysine residues, eliminating the positive charge of this amino acid and by that may alter the function of proteins by influencing their catalytic activity, their ability to interact with other proteins or their subcellular localisation.N-terminal regions of proteins affecting protein stability and subcellular localisation.N-terminal domains where it regulates chromatin structure and gene transcription, but with the development of more sensitive proteomic equipment, thousands of non-histone proteins have been identified acetylated in prokaryotes and eukaryotes, affecting a wide variety of cellular pathways.,,,,,Leishmania, but compared to higher eukaryotes, as humans, it is reduced and generally diverged, which make them excellent targets for chemical inhibitors.,Leishmania acetylation regulatory machinery is formed by seven, six and seven, KDACs, KATs and BRDs coding genes, respectively.,+-dependent or sirtuins with three genes; while two KATs families, MYST (four genes) and GNAT (two genes) are found.Leishmania, except the members of KDACs from the DAC family and the members of GNAT family of KATs. Although the scenario to explore KATs, KDACs and BDRs as drug targets in Leishmania is favorable, with several genes found to be essential for parasite survival, efforts have been focused on the sirtuin proteins (Sir2rp1-3), which had been proved to be crucial for both in vitro and in vivo growth of Leishmania.,,,,,,L. mexicana and found that five of seven are essential for the parasite and that BRD5 is important for chromatin structure regulation.Leishmania BDFs by drug repositioning. Moreover, the availability of different BDF protein structure will help to accelerate this process.Leishmania.We still have a great opportunity to better explore the regulatory machinery of protein acetylation for the development of new antileishmanial molecules by characterising the other members and also by testing the myriad of available KATs, KDACs and BDRs inhibitors using high content drug screenings.Considerable progress has been made in the last decades at unveiling KDACs and bromodomain proteins (BDPs) as promising therapeutic targets in Protein crotonylation - Lysine is by far the most modified amino acid with many different acylation marks, been reported to be present in the Nε-amino group of this residue, including succinylation, malonylation and crotonylation.,,,,,,Leishmania genome,L. mexicana procyclic form (unpublished data). The role of YEATS in pathogenic organisms is still poorly explored, but recent studies from Candida albicans YEATS observed that mutant knockout cell lines for the two genes encoding YEATS had an impairment growth and a significant reduction in virulence in vivo infection assays using murine models,Leishmania.,,Other modifications - Recent studies have demonstrated the importance of arginine methylation and ubiquitination for Leishmania biology. Arginine methylation is catalysed by PRMTs (Protein Arginine Methyltransferases) by the transfer of a methyl group from S-adenosyl methionine to the target arginine residue, which increases their hydrophobicity.Leishmania has seven PRMTs members (PRMT1-7) and it has been reported that PRMT7 could impact parasite infectivity in the mouse model animal and the RNA-binding capacity of target-proteins.,,Leishmania stage differentiation using different biochemical and molecular approaches, validating some of the members of these families as potential drug targets in the parasite.,Leishmania.Concluding remarksLeishmania adaptation between its two hosts. Progress in the field has unraveled many of these modifications and how they modulate crucial biological processes in trypanosomatids, but we still miss a lot of information regarding this in Leishmania. Thus, identifying the set of proteins that are modified and the PTMs present in Leishmania, as well as, the proper characterisation of the regulatory machinery of these PTMs will increase substantially the number of potential drug targets to be explored. Redox homeostasis can be considered a target for drug discovery and elucidation. In this case PTM on cysteine residues can produce distinct alterations and alter cell signaling mediated by redox-sensitive proteins and drug pharmacology.Current main challenges on drug discovery and development converges to standardise pre-clinical protocols worldwide and meet the requirements for a product market fit aligned with the pharmaceutical industry and specific features of the suffering areas such as drug resistance, cold chain (temperature stability) and price. Metallodrugs deserve more attention as antileishmanial entities and collaborative drug discovery is strongly encouraged. Advances have been made on elucidating PTMs as crucial mechanisms to maintain homeostasis being key to regulate the cellular mechanisms involved in the


Dictyostelium. RapA is a key regulator of cell adhesion and cytoskeletal rearrangement. Recently, RapC has been reported to be involved in cytokinesis, cell migration, and multicellular development. Here, we compare the functions of RapA and RapC using cells expressing or lacking Rap proteins, and confirm that RapA and RapC have opposite functions in cell spreading, adhesion, and migration. On the other hand, RapC has a unique function in cytokinesis and multicellular development. Activated RapA appears to stimulate spreading and adhesion of the cells to the substrate, possibly resulting in a decrease in the migration speed of the cells during chemotaxis without affecting the directionality, whereas RapC suppresses cell spreading and adhesion, thereby increasing the migration speed. Cells lacking RapC were defective in cytokinesis and multicellular development and showed multinucleation and formation of multiple tips from a mound during development. At the C-terminus, RapC has an additional stretch of amino acids, which is not found in RapA. The mechanism through which RapA and RapC perform their opposite functions in diverse cellular processes should be characterized further to understand the Rap signaling pathways in detail.There are three Rap proteins in ABBREVIATIONS: GAP; GTPase-activating proteins; GEF; guanine nucleotide exchanging factor; WT; wild type; CA; constitutively active; DN; dominantly negative Dictyostelium discoideum, a free-living social amoeba, has been used as a model organism to study basic cellular processes including cell migration, phagocytosis, and cytokinesis, as well as the Ras signaling pathways , three Rap , and one Rheb protein . The results demonstrated that RapA and RapC have opposite effects on cell spreading, adhesion, and migration.The Rap (RAs-Proximate) proteins were originally identified as Ras antagonists and consist of several isoforms (Kortholt and van Haastert Dictyostelium wild-type KAx-3 cells were grown in HL5 medium or in association with Klebsiella aerogenes at 22°C. Knock-out strains and transformants were maintained in 10 μg/mL blasticidin and 10 μg/mL G418. The full coding sequences of the rapA and rapC cDNA were generated by RT–PCR and were cloned into the EcoRI – XhoI site of the expression vector pEXP-4(+) containing a GFP fragment at the N-terminus . RapCCA contains a valine at amino acid 13 instead of a glycine in wild-type RapC and RapCDN has an aspartate at amino acid 18 instead of a serine in wild-type RapC. The rapC knockout construct and rapC null cells were described previously  was recorded at intervals of 1 min for 30 min using an inverted microscope  with a camera . The data were analyzed using NIS-Elements software (Nikon) and ImageJ software . The migration speed was calculated by dividing the total distance traveled of a cell with time. Directionality was calculated as the shortest linear distance between the start and end points of the migration path divided by the total distance traveled by a cell for 10 min.Chemotaxis toward cAMP was performed as described previously  for 20 min . P values less than 0.05 was considered as statistically significant.All data were collected from at least three independent experiments and expressed as the means ± standard deviation (SD). Statistical analysis was performed using Student’s Dictyostelium Ras subfamily comprises 15 proteins including three Rap proteins, RapA, RapB, and RapC  form of RapA and RapC. Cells expressing RapAWT, RapACA, RapCDN, and rapC null cells showed increased cell adhesion, whereas RapADN, RapCWT, and RapCCA cells displayed decreased cell adhesion, compared to wild-type cells. These results suggest that RapA and RapC have opposite roles in cell adhesion; RapA increases the strength of cell adhesion, whereas RapC decreases it.Next, to examine the roles of RapA and RapC in cell adhesion, we measured the percentage of attached cells per total number of cells in the plate after agitating the plates for 1 h followed by aspiration of the detached cells (rapC null cells was rescued by expression of RapA and RapC (rapC null cells were spread and adhesive to the substrate compared to wild-type cells. This phenotype of rapC null cells was complemented by expression of RapC but not RapA. The spread shape of rapC null cells was restored to the appearance of wild-type cells following expression of GFP-RapC, and rapC null cells expressing RapC showed approximately 1.5-fold weaker adhesion compared to the wild-type cells. In contrast, rapC null cells expressing RapA exhibited a highly spread morphology and slightly increased cell adhesion compared to rapC null cells (To further confirm the opposite functions of RapA and RapC in cell spreading and adhesion, we sought to determine whether the phenotype of and RapC . rapC nuWT, RapACA, RapCDN, and rapC null cells, moved slower compared to wild-type cells, while the cells showing weak adhesion, including RapADN, RapCWT, and RapCCA cells, exhibited increased speed of migration (Cell migration requires coordinated and dynamic regulation of cell adhesion. Our previous results indicate that cell adhesion is regulated by RapA and RapC in opposite ways. To determine the roles of RapA and RapC in cell migration, we examined the chemotactic ability of cells expressing RapA or RapC toward cAMP chemoattractants using a Dunn chemotaxis chamber . ConsistrapC null cells showed strong adhesion and decreased migration speed compared to wild-type cells. To confirm the opposite functions of RapA and RapC in cell migration, we examined whether the phenotype of rapC null cells during chemotaxis was complemented by expression of RapA and RapC (rapC null cells, the migration speed increased more than that of wild-type cells, whereas rapC null cells expressing RapA showed markedly decreased migration speed (and RapC . When RarapC null cells are multinucleated (Park et al. rapC null cells and cells expressing RapCDN contained approximately one nucleus per cell. In contrast, rapC null cells contained 2–3 nuclei per cell, and cells expressing RapCDN contained approximately two nuclei per cell (rapC null cells were rescued by expressing RapC but not RapA (rapC null cells expressing RapC had approximately one nucleus per cell similar to the wild-type cells. These results suggest that RapC, but not RapA, plays a specific role in cytokinesis.Loss of RapC causes defects in cytokinesis and not RapA . rapC nuDictyostelium cells are starved, the cells aggregate and differentiate into several types of cells. Then they finally form fruiting bodies consisting of a dead stalk with a mass of spores on the top (Chisholm and Firtel Dictyostelium development (rapC null cells (rapC null cells was rescued by the expression of RapC, but not RapA (Dictyostelium.When elopment . Wild-tyll cells . Cells lnot RapA . These rDictyostelium. Activated RapA appears to stimulate spreading and adhesion of the cells to the substrate, decreasing the migration speed during chemotaxis, whereas RapC suppresses cell spreading and adhesion, thus increasing the migration speed. On the other hand, RapC appears to have a unique function in cytokinesis and multicellular development. When the levels of wild-type or CA form of RapA were increased, the cells were flat and adhesive and moved slowly toward the chemoattractant. In contrast, cells containing elevated levels of wild-type or CA form of RapC were shrunk and displayed weak adhesion to the plate and high migration speed compared to the wild-type cells. In agreement with these findings, the phenotypes of rapC null cells that were similar to cells with high levels of RapA were restored by RapC but not RapA. rapC null cells expressing RapA showed increased spread morphology and stronger adhesion compared to rapC null cells or wild-type cells expressing RapA. We were unable to examine the phenotypes of cells lacking RapA since rapA null cells are unavailable, suggested that RapA is essential (Jeon et al. This study demonstrates that RapA and RapC have opposite functions in controlling cell morphology, adhesion, and migration in rapGAP1 or rapGAP9 null cells (Kortholt et al. RapA is a key regulator of integrin-mediated cell adhesion and cadherin-mediated cell–cell interaction and has been linked to several signaling pathways, including cell migration, osmotic stress responses, and phagocytosis; additionally, it functions as a regulator of cytoskeletal rearrangements during cell migration (Kortholt and van Haastert rapC null cells showed defects in multicellular development, forming multiple tips and structures from a single mound. These phenotypes were restored by RapC but not by RapA. In our study, cells expressing RapA or RapA-mutant proteins demonstrated no defect in cytokinesis and development, although another group reported that cells overexpressing CA form of RapA were defective in cytokinesis and multinucleated (Plak et al. rapC null cells resulting in the formation of multiple structures from a mound are likely to be generated by misregulation of cAMP waves. Tip formation from aggregates during multicellular development is controlled by cAMP waves, propagating from the top to the posterior of the multicelluar aggregates and inducing differentiation and sorting of the cells in the mounds into prespore and several cell types (Siegert and Weijer rapC null cells of multiple tip formation might be resulted from disturbed regulation of cell–cell adhesion in the multicellular organisms.In contrast to RapA, RapC has unique functions in cytokinesis and multicellular development. Cells lacking RapC or expressing a DN form of RapC displayed multinucleation. In addition, This study confirms that, despite their high sequence homology, RapA and RapC have opposite functions in cell spreading and adhesion. RapC appears to have a unique function in cytokinesis and multicellular development. At the C-terminus, RapC has an additional stretch of amino acids, which is not found in RapA. The mechanism through which RapA and RapC perform their opposite functions in diverse cellular processes needs to be characterized further to understand the Rap signaling pathways in detail.Click here for additional data file.


This popular polymer, widely researched since the 1970s, can be readily processed through various techniques such as 3D printing and electrospinning to create biomimetic and customized medical products. However, low mechanical strength, insufficient number of cellular recognition sites, poor bioactivity, and hydrophobicity are main shortcomings of PCL limiting its broader use for biomedical applications. To maintain and benefit from the high potential of PCL, yet addressing its physicochemical and biological challenges, blending with nature-derived (bio)polymers and incorporation of nanofillers have been extensively investigated. Here, we discuss novel additives that have been meant for enhancement of PCL nanofiber properties and thus for further extension of the PCL nanofiber application domain. The most recent researches (since 2017) have been covered and an updated overview about hybrid PCL nanofibers is presented with focus on those including nature-derived additives, e.g., polysaccharides and proteins, and synthetic additives, e.g., inorganic and carbon nanomaterials.As a low cost, biocompatible, and bioresorbable synthetic polymer, poly (ɛ-caprolactone) (PCL) is widely used for different biomedical applications including drug delivery, wound dressing, and tissue engineering. An extensive range of  PCL wasrganisms . Despiterganisms . On the rganisms . Afterwarganisms .PCL can be synthesized via ring-opening polymerization (ROP) of ɛ-caprolactone involving a diverse range of cationic, anionic, and co-ordination catalysts. Additionally, it can be prepared through the free radical ROP of 2-methylene-1-3-dioxepane . In orded,l-lactide acid (PDLA), it could be employed in drug delivery systems with a life span of over 1 year and in commercial suture materials (Maxon™) (pseudomonas lipase (PS) within a short time of 4 days . PCL is dissolved in a variety of solvents and is readily melted at low to moderate temperatures (59–64°C). Moreover, it can be properly blended with many polymers and biopolymers, thereby being proposed for diverse biomedical applications . Such fe(Maxon™) . The cap(Maxon™) . The hyd(Maxon™) . The bio(Maxon™) . PCL hasf 4 days . Such def 4 days . Among tf 4 days . As a ref 4 days . In thisf 4 days . Thus, sf 4 days .PCL can be processed in different physicochemical ways to produce biomedical systems at low cost compared to other aliphatic polyesters. Among all the production techniques, electrospinning is the most widely studied one for development of PCL nanofibrous materials for wound dressing and tissue engineering. The applicability of PCL nanofibers can be extended and their properties can be optimized by employment of benign solvents that assure eco-friendly processing  and/or iin vitro cell behaviour including migration, proliferation, and differentiation, and also cell shape  is an antibiotic that is placed within the World Health Organization (WHO)׳s list of Essential Medicines, which includes the vital medications necessary for any basic health system . As a pr2+ ions from ZnO when subjected to the aqueous medium  is a member of the TC antibiotics family that plays a bacteriostatic role via hampering the synthesis of bacterial protein. These antibiotics are lethal to an extensive range of both Gram-negative and Gram-positive bacteria . TC antis medium . In addis medium , among Zin vivo conditions thanks to its undesired biopharmaceutical characteristics such as poor solubility, fast metabolism, and insufficient chemical resistance. Accordingly, there is a need to creation of a carrier system that tackles the biopharmaceutical challenges and thereby maximizes the prophylactic and therapeutic capacity of RSV  is a natural substance with therapeutic effects against periodontal disease. It shows anti-oxidative and anti-inflammatory effects that alleviate the adverse consequences of the periodontal disease such as lower generation of NO , excessiy of RSV . In thisy of RSV . Additioy of RSV . One of y of RSV  includedin vivo and offers anticancer effects in vitro  infected wounds. According to the in vivo tests, in the presence of PCL/CS/CUR nanofibers, wounds are re-epithelized faster, thereby accelerating the wound closure. In fact, such a consequence enables the wound to be further healed and the damaged skin tissue to be regenerated  is derived from incomplete deacetylation of chitin. It contains many functional amine and hydroxyl groups and thus shows an exclusive polycationic nature and chelating and film-forming properties. As a proven fact, N-acetyl  process . Chitosa process . Causing process . Thanks  process . As a re process . Such blenerated . Particuin vivo interaction of the PCL nanofibers is crucial to avoid foreign body reactions such as infection, inflammation, embolization, and thrombosis. To tackle the mentioned bottlenecks and to achieve the desired biological features, PCL has been blended with hydrophilic biomolecules (proteins), e.g., collagen  are major shortcomings of PCL nanofiber mats that lead to lower cellular activities such as cell adhesion, proliferation, and migration, and thus hinder their extensive use in biomedicine . On the collagen , gelatincollagen , bovine collagen , and silcollagen  among otAs a general strategy to improve bioactivity of synthetic biomaterials including PCL, natural polymers with cell recognition sequences are hybridized to modulate the cell-material interactions . Many ECIn a recent study , we enhaOther than blending, surface treatment of PCL nanofibers and their decoration with biomolecules have been also investigated. For instance, the PCL nanofibers surface can be carboxylated through plasma treatment, allowing for adhesion of protein molecules based on a covalent bond between carboxyl and protein (platelet-rich plasma (PRP))’s amine groups . This siBombyx mori (B. mori) silk fibroin comprises two distinct chain polypeptides in terms of molecular weight  that are coupled via a disulfide bond ’s biocompatibility has justified its extensive applicability in biomedicine, e.g., pertaining to bone and skin tissue engineering . Bombyx ide bond . The SF ide bond . As provide bond , incorpoide bond .Gelatine (Gel), as a conventional natural polymer, is synthesized via incomplete hydrolysis of collagen. In comparison with collagen, Gel possesses a lower molecular weight rendering its electrospinning easier. On the other hand, its integrin anchorage zones are abundant, allowing for better cell adhesion, cell migration, and cell differentiation . By coupin vivo study also confirmed that after 21 days, all classes of Gel/PCL nanofibers but the blended type could stimulate re-epithelialization and develop a fully epithelialized tissue. Additionally, by immunohistochemical staining of βIII tubulin, it was proved that PCL-RGD and the Gel/PCL blend nanofibers induce formation of nerve fibers that re-innervate a notably larger length of the wound area compared to the gauze control.As a skin regenerating material, Gel/PCL nanofibers could stimulate endogenous wound healing and allow for controlled delivery of dermal progenitors (adult human skin-derived precursor cells (hSKPs)). As Gel/PCL blend nanofibers have also been studied for blood vessel tissue engineering, where inclusion of Gel leads to a more robust system with improved hydrophilicity, in contrast to the neat PCL nanofibers . DespiteFor particular tissue engineering applications, inorganic bioactive nanomaterials have been added to the PCL nanofibers. For instance, regarding bone tissue engineering, PCL nanofibers alone or as blended with natural polymers, e.g., chitosan, Col, etc. have been reinforced by addition of bioceramics such as hydroxyapatite (HA), tri-calcium phosphates (TCP), bioactive glass, silica , among o10 (PO4)6(OH)2]) and bioactive glass nanoparticles into polymeric materials or via surface deposition  of nHA on a polymeric substrate , thereby providing better conditions for chondrogenesis   .2) is another inorganic additive to PCL nanofibers that are meant to act as bone tissue engineering scaffolds, for example for nonunion injuries of bone  by Lary Hench in 1971 (2), 24.5 wt% sodium oxide (Na2O), 24.5 wt% calcium oxide (CaO), and 6 wt% phosphorous pentoxide (P2O5) and as the term “bioactive” implies, BG can form a robust interface with natural tissues as a consequence of a particular biological reaction taking place at the material surface (The other prominent inorganic additive to PCL nanofibers is bioactive glass (BG) that is meant to raise bioactivity of the composite made thereof. The era of inorganic bioactive materials started with the invention of 45S5 bioactive glass  and reduced graphene oxide (rGO) have been appealing for potential applications ranging from biosensors, membranes, and superconductive materials to drug delivery and tissue engineering . In biomPCL electrospun nanofibers are highly attractive for a plethora of biomedical applications spanning from wound dressing to various tissue engineering scaffolds. Such attractive capacity originates from biodegradability, easy processability, and the relatively low cost of this polymer. However, insufficient interaction with cells, hydrophobicity, and poor mechanical properties of PCL nanofibrous systems that might mismatch with the hosting tissue properties limit their further usability in biomedicine. The main solutions for such shortcomings are either the surface treatment of PCL nanofibers or their hybridization. Thanks to a large specific surface area that can be engineered to encompass many functional groups and to load hydrophilic, bioactive and/or therapeutic agents, PCL nanohybrid nanofibers can turn to a bioactive, hydrophilic nanostructured therapeutic/regenerative platform. On the other hand, inclusion of drugs and therapeutic agents in PCL nanofibers can lead to their controlled release while incorporation of nanofillers can guarantee improved physicochemical properties of the nanofibrous biomedical systems made thereof. Despite the significant progress taken place in the design, engineering, and application of PCL hybrid nanofibers, there are still several gaps that should be targeted in the future by researchers to address and thereby to further develop their potential applications.A simple glimpse in the literature reflects that applications of PCL hybrid nanofibers have been investigated for wound dressing and engineering of bone, nerve, cornea, vessels, cartilage, and meniscus tissues. Additionally, PCL hybrid nanofibers have been used in relation to cancer research and treatment of periodontal disease (periodontitis). Considering the versatility and high potential of PCL nanofibers, many more applications related to hard and soft tissue engineering can be aimed in the future. One potential area could be electroactive scaffolds that are typically used to restore the performance of destroyed cardiac tissue and in general muscle tissue engineering.With respect to soft tissue engineering, in addition to surface functionality, PCL nanofibrous systems should support cell anchorage signals mechanically thus enabling the cells to spread largely on the surface. The modulation of surface mechanical properties of PCL nanofibers within the range of soft tissues’ has been overlooked and insufficient attention has been paid to mechanotransduction to govern the cellular adhesion on these electrospun nanofibers. Typically, PCL hybrid nanofibers are highly stiff, while soft tissue engineering requires the nanofiber scaffolds much softer than those currently available. Otherwise, biomechanical disharmony will lead to failure of the tissue regeneration strategy based on such nanofiber scaffolds, as the cell adhesion is directly associated with cell signalling and gene expression of the cells depends on the cell interaction with the substrate.Synthesis approaches for PCL hybrid nanofibers are limited to a few techniques, mainly blend electrospinning. Co-axial and emulsion electrospinning techniques have been rarely employed probably due to their complexity compared to blend electrospinning. On the other hand, surface functionalization and in general post treatment of PCL nanofibers have been insignificantly taken into account. This might originate from poor functionality of the PCL nanofiber surface that mandates the involvement of supplementary techniques such as plasma oxidization. One significant challenge regarding blend electrospinning, particularly in the case of inclusion of nanofillers, is their entrapment within the nanofibers, rather than their surface residence. This issue definitely restricts the interactivity of bioactive nanofillers with the external medium and thus their efficiency. On the other hand, according to the Coffee stain effect, some therapeutic agents that are highly soluble in the electrospinning solvent might be driven towards the surface, while being loosely bound. This will lead to their burst release in a short time window and adverse toxic effects. The incorporation of delicate, sensitive agents into PCL nanofibers via core-shell electrospinning might be also challenging, considering the high shearing forces and high voltages applied where shell and core fluids meet. Taken together, the synthesis strategy should be properly regulated considering all the involved risks and concerns. One further issue is related to the porosity and pore size of PCL hybrid nanofiber mats that should be maintained properly and not sacrificed against inclusion of additives. As some additives are readily ionized, they can potentially reduce the nanofiber diameter during electrospinning. As a result, pore size can decline notably, cell penetration into the mat could be inhibited and permeability to nutrients/waste and air/water exchange could be exacerbated. On the other hand, some additives physicochemically interact with PCL and thus raise the viscosity of the PCL solution that is being electrospun. Consequently, the nanofiber diameter increases and pore size expands, resulting in less topographical cues and mechanical support for the cells. Conclusively, the formulation of the hybrid fibers should be so designed that does not lead to reduction of structural properties.In terms of formulation, PCL nanofibers hybridized with nature derived compounds that are inherently biocompatible and offer antibacterial and anti-inflammatory effects seem to be better candidates for biomedical applications compared to those hybridized with synthetic additives. Despite such promising potentials of this class of PCL hybrid nanofibers for biomedicine, certified via many relevant studies, there is no commercially viable product made of them in the healthcare market, to the best of the authors’ knowledge. This gap stems from the likely challenges of scalable co-electrospinning of nature-derived therapeutic agents or natural polymers with PCL, that will necessitate involvement of potentially hazardous cross-linkers and other functional impurities to assure their uniform distribution across the nanofiber. On the other hand, PCL is typically electrospun using organic, hazardous solvents whose trace residual amount in the nanofiber could lead to adverse biological and immunological consequences. This fact is generating increasing research efforts on benign solvents for PCL electrospinning.in vitro and in vivo testing of these hybrid nanofibers in long term studies are still necessary to assure their efficiency over the course of the therapeutic process. Similar consideration in terms of lack of long-term in vivo studies can be made for the promising technology of ion doped bioactive glass loaded PCL fibers. As a plausible possibility, the in vivo trial study might be unsuccessful. Even so, the negative data and the reason behind failure are as important as the positive data and can help the researchers fine tune their approach and material design to raise the potential success of a given therapeutic strategy.To provide an antibacterial effect, PCL nanofibers need to incorporate additives such as antimicrobial peptides, Ag ions, drugs (antibiotics), or nature-derived antibacterial compounds. Recalling the fast-paced emergence of antibiotic resistant bacteria, research is directed towards creation of alternative systems for traditional drug delivery PCL nanofibers. One solution could be the development of AMP functionalized PCL nanofibers. This strategy also prevails over metal nanoparticle loaded PCL nanofibers that could excessively release ions beyond the WHO limits. However, Taking into account the complexity of tissue regeneration and wound healing processes involving various biological steps such as inflammation, angiogenesis, and tissue remodelling, many proposed systems lack efficiency and are not multipurposed. They target only one particular healing goal and are designed to be interactive in that specific respect, e.g., inflammation, or infection. To fulfil realistic medical needs, the nanofiber system should be able to interact with the body from different standpoints and address various therapeutical needs concurrently. This need could be met through development of multifunctional PCL nanofiber systems that can co-release different therapeutic agents, e.g. drugs, bio-derived compounds, etc. over the therapeutic window.As other nanofiber systems, scalability of PCL nanofibers with a hybrid formulation for purpose of translation into medical technologies is a crucial hurdle. The production process must be scalable and yet cost efficient to justify the utilization of such nanofiber systems in competition with the commercial counterparts currently available in the medical product market.


Acute kidney injury (AKI) is a poorly defined syndrome characterized by sudden excretory dysfunction. AKI has a high and increasing incidence, a high mortality rate, especially in critically ill patients, and serious medium- and long-term consequences, including progression to chronic kidney disease and elevated cardiovascular morbimortalityThe creatinine-centered view of AKI neglects the etiopathologic granularity required for personalized diagnosis. Because of the heterogeneity of AKI and the complexity of the underlying biological processes, this limitation is inherent to all diagnostic methods based on a single parameter or biomarker. Accordingly, new (mostly urinary) biomarkers have emerged in the last two decades that capture pathophysiological information missed by creatinine testing, with earlier sensibility, purportedly complementing diagnosis,,Recently, the furosemide stress test (FST) has gained increasing attention as a potential candidate for the evaluation of tubular functionality. The FST was rescued a few years ago and standardized for potential nephrologic diagnosisIn this issue of JBN, Pon et al.renal functional reserve. The FST widens the concept of functional reserve to the tubular compartment, while highlighting the need to revise the nomenclature and ontology behind the term.Overall, the FST provides a prospective diagnostic parameter that contains some defined pathophysiological knowledge and complementary creatinine-insensitive information, but it needs further substantial contextualization. Furthermore, as a stress test, the FST fits perfectly with the strengthening concept of an acquired predisposition to AKI caused by a reduced functional reserve


Accumulating evidence implicates gut-microbiota-derived metabolites as important regulators of host energy balance and fuel homeostasis, the underlying mechanisms are currently subject to intense research. In this review, the most important executors, short chain fatty acids, which both directly and indirectly fulfill the interactions between gut microbiota and host will be discussed. Distinct roles of individual short chain fatty acids and the different effects they exert on host metabolism have long been overlooked, which compromises the process of clarifying the sophisticated crosstalk between gut microbiota and its host. Moreover, recent findings suggest that exogenously administered short chain fatty acids affect host metabolism via different mechanisms depending on the routes they enter the host. Although these exogenous routes are often artificial, they may help to comprehend the roles of the short-chain-fatty-acid mechanisms and signaling sites, that would normally occur after intestinal absorption of short chain fatty acids. Cautions should be addressed of generalizing findings, since different results have appeared in different host species, which may imply a host species-specific response to short chain fatty acids. For example, human serum SCFAs concentrations were reported to be ∼100–150, ∼4–5, and ∼1–3 μM for acetate, propionate and butyrate, respectively . The EC5per se, as a substrate of IGN, induced the activity of glucose-6-phosphatase and the expression of genes related to IGN via modulation of the FFAR3  in overweight adults also suppressed energy intake, this effect was observed together with increased levels of gut hormones peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) secretion, which might indicated that propionate suppressed energy intake via stimulating gut hormone release that may tie in to brain pathways enhancing satiety and anorexia .As mentioned above, oral butyrate administration in mice decreased food intake by upregulating the protein expression of tyrosine hydroxylase and inhibiting the proportion of c-Fos positive (orexigenic) neurons in the hypothalamus that expressed neuropeptide Y (NPY), however, this effect was not noted with intravenous application of butyrate . AnorexiHuman studies pointed out that distal, but not proximal, colonic acetate administration in obese individuals increased fat oxidation and fasting plasma PYY, which may be related to higher transfer of acetate to the circulation . SCFAs pin vitro effects on lipolysis in human white adipocytes. When human multipotent adipose tissue-derived stem cells were incubated with mixtures of acetate, propionate, and butyrate or individual SCFAs, basal and the β-adrenergic receptor-mediated glycerol release were significantly suppressed in incubation with only acetate, while butyrate increased glycerol release and propionate did not affect their release. Additionally, the antilipolytic effect of acetate was found to be prevented when GPR41 and GPR43 in the adipocytes were inhibited, indicating an underlying mechanism mediated by these SCFA receptors involving GPR41 and GPR43 (Jocken et al. demonstrated that specific SCFAs exerted distinct nd GPR43 . Taken tOverall, the reviewed data of distinct effects of specific SCFAs on host energy balance and fuel homeostasis suggest the following. Orally administered SCFAs may reach the small intestine and target local SCFA receptors, rather than being consumed by colonocytes. Intraperitoneally administered SCFAs may directly reach peripheral tissues and organs in supraphysiological concentrations and thus exert distinct impacts on host metabolism and physiology that probably are more profound compared to bacteria-derived SCFAs see . In spitAn important aspect in consideration of these findings is host species. Energy balance and fuel homeostasis in response to SCFA may be dependent on species specific factors such as diet, anatomical and physiological characteristics of alimentary systems and SCFAs production capacity. Even comparison among rodent species may yield different outcomes. For instance, supplementation of propionate and butyrate to high-fat-fed mice, but not to high-fat-fed rats, reduced cumulative energy intake . Such inDK wrote the first draft of the manuscript. DK, LS, and GD wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.LS is employed by Danone Nutricia Research, Utrecht, Netherlands. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Salmonella, are tripartite systems with an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB binding are also involved in AcrD binding. Resistance-nodulation-division (RND) efflux pumps are important mediators of antibiotic resistance. RND pumps, including the principal multidrug efflux pump AcrAB-TolC in acrD and acrE genes were expressed in a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined.The acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates, showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of Escherichia coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates.Co-expression of These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition. All plasmids were sequenced to check for the presence of the desired point mutations and to ensure there were no unwanted secondary mutations.The The MICs of various antimicrobials were determined using the agar dilution method according to CLSI guidance.S. Typhimurium SL1344 AcrD, especially in the context of PAP-RND interactions, which may provide modulatory effects on the specificity of the pump. Previously, it has been reported that E. coli AcrD exports aminoglycosides.,Salmonella and most of the features of Salmonella AcrD are inferred, based on close sequence similarity to E. coli AcrD  strain did not result in a significant increase in MICs of the aminoglycosides kanamycin, gentamicin, spectinomycin or streptomycin  in S. Typhimurium, and leucine to glutamine at position 565 (L565Q), both of which are likely to cause steric hinderance and impact the electrostatics of the access binding pocket, respectively.,A possible explanation for the differences in the substrate profiles of AcrD between s Figure . Due to ,acrAB, acrD and acrEF genes in S. Typhimurium SL1344 to create a strain without active RND-dependent efflux, as indicated by significantly increased susceptibility to AcrB, AcrF and AcrD substrates ,acrD or acrE alone in the Δ3RND strain did not significantly increase MIC values of the AcrD substrates tested  between these two PAPs.Salmonella AcrA, we identified several residues mapping to the β-barrel and membrane proximal domains that were important for AcrB binding.acrA and pacrE  and co-transformed with pacrD into the Δ3RND strain. Based on structural analysis, we chose F292G and G363F as target mutations due to their radical change of respective sidechain properties.AcrE has been shown to complement AcrA and is capable of interacting with AcrB.Consistent with the data obtained in co-expression with AcrB,Here, we report that the PAP AcrE can form a functional complex with the RND transporter AcrD, further validating the interchangeability between the homologous PAPs AcrA and AcrE. Furthermore, this interchangeability is likely to be due to the highly conserved and specific RND-binding sites between these two PAPs. Our report highlights that the redundancy between these two PAPs must be taken into account when targeting them for efflux inhibition. Therefore, the residues we identified here could inform future design of effective efflux inhibitors targeting PAPs or tripartite complex assemblies.dkab237_Supplementary_DataClick here for additional data file.


The breast experiences substantial changes in morphology and function during pregnancy and lactation which affects its imaging properties and may reduce the visibility of a concurrent pathological process. The high incidence of benign gestational-related entities may further add complexity to the clinical and radiological evaluation of the breast during the period. Consequently, pregnancy-associated breast cancer (PABC) is often a delayed diagnosis and carries a poor prognosis. This state-of-the-art pictorial review illustrates how despite currently being underutilized, technical advances and new clinical evidence support the use of unenhanced breast MRI during pregnancy and both unenhanced and dynamic-contrast enhanced (DCE) during lactation, to serve as effective supplementary modalities in the diagnostic work-up of PABC. Diffusion MRI may serve as a standalone modality during pregnancy.DCE MRI of the breast remains of significant value during lactation.Unenhanced DTI may increase PABC lesion conspicuity as compared with DCE.Non-fat suppressed T2 images can improve the delineating of non-mass DCIS lesions.Increased utilization of MRI may facilitate an earlier PABC diagnosis.Pregnancy-associated breast cancer (PABC) is traditionally defined as breast cancer diagnosed during pregnancy, in the first year postpartum, or anytime during lactation , and typMagnetic resonance imaging (MRI) and in particular, it’s workhorse sequence, dynamic contrast-enhanced (DCE) MRI, continues to serve in the mainstay of breast cancer diagnostic workup –13, and Nevertheless, despite its evident diagnostic superiority, inherent limitations of breast MRI surface during pregnancy and lactation, and as a result, restrictions are imposed on its utility , 19–21. In recent years, the advent of promising preliminary investigations and emergence of advanced MRI protocols, such as the increased clinical employment of unenhanced diffusion-based MRI techniques , has driThroughout pregnancy, the breast undergoes a series of unique structural and functional alterations in preparation for its eventual biological role in lactation. Within this process, called lactogenesis, the mammary gland grows with developed glandular tissue at the expense of shrunken adipose and connective tissues . RegulatBoth clinical and radiological evaluation of the breast are influenced by the physiological changes during pregnancy and lactation. Clinically, breast examination can be challenging due to the enlarged size of the breasts, their tenderness, and especially their harder, more nodular consistency . ImagingThese physiological changes of the breast are also reflected on the various MRI pulse sequences. Since contrast enhanced breast MRI scans are not performed during pregnancy, reports on breast MRI of pregnant patients have been limited to examinations performed prior to elected abortion  or usingFurther characterization of the lactating breast has been afforded by additional MRI sequences. On MR spectroscopy, a total choline peak, an established breast cancer biomarker , was eviAdvanced diffusion MRI models were also used to investigate the unique features of the breast during pregnancy and lactation. Intra-voxel incoherent motion (IVIM), a bi-exponential diffusion model, is based on acquiring multiple diffusion weightings in the fast and slow regimes in order to separate the fast perfusion-based “pseudo-diffusion” component from the slow diffusion process . Using IBenign entities account for the vast majority of findings among patients presenting with a palpable breast mass during pregnancy and lactation . In a stA galactocele, a Greek term meaning “milky pouch,” is a milk collection retained within the fibroglandular tissue because of duct obstruction. This etiology usually regresses spontaneously on follow-up and is the most common benign breast mass among lactating patients . CharactLactating adenoma represents a benign stromal alteration with a tendency to regress upon breastfeeding cessation . LactatiFibroadenomas are composed of epithelium and stroma and account for the most common benign tumor detected in young females . InteresDuct ectasia of the breast is among the benign processes that may affect the nipple-areolar complex during pregnancy and lactation . The cliMastitis is a common infectious condition that may affect up to one-third of lactating women  and is aDuring pregnancy, breast DCE-MRI is contraindicated due to the increased risk of a broad set of rheumatological, inflammatory, or dermal conditions, as well as stillbirth or neonatal death, associated with gadolinium-based contrast agents used during the MRI . The lonMoreover, the first attempt to utilize unenhanced diffusion MRI as a standalone modality for pregnant patients at high risk or with newly diagnosed PABC was recently reported . This woAn interesting and unusual case we encountered was of a pregnant patient with newly diagnosed mucinous breast carcinoma who underwent MRI prior to elected abortion. Pure mucinous carcinoma typically appears on MRI as a circumscribed mass with extremely high signal intensity on fat-saturated T2-weighted imaging and a benign-appearing persistent enhancement curve , 95. As Unenhanced diffusion MRI is also gaining recognition in the diagnostic workup of PABC for the systemic staging of pregnant patients, when the use of PET/CT is discouraged . For thiBreast MRI is much more common during lactation, due to the fact that injection of gadolinium-based contrast agent is considered safe for administration . Past stThe main concern regarding the use of breast MRI during lactation does not stem from safety worries, but rather reservations regarding its uncertain diagnostic performance. In light of the increased BPE, there are concerns that it may potentially obscure the presence of the underlying tumor . SeveralHerein, we present an assembly of representative cases, illustrating the spectrum of appearance and persevered diagnostic capabilities of breast MRI, even in lactating patients. The first case is a pre-operative MRI of a PABC patient who presented with a palpable mass after 3 months of lactation. IDC was diagnosed using US-guided biopsy, and MRI reassured the existence of a solitary lesion on top of the surrounding BPE Fig. . OccasioOne noteworthy type of cancer that deserves a specific mention is ductal carcinoma in situ (DCIS), which often displays overlapping radiological and pathological features with lesions with uncertain malignant behavior . Unlike Occasionally, PABC can manifest as multi-centric carcinoma, which is difficult to fully estimate its extent using conventional imaging. A representative case of a lactating patient with newly diagnosed IDC which turned to comprise no less than seven distinct malignant ipsi-lateral lesions is presented in Fig. Since PABC is often a delayed diagnosis, it is associated with more advanced tumor size at the time of diagnosis compared to non-PABC  and evenConsidering the difficulty of interpretation of DCE-MRI with marked BPE and the high likelihood of lactation-related benign entities, some authors suggest that it may be reasonable to delay the examination until several months after weaning to minimize false-positive results that may lead to unnecessary biopsies . ScreeniIn light of the marked physiological changes that the breast undergoes during pregnancy and lactation, clinical and radiological evaluation of the breast becomes extremely challenging. Considering the high incidence of gestational-associated benign breast entities, it is no surprise that PABC is often a delayed diagnosis . The delWhile there is wide agreement that US should be the first-line modality for breast imaging during pregnancy and lactation, and that mammography may have a supplementary additive role, the role of MRI remains controversial in the diagnostic work-up of PABC. In this pictorial review, we have illustrated how gestational-related physiological and benign processes are translated to MRI. Moreover, we have demonstrated the promising utility of unenhanced MRI to serve as a standalone breast imaging modality during pregnancy, and the more established utility of both contrast enhanced and unenhanced breast MRI during lactation. Specifically, it appears that since most cases of PABC reach the radiological work-up with a large palpable mass, the opportunity to facilitate an earlier diagnosis of PABC could be found among high-risk patients and BRCA mutation carriers, which account for up to 35% of PABC cases . In thisUnenhanced breast MRI using DWI variants has shown great strides to serve as a possible cost-effective, fast, and clinically effective alternative to DCE . NeverthFor DCE, it is safe to assume that during pregnancy it would remain unutilized. During lactation, however, the role of DCE may expand, possibly due to the implementation of novel acquisition schemes that may allow better separation between enhancing lesions and BPE. In recent years, developments in accelerated MRI using the application of compressed sensing  have allDuring pregnancy and lactation, the breast experiences substantial changes in morphology and function that affect its imaging properties and may reduce the visibility of concurrent pathological processes. Moreover, the high incidence of benign, gestational-related entities may further add complexity to the clinical and radiological evaluation of the breast during this period. Consequently, PABC is often a delayed diagnosis that carries a poor prognosis. Despite currently being underutilized, this state-of-the-art pictorial review illustrates how technical advances and new clinical evidence support the use of unenhanced breast MRI during pregnancy and both unenhanced and DCE during lactation. These modalities serve as effective supplementary options in the diagnostic work-up of PABC, especially among high risk populations, with the aim to facilitate an earlier diagnosis.


Metastasis of cancer cells from the primary tumor to other organs and tissues in the body is the leading cause of death in patients with malignancies. One of the principal ways cancer cells travel is through lymphatic vessels, and tumor invasion into the regional lymph nodes is a hallmark of early metastasis; thus, the formation of especially peritumoral lymphatic vessels is essential for tumor transportation that gives rise to further progression. In the past few decades, tumor-induced lymphangiogenesis has been testified to its tight correlation with lymphatic metastasis and poor clinical outcomes in multiple types of human malignancies, which warrants novel potential therapeutic targets for cancer treatment. As the understanding of underlying molecular mechanisms has grown tremendously over the years, an inexorable march of anti-lymphangiogenic therapy also aroused terrific interest. As a result, a great number of drugs have entered clinical trials, and some of them exhibited predominant contributions in cancer management. Herein, this review provides an updated summary of the current advances in therapies preventing lymphatic metastasis and discusses the validity of different applications. Lymphangiogenesis is known as the formation of lymphatic vessels from pre-existing lymphatic vasculature. It participates in various physiological settings like homeostasis, immunity, embryonic development, and wound healing, while pathologically, this process is ordinarily implicated in organ graft rejection, lymphedema, and cancer metastasis. Additionally, other than the conventional views, many novel functions and roles of the lymphatic vasculature were uncovered more recently. Research has shown that an alteration of lymphangiogenesis can result in human pathologies such as obesity, aging, cardiovascular diseases including myocardial infarction and atherosclerosis, ocular diseases especially glaucoma, and inflammatory bowel diseases like Chron’s disease. Some neurological disorders, including neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease, and multiple sclerosis, can also be regulated by meningeal lymphatics . The corvia different LEC surface receptors. Despite the participation of multiple receptors and the sophisticated process, it is commonly acknowledged that the main mechanism underlying lymphangiogenesis is through the VEGFR-3 signaling pathway  with Epirubicin, Cisplatin and Capecitabine (CX) as first-line therapy in advanced MET-positive gastric or gastroesophageal junction adenocarcinoma was taken. However, the trial was terminated with a disappointing result as the Rilotumumab group presented a lower median overall survival (OS) and 12-month survival rate compared to the placebo arm, and it was associated with frequent fetal events such as neutropenia 29%) and anemia (12%) . Filcatu% and aneFortunately, several agents inhibiting the interaction between Angiopoietin-1/-2 and the Tie2 receptor were demonstrated with promising efficacy. Trebananib, an angiopoietin neutralizing peptibody, prolonged the progression-free survival in a phase 3 trial of patients with recurrent epithelial ovarian cancer, proving the effectiveness of targeting this non-VEGF pathway . But somin vivo safety of topical use wasn’t tested yet  targeting VEGFRs for their significant improvement of progression-free survival in patients with renal cell carcinoma, thyroid cancer, hepatocellular carcinoma, and multiple other tumor types Table 2.sted yet . Interessted yet . An earlsted yet . Howeversted yet . The parConcurrently, there are extensive ongoing trials and preclinical studies of novel therapeutics. Investigations have been taken to test the synergistic effect of combining Vatalanib (750 mg BID) with the mTOR inhibitor, Everolimus (10 mg QD), and a 20% increase in the response rate was detected through this combination  in neuroendocrine tumors . SelectiThe other potent group of TKI is the MET kinase inhibitor Table 3.In vitro study of Fucoxanthin showed decreased lymph node metastases and significant reduction of in vivo LVD (lymphatic vessel density) in an MDA-MB-231 breast cancer model. Enhanced blocking of matrix metalloproteinases (MMP) secretion and increased TIMP-1 protein expression were also demonstrated in the experimental data of Fucoxanthin (A previous study utilizing the CI66 tumor model suggested that the siRNA-mediated VEGF-C gene silencing could efficiently suppress tumor lymphangiogenesis as well as recruitment of inflammatory cells in TME (tumor microenvironment) and calcium carbonate nanoparticle was believed to be a prosperous vector for it Table 4.oxanthin . Shikonioxanthin . As an eoxanthin . Meanwhioxanthin . Many otoxanthin , Aspirinoxanthin , Hedyotioxanthin , and Curoxanthin  were abloxanthin , 97. Simoxanthin . Newly eoxanthin  and Dimeoxanthin  both cauvia different mechanisms, shedding a light on a new group of potential therapeutics in this field.Tumor-induced lymphangiogenesis and therapeutics providing virtual management of lymphatic metastasis will continue their upward trend in advances based on the successive achievements. Targeting the VEGF-C/VEGFR-3 at different levels remained the major approach due to the increasing confirmation for its essential role in the lymphangiogenic mechanism and a growing understanding of the axis. Newly discovered targets still need further investigation for their accurate regulatory effects in various tumors, whereas some of them, like angiopoietin and genetic transcription factors, were already considered plausible in addition to the VEGF/VEGFR pathway to reduce the common adverse effects associated with VEGF therapies. Although there haven’t been any FDA-approved anti-lymphangiogenic drugs yet, accumulated favorable results, especially from small molecular VEGFR inhibitors, were presented in clinical studies. Most of the examined TKIs were selected from existing anti-angiogenic applications and exhibited promising therapeutic effects. However, single-agent treatment, for example, with monoclonal antibodies alone, received minor therapeutic responses; thus, current anti-lymphangiogenic investigations focus more or less on combining anti-lymphangiogenic methods with other standard anti-tumor drugs due to the extended involvement of different mechanisms in cancer progression. It is noteworthy that, apart from the medications from anti-angiogenic therapy, many preclinical studies Table 4 Since angiogenesis, lymphangiogenesis, and neoplasia share many similar mechanisms and pathways, it’s still necessary to continue to uncover some basic problems, such as what factors keep the blood vasculature apart from the lymphatic vessels after embryonic differentiation and the functional differences of each factor in blood endothelial cells and lymphatic endothelial cells. Also, some recent research pointed out that the LEC genetic pattern strongly influenced the lymphovascular response to the factors in different tissues, causing both functional and structural heterogeneity of tumor lymphangiogenesis. Hence, revealing specific molecular biomarkers in each particular cancer and tracing tumor LEC genetic lineage for their precise roles and mechanisms might be the upcoming challenges in future studies. Moreover, according to the paradoxical effects of lymphatic vessels in immune-cell trafficking, an accurate balance between the protumor and anti-tumor immune response is needed in order to improve the benefits of anti-lymphangiogenic therapies.Literature review and writing—original draft preparation: CW. Writing—review and editing: MC. Supervision and funding acquisition: MC. All authors have read and agreed to the published version of the manuscript.This work was supported by National Natural Science Foundation of China (81603119) and Natural Science Foundation of Beijing Municipality (7174316).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.