Driving in an adverse rain environment is a crucial challenge for vision-based advanced driver assistance systems (ADAS) in the automotive industry. The vehicle windshield wiper removes adherent raindrops that cause distorted images from in-vehicle frontal view cameras, but, additionally, it causes an occlusion that can hinder visibility at the same time. The wiper-occlusion causes erroneous judgments by vision-based applications and endangers safety. This study proposes behind-the-scenes (BTS) that detects and removes wiper-occlusion in real-time image inputs under rainy weather conditions. The pixel-wise wiper masks are detected by high-pass filtering to predict the optical flow of a sequential image pair. We fine-tuned a deep learning-based optical flow model with a synthesized dataset, which was generated with pseudo-ground truth wiper masks and flows using auto-labeling with acquired real rainy images. A typical optical flow dataset with static synthetic objects is synthesized with real fast-moving objects to enhance data diversity. We annotated wiper masks and scenes as detection ground truths from the collected real images for evaluation. BTS outperforms by achieving a 0.962 SSIM and 91.6% F1 score in wiper mask detection and 88.3% F1 score in wiper image detection. Consequently, BTS enhanced the performance of vision-based image restoration and object detection applications by canceling occlusions and demonstrated it potential role in improving ADAS under rainy weather conditions. Recently, the automotive industry has focused on the implementation of autonomous vehicles mounted with only cameras, which, like human drivers, primarily depend on visual perception ,2, as imDeraining ,14,15,16Although vision-based deraining approaches struggle with troublesome objects, vehicles have removed rain simply with a physical device, a windshield wiper, since 1903. This is one of the reasons why frontal view cameras for ADAS are integrated interior of vehicles to avoid ADAS performance degradation by the debris under rainy weather conditions. Nevertheless, although this intuitive and robust device brings clear visibility to an image, it creates unintentional occlusions, as shown in Even though wipers occlude images and deteriorate vision-based performances, only one study has been conducted on removing the wiper . In the Auto-labeling is an approach for obtaining approximate data to improve the efficiency of annotation. For auto-labeling windshield wipers, general vision-based object detection ,25, and We propose a behind-the-scenes (BTS) for windshield wiper-occlusion canceling by leveraging optical flow to maintain clear visibility of images while driving under rainy weather conditions. The main purpose of BTS is to extract the mask of a windshield wiper, a relatively fast-moving object in an image, by exploiting the predicted optical flows from a sequential image pair. Our method facilitates the extension of data diversity in optical flow datasets, pixel-wise detection of actual windshield wipers, and restoration of wiper-occlusion regions, as depicted in Real image data under rainy weather conditions were collected by driving a camera-mounted vehicle in a metropolitan city in Korea to address the absence of windshield wiper images. For data diversity, we acquired data considering various locations, precipitations, and seasons. Hand-crafted ground truth masks were generated only to evaluate the predicted wiper masks and scenes, while pseudo-ground truth masks and flows were extracted by auto-labeling using a pre-trained optical flow model. The idea of data synthesis is to mount a virtual wiper on a virtual camera lens of a synthetic dataset to implement intentional occlusions. Therefore, we overwrote the pseudo-data to MPI-Sintel , a commoWe employed the RAFT  as a basAcquisition of a real dataset of driving under adverse rainy weather conditions using windshield wipers;Implementation of a fine-tuning optical flow-based model with a synthesized dataset to detect precise windshield wiper-occlusion regions;Conception and realization of wiper-free rain images for autonomous driving datasets.The remainder of this paper is organized as follows: Until 2017, model-driven approaches were the prominent methods that rely on a statistical analysis of rain models and build a cost function to optimize. Kang et al. proposed a framework by formulating rain removal as an image decomposition into low/high-frequency parts based on morphological component analysis . Luo et With advancements in deep learning, such as convolutional neural networks (CNNs) or generative adversarial networks (GANs), data-driven methods have begun to solve more complicated tasks and improve deraining performance. Yang et al. constructed a CNN-based multi-task deep learning architecture that progressively detects and removes rain streaks and rain accumulation . Li et aAlthough deep learning-based approaches have enhanced deraining, performance degradation occurs when real rainy images are applied. In general, studies involving rain streaks and rain accumulation employ open datasets, such as Rain12 , Rain100Hnewa et al. employed various deraining models to real captured images under rainy conditions  from theAs mentioned previously, Qian et al. removed adherent raindrops with a GAN , but itsAdherent raindrops commonly appear on the windshield of an actual vehicle, and windshield wipers are mandatory devices to remove them, with apparent advantages and disadvantages. Although wipers clear adherent raindrops, they cause occlusion in the image data. Lin et al. classified wipers by training a principal component analysis (PCA) with extracted wiper masks from captured data using a constant camera position . Dalal eOpen datasets have driven significant developments in vision-based autonomous driving tasks. The quality, quantity, and diversity of data from various sensor modalities contribute intuitively to related techniques. Most datasets ,20,41,42Optical flow estimates the displacement vectors of all pixels in two images and is used for visual surveillance, robot navigation, image interpolation, or physical and metrological applications. Fischer et al. presented an end-to-end optical flow estimation network using CNNs and generated a synthetic dataset to train supervised learning-based networks . Ilg et Generating a precise pixel-wise ground-truth optical flow from real data is challenging because matching pixels between sequential images and obtaining displacement vectors is nearly impossible. For this reason, most optical flow datasets are composed of synthetic data, including FlyingChairs , FlyingTWe propose behind-the-scenes (BTS), a wiper-occlusion canceling model that detects windshield wipers in captured images from an in-vehicle frontal view camera sensor while driving in adverse rain environments. As depicted in We mounted a camera (Stereolabs ZED2 ) in the We extracted images from the recorded videos and acquired more than 150 k images at a resolution of WipersSpring, WipersSummer, and WipersFall and exploited them for different purposes.According to the Korea Meteorological Administration reports, our dataset contains image data under daily precipitation totals of 12.4 mm (27 August 2020), 47.7 mm (19 November 2020), and 56.2 mm (3 April 2021), while the maximum hourly precipitation ranged from 9.0 mm/h to 13.5 mm/h. Our data were collected during the daytime between 09:00 and 16:00 in three different seasons: spring (April), summer (August), and fall (November). We divided the datasets into Pseudo-ground truth wiper data for training and ground truth wiper scene for the quantitative evaluation were generated from WipersSpring. Hand-crafted ground truth wiper data for the quantitative evaluation were generated from WipersSummer and test images were selected from WipersSummer and WipersFall.true or false for wiper scenes based on the existence of severe truncation or occlusion by windshield wipers, and 6029 images were labeled as wiper scenes in the WipersSpring dataset. The ground truth data were utilized to evaluate wiper scene detection (WSD). As depicted in The main purpose of the ground truth is only to evaluate our prediction results regarding the wiper mask and scene, so we annotated the corresponding ground truths. We manually classified 34,960 images into The pseudo-ground truth in data synthesis is leveraged to address the limited data diversity of typical optical flow datasets and improve the wiper detection accuracy efficiently. Prior optical flow datasets, such as FlyingChairs , FlyingTWe initially searched for the optimized flow vector magnitude threshold for heuristically classifying wipers out of predicted optical flows in WipersSpring, and the optimized range was measured between 25.0 and 50.0 for an image size of Applying appropriate scenarios for sampling pseudo-ground truth for data synthesis is essential to our proposed method for enhancing data diversity. Unlike slow-moving objects in other typical optical flow datasets, Sintel is derived from a 3D animated short film composed of dynamic objects captured by a virtual shooting camera in 23 scenes of image sequences. Significantly, longer sequences are suitable for applying diverse sequential or random combinations of pseudo-data. Our idea is to mount virtual wipers to the virtual shooting camera lens to create similar occlusions by synthesizing the pseudo-ground truth wiper data into Sintel.S)starting returning ending  and 1041 flows (flow) in the training dataset. The model was trained with two TITAN RTX GPUs based on the following training schedule and parameters, as summarized in We adopted the Pytorch implementation of supervised learning-based RAFT as the baseline of our system because other networks such as FlowNet 2.0  and PWC-TP), false negative (FN), and false positive (FP) as follows:Images in WipersSummer and WipersFall were utilized for the quantitative and qualitative evaluations. For the quantitative evaluation, the structural similarity index measure (SSIM)  and binaBTS outperformed the other models in both SSIM and binary classification evaluations while producing 15 frames per second, as summarized in Raft-chairs scored the worst and seemed unable to overcome the domain mismatch because the model was trained with unrealistic and steady-moving synthetic objects different from the target object. Instead, raft-things, trained with more complex and diverse 3D objects, made significant improvements of +10.4%p SSIM, +59.7%p F1-WMD, and +42.6%p F1-WSD by adapting to the real domain. Even though raft-sintel was trained more with dynamic, diverse, and sequential frames of synthetic data, the performance was degraded. In contrast, fine-tuning with driving environment-based KITTI deteriorated by −18%p F1-WMD. However, by replacing Sintel with our generated synthetic datasets for fine-tuning, SintelWiper made the model much more accurate and balanced, proven by the +10.8%p F1-WMD and +7.6%p F1-WSD compared with raft-things. Higher F1 scores were accomplished by significantly improving recall in WMD and WSD. This means that BTS detects the pixels of wipers that other models missed, especially wipers in the ending states, which can be compared intuitively in the qualitative results.Moreover, the optical flow estimation EPE errors of BTS on the original datasets were slightly increased to less than +0.27%p, which is still an acceptable performance compared with the others, as summarized in As summarized in The predicted wiper mask for each model was overlapped with the original image for an intuitive qualitative comparison, as depicted in BTS demonstrated outstanding performance regardless of the speed of the wipers, as categorized by the bounding boxes in In Our main purpose of image restoration is to remove wiper-occlusions in images, restore information behind the wipers, and obtain clear visibility. We utilized a flow-based video completion application, FGVC , which rAs illustrated in Object detection is one of the core applications in ADAS because it outputs bounding boxes for surrounding objects to provide core information regarding confidence, location, size, number, and existence. Therefore, erroneous detection results may cause inaccurate decisions by ADAS to endanger safety. We tested a representative vision-based 2D object detection network, YOLOv4 , using tSince it is impossible to obtain corresponding ground truth images and requires expensive efforts to annotate bounding boxes of objects due to wiper-occlusions, we presented the test images with truncated or occluded objects whose predicted bounding boxes were qualitatively inaccurate. As illustrated in We achieved the detection of vehicle windshield wipers driving under rainy weather conditions, leveraging a synthesized optical flow dataset with generated pseudo-ground truth wiper data by auto-labeling acquired real datasets. Although the proposed method exploits optical flow for pixel-wise object detection, the brightness constancy assumption, which can deteriorate the optical flow functionality, was not considered. Empirically, the brightness under rainy weather conditions is stable compared with that under clear weather conditions because of the absence of the sun. Therefore, we tested our method, which was fine-tuned with rainy data only, to unseen images captured on a sunny day where fatal backlighting occurred. f), number of frames (n), and distance (d).Even though our datasets recorded ordinary driving circumstances for hours to capture various truncations or occlusions, there were no abnormal cases. However, it could be a concern when a wiper completely occludes an object in sequential frames. If our method provides such images to image restoration as inputs, the images would be recovered as if the object never existed. Assuming that such a situation occurred in the frames in The object should move at 198 km/h (approximately 123.8 mph) in the image through the calculation, which is uncommon and illegal in most driving environments so the authors believe that complete occlusions for sequential frames rarely occur. However, it would be a limitation of BTS if the case of a fast-moving object at the speed appears.Although the proposed method accommodates minor errors in the input images, limitations exist under heavy rain in the night-time. Higher hourly precipitation increases the number of adherent raindrops on a windshield that merge into larger droplets before the wipers operate. As a result, severe image deterioration occurs as a result of unpredictable light refraction and reflection in the droplets caused by countless random light sources in the darkness, including traffic lights, vehicle head/rear lamps, and street lamps. The authors believe that the complicated features of objects is one reason why classical hardware is still applied to vehicles in the 21st century. Resolving the complexity adherent raindrops for removal is our next step to overcome heavy rain without existence of wipers.The proposed method focuses on simple, efficient, and robust real-time wiper detection for driving under rainy weather conditions. As illustrated in the application demonstration in In addition, we expect to leverage our model to auto-label pseudo-ground truth for panoptic segmentation to observe capabilities and to apply various software techniques, such as pre-/post-processing, to develop our method in future work to search for a breakthrough. Developing the detection of unintentional occluding objects for vision-based tasks in various industries, including surveillance, anomaly detection, autonomous vehicles, and unmanned aerial vehicles (UAV), offers promising future applications for the proposed method.In this study, we developed a novel real-time windshield wiper-occlusion canceling model, BTS, for driving under adverse rain conditions by leveraging optical flow for data synthesis with pseudo-ground truth using auto-labeling. The purpose of the proposed technique is to provide a deep learning-based optical flow model to provide a precise mask for a fast-moving wiper that occurs in massive occlusion of image data. We acquired 150 k real images of driving in rainy environments in a metropolitan city in South Korea to augment a prior dataset for fine-tuning and to obtain hand-crafted ground truth wiper masks and scenes for objective evaluation. We synthesized the fast-moving object onto synthetic image sequences as if a virtual wiper was mounted on a virtual camera. We achieved accurate pixel-wise wiper mask detection by scoring an average of 0.962 (+0.025) with a standard deviation of 0.027 (−0.050) in SSIM, and an F1-score of 91.6% (+9.4%p) in binary classification, while wiper image scene detection showed an F1-score of 88.3% (+7.6%p). BTS demonstrated significant enhancements by applying it to vision-based applications to provide precise occluding regions for generating wiper-free images to enable object detection on previously invisible images. The proposed method also proved the compatibility, utility, ability, and extensibility to other vision-based applications can be leveraged to enhance their performance under adverse rain environments.


Chen and Segev share their perspective by highlighting quantum, topological, and AI photonics on  This perspective is written by two renowned scientists, Prof. Zhigang Chen and Moti Segev, who highlighted three major frontiers in photonics that will most likely undergo exciting growth in the next decade (2021–2030): integrated quantum photonics, topological photonics, and artificial-intelligence (AI) photonics.For graduate students looking for thesis topics, professionals keeping up with the latest directions, or funding managers identifying the next promising projects in photonics, a timely and well-crafted analysishttps://psiquantum.com) and Xanadu (https://xanadu.ai), are already on their way to produce quantum photonic chips.With the significant amount of funding from state governments as well as big companies, it is clear that quantum science will continue to take center stage in the near future. The holy grail of quantum technology is the long-term goal of making a fault-tolerant universal quantum computer. Among various approaches under keen investigations, Chen and Segev put their bets on integrated quantum optics. The main argument is the low coupling between photons and the environments, allowing room-temperature operations with the lowest error rates compared to other scalable platforms based on matter-qubits. At least two startup companies, PsiQuantum (Light: Science & Applications2). The field was initiated from a theory of the 2016 Nobel-laureate Duncan Haldane, who proposed the perfect (one-way) waveguiding mechanism at the edge of two-dimensional magnetic photonic crystals. Although time-reversal breaking is the necessary condition for complete back-scattering immunity, researchers have to relax this condition due to the weak magneto-optical effect at optical wavelengths. Although this comes with the penalty of reopening the back-scattering channels and conditional robustness, these non-magnetic topological waveguides are still stabler than their traditional counterparts. Chen and Segev emphasized the idea of using these edge channels to couple VCSEL arrays, addressing the long-standing issue of super-mode instability in phase-locked laser arrays that are intrinsically non-Hermitian and nonlinear devices.Topological photonics is a uniquely new field whose underlying principles are not covered in existing electromagnetics or optics textbooks. , Lightmatter (https://lightmatter.co), Luminous (https://luminous.co) among others.The sweeping success of two projects from Google’s DeepMind has reshaped our perception of AI, with Alpha-Go crashing the best go-players and Alpha-Fold predicting protein structures at unprecedented accuracy. We might be seeing the dawn of the AI age, as AI algorithms and products are penetrating our daily lives at an increasing rate, ranging from speech recognition, medical diagnosis to robotics. On one hand, AI can be used to predict photonic designs without solving Maxwell’s equations, on the other hand, photonic hardware can accelerate AI by performing machine-learning computations using photons with much higher throughput and lower energy consumption. A few startups are already building such optical AI-accelerators, including Lightelligence (3. The other is the hollow-core fiber4, whose propagation loss is expected to break the 0.14 dB/km record of silica fiber! Of course, what we anticipate the most are those ground-breaking discoveries that no one expects.Other than the aforementioned three areas featured by Chen and Segev, there are certainly many other exciting directions that are growing tremendously. Here in Fig.


Bacterial biofilms are aggregation or collection of different bacterial cells which are covered by self-produced extracellular matrix and are attached to a substratum. Generally, under stress or in unfavorable conditions, free planktonic bacteria transform themselves into bacterial biofilms and become sessile.Various mechanisms involving interaction between antimicrobial and biofilm matrix components, reduced growth rates, and genes conferring antibiotic resistance have been described to contribute to enhanced resistance. Quorum sensing and multi-drug resistance efflux pumps are known to regulate the internal environment within the biofilm as well as biofilm formation; they also protect cells from antibiotic attack or immune attacks. This review summarizes data supporting the importance of exopolysaccharides during biofilm formation and its role in antibiotic resistance.Involvement of quorum sensing and efflux pumps in antibiotic resistance in association with exopolysaccharides. Also, strategies to overcome or attack biofilms are provided. Leuconostoc mesenteriodes. In 1973, Characklis described these biofilms as persistent and resistant to disinfectant. The term “Biofilm” was coined by a Canadian microbiologist, Bill Costerton in 1978. He defined it as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface” [Mycobacterium tuberculosis [Pseudomonas aeuroginosa is one of the most well studied. It can serve as a model for understanding biofilm formation in bacteria. The exopolysaccharides produced by these bacteria can help them in immune invasion, increased pathogenicity, and enhance their survival. In patients of cystic fibrosis, biofilm forming Pseudomonas aeruginosa isolates can cause life threatening infections [Bacteria can produce a protective layer of a polymer during unfavorable conditions; it uses simple complex substrate to produce biopolymer of different characteristics. Microbial biofilm acts like a cooperative group made up of different microbial communities and shows structural as well as physiological complexity. Generally, the microbial biofilm develops on a substratum and is immersed in polymeric matrix that is secreted by the bacteria. Both auto and hetero-trophic microbes form stable biofilms for their survival in harsh conditions. They can develop in multiple environments such as sewage channels, bathrooms, laboratories, hospitals, biomedical equipment, hot springs, deep-sea vents, rivers, and rocks –3. Biofisurface” , 5, 6. Isurface” , 5. Biofsurface” . Cellulorculosis . The basrculosis . There hrculosis . There arculosis . The biofections . Althougfections  along wiBiofilm formation is a natural phenomenon, which occurs when the density of the population of bacterial cells reaches a certain threshold and so as a result leads to the formation of multiple micro-colonies. This process is further classified into four different phases, i.e., surface attachment, sessile growth phase which is controlled by intercellular interaction or colonization, biofilm maturation, and detachment –18  fimbriae, among which CupA is involved in adherence and auto-aggregation. Similarly, E. coli relies on type I pili assembled by CUP for surface adherence in a niche-specific manner. In E. coli along with type I pili, curli fimbriae and Antigen 43 are known to regulate attachment and inter-bacterial interactions. In the case of non-motile bacteria such as Enterococci, adhesins such as SagA, Acm (E. faecium) and Ace , and the surface protein Esp have been known to facilitate biofilm formation on abiotic surfaces [This is the first and the important step that involves the affixing of the bacteria to a solid substratum, and is highly dynamic and reversible depending on the situation. Initially, bacteria are introduced to the surface where it is going to be attached. This process is majorly carried out by Brownian motion and gravitational forces and is also impacted by hydrodynamic forces in the vicinity of the bacteria. As it settles down on a substrate, microbe encounter attractive and repelling forces which may differ depending on nutrient levels, pH, ionic strength, temperature, and also the microbe cell-surface composition affects the biofilm attachment by regulating its velocity and direction. Motile bacteria have an advantage over non-motile bacteria since it can use flagella to bridle hydrodynamic forces and repulsive forces and that has been observed in surfaces .Once the bacteria get attached to the surface, the attachment induces the differential gene expression, upregulation of factors favoring attachment. Factors identified to support sessility are ECMs such as cellulose, polyglucosamine (PGA), and colanic acid which also contribute to the architecture. The composition of the extracellular matrix varies within bacterial species depending on the environmental conditions .Pseudomonas species in response to high levels of c-di-GMP signal, which further binds to polysaccharide synthesis locus (PSL) and helps to maintain the biofilm structures. Alginate, an EPS found in P. aeruginosa, its synthesis is regulated by a surface-bound diguanylate cyclase MucR through high local concentrations of c-di-GMP. Along with EPS, e-DNA has been observed to regulate cell to cell interactions and stabilizes Pseudomonas biofilm at initial stages. e-DNA is organized in a well-defined pattern and is located in the stalk part of the biofilms which were shaped like mushrooms [Maintaining biofilm attachment is important to regulate pathogenicity. CdrA is an adhesin secreted by ushrooms .P. aeruginosa biofilms, detachment occurs due to increased carbon and nitrogen sources or due to action of the EPS degrading enzymes such as alginate lyase. Also, the void created within P. aeruginosa biofilms by cell death can facilitate biofilm detachment. In E. coli, the CsrA protein is known to inhibit PGA synthesis. The reduced c-di-GMP signal results in biofilm detachment in P. aeruginosa and E. coli [. Dispersing bacteria can form bacterial biofilm in suitable environmental conditions and thus result in chronic infections. So, this way it becomes difficult to treat biofilm-related infections.Biofilm detachment can occur due to changes in the availability of nutrients, instability in concentrations of available oxygen levels, an elevation in levels of toxins in the vicinity, or due to conditions that induce stress in the bacteria . Biofilm E. coli . DispersVibrio fischeri and Vibrio harveyi, and enzymes involved in QS were responsible for light production. The lux system of QS was first revealed in V. fischeri. Over 80% of bacterial species involved in biofilm formation exhibit this process. This process involves the production, release, and detection or recognition of extracellular signaling molecules called auto-inducers (AI) [Quorum sensing (QS) is a cell-cell communication process that enables bacteria to modify their behavior in response to changes in the cell density, pH, signal flow rates, and surrounding microbial community , 19, 20.ers (AI) , 19, 21.ers (AI) , 20, 22.ers (AI) .l-homoserine lactone (N-AHLs) as a chemical signal. Whereas in Gram-positive bacteria, chemical signals vary in nature which includes modified peptides, amino acids, and similar amino compounds bind to receptor histidine kinases [V. harveyi induced by AI-2, hydroxyl–palmitic acid in Ralstonia solanacearum, diketopiperazines in P. aeruginosa, methyl dodecanoic acid, and quinolone are examples of QS signaling molecules in Gram-negative bacteria. Gram-positive Bacillus subtilis and Staphylococcus aureus utilize eukaryotic-like Ser/Thr kinase-mediated regulation of biofilm formation [The QS mechanism differs from Gram-negative bacteria to Gram-positive bacteria. The Gram-negative bacteria uses low-molecular-weight acyl derivatives of  kinases . Oligopeormation .In gram-negative bacteria, LuxI/LuxR-type QS mechanism has been described to induce virulence. During the process, AHLAI synthesized by LuxI homolog diffuses through the cell membrane, and at high concentration it binds and stabilizes the cognate LuxR receptor. This AHL-LuxR receptor protein complex results in an alteration in the expression of several genes while auto-inducing LuxI expression in feedforward reaction to produce more AHL AI to amplify the signal , 17. QS Bacillus subtilis and Streptococcus pneumoniae, virulence in S. aureus, conjugation in E. faecalis and microcin production in Lactobacillus sake and Carnobacterium piscicola. Instead of LuxI/LuxR signaling circuit, Gram-positive bacteria consist of two-component signal transduction system (2CSTS); they secrete modified peptide signaling molecules that are usually dedicated by ABC (ATP-binding cassette) exporter protein. Secreted peptide signals are recognized by cognate two-component sensor kinase proteins that are known to interact with cytoplasmic response regulator proteins. Most of the sensor kinases found are membrane-bound proteins that auto-phosphorylate in the presence of ATP at a conserved histidine residue, from the phosphoryl are transferred to a conserved aspartate in the response regulator, thus this signal transduction mechanism is a phosphorelay cascade [S. aureus, since it can cause a tremendous range of disease, from simple skin infections to life-threatening ailments, such as sepsis, endocarditis, and osteomyelitis.In Gram-positive bacteria, there is a number of processes present that work in response to cell population density. In the absence of an outer membrane, the Gram-positive bacteria uses the cell wall as the scaffold for displaying a wide variety of surface molecules, which include teichoic acid, lipoteichoic acid, and several protein adhesins. Some of the surface proteins are bound to the cell wall non-covalently, and other proteins are anchored covalently to the peptidoglycan. Well-described QS system in Gram-positive bacteria includes competence for DNA uptake in  cascade , 24. Num cascade . In the P. aeruginosa biofilm development. Type IV pili plays a direct role in stabilizing interactions between the abiotic surfaces required to form a micro-colony and also helps the cell to migrate along the surface to form multi-cell aggregates. Strains defective in pili biogenesis expresses neither twitching motility nor micro-colony formation phenotypes. The pilBCD operon is thought to encode accessory factors required for type IV pili biogenesis [Both flagella and twitching motility play an important role in the initiation of biofilm attachment. Motility is important for the cells to make initial contact with an abiotic surface. Type IV pili and type V pili mediate twitching mobility in ogenesis .Efflux pumps are membrane proteins responsible for the extrusion of substances from within the cell into the extracellular space. These pumps are found in both Gram-positive and Gram-negative bacteria as well as in eukaryotic organisms , 20, 27.Campylobacter jejuni (CmeABC), E. coli , P. aeruginosa , Streptococcus pneumoniae (PmrA), Salmonella typhimurium (AcrB), and S. aureus (NorA) [Bacterial efflux pumps are classified into five families depending on the number of components, number of transmembrane spanning regions, the energy source utilized for the export, and the types of molecules that are exported: MF (major facilitator) superfamily, MATE (multidrug and toxic efflux) superfamily, RND (resistance-nodulation-division) superfamily, SMR  superfamily, PACE  superfamily, and ABC (ATP binding cassette) superfamily. The ABC family transporter makes use of ATP hydrolysis to stimulate the export of substances, whereas other family transporters make use of the proton motive force as an energy source. The expression of these pumps is tightly regulated , 27–31. Burkholderia pseudomallei dependent on BpeAB-OprB efflux pump [QS system are known to control biofilm formation by regulating expression of efflux genes. One such example is biofilm formation in lux pump   and helps to escape host immune system. Therefore, it is crucial for the stability and persistence of mucoidal strains [Staphylococci strains are one of the primary causes of nosocomial infections, specifically associated with infections on implanted medical devices and catheters. Staphylococcal biofilms mainly consist of PNAG or PIA EPS. In the case of S. epidermis, PIA protect by reducing the ability of phagocytes and from antimicrobial peptides [Bacterial species such as  and Psl , 41. Amo and Psl . Pel pol and Psl  and can  and Psl . Psl phy and Psl , 44. Whe strains  in cysti strains . Staphylpeptides  Fig. 2)S. aureusP. aeruginosa block the deposition of complement on the bacterial surface; consequently, this provides an advantage to biofilms and helps to avoid immune attack. Along with these benefits, it reduces the chance of phagocytosis, the release of ROS, and cell attack by neutrophils [Kingella kingae PAM galactan, a galactofuranose homopolymer, provides resistance to serum killing by preventing opsonin deposition and evading complement-mediated killing. The functions of EPS depend on the polymer structure and composition [The complement system, a multifunctional group of circulating proteins, is a key component of innate immunity that recognizes pathogens through chemotaxis and opsonization. Complement once activated deposits proteins on the bacterial surface and promotes lysis of that particular bacteria. However, the EPS produced by the bacterial biofilms protect the cells from the complement components of the immune system . For an trophils , 46. Sintrophils , 43, 46.trophils , 48. EPSposition  S. aureus, azithromycin increases the biofilm matrix production as well as sub-MIC of lactams led to increased e-DNA, and autoaggregation in biofilms. Antibiotics such as erythromycin, tetracycline, and quinpristin-dalfopristin when used at sub-MIC enhance ica genes expression in Staphylococcus epidermidis, which results in increased EPS production. In P. aeruginosa, sub-MIC tigecycline increased PNAG production and also the aminoglycoside, sub-MIC of impenem enhances the production of alginate and thus renders antibiotic resistance [P. aeruginosa biofilms [Generally, EPS are produced under antibiotic pressure or stressful conditions, since this is one of the mechanisms adapted to protect cells against different micro-organisms or antimicrobial agents . Sub-minsistance . The worsistance . Inactivsistance . In somebiofilms , 48.Acinetobacter baumannii biofilm-related infections, capsule structures in the biofilm matrix have been found responsible for tolerance to antibiotics such as AMPs. Also at sub-MIC, the transcriptional inhibitors such as Cm and Em cause rapid and reversible production of capsular EPS, thus conferring resistance to antibiotics as well as complement-mediated serum killing. One of the reasons for overproduction of EPS is the activation of BfmRS TCS. Capsular EPS also blocks the deposition or access of the serum complement components required for lysis. A study carried out by Vazquez-Rodriguez et al. showed that Rhodotorula mucilaginosa UANL-001L yeast strain was able to produce EPS under metal-stress conditions, and also when co-cultured with E. coli result in increased EPS synthesis [In the case of ynthesis .P. aeruginosa biofilm and thus overproduced biofilms protect from oxidative radicals [P. aeruginosa and E. coli. Iron used in the Fenton reaction stimulates the biofilm formation in E. coli, and sometimes the redox-active phenazine pyocyanin produced by P. aeruginosa also stimulates the biofilm formation. Oxidative stress, hypoxia generated by the host as defense, lead to drug-tolerant and persister cells in Mycobacterium tuberculosis biofilms. A. Trivedi et al. showed that reductive stress triggered by dithiothreitol (DTT) results in the formation of biofilm in M. tuberculosis. It was shown that reductive stress leads to envelope stress response wherein the envelope and periplasmic proteins unfolds and results in downregulation of ribosome biogenesis and translation machinery. Another way is that cellulose polysaccharide produced by bacteria combines with host polymers and create a complex and long-lasting shelter from leukocytes [Oxidative stress is one of the conditions that induce the formation of biofilm. When exposed to oxidative stress, alginate production increases within the radicals . ROS proukocytes  Fig. 4)P. aerugiCandida albicans EPS, β-1,3-glucan, can bind to ofloxacin; so, when C. albicans present with other bacteria such as E. coli increases the tolerance to ofloxacin for E. coli biofilms. Likewise, when S. aureus present within C. albicans biofilm can resist vancomycin treatment. Psl produced in P. aeruginosa biofilm confer resistance to non-Psl producing bacteria against colistin [E. coli and S. aureus, which are incapable of synthesizing Psl, can be provided with resistance if co-cultured in biofilm with bacteria expressing Psl [P. aeruginosa, P. protegenes and Klebsiella pneumonia, Psl polysaccharide produced by P. aeruginosa protected other two pathogens, while the alginate and Psl mediate surfactant tolerance such as SDS in the community [Moraxella catarrhalis and Streptococcus pneumonia, M. catarrhalis using quorum signal confer passive resistance to S. pneumonia against b-lactam antibiotics and bacterial clearance, whereas S. pneumonia provided protection to M. catarrhalis from azithromycin killing [The polymicrobial nature of biofilms and proximity between bacterial species helps the non-resistant bacteria to tolerate antibiotic treatment relative to biofilms formed by an individual species. EPS and other biofilm components that segregate the antibiotics can provide resistance to other species that lacks this property, mostly via horizontal transfer of resistance conferring genes. For example, colistin , 36. Bacsing Psl . Periasaommunity . A study killing  Fig. 5)Candida aC. albicans and Streptococcus mutans have been known to be associated with early childhood caries, which modifies the biofilm environment by enhancing the production of EPS and also increases the bulk of the biofilm. So, in this study, C. albicans stimulates the expression of glucosyltransferase B (GtfB), responsible for the formation of α-glucans biofilm matrix and its adhesion to the surface. When treated with a combination of drugs, EPS produced by the bacteria enhances tolerance to anti-fungal drugs by producing a dense layer of the polymeric matrix surrounding the fungal cells and decreasing the drug uptake [Polymicrobial interactions between g uptake . So, it Generally, the role of matrix components in mono-species biofilms is studied for their antibiotic tolerance, so it becomes challenging to understand the role of biofilm matrix in multispecies bacterial biofilm and thus it becomes difficult to treat bacterial biofilm in real. To treat multispecies biofilms, it is necessary to understand cell to cell interactions within biofilm to develop better strategies for the prevention of biofilm formation and increase the therapeutic values of typical antibiotics.N-acetylglucosamine (GlcNAc), produced by Staphylococcus species, Yersinia pestis, Bordetella species, and E. coli; Pel produced by P. aeruginosa is made up of GlcNAc and N-acetyl galactosamine , and β-1,4-linked N-acetylmannosamine polysaccharide produced by Listeria monocytogenes consists of terminal α-1,6-linked galactose  residues. All these polymers with hexosamine residues are partially deacetylated by polysaccharide deacetylases and as result, exposed amino groups are protonated and thus renders polysaccharide polycationic property. Many studies have shown the production of deacetylated polysaccharide can confer resistance and tolerance to host defenses and antimicrobial agents. For example, loss of PNAG deacetylation makes Staphylococcal species prone to neutrophil phagocytosis. PNAG inhibits the deposition of the complement on the cell surface of pathogens such as Bordetella species.A broad range of microbial species produces hexosamine-rich EPS such as PNAG, a homopolymer made up of S. epidermidis provides resistance to cationic LL-37 and β-defensin 3 AMPs through electrostatic interaction and also protects the biofilm integrity from the bactericidal activity of anionic human AMP dermcidin [Aggregatibacter actinomycetemcomitans confers resistance to the cationic detergent cetylpyridinium chloride and in S. epidermidis protects the biofilm from microbicidal action of vancomycin [E. coli, provides negative charge to outer cell surface and thus acts as a shield which prevents the entry of protons into the cells. This EPS act as protective barrier and enhances the pathogen survival in acidic condition like that of the human stomach [These cationic EPS protect biofilms from charged antimicrobial agents through electrostatic repulsion and deplete anionic molecules within the biofilm matrix to decrease the penetration of the agents . For exaermcidin . PNAG prncomycin , 56. Simncomycin . Extracencomycin . Colanic stomach .S. epidermidis and S. aureus protect the biofilm cells from the actions of cationic. In P. aeruginosa, alginate when bind to AMPs can generate helical conformational for AMPs such as magainin II and cecropin P1, which is kind of similar to their interactions with the cytoplasmic membranes. So it can be concluded that alginate even though they are hydrophilic in nature they can mediate hydrophobic interactions with the following AMPs [Mechanism for biofilm-associated resistance to AMPs mostly involves its interaction with EPS in the biofilm matrix. As seen earlier, the positively charged EPS can prevent entry of charged AMPs in the biofilm through electrostatic repulsion. For an example, PIA the polysaccharide produced by ing AMPs .P. aeruginosa produces several EPS such as alginate, Pel and Psl, PelD, PilZ, FleQ, and BcasA/BcsB. P. aeruginosa uses its QS system in the early stages of biofilm development, where it initiates the differentiation of individual cells into a complex multicellular biofilm. Two LuxI/R pairs  which function in tandem to control the expression of virulence factors exist in this bacterium [las and rhl QS system consist of the LuxRI homologs LasRI and Rh1RI respectively. The LuxI homologs LasI and Rh1I are important for the regulation of the synthesis of las and rh1 signals, N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) (PAI1), and N-butanoyl-l-homoserine lactone (PAI2), respectively. At low population density, LasI induces low production of 3OC12-HSL, whereas at high population density LasR binds its cognate AHL AI, and together they bind at promoter elements enhancing the production of virulence factors that are responsible for host tissue destruction during initiation of the infection process and also activates the transcription of lasI, rhlR. PAI1 is known to activate the Lux-R type transcription factor LasR and as a result, LasR-PAI1 triggers the production of various virulence factors such as LasB elastase, LasA protease, alkaline protease, exotoxin A, and LasR1. The las QS system is required for the biofilm development, so P. aeruginosa lasI mutants do not develop into mature biofilms and are completely flat and detergent sensitive and the rhlI mutant formed a colony that appeared hyper-wrinkled. Las QS system stimulates the transcription of the pel genes and also influences biofilm formation by P. aeruginosa PA14. PAI2 together with cognate regulator Rh1R enhances the production of secondary metabolites such as rhamnolipid, pyocyanin, LasB elastase, hydrogen cyanide, and cytotoxic lectins. The las QS system regulates the rh1 QS system both at the transcriptional and post-transcriptional levels, as both these QS systems comprised hierarchical cascade and any mutation in QS can result in reduced virulence and thus reduces the pathogenicity [acterium . The lasgenicity , 58, 59.lasB requires the cooperation of PQS and NAHL. PQS is synthesized at the late stationary phase and it determines the interaction between the las and rh1 irrespective of the population density [An intracellular signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) induces rh1I and the production of  density , 31. PQS density .S. aureus uses a QS system with auto-inducing peptides (AIPs) as signaling molecules to regulate the expression of several virulence factors as well as the synthesis of AIPs. In a S. aureus, only one copy of the QS system, i.e., agr system will be present on the chromosome, but the system can be any one of four types, each with the same basic components. The difference in each system will result in the formation of unique AIP signal such as AIP-I, AIP-II, AIP-III, and AIP-IV, and also cause corresponding changes in AgrB and AgrC to build and detect this signal, which is sometimes referred as the variable region. At high population cell density, signaling molecules produced and accumulated in the extracellular space are recognized by the bacteria and results in alterations in their transcriptional behavior. The Agr system is encoded by a 3.5-kb locus on the chromosome and consists of two transcriptional units, RNAII and RNAIII, driven by the two promoters P2 and P3, respectively [agrBDCA genes; these genes are known to encode proteins required for AIP biosynthesis, transport, signal perception, and subsequent regulation of target genes. First, the signaling transduction starts with the transcription and translation of AgrD, which is the pro-peptide precursor of AIP. Secreted AgrD is post-transcriptionally modified into the final peptide by AgrB, which is an integral membrane endopeptidase, and is also modified via the type I signal peptidase SpsB. At high AIP concentration, AIPs bind to membrane-bound histidine kinase AgrC; this process further leads to auto-phosphorylation and initiation of the signal transduction. Once the signal cascade is initiated, AgrC further phosphorylates AgrA, which triggers the expression of RNAIII from the promoter P3. RNAIII controls the upregulation of genes encoding for proteins such as toxins and exoenzymes, also it downregulates the expression of genes encoding for surface associated adhesins. However, RNAIII transcript is responsible for the regulation of the majority of the genes; there are some genes which are directly regulated by AgrA. Among these, the most prominent is the direct regulation of RNAII from the P2 promoter, leading to upregulation of AIP production and a positive feedback loop. The psm and psm transcripts are regulated by AgrA as well as the hld gene for toxin is encoded within RNAIII. QS system activity is low at the initiation of S. aureus biofilm formation since Agr QS system can repress the expression of surface associated proteins such as FnBPs and SdrC, which are important for cell attachment. Agr QS system is activated in local patches instead of uniform expression and results in detachment of bacterial cells; this Agr QS activity creates a void in the biofilm which is later filled by regrowing cells in those areas. This cell detachment process is characteristic of S. aureus biofilms. Upregulation of Agr-related proteases by the Agr QS system is a consequence of the negative regulation of Rot via the RNAIII effector, whereas the core genome-encoded PSMs are directly regulated via AgrA. Therefore, activation of the Agr QS system leads to upregulation of PSMs and matrix-degrading enzymes, which are responsible for biofilm structuring and detachment of staphylococcal cells from the mature biofilm. As a result, inactivation of the Agr QS system enhances biofilm formation on certain surfaces by obstructing or delaying the dissemination process of the biofilm [ectively , 25, 60.Staphylococcal adherence to biotic surfaces is mediated by cell wall anchored (CWA) proteins such as the fibrinogen-/fibronectin binding protein such as FnBPA and FnBPB and clumping factors A and B of S. aureus. ECM consists of large water filled channels, which accumulate antibiotic degrading enzymes such as β-lactamases and plays role in the adaptive resistance mechanisms due to e-DNA [to e-DNA .S. aureus biofilms. An early biofilm dispersal event, termed exodus, that is part of the five-phase biofilm model, takes place before tower formation in S. aureus biofilm maturation and is independent of Agr QS. With a set of elegant experiments, the expression of Nuc preceded the exodus event, which likely led to the degradation of e-DNA in the biofilm matrix. The exodus event could directly be linked to the 2CSTS system, as neither a sensor kinase nor a nuc mutant exhibited the exodus phenotype. As a result, it was found that nuc transcription was regulated by the 2C system in their biofilm model. Sensor kinases show polymorphism across S. aureus strains. For example, a point mutation in sensor kinase present in S. aureus Newman leads to hyperactivity of the SaeRS TCS, which was linked to the inability of this strain to form robust biofilms. In a recent study, it was also determined that several 2CSTS-regulated virulence genes in S. aureus are stochastically expressed in a subpopulation of cells in the nascent biofilm with an expression pattern matching the one of the thermonuclease Nuc. In S. epidermidis, mutation of saeRS leads to enhanced biofilm formation concomitant with higher Aap expression and e-DNA release. The derepression of SasG, a surface protein that promotes intercellular interactions and was implicated in the abiotic surface attachment [Sensor kinases recognize host signals such as human defensins and regulates the expression of various immune-stimulatory exo-proteins important for pathogenesis and biofilm generation, among them hemolysin (Hla), the MSCRAMMs FnbA and FnbB, and the extracellular thermonuclease Nuc are well studied. Fibronectin-binding proteins are important for biofilm formation at the level of intercellular accumulation, allowing cells to bind together via homophilic interactions between surface proteins, while Nuc is known to cleave e-DNA, a structural component of tachment , 60.S. aureus. The agr regulatory system controls a number of toxins and enzymes and also identifies the exact agents responsible for biofilm dispersal. Many studies have described that surfactant properties of δ-toxin can be responsible for the cell dispersal. δ-toxin was part of a larger family of PSM peptides, so multiple PSMs are critical in modulating S. aureus biofilm structure. Hence, most of the clinical isolates of S. aureus can make biofilms that are protease-labile, and also secretes many extracellular proteases that are known to self-cleave surface adhesins [When the level of the AIP signal is controlled, the entire biofilm could be dispersed and resensitized to antibiotics. This mechanism is highly conserved across strains and even functions in the emerging adhesins .S. mutans showed that expression of efflux transporter encoding genes increased when cultured with sub-MIC of tetracycline, also when exposed to heat shock or osmotic stress. Liu et al. reported that when S. mutans treated with sub-MIC of chlorhexidine (CHS) resulted in increased expression of differentially expressed efflux pump genes lmrB in order to survive in adverse environment. Inactivation of this efflux pump may result in overexpression of other homolog efflux pumps to compensate for the loss of lmrB as well as significant increase in the biofilm biomass as a consequence of enhanced thickness and EPS production was observed [E. coli treated with fluoroquinolones resulted in increased efflux and decreased penetration of the agent [A connection between the EPS diffusion barrier or the cell membrane and a multi-drug resistance (MDR) pump in providing resistance or tolerance to antibacterial agents has been observed in several bacterial pathogenesis . Drug efobserved . When E.he agent . It can A. baumannii, MDR pumps such as AdeABC, AdeFGH, and AdeIJK belonging to RND superfamily play an important role in biofilm formation. So, when exposed to sub-MIC of meropenem and tigecycline resulted in overexpression of adeB and adeG genes and increased biofilm formation. Also elevated expression of adeB and adeG genes is positively correlated with transcription level of adeI gene, thus displaying relation between the RND efflux pumps and QS. Also, the increased expression of AdeABC and AdeFGH transporters induces the export of AHLs which lead to increased biofilm formation [P. aeruginosa mutant lacking MexAB-OprM efflux pumps, reduced biofilm formation as well as reduced production of QS-controlled virulence factors have been observed due to intracellular accumulation of 3OC12-HSL, an important AHL that induces QS [P. aeruginosa treated with azithromycin showed reduced biofilm formation as a consequence of reduced production of 3OC12-HSL and N-butyryl-l-homoserine lactone (C4-HSL). So when both the molecules were added in the presence of azithromycin, it resulted in recovery of biofilm formation, thus confirming their role in biofilm formation [S. aureus, three genes which code for MFS efflux pumps, i.e., NorB and NorC pumps are known to export substances such as cetrimide, ethidium bromide, quinolones, and tetraphenylphosphonium, whereas MdeA pump export wide range of quaternary ammonium compounds and antibiotics. Acid shock and reduced aeration results in overexpression of NorB to protect biofilms [In ormation . In P. aduces QS , 31. Wheormation , 20. In biofilms .P. aeruginosa. Out of all the known efflux pumps, MexAB-OprM, MexCD-OprJ, MexEFOprN, and MexXY that have been mostly studied in P. aeruginosa [E. coli and is known to export substrates such as chloramphenicol, fluoroquinolones, fusidic acid, rifampicin, tetracycline, ethidium bromide, bile salts, etc. These efflux systems are overexpressed in bacterial biofilms and due to exposure to several antibiotic agents [P. aeruginosa [P. aeruginosa biofilm-related infections may show resistance to fluoroquinolones as a result of mutations in the genes encoding DNA gyrase, topoisomerase IV, or due to overexpression of efflux pumps which cause accumulation of antibiotics in the bacterial cell [E. coli export chloramphenicol, fluoroquinolone, tetracycline, novobiocin, rifampin, fusidic acid, nalidixic acid, and b-lactam antibiotics. On the other hand, the AcrAB-TolC efflux pump in S. typhimurium exports antimicrobial agents such as quinolones, chloramphenicol, tetracycline, and nalidixic acid. In the case of P. aeruginosa, MexAB-OprM efflux pumps which are the homolog of AcrAB-TolC and MexXY-OprM efflux pumps together export fluoroquinolones, tetracycline, and chloramphenicol [Several MDR pumps encoding genes belonging to RND superfamily have been known to play a role in antibiotic resistance in ruginosa  are resiruginosa . MexAB-Oruginosa . Anotherc agents . Overexpc agents . Overexpruginosa . P. aeruial cell . Efflux phenicol .V. cholerae to target phagocytic cells or bacterial cells hampering their growth and confer tolerance. There are few bacteria like P. aeruginosa which have to adapt a unique mechanism to tackle T6SS attacks by constructing its own T6SS apparatus. It was proven that a layer of EPS could act as a shield or physical barrier and protect the bacterial colonies from exogenous T6SS attacks. The defense mechanism by EPS depends on its structure like if the EPS consists of open channels that are perpendicular to the cells; the ability to block the attack would depend on the angles of the attacks. Another structure would be the presence of small gaps in the EPS layer, where the T6SS apparatus is localized at the gaps [One of the techniques used by most Gram-negative bacteria to survive in a mixed-species biofilm is the type 6 secretion system (T6SS). The T6SS is like a contact-dependent system which does not require a specific receptor to target cells and thus deliver toxic effector into surrounding cells. This system provides an advantage to bacteria like the gaps . This tyP. aeruginosa affects the expression of the T3SS genes negatively, as a consequence of reduced expression of the exsA gene that encodes for the transcriptional activator of T3SS [Salmonella enteritidis MDR mutants revealed that reduced expression of invasion genes in the SP-1 region encoding a T3SS and genes responsible for flagella biosynthesis reduced the ability of the bacterial cell as well as its invasion capability thus hampering the bacterial adherence [A connection between MDR efflux pumps and expression of the type III secretion (T3SS) system which has been implicated in several bacterial pathogenesis has been described. Continuous overproduction of MexCD-OprJ or MexEF-OprN efflux pumps in  of T3SS . Anotherdherence .Once biofilms are established, it becomes difficult to get rid of it. The main purpose of any research should be to find new molecules to target the biofilm mode of growth to prevent and treat chronic bacterial infections. Since EPS are known to protect against antibiotics and the immune system, the main focus should be on developing drugs that could weaken or disrupt the biofilm by targeting the EPS production and other extracellular matrix components.P. aeruginosa biofilms and as a result, they were capable of reducing biofilm formation. These compounds also showed anti-virulence activity. Out of all the compounds studied, acetylcholine, a known anti-biofilm compound, was confirmed repressor and there were compounds which were structurally similar and shared benzothiazole component. These compounds did not inhibit the bacterial growth but reduced the expression of Pel gene, thereby affecting the biofilm growth, and thus can be used for treating P. aeruginosa infections. Causing mutations in EPS expressing genes can reduce the ability to form a biofilm, by reducing the bacterial cell adherence and expression of EPS in the biofilm [High-throughput screening (HTS) approaches are being applied to look for new molecules which could reduce biofilm formation, and which could also possibly detach preformed biofilms in many species of bacteria. Van Tilburg Bernardes et al. found Pel repressors that could reduce the expression of the Pel genes in  biofilm .P. aeruginosa-related infections PslG and PelA, a glycosyl hydrolase can prevent biofilm formation as well as disrupt already established biofilms from a broad range of Pseudomonas strains at nano-molar concentrations [P. aeruginosa biofilm prone to colistin treatment [P. aeruginosa biofilms as it hydrolyzes the e-DNA with the extracellular matrix [cis-2-decenoic acid; anti-matrix molecules, or surfactant molecules such as chitosan, rhamnolipids, and urea; and sequestration molecules like EDTA and lactoferrin are used for surface detachment of the biofilms [Mainly detachment process is carried out by sing EPS degrading enzymes such as glucanohydrolases, dispersin B, or DNAse to eradicate biofilms  have shotrations . So pre-trations . Degradireatment . Anotherr matrix . The bior matrix . The antbiofilms . This maP. aeruginosa biofilms can be used to in combination with antibiotics to treat chronic infections effectively [Using different antibiotics in combination with biofilm disrupting drugs or antimicrobial agents has proven to be an innovative approach to treat biofilm-related infections. This way the effectiveness of antibiotics used can be enhanced and so together they could synergistically affect the biofilm formation as well as decrease the probability of resistance evolution. Nitric oxide known for disrupting ectively .S. aureus (MRSA) pathogens. In MDR P. aeruginosa, AMP like synthetic cyclolipopeptide analog of polymyxin in combination with carbapenems kills the established biofilm [N-acetylglucosamine, when used in combination with a silver-containing wound dressing, or with an AMP can synergistically affect chronic wound associated biofilm infections such as MRSA [S. aureus, P. aeruginosa, and E. coli [S. mutans and thus causing reduction in biofilm formation on surfaces like polystyrene or and saliva-coated hydroxyapatite. Another is the human liver-derived AMP hepcidin 20, which has the capability to reduce the mass of extracellular matrix and also can cause alterations in the biofilm structure of S. epidermidis by targeting PIA polysaccharide [AMPs have shown profound effects when used in combination with typical antibiotics and thus can be used in many antibiotic treatments. AMPs are produced by many multicellular organisms and hence can target a broad range of metabolic processes of bacterial cells. Since AMPs consist of a net positive charge, it can interact with the negatively charged component of the biofilm and gain access to it. Once AMP enters into biofilm, they result in membrane disruption via various mechanisms, also the cell integrity is lost and these lead to cell death and it also has the potential to kill dormant cells residing deep inside the biofilms. AMPs also exhibit intercellular inhibitory activity through various important processes such as cell division, nucleic acid metabolism, cell wall biosynthesis, as well as can modulate the immune response stimulating cytokine production, acting as chemokines, and promote wound healing. Cationic AMPs like LL-37, piperacillin, buforin II, ceprocin P1, indolicidin, nisin, and magainin II have shown promising effects when used in combination with antibiotics such as polymyxin E, piperacillin, azithromycin, daptomycin, linezolid, and clarithromycin; it increases the bioavailability of antibiotic against many MDR Gram-negative bacteria as well as methicillin-resistant  biofilm . Dispers as MRSA , 64. AMP E. coli . AMP likOchrobactrum pseudintermedium ciprofloxacin together gave rise to the possible highest antibacterial activity [actinomycetes have been shown to have an anti-biofilm activity against Vibrio and S. aureus. A combination of pigment prodigiosin having antibacterial property and a surfactant EPS extracted from Serratia marcescens together act as an effective antimicrobial agent. Spanò et al. showed that EPS1-T14, a EPS produced by Bacillus licheniformis strain T14, possesses potential antibacterial activity and can inhibit the formation of biofilms by multi-resistant bacteria such as E. coli, K. pneumoniae, P. aeruginosa, and S. aureus [.In rare occasions, a particular EPS produced by one bacterial biofilm has been shown to possess toxic activity against biofilm produced by some other bacterial species. This is one of the adaptive capabilities exhibited by bacteria to survive in a highly competitive environment. The use of biologically derived molecules with conventional antibiotics can be considered as a better therapeutic option. One such example is surfactant EPS (SEPS), a derivative of EPS, exhibits antimicrobial properties for a broad range of bacterial species in a non-specific manner. SEPS being anionic in nature binds to membrane cations, and disrupts cell membrane by altering the cell permeability. Sengupta et al. showed that SEPS extracted from activity . Bacteri. aureus .P. aeruginosa and MRSA. The combinatory therapy including bacteriophage and ciprofloxacin effectively treat P. aeruginosa associated infections [S. aureus-specific bacteriophage Sb-1 and subsequent treatment with antibiotics such as fosfomycin, rifampin, vancomycin, daptomycin, and ciprofloxacin was able to degrade the MRSA extracellular polysaccharide and target the persister cells. This combination can completely eradicate MRSA biofilm and thus can be used to treat biofilm infections [Bacteriophages, a virus that infects bacteria, used in combination with typical antibiotics eradicate the biofilm formed by pathogenic bacteria such as fections , 37. T. fections .Propionibacterium acnes, and acetylene exhibits both anti-biofilm and anti-virulence activity against Candida albicans [P. aeruginosa biofilms, by significantly reducing MIC of antibiotics. These extracts inhibit biofilm formation due to the presence of polyphenol compounds which are known to disable QS system, suppress bacterial cell adherence and motilities as well as inhibited polymeric matrix synthesis [Bacillus cereus, S. aureus, P. aeruginosa, E. coli, and Salmonella enterica serovar typhimurium [A new approach including the use of compounds of nature origin that could act against resistant bacteria is increasing. The natural product brazilin has been shown to have antibacterial activity against unrelated bacterium albicans . Abu El-ynthesis . Essentihimurium .Therefore, combining agents having some anti-bacterial potential with typical antibiotics not only enhance the efficacy of antibiotics but also reduces the MIC of the antibiotic used. This would reduce the side effects of high dosage as well as the development of antibiotic resistance.P. aeruginosa [E. coli, Klebsiella pneumonia, S. aureus, and P. putida [. The first identified inhibitor is PAβN which in combination with fluoroquinolones have shown inhibitory action against AcrAB-TolC, MexAB-OprM, MexCD-OprJ, and MexEF-OprN pumps [E. coli and K. pneumoniae [One of the promising strategies to treat bacterial biofilm infections is to use efflux pump inhibitors targeting broad range of efflux pumps that would inhibit the flux of substances from different bacterial species and as well as to increase the potential of the exported substances , 27. The. putida , 4. The rN pumps . Also useumoniae .N-(2-oxocyclohexyl)-3-oxododecanamide which works antagonistically toward the las QS system; N-(2-hydroxyphenyl)-3-oxododecanamide is known to interfere with the rh1 QS system as well as las QS system, and N-octanoyl cyclopentylamide (C8-CPA) moderately inhibited expression of the lasB-lacZ transcriptional fusion gene and also the expression of lasB which encodes for LasB elastase in P. aeruginosa PAO1. Another inhibitor N-decanoyl cyclopentylamide (C10-CPA) which is a stronger QS inhibitor is known to interfere with the expression of virulence genes regulated by both las and rh1 QS systems by inhibiting the interaction between their response regulators and AIs. The length of the acyl side chain and the ring structure is critical for C10-CPA to act as an inhibitor. C10-CPA analogs with cyclopropylamide, cyclobutylamide, cyclohexylamide, cycloheptylamide, and cyclooctylamide rings exhibit little inhibitory activity [P. aeruginosa. Lactoferrin stimulates twitching which causes the bacteria to wander instead of forming biofilms [Many QS inhibitors have been discovered such as activity . Lactofebiofilms .P. aeruginosa isolates and is an essential physiological event for subsequent differentiation and dispersal of a subpopulation of surviving biofilm cells. Cell death in this bacterium requires a functional rpoN gene which regulates two types of cell structures, i.e., type IV pili (T4P) and flagella. Water channels around P. aeruginosa micro-colonies are regulated by the QS-controlled production of rhamnolipid surfactants. Dispersal of the bacterial cells from voids present within the biofilm has been described. The LasR-dependent AI, N-(3-oxododecanoyl)-homoserine lactone, prevents the binding of the RhlI-dependent AI, N-(butryl)-homoserine lactone, to its cognate regulator RhlR. This control of RhlI/RhlR autoinduction by the LasI/LasR system assures that the two systems initiate their cascades sequentially and in the appropriate order additional AI has recently been demonstrated to be involved in QS in P. aeruginosa. This signal is noteworthy because it is not of the homoserine lactone class [Programmed cell death through autolysis has been observed in ne class .P. aeruginosa, and further can make bacteria susceptible to antibiotic treatment or host immune responses. As a result, efficiency of many antibiotics can be increased when used in combination with sub-MIC bismuth thiols. Medical implants coated with a metal such as silver are also efficient in inhibiting biofilm formation [Apart from antibiotic treatment, it is also important to make strategies to prevent biofilm formation. One way is to modify the abiotic surface properties by layering the surface with antimicrobial agents which could inhibit the bacterial biofilm adhesion and development. For example, surfaces of the medical devices or surgical implants can be altered such as making the surface hydrophilic and incorporating antimicrobial agents such as copper, probiotics to prevent the bacterial biofilm adherence and also control the transmission of bacterial pathogens into the environment . Bismuthormation . HoweverBiofilm formation is evolutionary adaptability gained by bacterial species to tolerate and survive in extreme conditions including mechanical stress and antibiotic treatments. Biofilm is an aggregation of micro-organisms that are irreversibly attached to a substratum and are enclosed in a self-produced extracellular polymeric substance composed of EPS, lipids, e-DNA, and proteins. Antibiotic resistance is a natural phenomenon observed in bacterial species. The formation of bacterial biofilms is considered one of the major reasons for several chronic infections. A deeper understanding of how biofilm is formed, the antibiotic resistance mechanism, and the interaction of the biofilms with the host are important. However, antibiotic treatment involving the use of conventional antibiotics is repeatedly proven to be inadequate to eliminate biofilm-related infections . It has


Advances in electronic technology have created opportunities for new instructional designs of medical curricula.We created and evaluated a 4-week online elective course for medical students to teach the cognitive basis for interviewing skills.Ten students, from 2 medical schools, studied online modules on interviewing concepts and viewed videos illustrating the concepts. They then participated in asynchronous discussion groups designed to reinforce course concepts, stimulate reflective learning, and promote peer learning.In qualitative evaluations, learners reported improvements in self-awareness; increased understanding of interviewing concepts; and benefits of online learning vs face to face learning. Participants reported high levels of satisfaction with online learning and with achievement of course objectives. Self-reported knowledge scores increased significantly from pre-course completion to post-course completion.Online education has significant potential to augment curriculum on the medical interview, particularly among students trained in community settings geographically distant from their academic medical center. A number of organizations - 3 have The instructional design we use for online courses  has the Blackboard, a web-based learning system  was usedStudents received access to a moderated, asynchronous discussion board and were required to post their impressions and observations each week. If necessary , they were reminded by the moderator. Using established principles - 9 .Eleven formative evaluation questions  were preStudents also completed pre-course and post-course Web-based questionnaires with 21 items , each scr= 0.9412, P< .001), providing support for the construct validity of the self-reported knowledge measures  and opportunities to apply the concepts to real patients (Theme 4).Open-ended comments on the course evaluation form supported these themes, and provided more detail about advantages of online learning in this course over more conventional methods. Two students provided representative viewpoints:Student 1: ". . . interacting with students in the on-line format allowed for well thought-out, comprehensive responses and much more insightful comments than sometimes heard in a classroom. I attribute this to the time one has to sit and think through a response, choose the words carefully, and elaborate uninterrupted. There's less pressure on-line, so you can piece together your thoughts with less stress and greater sincerity."Student 2: "The strengths are the high level of participation and interaction and conversation (more so than in any other course so far.)"P< .01) at the end of the course   .r= 0.72, P=.02), suggesting that greater educational effort was correlated with greater self-reported gain in knowledge , in a speech once exhorted medical educators to seize "the potential of the technological revolution to transform the way students learn" . In resp


Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death.Programmed cell death is used to remove excess cells between ommatidia in the echinus alleles, some likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases. These proteins cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss.We generated a number of new echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye.The  Drosophila eye consists of 750–800 individual unit eyes, known as ommatidia, which are arranged in a hexagonal lattice. Each ommatidium consists of 8 photoreceptors, 4 lens-secreting cone cells and 2 primary pigment cells. Ommatidia are separated from each other by secondary and tertiary (2° and 3°) pigment cells, and by sensory bristles. Each of these cell types occupies a stereotypic position within the lattice. Pattern formation in the eye is initiated in the 3rd larval instar as a wave of morphogenesis sweeps across the epithelial cell layer in the eye imaginal disc. First, eight photoreceptor cells and four lens-secreting cone cells are specified through sequential inductive interactions. During early pupal stages, cone cells come to cover the photoreceptors. They also recruit two primary pigment cells, which surround the cone cells. Cells that have not been specified at this stage form the interommatidial cell (IOC) lattice, which will ultimately be composed of secondary pigment cells, tertiary pigment cells, and bristles. These cells initially appear undifferentiated and unpatterned, with several layers of IOCs often separating neighboring ommatidia. Reorganization begins with presumptive lattice cells maximizing their contacts with primary pigment cells rather than with other lattice cells. This results in each lattice cell being connecting to at least two primary pigment cells, and with each ommatidia being separated by a single layer of lattice cells, arranged in an end-to-end chain. About two-thirds of these cells will go on to develop as secondary pigment cells, each of which makes up one face of the ommatidial hexagon, or tertiary pigment cells, which make up alternative vertices, with bristle groups making up the other vertices. The remainder of the IOCs are eliminated by apoptotic cell death . Ex. Exechinype Fig. . We cannping eye ,56,57. Aechinus cDNAs and identified three splice variants   Fig. . In eachechinus in multiple Drosophila species as well as several other insects. Outside of the arthropods Echinus shows most homology with the mammalian DUBs USP53 and USP54, with essentially all of this homology occuring within the USP domain. Most importantly for the purposes of this report, Ec-SF2 and Ec-SF3 encode proteins with truncated USP domains and lack residues important for catalysis. Specifically, Ec-SF2 and Ec-SF3 contain the catalytic histidine and aspartic acid residues found in the USP His box, but they lack the Cys box and its catalytic cysteine. Ec-SF2 and Ec-SF3 instead have alternative N-termini that are conserved in Drosophilia and show no sequence similarity to the Cys box motif . USP-containing DUBs are cysteine proteases that are capable of removing ubiquitin or ubiquitin-like proteins from substrates ,59. The f Figure . The homechinus-like adult rough eye phenotype, and pupal eyes contained extra IOCs . Nonetheless, our results from targeting Ec-SF1 demonstrate that this splice form, at least, is important for bringing about IOC death, and suggest that it may be the most important for regulating IOC survival. Also consistent with this hypothesis is our observation that GMR-driven RNAi that targets all splice forms (GMR-CG2904-RNAi) results in a phenotype similar to that observed in flies in which only ec-SF1 was targeted , separated by an arginine (Arg) residue . Nfa-g removes Notch activity specifically in pigment cells [85], leading to a failure of 1° pigment cells to differentiate, and a decrease in IOC death [86]. Notch and echinus are both on the X chromosome. To search for interactions between Nfa-g and echinus loss-of-function mutations we took advantage of flies carrying an autosomal insertion of GMR-CG2904-RNAi that targets transcript sequences common to all echinus splice forms, and that phenocopies echinus  and loss-of-function (GMR-ec-RNAi) of echinus mutants were introduced into gain (GMR-GAL4-UAS-rst) and loss-of-function (rstCT) roughest mutant backgrounds. No significant interactions were observed between these genes.Click here for file


The worse prognosis in patients without ST-elevation (non-STEMI) as compared to ST-elevation myocardial infarction (STEMI), may be due to treatment differences. We aimed to evaluate the differences in characteristics, treatment and outcome in patients with non-STEMI versus STEMI in an unselected patient population.Individual patient data from all patients in our hospital with a discharge diagnosis of MI between Jan 2001 and Jan 2002 were evaluated. Follow-up data were obtained until December 2004. Patients were categorized according to the presenting electrocardiogram into non-STEMI or STEMI.A total of 824 patients were discharged with a diagnosis of MI, 29% with non-STEMI and 71% with STEMI. Patients with non-STEMI were significantly older and had a higher cardiovascular risk profile. They underwent less frequently coronary angiography and revascularization and received less often clopidogrel and ACE-inhibitor on discharge. Long-term mortality was significantly higher in the non-STEMI patients as compared to STEMI patients, 20% vs. 12%, p = 0.006, respectively. However, multivariate analysis showed that age, diabetes, hypertension and no reperfusion therapy (but not non-STEMI presentation) were independent and significant predictors of long-term mortality.In an unselected cohort of patients discharged with MI, there were significant differences in baseline characteristics, and (invasive) treatment between STEMI and non-STEMI. Long-term mortality was also different, but this was due to differences in baseline characteristics and treatment. More aggressive treatment may improve outcome in non-STEMI patients. Myocardial infarction (MI) is usually categorized into non-ST-elevation myocardial infarction (non-STEMI) and ST-elevation myocardial infarction (STEMI). Patients with STEMI should be treated immediately with reperfusion therapy by either percutaneous coronary intervention (PCI) or thrombolysis, if admitted within 12 h of symptom onset . PatientA previous study has shown that in unselected patients, mortality was significantly higher in the non-STEMI as compared to STEMI patients . HoweverFrom January 2001 to January 2002, individual patient data from all patients with the discharge diagnosis of acute myocardial infarction at the Isala klinieken  were recorded. To avoid double inclusion of patients, only the first admission for MI during the study period was used.Non-STEMI patients consisted of only patients admitted to our center, however, STEMI patients included also those referred and those diagnosed by paramedics in the ambulance and transported directly to our center.According to the presenting ECG, patients were categorized as non-STEMI or STEMI. Patients were diagnosed with non-STEMI if they had ischemic chest pain classified as Braunwald class 3 and the presence of at least 1 of the following criteria: (new) ST depression of more than 1 mm in at least 2 ECG leads or a positive biomarker . STEMI was defined as chest pain of > 30 minutes' duration and ECG changes with ST segment elevation of > 2 mm in at least 2 precordial and > 1 mm in the limb leads.We collected the following variables from the patient files: age, gender, history of hypertension, diabetes, hyperlipidemia, smoking, previous myocardial infarction and discharge medication. Follow-up information was obtained from the patient's general physician or by direct telephone interview with the patient.The investigation conforms with the principles outlined in the Declaration of Helsinki . The study was approved by the Committee on Research Ethics of the Isala Klinieken, Zwolle.Statistical analysis was performed with the Statistical Package for the Social Sciences  version 12.0.1. Continuous data were expressed as mean ± standard deviation of mean and categorical data as percentage, unless otherwise denoted. The analysis of variance and the chi-square test were appropriately used for continuous and categorical variables respectively. Cox proportional hazard regression procedure was performed to estimate the hazard ratio of mortality of the findings. Significant variables analyzed are reported with their respective Hazard ratio (HR) and 95% confidence intervals (CI). For all analyses, statistical significance was assumed when the two tailed probability value was < 0.05.During the study period, 824 patients were discharged with a diagnosis of MI and categorized as non-STEMI 29% (N = 241) and STEMI 71% (N = 583).Baseline characteristics are summarized in Table Cardiac catheterization and percutaneous coronary intervention (PCI) were significantly less often performed in non-STEMI patients Table . CoronarAt 3 year follow up, mortality was 20% in the non-STEMI and 12% in the STEMI patients, p = 0.006 Figure . The excUnivariate predictors of mortality in the STEMI patients were age, diabetes, previous myocardial infarction, hypertension, previous myocardial infarction and no reperfusion Table .The following factors were included in the Cox-regression analysis: age, gender, diabetes, previous MI, hypertension, smoking, non-STEMI (STEMI reference) and no reperfusion therapy. This analysis revealed that age, diabetes, hypertension and no reperfusion therapy were significantly associated with increased risk of mortality Table .This study identifies important differences in patient characteristics and management among patients discharged with the diagnosis of non-STEMI versus STEMI in a cohort of unselected patients. The higher mortality of patients with non-STEMI was due to differences in baseline characteristics and treatment.The ratio of STEMI/non-STEMI is higher in our study as compared to other studies . The is Our results are in accordance with a previous study , showingFurthermore, compared to our study, many large scale randomized trials have reported lower mortality rates at one year follow up after MI -14. The The fact that non-STEMI patients were older, had higher risk profiles, were more often treated conservatively and less frequently received guideline recommended medication on the time of discharge, might possibly explain the difference in mortality rate. These results are in accordance with a previous study, showing that an early invasive management strategy is not utilized in many high-risk patients. An invasive strategy appears to be reserved for patients without significant co-morbidities and with a lower risk of in-hospital mortality . HoweverThe outcomes in patients with acute coronary syndromes have improved over time. This was associated with the use of antiplatelet and antithrombotic therapies and the increased utilization of revascularization . HoweverThe question remains how we can further improve the outcome of patients with non-STEMI. This may be achieved by a multifactorial approach; first, identifying high risk patients, which can be done through a good clinical evaluation of medical history, physical examination, electrocardiogram (ECG) and cardiac biomarkers e.g. myoglobin, cardiac troponin and CK-MB ,27. SecoAll the above mentioned trials have increased our knowledge with regard to how we should treat high-risk patients with non-STEMI. However, very early aggressive therapy in non-STEMI patients, as is performed in STEMI, remains a field for further research. Further studies are warranted to evaluate whether immediate cardiac catheterization and reperfusion in high-risk non-STEMI patients will further improve the outcome.Although we tried to register all patients with AMI, it is however, not possible to include all patients e.g. STEMI patients that are not referred for atypical chest pain or are presented too late. However, when comparing only patients admitted directly to our hospital, results remained unchanged. Other factors such as co-morbidity or contraindications for each considered treatment may account for differences in outcome. Thus selection bias may play a role in our study. Other factors such as history of stroke, neoplasm and depression mat affect both treatment and outcome, however, we have no data regarding these variables. Furthermore, we have no separate date on bundle branch block MI (BBBMI), as they seem to have worse outcome . HoweverThis study shows important differences in baseline characteristics, treatment and prognosis between non-STEMI and STEMI patients. A more invasive therapy and guideline recommended treatment might further improve outcome, especially in the non-STEMI population.The author(s) declare that they have no competing interests.SR: participated in the design of the study, performed statistical analyses and drafted the manuscript. JPO participated in the study design and coordination and helped draft the manuscript. J-HD, M-JB, JH, MG, FZ and HS participated in the design of the study helped draft the manuscript. AH is the principle investigator, has made substantial contributions to conception and design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We investigated lipid profiles and lipoprotein modification after immuno-intervention in patients with early rheumatoid arthritis (ERA). Fifty-eight patients with ERA who met the American College of Rheumatology (ACR) criteria were included in the study. These patients had disease durations of less than one year and had not had prior treatment for it. Smokers or patients suffering from diabetes mellitus, hypothyroidism, liver or kidney disease, Cushing's syndrome, obesity, familiar dyslipidemia and those receiving medications affecting lipid metabolism were excluded from the study. Sixty-three healthy volunteers (controls) were also included. Patients were treated with methotrexate and prednisone. Lipid profiles, disease activity for the 28 joint indices score (DAS-28) as well as ACR 50% response criteria were determined for all patients. The mean DAS-28 at disease onset was 5.8 ± 0.9. After a year of therapy, 53 (91.3%) patients achieved the ACR 20% response criteria, while 45 (77.6%) attained the ACR 50% criteria. In addition, a significant decrease in the DAS-28, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were observed. ERA patients exhibited higher serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides, whereas their serum high-density lipoprotein cholesterol (HDL-C) levels were significantly lower compared to controls. As a consequence, the atherogenic ratio of TC/HDL-C as well as that of LDL-C/HDL-C was significantly higher in ERA patients compared to controls. After treatment, a significant reduction of the atherogenic ratio of TC/HDL-C as well as that of LDL-C/HDL-C was observed, a phenomenon primarily due to the increase of serum HDL-C levels. These changes were inversely correlated with laboratory changes, especially CRP and ESR. In conclusion, ERA patients are characterized by an atherogenic lipid profile, which improves after therapy. Thus, early immuno-intervention to control disease activity may reduce the risk of the atherosclerotic process and cardiovascular events in ERA patients. Rheumatoid arthritis (RA) is a chronic systemic disease affecting primarily the synovium, leading to joint damage and bone destruction . RA causIn general, and with some variations between different studies, the lipid profile of patients with active or untreated RA is primarily characterized by a decrease in serum levels of HDL-C whereas contrasting results have been published on the serum levels of TC and LDL-C -20. ImpoThe cholesterol ester transfer protein (CETP) has a central role in HDL metabolism and in the regulation of HDL-C levels in serum. CETP exchanges cholesterol esters with triglycerides between HDL and apolipoprotein B-containing lipoproteins and thus significantly contributes to the reverse cholesterol transport pathway. High levels of CETP activity lead to a reduction in HDL-C levels and an atherogenic lipoprotein profile ,22. ThusOur knowledge about the effect of treatment on the lipid profile of patients with RA is limited and only cross-sectional and short term uncontrolled studies have been performed ,17-20. IFifty-eight consecutive, unselected patients who were referred to the outpatient rheumatology clinic between January 2004 and September 2005 were investigated. All patients fulfilled the American College of Rheumatology (ACR) 1987 criteria for RA , had earSmokers or patients suffering from conditions that affect the lipid profile, such as diabetes mellitus, hypothyroidism, liver or kidney disease, Cushing's syndrome, obesity (body mass index >30) and a history of familial dyslipidemia, were excluded. In addition, patients receiving medications affecting lipid metabolism, such as lipid-lowering drugs, beta-blockers, oral contraceptives, estrogen, progestin, thyroxin and vitamin E, were excluded from the study.Sixty-three apparently healthy, non-smoking volunteers also participated in the study and were used as a control group. These subjects were selected from blood donors during the last two months of the patients' selection period and fulfilled the same exclusion criteria reported for the patient group. None of the subjects participating in the control group had a history of CAD. The control group was proportionally matched for age and sex to the patient group. All controls reported no significant changes in their body weight for at least three months before entry to the study. All patients and controls gave informed consent and the study protocol was approved by the Institutional Ethics Committee.Patients were treated with methotrexate  and prednisone (7.5 mg/day). The dose of MTX remained stable during the study while the dose of prednisone was tapered according to a patient's clinical response. Disease activity was assessed by measuring the disease activity for 28 joint indices score (DAS-28) , while tOvernight fasting blood samples were obtained at baseline and after 12 months follow-up from both ERA patients and the control group. Serum lipids were determined within six hours of blood sampling. TC, triglycerides and HDL-C were determined on an Olympus AU560 Clinical Chemistry analyser  as previously described . LDL-C wt test. Correlation between variables was examined using the Pearson's correlation coefficient. A p value < 0.05 was considered statistically significant.All data were analyzed with the STATISTICA 5.1 program. Comparisons between groups were conducted using the During the selection period (January 2004 to September 2005), a total of 72 patients were recruited. Of these, 14 were excluded; 7 who were current smokers, 2 with diabetes mellitus, 4 who had hypertension treated with propranolol and diuretics and 1 who had hypothyroidism. Thus, 58 patients were included in the study. None of the patients reported a modification of dietary habits or experienced significant changes in body weight during the study. The clinical characteristics and lipid profiles of patients before and after therapy and normolipidemic controls are described in Table ERA patients exhibited mild but significant higher baseline serum levels of TC, LDL-C, nonHDL-C, triglycerides and apoB Table . ImportaAfter 12 months of therapy, a significant decrease in the DAS-28 and ESR values as well as in the CRP levels were observed  and higher TC/HDL-C and LDL-C/HDL-C ratios in active and/or untreated disease than in the general population ,20,31,32Systemic inflammation may also play a role in the development of atherosclerosis ,33. In fAn important observation of the present study is that ERA patients exhibit low HDL-C serum levels, which are significantly increased after immuno-intervention in parallel with the reduction of CRP levels and ESR values. Importantly, the increase in HDL-C levels was inversely correlated with the reduction in either CRP levels or ESR values. This suggests that inflammation is an important determinant for the reduced HDL-C levels observed in ERA patients. By contrast, immuno-intervention did not influence the serum levels of LDL-C and triglycerides and it is thus unlikely that inflammation is responsible for the elevation of these lipid parameters observed at baseline in our patients.In an effort to investigate the biochemical basis for the reduced HDL-C levels exhibited by ERA patients at baseline, we determined the serum levels of CETP activity. CETP exchanges cholesterol esters with triglycerides between HDL and apoB-containing lipoproteins, leading to the reduction in HDL-C levels ,22. IncrAccording to our results, ERA patients exhibited significantly higher CETP activity at baseline compared to controls. Thus, we may hypothesize that the reduced HDL-C levels, and consequently the atherogenic lipid profile observed in our patients before treatment, could be attributed at least partially to the higher baseline levels of CETP activity. This hypothesis is further supported by the finding that immuno-intervention significantly reduced CETP activity in parallel with the elevation of the serum HDL-C levels. However, we cannot exclude the possibility that the increased CETP activity and the reduced HDL-C levels observed in our patients at baseline could also be attributed to the fact that several inflammatory mediators inhibit cholesterol efflux from cells by reducing expression of the ATP-binding cassette A1 gene . This poThe use of DMARDs in ERA for controlling the disease activity may reduce articular damage. Several DMARDs exist for treating RA, but low dose MTX is usually the main choice. Long-term observational studies for periods up to 10 years have shown a sustained clinical response and an acceptable toxicity profile for MTX treatment -46. In aCorticosteroids, on the other hand, have a potentially atherogenic effect, given that they cause dyslipidemia and hypertension ,16. In sIt is possible that RA patients may have some classic risk factors for atherosclerosis development. However, it is not correct to attribute the increased prevalence of atherosclerosis observed in RA patients to these factors. In our study, we tried to exclude patients with classic risk factors for atherosclerosis and we found that ERA patients with high disease activity showed an adverse lipid profile before the commencement of therapy. After one year of treatment, a significant clinical and laboratory improvement was noted. This was correlated with changes in the lipid profile, mainly the increase of HDL-C. HDLs are particles with numerous atheroprotective functions, including facilitation of reverse cholesterol transport, improvement of endothelial function, protection of LDL from oxidation, limitation of hemostasis and retardation of inflammatory activity related to the vascular wall . Since oThese findings provide evidence that early immuno-intervention with MTX and corticosteroids controlling the inflammatory process may reduce the risk of atherosclerosis and cardiovascular events in ERA patients. Further, long term longitudinal studies are needed to demonstrate if early treatment in RA patients reduces the risk of cardiovascular events.ACR = American College of Rheumatology; ApoA-I = apolipoprotein A-I; ApoB = apolipoprotein B; CAD = coronary artery disease; CETP = cholesterol ester transfer protein; CRP = C-reactive protein; DAS-28 = disease activity for 28 joint indices score; DMARDs = disease modifying antirheumatic drugs; ERA = early rheumatoid arthritis; ESR = erythrocyte sedimentation rate; HDL-C = high density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; MTX = methotrexate; RA = rheumatoid arthritis; TC = total cholesterol.The authors declare that they have no competing interests.ANG wrote the paper and participated in the collection of the data. ECP and ESL performed the biochemical and lipoprotein profile. YA carried out the statistics. CK performed the assays for ApoA-I and ApoB. ADT performed the control of the statistics and the lipoprotein profile. AAD conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.