Peroxisome-driven ether-linked phospholipids biosynthesis is essential for ferroptosis

It is well established that ferroptosis is primarily induced by peroxidation of long-chain poly-unsaturated fatty acid (PUFA) through nonenzymatic oxidation by free radicals or enzymatic stimulation of lipoxygenase. Although there is emerging evidence that long-chain saturated fatty acid (SFA) might be implicated in ferroptosis, it remains unclear whether and how SFA participates in the process of ferroptosis. Using endogenous metabolites and genome-wide CRISPR screening, we have identified FAR1 as a critical factor for SFA-mediated ferroptosis. FAR1 catalyzes the reduction of C16 or C18 saturated fatty acid to fatty alcohol, which is required for the synthesis of alkyl-ether lipids and plasmalogens. Inactivation of FAR1 diminishes SFA-dependent ferroptosis. Furthermore, FAR1-mediated ferroptosis is dependent on peroxisome-driven ether phospholipid biosynthesis. Strikingly, TMEM189, a newly identified gene which introduces vinyl-ether double bond into alkyl-ether lipids to generate plasmalogens abrogates FAR1-alkyl-ether lipids axis induced ferroptosis. Our study reveals a new FAR1-ether lipids-TMEM189 axis dependent ferroptosis pathway and suggests TMEM189 as a promising druggable target for anticancer therapy.Subject terms: Phospholipids, Cancer metabolism

Ether phospholipids represent an important group of phospholipids containing a glycerol backbone with an alkyl or a vinyl bond connecting a fatty alcohol at sn-1 position, usually polyunsaturated fatty acid (PUFA) including docosahexaenoic acid and arachidonic acid at sn-2. Ether phospholipids are initially synthesized in peroxisomes and processed in the endoplasmic reticulum (ER) [1–3]. Plasmalogens are the most abundant form of ether phospholipids which have a vinyl ether bond, enriched in the brain and heart tissues [1–3]. The plasmalogens have been found as endogenous antioxidants with vinyl ether bond susceptible to cleavage by reactive oxygen species (ROS). The deficiency of plasmalogens correlates with various human disorders, including Alzheimer’s disease and cancer [1, 2, 4].Ferroptosis is an iron-dependent form of non-apoptotic cell death induced by excess accumulation of peroxidized phopholipids, generated through oxidation of the PUFA moieties at sn-2 position of membrane phospholipids [5–9]. Ferroptosis is morphologically, biochemically and genetically distinct from other forms of cells death [5], which is tightly regulated by glutathione peroxidase 4 (GPX4) via converting lipid hydroperoxides (PUFA-OOH) into non-toxic lipid alcohols (PUFA-OH) [10, 11]. Emerging evidence indicates that ferroptosis is implicated in ischemia–reperfusion injury (IRI), neurodegeneration, antiviral immunity, cancer immunotherapy and tumor suppression [11–19].Accumulating evidence reveals a robust link between lipid metabolism and ferroptosis [14, 20–24]. However, little is known about the role of ether phospholipids in ferroptosis. In the present study, we revealed the FAR1-TMEM189 axis as a central pathway to drive the susceptibility of ferroptosis. FAR1-TMEM189 axis specifically synthesizes alkyl and vinyl ether phospholipid, where the two isoforms of ether phospholipid play distinct role in ferroptosis. Our findings provide an insight into the mechanism of ether phospholipid-mediated ferroptosis, with implications for novel treatment options for cancer therapy.     

The NOTCH signaling pathway in normal and malignant blood cell production

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251–264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115–134, 2012), proliferation (VanDussen et al. in Development 139:488–497, 2012) and apoptosis (Cao et al. in APMIS 120:441–450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438–1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649–61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376–389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537–542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23–35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877–881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.     

Organochlorine contamination enriches virus-encoded metabolism and pesticide degradation associated auxiliary genes in soil microbiomes

Viruses significantly influence local and global biogeochemical cycles and help bacteria to survive in different environments by encoding various auxiliary metabolic genes (AMGs) associated with energy acquisition, stress tolerance and degradation of xenobiotics. Here we studied whether bacterial (dsDNA) virus encoded AMGs are enriched in organochlorine pesticide (OCP) contaminated soil in China and if viral AMGs include genes linked to OCP biodegradation. Using metagenomics, we found that OCP-contaminated soils displayed a lower bacterial, but higher diversity of viruses that harbored a higher relative abundance of AMGs linked to pesticide degradation and metabolism. Furthermore, the diversity and relative abundance of AMGs significantly increased along with the severity of pesticide contamination, and several biodegradation genes were identified bioinformatically in viral metagenomes. Functional assays were conducted to experimentally demonstrate that virus-encoded L-2-haloacid dehalogenase gene (L-DEX) is responsible for the degradation of L-2-haloacid pesticide precursors, improving bacterial growth at sub-inhibitory pesticide concentrations. Taken together, these results demonstrate that virus-encoded AMGs are linked to bacterial metabolism and biodegradation, being more abundant and diverse in soils contaminated with pesticides. Moreover, our findings highlight the importance of virus-encoded accessory genes for bacterial ecology in stressful environments, providing a novel avenue for using viruses in the bioremediation of contaminated soils.Subject terms: Metagenomics, Soil microbiology, Microbial ecology

As the most abundant biological entities on earth, viruses of bacteria (bacteriophages referred as viruses from here on) play a critical role in modulating the ecology of microbial communities through lytic infection and lysogenic conversion of their bacterial hosts [1, 2]. Viruses significantly influence the biogeochemical cycles via the release of organic carbon and nutrients through host cell lysis, and in addition to core viral genes (i.e., genes encoding viral structural proteins [3]), they also encode various auxiliary metabolic genes (AMGs [4, 5]), which contribute the metabolic capacity and survival of their bacterial hosts. The role of AMGs has been especially well demonstrated with marine viruses that encode a diversity of AMGs involved in photosynthesis [6], translation machinery [7], carbon metabolism [8], phosphate metabolism [9] and sulfur cycle [10, 11]. Furthermore, sequencing of whole marine viral communities has revealed a clear involvement of viral AMGs in central carbon metabolism of host bacteria [10, 12]. Compared with the study of viral communities in marine ecosystem, the diversity and functional role of viral AMGs in soils are less well understood.In soils, viruses reach abundances of up to ~10⁹ per gram of soil leading to frequent encounters with their host bacteria [13]. Similar to aquatic environments, viruses can regulate host bacterial densities, leading to indirect changes in the relative abundance of non-target bacterial taxa likely via release of niche space [14, 15]. Moreover, over longer time periods, viruses can coevolve with their host, following fluctuating selection dynamics [16] or patterns of local adaptation [17]. Viruses are also important mediators of horizontal gene transfer, promoting the transfer of antibiotic resistance genes, virulence factors and AMGs [18, 19]. However, these effects are less well understood at viral community level. Recent advances in viral purification have enabled a glimpse into soil viral communities of permafrost peatland [20, 21] and agricultural ecosystems [22, 23] based on metagenomics. These studies have demonstrated that viruses may alter the biogeochemical nutrient cycling [1, 2] and bacterial adaptation and evolution by carrying genes linked to carbon and nitrogen metabolism [20, 21]. Moreover, recent identification of atrazine chlorohydrolase trzN [24] and arsenic methyltransferase arsM [25] genes in soil-associated lysogenic viruses suggest that virus-encoded AMGs could shape bacterial metabolism under pollutant exposure. Therefore, we hypothesize, that contaminated soil microbiomes could contain a relatively higher abundance of viruses carrying AMGs linked to the degradation of pesticides and xenobiotics due to their potential benefit for the host bacteria.Pesticide contamination imposes a serious threat to natural ecosystems and public health globally. China is the leading producer of organochlorine pesticides (OCPs), which are synthetic pesticides with vast applications in chemical and agricultural industries. OCPs are especially notorious due to their high toxicity, slow degradation and bioaccumulation [26]. Following the implementation of the Stockholm Convention, hundreds of pesticide plants in China were closed or re-located, and contaminated soils around the plants left untreated. As microbial communities are often capable of degrading OCPs, there is growing biotechnological interest to identify important genes and microbial taxa behind pesticide biodegradation. Heavy OCP contaminations have previously been shown to adversely impact soil bacterial diversity, composition, and activity [27, 28]. Prolonged exposure to contaminants has resulted in selection for bacteria that have evolved their own degradation enzymes, such as dehalogenases, which protect from the toxic effects of pesticides [29]. Interestingly, if also viruses can carry and encode such genes, pesticide exposure could create a strong positive selection for virus-encoded AMGs associated with pesticide degradation, potentially shifting soil microbiome community composition [30] by favoring bacterial and virus taxa that carry these genes.To address this, we used a combination of metagenomics and direct experimentation to explore how pesticide exposure affects the abundance and type of bacterial and virus-encoded AMGs in the soil of former OCP production factory in Yangtze River Delta (China). We found that contaminated and clean control soils harbored very distinct bacterial and viral communities, and crucially, pesticide exposure was linked to higher diversity and abundance of virus-encoded metabolism and pesticide degradation AMGs. The functional activity of one candidate viral AMG, L-2-haloacid dehalogenase (L-DEX), was experimentally shown to improve bacterial growth at sub-inhibitory concentrations of haloacid, which is an important precursor of herbicides and insecticides. Together, our findings suggest that virus-encoded auxiliary genes could help bacteria to counteract pesticide stress, potentially explaining the benefits of virus carriage in stressful soil microbiomes.     

Investigating dynamic interdomain allostery in Pin1

Signaling proteins often sequester complementary functional sites in separate domains. How do the different domains communicate with one another? An attractive system to address this question is the mitotic regulator, human Pin1 (Lu et al., Nature 380:544–547, 1996). Pin-1 consists of two mutually tethered domains: a WW domain for substrate binding and a catalytic domain for peptidyl-prolyl isomerase (PPIase) activity. Pin1 accelerates the cis–trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs within proteins regulating the cell cycle and neuronal development. The early X-ray (Ranganathan et al., Cell 89:875–886, 1997; Verdecia et al., Nat Struct Biol 7:639–643, 2000) and solution NMR studies (Bayer et al., J Biol Chem 278:26183–26193, 2003; Jacobs et al., J Biol Chem 278:26174–26182, 2003) of Pin1 indicated inter- and intradomain motions. We have explored how such motions might affect interdomain communication, using NMR. Our accumulated results indicate substrate binding to Pin1 WW domain changes the intra/interdomain mobility, thereby altering substrate activity in the distal PPIase domain catalytic site. Thus, Pin1 shows evidence of dynamic allostery, in the sense of Cooper and Dryden (Eur J Biochem 11:103–109, 1984). We highlight our results supporting this conclusion and summarize them via a simple speculative model of conformational selection.     

New Method for Monitoring the Process of Freeze Drying of Biological Materials

A capacitive sensor was proposed and tested for the monitoring and control of a freeze drying process of a vaccine against the Newcastle disease of birds. The residual moisture of the vaccine was measured by the thermogravimetric method. The vaccine activity was determined by titration in chicken embryos. It was shown that, at the stages of freezing and primary drying, a capacitive sensor measured the fraction of unfrozen liquid phase in a material and allowed one to control the sublimation stage of drying in an optimal way. This prevented the foaming of the material and shortened the total drying time approximately twice. The control range at the sublimation stage of drying expanded up to −70°C. It was found at the final stage of drying that the signal of a capacitive sensor passed through a maximum value. We supposed that this maximum corresponds to the minimum of intramolecular mobility of biological macromolecules and hence to the optimal residual moisture of the material, which ensures long-term preservation of its activity. We also suppose that using the capacitive sensor at the final stage of drying allows one to more precisely detect the time when the residual moisture of dried material reaches the optimal value.KEY WORDS: biological materials, capacitive sensor, freeze drying, optimal residual moistureAt present, most biological materials containing live viruses or bacteria are exposed to lyophilization (i.e., drying from the frozen state); this ensures long-term preservation of their activity. Typically, this process consists of preliminary freezing and subsequent freeze drying. The latter process, in turn, consists of two stages: primary drying and secondary drying. During primary drying or sublimation, frozen water is removed from a biological product under vacuum and at temperatures below 0°C. At this stage, the drying rate is limited because of the foaming of a product that occurs due to its high temperature and the excess amount of liquid phase in it. The secondary drying, or final stage, begins after the end of the sublimation stage and occurs at temperatures above 0°C. The goal of the secondary drying is to bring the residual moisture of a biological product to an optimum level, which provides long-term preservation of its activity. Note that the moisture content both above and below the optimum value reduces the effective life of biological materials (1,2)To increase the shelf life of biological products, the following should be investigated: (1) the influence of the composition of the dried biological product and the residual moisture on the change in its activity over the time (3); (2) it is needed to optimize the sublimation drying process for different types of biological products (4). For the investigation of the of the state of water in the dried biologic drugs and the influence of the humidity of the biological on the change in their activity during shelf life, different physical methods are used such as neutron scattering (5), nuclear magnetic resonance (NMR) (6,7), Raman spectroscopy (8), infrared spectroscopy, differential scanning calorimetry, thermal activity monitor (9), and gravimetric sorption analysis (10). The investigations using these methods allow to find an optimum composition of a protective medium for biologics and to determine its optimal residual moisture.At all stages of the freeze drying, the parameters of the material and the parameters of the drying process (temperature of a material, the shelf temperature, the condenser temperature, the pressure in the sublimation chamber, etc.) are also monitored. According to these data, the mode of the process is selected to conduct him for the minimum time and get the best product quality (11). Usually during the drying process, the temperature is measured in several vials with biologic located on different shelves. The sharp increase of the temperature indicates the end of primary drying and the beginning of the secondary drying. The finish of the sublimation stage is revealed by a sharp decrease of the partial pressure of water vapor in the sublimation chamber (12,13). Note that the partial pressure of water vapor in the sublimation chamber does not characterize the state of the biological product to be dried and it is an indirect parameter. For monitoring and controlling the process of freeze drying, it is important to use the own properties of biological materials. In (14), a resistivity sensor placed in a frozen biological material was proposed to control the primary stage of freeze drying. A disadvantage of this method is that one cannot establish an unambiguous relationship between the amount of liquid phase in the frozen material and the value of resistivity: the resistance of the sensor depends not only on the amount of liquid phase but also on the concentration of dissolved salts. Another disadvantage of the resistivity sensor is that, when the temperature decreases, the resistivity of the material sharply increases to values that are difficult to measure, which makes impossible the control of the sublimation stage with this sensor.In (15,16), the interesting methods for determining the moisture of biological materials during secondary drying were proposed. These methods are based on the measurement of the partial pressure of water vapors in the sublimation chamber by NIR spectroscopy or Raman spectroscopy. Note that this method is indirect and requires laborious calibration to establish a correspondence between the current moisture of the biological material in vials and the pressure of water vapor in the sublimation chamber.It should be noted that one has to carry out a series of long-term experiments to find the optimal residual moisture of a biological product. These experiments result in the lifetimes of biological samples with various residual moistures. As the optimal residual moisture of a biological product, one takes the value that provides the longest term preservation of its activity.However, finding the optimal conditions of freeze drying has traditionally been a process of trial and error and required several experimental runs (17). Note also that the freeze drying process is time-consuming and labor intensive.A promising method for the investigation of the properties of biological materials is dielcometry (18,19). This method is relatively simple and very informative since it gives information about the structure of biological macromolecules and the state and role of water in the biological material, etc. This method was used in (20–22) for monitoring biological materials at the primary stage of freeze drying. In (20), authors had found an anomalous low-frequency dispersion of the dielectric permittivity in the biological under study and explain this phenomenon by the proton transfer among water molecules, connected by hydrogen bonds The dielectric relaxation time turned out to be sensitive to the loss of moisture content in the product, and the authors suggested to use of this phenomenon to determine the end point of the freeze drying process. The authors mounted the electrodes of the capacitive sensor on the outer surface of vials with the material to be dried. This approach allows monitoring the sublimation rate and determining the end of the primary stage of freeze drying. Unfortunately, the sensitivity of the capacitive sensor of this design is not enough for the reliable monitoring of the stage of secondary drying.In this paper, a new design of a capacitive sensor and measurement technique are proposed that enable monitoring all stages of the drying process: the freezing stage, the sublimation stage, and the final stage. During freezing and the sublimation stages, the sensor monitors the amount of liquid phase in the frozen material. This allows an optimal control during the whole sublimation stage which prevents the foaming of the material and significantly reduces the total drying time. The sensor also fixes the end of the sublimation stage and the beginning of the final stage of drying. At this stage, the high sensitivity of the measuring system enables one to discover that there is a certain time interval when the signal of the capacitive sensor passes through a maximum. We believe that this maximum corresponds to the minimum of the molecular mobility of biological macromolecules and the optimal residual moisture of the material to be dried.     

Phospholamban phosphorylation,mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca²⁺ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca²⁺ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation, by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012), is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders.     

Targeting subchondral bone in osteoporotic osteoarthritis

The identification of well-defined phenotypes along the course of the disease may open new avenues for personalized management in osteoarthritis (OA). In vivo research carried out in various animal models as well as epidemiological and clinical data support the existence of a particular phenotype – osteoporotic OA. In fact, subchondral bone has become a potential therapeutic target in OA. Depending on the ratio between formation and resorption, subchondral bone remodeling can culminate in either a sclerotic or an osteoporotic phenotype. Patients with osteoporotic OA may thus achieve clinical and structural benefit from treatment with bone-targeted interventions.Subchondral bone has become a potential therapeutic target in osteoarthritis (OA). In a previous issue of Arthritis Research & Therapy, Wang and colleagues demonstrate that osteoporosis aggravates cartilage damage in an experimental model of knee OA in rats [1]. Interestingly, the authors also describe that extracorporeal shockwave therapy (ESWT), a mechanical therapeutic intervention probably acting at subchondral bone, may reduce OA progression [1]. The significance of these findings in experimental osteoporotic OA relates to the search for well-defined phenotypes in human OA that will lead to personalized therapy.The controversy regarding the relationship between subchondral bone quality and cartilage integrity originates from the complex biological and mechanical nature of the osteochondral junction [2]. OA progression is often accompanied by increased subchondral bone remodeling that enables mechanical forces to dynamically modify its structure. Depending on the ratio between formation and resorption, subchondral bone can exhibit either a sclerotic or an osteoporotic phenotype [3]. These phenotypes may represent up to 70% and 30% of patients in daily practice, respectively [4]. Furthermore, OA in females can display a different pathogenic profile from OA in males. In this sense, it is reasonable to underline the consequences of estrogen deficiency during menopause [5]. A low estrogen state could induce a deleterious effect on all articular tissues of the knee joint, the subchondral bone being particularly affected due to its capacity for high bone turnover. Thus, during early post menopause, estrogen deficiency may be a risk factor for the development of knee OA. Taking all these facts into consideration, the characterization of patients with either sclerotic or osteoporotic OA phenotypes may enable individualized targeted therapy [3].The effects of estrogen deficiency on the knee joint have been reported in various experimental animal models of OA. The findings obtained by Wang and colleagues on subchondral bone quality and articular cartilage damage support previous research carried out in rabbits, in which osteoporosis aggravated instability-induced OA [6]. In this combined model, the induction of systemic and subchondral osteoporosis associated with increased bone remodeling resulted in worse cartilage damage compared with control animals. Greater fragility of the subchondral bone was suggested to account for the aggravation of cartilage damage when early OA and osteoporosis coexist [7]. In a further study carried out in the same model, the intermittent administration of parathyroid hormone 1-34, a bone-forming agent, was used to increase subchondral bone density and quality [8]. As a consequence, the improvement of subchondral bone integrity was associated with reduced progression of cartilage damage in OA preceded by osteoporosis. In a similar approach, the inhibition of bone resorption by pamidronate in osteoporotic mice alleviated the instability-induced OA histological score with a reduction in the expression of aggrecanases [9]. Several experimental models therefore indicate that osteopenia/osteoporosis induces an accelerated progression of knee OA that can be reversed not only by bone-forming agents but also by antiresorptive drugs.These findings in animal models could be translated to humans, and together with epidemiological and clinical data they support the existence of a particular phenotype – osteoporotic OA [10]. Indeed, this phenotype characterized by decreased density and high remodeling at subchondral bone defines a subgroup of patients treatable with specific agents. In fact, beneficial effects of bone-acting drugs in OA are increasingly reported, but reliable conclusions regarding their efficacy are hindered by methodological drawbacks in study design [10]. Identifying patients with osteoporotic OA may improve the success of bone-directed agents.The original approach of using ESWT in OA by Wang and colleagues remains intriguing. These authors have reported previously that the application of ESWT to subchondral bone of the proximal tibia showed a chondroprotective effect in the initiation of knee OA and regression of established OA of the knee in rats. These effects were attributed to the ESWT multifunctional actions on cartilage and bone. Yet achieving such beneficial effects in this osteoporotic OA model suggests that the main mechanism of action of ESWT may be improving subchondral bone structure [1]. However, some limitations on the study design and the lack of adequate standardization of dosages and optimal frequency, as well as little information regarding the molecular mechanisms underlying the effects of ESWT, hold back the achievement of solid results. In any case, this study points out the potential benefit of nonpharmacological interventions aiming to improve mechanical properties of articular tissues in OA.In summary, the study by Wang and colleagues further supports the existence of the osteoporotic OA subtype and the potential benefit of bone-acting therapeutic interventions. Consequently, the identification of patient phenotypes along with the discovery of specific therapeutic interventions targeting relevant pathogenic mechanisms during the course of the disease could lead to a personalized approach to the management of OA.     

Irreversible Electroporation for Microbial Control of Drugs in Solution

The purpose of this study was to examine the feasibility of using irreversible electroporation (IRE) as a non-chemical method for eliminating microorganisms of liquid drugs. The studied drug was a topical ophthalmic medication, a pharmaceutical field in which the problem of microbial contamination has not yet been adequately solved, especially in the case of eye drops prescribed for chronic use. Commercially available Hylo-Comod® preservative-free eye drop solution was subjected to contamination with Escherichia coli bacteria (10⁶ colony forming units/mL). Electroporation parameters for bacterial control were investigated by comparing the effects of electrical fields of 5.4, 7.2, and 10 kV/cm, delivered as 100-µs square pulses at 1 Hz in sequences of 10 pulses, 20 pulses, or 20 pulses delivered as four sets of five pulses with 1-min intervals between each set. Microorganism survival after treatment was determined by pour plate counting. Effects of the treatment parameters on temperature and pH were recorded. Bacterial survival was lowest (0.14% ± 0.03%) after application of 20 pulses delivered as four separate sets. With that application mode, the solution remained at pH 7.5 and the temperature rose to 35.6° ± 0.2°C. Because IRE can be efficiently delivered under conditions that avoid the potentially deleterious effects of electrical pulses on temperature and pH, it appears to be a feasible method for bacterial control of drugs in solution. The principles established in this study can be applied to any drug in solution and optimized individually according to the solution''s composition.Key words: contamination, eye drops, irreversible electroporation, microorganisms, preservatives, sterilization of drugsContamination of liquid drugs can have substantial detrimental effects on the health of patients using drugs (1,2), necessitating the addition of preservatives in many pharmaceutical preparations. A particularly significant problem is the presence of preservatives in pediatric vaccination and the possible association with neurodevelopmental disorders such as autism (3,4). While this association is highly controversial, eliminating the need for preservatives in the vaccination will serve to allay the apprehension among parents and may increase the use of vaccinations. We focus here on the contamination of topical ophthalmic medications. The problem of infections engendered by microorganisms in eye drops has not yet been adequately solved and is especially troublesome when eye drops are used chronically for many years, as in glaucoma patients. In addition, patients who suffer from dry eyes and do not use the more expensive single-unit dose preparations are exposed to similar risks of infection, as are contact lens users. The problem of contamination can also arise in cases of acute eye drop treatment spanning days or weeks.The prevalence of bacteria in anti-hypertensive glaucoma drops in the community setting has been documented in a number of studies. Geyer et al. found bacteria in more than 28% of in-use topical medications (bottle tips and drops) of 109 treated glaucoma patients (5). The contamination rate was significantly related to the time since the container was first opened; bacteria were detected in 40% of eye drops from bottles that had been opened more than 8 weeks earlier compared to 19% in bottles in use for less time. Similar findings were reported by Schein et al. (6) in drops used by patients suffering from ocular surface diseases. Lower rates (12.8% and 12.9%) have also been reported (7,8). The high contamination rate is not surprising given the way in which eye drop containers are handled by patients. More than half of all elderly patients in one study were found to touch the eyelid or conjunctiva with the container, undoubtedly causing the solution to become infected by flora of the skin and conjunctiva (9). In contrast, a much lower contamination rate (2.3%) was measured in drops used by medical personnel in a clinic (10).Growth of microorganisms in ophthalmic medications can be reduced to some extent by adding preservatives to the solution, typically benzalkonium chloride (BAK). However, since the contamination rates cited above were found in eye drops that contain preservatives, their presence obviously does not solve the problem. Moreover, all preservatives have considerable side effects, particularly when the medications are used on a chronic basis. BAK, which is used in most topical ophthalmic preparations, harms the surface of the eye and probably accounts for the finding that well over half of treated glaucoma patients suffer from symptoms and signs of dry eyes (11). This compound can actually be used to induce inflammation when producing a dry eye model in rabbits (12). Not only does BAK damage the superficial eye tissues but its chronic administration apparently also harms the trabecular meshwork and thus may counteract the anti-hypertensive ocular drugs in which it helps control the bacterial load (13). Newer preservatives might be less injurious to the eyes than BAK, but they too are not free of complications (14), and they have not been in use long enough for their possible effects to be precisely determined. As expected, preservative-free medications seem to produce the least complications (15). One possible way to overcome the problem might be through the use of eye drops packaged in single-unit dose containers. These, however, are expensive and are not generally used for the glaucoma drugs financed by health maintenance organizations. Furthermore, many elderly patients find the containers difficult or impossible to manipulate properly (9).Clearly, then, it is important to find a non-chemical, practical method of bacterial control in liquid fluids in their delivery containers. Accordingly, the aim of this study was to examine the feasibility of using irreversible electroporation (IRE) as a method for controlling bacterial contamination in liquid drugs. Electroporation is a physical phenomenon in which a cell membrane becomes permeabilized by application of short (microsecond-scale) electrical pulses across the living cell. The mechanism presumably operates by forming nanoscale defects in the cell membrane. The overall effect of the electrical pulses is a function of various pulse parameters such as pulse length, pulse amplitude, and number of pulse repeats. These parameters determine whether the cell membrane will remain intact or will become permeabilized, either reversibly (reversible electroporation) or irreversibly (IRE). The nature of both reversible and irreversible electroporation, and their uses, most widely in the food industry, are well documented and have been comprehensively reviewed in the scientific literature (16–26).We postulated that IRE can be used as a means of bacterial control in fluid drug containers, either for the whole volume or during the passage of fluid into and out of the container. The issues to be addressed when treating drugs by IRE are different from those documented in the case of foods. With drugs, the volume of the solvent is significantly smaller, its ionic content is proportionately much larger (25), and the solution conditions after IRE [in particular temperature and pH (25,26)] should remain unchanged to avoid their potentially undesirable effects on the drug. These issues are addressed in this preliminary study on the use of IRE in bacterial control in liquid drugs. We first studied the effects of IRE in a liquid ophthalmic preparation and at a volume typical of eye drop containers. We next investigated what are the IRE pulse modes capable of maximal reduction in bacterial contents of the solution in these small containers without substantially affecting its temperature. Finally, we examined the effects of the IRE pulses on the pH of a small volume of solution.     

Borderline pulmonary pressures in scleroderma - a ‘pre-pulmonary arterial hypertension’ condition?

Patients with systemic sclerosis may develop borderline pulmonary arterial pressure. The clinical relevance of this condition is not always clear. Reported data support the evidence that this subgroup may represent an intermediate stage between normal pulmonary arterial pressure and manifest pulmonary arterial hypertension, a serious complication in scleroderma. Recognizing the clinical relevance of borderline pulmonary arterial pressure increase in scleroderma patients, future studies should aim for clear evidence for diagnostic and therapeutic algorithms for this population.In their recent study, Visovatti and colleagues [1] present a detailed analysis of patients with borderline pulmonary arterial pressure (PAP) as a subgroup analysis of the DETECT study, providing important clinical data for understanding early pulmonary vasculopathy in patients with systemic sclerosis.In fact, every physician who has observed the dramatic deterioration of patients with pulmonary arterial hypertension (PAH) and successive right ventricular failure would urge for the earlier recognition and therapy of this devastating condition. About 10% of all scleroderma patients may develop PAH [2], which - besides lung fibrosis - represents the most frequent cause of death in this patient population [3]. But can PAH be recognized at an early stage and maybe even prevented?If we assume that the increase of PAP is a process lasting for a longer period of time, there must be a phase of transition from normal (mean PAP ≤20 mmHg) pulmonary hemodynamic conditions to PAH (mean PAP ≥25 mmHg). Patients in this so-called ''borderline'' range may represent the early stage of PAH. Earlier studies found that such patients were more likely to develop pulmonary hypertension than patients with mean PAP ≤20 mmHg, with a hazard ratio of 3.7 [4]. The rate of borderline patients developing PAH was 19% after 3 years and 27% after 5 years. Accordingly, we may argue that borderline PAP is a ''pre-PAH'' condition in scleroderma. Of course, borderline elevation of PAP may be caused not only by pulmonary vasculopathy but also by cardiac or pulmonary co-morbidities [5]. In these cases borderline elevation of PAP may be considered as a general prognostic marker [5,6].The analysis of Visovatti and colleagues [1] includes several clinical (for example, current/past telangiectasis, presence of peripheral edema), laboratory (for example, ACA antibody, NT-proBNP), lung functional (for example, forced vital capacity (percentage predicted)/diffusion capacity for carbon monoxide ratio) and cardiac (for example, tricuspid annular plane systolic excursion) markers that may distinguish scleroderma patients with borderline PAP elevation from those with normal PAP or with manifest PAH. According to this analysis, borderline elevation of PAP in scleroderma patients may represent an intermediate stage in the continuum between normal PAP and manifest PAH.Among the DETECT population, 15% of all patients presented with borderline PAP hemodynamics. Although this number may be different in the general scleroderma population, due to the strict inclusion and exclusion criteria of the DETECT study [7], the borderline population seems to be a substantial subgroup. Unfortunately, follow-up data of the described patients in comparison with normal PAP and manifest PAH patients have not been provided. Such data might impact the development of clinical algorithms regarding further follow-up and treatment of these patients.In addition to the borderline elevation of resting PAP, another specific hemodynamic situation in scleroderma patients needs careful interpretation: exercise-induced PAP increase. Earlier studies showed that this may be a frequent condition among scleroderma patients and clinical deterioration and the development of PAH are frequent in this population [2]. In a recent analysis, a strong correlation between resting and exercise PAP values was evident [5], suggesting that patients with borderline hemodynamics and those with a strong PAP increase during exercise may strongly overlap, closing the gap between these two hemodynamic conditions.The most important question remains open: should targeted PAH therapy be offered to scleroderma patients with borderline PAP or exercise-induced PAP increase? Unfortunately there has been no clinical study investigating patients with borderline PAP so far and only two small studies have selected patients with exercise-induced PAP increase [8,9]. The results of these studies are promising, but need to be confirmed in adequately powered, randomized, prospective trials.Based on a series of studies indicating borderline hemodynamics has an important role in scleroderma patients with regard to the development of PAH and potentially for early treatment, future studies should aim for clear evidence for diagnostic and therapeutic algorithms for this patient population. This may contribute to a substantial prognostic improvement for patients with scleroderma who develop pulmonary vasculopathy     

Solutions in microbiome engineering: prioritizing barriers to organism establishment

Microbiome engineering is increasingly being employed as a solution to challenges in health, agriculture, and climate. Often manipulation involves inoculation of new microbes designed to improve function into a preexisting microbial community. Despite, increased efforts in microbiome engineering inoculants frequently fail to establish and/or confer long-lasting modifications on ecosystem function. We posit that one underlying cause of these shortfalls is the failure to consider barriers to organism establishment. This is a key challenge and focus of macroecology research, specifically invasion biology and restoration ecology. We adopt a framework from invasion biology that summarizes establishment barriers in three categories: (1) propagule pressure, (2) environmental filtering, and (3) biotic interactions factors. We suggest that biotic interactions is the most neglected factor in microbiome engineering research, and we recommend a number of actions to accelerate engineering solutions.Subject terms: Community ecology, Microbial ecology

Microbiome engineering is a rapidly evolving frontier for solutions to improve human health, agricultural productivity, and climate management. Microbiome engineering seeks to improve the function of an ecosystem by manipulating the composition of microbes. Two major challenges for successful microbiome engineering are (1) the design of a microbiome with improved function and (2) the establishment of an improved microbiome in a recipient system of interest. While multiple articles and reviews have addressed functional design [1–3], microbiome establishment has received less attention. Here, we propose a strategy to improve microbiome engineering by focusing on microbial establishment and leveraging insights from macrobial ecology.Two general engineering strategies are to manipulate indigenous microbes [4] or to introduce new members [5]. The latter involves the design and delivery of inoculants (a.k.a., probiotics in medical and agricultural arenas) and is a rapidly growing biotechnology sector. In their most general form, both strategies have been practiced crudely for thousands of years in human health [6] and agriculture [7]. However, despite current technical advances, inoculants frequently still fail to establish or confer long-lasting (months to years) modifications to ecosystem function [8]. We argue that this repeated failure is in part driven by lack of emphasis on establishment of inoculants.The problem of organism establishment in recipient ecosystems is not unique to microbiome engineering; it has roots in macrobiology, particularly invasion biology and restoration ecology. We propose that adopting a cross-disciplinary conceptual framework to identify barriers to organism establishment, and then prioritizing these barriers through targeted research will accelerate successful microbiome engineering. In addition, recognizing differences in terminology and experimental design within and across disciplines will facilitate research integration across diverse ecosystems and scales. The components of a more holistic strategy are discussed below.     

Predicting stable functional peptides from the intergenic space of E. coli

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make ‘user-defined’ genes from non-coding genome—how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40–48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into ‘protein sequence equivalents’ and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.     

Intrinsic microtubule GTP-cap dynamics in semi-confined systems: kinetochore–microtubule interface

In order to quantify the intrinsic dynamics associated with the tip of a GTP-cap under semi-confined conditions, such as those within a neuronal cone and at a kinetochore–microtubule interface, we propose a novel quantitative concept of critical nano local GTP-tubulin concentration (CNLC). A simulation of a rate constant of GTP-tubulin hydrolysis, under varying conditions based on this concept, generates results in the range of 0-420 s⁻¹. These results are in agreement with published experimental data, validating our model. The major outcome of this model is the prediction of 11 random and distinct outbursts of GTP hydrolysis per single layer of a GTP-cap. GTP hydrolysis is accompanied by an energy release and the formation of discrete expanding zones, built by less-stable, skewed GDP-tubulin subunits. We suggest that the front of these expanding zones within the walls of the microtubule represent soliton-like movements of local deformation triggered by energy released from an outburst of hydrolysis. We propose that these solitons might be helpful in addressing a long-standing question relating to the mechanism underlying how GTP-tubulin hydrolysis controls dynamic instability. This result strongly supports the prediction that large conformational movements in tubulin subunits, termed dynamic transitions, occur as a result of the conversion of chemical energy that is triggered by GTP hydrolysis (Satarić et al., Electromagn Biol Med 24:255–264, 2005). Although simple, the concept of CNLC enables the formulation of a rationale to explain the intrinsic nature of the “push-and-pull” mechanism associated with a kinetochore–microtubule complex. In addition, the capacity of the microtubule wall to produce and mediate localized spatio-temporal excitations, i.e., soliton-like bursts of energy coupled with an abundance of microtubules in dendritic spines supports the hypothesis that microtubule dynamics may underlie neural information processing including neurocomputation (Hameroff, J Biol Phys 36:71–93, 2010; Hameroff, Cognit Sci 31:1035–1045, 2007; Hameroff and Watt, J Theor Biol 98:549–561, 1982).     

A continuum mechanics model for the Fåhræus-Lindqvist effect

The decrease in apparent relative viscosity that occurs when blood is made to flow through a tube whose diameter is less than about 0.3 mm is a well-known and documented phenomenon in physiology, known as the Fåhræus-Lindqvist effect. However, since the historical work of Fåhræus and Lindqvist (Amer. J. Physiol. 96(3): pp. 562–568, 1931), the underlying physical mechanism has remained enigmatic. A widely accepted qualitative explanation was provided by Haynes (Amer. J. Physiol. 198, pp. 1193–1200, 1960) according to which blood flows in microvessels with a core-annulus structure, where the erythrocytes concentrate within a central core surrounded by a plasma layer. Although sustained by observations, this conjecture lacks a rigorous deduction from the basic principles of continuum dynamics. Moreover, relations aimed to reproduce the blood apparent relative viscosity, extensively used in micro-circulation, are all empirical and not derived from the analysis of the fluid mechanical phenomena involved. In this paper, we apply the recent results illustrated in Guadagni and Farina (Int. J. Nonlinear Mech. 126, p. 103587, 2020), with the purpose of showing that Haynes’ conjecture, slightly corrected to make it more realistic, can be proved and can be used to reach a sound explanation of the Fåhræus-Lindqvist effect based on continuum mechanics. We propose a theoretical model for the blood apparent relative viscosity which is validated by matching not only the original experimental data reported by Fåhræus and Lindqvist (Amer. J. Physiol. 96(3), pp. 562–568, 1931), but also those provided by several subsequent authors.     

Down on one knee: soft tissue knee injuries across the lifespan

Joint injury is a potent risk factor for osteoarthritis, the most important musculoskeletal disease affecting humankind. Yet the population incidence of soft tissue knee injury is not well documented. Using health-care register data from Sweden, Peat and colleagues report that soft tissue knee injuries are common, peak in adolescence and early adulthood, have a second spike in women who are 35 to 49 years old, and continue throughout the lifespan. The study highlights the need for more knowledge on the natural history of knee injuries, their impact on knee osteoarthritis development and progression, and the potential for prevention programs to reduce the incidence of these injuries.Joint injury is a potent risk factor for osteoarthritis (OA), the most important musculoskeletal disease affecting humankind. Although evidence is mounting that knee joint injury rates are high and increasing, it is also perhaps the lowest hanging fruit for primary OA prevention; several randomized clinical trials have shown that knee injuries can be dramatically reduced with relatively straightforward interventions. Yet outside of anterior cruciate ligament (ACL) injury and despite its potential public health impact, the population incidence of soft tissue knee injury requiring medical attention is not well documented: we have not known the extent or the nature of the problem, until now.In a recent issue of Arthritis Research & Therapy, Peat and colleagues [1] provided population-wide estimates of clinically diagnosed soft tissue knee injuries across all ages on the basis of an entire region of Sweden (approximately 1.3 million people). The opportunity to report and classify all clinically diagnosed knee injuries across the lifespan arises from unique and detailed health-care registries typical to Scandinavian countries. This overcomes weaknesses of previous epidemiological evaluations of knee injuries, which are limited to specific health-care settings, subgroups of people, and specific injury types. Of note, the findings of Peat and colleagues [1] have convergent validity - largely agreeing with previous reports of incidence for specific injury types and subgroups where data overlap.What emerges is that population exposure to soft tissue knee injury is a common problem; the annual incidences for males and females are 766 and 676 per 100,000 persons per year, respectively. This is approximately 10 times higher than ACL injuries alone. If these ‘less catastrophic’ but more common injuries are a risk for OA development (as risk factor studies measuring self-reported injury suggest [2]), then this study may be uncovering and detailing critical new exposure data. They are clearly more numerous though more difficult to accurately diagnose. This study begins to shed light on this challenge.Also revealed is new information on age and gender differences. The incidence of soft tissue knee injuries peaks in adolescence and early adulthood and is likely sports-related, matching seasonal fluctuations in popular sports in Sweden. The rates after this period decline over the lifespan with a notable exception: females from 35 to 49 experience a second peak. This is intriguing and the reasons are not clear, although the authors propose that the previously reported link between parity/child-bearing and knee OA may be mediated by injury. Although the reasons remain obscure, the finding is compelling and may help elucidate the consistently reported, but unexplained, higher prevalence of knee OA in females.Peat and colleagues [1] show that, although incidence rates are highest in the second and third decades of life, considerable rates of contusion, collateral ligament sprain, and other soft tissue strains continue into middle and old age. These injuries coincide with the age of onset of knee OA symptoms and illustrate the challenge of differentiating what is truly an injury from what is part of a previously latent or degenerative process or both. This also applies to meniscal injuries. Surgeries for meniscal tears peak in the mid to late 40s [3–5]. In contrast, Peat and colleagues [1] report a high incidence of meniscal tears in adolescents and young adults. As acknowledged by the authors, less severe injuries such as meniscal tears likely suffer from some misclassification. However, the relationship between diagnosis and surgery for meniscal tears requires further investigation.The high injury incidence among adolescents and young adults, together with the known risk of OA incidence from ACL and meniscal injuries, provides further impetus for implementing knee injury prevention programs, for which there is a strong body of level 1 evidence [6–11]. Efficacy has been demonstrated primarily in the sports team setting, implemented as novel 10- to 15-minute team warm-ups consisting of neuromuscular exercises to train athletes to land, decelerate, and push off with better lower limb alignment and improved trunk control, balance, and proprioception. The reported risk reductions range from 41% to 88% [7,8,11]. Given the age and frequency at which these injuries most often occur and their potential sequelae, perhaps targeting injury prevention programs to physical education classes in public schools could address a growing public health problem.The study by Peat and colleagues highlights several areas for further study. Knowledge is needed on the natural history of knee injuries in the development of knee OA as well as the potential for prevention programs to reduce the incidence. The spike of injuries in females between 35 and 49 requires confirmation and further investigation as to its causes, prevention, and potential role in OA development or progression. The same is true for injuries that occur in middle and older age, often coinciding with a time when knee OA has been diagnosed. Further clarity is needed around meniscal injury: what is traumatic injury and what is degenerative knee disease? There is still much to discover about the different knee injury types throughout the lifespan and the initiation and progression of knee OA. The study by Peat and colleagues [1] provides a good platform for this to be pursued.     

A mathematical model of the tripartite synapse: astrocyte-induced synaptic plasticity

In this paper, we present a biologically detailed mathematical model of tripartite synapses, where astrocytes modulate short-term synaptic plasticity. The model consists of a pre-synaptic bouton, a post-synaptic dendritic spine-head, a synaptic cleft and a peri-synaptic astrocyte controlling Ca^2 + dynamics inside the synaptic bouton. This in turn controls glutamate release dynamics in the cleft. As a consequence of this, glutamate concentration in the cleft has been modeled, in which glutamate reuptake by astrocytes has also been incorporated. Finally, dendritic spine-head dynamics has been modeled. As an application, this model clearly shows synaptic potentiation in the hippocampal region, i.e., astrocyte Ca^2 + mediates synaptic plasticity, which is in conformity with the majority of the recent findings (Perea and Araque (Science 317, 1083–1086, 2007); Henneberger et al. (Nature 463, 232–236, 2010); Navarrete et al. (PLoS Biol. 10, e1001259, 2012)).

Electronic supplementary material

The online version of this article (doi:10.1007/s10867-012-9267-7) contains supplementary material, which is available to authorized users.     

High-resolution genomic analysis: the tumor-immune interface comes into focus

A genomic analysis of heterogeneous colorectal tumor samples has uncovered interactions between immunophenotype and various aspects of tumor biology, with implications for informing the choice of immunotherapies for specific patients and guiding the design of personalized neoantigen-based vaccines.Please see related article: http://dx.doi.org/10.1186/s13059-015-0620-6Immunotherapy is a promising new approach for treating human malignancies. Approximately 20% of melanoma and lung cancer patients receiving immune checkpoint inhibitors show responses [1,2]. Current major challenges include identification of patients most likely to respond to specific therapies and elucidation of novel targets to treat those who do not. To address these problems, a detailed understanding of the dynamic interactions between tumors and the immune system is required. In a new study, Zlatko Trajanoski and colleagues [3] describe a powerful approach to dissecting these issues through high-resolution analysis of patient genomic data. This study represents a significant advance over previous work from this group, which defined 28 immune-cell-type gene expression signatures and identified specific cell types as prognostic indicators in colorectal cancer (CRC) patients [4]. Here, the authors [3] integrate genomic analyses of CRC tumor molecular phenotypes, predicted antigenicity (called the ‘antigenome’), and immune-cell infiltration derived from multiple independent cohorts to gain refined insights into tumor-immune system interactions.     

Modeling effect of GABAergic current in a basal ganglia computational model

Electrical high frequency stimulation (HFS) of deep brain regions is a method shown to be clinically effective in different types of movement and neurological disorders. In order to shed light on its mode of action a computational model of the basal ganglia network coupled the HFS as injection current into the cells of the subthalamic nucleus (STN). Its overall increased activity rendered a faithful transmission of sensorimotor input through thalamo-cortical relay cells possible. Our contribution uses this model by Rubin and Terman (J Comput Neurosci, 16, 211–223, 2004) as a starting point and integrates recent findings on the importance of the extracellular concentrations of the inhibiting neurotransmitter GABA. We are able to show in this computational study that besides electrical stimulation a high concentration of GABA and its resulting conductivity in STN cells is able to re-establish faithful thalamocortical relaying, which otherwise broke down in the simulated parkinsonian state.