Molecular diagnosis is the field that aims to develop nucleic-acid-based analytical methods for biological markers and gene expression assessments by combining laboratory medicine and molecular genetics. As it gradually becomes a clinical reality, molecular diagnosis could benefit from improvements resulting from thorough studies that could enhance the accuracy of these methods. The application of magnetic particles in molecular diagnosis tools has led to tremendous breakthroughs in terms of specificity, sensitivity, and discrimination in bioassays. Therefore, the aim of this review is to highlight the principles involved in the implementation of magnetic particles for sample preparation and targeted analyte isolation, purification, and extraction. Furthermore, the most recent advancements in the area of cancer and infectious disease diagnosis are presented, with an emphasis on screening and early stage detection. The practice of medicine is currently focusing on the development of quick and accurate tests for assessing the health of an individual at the cellular and molecular levels. Such tests have been previously reported as point of care devices, which are instruments designed to perform analytical and diagnostic tests at the site of interest, including the hospital, but also in the field ,2,3,4. AAccordingly, molecular diagnostics has emerged as the field that combines laboratory medicine and molecular genetics ,9 to devThe second half of the twentieth century represented the birth of molecular diagnostics, through the discovery of the Sanger sequencing method in 1977, also known as the chain termination method, which provides high-quality sequencing for long fragments of DNA of up to 900 bp ,17; and Although it has firstly involved targeted searches in one specific gene or region, the field of molecular diagnostics is growing rapidly towards techniques that allow for the simultaneous survey of the entire genome . MoreoveNucleic acid testing generally involves techniques for DNA and/or RNA isolation, amplification, detection, and discrimination . NucleicThese techniques have set the standards for the development of high-throughput systems, such as microarrays for large-scale single nucleotide polymorphism genotyping and DNA diagnostics of inherited, acquired, or infectious diseases . HoweverFast, selective, and accurate disease diagnosis based on molecular tools is fundamental for providing more precise preliminary medical assessments. However, as biological samples are highly complex, due to the presence of multiple components, the specific target entity must be isolated from the raw sample prior to analysis . TraditiOwing to their advantageous surface properties and versatility, magnetic particles have been studied for a variety of biomedical applications, from targeted drug delivery, bioimaging systems, and hyperthermia for cancer therapy, to separation techniques and bioassays, such as blood screening, cell labeling, sorting, and identification, nucleic acid, bacteria, and virus capture, isolation, concentration, and detection, and immunosensors and immunoassays ,33,34,35In the context of molecular diagnosis, magnetic particles have been applied for mixing fluids, selectively capturing, concentrating, transferring, and labelling targeted analytes, performing stringency and washing steps, and probing biophysical properties of analytes ,46,47,48Sample preparation is the initial and a key step for any analytical workflow that aims to qualitatively and quantitatively determine the presence of trace analytes in complicated matrices . SpecifiWith the rapid development of nanotechnology in materials science, various nanomaterials useful for analytical chemistry have been manufactured. Their potential for rapid and efficient sample preparation is based on their high specific area, increased surface activity, and unique physicochemical properties . FurtherThe general steps involved in the extraction of target analytes are effective cell or tissue disruption, the removal of membrane proteins, lipids, and other contamination through the denaturation of nucleoprotein complexes and the inactivation of nucleases, and nucleic acid purification and concentration ,56. In t9 cells/hour, and, to reduce the detection time and enhance the sensitivity, larger sample volumes are required. Moreover, as the resulting culture is not highly pure, magnetic separation must often be coupled with other methods to improve detection [The other direction of applications in molecular diagnosis is represented by the molecular detection of biomolecules labeled with magnetic particles. Commonly used biosensors based on magnetic particles with various sensing properties include magnetic-particle-relaxation-based sensors, magnetoresistive sensors, and magnetic relaxation sensors . Moreoveetection ,62. AnotRecent years have witnessed a tremendous interest in applying microfluidics for sample preparation, cell separation, cancer diagnosis, and delivery and screening of therapeutic drugs. Microfluidic systems, known as lab-on-a-chip, biochips, or bio-microelectromechanical systems integrate and implement multilaboratory functions, such as chemical synthesis, biochemical operations, or nucleic acid sequencing, onto a single microdevice ,63,64. SThere are several parameters that must be considered when designing magnetic-particle-based molecular diagnosis applications. Specifically, these parameters affect the outcome of the analysis, and they include the size and composition of both the magnetic core and the shell, the surface chemistry, and the magnetic responses to the sensor . While mDefined by the World Health Organization (WHO) as the ‘presumptive identification of unrecognized disease or defect by the application of tests, examinations or other procedures, which can be applied rapidly to the target population’ , cancer Biomarkers are biological molecular entities that can be objectively measured and are associated with physiological functions, disease progression, treatment efficiency, and adverse effects to a therapeutic agent ,76. BiomMagnetic particles have proved their important potential in cancer imaging, diagnosis, and treatment. Conventional cancer imaging techniques include magnetic resonance imaging, magneto-acoustic tomography, computed tomography, and near-infrared imaging. Although they offer the possibility of non-invasive diagnosis, their application is limited in the context of detecting subtle invasion, micrometastases, and early stage detection ,85. ConsIn the context of molecular diagnosis, there are many studies described in the literature reporting the use of magnetic particles for the specific detection of cancer biomarkers. Thus, the development of a next generation, aptamer-based bio-barcode assay to detect cytochrome-c, a biomarker released from cancer cells, has been reported. Specifically, magnetic microparticles coated with capturing antibodies and a specific aptamer against cytochrome-c led to the formation of sandwich structures. In this manner, the detection process can be completed within three hours, thus providing a fast, sensitive, and robust tool for anti-cancer drug screening . SimilarIn addition, magnetic particles have proven to be efficient drug carriers for cancer therapy, as they tend to accumulate at specific locations through the application of an external magnetic field . MoreoveResponsible for highly diverse processes such as photosynthesis, nitrogen fixation, vitamin production, and organic matter decomposition, microorganisms play crucial roles in the existence of humans. While there are some highly specialized microorganisms that can survive in harsh environments, root-colonizing bacteria rely on the abundant resources provided by higher organisms . In someInfectious diseases and their worldwide spread have become a serious public health concern, as they affect millions of lives daily ,108. TheThere have been several studies in the literature reporting the use of magnetic particles for the detection of viruses. Specifically, hepatitis A virus and hepatitis B virus were captured and detected using carboxyl-derivatized magnetic beads  and magnL. monocytogenes, have been efficiently captured using vancomycin-functionalized polyethylene glycol-modified magnetic nanoparticles, due to their surface moieties [Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Klebsiella pneumoniae was reported using systems based on magnetic particles and fluorescent nanoparticles, namely quantum dots, proving its potential in detecting very low concentrations [Staphylococcus aureus involve the use of dimer-like iron oxide–silver hybrid nanoparticles, which could also be applied for the effective treatment of infectious diseases through their bactericidal properties [Staphylococcus aureus could be detected using magnetic particles coated with a layer of molecularly imprinted polymer [Escherichia coli and Salmonella typhimurium [Escherichia coli in urinary tract infections [Moreover, the detection of bacteria has witnessed considerable advancements through the use of magnetic particles. Thus, Gram-negative bacteria, especially moieties . Moreovetrations . Other moperties . Additio polymer , aptamer polymer , or apta polymer . Other bhimurium ,124,125.himurium . FurtherCandida spp. has been evaluated using a liquid crystalline system containing iron oxide magnetic nanoparticles for the controlled release of propolis in the intra-periodontal pocket [Antifungal activity against l pocket . Additiol pocket .Molecular diagnosis has emerged as an innovative field encompassing a variety of methods and techniques to identify variations at the gene, DNA, RNA, or protein levels. Although there have been tremendous advancements in this area since the discovery of the Sanger sequencing method in 1977, these methods are still limited by low sensitivity and specificity, laboriousness, and time consumption. The aim of this paper is to emphasize the importance of magnetic particles’ application in molecular diagnosis, for sample preparation and targeted analyte isolation, purification, and extraction. The basic principle for using magnetic particles in sample preparation is based on the attachment of the targeted analytes onto the particles using specific ligands and receptors, and their subsequent magnetic separation by applying an external magnetic field. Such systems are of key interest in the diagnosis of cancer and infectious diseases, as they allow for the detection of extremely low concentrations of biomarkers and pathogens in biological fluids. There is still room for improvement, as there is a fundamental need to discover new, cancer-specific biomarkers that could be detected using magnetic molecular diagnosis. Thus, cancer screening and early stage detection could be improved and could possibly lead to the cure of cancer. Additionally, more pathogen-specific methods for diagnosing infectious diseases could lead to enhanced diagnostic tools that could prevent further spreading. In this manner, point of care devices could be widely implemented as standard tools to assess various disorders of genetic, environmental, or microbial origins.


Improved post-ischemic functional recovery and better mitochondrial respiration were observed in both sEH null hearts or WT hearts perfused with t-AUCB. Inhibition of sEH markedly attenuated the activation of the NLRP3 inflammasome complex and limited the mitochondrial localization of the fission protein dynamin-related protein-1 (Drp-1) triggered by IR injury. Cardioprotective effects stemming from the inhibition of sEH included preserved activities of both cytosolic thioredoxin (Trx)-1 and mitochondrial Trx-2 antioxidant enzymes. Together, these data demonstrate that inhibiting sEH imparts cardioprotection against IR injury via maintaining post-ischemic mitochondrial function and attenuating a detrimental innate inflammatory response.Activation of the nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome cascade has a role in the pathogenesis of ischemia/reperfusion (IR) injury. There is growing evidence indicating cytochrome p450 (CYP450)-derived metabolites of n-3 and n-6 polyunsaturated fatty acids (PUFAs) possess both adverse and protective effects in the heart. CYP-derived epoxy metabolites are rapidly hydrolyzed by the soluble epoxide hydrolase (sEH). The current study hypothesized that the cardioprotective effects of inhibiting sEH involves limiting activation of the NLRP3 inflammasome. Isolated hearts from young wild-type (WT) and sEH null mice were perfused in the Langendorff mode with either vehicle or the specific sEH inhibitor  Ischemic heart disease is a leading cause of cardiovascular death and disability worldwide ,2. In paExperimental evidence has demonstrated that during reperfusion a surge of reactive oxygen species (ROS) is rapidly generated from damaged mitochondria. This ROS burst triggers a series of inflammatory reactions, which induce the formation and activation of inflammasomes aggravating myocardial injury ,7,8. InfSoluble epoxide hydrolase (sEH) catalyzes the hydrolysis of lipid epoxides to their corresponding diol derivatives by the addition of water . sEH is Ephx2) or pharmacological inhibition of sEH enzyme could attenuate myocardial IR injury through maintaining mitochondrial function and consequently limiting NLRP3 inflammasome activation. To the best of our knowledge, the current study also provides the first evidence that the cardioprotective effects associated with sEH inhibition against IR injury is sex-independent in young subjects. Inhibition of sEH has emerged as an intriguing approach to limit cardiac damage in different cardiovascular settings ,27. Desptrans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) and subjected to IR showed significantly improved post-ischemic recovery of left ventricular developed pressure (LVDP) compared to the WT IR control group  and relaxation (dP/dt min) in comparison to the corresponding IR control mice  of sEH on the mitochondrial respiration in fibers separated from hearts subjected to IR injury. Notably, basal respiration rates did not differ significantly between all groups -stimulated respiration and RCR values, suggesting better mitochondrial function in post-ischemic hearts , play a role in the cascade of myocardial IR injury ,35. In rof Drp-1 A, supporEphx2 or pharmacological inhibition of sEH preserved both Trx-1 and Trx-2 catalytic activities as well as significantly blunted the accumulation of MDA in post-ischemic hearts  are important antioxidant proteins that play a cytoprotective role against various oxidative stresses in a variety of systems. Trxs are important for maintaining the reducing environment in the cell, protecting against oxidative stress and thus limiting cardiomyocyte cell death ,38. In mc hearts B–D, suggEphx2) in sEH null animals  is an upstream trigger in the NLRP3 inflammasome cascade that binds these two proteins together, which is essential for downstream activation. Furthermore, Txnip acts as a pro-oxidant protein by preferentially binding to and inhibiting the antioxidant activities of both Trx-1 and -2. Thus, Txnip serves as a central protein linking oxidative stress to NLRP3 inflammasome formation ,45,46. Iractions A,B. Ephx2 was genetically deleted (Mitofusins (Mfn)-1 and -2 are integral proteins that localize to the outer mitochondrial membrane and have a role in dynamic events regulating mitochondrial quality . In the  deleted B. Althou deleted B. OveralEphx2 or pharmacological inhibition of sEH enzyme with t-AUCB limits mitochondrial damage, abrogates activation of the NLRP3 inflammasome cascade, improves post-ischemic functional recovery and thus imparts cardioprotection in the setting of IR injury. The cardioprotective effects associated with sEH inhibition were independent of sex or hemodynamic changes in young mice. Together, the current data suggest a novel cardioprotective mechanism for inhibition of sEH that limits an innate inflammatory response.In the current study, we demonstrate that genetic deletion of Ephx2) has been identified in numerous species such as mouse [The gene encoding mammalian sEH  mainly during the ischemic period . As suchEphx2 and pharmacological inhibition of sEH enzyme significantly inhibited the translocation of Drp-1 to the mitochondria, correlating with improved cardiac functional recovery. These results support our previous findings demonstrating post-MI mitochondrial ultrastructure and function in young WT mice displayed a complete loss of cellular organization, cristae and function that was maintained in the young sEH null mice [Proteins regulating mitochondrial dynamics are involved in the pathogenesis of myocardial IR injury. Under normal conditions, Drp-1 is localized in the cytosol in an inactive phosphorylated form, which becomes dephosphorylated following a stress event and translocates to the outer mitochondrial membrane initiating mitochondrial fission, reducing the number of functional mitochondria and accelerating myocardial injury ,107. Sevull mice ,72.Evidence demonstrates Txnip, a pro-oxidant and a well-known activator of NLRP3 inflammasome, has a negative role in the pathogenesis of IR injury ,108. UndEmerging evidence for the non-canonical roles of Mfn-2 in the activation of different innate immune components and pathogenesis of IR injury indicates that mitochondrial dysfunction is associated with increased Mfn-2 expression, which accelerates cardiomyocyte death ,114. TheIn conclusion, our results demonstrate that inhibition of sEH protects against myocardial IR injury by preserving mitochondrial function and inhibiting NLRP3 inflammasome activation. Although the exact molecular mechanisms remain unknown, we propose inhibiting sEH results in altered cardiac levels of bioactive epoxylipids EDPs and EETs together with reduced DiHOME levels, which collectively maintain an optimally functioning mitochondrial pool, inhibit a detrimental innate inflammasome response and thereby promote cell survival. It is recognized the current study was limited by not directly assessing changes in the levels of epoxylipids  as well as the diol metabolites (DiHOMEs) in response to IR injury. However, it has been shown that cardiac ischemic insults decrease levels of the cardioprotective epoxylipids EETs and EDPs and cardiotoxic DiHOMEs accumulate aggravating the injury ,118. TheEPHX2 (sEH null) and wild-type (WT) littermates on a C57/Bl6 background were maintained in a colony at the University of Alberta [w/w)), more specifically linoleic acid (2.12%), linolenic acid (0.27%), arachidonic acid (0.01%), omega-3 fatty acid (0.45%), total SFA (0.78%) and total MSFA (0.96%)  and housed under conditions of constant temperature and humidity with a 12:12-h light–dark cycle. All animal experimental protocols were approved by the University of Alberta Health Sciences Welfare Committee  and conducted according to strict guidelines provided by the Guide to the Care and Use of Experimental Animals . Mice with targeted disruption of  Alberta  and used–/–) and wild-type (WT) mice of both sexes  were anesthetized by an intraperitoneal injection of sodium pentobarbital . Following complete non-responsiveness to external stimulation, hearts were quickly excised and perfused in the Langendorff mode with Krebs–Henseleit buffer containing (in mM) 120 NaCl, 25 NaHCO3, 10 Dextrose, 1.75 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 4.7 KCL, 2 Sodium Pyruvate (pH 7.4) and bubbled with 95% O2 and 5% CO2 at 37 °C [2O were excluded from the experiment. Mouse hearts were perfused in the retrograde mode at a constant flow rate for 40 min of baseline (stabilization) and then subjected to 30 min of global no flow ischemia followed by 40 min of reperfusion. In a group of WT mice, the specific sEH inhibitor t-AUCB (0.1 µM)  [Soluble epoxide hydrolase null   was addeg for 10 min at 4 °C to separate the cellular debris. The collected supernatant was then centrifuged at 10,000× g for 20 min. The pellet was resuspended in homogenization buffer to obtain a mitochondrial-enriched fraction. The supernatant was ultra-centrifuged at 105,000× g for 60 min and the subsequent supernatant was used as the cytosolic fraction. Protein concentrations in both cytosolic and mitochondrial fractions were measured by the Bradford assay. Western blotting was done as previously described [Frozen mouse hearts were ground, homogenized and then fractionated into mitochondrial and cytosolic fractions as previously described ,69. Brieescribed ,104. ProEnzyme-linked immunosorbent assay (ELISA) was used to quantify the cardiac cytosolic levels of the cytokine IL-1β where mouse IL-1β ELISA kit  was used according to the manufacturer’s recommendations. Briefly, cytosolic samples were pipetted into a 96-well plate where IL-1β present in a sample became attached to the wells by the immobilized antibody specific for mouse IL-1β that is coated on the wells. The wells were then washed, and biotinylated anti-mouse IL-1β antibody was added. Horseradish peroxidase (HRP) conjugated streptavidin was added to the wells after washing away unbound biotinylated antibody. A 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was then added to the wells. Afterwards, the stop solution was pipetted into the wells and the intensity of the color was measured at 450 nm. IL-1β concentration in the different samples was calculated by using a linear standard curve created with different concentrations of the standard IL-1β.The level of MDA was assessed in the cardiac tissue using a lipid peroxidation (MDA) colorimetric assay kit  according to manufacturer’s instructions . In this2EGTA, 20 mM imidazole, 20 mM taurine, 49 mM K-MES, 3 mM K2HPO4, 9.5 mM MgCl2, 5.7 mM ATP,1 µM leupeptin, 15 mM phosphocreatine). A 3–5 mm strip of the anterior left ventricle was isolated and the remaining fats and vessels were removed. Afterwards, myocardial strips were disassembled into bundles containing 6–8 fibers each, 1 mm wide and 3–4 mm long. Fresh fibers were then permeabilized in isolation buffer containing 100 µg/mL saponin, washed three times for 5 min in ice-cold respiration buffer and immediately added to the respiration chamber containing 1.8 mL respiration buffer. The rate of oxygen consumption was measured at 30 °C before and after addition of 0.5 mM ADP in the presence of 5 mM malate and 10 mM glutamate as respiratory substrates to initiate basal respiration. RCR was calculated as the ratio between basal and ADP-stimulated respiration rates to estimate mitochondrial respiration efficiency. Clark electrode connected to an Oxygraph Plus recorder  was used to measure mitochondrial oxygen consumption in permeabilized cardiac fibers. Fresh cardiac fibers were isolated from the left ventricles of the perfused hearts at the end of reperfusion as previously described ,120. BriCleavage of the caspase-1 specific fluorogenic substrate Ac-YVAD-AMC  was used to assess functional caspase-1 activity in cytosolic fractions of the heart homogenates . The assThe insulin disulfide reduction assay was conducted to measure thioredoxin (Trx) activity as previously described ,123. In p < 0.05 was considered statistically significant.Values are expressed as mean ± standard error of mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA) with a Tukey post hoc test to assess differences between groups;


We describe a case of recurrence of KS with a rare presentation of breast and musculoskeletal involvement in the setting of a CD4 count of 374 cell/mm3 and an undetectable viral load. The patient was on highly active antiretroviral therapy for 5 years at the time of the second presentation.Kaposi sarcoma (KS) is the most common malignancy associated with HIV infection. It usually affects the skin, the gastrointestinal tract and the lungs. It is generally described in the setting of CD4 counts < 150 cells/mm She had a past medical history of KS with skin lesions on the face and in the lower limb, treated with chemotherapy 5 years previously. No skin lesions were noted at this presentation. She was on highly active antiretroviral therapy (HAART) since her previous presentation.A 42-year-old HIV-positive woman with a CD4 count of 374 cell/mmMammography revealed a large round mass in the superolateral quadrant of the left breast . It had Immunohistological findings of the masses were consistent with KS. HHV stain was strongly and diffusely positive.The patient then had a computed tomography (CT) scan from the base of the skull to the symphysis pubis, which showed a heterogeneously enhancing left breast mass, a left lower back mass infiltrating the iliocostalis and quadratus lumborum muscles with encasement and destruction of the 11th and 12th ribs  as well 3 Sub-Saharan Africa remains the most severely affected, with nearly one in every 25 adults (4.4%) living with HIV and accounting for nearly 70% of the people living with HIV worldwide.3 The estimated overall HIV prevalence rate is approximately 12.7% of the total South African population. The total number of people living with HIV in South Africa is estimated at approximately 7.03 million in 2016.4In 2015, the World Health Organization (WHO) reported that 36.7 million people were living with HIV.5The risk for KS in acquired immunodeficiency syndrome (AIDS) patients is estimated to be 20 000 times greater than the general population and 300 times greater than in other immunocompromised populations.2 KS is the most common malignancy associated with HIV infection, and it is an AIDS defining malignancy.5 It is associated with HHV-8, and the virus infects the KS spindle cells.5 AIDS-related KS develops in the setting of a low CD4 lymphocyte count < 150 cells/mm3–200 cells/mm3.2 However, there is a single article in the literature that mentions that it can manifest at any time in the course of HIV infection.5Kaposi sarcoma is a low-grade vascular tumour that typically manifests as one of the four variants: classic KS, endemic KS (Africa), iatrogenic KS (organ transplant-related) and AIDS-related KS.1 The lymph nodes and visceral organs, most commonly respiratory and gastrointestinal tracts, can also be involved.1 The manifestations of KS have changed considerably with the advent of HAART. As a result of HAART, a smaller proportion KS patients present with visceral disease.5Frequently KS manifests in mucocutaneous sites, typically the skin of the lower extremities, face, trunk, genitalia and oropharyngeal mucosa.6 Disseminated disease, however, would require a combination of HAART as well as chemotherapy.6In cases of localised disease, treatment with HAART alone could result in improvement or resolution of disease. This is thought to be directly attributable to the suppression of HIV replication and a decrease in viral load with an increase in CD4 lymphocyte count.7 This was unlikely to be the cause of recurrence in our patient as she had been on HAART for 5 years before her second presentation.An immune reconstitution inflammatory syndrome (IRIS) has also rarely been reported in patients who are HAART naïve at the time of KS diagnosis. This may vary in severity, and fatal cases have been reported.8 and one with isolated breast involvement.5 Only one of the cases reported had concurrent bone involvement1 and one case occurred in the setting of low CD4 counts < 100 cells/mm5 Our case presented with the involvement of two unusual sites: breast and musculoskeletal system in the setting of good immunity with a CD4 count of 374 cells/mm3 and an undetectable viral load. The patient was also on HAART at the time of this presentation.Kaposi sarcoma of the breast is rare. In our literature search, we found three reported cases of KS in the breast, two with disseminated diseaseThe HIV pandemic has resulted in an increased incidence of KS occurring in common sites described above. Our patient presented with multiple rare findings that included involvement of the breast and musculoskeletal system in the setting of good immunity. There are minimal reports in the literature of such cases.


DS) is a noninvasive electromagnetic‐based technology intended to quantify lung fluid content.Right heart catheterization is the gold standard in clinical practice for the assessment of cardiovascular hemodynamics, but it is an invasive procedure requiring expertise in both insertion and reading. Remote dielectric sensing (ReDS readings were obtained in supine position just before right heart catheterization procedure in patients with heart failure. Agreement between ReDS and pulmonary artery wedge pressure (PAWP) was analyzed. Of all, 139 patients with heart failure received hemodynamic assessment and ReDS measurement. A good correlation was found between ReDS and PAWP measurement . Receiver operating characteristic analysis of the ability to identify a PAWP ≥18 mm Hg resulted in a ReDS cutoff value of 34%, with an area under the curve of 0.848, a sensitivity of 90.7%, and a specificity of 77.1%. Overall, ReDS <34% carries a high negative predictive value of 94.9%.In this prospective single‐center study, ReDS, correlates well with PAWP. The high sensitivity and specificity and especially the high negative predictive value make ReDS a reliable noninvasive tool at the point of care, to rule out elevated PAWP in patients with heart failure and to help with medical management of patients with heart failure. Further studies are warranted to compare this tool with existing tests and to relate the findings to the clinical outcomes.Lung fluid content, as measured by Re We demonstrated the relationship between the remote dielectric sensing technology and invasively measured pulmonary artery wedge pressure.Remote dielectric sensing <34% had a high negative predictive value of 94.9% to exclude patients with pulmonary artery wedge pressure >17 mm Hg.Remote dielectric sensing technology may be a reliable noninvasive tool to rule out ambulatory patients with elevated pulmonary artery wedge pressure.The heart failure (HF) epidemic is increasing, with >23 million patients worldwide and 6 million people in the United States.Dyspnea is a common symptom that can be attributed to HF, lung disease, anemia, and other conditions. In patients with HF, dyspnea is the result of salt and water retention as a consequence of the increased neurohormonal activity.A reliable, safe, and noninvasive tool for the assessment of pulmonary fluid status could replace the need for an invasive approach and would be of great value in the long‐term management of patients with HF, in providing an actionable measure that triggers HF‐specific therapy to patients evaluated in urgent care settings because of shortness of breath. Besides a better safety profile, a single noninvasive device could be used in the management of many patients cost‐effectively.Accordingly, the purpose of the current study was to evaluate the relationship between remote dielectric sensing (ReDS) assessment of lung fluid volume and invasive hemodynamic assessment during RHC.The data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure from the corresponding author on reasonable request. This was a single‐center, prospective, observational study performed in patients scheduled for an RHC on the basis of clinical need. The University of Chicago Institutional Review Board approved this study, and all patients signed informed consent before the enrollment.https://www.youtube.com/channel/UCeTy3Ifw980KCHd5_hcnKEQ). The analyzed signal reflects the dielectric properties of the portion of the lung between the sensors. The dielectric coefficient of a material is represented by a frequency‐dependent complex number describing its interaction with electromagnetic energy, including the degrees of absorption, reflection, and transmission of the energy. Because water has a high dielectric coefficient and air has a low dielectric constant, the dielectric coefficient of a tissue is determined predominantly by its fluid content. For example, healthy fat tissue, which has low fluid content, is characterized by a relatively low dielectric coefficient. Blood is relatively rich in water and is characterized by a higher dielectric coefficient. The measurement takes 90 seconds to obtain. The overall procedure to complete the ReDS measurement takes within 5 minutes. We can obtain ReDS data just by imputing patient information and pushing a start button. The normal range for the ReDS value is from 20% to 35%.ReDS technology has been described in detail previously.1Body habitus out of range because of ≥1 of the following: aHeight <155 cm or >190 cm or b2 or >36 kg/m2.Body mass index of <22 kg/m2Physical deformity of the thorax or lesion that may prevent proper vest application or adjustment.3Illness or condition that may be aggravated or cause significant discomfort by the application of the vest .4 Congenital heart malformations or intrathoracic mass that would affect the right lung anatomy (dextrocardia and lung carcinoma).5 Pacemaker generator on the right side of the chest.6 Vulnerable populations, such as those unable to provide informed consent and women who are pregnant or lactating.All consecutive patients with HF, which was diagnosed by Framingham criteria, who underwent RHC (between September 2016 and February 2017) as part of their standard of care  were enrolled in this study, unless they met any of the following exclusion criteria:Patients being catheterized after heart transplantation were also included.After informed consent, baseline demographic data were collected. ReDS system readings were obtained in supine position just before transport into the catheterization laboratory for the RHC, where standard invasive hemodynamic testing was performed. In addition to quantifying mean values of central venous pressure (CVP) and PAWP obtained at the end of the expiratory phase, we also quantified large v‐waves, which were defined as a v‐wave with a magnitude of 7 mm Hg above mean PAWP. All hemodynamic parameters were measured automatically and then overread independently by 2 qualified investigators (G.S. and G.K.). When differences between values provided by the 2 investigators were within 3 mm Hg, the value of the first investigator was used. When the difference was >3 mm Hg, the one that a third independent investigator read was used as a final value. All investigators were blinded to ReDS readings and to each other. Interobserver variability in each hemodynamic variable was assessed by the Ebel intraclass correlation coefficient.The primary end point of this study was an assessment of the correlation between invasive PAWP and noninvasive ReDS system measurement in patients with HF. A test for normality of data distribution was conducted using a Shapiro‐Wilk test. The PAWP‐ReDS correlation assessment was performed via Pearson's correlation coefficient.A sensitivity and specificity analysis was performed using a receiver operating characteristic analysis to determine the ReDS value that most accurately identified whether PAWP was >18 or <15 mm Hg, a clinically meaningful cutoff value for adjustment of medical therapy. We also assessed the influence of CVP on ReDS measurement by dividing patients into 4 groups: normal CVP and PAWP, elevated PAWP with normal CVP, elevated CVP with normal PAWP, and elevated PAWP and CVP. Comparison of the ReDS values in these groups was performed with analysis of variance with a post hoc Tukey's test. For the assessment of association of variables, including ReDS measurement on PAWP, univariate and multivariate linear regression analyses were performed. Variables significant in the univariate analyses were enrolled into the multivariate analyses to construct an ideal model. In repeated analyses, paired data were compared by using the Wilcoxon signed‐rank test considering the small sample size.P<0.05 was considered significant.All calculations were performed in SPSS Statistics 22 software , and 2‐sided 2) met inclusion criteria and were enrolled in this study. Fifteen patients with a body mass index >36.0 kg/m2 were excluded, and 40% of subjects were post–heart transplant. Baseline characteristics of the study participants are summarized in Table Of all, 154 patients were screened, and 139 patients , and the linear regression was PAWP=−2.392+0.538×ReDS . The relationships between the similar variables (ReDS versus PAWP and ReDS versus CVP), standardized per SD, are also shown in Figure There was a positive correlation between ReDS measurement and PAWP  and high or normal ReDS (with 34% as the cutoff), we found that 113 of 139 patients (81.3%) had concordant PAWP and ReDS measurements , whereas only 26 of 139 patients (18.7%) had discordant measurements .Receiver operating characteristic analysis of the ability to use ReDS to identify a PAWP ≥18 mm Hg resulted in a ReDS cutoff value of 34% Figure .r=0.711, P<0.001). Group 1 patients were those with normal CVP (<12 mm Hg) and normal PAWP (<18 mm Hg), group 2 patients had high CVP and normal PAWP, group 3 patients had normal CVP and high PAWP, and group 4 patients had elevated CVP and PAWP. ReDS was significantly higher in groups with elevated PAWP  compared with groups with PAWP <18 mm Hg   Figure B. On the2; P<0.001) and more patients were in the hospital .Twenty‐two patients had ReDS ≥35% (suggesting a hypervolemic state) and PAWP <18 mm Hg. The clinical characteristics of those patients were similar to the overall patient population, except that they had a significantly higher body mass index (30.9±4.2 versus 27.5±4.5 kg/mP<0.05) were included in a subsequent multivariate analysis, which was followed by a second multivariate analysis with parameters that continued to show significance at P<0.05. Only ReDS, mean PA pressure, and CVP were found to be related to PAWP .Of the total cohort, 38 patients had repeated RHC. Changes in PAWP and ReDS between the first and the second measurements were shown in Figure P=0.60), whereas ReDS decreased significantly from 42.9±5.9% to 33.9±3.9% (P=0.018). Consistent with this observation, paired data from these 7 patients showed the downward movement of the PAWP‐ReDS relationship , 7 had repeated RHC during the study period, and second sets of measurements of ReDS were taken Figure . Of themp Figure , with 5 P<0.05 for both). Despite the differences in pretest probability, sensitivity, specificity, and negative predictive value for the ReDS, measurement of predicting a PAWP ≥18 mm Hg was similar in the 2 cohorts.Transplant patients were included in this study to enrich the overall cohort with patients who were more likely to have normal hemodynamics, as demonstrated in Table We evaluated the correlation between ReDS assessment of lung fluid volume and invasive measurement of PAWP in patients undergoing RHC for any indications. Our main findings are as follows: (1) A good correlation exists between ReDS and PAWP. (2) ReDS has high sensitivity and specificity to detect abnormally high PAWP that would trigger changes in medical therapy. (3) ReDS reading in the normal range has a high negative predictive value of (95%) in ensuring the PAWP is <18 mm Hg.The current available noninvasive tools for the HF practitioner to assess fluid congestion include physical examination, chest x‐ray, and B‐type natriuretic peptide (BNP). Both physical examination and chest x‐ray are prone to operator experience, subjective interpretation, and basic limitations, which renders them relatively unreliable.A reliable, safe, easy‐to evaluate, and noninvasive tool for the assessment of pulmonary fluid status could replace the need for an invasive test or permanent implantable device and would be of great value in both clinical and economic terms in the long‐term management of ambulatory patients with HF by providing an actionable measure that can trigger HF‐specific therapies.HF practitioners could benefit from an HF‐related vital sign, such as ReDS, much like blood pressure or glucose for systemic hypertension and diabetes mellitus, respectively. ReDS technology is an actionable day‐to‐day parameter that is directly related to fluid volume, according to which the HF treatment can be titered.In 15.8% of patients, ReDS measurements indicated that patients’ lung fluid was higher than normal, yet PAWP was normal. In a smaller number of patients (2.8%), ReDS was normal, yet PAWP was elevated. There are some potential reasons for such discordance. First, a perfect correlation between ReDS and PAWP is not expected, because lung fluid content and intravascular pressures are related, but they measure different properties. Extravascular volume is a complex and variable function of vascular volume, vascular tone, hematocrit, and blood oncotic pressure; the elements of time, especially in the context of changing fluid status because of volume retention, and treatments, such as diuretics, further complicate the situation.Accordingly, several studies highlight the low expected correlation between these 2 entities.Thus, the stability of a patient's fluid status and timing from diuretic dosing can influence the ReDS‐PAWP relationship.Second, depending on the chronicity of the HF state, there can be changes in pulmonary vasculature that influence flux of fluid between compartments and the equilibrium between intravascular and extravascular fluid volume.However, the mechanism of an elevated ReDS measurement in the presence of normal PAWP in several populations is unclear. Currently, we believe that it is important to present the data as is and continue to learn and explore more about this technology. Nevertheless, on the basis of ReDS correlation with PAWP and its high negative predictive value, ReDS measurements as a point‐of‐care assay can help guide clinical decision making, such as treatment and patient disposition, and can also exclude elevated filling pressures that would warrant hospitalization. When a patient with shortness of breath has a low ReDS, <35%, other causes should be explored. In addition, predictability of ReDS for PAWP >15 mm Hg, which is one generally accepted threshold for defining pulmonary congestion attributable to left heart disease in patients with pulmonary hypertension, was similarly high compared with the results when using a cutoff of 18 mm Hg. Among patients with pulmonary hypertension, ReDS technology may be helpful to distinguish noninvasively whether the cause is attributable to left heart disease or not.This study enrolled all comers to the catheterization laboratory, which included in‐hospital and out‐of‐hospital patients with HF and posttransplant patients. This provided a cohort with a wide range of hemodynamic profiles with different pretest probabilities of PAWP ≥18 mm Hg Table . As withIn the context of post–heart transplant management, the high negative predictive value of a normal ReDS reading can be extremely beneficial as an adjunct to other noninvasive tests to reduce the number of RHCs. As more noninvasive options to detect rejection become available, such as routine gene expression testing  and cell‐free DNA testing , the need for routine endomyocardial biopsy will be reduced; however, these tests do not provide an assessment of filling pressures. By combining ReDS technology with noninvasive assessment of a rejection episode, we may undergo both the fluid status assessment and rejection surveillance without the need of any invasive testing.r=0.492). ReDS technology cannot estimate absolute value of PAWP accurately. The great advantage of this technology is a high negative predictive value (94.9%) to exclude the patients with PAWP >17 mm Hg, particularly in an ambulatory setting. The outpatients with ReDS >34% would receive more intensive assessment (including invasive hemodynamic tests) and treatments. Nevertheless, we should understand that this high negative predictive value (94.9%) was derived from the combination of HF population (88.9%) and transplant population (100%). We cannot simply adopt our results into other background populations. Third, because this was a registry study, we did not have access to other clinical information that could have corroborated fluid status aside from PAWP. For example, physical examination findings, BNP values, chest x‐ray films, and chest computed tomography scans could have provided insights in cases of discordance between ReDS and PAWP measurements. Also, these procedures, in addition to ReDS measurement, would strengthen the utility to understand patient physiological condition via different perspectives (volume status or filling pressure) and adjust medications. Other noninvasive measurements, such as transthoracic echocardiography, lung ultrasound, and intrathoracic impedance, are also known to estimate fluid status; however, these technologies may require expertise technique or special attention for the interpretation of the obtained value. The ReDS system is the first technology that uses electromagnetic energy that travels between the sensors (chest and back) and provides the absolute value of the fluid level as a percentage. Comparison or combination with these modalities in classifying patients or predicting clinical outcomes is strongly warranted. The current study is cross‐sectional, and the trend comparing between preinterventions and postinterventions may suggest one possible explanation for the discordance between ReDS and PAWP measurements.First, this was a single‐center study. Second, there was not so strong correlation between PAWP and ReDS reading , which is a randomized open‐label study to assess the utility of daily ReDS measurements on HF readmission will be able to provide some insights into the role of ReDS in titrating and managing medication, although hemodynamics data will not be available. Combination with other traditional procedures, including BNP measurement, is a next concern.Lung fluid content, as measured by ReDS, correlates well with invasively measured PAWP. The high sensitivity and specificity, and especially the high negative predictive value, make ReDS a reliable noninvasive means to rule out elevated PAWP in patients with HF, with low burden to both patients and healthcare providers. This simple noninvasive test could help detect early fluid retention in patients with HF before acute decompensated HF symptoms develop, allowing caregivers to manage patients with HD in early stages of volume accumulation. Further investigations, especially longitudinal studies, are warranted.The registry data collection was sponsored by Sensible Medical Innovations Ltd.Imamura receives financial support from a Postdoctoral Fellowship for Research Abroad of Japan Society for the Promotion of Science. Burkhoff is a consultant to Sensible Medical Innovations Ltd. Abbo is an employee of Sensible Medical Innovations Ltd. The remaining authors have no disclosures to report.Table S1. Predictability of ReDS for PAWP >15 mm Hg at a Cutoff of 35Figure S1. Relationships between ReDS and PAWP (A) and between ReDS and CVP (B) (variables are standardized per standard deviation).Figure S2. Changes in PAWP and changes in ReDS between the first tests and the second tests.Figure S3. Trend of ReDS and PAWP between the first and second measurements in 7 patients with ReDS >35 and PAWP ≥18 mm Hg at the first measurement.Click here for additional data file.Video S1. Explanation of how to measure ReDS. ReDS indicates remote dielectric sensing.Click here for additional data file.


Porous Ag and Ag-NiO nanofibers were successfully obtained after a 3-h calcination at 500 °C under air atmosphere, and analyzed using various material characterization techniques. Synthesized, quasi-three-dimensional porous Ag and Ag-NiO nanofibrous mats were applied as SERS substrates, to measure the model compound Rhodamine 6G (R6G), and investigate the corresponding signal enhancement. Furthermore, porous Ag and Ag-NiO nanofibrous mats were employed as SERS substrates for melamine and methyl parathion respectively. Sensitive detection of melamine and methyl parathion was achieved, indicating their feasibility as an active SERS sensing platform, and potential for food safety and environmental monitoring. All the results suggest that the electrospinning-calcination, two-step method offers a new, low cost, high performance solution in the preparation of SERS substrates.In this study, two new quasi-three-dimensional Surface Enhanced Raman Scattering (SERS) substrates, namely porous Ag and Ag-NiO nanofibrous mats, were prepared using a simple, electrospinning-calcination, two-step synthetic process. AgNO Raman spectroscopy is an accurate and attractive molecule identification and monitoring method. It probes the chemical contents through molecular vibration, providing a unique, specific chemical or vibration “finger-print” for molecules ,2,3,4. HThe uniqueness of SERS technology can be ascribed to its ability to obtain structural information through molecule vibrations within a broad wavelength range. Therefore the discovery of SERS has resulted in a new sensor research field. SERS based sensors demonstrated several benefits, especially in ultra-sensitivity  and excellent selectivity (fingerprint spectrum) in real applications, compared with traditional analytical methods ,10,11,12For SERS sensors, fabrication of SERS substrates is of paramount importance in effective, sensitive and reproducible detection of targets. Consequently, the preparation of highly efficient SERS substrates has drawn considerable attention . AggregaElectrospinning provides a novel, simple and template-free strategy to prepare quasi-3D polymer nanofibrous membranes ,25,26. N3 and AgNO3/Ni(NO3)2 served as precursors [Previously, we prepared quasi-3D porous Ag and Ag-NiO nanofibrous mats simply by an electrospinning-calcination, two-step synthesis route, in which AgNOecursors . The nan3)2·6H2O) and silver nitrate (AgNO3) were bought from Acros Organics. Rodamine 6G (R6G), methyl parathion (C8H10NO5PS), melamine (C3H6N6) and poly(vinyl pyrrolidone)  were acquired from Sigma-Aldrich. All chemicals were of analytical grade and used without any pretreatment. Ultrapure water (18.2 MΩ∙cm resistivity) was employed to prepare aqueous solutions.Nickel nitrate hexahydrate (Ni(NOScanning electron microscopic (SEM) images were recorded using FEI Tecnai G2 Spirit BioTWIN and FEI Nova NanoSEM 450. X-ray Diffraction (XRD) pattern was recorded by a Rigaku Ultima IV diffractometer. A portable Raman spectrometer  was used to collect the Raman spectra coupled with a 785 nm, 499 mW laser. For each measurement, the Raman spectrum was obtained with an integration time of 5 s.3, 0.2 g Ni(NO3)2·6H2O and 0.8 g PVP were dissolved in 4 mL dimethyl formamide (DMF). The mixture was then stirred for 4 h to form an homogenous solution. 3/Ni(NO3)2/PVP nanofibers were prepared using the electrospinning setup, in which a 23-gauge needle and flow rate of 0.3 mL/h was employed, with an applied voltage of 20 kV. The collection distance between the needle tip and aluminum foil (serving as nanofibers collector) was 15 cm. In order to acquire the Ag-NiO nanofiber mats, as-synthesized AgNO3/Ni(NO3)2/PVP nanofibers were then thermal-treated at 500 °C for 3 h under air atmosphere. Preparation of porous Ag followed a similar procedure except for the absence of nickel salt (Ni(NO3)2·6H2O).Quasi-3D porous Ag and Ag-NiO nanofibrous mats were synthesized following the procedure in our previous report, with a minor revision . In a tyA methyl parathion ethanolic solution (0.01 M), R6G (0.01 M) and melamine (0.01 M) aqueous solution were prepared and used as stock solutions. Methyl parathion, R6G and melamine solutions with various concentrations were obtained by diluting corresponding stock solutions. Target molecule solutions (2 μL) with certain concentrations were directly dropped onto the surface of porous Ag. After drying of the target solutions the SERS spectra were recorded. To fabricate Ag-NiO nanofiber mats-based substrate, 0.2 mg of Ag-NiO nanofibers was dispersed into 2 mL water and treated with an ultrasonic bath for 30 s. Then, 5 μL of suspension was dropped onto a silicon wafer and left to dry. Two microliters of target solution with various concentrations were dropped onto the Ag-NiO nanofibers SERS substrate, and after being dried on the surface the SERS spectra was recorded.3/PVP nanofibers. The AgNO3/PVP nanofibers possess smooth surface and good uniformity. The inset of 3/PVP nanofibers was about 200 nm. 3/Ni(NO3)2/PVP nanofibers with a smaller average diameter (ca. 150 nm). However, there are some very tiny nanowires intertwined with large nanofibers. These observations can mainly be attributed to the difference in electrical conductivity and viscosity between AgNO3/PVP and AgNO3/Ni(NO3)2/PVP precursors. These results indicate the successful synthesis of AgNO3/PVP nanofibers and AgNO3/Ni(NO3)2/PVP nanofibers using electrospinning method.To study the morphology of the electrospun nanofibers before and after calcination, SEM characterization was first conducted. 3 and Ni(NO3)2 were degraded to yield metal Ag and NiO, respectively, following the reactions below [After 3 h of thermal treatment of the precursory nanofibers at 500 °C in air, the polymer completely decomposed and disappeared. The AgNOns below ,36:(1)2A3/PVP nanofibers. However, due to the high temperature applied during calcination, some Ag merged together to form 3D porous structure with a rough rather than a fibrous surface, shown in Quasi-3D porous Ag mat was obtained after calcination of AgNOTo study the chemical composition and crystallinity of porous Ag network and Ag-NiO nanofibers, XRD study was conducted. −3 M, SERS spectra of R6G with concentration of 5 × 10−8 M and 3 × 10−7 M recorded on the porous Ag and Ag-NiO nanofibers mats, respectively (Raman shift in the range from 670 cm−1 to 1670 cm−1 was collected). There was no obvious Raman scattering for both porous Ag and Ag-NiO nanofibers without casting R6G, indicating negligible background interferences from as-fabricated SERS substrates. Raman spectrum of high concentration R6G shows three relatively weak peaks at 1311 cm−1, 1361 cm−1 and 1509 cm−1. By contrast, SERS spectrum of R6G with much lower concentration was collected on the porous Ag. Besides three aforementioned peaks, four other distinct peaks appeared at 769 cm−1, 1123 cm−1, 1194 cm−1 and 1647 cm−1, accompanied with a significantly enhanced Raman signal. All these molecule vibration assignments were listed in −1 were extracted to serve as 5 and 2.89 × 104 for R6G. A relative lower EF obtained for Ag-NiO nanofibers can be attributed to the distribution of NiO phase on the surface of Ag-NiO nanofibers [To study the SERS activity of as-synthesized porous Ag and Ag-NiO nanofibrous mats, R6G was used as a model Raman dye. nofibers .−4 M) recorded on porous Ag. One can see that after loading 2.5 × 10−6 M of melamine on the SERS substrate, a prominent peak at 684 cm−1 was observed, corresponding to the characteristic peak of melamine (ring breathing) [−1 gradually increased with the increasing of melamine concentration. The results demonstrated that the porous Ag network displayed good sensitivity (down to micromolar level) towards melamine detection. SERS spectra of methyl parathion at various concentrations on Ag-NiO nanofibrous mat were collected and are shown in −1 (bending vibration of C-H) [−5 M of methyl parathion. Raman intensities increased significantly with the increase of methyl parathion concentrations. At higher concentrations, a new peak at 1111 cm−1 was also observed, corresponding to the stretching vibration of C-N [To further demonstrate the applicability of porous Ag and Ag-NiO nanofibers mat in SERS sensing, porous Ag was employed for melamine detection, while Ag-NiO nanofibrous mat was used for methyl parathion monitoring. eathing) . The Ram of C-H)  appearedn of C-N . An acce3/PVP and AgNO3/Ni(NO3)2/PVP as precursors, respectively. Formation of porous Ag was attributed to partial melting of silver at 500 °C. By contrast, the introduction of Ni(NO3)2 maintained the nanofibrous structure due to the formation and presence of NiO. The good SERS performances of as-synthesized quasi-three-dimensional porous Ag and Ag-NiO nanofibrous mat were first demonstrated using R6G as a model compound. The feasibility of a porous Ag and Ag-NiO nanofiber-based SERS sensing platform was further demonstrated for monitoring melamine and methyl parathion, respectively, indicating their potential application in food safety and environmental monitoring. These results demonstrate that the electrospinning-calcination two-step method offers a new strategy in the preparation of highperformance SERS substrates.In conclusion, we fabricated two new SERS substrates, porous Ag and Ag-NiO nanofiber, by using a simple, electrospinning-calcination two-step method with AgNO


Fluorescent silk fibroin nanofibers were fabricated via electrospinning method with three kinds of fluorescent dyes. Electrospun fluorescent nanofibers showed smooth surfaces and average diameters of 873 ± 135 nm, 835 ± 195 nm, and 925 ± 205 nm, respectively, for silk fibroin-fluorescein sodium, silk fibroin-rhodamine B, and silk fibroin-acridine orange nanofibers containing 2.0 wt% fluorescent dyes. At the same time, the secondary structure of silk fibroin in fluorescent nanofibers was predominantly amorphous conformation without influence by adding different concentrations of fluorescent dyes, as characterized by Fourier transform infrared spectroscopy and X-ray diffraction. Thermal degradation behavior of fluorescent silk fibroin nanofibers with a dramatic decrease in weight residue was observed at around 250 °C. The fluorescence effect of fluorescent silk fibroin nanofibers was changed by changing the concentration of different fluorescent dyes. These fluorescent nanofibers may make promising textile materials for large scale application. Nanomaterials exhibit excellent characteristics, such as large surface area and high porosity, compared with other known forms of materials . With thN-isopropylacrylamide)-co-(N-hydroxymethyl acrylamide)-co-(4-rhodamine hydrazonomethyl-3-hydroxy-phenyl methacrylate)] (PNNR) copolymers were synthesized by free radical copolymerization of three monomers. In acidic environments, PNNR copolymers showed highly selective and sensitive recognition and displayed “ON-OFF” fluorescence toward Cu2+, both in solution and in solid state [Fluorescent electrospun nanofibers are attracting increasing interest for their potential exploitation in textiles, photonic applications, and so on. For example, Li et al. showed fluorescent CdTe quantum dots were dispersed into poly (PVA) nanofibers by electrospinning, generating a highly fluorescent 1D material with dramatically high aspect ratios . Wang etid state . Hu et aid state . Wang etid state . In addiSilk fibroin (SF), a natural fibrous protein produced by Bombyx mori, has been used for textile and biomedical applications ,13. To sAs is commonly known, fluorescent nanofibers have been the subject of continued interest. There have been several reports on the biomedical and biotechnological applications using fluorescent silk fibroin. However, there is almost no application in the field of textiles, such as fluorescent strips and fluorescent icons for apparel. In this paper, we report herein the demonstration of electrospun fluorescent silk nanofibers with stable fluorescence performance for textile applications. The functionalities of fluorescent SF nanofibers depend on organic dyes doped in the silk solution. High-quality fluorescent nanofibers with spatial homogeneities in diameter and fluorescent emissions were obtained for three different fluorescent dyes. Furthermore, the effect of SF and fluorescent dye concentration on the fiber morphology and size distribution are systematically studied. The thermal stability and fluorescence properties of these composite fibers are investigated.Bombyx mori silkworm cocoons were purchased from Shandong province, China. Fluorescein sodium (FS) and rhodamine B (RB) were obtained from Sinopharm Chemical Reagent Co. Ltd. . Acridine orange (AO) was purchased from Shanghai Macklin Biochemical Co. Ltd. Other reagents  were also bought from Sinopharm Chemical Reagent Co. Ltd. , and directly used without purification.2 (FA-CaCl2) solvent at room temperature. This solution was evenly poured into culture dishes and dried for film formation, and then, films were soaked in deionized water to remove Ca2+ ions. After removing Ca2+ ions, films were dried at room temperature.Bombyx mori silk fibroins were prepared according to published procedures . DegummeDried films were re-dissolved in formic acid (FA) solution and stirred at room temperature to obtain the base solution. Three types of fluorescent agents, including RB, AO, and FS were used, and their chemical formulas are shown in −1. After electrospinning, fluorescent SF nanofibers were dried at room temperature overnight to dry off the remaining solvent.Fluorescent SF nanofibers were produced and the preparation parameters were as follows. The spinning voltage was 15 kV. The tip-to-collector distance was fixed to 15 cm. The spinning solutions were loaded into a 10 mL syringe, and the inner diameter of the metal needle was 0.9 mm. The flow rate of the solution was 0.2 mL hScanning electron microscope (SEM): The surface morphology of fluorescent nanofibers was observed by SEM . All samples were sputter coated with gold prior to imaging. The average diameter of fluorescent SF nanofibers was obtained by analyzing SEM images using image J analysis software, and the value of each sample was calculated by a diameter of one hundred nanofibers.Structural analysis: The secondary structures of fluorescent SF nanofibers were analyzed by FTIR on Nicolet5700  in absorbance mode. For each measurement, each spectrum was obtained by the performance of 32 scans with the wave number ranging from 400 to 4000 cm−1 with a resolution of 4 cm−1. Meanwhile, in order to investigate the crystalline structure of the fluorescent SF nanofibers, X-ray diffraction experiments were measured on X Pert-Pro MPD  in transmittance mode. The incident beam wavelength was 0.154 nm. The intensity was finally corrected for changes in the incident beam intensity, sample absorption, and background.Thermal analysis: The thermal stability of fluorescent SF nanofibers was characterized using SDT Q600  under nitrogen gas flow of 40 mL min−1. Samples were heated from room temperature to 600 °C, and the heating rate was 10 °C min−1.Fluorescence analysis: The fluorescence emission spectra of fluorescent nanofiber samples were carried out using Hitachi F-4600 Spectrofluorometer . During the test, every sample was folded into two layers and laid on a solid support of a fluorescence spectrophotometer. The operating voltage was set to 250 V. The slit width of excitation light and emitted light was 5 nm. The excitation wavelength was chosen to be 450 nm and the emission wavelength range was between 450 and 800 nm.U test .All values were expressed as mean ± standard deviation. Statistical differences were determined by a Mann-Whitney For fluorescent dyes involving nanofiber electrospinning, electrospinnability of nanofibers played a critical role. In this study, fluorescent SF nanofibers with different fluorescent dyes could be readily electrospun into fibrous form  owing toIn addition, fluorescent nanofibers were produced by electrospinning method, and influenced by three kinds of fluorescent dyes . The morphology of these fluorescent nanofibers exhibited a circular cross-section with smooth surface e. At the−1 (amide I), 1536 cm−1 (amide II), and 1239 cm−1 (amide III), corresponding to amorphous conformation (FTIR spectrum was carried out to confirm the functional groups in the fluorescent SF nanofibers . Figure ormation Ab. When ormation A–C, demo−1, 1536 cm−1, and 1239 cm−1, corresponding to the amorphous structure (−1, 1528 cm−1, and 1243 cm−1 were observed, corresponding to amorphous conformation (tructure a. When tormation b. For FSormation c,d. At tThe crystal structure of fluorescent SF nanofibers containing different types and concentration of fluorescent dyes was also analyzed by XRD . In prevFrom thermogravimetric curves results, a weight loss of about 7.0 wt% around 100 °C was detected in pure SF nanofibers, which was ascribed to the evaporation of water Aa. As thFluorescent SF nanofibers were electrospun by adding different types and concentrations of fluorescent dyes. These nanofibers were placed in a dark place and irradiated with an ultraviolet lamp to observe their fluorescence effect . The resFor fluorescent SF-RB nanofibers, the maximal emission was located at 615 nm B1a,b. FiFluorescent SF nanofibers were prepared via electrospinning method with different fluorescent dyes. Fluorescent SF nanofibers containing 2.0 wt% fluorescent dyes could be electrospun into the continuous fibrous structure, although fluorescent SF nanofibers showed larger diameters and broader diameter distribution than pure SF nanofibers. SEM results showed the average diameters were 873 ± 135 nm, 835 ± 195 nm, and 925 ± 205 nm, respectively, for SF-FS, SF-RB, and SF-AO nanofibers containing 2.0 wt% fluorescent dyes. Different fluorescent dyes had no significant effect on the morphology and diameters of fluorescent nanofibers. At the same time, the secondary structures of fluorescent SF nanofibers were characterized by FTIR, XRD, and thermal analysis. Comparing with pure SF nanofibers, the structure of SF in fluorescent nanofibers was predominantly amorphous conformation, without being influenced by adding different concentrations of fluorescent dyes. The crystal structure was confirmed by FTIR and XRD. Thermal analysis results showed thermal degradation behavior of fluorescent SF nanofibers with a dramatic decrease in weight residue was observed at around 250 °C. Furthermore, the fluorescence effect of fluorescent SF nanofibers was changed by changing the concentrations of different fluorescent dyes. These fluorescent nanofibers may make promising textile materials for large scale application.


Pathology has evolved from a purely morphological description of cellular alterations in disease to our current ability to interrogate tissues with multiple ‘omics’ technologies. By utilising these techniques and others, ‘molecular diagnostics’ acts as the cornerstone of precision/personalised medicine by attempting to match the underlying disease mechanisms to the most appropriate targeted therapy.Despite the promises of molecular diagnostics, significant barriers have impeded its widespread clinical adoption. Thus, the National Cancer Research Institute (NCRI) Cellular Molecular Pathology (CM-Path) initiative convened a national Molecular Diagnostics Forum to facilitate closer collaboration between clinicians, academia, industry, regulators and other key stakeholders in an attempt to overcome these.We agreed on a consensus ‘roadmap’ that should be followed during development and implementation of new molecular diagnostic tests. We identified key barriers to efficient implementation and propose possible solutions to these. In addition, we discussed the recent reconfiguration of molecular diagnostic services in NHS England and its likely impacts.We anticipate that this consensus statement will provide practical advice to those involved in the development of novel molecular diagnostic tests. Although primarily focusing on test adoption within the United Kingdom, we also refer to international guidelines to maximise the applicability of our recommendations. In the field of cellular pathology, it is now possible to supplement traditional light microscopic assessment of tissue samples with a vast array of information at genomic, epigenomic, transcriptomic, proteomic and metabolomic levels. Thus, molecular diagnostics is now the cornerstone of precision/personalised medicine, in which individual patients receive customised healthcare on the basis of their specific test results, and has the potential to revolutionise patient care and improve outcomes, as exemplified by its use in haematological malignancies.2 The application of molecular diagnostics is currently being expanded into other clinical areas; for example, in the United Kingdom (UK), the 100,000 Genomes Project has brought whole-genome sequencing into routine clinical practice by initially applying this technique to cancer and rare diseases.3Pathology—the study of disease—has evolved significantly since its beginnings with Virchow and a purely morphological description of cellular alterations, to our current ability to make fine-resolution observations at the subcellular/molecular scale.4 Until recently, there has been a lack of national strategy for molecular diagnostic testing with complex commissioning and funding arrangements.5 Moreover, the National Health Service (NHS) is currently poorly equipped to embrace fully this healthcare revolution. In particular, the substantial attrition of academic pathology in the UK over the past two decades, coupled with the increasing service demands placed on pathologists, means that many diagnostic laboratories lack the knowledge, expertise and capacity to introduce these new tests efficiently.6 In addition, the interaction between clinicians, academia, industry and regulators required to expedite the development of new molecular diagnostic tests and their introduction into clinical practice has not been uniformly present to date.Despite the promises of molecular diagnostics, significant barriers have impeded its widespread clinical adoption.https://cmpath.ncri.org.uk). To advance pathology in the UK, and thus ensure that patients receive the highest quality of care possible, CM-Path recognises the value of collaborating with industry, regulators and other key stakeholders. To this end, members of CM-Path workstream 4 (‘Technology and Informatics’) convened the first meeting of the CM-Path Molecular Diagnostics Forum on 26th January 2018 at the Royal Society of Medicine in London. The overarching aims of the forum are as follows:To define infrastructure, regulatory and workflow requirements for the adoption of molecular diagnostics in NHS pathology laboratories;To develop protocols to ensure faster and more efficient implementation of emerging technologies and novel bespoke and validated molecular panels;To assist in the education/training of the workforce required to provide high-quality, nationwide molecular diagnostic services;To actively engage pathologists with industry and regulators to develop the next phase of molecular diagnostic tests;To form links with companies developing software to assist in test interpretation and correlation between molecular findings and clinical outcomes.In 2016, the National Cancer Research Institute (NCRI) launched its Cellular Molecular Pathology (CM-Path) initiative with the aim of supporting modernisation of pathology in the UK and, in so doing, to help to develop the workforce and infrastructure required to provide nationwide molecular diagnostic services  that are likely to be encountered during these processes. The meeting commenced with invited case presentations on the development and implementation of new molecular diagnostic tests in rare ophthalmic disease  and bladder cancer , providing illuminating ‘real world’ insights into these processes. Summaries of the perspectives of industry and of the National Institute for Health and Care Excellence (NICE) on the current state of affairs were also presented by Jane Coppard (public affairs manager at Roche) and Rebecca Albrow , respectively. It was highlighted that NICE diagnostics guidance recommendations are typically made by the Diagnostic Advisory Committee (DAC), an independent decision-making body that bases its recommendations on review of clinical and economic evidence. Once recommendations are made, NICE diagnostics guidance is published on the NICE websiteIn a subsequent breakout session, delegates were grouped by professional background and tasked to create a roadmap describing the stages in the development of a new molecular diagnostic test, from initial concept to clinical implementation. This is particularly important as, compared with therapeutics, the validation and approval processes for diagnostic tests are poorly defined. It quickly became clear that no single group was able to map the entire pathway, immediately justifying the value of arranging this multidisciplinary meeting. Ultimately, a final roadmap was agreed by consensus between the groups Fig. ; access The groups were then mixed and asked to identify challenges that are likely to be encountered within the roadmap. Several key themes emerged during this discussion; importantly, a number of innovative solutions were also suggested Table .Table 1C8 By creating a single national testing network co-ordinated through seven Genomic Laboratory Hubs (GLHs), this reconfiguration aims to expedite widespread adoption of molecular diagnostics into routine clinical practice and to ensure that such tests are conducted to uniform standards, thus providing consistent and equitable care across the country. Building upon the success of the 100,000 Genomes Project, this project forms part of the Government’s Life Sciences Strategy,9 and aims to develop a world leading Genomic Medicine Service within the NHS, as well as to support scientific research and innovation more broadly. The new service now includes a National Genomic Test Directory for both cancer and rare and inherited diseases.10 This directory specifies which tests are available within the NHS and how they are funded, which patients are eligible to receive these tests and which technology platforms should be used to perform each test. The directory will be updated annually, based on recommendation from a Clinical and Scientific Expert Panel that will evaluate new genomic tests and determine which existing tests should be retired or replaced. The authors believe that this positive development will help with many of the challenges that we have identified but, crucially, it only currently covers genetic testing and not other forms of molecular diagnostics (e.g. infectious disease).A follow-up meeting was held in October 2018 to discuss these challenges in greater detail, and to consider how our roadmap will likely be impacted by the reconfiguration of genomic laboratory services within NHS England that took place that month.11 Likewise, lung cancer mutation status can have a significant impact upon immediate clinical management and rapid in-house testing can be very useful, particularly in the context of acutely unwell patients or where a prompt initial screening test result can avoid the need to perform further unnecessary tests .12 Initially, MDTs may also wish to arrange local funding for specific tests, rather than incur the time penalty involved in sending samples away. Nevertheless, the majority of molecular diagnostic tests are generally not urgent 13 and are therefore likely to be best performed in a centralised reference laboratory. Furthermore, over time, we hope that the GLHs will generate evidence to demonstrate that centralised testing can return results in a clinically relevant timeframe for most indications. Another reason to retain local testing might be when a centre has already developed expertise in the performance and interpretation of a specific test, which could not be delivered to the same standard through an associated GLH.Whilst this new system should help to deliver more uniform nationwide access to molecular diagnostic tests, some scope for local flexibility in testing strategy is likely to be of benefit to patient care. A crucial issue to consider when ordering a molecular diagnostic test is how this test is best integrated into each patient’s individual care pathway and we envisage that local multidisciplinary team (MDT) meetings will continue to play an important role in making such decisions. Some test results are needed more urgently than others and this can influence the type of test selected and whether this is performed locally or sent externally. For example, one-step nucleic acid amplification (OSNA) testing to detect cytokeratin 19 (CK19) mRNA copy numbers in homogenised axillary lymph node samples, as a marker of breast cancer sentinel lymph node metastasis, has been performed in some UK centres for many years, with rapid intraoperative results determining the requirement for nodal clearance as part of a one-step procedure.14 This may also entail the incorporation of digital pathology, which is currently being promoted via an Innovate UK initiative with the establishment of five centres of excellence for digital pathology, image analysis and artificial intelligence.16 Such approaches are likely to become part of integrated reporting, bringing together the clinical, morphological, immunohistochemical and molecular data, in order to improve diagnostics and patient management.It was felt by forum participants that GLHs could play an important role in the development of novel molecular tests by providing access to high-quality human tissue samples via linked academic biobanks and by assisting in test validation, particularly by facilitating rigorous comparison with established tests and by recruiting patients into clinical trials. Once an evidence base has been established, a key milestone for any new molecular test will be inclusion in the test directory and it is envisaged that this step could be aligned with approval by the NICE DAC. GLHs will also have responsibility for implementing newly approved tests, ideally working in collaboration with each other to ensure optimal quality control, and in monitoring test uptake and downstream clinical effects, for example by transmitting relevant information derived from genomic MDT meetings to a centralised repository of outcome data. Likely future challenges for the GLHs include extending molecular tests to include other ‘omics’ approaches  whilst at the same time ensuring standardised, high-quality performance of established techniques .17 Whilst earlier consolidations have produced cost savings,18 a large initial financial investment is often required, for example to cover the cost of new transport networks and to develop the information technology (IT) infrastructure required to connect different hospitals/laboratories. Critically, the NHS workforce remains central to the provision of high-quality diagnostic testing and there is a risk of loss of valuable expertise amongst staff who are not based in GLHs. Furthermore, sending tissue samples away for testing may negatively impact upon the ability of ‘non-hub’ centres to contribute to biobanking activities that are critical to support biomedical research. Given these risks, and to foster a new molecular medicine culture within the NHS, it is imperative that the seven GLHs  adopt a collaborative, rather than competitive, approach to service delivery. Importantly, shared leadership by pathology, genetic and clinical teams will be needed to deliver a truly integrated service.Centralised testing offers many benefits but there are also potential downsides to such an approach, and lessons should be learnt from previous reconfigurations of pathology services.20 Importantly, a requirement for formal molecular pathology teaching is now included in the Royal College of Pathologists (RCPath) ‘Curriculum for Specialty Training in Histopathology’;21 a 2-week molecular pathology attachment for histopathology trainees is now advocated22 and trainee knowledge of this area will be evaluated both through workplace-based assessment and formal professional examinations. The curriculum is currently undergoing further revision and it is envisaged that molecular pathology will feature even more prominently in the next iteration. In parallel, Health Education England (HEE), in partnership with several leading UK universities, provides formal postgraduate qualifications in genomic medicine as part of its Genomics Education Programme, as well as numerous other online-learning resources (https://www.genomicseducation.hee.nhs.uk). In addition, a range of professional training courses in molecular pathology are also available: ‘Molecular Pathology and Diagnosis of Cancer’ delivered by the Wellcome Genome Campus and RCPath,23 ‘UK Molecular Diagnostics Training School’ delivered by the Nottingham Molecular Pathology Node,24 ‘Molecular Pathology Study Day’ organised by the British Division of the International Academy of Pathology (BDIAP)25 and ‘Getting to Grips with Genomics’ which is a joint initiative between CM-Path, RCPath and HEE, and importantly, provides education in molecular pathology to both trainees and trainers alike.26Nationwide delivery of a ‘cutting-edge’ molecular diagnostic service will require large-scale upskilling of the current laboratory workforce, as well as amendments to the training of medical students, junior doctors and clinical scientists. With this requirement in mind, CM-Path, in collaboration with other relevant organisations, is actively working to develop training opportunities in molecular pathology.27 and the European Union (EU).28 In the UK, the Medicines and Healthcare products Regulatory Agency (MHRA) is responsible for ensuring that medical devices are safe for clinical use. Currently, there is a Europe-wide transition to the new EU Regulation on In Vitro Diagnostic Medical Devices 2017/746.29 This regulation sets out a new pathway for certification that will be carried out by approved notified bodies and Conformité Européenne In Vitro Diagnostic (CE IVD) approval is a sign of conformity with European standards. Whilst still to be confirmed, it is likely that these changes will apply in the UK even after its withdrawal from the EU. In the UK, all molecular assays and laboratory processes must also be accredited by the United Kingdom Accreditation Service (UKAS) through meeting a range of different International Organization for Standardization (ISO) requirements. UKAS also requires that IVDs undergo external quality assessment (EQA), with such quality control exercises most commonly conducted by the United Kingdom National External Quality Assessment Service (UK NEQAS). In the United States of America, IVDs are classified based on likely patient risk and are usually required to undergo premarket approval (PMA), unless there is a specific exemption.30 Through the Molecular Diagnostics Forum, for example, CM-Path is working closely with the MHRA and The British In Vitro Diagnostic Association (BIVDA) in order to ensure that regulators are involved at an early stage in the development of new diagnostic tests.Finally, legal, accreditation and regulatory frameworks must be considered when selecting or developing new molecular diagnostic tests. New in vitro diagnostic devices (IVD) must be approved before clinical adoption; regulatory guidelines for such approval exist both within the UKOur NCRI CM-Path Molecular Diagnostics Forum meetings proved to be highly constructive in identifying strengths and weaknesses in the application of molecular pathology across the NHS and the group is committed to facilitate continued collaboration between pathology (in both the NHS and academia), industry and regulators. To our knowledge, this is the first cross-sector attempt at defining the roadmap for molecular diagnostic tests, from conception through to deployment and use in accredited laboratories within the NHS. Whilst this process is currently complex, we believe that many of the challenges that we have identified can be overcome through closer collaboration between key stakeholders and with the network of GLHs. The next forum meeting will have a specific emphasis on addressing optimal sample handling for molecular testing, how the new ‘hub and spoke’ arrangement of GLHs will impact upon specimen journey from patient to laboratory and how molecular testing at GLHs can be potentially integrated with digital pathology being performed at the above-mentioned five new centres. Lessons learned will be integrated into the roadmap, further developing molecular diagnostic capabilities in the UK.CM-Path would be delighted to hear from any individual or group who feel that the Molecular Diagnostics Forum is relevant to their work and who would like to attend future meetings—please email cmpath@ncri.org.uk to get in touch.


Kaposi’s sarcoma (KS) is a vascular tumour of endothelial origin that is associated with human herpes virus-8 infection. In sub-Saharan Africa, AIDS-KS remains the most common HIV-associated malignancy, and hence it poses a huge burden to the already constrained health-care systems. KS has four clinical variants, namely, classic, endemic, iatrogenic and epidemic KS. The histopathology in these different KS forms is essentially identical; however, they have different clinical patterns. Expanding knowledge of KS biology increases hope for prevention, disease control, and hence better quality of life among patients. Primary prevention strategy for KS-associated herpes virus and management of disease complication, such as lymphoedema should be the focus of disease-prevention and -control research. Kaposi’s sarcoma (KS) is a multifocal angioproliferative disorder of vascular endothelium . There aFor decades, the aetiology and pathogenesis of KS was unknown. In 1994, Chang et al. , reporteAlthough KS lesions may be clinically typical, biopsy of the lesion is needed for diagnosis and before patients are subjected to cytotoxic treatment. The histologic features characteristically comprise of large endothelial cells spindle shaped tumour cells. Presence of erythrocyte extravasation, inflammatory mononuclear cell infiltration, lymphocytes macrophages and plasma cells tend to be diagnostic . AlthougKS most frequently presents as cutaneous lesions, which may occur in any site on the body . The lesThe most common differential diagnosis of cutaneous KS lesions in Uganda is bacillary angiomatosis; tissue evaluation is important in differentiating the two. However, microscopic features are quite similar showing abundance of proliferating mononuclear inflammatory and spindle cells, ill-defined vascular channels, haemorrhage and oedema. The best differentiator is, therefore, additional tests for KSHV DNA which is negative in bacillary angiomatosis . Since tCurrently, there is no vaccine against KSHV and no primary prevention. Given the lack of knowledge of the route of acquisition, no proper prevention message can be provided to the public. Riding on the back of the HIV prevention success, the measures in place for HIV can be used in the prevention of a KS epidemic. From an epidemiological perspective, KSHV presents a case for preventable vaccine for stopping transmission.From the clinical perspective, since the majority of tumour cells are latently infected with KSHV, lytic replication plays a major role in disease progression and virus dissemination . TherefoAt a population level, a key preventive strategy would be breaking the transmission of KSHV through exchange of saliva.Currently, KS has no cure. The goals of treatment are to control disease symptoms and prolong life, and therapy is tailored to the clinical variant of KS and disease stage. However, apart from highly active anti-retroviral therapy (HAART), treatment options are similar for the different epidemiologic forms of KS. The majority of patients with KS in sub-Saharan Africa present with advanced stage of the illness, and hence the aim of treatment is to control symptoms, palliate pain and improve their quality of life –25). SurCo-existing opportunistic infections, mainly TB are common among patients, with AIDS-related KS reflecting mainly the degree of immunosuppression, requiring appropriate treatment. Extensive lymphedema resulting from KSHV-induced exuberant proliferation of endothelial cells may lead to the occlusion of lymphatic vascular lumens leading to lymphedema and lymphadenopathy . There iKS is a vascular tumour of endothelial origin that is associated with HHV8 infection. In sub-Saharan Africa, AIDS-KS remains the most common HIV-associated malignancy and hence it poses a huge burden to the already constrained health-care systems. KS has four clinical variants, namely, classic, endemic, iatrogenic and epidemic KS. The histopathology in these different KS forms is essentially identical; however, they have different clinical patterns. Even though there is no cure at present, the expanding knowledge of KS biology increases hope for prevention and control of the disease hence better quality of life among patients with KS. Primary prevention strategy for KSHV and management of disease complication such as lymphedema should be the focus of research.The author has no conflicts of interest to declare.This work was supported by the Uganda Cancer Institute.


Escherichia coli and examined the effect of porins on antibiotic resistance and membrane integrity. The OmpF-defective mutant was resistant to several antibiotics including β-lactams, suggesting that OmpF functions as the main route of outer membrane penetration for many antibiotics. In contrast, OmpA was strongly associated with the maintenance of membrane integrity, which resulted in the increased susceptibility of the ompA mutant to many antibiotics. Notably, OmpC was involved in both the roles. Additionally, our systematic analyses revealed that other porins were not involved in the maintenance of membrane integrity, but several porins played a major or minor role in the outer membrane penetration for a few antibiotics. Collectively, these results show that each porin plays a distinct role in antibiotic resistance and membrane integrity, which could improve our understanding of the physiological function and clinical importance of porins.A defining characteristic of Gram-negative bacteria is the presence of an outer membrane, which functions as an additional barrier inhibiting the penetration of toxic chemicals, such as antibiotics. Porins are outer membrane proteins associated with the modulation of cellular permeability and antibiotic resistance. Although there are numerous studies regarding porins, a systematic approach about the roles of porins in bacterial physiology and antibiotic resistance does not exist yet. In this study, we constructed mutants of all porins in  The outer membrane (OM) of Gram-negative bacteria is a unique architecture which is composed of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrel porins . The OM ompF mutant was resistant to several β-lactam antibiotics in some Gram-negative pathogens, including Escherichia coli , membrane integrity-related non-specific porin (OmpA), and non-specific porins involved in both antibiotic transport and membrane integrity (OmpC and OmpF). OmpF and OmpA play a major role in antibiotic transport and the maintenance of membrane integrity, respectively. OmpC is important for both of the two functions. These results suggest the functional diversity of porins and explain why the effect of porins on antibiotic resistance is diverse depending on the kind of porin.In this study, we analyzed how all porins of The bacterial strains and plasmids used in this study are listed in ompAΔC mutant was constructed by replacing theregion between 197th codon and the stop codon of the ompA gene with the kanamycin-resistance gene containing its promoter region. The kanamycin-resistance gene was amplified using a forward primer with a synthetic linker (underlined) and FLAG-tag (in boldface type), 5′-ACGGCATGCTGAGCCTGGGTGTTTCCTACCGTTTCGGTC AGGGCGAAGCAGGCAGCGGCGACTACAAAGACGATGACGACAAGTAGCTTAGACGTCAGGTGGCACT-3′ and a reverse primer, 5′-AAAGGCAAAAAAAACCCCGCAGCAGCGGG GTTTTTCTACCAGACGAGAACACGCTCAGTGGAACGAA AAC-3′. The purified PCR product was electroporated into MG1655 cells harboring the pKD46 plasmid, and the deletion mutant was selected on LB plates with kanamycin at 37°C.The E. coli cells were grown in the Mueller-Hinton broth to a McFarland turbidity standard of 0.5 (approximately 1.5 × 108 cells/ml). Cultures were diluted 10-fold with Mueller-Hinton broth to reach a final concentration of 107 cells/ml. A 10 μl volume was spotted on the plates. After incubation at 37°C for 20 h, the MIC value was determined. The MIC is defined as the lowest concentration of an antibiotic preventing the lawn growth of cells.The MIC values were examined on agar plates with the agar dilution method according to the Clinical Laboratory Standards Institute guidelines . The Mül8 to 104 cells/ml in 10-fold dilutions. Aliquots of 2 μl were spotted onto LB plates supplemented with 2% SDS, 6% ethanol, 750 mM NaCl, 64 μg/ml vancomycin, or 20 μg/ml chlorophenyl red-β-d-galactopyranoside (CPRG). To determine the effect of the C-terminal domain of OmpA on antibiotic sensitivity, cells were spotted onto LB plates containing 6 μg/ml ampicillin, 2.5 μg/ml cefalotin, 6 μg/ml choramphenicol, 150 μg/ml clindamycin, or 5 μg/ml rifampicin. After 8–20 h incubation at 37°C, photographs of the plates were taken with a digital camera EOS 100D . Isopropyl-β-d-thiogalactopyranoside of 50 μM was added when experiments using CPRG were performed.Stationary phase cells grown in LB medium were serially diluted from 10ompA ompC ompF triple mutants were inoculated into LB medium. When the A600 reached 2, cells were stained with FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium dibromide], propidium iodide (red), DAPI  (blue), and SYTOX green (green), and then spotted on a 1% agarose pad prepared in phosphate buffered saline (PBS). Cells were visualized using a Nikon Eclipse Ni microscope .For fluorescence imaging, stationary phase cells of wild-type and E. coli, we constructed the deletion mutants for all porin genes of E. coli and measured the corresponding MIC values of various antibiotics from different classes  that is chromosomally deleted for the C-terminal domain of OmpA. Notably, all of the phenotypes of the ompA mutant were phenocopied by the ompAΔC mutant , membrane integrity-related non-specific porin (OmpA), and porins associated with both antibiotic transport and membrane integrity (OmpC and OmpF). The functional diversity of porins might indicate why the effects of porins on antibiotic resistance differ so widely depending on the kind of porin. The efflux systems strongly affect antibiotic resistance (In this study, we performed a systematic analysis examining the roles of all porins of ibiotics . The C-tibiotics . Althougenotypes . Increasenotypes . OmpF isenotypes , which iibiotics . AdditiompF gene . These rmpF gene , 4, 5. Dibiotics . OmpC seibiotics , which iibiotics . Furtheribiotics . Therefoibiotics . Our resresponse . Thereforesponse . Severalresponse . Therefosistance . TherefoE. coli specific porins, we identified a novel porin YddB which seems to be involved in the passive transport of novobiocin across the OM. YddB is an outer membrane protein that is predicted to have a β-barrel structure, but its function is unknown. The yddB mutant displays a dramatically decreased susceptibility to novobiocin compared to the wild type strain (Through our systematic study on e strain . The MICe strain ; therefoompC increases under high osmolarity conditions (ompC mutant was sensitive to various envelope stresses (In this study, we find that all non-specific porins, OmpA, OmpC, and OmpF, are involved in the maintenance of membrane integrity, despite a large diversity of their roles and influences. OmpA is known to affect membrane integrity through non-covalent interaction of its C-terminal periplasmic domain with peptidoglycan . The rolnditions . The ompstresses , but thestresses . MlaA isstresses . Becausestresses , the rolstresses . BecauseIn conclusion, our systematic study shows two possible roles of porins, the transport of antibiotics and the membrane integrity regulation, which differently affect antibiotic resistance, and the distinct role of each porin. This provides an important insight to understand not only the role of each porin as a functionally important component of the outer membrane, but also its specific role in the survival of the pathogen under the action of antibiotics.All datasets generated for this study are included in the manuscript and/or the C-RL contributed to the conception and design of the experiments. C-RL and UC researched and wrote the manuscript.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.