Cholera causes an estimated 100,000 deaths annually worldwide, with the majority of burden reported in sub-Saharan Africa. In May 2018, the World Health Assembly committed to reducing worldwide cholera deaths by 90% by 2030. Oral cholera vaccine (OCV) plays a key role in reducing the near-term risk of cholera, although global supplies are limited. Characterizing the potential impact and cost-effectiveness of mass OCV deployment strategies is critical for setting expectations and developing cholera control plans that maximize the chances of success.We compared the projected impacts of vaccination campaigns across sub-Saharan Africa from 2018 through 2030 when targeting geographically according to historical cholera burden and risk factors. We assessed the number of averted cases, deaths, and disability-adjusted life years and the cost-effectiveness of these campaigns with models that accounted for direct and indirect vaccine effects and population projections over time. Under current vaccine supply projections, an approach optimized to targeting by historical burden is projected to avert 828,971  cases . An approach that balances logistical feasibility with targeting historical burden is projected to avert 617,424  cases. In contrast, approaches optimized for targeting locations with limited access to water and sanitation are projected to avert 273,939  and 109,817  cases, respectively. We find that the most logistically feasible targeting strategy costs US$1,843  per DALY averted during this period and that effective geographic targeting of OCV campaigns can have a greater impact on cost-effectiveness than improvements to vaccine efficacy and moderate increases in coverage. Although our modeling approach does not project annual changes in baseline cholera risk or directly incorporate immunity from natural cholera infection, our estimates of the relative performance of different vaccination strategies should be robust to these factors.Our study suggests that geographic targeting substantially improves the cost-effectiveness and impact of oral cholera vaccination campaigns. Districts with the poorest access to improved water and sanitation are not the same as districts with the greatest historical cholera incidence. While OCV campaigns can improve cholera control in the near term, without rapid progress in developing water and sanitation services or dramatic increases in OCV supply, our results suggest that vaccine use alone is unlikely to allow us to achieve the 2030 goal. In this modeling study, Elizabeth Lee and colleagues estimate the effectiveness of different strategies for targeting oral cholera vaccination in sub-Saharan Africa. Cholera is a waterborne infectious disease with substantial disease burden in sub-Saharan Africa.Oral cholera vaccines are safe and effective, but they have not often been considered for routine, preventive use because global supplies are limited.A recent World Health Assembly resolution declared a commitment to reduce cholera mortality by 90% by 2030, raising the importance of understanding how geographic targeting of routine mass cholera vaccination campaigns can maximize the health impact and cost-effectiveness of these programs.We developed a phenomenological model that examines the projected health impacts and cost-effectiveness for different vaccination campaign targeting strategies across sub-Saharan Africa from 2018 to 2030.The model leverages previous high-resolution estimates of cholera incidence and accounts for direct and indirect vaccine effects and population changes over time.The model suggests that targeting vaccination to districts based on historical cholera burden is significantly more effective than targeting districts based on indicators of access to water and sanitation infrastructure.We found that targeting vaccination campaigns geographically improved cost-effectiveness more than increasing vaccine supply or vaccination coverage.Effective geographic targeting of mass cholera vaccination campaigns optimizes the use of limited global cholera vaccine supplies.Targeting districts with the highest historical cholera risk leads to greater reductions in cholera incidence than targeting districts with poorest access to improved water and sanitation infrastructure.Despite a substantial projected health impact, mass vaccination campaigns cannot achieve the ambitious 2030 goal without large increases in access to improved water and sanitation. Cholera remains a significant global public health threat, causing more than 100,000 deaths per year globally, with sub-Saharan Africa bearing the majority of the burden . In May Due to successful campaigns conducted in over 15 countries between 2013 and 2018  and geneNevertheless, OCV presents several challenges to traditional approaches to vaccine deployment. OCV does not provide lifelong immunity; while the length of protection is uncertain, it is thought to wane significantly after 5 years . FurtherThe limited supply of OCV further complicates its integration into routine cholera control activities. In 2018, 23 million doses of OCV were produced globally (enough to provide the recommended 2-dose course to 11.5 million people) . Wit. Wit3]. These are the same as their rate-optimized counterparts, except districts were ranked according to raw cholera case numbers instead of estimated cholera incidence rates  only.We ordered districts across all countries according to their estimated coverage of access to improved water, improved sanitation, and improved watsan  only, anWe estimated the public health impact of conducting OCV campaigns from 2018 to 2030 as the number of cholera cases, deaths, and disability-adjusted life years (DALYs) averted over the period from 2018 to 2030 see .We reviewed 4 cost surveys for mass OCV campaigns that reported the vaccine delivery and vaccine procurement costs per fully vaccinated person  in non-refugee African settings –33 Tabl. One outProgram costs were measured as the cost per DALY averted (2017 USD), and discount rates for health benefits and costs were set to 0% and 3%, respectively . We calcWe performed 1-way sensitivity analyses of variability in vaccination deployment strategy, vaccine efficacy, indirect vaccine protection, vaccination campaign coverage, vaccine supply, net loss of vaccinated individuals due to migrations and deaths , and the time between vaccination campaigns in a given location  represents the direct vaccine efficacy n years after vaccines were administered to the population. We modeled demographic changes in the population aspi,k,t is the proportion of the population in location i from year k that is still present in year t. This proportion is calculated as a function of the population N in year k and location i, and the location’s net loss of vaccinated individuals due to migrations and deaths, μ. We assumed that the net loss of vaccinated individuals due to migrations and deaths (population turnover rate) was the same for all locations and tied roughly to median life expectancy across African countries in our data for 2018–2030 (65 years−1) [t and location i, Yi,t, is then calculated asSi,t is the susceptible proportion of the population after accounting for both direct and indirect vaccine effects, and λi is the projected baseline cholera incidence for location i. The total proportion of effective susceptibles Si,t may be represented asgv is a function that models the indirect effects of vaccination, as described in the Vaccine Properties section above , in location years−1) . The expFig C in . To estiAbsent substantial changes to cholera prevention and control, or secular trends in incidence, we expect 2.44 (95% CI 2.41–2.48) million reported cholera cases from 2018 through 2030 in sub-Saharan Africa, with nearly 50% of these cases in just 4 countries—the Democratic Republic of the Congo, Nigeria, Somalia, and Sierra Leone. Given this clustering of disease burden, we examined practical deployment strategies targeting the highest risk districts across sub-Saharan Africa according to historical cholera burden (rate optimized and rate-logistics optimized) and to water and sanitation coverage (water optimized and sanitation optimized) and assessed the sensitivity of our results to different vaccine-related assumptions , 34.0% (95% CI 33.2%–34.8%) of cases that would have otherwise occurred without vaccination from 2018 through 2030 were averted . This reTaking a logistically simpler approach to incidence-rate-based targeting, where high-risk districts within high-risk countries are targeted together (rate-logistics optimized), 25.3% (95% CI 24.8%–26.1%) of cases that would have otherwise occurred without vaccination were averted cumulatively, translating to 617,424  cases, 24,189  deaths, and 577,533  DALYs averted after 13 years of vaccination campaigns .Targeting districts geographically by lack of access to improved water and sanitation was not as effective as targeting by historical cholera disease burden. When targeting districts by lack of access to improved water (water optimized), 11.2% (95% CI 11.1%–11.4%) of cases were averted; this translates to 273,939  cases, 10,672  deaths, and 255,090  DALYs averted after 13 years of vaccination campaigns . TargetiAcross all years with vaccination campaigns, the rate-optimized strategy averted 20%–47% of annual cholera cases (range of mean estimates across years from 2018 to 2030) and the rate-logistics-optimized strategies averted 9%–31% of annual cholera cases  and $2,383  per DALY averted (2017 USD) for the rate-optimized and rate-logistics-optimized strategies, respectively . TargetiWe examined the 1-way sensitivity of our results to alternate parameters for vaccine efficacy, indirect vaccine protection, vaccination campaign frequency, vaccination coverage, vaccine supply, population turnover, and campaign deployment strategy, taking the rate-logistics-optimized strategy as the primary scenario ; Fig 3. This study suggests that geographic targeting is critical to ensuring that extended cholera vaccination campaigns across sub-Saharan Africa have a measurable impact on cholera incidence. When considering the direct and indirect protective effects of OCVs, our results suggest that, under projected resource constraints, campaigns that are geographically targeted according to disease burden may avert over 8 times more cholera cases, deaths, and DALYs than untargeted  approaches. Vaccination deployment strategy can have a greater impact on health impact and cost-effectiveness than substantial improvements to vaccine efficacy, vaccination campaign frequency, and vaccination coverage.Recent discussions and guidance by the GTFCC have suggested that priority areas for preventive cholera control (burden hotspots) should be ranked according to mean annual incidence, and that risk factors like access to water and sanitation should have a secondary role. Decision-making around OCV allocation and targeting is complex, and different actors are concerned with maximizing public health impact, navigating global and local politics, and considering the ethics of balancing preventive, responsive, and humanitarian uses of vaccine. Our results contribute to the discussion around maximizing the public health impact for preventive and potentially routine OCV campaigns, but our results should not be generalized to identify locations at risk for new cholera introductions. Seventy percent of the targets in the best-performing strategy are in countries with endemic cholera, and it is likely that reactive campaigns would occur in locations with less predictable cholera patterns  does prevent more cases, the resulting benefit would not likely outweigh the unmeasured added costs in logistical implementation. Our study suggests that vaccination targeting the highest-risk geographic locations can be a cost-effective cholera treatment strategy, thus complementing more comprehensive analyses that suggest the cost-effectiveness of targeting high-risk demographic groups .Improved access to safe water and sanitation is necessary for long-term cholera control and reductions in overall diarrheal disease burden –41. We eWe examined the sensitivity of our model to different assumptions about cholera and cholera vaccine dynamics in our models, but there remain several limitations outside the scope of these analyses  survey data collected under the new JMP definitions, combined with higher quality cholera surveillance data, may make nuanced treatment of complex epidemiologic interactions possible. For instance, projections of OCV impact could employ more complex targeting strategies that account for interactions between cholera immunity due to recent outbreaks and the probability of epidemic cholera introduction and propagation due to limited WASH access.Our results show how geographic targeting can play an essential role in ensuring that mass OCV use leads to substantial reductions in the global burden of cholera, even under current supply constraints. Strategic targeting of resources can play an essential role in making cholera control efforts cost-effective, a message that may be generalized to the entire suite of cholera control activities, including those to increase access to safe water and improved sanitation. Continued increases in global OCV production would enable a greater proportion of high-risk populations to be targeted with vaccination with greater regularity, but our results suggest that even the most effective geographic targeting strategies paired with optimistic OCV supply projections will not be enough to achieve the global cholera burden reduction goals set forth by the World Health Assembly resolution and the GTFCC roadmap to 2030. Substantial improvements to other sectors of the roadmap, including broad investments and progress in improving water and sanitation infrastructure and cholera surveillance, will be needed to make headway in this ambitious initiative.S1 AppendixThis content includes Tables A to Q and Figs A to O.(PDF)Click here for additional data file.


Frontiers of Immunology selected a series of articles focusing on how immune and tissue cells respond to crystals, microparticles, and nanoparticles. Our authors shed light on the recent and ongoing research efforts to unravel the molecular mechanisms underlying particle-related tissue injury, inflammation, and remodeling. We hope the readers of this Research Topic find the content useful to further develop the field, to identify common and particle-specific mechanisms of crystallopathies.Crystals, fibers, and other microparticles form inside the body from nutrients or metabolites but also enter the body from outside during environmental or occupational exposures. Altogether crystal and microparticle-related diseases account for numerous medical disorders . In someNano- and microparticles induce a broad range of cellular responses when exposed to immune and non-immune cells. These crystalline particles could be either of environmental origin e.g., silica, asbestos, air pollutants, or biological origin e.g., cholesterol, calcium, and monosodium urate (MSU) crystals.Shu and Shi gave a systematic overview of the physical and chemical properties of solid particles and their host responses. For example, phagocytosis of solid particles is mainly governed by their size, which regulates the inflammatory and cell death responses. Crystals with sharp and pointy edges induce direct injury as compared to spherical crystals and rod-shaped crystals. Accordingly, Köppert et al. also found that primary calciprotein particles, which are spherical, amorphous and soft, were rapidly cleared by liver sinusoidal endothelial cells, whereas secondary calciprotein particles, which are longer, more crystalline, and less soluble, are cleared by Kupffer cells and macrophages.In the current issue, Shu and Shi). In addition, MSU was reported to activate Syk and Src pathways leading to cell death and inflammation in phagocytosis-independent manner (Shu and Shi). Similarly, Shu et al. demonstrated that microscopic cholesterol crystals extract cholesterol from the cell surface upon physical interactions with the plasma membrane, which results in catastrophic plasma membrane rupture and necrosis independent of any known cell death-inducing pathway. Furthermore, in response to stress, a self-cannibalization process termed autophagy is activated, which might be associated with cell death. Frauscher et al. demonstrated that uremia-induced autophagy in vascular smooth muscle cells protects from vascular calcification. Interestingly, pharmacological activation of autophagy protected mice from vascular calcification during chronic kidney disease (CKD).Cytotoxicity of crystals mainly involves lysosomal rupture, activation of multiple cathepsins, mitochondrial dysfunction leading to either apoptosis or regulated necrosis—viz. mitochondria permeability transition-related necrosis, necroptosis, and pyroptosis . Silica nanoparticles when ingested during pregnancy cause pregnancy complications. Bilyy et al. demonstrated that agglomerations of non-stabilized superparamagnetic iron oxide nanoparticles (SPIONs) induce NET formation both in vivo and in vitro. Interestingly, they observed that inert coating of SPIONs with biocompatible albumin or dextran reduced agglomeration and NET formation by neutrophils and prevented vascular occlusion in vivo. These findings again confirm that the physical and chemical properties of solid particles drive host response (Shu and Shi).A wide range of solid particles is known to induce the formation of NETs , which rLi et al. summarizes findings related to NETs formation upon the contact of neutrophils with crystals from MSU, calcium pyrophosphate dihydrate, cholesterol, as well as calcium carbonate and silica. Hydrophobic nanoparticles cause NET formation by membrane disruption and lysosomal leakage. However, for some micro- and nanoparticles the mechanisms of action for the formation of NETs still remains elusive.At their discovery NETs were considered effective weapons aimed to capture, immobilize, and kill bacteria. Later the list of targets was extended to fungi, viruses, and further pathogens. Recent discoveries have clearly demonstrated that nano-and microparticulate matter is also prone to stimulate NETs formation. The minireview of Fernandes and Naccache. The data suggest that interaction with receptor not only induces mechanical grouping of these cell surface molecules but initiate more complex regulatory processes with distinct effects on leukocyte activation. Targeting inhibitory receptors is a potential therapeutic option to evaluate existing drugs and future drug candidates. However, the list of nanoparticles triggering NET formation can be quite exotic, and very selective.Nanocrystals e.g., those from MSU or cholesterol are prone to cluster cell surface receptors to trigger cellular responses. The inhibitory receptors for MSU-induced inflammation CLEC12A and SIRL-1, are thoroughly discussed in the minireview of Damby et al.). Inflammasome activation through inhalation of volcanic ash again links air-born microparticles with neutrophil activation and pulmonary diseases. Interestingly, cellular uptake of ash results in lysosomal destabilization, as was reported earlier size dependently for 10 nm diamonds , was identified as a trigger in macrophages of NLRP3 inflammasome activation with subsequent release of IL-1β , and thus anti-TGFβ antibodies are widely used in treatment. Dominguez-Gutierrez et al.). In addition, the properties of a putative calcium oxalate receptor are discussed in the paper.Calcium oxalate nanoparticles were also found to activate human monocyte and enhance local tissue inflammatory responses governed by the production of TNF-a, IL-1b, IL-8, and IL-10. Contact with calcium oxalate crystals but not with zinc oxide nanoparticles stimulated macrophage differentiation into the inflammatory M1-type , where long pong pentraxin 3 (PTX3), an opsonin known to interact with dead cells and other extracellular microparticles, was identified as an endogenous factor inhibiting growth of calcium oxalate crystals. By limited CaOx crystal aggregation and adhesion to tubular cell membranes it serves as one of several endogenous inhibitors of stone formation in nephrocalcinosis and potentially other crystallopathies.Another natural defense mechanism against nanoparticles was reported in the work . Effect of the nanoparticle engulfment can be quite different. Silver and gold nanoparticles when covered with tannic were able to induce maturation of dendritic cells and stimulate the uptake of viral particles (HCV-2) .When it comes to immune response the first task of the immune system is to recognize the invader, and micro-and nanoparticles are hard to be recognized, since usually they are the same compounds the body encounters daily in smaller concentrations or in a different soluble form . The review of Mahon and Dunne summarize the role of particles associated with gout, calcium pyrophosphate deposition (CPPD) disease, and osteoarthritis (OA). As well as wear-debris particles generated from prosthetic implants and contributing to joint destruction through the production of cartilage-degrading enzymes and pro-inflammatory cytokines, driving periprosthetic osteolysis which impacts on the longevity of total joint replacements. Understanding the danger of implanted material will allow us to better define molecular targets to inhibiting side effects of implantation-associated dangerous micro and nanoparticles .Unfortunately, there are much more of them causing harmful effects. The minireview by Mayeux et al. this limitation was overcome by in-depth analysis of diversity outbred mice, revealing not only association of silicosis with lung autoimmune markers, bronchoalveolar lavage fluid cells, IL-6 and anti-ENA5, but also stronger silica-induced inflammation between mal mice. We should remember that cellular remnants and “apoptotic blebs” are also of nano- and microscale, and usually engulfment is silent and anti-inflammatory process . Apoptotic cells were known to produce different type of micro and nano extracellular vesicles (EV) they were known to be differently glycosylated and distinctly processed by macrophages . Another important finding suggests that release of large extracellular vesicles, 200 to 1,000 nm in diameter, involve proteasome action .Some nanoparticles like silica are known for decades to cause disease and autoimmune disorder, but it is not that easy to study the conditions in experimental setup due to the fact that most established animal (mice) cell line are prone to develop only a part of the whole repertoire of interactions attributable to autoimmune conditions in human. In the work of rophages . HoweverChulpanova et al.).But when it comes to cancer, it looks that EV are becoming powerful communicating and instructing tools. In minireview “Role of macrovesicles produced by cancer cells,” immune cells and mesenchymal stem cells are discussed in details, demonstrating how modification of the extracellular vesicle cargo can target specific tumor mechanisms responsible for tumor formation and progression to develop new therapeutic strategies and to increase the efficacy of antitumor therapies .Immunosuppression is a key factor in silent clearance of dying cell microdebris, thus it's role in dealing with disease associated nano- and micro particles is crucial. The review by The studies of this issue illustrate that crystals and microparticles induce diverse biological responses beyond triggering the NLRP3 inflammasome and the secretion of IL-1β from myeloid cells . CrystalAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic rhinosinusitis (CRS) is nearly ubiquitous in patients with cystic fibrosis (CF). CF CRS is a challenging entity to define, diagnose, and treat, as patients often have severe refractory sinus disease in addition to complex medical comorbidities. The purpose of this article is to review the literature on the medical management of CF CRS and determine how to best identify, diagnose, and manage CF CRS. Ultimately, the treatment of these patients requires a multi-disciplinary approach involving the pulmonologist and otolaryngologist. It is a systemic disease that can affect the sinopulmonary, gastrointestinal, and genitourinary systems [Cystic fibrosis (CF) is an autosomal recessive genetic disorder characterized by poor chloride ion . Hopkins et al. showed that the Sinonasal Outcome Test-22 (SNOT-22) is a validated instrument that includes rhinologic, extra-nasal rhinologic, aural/facial, psychologic dysfunction, and sleep dysfunction domains that can be used to determine the effect of CRS on a patient’s QOL as well as assess the outcome of surgical therapy . Habib eAnother QOL instrument studied in the pediatric CF population is the Sinus and Nasal Quality of Life Survey (SN-5). The SN-5 has been validated as a reliable health-related QOL survey in children ages two to fourteen with sinonasal symptoms . It asksNasal endoscopy is another tool commonly used by otolaryngologists in diagnosing paranasal sinus pathology. For CF patients with sinonasal disease, nasal endoscopy can reveal thick nasal drainage, mucosal edema, and nasal polyposis ,8,15. HoComputed tomography without contrast is indispensable in evaluating patients for CF CRS. Common findings include sphenoid or frontal hypoplasia/aplasia, medial bulging of the lateral nasal wall, demineralization of the uncinate process, sphenoethmoidal recess inflammation, sinus opacification, osteitis and neogenesis, and sclerosis of paranasal sinus bone ,17,18. HCFTR gene with categories ranging from defective synthesis of CFTR in class I mutations to reduced synthesis or stability of active CFTR in class V mutations [The relationship between CF patients’ genotype and resultant severity of sinus disease has also been assessed. Abuzeid et al. found that patients with high risk CF genotypes, such as class I–III mutations which lead to markedly diminished presence or activity of the cystic fibrosis transmembrane regulator chloride channel, have no statistically significant difference in their SNOT-22, Lund–Mackay, and LKNE scores when compared to low-risk CF genotypes, such as class IV–V mutations, after controlling for confounding variables [utations . They noutations .Based on the above findings, both nasal endoscopy and computed tomography should be used concurrently to identify objective characteristics of sinonasal disease in CF patients presenting with sinonasal symptoms. The symptomatic patient with evidence of nasal polyposis, sinonasal mucopurulence, and mucosal edema on nasal endoscopy as well sclerotic bone and opacified paranasal sinuses on CT should possibly be treated for CRS.In treating CF-related sinonasal disease, a number of systemic and topical medical therapies have been identified, with varied data supporting their efficacy as seen in Nasal saline irrigations are also commonly used to treat chronic sinusitis. Mainz et al. performed a randomized controlled trial in which CF patients used both 28 days of isotonic saline irrigations and 28 days of hypertonic (6.0%) saline irrigations with a washout period in-between . In totaTopical corticosteroids are another option available in treating CF sinonasal disease. Hadfield et al. performed a randomized controlled trial in which CF patients with nasal polyps were given 6 weeks of either twice daily topical betamethasone or placebo . After sPseudomonas aeruginosa and Staphylococcus aureus are common in CF patients, topical antibiotics are yet another therapy for treating CF-related sinus disease [P. aeruginosa growth as well as a statistically significant improvement in SNOT-20 scores when compared to a placebo [As chronic infection in the upper and lower airways with  disease . One stu disease . However placebo . Yet ano placebo . This evCFTR modulator that acts by restoring CFTR ion transport function at the cellular level in patients with specific CFTR mutations known as gating mutations [CFTR modulator therapies approved for an ever expanding list of CFTR genotypes. Though a surplus of data on the modulators’ effect on CF CRS is still lacking, it is possible that these groundbreaking drugs, with their ability to restore chloride transport to improve mucociliary clearance in CF, will modify the course and attenuate the severity of disease of the upper airway as it does in the lower airways.Ivacaftor is a recently developed novel therapy which works as a utations . In CF putations ,40. Withutations . Additioutations . Furtherutations . There hIbuprofen is yet another medical therapy that some have trialed for the treatment of CF-related sinonasal disease. One study showed that 12 patients with CF and nasal polyposis had resolution of their nasal polyps at some point during high-dose ibuprofen therapy . HoweverCFTR modulator.At our institution, we use a variety of methods to diagnose and treat patients with CF CRS. Our main diagnostic strategies include history, physical exam, nasal endoscopy, and CT of the paranasal sinuses to evaluate for mucosal inflammation and presence of sinus hypoplasia/and osteitic bone. Together, our otolaryngologists and pulmonologists additionally discuss recent pulmonary function and frequency of pulmonary infections to help determine which patients may require treatment. Our therapeutic strategy includes a combination of surgical and medical therapy; the details of surgical therapy are outside the scope of this review. The medical management includes a combination of saline irrigation compounded with a corticosteroid and antibiotics, delivered with a sinus rinse bottle, which is readily available at local pharmacies. Our experience is consistent with the benefits demonstrated in the literature regarding similar strategies. Additionally, many patients referred to our office are concurrently using systemic therapies such as a Cystic fibrosis is a multisystem disease with a high-level of pulmonary morbidity and mortality that also has a considerable impact on the paranasal sinuses. CRS negatively affects quality of life in individuals with CF. Defining CF-related sinonasal disease and determining the indications for medical and surgical intervention remain a challenge. For primary medical therapy to treat CF-related sinus disease, a range of options are available, with the most data supporting the use of dornase alfa, topical corticosteroids, and topical antibiotics. Further prospective trials are needed to determine the optimal medical management for these patients.


Worldwide traveling air pollution particulate matter is now considered as a possible trigger factor for the development of a variety of neuropathologies. Micro- and nano-sized particles can reach the central nervous system during inhalation avoiding the blood–brain barrier, thereby making synaptic neurotransmission extremely sensitive to their influence. Neurosafety of environmental, engineered and planetary particles is difficult to predict because they possess other features as compared to bulk materials from which the particles are composed of. The capability of the particles to absorb heavy metals and organic neurotoxic molecules from the environment, and moreover, spontaneously interact with proteins and lipids in organisms and form biomolecular corona can considerably change the particles‘ features. The absorption capability occasionally makes them worldwide traveling particulate carriers for delivery of environmental neurotoxic compounds to the brain. Discrepancy of the experimental data on neurotoxicity assessment of micro- and nano-sized particles can be associated with a variability of systems, in which neurotoxicity was analyzed and where protein components of the incubation media forming particle biocorona can significantly distort and even eliminate factual particle effects. Specific synaptic mechanisms potentially targeted by environmental, engineered and planetary particles, general principles of particle neurosafety and its failure were discussed. Particle neurotoxic potential depends on their composition, size, shape, surface properties, stability in organisms and environment, capability to absorb neurotoxic compounds, form dust and interrelate with different biomolecules. Changes in these parameters can break primary particle neurosafety.Nerve cells take a special place among other cells in organisms because of their unique function mechanism. The plasma membrane of nerve cells from the one hand performs a classical barrier function, thereby being foremost targeted during contact with micro- and nano-sized particles, and from the other hand it is very intensively involved in nerve signal transmission, i.e., depolarization-induced calcium-dependent compound exocytosis realized via vesicle fusion following by their retrieval and calcium-independent permanent neurotransmitter turnover via plasma membrane neurotransmitter transporters that utilize Na Growing number of patients with neurological and neurodegenerative disorders and diseases, etiology of which is far from being clear, is burden of modern society. An increasing amount of environmental pollutants, especially particulate matter components of air pollution, can be responsible for the development of such pathologies . ImportaTons of cosmic dust from meteorites, comets and other objects of the solar system fall on the Earth daily. Also, beside problems associated with altered gravity, radiation, etc, threat to the nervous system during long-term space flight and planet colonization can come from planetary and interstellar dust exposure.Figure 1A) is quite uncertain and still remains almost undeveloped. From the one hand, this is because of both the relatively recent recognition of nanotechnology and also new consideration regarding involvement of air pollution particulate matter in the etiology of different neuropathologies, and from the other hand – because of enormous micro- and nano-sized particle diversity. Precise prognosis regarding neurotoxicity of the particles cannot be made even when they originate from well-characterized materials. It is so because bulk materials change their features when they act in a micro- or nano-sized form. Then, the particles change their properties during interference with biological objects, entrance to living organisms and interaction with proteins and lipids that spontaneously form biocorona at the particle surface. Also, properties of micro- and nano-sized particles can be changed by spontaneous absorption of a variety of neurotoxic compounds from the environmental surrounding.Novel experimental data demonstrate that the central nervous system (CNS) is extremely sensitive to the influence of micro- and nano-sized particles . CurrentNormal brain functioning requires maintenance of precise homeostasis of ions and molecules between the blood and brain by tight control and regulation of their transportation. It is realizing through a unique blood–brain barrier (BBB), which comprises a highly specialized endothelial cells adjoined by tight cell-to-cell junction forming a seal between opposing endothelial membranes. This barrier is characterized by specific transport mechanisms, a low pinocytosis rate and a weak permeability with restriction of paracellular solute diffusion from the blood . SpecifiNumerous neuropathologies, e.g., stroke, multiple sclerosis, brain tumors, Parkinson’s and Alzheimer’s diseases and neuroinflammation are accompanied by BBB dysfunction . DisturbThe CNS can be classified as a secondary exposed tissue during organism contact with micro- and nano-sized particles as it is not subjected to direct interaction with them. In the circulation, nano-sized particles can switch on cascade mechanisms disrupting the endothelial cell tight junctions in the BBB and change the membrane permeability. It has been demonstrated that intravenously, intraperitoneally and intracerebrally administrated gold, cuprum and aluminum nano-sized particles disrupt the BBB . Nano-si2 with thin cellular layer of ∼0.1–0.2 microm , which also aids in filtering inspired air . Intranasally instilled nano-sized maghemite particles were transported into the brain via the olfactory route .Despite the CNS is effectively protected by the BBB from the action of different substances in the molecular form, we can conclude from the previous subsection that this barrier does not strand against evolutionary new challenge of micro- and nano-particle exposure. This secure barrier is mainly unsafe and does not completely protect the CNS functioning during particle exposure associated primarily with modern and industrial anthropogenic activity .Between periodically occurred exocytotic events, the main activity is underscored by maintenance of the proper ambient level of fast neurotransmitters, for example glutamate, GABA, glycine, aspartate, through their permanent turnover across the plasma membrane driven by Nasporters . The funsporters  and any So, from the one hand the plasma membrane of nerve cells is involved extremely intensively in nerve cell functioning and signal transmission, and from the other hand it simultaneously performs the classical barrier and compartmentalization function. The plasma membrane is a first target for micro- and nano-sized particle influence when they reach and contact with nerve cells. In this context, nerve signal transmission and the CNS functioning in a whole can be extremely susceptible and sensitive to particle impact. Importantly, changes in plasma membrane properties, e.g., in the cholesterol level, can influence its barrier function regarding resistance to micro- and nano-sized particle exposure.Nowadays, general principles and methods for neurotoxicity risk assessment of environmental, engineered and planetary micro- and nano-sized particles are quite uncertain and still undeveloped. The neurotoxic potential is difficult to predict because the particles possess different features in comparison with bulk precursor materials from which they are derived from. Well-characterized compounds change their properties when they are transformed into the micro- and nano-sized forms. For engineered particles, it occurs during their synthesis and surface functionalization and for environmental ones – through exposure of bulk materials to environmental factors and during anthropogenic activity. The particles can change their properties during further interaction with neuroactive environmental pollutants and then with proteins, lipids, nucleic acids, etc., in living organisms being coated with biocorona. This unpredictable path can haphazardly and tremendously modify particle features making almost impossible integrated neurotoxicity risk prognosis.The particle size is very important parameter that can significantly contribute to their neurotoxic potential and determine their interaction with cells and cellular internalization. Nanoparticles, the size of which is less than 5 nm, are considered to be the most hazardous because of a nuclear access possibility and an extremely large ratio of surface parameters related to volume ones. Nanoparticles, the size of which is higher than 40 nm demonstrated a reducing internalization efficiency and cytotoxic effects , 2015. NFeatures of particle surface area can also considerably influence their neurotoxicity. Carbon nanodots synthesized from different precursors, i.e., sulfur-containing and sulfur-free ones, thiourea and β-alanine, respectively, possessed diverse surface characteristics, and therefore displayed a diverse level of efficiency; however, their effects were unidirectional in brain nerve terminals . NanodiaL-[14C]glutamate binding, whereas volcanic dust particles of spherical form did not show this effect , stability in organisms (including release of toxic substances), dust-forming capability of micro- and nano-sized particles can unexpectedly convert them from non-neurotoxic to potentially neurotoxic ones and vice versa. So, the exact effects of abovementioned factors on particle neurotoxicity need thorough understanding.The amount of death associated with ambient air pollution originated from modern industrial and urban development is growing. The total number of death attributable to air pollution particulate matter the size of which is 2.5 μm (PM2.5) increased by 20% from 3.5 million in 1990 to 4.2 million in 2015. A mortality prognosis associated with ambient PM2.5 air pollution has revealed that the number of death will increase over the next three decades by more than 50% upto 6.6 million in 2050 .Essential sources of air pollution carbon-containing particles are coal consuming technologies, operation of diesel engines, wood combustion for residential heating in the cold season and other areas of anthropogenic activity and natural disasters, that is, forest fires, oil, biomass and garbage burning. Also, tons of the particles including carbon-containing ones fall on the Earth daily . Wood coPM2.5 air pollution was demonstrated to be associated with a wide range of diseases, among which cardiovascular and pulmonary diseases are in the first place and are well-characterized showing the strongest causal PM2.5 associations. Emerging evidences promise that a causal association may exist between PM2.5 pollution and lowered cognitive function, autism and attention-deficit or hyperactivity disorder in children, neurodegenerative disease, including dementia in adults and stroke . It has The feasible brain health hazard from environment-derived micro- and nano-sized particles was demonstrated in recent study, where the magnetite nanoparticles were found in the human brain . GrowingSurface functionalized nanoparticles for targeted CNS drug delivery, neuroimaging, neurological disease treatment and neurotheranostics are in the main stream of modern research and development in the field of nanotechnology and nanomedicine. A lot of studies show a great progress in this valuable and prospective field. However, literature data examination reveals a lack of systematic analysis of neurosafety of different types of engineered micro- and nano-sized particles. This fact underscores importance and necessity of parallel comparative assessment of particle neuroactive features performed using similar methodological approaches.Titanium oxide nanoparticles have been used extensively and broadly. Their accumulation in the brain influenced metabolism and transport of neurotransmitters norepinephrine and serotonin . Animals+ increasing its efflux and induced neuronal apoptosis . This data has shown that synaptic neurotransmission and inhibitory/excitatory balance are exceptionally susceptible to nanoparticle administration.Comparative analysis of neurosafety levels of different types of micro- and nano-sized particles was carried out in parallel experiments using similar methodological approach. Unique dynamic balance of inhibition and excitation determines brain functioning of individuals and its disturbance contributes to the pathogenesis of main neurological disorders. Individual ambient concentrations of GABA and glutamate in the synaptic cleft are maintained due to neurotransmitter uptake by the plasma membrane neurotransmitter transporters in nerve terminals . Changesith Eu3+ , maghemiu3+  polychlorinated biphenyls and (∗∗) methyl mercury that themselves caused cognitive impairments especially in children exposed prenatally (∗∗∗) organophosphate pesticide, (∗∗∗∗) chlorpyrifos, which caused microcephaly at birth, delays in brain development and autistic behaviors in children as a result of prenatal exposure (∗∗∗∗∗) polycyclic aromatic hydrocarbons associated with neurodevelopmental delays in children exposed prenatally  oexposure , and , excess of which jeopardizes brain health . This approach can prevent masking effects of the primary biocorona consisted of incubation medium protein components that cover authentic neurotoxic potential of the particles. Factual membrane activity of the particles and their capability to form biocorona directly from plasma membrane proteins (and lipids) can be analyzed using this approach. We suggest that neurotoxicity of carbon dots, nanodiamonds, maghemite nanoparticles, nanocrystals shown in the experiments with the nerve terminal preparations (We suppose that exact acute effects of micro- and nano-sized particles on the cellular plasma membrane and in general particle-cellular interaction can be accurately monitored using different cell and tissue preparations, the incubation media of which do not contain additional protein components (arations  can be ain vitro system.Comparative analysis of neurotoxicity of different types on micro- and nano-sized particles in order to have less toxic ones or evaluation of the particle size, shape and surface effects can be properly made using identical During intranasal entry, the primary environment-derived biocorona of the particle surface can be spontaneously replaced by the secondary one consisted of mucosa proteins, and in case of particle access to the circulation the secondary biocorona can be formed from serum proteins. The important question is to what extent existed environmentally-derived particle biocorona can be replaced by the physiological one in organism. How easy neurotoxic substances and heavy metals absorbed at the particle surface can be released in organisms and whether or not biocorona changes the particle distribution and clearance in organism.Figure 4 represents a timeline diagram of important milestones.Environmental, engineered and planetary micro- and nano-sized particles can reach the CNS during inhalation due to their unique ability to bypass the BBB, affect neuronal functioning and provoke development of neuropathologies. An airborne pollution particulate matter disperses globally and crosses state boundaries, oceans and continents, thereby significantly widening the negative health consequences and enhancing the importance of its neurotoxicity risk assessment. Moreover, heavy metals and various neurotoxic substances from environment can be absorbed at the particle surface and occasionally use them as particulate carriers for brain access. The particles originated from highly polluted regions can be enriched with local neurotoxicants, thereby not only spreading them worldwide, but also being simultaneously brain delivery composite carriers during their traveling. Spontaneous particle-biomolecule interactions in organism and environment, and so biocorona formation can modify particle features and their distribution and clearance from organism. Particle-biomolecule interaction mechanisms are far from being clear and can be clarified by separate analysis of each type of these temporal complexes. A common unified methodology for assessment of neurosafety of environmental, engineered and space micro- and nano-sized particles still remains almost undeveloped thereby making difficult predictive modeling. Prognosis of particle neurosafety potential is complicated because precursor materials, from which the particles are synthesized from, have different properties as compared to those in the micro- and nano-sized forms. Experimental data on particle neurotoxicity varied in dependence on the systems, where it was analyzed. Exogenously added protein components during experimental procedures can significantly mask factual particle effects. The direct acute effects of the particles on the cellular plasma membrane can be uncovered using preparations, the incubation media of which do not contain exogenously added protein components.Neurosafety level of micro- and nano-sized particles depends on their composition, size, shape, surface properties, stability in organism and environment, capability to absorb neurotoxic substances, aggregate, form dust, and interrelate with different biomolecules. Newly arising changes in one of these parameters can break their initial authentic neurosafety.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MS and handling Editor declared their shared affiliation.


OBJECTIVES/SPECIFIC AIMS: Describe the process used to develop job descriptions and how this translates into consistent hiring practices. Describe how competencies are used to provide transparency into professional development opportunities. Discuss planned incorporation of competencies into efforts to train the clinical research workforce. METHODS/STUDY POPULATION: These processes were developed at Duke, an academic medical center with over 2000 active clinical research protocols and 300 new clinical trials per year. Over 1000 employees were evaluated for mapping into clinical research positions, with 685 mapping into new research positions (makeup of workforce to be depicted). RESULTS/ANTICIPATED RESULTS: Prior to this initiative, the clinical research workforce was not well-defined. Through the mapping process, employees were mapped from over 80 different positions into 10 (figure), resulting in a workforce that allows for visible career ladders and greater opportunity for development. As the initiative evolves and grows to include competency-driven performance evaluations, training modules, and assessments, we anticipate the ability to see the relationship between the competencies and high-quality clinical research support. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of competencies in the context of workforce development is not new, yet in clinical research, they provide a much-needed framework for an ever-evolving profession. This comprehensive use of competencies throughout a workforce development initiative is key to ensuring strong support of high-quality clinical research.


Conoidea venom is used for both predation and defense, and therefore under strong selection pressures. The need for conoidean venom peptides to be potent and specific to their molecular targets has made them important tools for investigating cellular physiology and bioactive compounds that are beneficial to improving human health. A convincing case for the potential of Conoidean venom is made with the first commercially available conoidean venom peptide drug Ziconotide , an analgesic derived from Conus magus venom that is used to treat chronic pain in HIV and cancer patients. Investigation of conoidean venom using -omics technology provides significant insights into predator-driven diversification in biodiversity and identifies novel compounds for manipulating cellular communication, especially as it pertains to disease and disorders.Venomous organisms used in research were historically chosen based on size and availability. This opportunity-driven strategy created a species bias in which snakes, scorpions, and spiders became the primary subjects of venom research. Increasing technological advancements have enabled interdisciplinary studies using genomics, transcriptomics, and proteomics to expand venom investigation to animals that produce small amounts of venom or lack traditional venom producing organs. One group of non-traditional venomous organisms that have benefitted from the rise of -omic technologies is the Conoideans. The Conoidean superfamily of venomous marine snails includes, the Terebridae, Turridae  Naja) has been used to identify 40% of all three-finger snake venom toxins (3FTxs) sequenced. Only three studies have used harder to milk and less studied, non-front-fanged snakes to investigate 3FTx bioactivity  are the most studied in the Conoidea  family, which has more recently been divided into seven family groups . The majority of conotoxins have been classified into venom gene superfamilies by examining the sequence identity of the signal sequence  vary significantly from the venoms of cone snails  rapid acting, (2) highly selective, and (3) very potent , calcium (Ca2+), and sodium (Na+) channels, as well as nicotinic acetylcholine receptors, and noradrenaline transporters (Conus magus, was approved for commercial use by the Food and Drug Administration in 2004 and is the first non-opiod analgesic (Conus that are currently undergoing clinical trials to determine of their potential as pain therapeutics (Conus peptides in pharmaceutical development are being used as analgesics, PVIIA shows promise as a therapy for myocardial infarction, and Conantokin-G for epilepsy (The >1 million estimated venom peptides expressed in conoidean venom are an immense resource for discovering novel compounds for therapeutic drug development. The majority of conoidean venom peptides are disulfide-rich molecules that have been shown to manipulate voltage and ligand gated potassium (Ksporters ; Fig. 4.sporters . Ziconotnalgesic . Similarapeutics . Althougepilepsy .Fig. 4.SThe progress of -omics technologies triggered a domino effect in venom research. An integrated venomics strategy has enabled a broader range of venomous organisms, many of which are non-model organisms, difficult to acquire, and contain limited amounts of venom, to be examined and ultimately contribute to the understanding of venom evolution in biodiversity. Without the vast technological molecular improvements in the last decade, studies would most likely still revolve around snakes, spiders, and scorpions. The increasing amount of venom peptides identified from Conoideans that are now in clinical trials, demonstrates the importance of expanding the diversity of venomous species examined . As -omi


To explore how access to transportation and technology/social media influence social connectivity among an ethnically diverse group of vulnerable low-income older adults, six focus groups were conducted (N=48) in English, Spanish, and Korean at a senior services agency. Qualitative thematic analyses revealed overarching themes that fit within the World Health Organization’s Age-Friendly Domains of Livability. The sub-theme “barriers and facilitators to accessibility” ran through each of the overarching themes, demonstrating how specific factors of the built, social, and community health environments intersect to promote or hinder social connection. Although transportation and technology uses were linked to social engagement, challenges with the built environment and limited financial resources hindered older adults’ abilities to remain engaged in their communities, both in-person and electronically. Age-Friendly initiatives must continue to consider the community-specific barriers and facilitators for older adults to remain physically and socially connected to the community.


Deep brain stimulation has developed into an established treatment for movement disorders and is being actively investigated for numerous other neurological as well as psychiatric disorders. An accurate electrode placement in the target area and the effective programming of DBS devices are considered the most important factors for the individual outcome. Recent research in humans highlights the relevance of widespread networks connected to specific DBS targets. Improving the targeting of anatomical and functional networks involved in the generation of pathological neural activity will improve the clinical DBS effect and limit side-effects. Here, we offer a comprehensive overview over the latest research on target structures and targeting strategies in DBS. In addition, we provide a detailed synopsis of novel technologies that will support DBS programming and parameter selection in the future, with a particular focus on closed-loop stimulation and associated biofeedback signals. Deep brain stimulation (DBS) has become the treatment of choice for movement disorder, such as Parkinson's disease (PD), medically intractable essential tremor (ET) and complicated segmental and generalized dystonia . In addiDBS surgery involves implantation of electrodes into one of several target regions and administering electrical current pulses that are generated by an implanted impulse generator. Although the effects of DBS on for example Parkinsonian symptoms and quality of life are generally satisfying , the cliThe setting of DBS parameters to optimize therapy is time-consuming and will likely get more complicated with new technological developments, introducing an ever increasing combination of parameters like pulse duration, stimulation frequency, stimulation contacts and so forth. In open loop DBS, which is the current standard protocol, these stimulation parameters are set by a clinician in a trial and error procedure and remain constant until manually updated, irrespective of disease fluctuations. In a closed loop DBS system, a sensor continuously records a feedback signal, a so-called biomarker, which is ideally correlated or causally linked to a clinical symptom. A second major point of interest in DBS research therefore is to develop more sophisticated strategies and automated algorithms on how to program and adjust stimulation parameters in a precise and effective manner.Contemporary research in humans features investigations into different network structures connected to individual DBS targets and explores structural networks , 14 invoStudies in dystonia patients have shown that ventral GPi stimulation is more efficient in alleviating dystonic symptoms . Using dWhile PD patients show similar improvement in motor function after GPi- and STN-DBS , STN DBSIn summary, these results highlight the relevance of targeting specific (sub)-structures and networks in improving the clinical outcome after DBS surgery.Functional neurosurgery has been driven by technological innovations and DBS has evolved over the years, including new approaches to surgical targeting, evaluation and in the delivery of therapy at the target. For a detailed overview see Gross and McDougal . ImproviNew software now allows for a patient-specific reconstruction of DBS leads based on MRI and post-operative CT imaging, the reconstruction of nuclei and fiber tracts adjacent to stimulation sites and the mapping of intra- and perioperative electrophysiological recordings , 35. ForThe VTA is a concept to model the spatial dimension of stimulation for a given set of stimulation parameters , 41–43. As the electric fields generated during multi-contact stimulation become more complex, new approaches are needed minimize the prediction error for VTA and to quantify axonal and pathway responses in patients-specific models , 47. TheAdvancements in imaging methods, such as ultra-high field MRI and new learning algorithms , 56–59 pToday, DBS systems stimulate in an open-loop manner, meaning that stimulation parameters are pre-programmed and are not responsive to changes in the patient's clinical symptoms or in the underlying physiological activity. Although open-loop stimulation is state of the art, limitations like overall efficiency, reduction of efficiency over time or side-effects have become more obvious with growing clinical experience. DBS therapy adjustment also remains time-consuming, requiring clinicians to evaluate numerous combinations of stimulation parameters in order to achieve the optimal outcome. Selecting the right combination among many possibilities can have a major impact on the therapeutic effect . DBS praMedtronic now offers the implantable and rechargeable neurostimulator Medtronic® ACTIVA RC + S, a research system following the Activa PC + S system, which records electrophysiological signals from the implanted DBS electrodes and also offers inertial measurements. New miniature implants  with namLooking forward, adaptive closed-loop stimulation systems that integrate feedback signals will ideally be able to rapidly respond to real-time patient needs and make human programming unnecessary . NeuroPaOptimally, biomarkers for adaptive closed loop DBS should be usable continuously after DBS implantation to make them applicable for clinical practice. Local field potentials and network connectivity measures based on electrophysiological signals with their high temporal resolution can already be measured with sensing DBS electrodes or other implanted neural sensors and hold great promise as biomarkers.Regarding closed loop adaptive DBS, a distinction has to be made between feedback signals (biomarkers) and mechanisms of control. A biomarker describes a correlative or causal relation to a clinical symptom. Adaptive control mechanisms then define how to adjust stimulation based on the evolution of biomarkers.Recordings of LFPs in the basal ganglia of PD patients show oscillations at several frequencies, including oscillations at low frequencies in the delta and theta band (1~7 Hz), alpha and beta band (8–35 Hz), gamma band (35–200 Hz) and high frequency oscillations (>200 Hz). It has been demonstrated that the beta activity amplitude is correlated with motor symptom severity without medication –68. MoreEarly approaches using local field potentials (LFP) as feedback signals for adaptive DBS incorporated the beta frequency amplitude as a mechanism to trigger the stimulation  demonstrThe use of electrophysiological biomarkers in aDBS is also restricted due to an often unfavorably low signal-to-noise ratio and interference with external artifacts like movement, speaking and cognition . Also, sFor a detailed overview of oscillatory features related to pathological and physiological states in DBS patients, see Neumann et al. .in vivo neurotransmitter concentration across multiple anatomical targets using implanted neurochemical sensors. These devices provide real-time neurochemical feedback for closed loop control  and actual clinical state . With such an approach, the optimal stimulus could be learned and adjusted based on feedback signals, closing the loop. Alternative stimulation protocols and parameters  could then be evaluated within a clinically acceptable range of stimulation energy. However, the vast amount of free parameters in DBS programming introduces a potentially very large search space to evaluate during reinforcement learning, even when constraining the search space to clinically acceptable parameters. Algorithms for reinforcement learning are commonly either model-free or model-based. While in model-free learning, the agent simply relies on trial-and-error experience to learn a policy that optimizes immediate and future reward, in model-based learning, the agent exploits previously learned lessons . Althougantially .This review is based on expert opinions and does not follow a systematic searching strategy.FH, JM, TK, and KB conceived the project. FH and CP did the literature search and wrote the manuscript. TK, JM, and KB wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Podarcis wall lizards to test whether phenotypic diversity within and divergence across populations follow concordant patterns, and to examine how phenotypic variation responds to genetic and environmental variability across different hierarchical levels of biological organization, in an explicit geographic framework.Understanding how phenotypic variation scales from individuals, through populations, up to species, and how it relates to genetic and environmental factors, is essential for deciphering the evolutionary mechanisms that drive biodiversity. We used two species of We found a general concordance of phenotypic variation across hierarchical levels . However, we also found that within-population diversity does not exhibit a coherent geographic structure for most traits, while among-population divergence does, suggesting that different mechanisms may underlie the generation of diversity at these two levels. Furthermore, the association of phenotypic variation with genetic and environmental factors varied extensively between hierarchical levels and across traits, hampering the identification of simple rules to explain what yields diversity.Our results in some cases comply with general ecological and evolutionary predictions, but in others they are difficult to explain in the geographic framework used, suggesting that habitat characteristics and other regulatory mechanisms may have a more substantial contribution in shaping phenotypic diversity.The online version of this article (10.1186/s12862-018-1237-7) contains supplementary material, which is available to authorized users. Variation is the cornerstone of evolution. It is the raw material for the action of selection and a central component for identifying how phenotypic diversification occurs across all levels of biological organization . The uniunder the null hypothesis of random accumulation of variation. Of course, evolution, and particularly that of the phenotype, rarely occurs under stochastic processes alone. Instead, phenotypic traits are usually also correlated with the environment where organisms live, through effects on physiological, functional and ecological performance ),Podarcis to be informative  and ventral (VSN) scales, femoral pores (FPN), and supraciliary granules (SCGN) Fig. . These tot fixed ). All scP. bocagei) and Additional file P. vaucheri).Based on each of the above sets of traits, we quantified the level of diversity within each population using the mean distance to the population centroid . In addiP. bocagei [To characterize recent population dynamics, we genotyped all individuals examined for morphological analyses for nine microsatellite loci, initially developed for  bocagei . Based o bocagei  genetic  bocagei .In addition, we examined a 623 bp fragment of the NADH dehydrogenase subunit 4 (ND4) mtDNA gene to describe the phylogeographic structure and deeper evolutionary history of the populations under study. Based on these data, we described the genetic distance among populations using Dxy, the average number of nucleotide substitutions per site between populations ; and estA detailed account of laboratory and analytical procedures followed to obtain genetic data, and the corresponding descriptors of population diversity and differentiation, are provided in Additional file R < 0.7), and which are known to be biologically informative for the species under study [http://glam1.gsfc.nasa.gov/; at 250 m of resolution) for the presumed activity period of wall lizards during our sampling periods . Furthermore, to characterize topographic conditions of each locality we derived the slope variable from altitude , using the “slope” function of ArcGIS [To characterize the average climatic environment of the studied populations, and fluctuations to it, we clipped the 19 bioclimatic variables (at 30 arc-seconds of resolution) included in the Worldclim database  to our ser study , 63, incer study ). We obtf ArcGIS . Finallyf ArcGIS . FurtherTo investigate whether the structure of morphological variation was concordant across individuals and across populations of each species, we performed principal components analyses of each multivariate trait block separately , considering a) the matrix of individual observations and b) population means for each trait. We then permuted population-centered residuals of individual observations across populations to examine whether the first principal component of individual morphological variation aligned with that of population divergence, by testing whether the angle θ between these two PC vectors was larger than that expected at random.To visualize the spatial structure of morphological diversity within populations, we spatially interpolated within-population mean distance to centroid for each of the four explored trait sets for each species, using the kriging interpolation method in ArcGIS . A detaiTo evaluate which genetic and environmental factors may contribute in shaping levels of intrapopulation phenotypic diversity, we used Generalized Least Squares (GLS) to fit single-predictor linear models while taking the spatial structure of our sampling into account. Specifically, we estimated GLS regressions with population phenotypic diversity  for each of the morphological traits examined as the response; and each of the genetic diversity and environmental factors described above as predictors. We preferred this simple, pairwise approach, instead of fitting multiple-predictor GLS models, to obtain an overall view of which factors may influence intraspecific phenotypic diversity without confounding statistical interactions among predictors.The most adequate autocorrelation structure for each response-predictor combination was determined by fitting models using a linear, exponential, Gaussian, or rational quadratic structure, and then comparing their fit to the data using Akaike’s Information Criterion (AIC). The model with the lowest AIC for each combination was considered to optimize the expected covariance among observations due to spatial proximity. When GLS models suggested a significant contribution of a genetic or environmental predictor on the level of population diversity for a trait, we examined the strength of such an association by inspecting the Pearson correlation between response and predictor variables.Similar to what we did for intra-population diversity, we used kriging of inter-population phenotypic distances to explore the geographic structure of phenotypic divergence among populations Additional file 2:Results of Generalized Least Squares analysis performed to identify environmental and genetic variables that contribute significantly in explaining differences across populations in levels of intra-populational morphological diversity while taking geographic distance into account. (DOCX 73 kb)Additional file 3:Geographic coordinates in the WGS1984 system, number of individuals sampled per sex  and in total (Ntot), and abbreviation (CODE) for the examined populations. (DOCX 16 kb)Additional file 4:Geometric morphometric procedures for quantifying variation in dorsal head shape, provides a detailed description of all steps involved in obtaining head shape data, including landmark definitions and digitizing, superimposition of landmark coordinates and other pre-processing applied to GM data. (DOCX 13 kb)Additional file 5:P. bocagei, provides the data used for all analyses for this species, after mean-centering by sex, including biometric and scalation variables as described in Methods, and superimposed, sex-mean-centered landmark coordinates (X1 – Y24), as well as the population of origin in accordance with Additional file Sex-corrected individual values for all morphological traits for populations of Additional file 6:P. vaucheri, provides the data used for all analyses for this species, after mean-centering by sex, including biometric and scalation variables as described in Methods, and superimposed, sex-mean-centered landmark coordinates (X1 – Y24), as well as the population of origin in accordance with Additional file Sex-corrected individual values for all morphological traits for populations of Additional file 7:Genetic analyses, provides a detailed account of of laboratory and analytical procedures followed to obtain genetic data, and the corresponding descriptors of population diversity and differentiation. (DOCX 81 kb)Additional file 8:Individual multilocus genotypes for the nine microsatellite loci. Each allele is coded as its length, given in bp. Missing data is represented by “0”. (XLSX 66 kb)Additional file 9:Maxent models for quantifying environmental suitability, provides a description of methods and results related to ENM used to obtain habitat suitability maps for each species. (DOCX 727 kb)


The aims of this paper are: (1) to review the current state of the art in the field of cartilage substitution and regeneration; (2) to examine the patented biomaterials being used in preclinical and clinical stages; (3) to explore the potential of polymeric hydrogels for these applications and the reasons that hinder their clinical success. The studies about hydrogels used as potential biomaterials selected for this review are divided into the two major trends in tissue engineering: (1) the use of cell-free biomaterials; and (2) the use of cell seeded biomaterials. Preparation techniques and resulting hydrogel properties are also reviewed. More recent proposals, based on the combination of different polymers and the hybridization process to improve the properties of these materials, are also reviewed. The combination of elements such as scaffolds (cellular solids), matrices (hydrogel-based), growth factors and mechanical stimuli is needed to optimize properties of the required materials in order to facilitate tissue formation, cartilage regeneration and final clinical application. Polymer combinations and hybrids are the most promising materials for this application. Hybrid scaffolds may maximize cell growth and local tissue integration by forming cartilage-like tissue with biomimetic features. The aim of this paper is to review the current state of the art of materials for cartilage substitution and regeneration. Several reviews about hydrogels for cartilage regeneration have been published for the last 10 years, focusing on preparation and characterization, natural and synthetic polymer precursors, gelation kinetics, cell and drug delivery, growth factors, mechanical properties and biocompatibility. Nevertheless, most of those reviews do not propose new alternatives to improve hydrogels properties which can fulfill the real clinical needs in terms of tissue regeneration, mechanical properties and degradation kinetics. Therefore, this paper reviews relevant literature published during 2013–2017, related to the application, fabrication, characterization, in vitro and in vivo assays of biomaterials based on hydrogels for cartilage regeneration. The studies selected are articles and reviews written in English.Currently, there are no clinical satisfactory solutions for cartilage tissue regeneration ,2,3. TheDuring the last few years, material scientists and tissue engineers have tried to help clinicians by confronting the challenge of manufacturing porous 3D scaffolds which resemble the chemical composition and architecture  of the eSeveral natural and synthetic polymers are being used to create novel materials, as collected in However, the complexity of the physical structure and properties of cartilage, including mechanical ,52,53,54In the case of articular cartilage repair, the required materials must provide successful mechanical properties, biological delivery, fixation of the device in situ and stability to the joint. Besides, the assays done to these materials need to be based on the intended biological effect and potential risks which have to be evaluated, such as toxicity, dedifferentiation, immunogenicity and contamination. Some preclinical trials with little animal models, such as rats and white New Zealand rabbits, are necessary to predict how biomaterials may behave during clinical assays. Moreover, some preliminary studies can be done in cadaveric human bodies; however extensive trials with large animal preclinical models are mandatory to obtain market approval. Therefore, just a few of the novel materials or tissue protocols are allowed to have clinical application or even to be commercialized. When trying to compare different studies of novel materials once they are introduced into clinical practice, there are other problems: (1) lack of homogeneity due to the different studied population; (2) short- and mid-follow-ups; (3) use of different evaluation systems; (4) new scaffold-based strategies for cartilage regeneration, either cell seeded or cell-free biomaterials; (5) procedures which differ in scaffold fixation methods, surgical approaches and postoperative rehabilitation phases. Therefore, there is the need among scientists, clinicians, industry and regulatory experts to improve communication and collaboration in order to overcome all the barriers in tissue engineering and to establish a defined road map to reach clinical application.Hydrogels are emergent candidates for applications in cartilage regeneration. Hydrogels are three-dimensional hydrophilic polymer networks made up of water-soluble polymers, crosslinked by either covalent or physical methods  Figure  to form One important reason for the choice of hydrogels in cartilage applications is the possibility of making them injectable which offers advantages over solid scaffolds such as the possibility of using a non-invasive approach. Injectable hydrogels can fill any shape defect and they may provide a homogeneous cell distribution within any defect size or shape prior to gelation ,110,111.Collagen II and glycosaminoglycans (GAGs) are cartilage-specific extracellular matrix components; they play a crucial role in regulating the expression of chondrocytic phenotype and in supporting chondrogenesis. They have been used for in vitro and in vivo assays ; many atInvestigations based on cell-free hydrogel scaffolds focus on their physico-chemical characterization and mechanical properties . These sw/w, 15% w/w and 20% w/w of PVA/PVP. It was found that the inner structures of hydrogels tend to be denser when polymer concentration and polymerization degree of PVA increase. While the friction coefficient increases (from 0.037 to 0.044) with an increment in the polymerization degree of PVA (average increase rate is approximate 3%), the friction coefficient decreases  with an increment in the polymer concentration (from 10% to 20%) in the low load region and under liquid lubrication. Thus, there is the need to keep friction coefficients stable under lubricated conditions [Poly(ethylene glycol) (PEG) and derived hydrogels are ones of the most widely used synthetic polymer for tissue engineering. The modulus for bovine articular cartilage, measured in compression mode, is 950 KPa, which is close to the value of the fully hydrated polyethylene glycol diacrylate (PEG-DA) hydrogel . Polyvinnditions .G′ changed from 0.01 MPa (0.01 Hz) to 0.02 MPa (15 Hz)) similar to that of tibia cartilage  [Another study using PVA-based hydrogels, crosslinked with trimetaphosphate (STMP), revealed that fully hydrated hydrogels were covalently crosslinked systems when mechanically tested, with a rheological behavior (the  tissue) . As prev tissue)  reportedSince many materials do not exhibit a low friction coefficient or withstand several loading cycles, some of them are infiltrated with an interpenetrating network hydrogel to form functional scaffolds which provide load-bearing and tribological properties, similar to native cartilage ones. For example: (1) a porous three-dimensionally woven poly(ε-caprolactone) fiber scaffold was infused with a “tough-gel” made of alginate and polyacrylamide ; (2) a bp < 0.0005; NHAC-kn = 136% and hMSC = 263%). Furthermore, a biohybrid composite scaffold, composed by combining a decellularized Wharton’s jelly extracellular matrix with the polyvinyl alcohol (PVA)-based hydrogel, demonstrated its ability in promoting chondrocyte adhesion and scaffold colonization [Cell-based hydrogel scaffold therapy is one of the main strategies being investigated in cartilage regeneration. Several scaffolds and materials are being evaluated. These studies focus on whether or not the hydrogels provide an appropriate biochemical and biomechanical environment for a long-lasting hyaline-type cartilage regeneration. Decellularized extracellular matrices from natural tissues like dermis or adipose are being studied as functional biologic scaffolds . It is pnization . Other snization . Althougnization .One of the problems being reported when using cell therapies is the dedifferentiation of chondrocytes when cultured in two-dimensional cultures, making them less functional for cartilage repair. Wu L. et al.  hypothesSome other studies are meant to analyze how the structure and fabrication methods of the scaffolds influence cells behavior. Due to their intimate contact with chondrocytes, scaffolds are important components of cell niche. Investigations into micro-architecture of scaffolds have revealed that mean pore size is cell-type specific and influences cellular shape, differentiation and extracellular matrix secretion . Studies3 and 1.0 mm3 porogens. They demonstrated that bilayered scaffolds can closely mimic some of the structural characteristics of native cartilage due to: (1) the addition of aligned fiber membranes enhances the mechanical and surface properties of scaffolds; (2) zonal analysis of scaffolds showed region-specific variations in chondrocyte number, sulfated GAG-rich extracellular matrix and chondrocytic gene expression; (3) smaller porogens (0.03 mm3) yield higher GAGs accumulation and aggrecan gene expression.Furthermore, each zone of cartilage tissue varies in regard to biochemical content, morphology and biomechanical function. Deeper cartilage zones present higher stiffness, higher proteoglycan concentration but lower cellular density and same collagen concentration along cartilage tissue. In a general structural perspective, cartilage can be simplified into two main regions: (1) the superficial zone which exhibits a high tensile strength and low friction coefficient to keep a smooth articulation; (2) a dense ECM region rich in proteoglycan molecules which give the tissue adequate compressive mechanical properties by producing a high osmotic pressure within the tissue. Therefore, when fabricating a bilayer or three-layer scaffold, pore size and fabrication method of each layer influence the microenvironment of chondrocytes. As shown by Steele J.A.M. et al. , tissue It is important to understand the multi-scale biomechanical behavior of cartilage tissue, in order to realize the connection among joint kinematics, tissue-level mechanics, cell mechanics and mechanotransduction, matrix mechanics and the nanoscale mechanics of matrix macromolecules. Therefore, understanding mechanical behavior at each scale helps to correlate cell biology, matrix biochemistry and tissue structure/function of cartilage .Moreover, the combination of cell-based therapies with growth factor delivery, which can locally signal cells promoting their function, is also being investigated. Since morphogenetic protein (BMP-2) and transforming growth factor (TGF-β) are cytokines proposed as stimulants for cartilage repair, it is necessary to undertake a detailed comparative analysis of their biological effects on chondrocytes . As an eEvaluating cell-free and cell-based hydrogels reviewed above, only a few of these biomaterials have been used in clinical applications ,178,179 Since some of the reactions used to synthesize hydrogels are limited due to their complexity, the use of cytotoxic reagents, instability of some functional groups, possible side reactions and low coupling efficiency, there is the need to explore and exploit simple and highly efficient methods which may be applicable to a great variety of biodegradable polymeric precursors.The degradation rate and mechanical properties of manufactured hydrogels must be compatible with the growth of new tissue . As mentDepending on the polymer precursors, hydrogels can be synthesized in different ways. In the first step a polymer is modified with a functional group; then the polymer is crosslinked, either physically or chemically, to form a three-dimensional structure. While chemical hydrogels are covalently crosslinked, physical hydrogels are not. Crosslinking can take place at the same time or after the copolymerization . In situThe morphology and physico-chemical structure of hydrogels also depend on processing conditions applied during their formation, for example using electrospinning or cryogelation techniques. Hydrogel morphologies may range from fibrils, characteristic of protein-based hydrogels such as collagen and fibrin, to amorphous, characteristic of synthetic hydrogels such as PEG. When using the electrospinning technique, it is possible to obtain hydrogels with aligned fibrils morphologies. Mirahmadi F. et al.  added deSince conventional fabrication techniques may not provide a precisely control of pore size, interconnectivity or pore geometry for scaffolds, solid freeform fabrication (SFF) techniques are now being used to produce 3D scaffolds with an organized interconnected pore structure which ensures good functionality and good mechanical strength, necessary to maintain new cartilage formation.Bioprinting and plotting are being used as freeform fabrication techniques. These emerging techniques are used to fabricate viable 3D tissue constructs through a precise deposition of cells in hydrogels . HoweverNovel techniques of tissue scaffold fabrication, as ultrafast pulse DLW lithography, are attractive due to their 3D structuring capability, spatial resolution, scaling flexibility and diversity of working materials.Hydrogels degradation kinetics should be compatible with new tissue formation kinetics, in order to ensure a good integration of the construct. If hydrogels degrade very fast, it will trigger occurrence of defects in the formed tissue, such as cysts. On the other hand, if a very slow degradation occurs, hydrogels will inhibit the formation of new biological material and their integration with the surrounding tissue. Degradation of hydrogels can take place by either hydrolytic or enzymatic mechanisms. The hydrolytic degradation happens when hydrogel is kept in contact with fluids by breaking the polymer chains or the crosslinked network. This type of degradation mechanism can be controlled by limiting the amount of degradable precursor, used to synthesize hydrogels. The enzymatic degradation is caused by cells when they begin to invade the hydrogel or when encapsulated cells within the hydrogel start to proliferate or migrate throughout it. Many natural origin proteins have sites of cleavage in the protease, allowing hydrogels to degrade during the replacement of the ECM. This type of degradation may also depend on the degree and the type of chemical crosslinking in the hydrogels used as scaffolds .When trying to manufacture biomimetic scaffolds for cartilage tissue regeneration, naturally-derived hydrogels are widely used, due to their macromolecular properties and because the employed biopolymers are part of the natural tissue that needs to be healed. Most of the studied naturally-derived hydrogels are based on biopolymers such as collagen, gelatin, chitosan, hyaluronic acid, chondroitin sulfate, agarose, alginate and fibrin ,208.Since hyaluronic acid is a fundamental component of natural cartilage matrix, some studies have shown its importance and its good qualities as an excellent naturally derived polymer. Injections of hyaluronan into osteoarthritic joints have proved to restore viscoelasticity, augment joint fluid flow, normalize endogenous hyaluronan synthesis, and provide joint function ,112. SomFurthermore, when combining both naturally-derived and synthetically-derived polymers, adequate degradation kinetics and biological response can be achieved. Park H. et al.  created,Identified problems hindering the application of hydrogels in cartilage regeneration, as described along this paper, include: mechanical properties  and mechco–ε–caprolactone)/poly(ε-caprolactone) thermoplastic polymer scaffold, functionalized with methacrylate groups and covalently linked to a chondrocyte-laden gelatin methacrylamide hydrogel), the binding strength between the materials improved significantly, resulting in the enhanced mechanical integrity of the reinforced hydrogel. Embedded chondrocytes in hydrogel scaffolds also showed significant cartilage-specific matrix deposition, both in vitro and in vivo assays.Mechanical instability inhibits the integration of hydrogels with the surrounding native cartilage tissue when they are implanted ,211. In Another promising option to enhance mechanical stability is by regenerating cartilage and bone tissue simultaneously using a two-phased scaffold, since ceramic-to-bone interface has a better and faster integration compared to hydrogel-to-cartilage interface . AdditioIt is possible to have a stable fixation of a cartilage scaffold by exploring a fixation technique with the subchondral bone . One wayFollowing this trend, Yang S.S. et al.  developeAnother alternative to fulfill the inadequate mechanical strength of hydrogel is constructing a solid-supported thermogel, comprising hydrogel systems or demineralized bone matrix. Huang H. et al.  combinedUsing fibers of different natural materials to reinforce hydrogels is another way to offer mechanical strength to hybrid scaffolds ,211. MecIt can be deduced, from the reviewed studies, that combination of components yield up reinforced mechanical properties. However, to overcome the other requirements mentioned above, something different has to be done from the already investigated methods to fabricate materials. In authors’ opinion, there is a barely explored alternative in material science: studying the potential of hybrid hydrogels based on natural polymers and inorganic components. Hybrids are considered to be materials formed by two components bonded at a molecular level. Commonly one of these components is organic and the other one is inorganic. The new hybrid materials  may showSince degradation rate and mechanical properties can be fine-tuned through chemical and/or physical modifications on either naturally or synthetically derived scaffolds, an excellent opportunity is given to hybrids to emerge as a promising solution for cartilage regeneration. When fine-tuned, hybrids can acquire amorphous, semicrystalline, hydrogen-bonded or supramolecular physical structures ,210.2, and –SO3H) found in the polymeric components. These molecules give hydrogels different absorption potential [2, or demineralized bone matrix), which restrict the number and type of hydrophilic molecules.Hydrogels made from purely organic precursors are being used as viable materials for cartilage repair due to their ability to retain grate portions of water, swollen, distend and exhibit large changes of dimensions  ,217. Theotential  and the l-lactic acid) (PLLA) [A further step may be the integration of nanotubes with different chemical composition into hybrid scaffolds which may provide them with bioactive and mechanical properties. This is the case of three-dimensional porous collagen sponges where single-walled carbon nanotubes are incorporated into . The inc) (PLLA) . It was ) (PLLA) .The association of silica and polysaccharides within composites or hybrids has demonstrated therapeutic benefits in a wide range of bio-inspired silica-collagen materials . AlthougCurrently, there are no clinical satisfactory solutions for cartilage tissue regeneration. From this problem, it raises the need for developing new types of materials and designing more suitable scaffolds which can be used for cartilage regeneration. Investigations based on cell-free hydrogel scaffolds have focused on the optimization of physical-chemical and mechanical properties of matrices. Whereas, cell-seeded hydrogel scaffold studies focus on whether or not they provide an appropriate biochemical and biomechanical environment for regenerating a long-lasting hyaline-type cartilage. Since many materials neither exhibit a low friction coefficient nor withstand several loading cycles, many combinations of polysaccharides and synthetic hydrogels have been assayed to obtain load-bearing and tribological properties similar to native cartilage tissue ones. However, some unsolved problems hinder the application of these materials to the clinic: (1) toxicity of some crosslinking agents; (2) lack of mechanical integrity; (3) poor control of gelation kinetics; (4) unsuitable degradation kinetics.Among the most promising options, to synthesize mechanically stable hydrogels which support matrix formation, there is the combination of materials to regenerate cartilage and bone tissue simultaneously, using a two/several-phased scaffold. These combinations can be biologically and physically tuned to yield, within a range of different cell responses and according to these responses, combined hydrogels which may show potential therapeutic possibilities to treat either chondral or osteochondral lesions. Osteochondral treatment introduces a new problem and requires the use of a chondrogenic stimulator, such as the recently described kartogenin, to induce chondrogenic differentiation and suppress hypertrophy. From materials science perspective, hybrid hydrogels based on natural polymers and inorganic components may offer a fine tuning of mechanical and biological response, required to reproduce the complexity of the cartilage tissue environment. Silica, hydroxyapatite or demineralized bone matrix/polysaccharide-based hybrids restrict swelling in hydrogels in certain positions or directions by reducing the number and the type of hydrophilic molecules. The association of silica and polysaccharides within composites or hybrids has demonstrated therapeutic benefits in a wide range of bio-inspired silica-collagen materials. To fully comprehend these potential materials and to raise their value for the development of innovative biomedical devices for cartilage regeneration, it is important studying the interplay between the organic-inorganic precursors, which is to follow carefully the polymer self-assembly process and the inorganic condensation mechanisms. Therefore, biological, mechanical and degradation properties can be modulated, bioactivity and cytocompatibility guaranteed and biocompatibility eventually achieved.


A 74-year-old female with diabetes mellitus type 2 was admitted to the emergency department due to fever, severe pain and oedema in her left forearm, with two days of evolution, after she has done gardening, but no clear history of trauma. At clinical examination, the patient presented hypotension, fever and a hematic phlycten on her left forearm, which was tense to palpation . LaboratStreptococcus pyogenes as causal pathogen. This confirmed the suspicion of streptococcal toxic shock syndrome (STSS) with necrotizing fasciitis given the persistent hemodynamic instability. As the criteria for STSS were met, clindamycin was maintained for 13 days and concomitant intravenous immunoglobulin was also administered during 3 of these days. Piperacillin/tazobactam and vancomycin were substituted by penicillin G according to Streptococcus pyogenes antibiotic susceptibility test. The patient died 13 days after the admission to UCI due to multiple organ dysfunction. Blood cultures identified STSS is an acute and severe life-threatening illness associated with invasive infections by streptococci, mainly group A streptococcus (GAS) . It reprThe admission to the ICU and the initiation of supportive treatment of several dysfunction organs is usually necessary. In case of necrotizing fasciitis, aggressive surgical debridement of infected and necrotic tissues associated with systemic antibiotic therapy should be given rapidly, involving high doses of parental beta-lactams, including penicillin G, an agent which is considered first line therapy, in addition to clindamycin for its toxin-neutralising effect . In patiNone declared.