Antimicrobial resistance (AMR) is the acquired ability of pathogens to withstand antimicrobial treatment. To bridge the gap in knowledge for implementing effective and targeted interventions in relation to the AMR in Armenia, we designed this study to explore the performance of AMR diagnostics and the profile of AMR in the Nork Infection Clinical Hospital (NICH) for the period of 2016–2019, particularly to (i) determine the proportions of antimicrobial resistance among all samples tested at the hospital laboratory, (ii) determine the proportion of resistance against specific antimicrobials, and (iii) identify factors associated with AMR. A cross-sectional study was conducted with a secondary data analysis that included all the patients tested for AMR in the laboratory of the NICH for the period of 2016–2019. For this period, only 107 (0.3%) patients out of 36,528 had their AMR test results available and of them, 87 (81%) had resistance at least to one tested antimicrobial. This study has provided some valuable information on the AMR situation in Armenia. The results call for immediate actions to control the access to and the use of antimicrobials, strengthen AMR surveillance, and improve laboratory capacity for the proper and fast identification of drug resistance through a comprehensive system. Antimicrobial resistance (AMR) is the acquired ability of pathogens—whether they be viruses, bacteria, parasites, or fungi—to withstand antimicrobial treatment. This ability is acquired rather than innate and is secondary to inadequate exposure to treatment, which provides time for the pathogen to mutate and develop a mechanism to resist pharmacological therapy. However, the transfer of the resistant strains is possible, and patients can get a resistant infection from others. AMR leads to ineffective standard therapies, which results in longer and more complicated treatment courses, requiring additional tests and more expensive treatment regimens ,2. As a The genetic changes that result in AMR are a part of a natural process that is constantly occurring—however the process is accelerated when antimicrobials are misused. Misuse can take many forms, such as incomplete treatment courses, overuse of medicines when not indicated, poor or non-existent infection prevention and control programs, poor laboratory capacity, and inadequate surveillance and regulation ,3. AMR iArmenia is not an exception. The purchase of antimicrobial drugs without a doctor’s prescription is highly prevalent within the country. In 2015 the Armenian government implemented an AMR strategy and action plan in line with World Health Organization (WHO) global efforts to tackle AMR ,5. This While antimicrobial resistance is poorly understood in Armenia, several studies have attempted to assess the rates of self-medication in the country. In 2005, the prevalence of intended self-medication and the actual prevalence of self-medication were found to be 53.1% and 12.5%, respectively . In 2016The present available evidence indicates that the AMR is a major public health problem within Armenia, however little research exists for public policy to tackle the issue. In particular, the assessment of the performance of AMR diagnostics and the profile of AMR in the main facilities in charge of controlling infectious diseases in Armenia can help bridge the gap in knowledge in order to implement effective and targeted interventions. Thus, we designed this study to explore the performance of AMR diagnostics and the profile of AMR in the Nork Infection Clinical Hospital (NICH) in Armenia for the period of 2016–2019. The specific objectives of this study were to (i) determine the proportions of antimicrobial resistance among all samples tested at the hospital laboratory, (ii) determine resistance profile against specific antimicrobials, and (iii) identify factors associated with AMR.This was a cross-sectional study with a secondary data analysis that included all the patients tested for AMR in the laboratory of the NICH for the period of 2016–2019.Armenia is a land-locked country in the South Caucuses region with a population of about three million. There are 11 regions in the country and the capital city Yerevan hosts around 1/3 of the total population. About 30% of the population are below poverty threshold, while 95% of the population are literate.The national strategy for AMR surveillance and prevention was approved by the government of Armenia in 2015. The strategy includes seven directives, as follows: (1) the creation of management mechanisms for the use and consumption of antimicrobial agents; (2) the creation of an epidemiological surveillance system for AMR; (3) the enhancement of infection control measures and of the information systems for monitoring and controlling AMR at a health facility level; 4) the enhancement of AMR testing capacity; (5) the improvement of laws and regulations related to medication use and selling; (6) the raising of awareness within the general public on AMR and the provision of continuous education on AMR for health professionals; (7) the prevention of AMR formation in agricultural settings [ the enhaWith regard to the treatment of infectious diseases, the NICH is the only tertiary level hospital specialized in infectious diseases. All potentially dangerous infections and most of the severe cases of infections are referred (or directly brought to by ambulances) to this hospital for treatment.Most of the services provided in the hospital are covered by the state budget, while there are some services for which patient can pay (out of pocket payments). AMR testing is done using the standard Kirby–Bauer disk diffusion technique. Broad spectrum antimicrobials are prescribed usually at the beginning of the treatment. If the resistance to specific antimicrobials is identified during the treatment, then those antimicrobials are discontinued and replaced with an available substitute. The list of antimicrobials covered by the state budget that can be used for treating infections at the NICH is extremely limited. Thus, the majority of patients receive broad spectrum antimicrobials available on the list without the results of AMR testing. This approach creates a situation where use of an ineffective antibiotic (misuse) can increase the risk of AMR ,7.The practice described above has been in place for decades and potentially adds to the uncontrolled selling of antimicrobials (without prescription), thus there may already be a critical situation related to AMR in the country. The antimicrobial medication market is not well regulated. After registering the antimicrobials in the country, pharmacies are allowed to sell them without prescription. Pharmacists may suggest these medications to potential patients without consulting with a doctor or testing.The study population included all hospitalized patients who received treatment at the NICH for the period of 2016–2019 and were tested for AMR in the laboratory of the hospital. A database was developed based on the review of the available medical forms and data relevant for this study. Anonymized data was directly abstracted and entered into the database using an online google form linked with the database. Data variables included basic demographics , and clinical data including initial and final diagnosis, sample type, infectious agent identified, individual sensitivity to 22 antimicrobials, and antimicrobials used for the treatment. The working definition of AMR in this study was the following: AMR is the ability of pathogens to withstand antimicrobial treatment identified by the laboratory testing at the NICH laboratory.https://easystat.app/, accessed on 9 February 2021), developed on the basis of R statistical software, was used for the final analysis [EasyStat online statistical platform  patients out of 36,528 in total with available AMR test results and all of them were included in the study. The population was very diverse. The geography of patients covered the whole country, with the majority being from the capital region (37%). The average age of the patients was 18.7 years  and ranged from 0 to 78 years old with a skewed distribution. The proportion of males was 63%. Out of eight different types of tested specimens, urine was the most common (33%) followed by blood (21%). The demographic and clinical data of the study population are summarized in more detail in Escherichia Coli (E. Coli) (22%) being the most prevalent followed by Staphylococcus Aureus (S. Aureus) (18%).The identified pathogens were diverse (16 different species), with Patients had more than 50 different diagnoses or a combination of diagnoses.Only 20 (19%) out of 107 tested samples had no resistance, while 87 (81%) had resistance at least to one tested antimicrobial.The resistance profiles/proportions of the specific antimicrobials are presented in p values > 0.2). However, the risk factor analysis was indefinite due to the small number of sensitive samples . This disaggregation by different factors and categories resulted in small observations, which made an adjusted analysis not possible. The performed analysis yielded errors or indefinite results in the statistical software. Thus, the risk factor analysis needs to be repeated in future studies with larger samples.No risk factors were found to be associated with the AMR in a non-adjusted analysis. As presented in This is the first study in Armenia to explore the situation related to AMR. The study revealed some disturbing findings.shigellosis and intestinal salmonellosis is reported annually at the NICH. Most of these are patients receiving antimicrobial treatment. It is unclear how the selection of the patients who need AMR test results is carried out. This lack of testing may be the result of not having national treatment guidelines or having inadequate laboratory testing capacity. In latter case, it is likely that those tested for AMR and thus included in our study were hard-to-treat patients. Among all (107) specimens tested during the period of 2016 to 2019, 81% were resistant to at least one antimicrobial. A study from Israel that covered the period from 2014 to 2017 showed similar findings. In this study, the percentage of carbapenem-resistant Acinetobacter baumannii (A. baumannii) was 75–80% for different years, while the percentage of resistance for other species ranged from 0% to 32% [A. baumannii being resistant [S. Aureus was the second most identified pathogen, but we do not have data regarding Methicillin Resistant S. Aureus (MRSA). The population-weighted average of the MRSA percentage was 15.5% in 2019 for the countries in the European Union/European Economic Area region [First, only 0.3% of patients had AMR testing results available. This is a very alarming situation because as the vast majority of patients at the NICH do not have results of AMR testing, they may be receiving antibiotics that are not effective against potentially resistant infections. As an example, there were only 12 feces samples tested during the four year study period, while more than 400 cases of mong all 7 specimeesistant ,12. It ia region .E. coli and Klebsiella pneumoniae (K. pneumoniae), while for Cotrimoxazole the resistance ranged from around 2% for S. Aureus to 90% for A. baumannii. While these resistance patterns have some similarities, for cephalosporines the resistance pattern was worse in Israel, ranging from 10% for Pseudomonas aeruginosa (P. aeruginosa) to 98% for A. baumannii [Second, the proportion of AMR for the tested antibiotics ranged from 7% for Moxifloxacin to 83% for Chloramphenicol, making the latter almost unusable. Antifungals showed a similar pattern, with AMR ranging from 7% for Itraconazole to 88% for Nystatin. The continuation of the misuse of antimicrobials can be extremely dangerous and create a situation whereby many antibiotics can become ineffective. The percentage of resistance for some of the widely used, broad-spectrum antibiotics was around 40–50%, for example as seen with Amoxicillin with Clavulanic Acid and Cotrimoxazole. The resistance proportion was around 25% for cephalosporines. According to the study in Israel the proportion of resistant strains for specific antibiotics varied depending on the pathogens. The proportion for Amoxicillin with Clavulanic Acid was around 37% to 50% for E. coli, factors associated with AMR were travel to India and Southeast Asia, diarrhea, and being on a vegetarian diet [P. aeruginosa identified factors such as the prior use of antibiotics , indwelling urinary catheters, and prior invasive procedures [Third, the risk factor analysis in our study did not yield any significant results due to the small size and diversity of the sample. In the literature there are many known risk factors for developing AMR which may be different depending on the study context, species, and the diseases they cause. According to the WHO, the main risk factors include the misuse and overuse of antimicrobials; inadequate infection control in health care facilities and farms; a lack of access to clean water, sanitation, and hygiene for both humans and animals; low awareness and knowledge; a lack of access to medicines, vaccines, and diagnostics; and a lack of enforcement of legislation ,14. In tian diet . Anotherocedures .The strength of this study was the conduct and reporting of the study using the strengthening the reporting of observational studies in epidemiology (STROBE) statement . There wEven with limitations, our study sheds light on a major public health issue that Armenia is facing and there is an implication. The practice of the uncontrolled sale and misuse of antibiotics has resulted in a drastic situation related to AMR in Armenia. The findings call for as immediate control of the sale and use of antibiotics, to stop resistance formation or at least decrease its speed. One of the key approaches to optimize the use of antimicrobials is the implementation of antibiotic/antimicrobial stewardship programs (ASP) in hospitals. Stewardship not only requires the use of the appropriate drug, but also timely and appropriate dosing for the proper treatment of infections. To be effective, such a system requires an individualized patient approach using a complex dataset and precision antibiotics for personalized treatment. Factors, such as poor integration of such software with medical records, represent common barriers for the effective performance of such systems. Additionally, the benefits of rapid susceptibility testing in hours and not days requires close collaboration between the microbiology laboratory, physicians, and the wider ASP team to ensure timely action ,20,21,22The available data leads us to the conclusion that there is a substantial prevalence and significant burden of AMR in Armenia. This data gives us valuable information to confirm our hypothesis that the human diseases caused by most AMR pathogens are probably undiagnosed and significantly under-recognized, hence their public health importance is vastly underestimated. It creates a crucial need to strengthen AMR surveillance, increase awareness among health care workers to enable them to detect AMR pathogens in a timely manner, improve laboratory capacity to accurately identify the spectrum of drug resistance, and support accurate treatment. National guideline development may be required to set standards of care and ensure the proper diagnosis and treatment of infections and prevent the spread of AMR.Additionally, these infections may spread to other countries through migration and the short-term travel patterns of patients and carriers. Thus, to prevent and control outbreaks of resistant infections in Armenia, as well as to limit the pathogens’ spread to other countries, it is of critical importance for the country to have an infrastructure for accurately and rapidly identifying AMR pathogens of public health importance including in animals and other environmental sources.There is a need to have a comprehensive system to monitor antibiotic resistance and develop effective reporting at a national as well as an international level, for quick decision making and in order to comply with the International Health Regulations.This study has provided some valuable information on the situation related to AMR in Armenia. The proportion of resistance to at least one antimicrobial in the samples is as high as 81% among those tested, while the proportion of testing remains very low. This calls for immediate actions to control the access to and use of antimicrobials, strengthen AMR surveillance, increase awareness among health care workers to enable them to detect AMR pathogens in a timely manner, and improve laboratory capacity for the proper and fast identification of drug resistance.


Knee osteoarthritis (KOA) is the most common form of joint degeneration with increasing prevalence and incidence in recent decades. KOA is a molecular disorder characterized by the interplay of numerous molecules, a considerable number of which can be detected in body fluids, including synovial fluid, urine, and blood. However, the current diagnosis and treatment of KOA mainly rely on clinical and imaging manifestations, neglecting its molecular pathophysiology. The mismatch between participants’ molecular characteristics and drug therapeutic mechanisms might explain the failure of some disease-modifying drugs in clinical trials. Hence, according to the temporal alteration of representative molecules, we propose a novel molecular classification of KOA divided into pre-KOA, early KOA, progressive KOA, and end-stage KOA. Then, progressive KOA is furtherly divided into four subtypes as cartilage degradation-driven, bone remodeling-driven, inflammation-driven, and pain-driven subtype, based on the major pathophysiology in patient clusters. Multiple clinical findings of representatively investigated molecules in recent years will be reviewed and categorized. This molecular classification allows for the prediction of high-risk KOA individuals, the diagnosis of early KOA patients, the assessment of therapeutic efficacy, and in particular, the selection of homogenous patients who may benefit most from the appropriate therapeutic agents. Osteoarthritis (OA) is a common and disabling condition globally, within which knee OA (KOA) accounts for a large proportion and manifests several symptoms that weaken the quality of life, such as pain, stiffness, dysfunction, and even deformity . CombineAmong the drugs applied clinically, some of them show acceptable outcomes in pain relief and joint function improvement, whereas, a fair portion of them with unclear results . ClinicaIndeed, patients with KOA exhibit early molecular and structural changes before the disease shows any clinical manifestations . RecentlHerein, according to the temporal alteration of representative molecules in body fluids, we propose a novel molecular classification of KOA , with a focus on its role in KOA prediction, diagnosis, and treatment efficacy evaluation. This classification criteria may allow for a molecule-based diagnosis and treatment algorithm , for insTraditionally, KOA has been considered as a disorder of articular cartilage. However, the current view is that KOA is a whole-joint disease, or even a systemic disorder since it could be affected by various local and systemic risk factors. The major pathogenesis in KOA contains the molecular crosstalk between articular cartilage, synovium, subchondral bone, meniscus, tendon, muscle and infrapatellar fat pad (IFP) . For insFrom a practical point of view, less invasive or non-invasive methods to obtain the molecular spectrum will have greater clinical significance. Interestingly, along with intense molecular crosstalk, the opportunity to assess molecular changes is presented by the molecular carrying medium, in which synovial fluid (SF), blood, and urine are included . SF is aAlthough the identification of early KOA has gained widespread popularity in the past decade , there iObesity is a well-established risk factor for KOA. Lifetime risk of KOA rises with increasing body mass index, and longitudinal study shows that two-thirds of obese adults will develop symptomatic OA . DespitePrevious injury is a predominant joint-level risk factor for KOA initiation. Among the injuries, direct articular cartilage damage, anterior cruciate ligament (ACL) injury, and meniscal tear have been reported to be closely associated with KOA development . Both ACFemale sex is another major causal risk factor of KOA, with a prevalence 1.2–2.8 times higher than males . TherefoAlthough KOA is not considered as an autoimmune disease, activated molecular and cellular processes may stimulate maladaptive repair responses, which often include pro-inflammatory pathways of innate immunity and subsequent production of autoantibodies (AAbs) . RecentlTaken together, these predictive molecules will help to discriminate individuals with high risk of KOA initiation (pre-KOA) and may suggest the molecular based interventions to prevent KOA occurrence.The molecular disorder is a consequence of disturbed gene expression landscape, which is fine tuned by small, non-coding RNAs named microRNAs (miRNAs). By inhibiting the function of protein-coding transcripts, miRNAs alter multiple aspects of cell structure and function, including chondrocytes phenotype . DysreguAmong the miRNAs in early KOA, miR-140 and miR-210 are probably the most representative. Increasing evidence shows that miR-140 is capable for maintaining both cartilage and bone formation and homeostasis . Also, ide novo gene transcription or stabilizing target mRNA transcripts, IL-17 upregulates inflammatory gene expression, including IL-1β and TNFα, two major players of KOA pathophysiology  in serum have shown positive association with knee structural changes assessed by KL grades . In a st2, which results from the nitration of aromatic amino acids by peroxynitrite under oxidative condition in KOA chondrocytes  is the most abundant protein in bone, accounting for 90% of total bone protein . AttachiOsteoblasts express the highest concentration of collagen during their proliferative phase and of bone alkaline phosphatase (ALP) during matrix maturation . N-termiSeveral clinical trials have been focused on evaluating the potential therapeutic efficacy of anti-absorption agents on KOA, regrettably, with scarce data on the beneficial effects. For instance, intravenous zoledronic acid, a classical antiresorptive agent, did not show a significant improvement of pain score and the reduction of cartilage volume loss in KOA patients, even in those with bone marrow lesions . The mosAccumulating evidence supports that the inflammation in KOA is chronic, comparatively low-grade, and primarily mediated by the innate immune system . ClinicaHighlighting the role of inflammation, much interest has been expressed in identifying secreted inflammatory cytokines in the pathophysiology of KOA progression. The most widely studied biomarkers are IL-1β and TNF-α, two major players of KOA inflammation. Increasing evidence shows that both the serum levels of IL-1β and TNF-α are highly associated with the symptomatic and radiographic progression of KOA . Their eSynovitis is highly associated with KOA progression. Indeed, growing evidence supports the role of immune cells, particularly macrophages, in KOA pathophysiology . Some moIn addition to local inflammation of the joint, systemic inflammation may also have a vital role in KOA pathogenesis . For exaIdentifying the molecular profiles, including the local and systemic inflammatory cytokines, would help reveal the major inflammatory mechanisms and stratify KOA patients with different molecular characteristics. This strategy could facilitate the diagnosis of inflammation-driven subtype of KOA and the development of molecular based treatment strategies.Pain is a major driver of health service use and clinical decision making of KOA . HoweverNumerous works focused on the anti-inflammatory strategies that might be capable of alleviating KOA pain. Among the inflammatory molecules related to KOA pain, CRP is extensively and long-term used in reflecting the inflammatory response. Jin et al. included 32 studies in their meta-analysis and found that serum high sensitive CRP (hs-CRP) levels were significantly correlated with knee pain and physical function decline, but not with radiographic changes , indicatBradykinin is another promising peptide involved in KOA inflammatory pain by exciting and sensitizing sensory nerve fibers . As a va+ to induce an acidic microenvironment and then activate the acid-sensing receptor transient receptor potential vanilloid 1, which is responsible for the transcriptional activation of calcitonin gene-related peptide (CGRP), a well-established pain responder  has become a useful, economical and simple tool for reflecting inflammation. In a study enrolling 176 KOA patients, blood NLR was significantly higher in the severe (KL grade 4) group (2.18 ± 1.04) than in the mild to moderate group (1.79 ± 0.8), suggesting NLR as an indicator of end-stage KOA . FurtherTo explore the relationship between serum miRNA levels and the occurrence of severe KOA, Beyer et al. identified differentially expressed miRNAs in a population-based cohort including 816 individuals . They foGiven the emerging evidence demonstrating biomarkers for KOA , it is cIn the past decade, MRI has been rapidly evolved due to technical advances, and its application in clinical research has provided sufficient evidence regarding the feature of disease . AccordiIn addition, in order to prevent the influence of other diseases, population with similar baseline conditions should be preferred in clinical trials evaluating the KOA molecules. Future studies should also focus on verifying the efficacy and threshold values of a single molecule, or a panel of molecules, in clinical settings, and exploring new molecules to improve the molecular classification to promote its clinical application.In KOA, different interlinked molecules cause and sustain the pathogenesis, as early as before the clinical and radiographic manifestations are available. As such, based on the representatively investigated molecules, we proposed the novel KOA molecular classification , which oWe searched titles and abstracts in PubMed, using the search term “osteoarthritis” in combination with, but not limited to, “knee,” “biomarker,” “diagnosis,” “stage,” “progression,” “classification,” “body fluid,” “synovial fluid,” “blood,” “serum,” “urine,” “pathophysiology,” “subtype,” “phenotype,” and “clinical trial.” We mostly selected publications from the past 10 years, although commonly referenced and important older publications were not excluded. English articles presenting data on soluble biomarkers in body fluids  of human KOA were included.All 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mycoacterium bovis molecular types in cattle suggests that local factors are the primary drivers of spread. Northern Ireland’s agricultural landscape is comprised of highly fragmented farms, distributed across spatially discontinuous land parcels, and these highly fragmented farms are thought to facilitate localised spread. We conducted a matched case control study to quantify the risks of bovine tuberculosis breakdown with farm area, farm fragmentation, fragment dispersal, and contact with neighbouring herds. Whilst our results show small but significant increases in breakdown risk associated with each factor, these relationships were strongly confounded with the number of contiguous neighbours with bovine tuberculosis. Our key finding was that every infected neighbour led to an increase in the odds of breakdown by 40% to 50%, and that highly fragmented farms were almost twice as likely to have a bTB positive neighbour compared to nonfragmented farms. Our results suggest that after controlling for herd size, herd type, spatial and temporal factors, farm fragmentation increasingly exposes herds to infection originating from first-order spatial neighbours. Given Northern Ireland’s particularly fragmented landscape, and reliance on short-term leases, our data support the hypothesis that between-herd contiguous spread is a particularly important component of the region’s bovine tuberculosis disease system.Bovine tuberculosis remains a challenging endemic pathogen of cattle in many parts of the globe. Spatial clustering of  Mycobacterium bovis infection, is a complex and challenging disease of cattle, endemic in many countries across the globe [Bovine tuberculosis (bTB), caused primarily by he globe . The dishe globe ,3,4, anche globe ,6,7, andhe globe ,9. Failuhe globe .M. bovis can be introduced into a herd via processes operating over relatively long distances ,  factors ,15,16,17 factors ,19,20,21 factors ,23, and  factors . Farm fragmentation, whereby farms are distributed across multiple, spatially discontinuous land parcels, is particularly common on the island of Ireland ,26. In NContiguous cattle-to-cattle spread can be counteracted with robust biosecurity measures such as double fencing between parcels with a 3 m gap between neighbours . There iDespite earlier studies alluding to the potential for fragmented farms to disseminate infection ,34 this The final dataset consisted of 19,008 herds, from which 4,637 (24.4%) had at least one confirmed bTB breakdown during the three year study period; the spatial distribution of case herds in the whole cattle population is illustrated in p < 0.05).Moderate to strong correlation was observed between farm area, farm fragmentation, fragment dispersal and contact with neighbouring farms (R-Markdown p < 0.05).BTB breakdown risk was also associated with fragment dispersal , see LOESS plot in p < 0.05). The odds of bTB breakdown increased by between 6% and 9% for every additional km of shared boundary with neighbouring cattle farms ; see LOESS plot The number of bTB positive neighbours was also positively associated with the odds of bTB breakdown; for every additional neighbour, the odds increased by between 40% and 50% , see LOESS plot It was not possible to build a model with each of the exposures included, in part because of the extent of correlation between explanatory variables, and also because such a model has very sparsely populated categories. Instead, we assessed how each of the factors of interested changed or “adjusted” the Odds Ratio (aOR) when the number of bTB positive neighbours was considered as a confounding factor. Confounding can make the relationship between an exposure and the outcome appear stronger or weaker than actual. We were particularly interested in whether the number of bTB positive neighbours was a positive confounder, which made the relationship between the factor of interest an bTB positive status appear stronger than it is, or whether it was a negative confounder making the relationship between the factor of interest an bTB positive status appear weaker. Positive confounding means that the aOR is closer to the null value  compared to the crude OR following the inclusion potential confounding factor, whereas negative confounding means that the aOR is further from the null value, compared to the crude OR. Confounding effects were also found between farm fragmentation and bTB positive neighbours; in very highly fragmented farms, the OR for bTB positivity was reduced by 46% once the number of infected neighbours was included in the model. Indeed, we observed up to 14 bTB positive neighbours in very highly fragmented farms, compared to a maximum of 5 in nonfragmented farms; The relationship between fragment dispersal and the odds of bTB breakdown was positively confounded with the number of bTB positive neighbours, Considerable confounding was also identified between neighbour contact category and the presence of bTB positive neighbours. After accounting for the number of infected neighbours, the OR for the category representing high contact between neighbours was reduced by 11%, whilst the OR of the category representing very high contact between neighbours was reduced by 41%. Thirty-six percent of farms with low contact metrics had at least one bTB positive neighbour, compared to 79% of farms with very high contact, and farms with very high contact metrics also had the largest number of bTB positive neighbours; see M. bovis genetic types in host populations, thereby affirming the important role of geographically localised processes in maintaining the bTB epidemic [Previous molecular studies from NI have revealed considerable spatial clustering of epidemic ,36,37,38M. bovis genetic types in NI, along with the relatively small median distances between fragments in the vast majority of farms, means that the importance of long range introductions is not evident. We do acknowledge that moving cattle between fragments could be a potential mechanism of disease spread within the herd, but because the distances involved are small, we argue that disease associated with intra-parcel movements between fragments should be practically indistinguishable from other short-range processes. Simulations of food-and-mouth spread between separate but associated premises in Scotland also suggest a diminished role for long-range intra-herd movements, but highlight the need to better understand land parcel occupancy to fully gauge the impact of intra-premises spread [In our univariable models, we found that farm fragmentation, fragment dispersal and contact with neighbouring farms are each associated with increased odds of bTB breakdown, which is in concordance with previous studies. In the ROI, Byrne et al. (2020) observed an increase in bTB breakdown length of 6.6% in herds residing on farms with four parcels, compared to herds in farms with one parcel . In the s spread . “Farm area” was the only variable where residual bTB breakdown risk remained after controlling for the presence of bTB positive neighbours; in very large farms (>59.1 ha) the odds of breakdown remained elevated by between 20% and 52% compared to smaller farms. This suggests that herds in very large farms experience additional risk beyond that presented by infection from neighbours. It may be that larger farms simply have more badgers on the land, as suggested by Vial et al. , or reprThe number of first-order spatial neighbours with bTB exhibited a particularly strong association with bTB breakdown risk; indeed, the presence of at least one neighbour with bTB was associated with more than double the odds of bTB breakdown compared to having no bTB positive neighbours. In some breakdown herds, up to 14 bTB positive first-order spatial neighbouring herds were identified. The importance of neighbourhood has been highlighted in an ROI study, where 35% of bTB animal incidence was associated with being within 1 km of other infected herds . In a NoUltimately, tackling lateral spread of infection, requires understanding the drivers of fragmentation itself. Around 30% of NI’s farmland is dominated by the practice of informal short-term land-leasing called “conacre”, whereby a landowner may rent out only a portion of their farm for contracts <12 months. Leasing conacre is understood to be a pervasive mechanism driving increasing farm fragmentation, and the short-term nature of the lease means that land owners may be less likely to invest in conacre holdings, with subsequent poorer soil, reduced animal productivity and poorer biosecurity ,46. The As this was a large scale computation study and not a field study, we were unable to accurately geo-reference herds within holdings. Potentially therefore, herds could occupy only one area within a very fragmented farm. However, a small study quantifying intra-herd cattle movements in NI shows that even fragmented land was grazed frequently, especially by beef animals, and that dairy animals in particular were frequently moved between pastures . This suA limitation on determining the true extent of farms is introduced as a consequence of how the Basic Payment Scheme (BPS) system is administered. Either the land owner or tenant can claim for BPS and it is not possible to discern who is claiming for what parcels, and thus it is more challenging to be certain what land is being used and by whom. However, the BPS claimant is more often the farmer leasing and farming the land, and not the owner. This means that in practice, our estimations of farm area should largely reflect who is actively using the land parcels. One constraint to our conclusions surrounds the lateral testing process. Because bTB disclosure triggers an intensive epidemiological investigation into the herds and parcels surrounding a breakdown herd, surveillance efforts are not homogenous. Thus, there may be increased likelihood of disease being identified proximal to case herds, based on this factor alone. Whilst we hypothesise that high odds ratio associated with the presence of neighbours reflects the contiguous spread of disease; it could be debated that we are observing a consequence of enhanced surveillance and increased intensity of testing. We, however, argue against this interpretation; due to the annual testing schedule, bTB would be eventually detected in the herds surrounding control herds if it were present.The issue of fragmented farms poses an epidemiological risk for a wide range of pathogens in addition to bTB , however highly fragmented farms may also introduce logistical complexities into production, thereby decreasing the technical efficiency of dairy farms by . Future We conducted a retrospective matched case-control observational study at the herd level to quantify the strength of association between the risk of bTB breakdown, and key metrics of farm fragmentation, fragment dispersal, and contact with contiguous farms. The study period ran between 1 January 2015–31 December 2017 inclusive. 2) is situated in the northeast of the island of Ireland, with a national herd of 1.6 million cattle distributed throughout approximately 20,000 farms. Approximately 13% of these holdings are dairy farms (n = 2600) and 70% are beef , with a number of other herd types making up the remainder  [M. bovis require that all bovines in NI over 42 days of age undergo annual testing using the Single Intradermal Comparative Cervical Tuberculin (SICCT) test, whereby SICCT-positive animals are removed from the herd and culled. Surveillance also involves the routine inspection of animal carcasses for lesions consistent with tuberculosis (LRS); suspect lesions are confirmed either way via histology or bacteriological investigations. We followed DAERA’s policy on bTB breakdown confirmation at the time [Northern Ireland  . APHIS vLand parcel spatial boundaries for every land parcel associated with each cattle farm were made available from the DAERA Land Parcel Identification System, and used to derive model variables. The sum of all land parcels was used to derive the total farm area (ha). We also calculated the number of “fragments” associated with each cattle business, with a fragment defined as a spatially distinct functional unit of land parcels. This was more appropriate than using the total number land parcels, as all the land parcels within a fragment are epidemiologically linked, as farmers permit cattle to move between adjoining fields. Fragments were defined in a previous study , but brihttps://www.ceh.ac.uk/, accessed on 6 December 2021) at 25 m2 resolution [Fragment dispersal was defined using the median distance between fragments, calculated by measuring the Euclidian distance in km between fragment centroids. Contact with neighbouring farms was the length of shared perimeter between a cattle farm and its first-order spatial neighbours . As it isolution . Farm frWhilst similar, each of farm fragmentation, fragment dispersal and contact with neighbouring farms represent different exposures. We argue that number of first-order spatial neighbours with bTB is the most specific measure of direct risk from contiguous herds, however we included the farm fragmentation variable as a separate measure as highly fragmented farms may be materially different from nonfragmented farms via management approaches related to grazing regime e.g., rotational grazing across multiple parcels. Calculating the length of shared boundary with neighbouring cattle farms is a more precise measure of contact with neighbouring herds, given that this metric was derived from boundaries where both internal and external parcels were potentially suitable for cattle farming. We also included fragment dispersal, as unrecorded, intra-herd cattle movements could lead to animals being moved from lower incidence areas to higher incidence areas, thereby increasing the risk of breakdown. In addition to this, widely dispersed land parcels may capture more landscape heterogeneity on-farm, with potentially better habitat provision for potential reservoir species via availability of rough pasture and woodland .Case herds were those with at least one confirmed bTB breakdown that started and ended during the dates of the study. For herds that had more than one confirmed breakdown, we included only the earliest breakdown . Eligible control herds were herds that remained bTB free throughout the study period. We matched on production type ; herd size ; and Divisional Veterinary Office (DVO), a local spatial variable associated with bTB administration , and year of bTB test. Herd size was the only noncategorical matching variable, and here we matched on herd size +/− 10 head of cattle. Cases and controls were matches in a 1:1 ratio on potential confounding variables; we used a higher ratio of controls to cases as it was not possible to match on all criteria.survival package [ggplot2 [Descriptive statistics for each explanatory variable  for continuous variables) were generated for case and control herds. Locally estimated scatterplot smoothing (LOESS) plots and boxplots were created for exploratory analysis. Correlations between explanatory variables were assessed using Spearman’ Rank Correlation Coefficient. Univariable and multivariable parameter estimates were derived using conditional GLMs via the clogit function in the  package . Pair ID package . We were[ggplot2 . The fulNorthern Ireland’s agricultural system means that many farms are very fragmented, resulting in herds with high levels of direct contact with neighbouring farms, and sometimes considerable distances between fragments. We were ultimately interested in whether fragmentation was associated with odds of bTB breakdown, and whilst we found that farm area, farm fragmentation, fragment dispersal and contact with neighbouring farms were indeed associated with increased odds of breakdown, these factors were also confounded with the number of bTB positive neighbours. We found that highly fragmented farms were around twice as likely to have at least one bTB positive neighbour compared to nonfragmented farms, and that every bTB positive neighbour increased the odds of bTB breakdown by 40–50%. After accounting for the number of bTB positive neighbours in models of how bTB breakdown risk is associated with farm fragmentation metrics, we find no compelling evidence of a relationship between odds of bTB breakdown and fragmentation. This signifies that these farm fragmentation metrics are probably capturing the effects of “over the fence” cattle-to-cattle transmission. Although it is possible that similar observations arise if herds in the area were exposed to a shared infection source such as infected wildlife, our geographical matching criteria make this less likely. Tackling local spread will require a deeper understanding of the patterns, drivers and characteristics of NI’s conacre rental system, which is a key factor influencing land fragmentation.


Transmission of SARS-CoV-2, the virus that causes COVID-19, is common in congregate settings such as correctional and detention facilities  testing while isolated. On September 14, 2020, a visiting DHCP treated 10 incarcerated persons in a dental clinic at facility A . At entrThe outbreak investigation started on September 30, 2020. To better understand factors contributing to the outbreak, investigators interviewed facility dental and medical staff members during September 30–October 9, 2020. Investigators also reviewed case records of staff members who reported onset of COVID-19–compatible symptoms during September 14–September 24, 2020 and who worked in block 1 or in other areas where possible exposure to block 1 incarcerated persons or staff members might have occurred. This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.On September 14, 2020, DHCP1 wore a valveless N95 respirator face mask at entry to facility A, during temperature and symptom screening, and in transit to the dental clinic. During treatment, DHCP1 wore the N95 as well as a gown, gloves, and goggles, and changed gowns and gloves after each patient. Among 10 incarcerated persons (residents) who received treatment, six (including a resident who subsequently developed COVID-19 [patient R2]) had surgical tooth extractions (a 15-minute procedure), one (patient R3) had a combined evaluation and biopsy (12-minute procedure); and three had 10-minute patient evaluations. All 10 incarcerated persons were interviewed for 5 minutes each by one of five facility dental clinic staff members, all of whom wore recommended PPE. On the day of treatment, none of the incarcerated persons was tested for SARS-CoV-2, screened for fever or symptoms, or wore masks or gloves during treatment.https://stacks.cdc.gov/view/cdc/104506). Patients R1 and R2 were moved from block 1 to an unoccupied isolation block, and staff members began wearing N95 respirators and eye protection. On September 24, 2020 the nine remaining incarcerated persons treated by DHCP1 were tested. Patient R3, who also lived in block 1, received a positive result and was isolated.On September 21, 2020, patient R1  experienced symptoms and visited the infirmary the next day. On September 23, 2020, patients R1 and R2 both received positive SARS-CoV-2 RT-PCR test results; patient R2 was tested because of his close contact with patient R1, despite being asymptomatic at the time (he retrospectively reported a headache with indeterminate onset)  through which staff members occasionally travel. Among 174 incarcerated persons in block 2, three with COVID-19–compatible symptoms were moved to the isolation area on October 1, 2020 after receiving positive test results. On October 3, 2020, RT-PCR testing of specimens from the 171 remaining block 2 incarcerated persons identified an additional 89 cases  Table 2Table 2. https://stacks.cdc.gov/view/cdc/104506). Cumulatively, as of January 31, 2020, 88 (16%) cases among 550 staff members were reported in facility A.Investigations of six cases in staff members occurring during the period from the potential introduction of infection into facility A to the detection of COVID-19 cases in the first three incarcerated persons suggested likely acquisition at work for two ; staff mAfter 46 cases were detected with mass testing in block 1 on September 27, 2020, facility A notified the Salt Lake County Health Department and the Utah Department of Health. A team from both departments, with technical assistance from CDC, implemented twice-weekly calls with the facility to review infection control guidance, including protocols for cohorting, quarantine and isolation of incarcerated persons and repeated mass testing to identify new cases. The entire facility was placed under a quarantine restriction to limit mobility of staff members among residential units. The state mobile testing team supported mass testing of incarcerated persons and staff member testing events. As of March 2, 2021, the outbreak was ongoing; 1,545 cases  have been reported, as well as 31 hospitalizations and 12 deaths among incarcerated persons with COVID-19.SARS-CoV-2 might have been introduced into correctional facility A by DHCP1 or other staff members with community-acquired infection. The detection of 46 cases just 11 days after the first potential introduction by DHCP1 suggests that infection spread quickly. Infection might also have spread through undetected chains of transmission from staff members working in block 1 to other areas, especially because N95 respirators and eye protection were not usually worn before September 23, 2020.The possibility of transmission from staff members to incarcerated persons at facility A indicates a need for serial testing for both staff members and incarcerated persons (Ten incarcerated persons were exposed to the index patient (DHCP1) on the date of the index patient’s symptom onset and were not immediately quarantined or isolated; two of 10 appeared to be infected by DHCP1. The interval between patient R2’s exposure to R1 (his roommate) and R1’s symptom onset , suggests a mean 3.5-day incubation for these cases, consistent with previous estimates  of 2,632 residents  and an estimated 88 (16%) of 550 staff members.Quarantining and monitoring potentially exposed persons are important in preventing the introduction and spread of SARS-CoV-2 infection in correctional facilities and other congregate settings. Vaccination of incarcerated persons might help prevent or limit the spread of infection in these facilities.


The aim of this study was to assess the utility of inexpensive techniques in evaluating the interactions of risperidone (Ris) with different traditional π-acceptors, with subsequent application of the findings into a Ris pharmaceutical formulation with improved therapeutic properties. Molecular docking calculations were performed using Ris and its different charge-transfer complexes (CT) with picric acid (PA), 2,3-dichloro-5,6-dicyanop-benzoquinon (DDQ), tetracyanoquinodimethane (TCNQ), tetracyano ethylene (TCNE), tetrabromo-pquinon (BL), and tetrachloro-p-quinon (CL), as donors, and three receptors  as acceptors to study the comparative interactions among them. To refine the docking results and further investigate the molecular processes of receptor–ligand interactions, a molecular dynamics simulation was run with output obtained from AutoDock Vina. Among all investigated complexes, the [(Ris) (PA)]-serotonin (CTcS) complex showed the highest binding energy. Molecular dynamics simulation of the 100 ns run revealed that both the Ris-serotonin (RisS) and CTcS complexes had a stable conformation; however, the CTcS complex was more stable. Risperidone (Ris) is a second-generation antipsychotic that has been used to treat psychotic disorders, including schizophrenia, since the 1990s . CompareAccording to the literature, it being a safe, effective, and tolerable molecule categorized it into the World Health Organization’s List of Essential Medicines . MoreoveRis is an antagonist for a number of receptors, including dopaminergic , serotonergic (of 5-HT2A), and adrenergic  receptors. Its high affinity to 5-HT2A in comparison to D2 is behind its beneficial effects in ameliorating the undesirable symptoms of schizophrenia ,9. FurthIn biochemical and bioelectrochemical energy transfer processes, donor–acceptor interactions are critical and significant . The forCT interactions between electron donors and acceptors are also crucial in drug–receptor binding mechanisms , surfaceIn this study, Autodock Vina was used for molecular docking to study the interactions between the ligand (Ris and synthesized CT complexes) and receptors . Binding energies, along with hydrophobic and hydrogen bond surface interactions, were also determined. To provide a more effective mechanism for demonstrating receptor–ligand interactions, the best molecular docking data were subjected to a molecular dynamics simulation at 300 K for 100 ns. This kind of modelling is endorsed in the literature for further understanding and enhancing the therapeutic benefits of such antipsychotics . In termn-donor to form a charge transfer complex with π-acceptors.Because Ris has many electron density sites, it could be a good electron donor. After the protonation of N(1)-H , the preAccording to the generation of positive and negative dative anions under donor–acceptor chelation , the conThe way of charge carriers between valence and conduction bands determines the absorption process of photons in charge transfer. The band structure concept, which is utilized in semiconductor electronic transitions, was adapted to optical absorption in organic systems. The highest occupied molecular orbital  contributes to the valence band of a molecular crystal, whereas the lowest unoccupied molecular orbital  contributes to the conduction band .The band gap (Eg), which can be calculated from variations in optical absorption at the basic absorption edge, separates these bands. The following Bardeen formula  can be umer film .n = 1/2) and indirect (n = 2) permissible transitions are defined by the value of the constant n. (αhv)1/2 was plotted as a function of hν using the indirect transition (n = 2) as shown in g. The probability of transition decreases as the number of charge carriers on localized states increases, necessitating more absorption in these locations, thereby narrowing the band gap [Constant C is a transition probability-dependent parameter. The direct  (PA)], [(Ris) (DDQ)], [(Ris) (TCNQ)], [(Ris) (TCNE)], [(Ris) (BL)], and [(Ris) (CL)] were docked against serotonin (PDB ID: 6BQH), dopamine (PDB ID: 6CM4), and adrenergic (PDB ID: 6KUW) receptors, and the best docking poses obtained. Ris (donor moiety) was used as a control for comparative purposes. Molecular docking of the aforementioned six CT complexes revealed that their potential binding energy was higher than that of Ris at all receptors .Among the six CT complexes screened, [(Ris) (PA)] showed the highest docking energy compared to Ris. Molecular docking of [(Ris) (PA)] with serotonin, dopamine, and adrenergic receptors revealed potential binding energies of −11.4, −10.6, and −10.2 kcal/mol, respectively. The highest binding energy value of [(Ris) (PA)]-serotonin (CTcS) signifies a stronger interaction than that between dopamine and adrenergic receptors. The interactions between [(Ris) (PA)] and the CTcS complex with the receptors are depicted in Analysis of the best-docked pose of [(Ris) (PA)]-serotonin revealed that the amino acid residues, including His182, Asn187, Asn384, Lys320, and Arg173, formed hydrogen bond interactions. In addition, Leu325, Ala321, Ala108, and Ala176 established π-alkyl interactions while Asp172 formed a halogen (fluorine) interaction ,28. The The best-docked pose (RisS and CTcS) data with the highest docking score generated from AutoDock Vina was utilized as the starting structure for the 100 ns molecular dynamics (MD) simulation run. Only the best-docking output was employed to build up this method in a high-throughput manner to study the binding mechanism of the ligand at the active site of the protein under clearly defined water environments. The different structures represented in To examine structural stability, MD data were processed by calculating the root mean square deviation (RMSD). RisS and CTcS formed stable conformations after ~75 and ~62 ns, respectively, with RMSD values of 2.61 and 2.21 Å, respectively, as seen in the RMSD plot .The most acceptable RMSD value range is <3.0 Å, as low RMSD values indicate superior stability of the system . Our finTo evaluate and compare protein structures, RR distance maps  representing the average distance and standard deviation for all amino acid pairings between two conformations are employed . The RR A grid-search on 25 × 11 × 14 grids, rcut = 0.35, revealed the number of hydrogen bond interactions between ligand and receptor combinations (RisS and CTcS), which were plotted against time . When caOverall, adding PA to Ris resulted in a higher binding affinity to serotonin compared to dopamine and adrenergic receptors. This finding, in the context of the known benefits of Ris, could enhance the therapeutic benefits of this compound in terms of improving side effects associated with lower Dopamine activity as well as higher affinity for serotonin receptors, which could aid in alleviating comorbid depression. Therefore, such findings could potentially bridge the literature gap in terms of designing a multiple receptor profile drug targeting the relevant receptors .Previous animal studies showed that higher Ris affinity to dopamine receptors positively affects the molecular brain-to-plasma ratio . Having 2.The solid six risperidone solid charge transfer complexes with general formula [(Ris) (π–acceptor)] were produced as previously reported . A totalhttps://www.3ds.com/products-services/biovia/) that was accessed on 1 December 2021. The overall docking experiment was run on a processor (Intel(R) Core(TM) i5-4200U CPU @ 1.60 GHz 2.10 GHz 2.30 GHz, 64-bit).Structures of Ris and CT complexes, viz. [(Ris) (PA)], [(Ris) (DDQ)], [(Ris) (TCNQ)], [(Ris) (TCNE)], [(Ris) (BL)], and [(Ris) (CL)] were obtained in PDBQT format using OpenBabelIGUI software (version 2.4.1) . The ene+ and 27 Cl− ions  were added to neutralize the systems  version 2019.2 package was used to perform MD simulation analysis with the GROMOS96 43a1 force field. The parameter files and topology of both ligands were generated using the latest CGenFF via CHARMM-GUI . SPC wat systems . In the Molecular docking revealed that the [(Ris) (PA)] CT complex interacted with all three receptors more efficiently than the reactant donor (Ris) did, and among all complex–receptor interactions, the CTcS combination had the highest binding energy. A MD simulation of the 100 ns run revealed that the RisS and CTcS complexes both possessed a stable conformation; however, CTcS formed a more stable complex with the serotonin receptor. Therefore, we present theoretical support for augmenting Ris with PA to enhance serotonergic receptor affinity, since lower binding affinity to dopamine receptors was observed compared with pure Ris. This enhancement of serotonergic binding according to the serotonin deficiency theory is believed to reduce the highly prevalent depressive episodes associated with depression. Therefore, these results could pave the way for the further optimization of risperidone for comorbid depression.


Listeria spp. invasion proteins [context awareness is a computational discovery framework [Rapid detection of proteins is critical in a vast array of diagnostic or monitoring applications ,2,3. Devproteins ,10). In ramework , and hasramework , biologiramework , and synramework . For appramework . CADS reramework ) with thResearch on engineering molecular systems to optimize affinity between receptor(s) and target molecules is the most granular aspect of diagnostic sensing. Molecular events must also be scaled to the device level, where techniques such as embedded controls and multiplexing  may be nThe current pandemic is an example of the development of devices focused on public health for clinical diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) within the concept of CADS. The S-protein-ACE2 binding has been the model of study for molecular interactions and development of both rapid diagnosis tools  and screThe transition of a biosensor technology from a laboratory prototype to a finished diagnostics tool in the hands of users can be a intrincate process that most researchers have not succesfully navegated, as demostrated by the disproportionate ratio between the large number of “novel” biosensors published in the scientific literature in the past decades, and the compartively low number and variety of biosensing tools available to the public, particularly for non-clinical applications. This situation is unfortunate because diagnostic tools that can be deployed outside of clinical settings, such as in remote rural communities, are still urgently needed.CADS intentionally casts a wide net in an attempt to capture transdisciplinary research that aims to advance the field of diagnostic biosensors. Research focused on more than one aspect are sorely needed in order to drive convergence toward meaningful outcomes.


The goal of this paper is to compare the different anaphoricstrategies that Catalan and Catalan Sign Language (LSC) use by means of a parallelcorpus. In particular, our comparison is focused in an examination of the uses ofovert subject pronouns in Catalan and how these uses are rendered in a language thatexploits the visual-manual modality, such as LSC. As far as we know, this is one ofthe first studies to compare reference-tracking devices in a spoken and a signedlanguage by means of a parallel corpus and incorporating both a descriptive and atheoretical perspective. All instances of overt pronouns in Catalan were analyzedand most of the data can be accounted with three factors: topic change, focus andcontrast. As for LSC, the use of pronouns is rare and only few instances were found.Instead, other anaphoric strategies are used: while topic change and focus areprimarily encoded with bare nouns, the expression of contrast relies onmodality-specific features. In particular, our comparison isfocused in an examination of the uses of overt subject pronouns in Catalan and howthese uses are rendered in a language that exploits the visual-spatial modality,such as LSC.Natural languages are externalised in two different modalities. On theone hand, the auditory-oral modality is produced by the vocal tract and perceived bythe auditory channel; on the other, the visual-spatial modality is produced with thehands and the upper body and perceived visually. Linguistic research has proventhat, regardless of their inherent modality, spoken and signed languages share basiclinguistic properties on the different grammatical levels  to establish coreferential relations.Signing space is used for articulatory reasons; that is, it is the area where thehands and the arms move, like the tongue is accommodated in the mouth in spokenlanguage. But this is not the only function, since signing space also carrieslinguistic meaning. When a discourse referent is introduced, it is associated with aspatial area. This spatial area is used to further refer back to the discoursereferent by means of a pointing sign directed to it, which may function as a pronounor a determiner. Research into the mechanisms displayed in sign languages forreference-tracking has been so far rather limited, and it has partly concentrated ontheir acquisition. One of the few works is Morgan and null pronouns (in 1b).Catalan, as other Romance languages like Italian or Spanish, is anull-subject language and has a double system of pronouns Rigau . In subjThere are cases in which the overt pronoun is ungrammatical, as in2a, cases in which it is optional, as in 2b, and cases in which it is mandatory, asin 2c . Thus, the use of overt pronouns is quiterestricted and clearly the default pronominal expression is the null pronoun. Still,the question remains of when the overt pronoun is used in a null-subject language.Many proposals address precisely this question. In this section we will review thethree main factors, which can account for 87% of the data. These three factors arethe expression of (i) topic change, (ii) focus, and (iii) contrast. We will alsoreview some of the problematic cases in our data.It is a recurrent idea in the literature that while null pronounsmark topic continuation, overt pronouns mark topic change in some Romancelanguages. For instance, variationist studies that take into account severalfactors to account for overt pronouns systematically signal topic change  as one of the most important factors, as in the work byCameron  and SilvThe hypothesis in Vallduví  is that  Carminati proposesVallduví’s and Carminati’s ideas are similar, but not identical,since the former talks about topic change and the latter about subject change.Since subjects are often topics, both accounts will very often make the samepredictions, but the topic change proposal can account for more cases than thesubject change proposal.This factor can account for 30% of the examples in our corpus (21examples). (3) is such a case. In this example, the two main discourse referentsare two frogs: a big frog and a small frog.At the beginning of the fragment, the speaker is talking about thebig frog, which is the topic at that point. In the next clause, the subject of theverb ’to fall’ refers to the little frog and is realized through a null pronoun,which indicates that overt pronouns are not compulsory for a topic change if thereis enough contextual bias. In the following clause, there is an overt pronoun,which switches the reference again (it refers back to the big frog). Since thereis no contextual biasing in this example, the overt pronoun is needed to switchreference again. If there was a null pronoun instead, the hearer would interpretit as referring to the previous subject and not to the referent the speakerintended.Return to main topic: the pronoun may serve to close asegment and return to a previous main topic. For example, after an embeddedclause , which introduces a secondarytopic, the pronoun may serve to go back to the main topic. In example (4),the boy is the main topic, which is temporarily overriden by a secondarytopic (the boat), about which some information is added through two relativeclauses. When the speaker wants to go back to the main topic, an overtpronoun is used to mark the switch.Reference to a previous object: these are the cases thatwould be covered by the Position of Antecedent Hypothesis. The overt pronounrefers to the previous object and thus is used to change the topic of thesentence. (5) illustrates such a case:Of the overt pronouns used in a topic change situation, we candistinguish several special cases.There are 16 cases (23%) in our corpus in which the pronounrepresents focal information. In these cases, there is no real choice between thetwo types of pronouns. Focal information is placed at the end of the main clausein Catalan, which is where the main pitch of the sentence is located. If thesubject is focal information, the speaker cannot use a null pronoun: only a fullpronoun can host the main pitch of the sentence in the sentence-final focalposition. Otherwise, if a null pronoun were used, the main pitch would be placedon some other constituent and this would yield a different informationalstructure.Apart from pragmatic considerations, there is independent evidencefor considering these subjects as conveying focal information.As mentioned, in none of these cases can a null pronoun be used.There is, however, a choice between using the pronoun or using a definitedescription. For example, in (7) above, the choice would be between the pronoun‘ell’ and the noun phrase ‘el nen’ (‘the child’) (note that the same choice wouldbe present in the English translation of the cleft).It is well known that null-subject languages resort to overt pronounsin order to convey contrast. In our data, contrast accounted for 34% of the data (24examples). We identified three different types of contrast: double contrast,implicit contrast and weak contrast . In cases of double contrast, then, the alternatives being comparedare explicit and, for each of the relevant discourse entity, it is conveyed whetherthey did (or did not do) whatever is predicated of them. This is different from whathappens in instances of implicit contrast, in which the two contrasting alternativesare not explicit, but there is an implicit contrast between the antecedent of thepronoun and another entity, highly salient and identifiable in the context. This iswhat happens in (10): the second overt pronoun evokes an implicit contrast betweenthe boy and the rest of the family and it is conveyed that the rest of the family,unlike the boy, was looking forward to the dinner. Finally, there is a third type of contrast, weak contrast, in whichit is conveyed that the speaker ignores or does not want to commit himself towhether the predicate is true of anyone else than the antecedent of the pronoun.This is illustrated in example (11): a waiter is asking a group of people what theywant for dinner. The mother’s answer contains an overt subject pronoun, not becauseshe is opposing her eating chicken to someone else not eating chicken, but ratherbecause her answer is just a partial one: she has no information about what otherpeople will eat. In this paper we follow the proposal in Mayol  accordinThis implicature is one of uncertainty; the speaker does not knowwhether the other alternatives are true or not. Thus, this implicature directlyaccounts for the weak contrast illustrated above. Furthermore, this uncertaintycontrast can be coerced into a stronger exhaustive contrast, which conveys theimplicature that the relevant topic alternative is false. This strengthening takesplace if there are enough contextual cues: namely, there needs to be a salientrelevant alternative either in the context [as in the implicit contrast cases suchas (10)] or in the discourse [as in the double contrast cases such as (9)]. Thisexhaustive contrast is formalized in (13): There are still some examples (around 13%) in the corpus data thatare not covered by any of the previous factors. In these examples, the pronounrefers to the previous topic, it is not focal and there is no contrast, and stillit is felicitous.For example, consider (14)–(17). In all of them the pronoun isreferring to the previous subject, it is not part of the focus and there is nocontrast. The examples discussed in this section would still be felicitouswithout the overt pronoun and there would not be a significant change in meaning.A possible future line of research would be to investigate how pronouns cancontribute in signalling and processing certain rhetorical relations in order toexplain these unexpected uses. For instance, note that in (14)–(16), the overtpronoun is present in a segment that marks a rhetorical relation of violatedexpectation Kehler . One wayFrog Storiesthe anaphoric strategies found in LSC may be divided in two main groups,namely fully specified referring expressions and underspecified ones. While theformer may both introduce and refer back to a discourse referent already introduced,the latter are anaphorically dependent to the previously introduced antecedent. Thecategories included in each group are defined and exemplified below.(i)Noun phrases (NPs): Nouns followed or preceded by anindex-handshape (fist closed and index finger extended) directed tosigning space, which functions as the equivalent to determiners. InLSC, the prenominal or postnominal position of the determiner isirrelevant, as shown in (18).(ii)Bare nouns (BNs): Nouns used without any determineror quantifier. Once the discourse referent has been introduced, arepetition by means of a BN triggers a definite reading. 2.Underspecified referring expressions:(i)Pronouns: Signs articulated with an index handshapeand directed towards a spatial area in signing space, whichsubstitute an entity already introduced. In some cases, LSC alsouses thumb-handshape signs (fist closed and thumb extended), typicalin contexts with non-present discourse referents. (ii)Entity classifiers: Complex morphemes conveyingmovement and location information that function as an anaphoricdevice . LSC shows a rich array ofstrategies used in anaphoric contexts. In the Classifiers incorporate two functions: a predicative function and ananaphoric function. For the predicative function one needs to consider the wholeclassifier construction; that is, not only the handshape but also the movement andthe location of the hands. For the anaphoric function, only the handshape is takeninto account, because it serves as the link with the antecedent and is the relevantpart for the anaphoric chain (Barberà and Quer in press). This anaphoric function isthe one crucial for the present proposal, so we have only considered these cases. Inorder to distinguish them from the purely predicative function, only contexts withclassifiers with some intervening linguistic material between the noun and theclassifier, as shown in the configuration in (22), have been considered. Our LSC corpus contains 69 instances of anaphoric strategies. The useof BNs is the strategy most commonly used, which accounts for 55.1% of the cases.The use of entity classifiers is the device that follows, which accounts for 29%,followed by NPs, which represent 11.6% of the data. In the last place, pronounsrepresent 4.3% of the anaphoric strategies used. These amounts show that pronounsrepresent the least used strategy. In our analysis for LSC we have considered allfour strategies with the goal of offering a broader picture of how anaphoric chainsare conveyed in LSC.In the following section, we analyse the LSC data considering thethree main factors that trigger overt marking in spoken Catalan. Each factor isdeveloped and analysed considering the four strategies just presented.In LSC topic change may be achieved with all four anaphoricstrategies. The LSC corpus contains 13 instances of topic change, from which 69.2%are expressed with BNs, 15.4% with classifiers, 7.7% with NPs and 7.7% withpronouns. These percentages show that the use of the four strategies is notequivalent, as there is a clear preference for the use of BNs. If we consider therelationship between the kind of strategy used and the possible modality-specificaspects of each strategy , we observe that in topic change contexts LSC does notrely on modality-specific strategies. In this respect, determiners directed tospace and classifiers are very productive strategies typical from thevisual-spatial modality, which contribute to building anaphoric chains in signeddiscourse. On the one hand, both NPs and pronouns rely on the use of signing spacefor coreferential purposes. Once the antecedent has been introduced and associatedwith a spatial location, determiners and pronouns directed to that location areunderstood as coreferential. A pointing sign to a particular location is enough torefer back to the discourse referent. On the other hand, the particular handshapeused in classifiers is linked to the antecedent previously introduced and itincorporates rich referential information. Again, the articulation of theclassifier is enough to pick up the discourse referent. However, despite theavailability of these productive procedures, LSC narratives show a preference forthe repetition of the BN in contexts of switch reference. Such an example is shownbelow. The previous context in example (23) is centered on the boy, who isthe main topic of the fragment. In the second sentence in (23) the BN for ‘frog’is enough to change the topic of the first sentence. As the frog was the object inthe first sentence, this is an instance of reference to a previous object, whichis one of the special cases of topic change as shown also for Catalan in thesection ”Topic Change”. The BN refers to the previous object and it is thus usedto change the topic of the sentence. Furthermore, the BN is co-articulated withraised eyebrows, as indicated with the horizontal line in the glosses. Eyebrowraise has been considered a characteristic nonmanual marking associated to topicmarking Aarons  but it aThe second most frequent strategy in topic change contexts is theuse of classifiers, where the hand adopts a handshape that corresponds to somegeometrical and visual features of the antecedent. This is a strategycharacteristic from the visual-spatial modality. The following example is aninstance of a topic change returning to the main topic. The anaphoric strategy,which in this case is expressed with a classifier, serves to close a fragmentcentered in the bird and to refer back to the previous main topic, which is theboy. Both the classifier for the boy and the classifier for the bird are expressedwith the same handshape (8-handshape). However, the movement of the classifierhandshape (which fulfils the predicate function), together with the contextualbias are enough to capture the topic change. Focal information has been found in 8 instances in our LSC data,which are all conveyed with BNs. The basic sign order in LSC is S–O–V , followed by pronouns (17.6%), NPs (17.6%) and in the last place,classifiers (5.9%). When we look at the referring expression used in each type ofcontrast, it is interesting to note that double contrast is mostly expressed withBNs (60% of the cases) and pronouns (40%). In this respect, the use of signingspace does not play a role in contexts of double contrast since BNs (which do notinclude any pointing sign) and pronouns (which are directed to signing space) showa similar behaviour, with the former being slightly more frequent.In the example of double contrast expressed with pronouns shownbelow, we find a two-clause discourse in which the subject position is occupied bytwo different referents (‘we’ and ‘you’) about which opposite actions arepredicated, such as ‘go sailing’ and ‘stay’. a, as indicated in the glosses, and the boy is associated withlocation b. In the last clause, the implicitcontrast is conveyed by the use of the NP ‘the boy’, which includes a pointinglocalised to the same b area, where it waspreviously associated. This spatial association of the NP evokes an implicitcontrast between the boy and the rest of the family, who are associated at theopposed spatial area but not explicitly mentioned. As for implicit contrast contexts, NPs represent 58.3% of thecases, which differs in great measure with the rest of the referring expressions:BNs represent 25% of the cases, and pronouns and classifiers represent 8.3% each.The great amount of NPs used in implicit contrast shows that the use of space isrelevant in conveying implicit contrast. The NP includes a noun followed orpreceded by a pointing sign, which implies that another contrasting alternative,which is highly salient in the context, is present although not explicitlyexpressed. In order for the discourse referent to be contrasted with anotherentity identifiable in the context, which most likely is spatially established atthe opposite lateral area in signing space, BNs alone are not felicitous andsigning space is thus very much needed. In the following fragment, the discoursereferent ‘family’ is associated with location It should be noted that examples (26) and (27) are very similar tothe Catalan examples discussed in (9) and (10): in all these cases pronouns areused to convey either double or implicit contrast. However, such use of pronounsis rare in LSC, where not many cases have been found.Frog Stories, where the signer had to visualise herselftogether with the members of the family at a restaurant. When the waiter wouldcome, she had to play the role of each character. Members had to answer what theywanted without committing themselves about other member’s choice. In the signedcontexts obtained, the signers did use pronouns to refer to each member, whichwere always aligned with particular nonmanual marking. These nonmanuals werearticulated on the lower part of the facial expression and they consisted insucking the cheeks in and pulling the mouth ends down, always combined with ashrug. Therefore, while implicit contrast is conveyed in LSC with pointing signsdirected to signing space, weak contrast, and therefore the strongest version ofthe uncertainty implicature, is captured with nonmanual marking.Finally, there are no examples in our LSC sample data instantiatingthe third type of contrast, weak contrast. In order to study how weak contrast isconveyed in LSC we designed some elicitation tasks and provided the signers withcontexts that were expected to trigger a weak contrast context. One of thecontexts provided consisted in the same setting found in the Once the three factors that trigger an overt marking in LSC havebeen presented, it is interesting to note that there are some examples in the datawhich do not fit in any of the three categories, but rather form a fourth group.They are instances of overt marking in contexts of topic continuation.LSC behaves as other signed languages, like American Sign Language,which have been analysed as null-subject languages are not an expected strategy, as they stand as a repetition of the NP introducedas an antecedent. We hypothesize that this repetitive use of BNs and NPs is genrespecific, since it is also attested in narratives directed to deaf children, aswell as in narratives directed to adults, such as the Aesop fables (Barberà andQuer in press). Besides the productive strategies typical from the visual-spatialmodality, such as the use of space to refer back to discourse referents and thevisual-geometrical import of classifiers, in narrative discourse LSC opts for arepetition of BNs and NPs even in contexts of topic continuation, although to alesser degree compared to classifiers.As already mentioned, the use of pronouns is not a felicitousstrategy in topic continuation contexts. One possible reasoning would be toconsider that an overt pronoun always triggers a topic change context, rather thana topic continuation one. In LSC, topic change would be achieved when thedirection of the pronoun to signing space is opposed to the previously establishedone. That is, if for instance the previously introduced antecedent is establishedin the ispsilateral area  and the overt pronoun in subsequentdiscourse is directed to the contralateral area (the area close to thenon-dominant hand), a topic change context would arise. Therefore, the overtpronoun together with the corresponding spatial location associated is a strategynot found in topic continuation contexts, but it is more productive in topicchange ones.This article has presented an examination of the uses of overtsubject pronouns in Catalan and how anaphoric contexts are rendered in a languagethat exploits the visual-spatial modality, such as LSC. A correspondence in theclassification of functions of anaphoric strategies that the two languages use hasbeen shown, taking into account the different instantiations that serve areference-tracking function.One of the main findings of the comparison concerns the use ofpronouns in LSC. We were expecting to find pronouns in LSC in the contexts in whichpronouns are used in Catalan: that is, topic change, focus and contrast. However, wefound that the frequency of pronouns in LSC is very low. This is particularlysurprising given the fact that sign language pronouns are signs directed towards aparticular area in signing space and this use of space is a very productivestrategy: once the discourse referent is introduced into the discourse, andtherefore associated with a spatial location, a pointing sign directed to it shouldbe enough to refer back to it. However, the use of pronouns is the least usedstrategy. A possible explanation for this is that the particular discourse genrelicenses the use of anaphoric strategies that are informatively richer.Another surprising result is that the LSC data has shown an importantpreference for the use of BNs in both topic change and focus contexts. Again theresults for LSC in these contexts show a preference for strategies that are not tiedto the visual-spatial modality. The great use of BNs could be considered anon-expected strategy in cohesive narrative discourse. However, we have argued thatthis repetition may be due to a genre-specific strategy.An important distinction has been found in contexts of contrastivemeaning, and more concretely when dealing with the three kinds of contrast. BNs arethe primary means to convey double contrast. Implicit contrast relies in the use ofpointing signs directed to space co-articulated with the noun. The use of signingspace, as a modality-specific feature characteristic of sign languages, evokes acontrastive set of alternatives that are not explicitly expressed. Finally,uncertainty in weak contrast contexts is conveyed with particular nonmanual markingarticulated in the lower part of the facial expression.As the first comparison study between overt anaphoric strategies intwo languages of different modality, some issues have not been included in thisarticle. On the one hand, role shift structures, a mechanism typical from signedlanguages where the signer adopts the role of a character of the story, have notbeen taken into account. Role shift is a way of maintaining an active topic and itis mainly expressed with change in the nonmanuals. In its quotational use, it isused to directly report the speech or the thoughts of a character. This strategywould then need to be compared with spoken Catalan intonation to represent thespeech of the character and has been left for future research. Another strategy thathas not been treated here is simultaneous constructions; that is the use of thedominant and the non-dominant hand to express two actions that are happeningsimultaneously. This strategy allows preserving a topical sign across severalsentences (mainly expressed with the non-dominant hand) and may affect the use ofdifferent referring expressions. Forthcoming research should take into accountsimultaneous constructions.Finally, another issue we have left for future research is the studyof the role that rhetorical relations have in licensing a particular anaphoricexpression. Kehler  shows ho


Cell responses to changes in their redox state are significantly mediated by reversible oxido-reductive post-translational modifications of proteins, potentially altering their activities or interactions. These modifications are important for the homeostatic responses of cells to environmental changes that alter their redox state. Such redox regulatory mechanisms not only operate to maintain health, but can become dysregulated and contribute to pathophysiology. In this review, we focus on the redox control of soluble epoxide hydrolase (sEH), which is widely expressed, including in blood vessels and cardiomyocytes. We review the different types of oxidative modifications that regulate sEH and how they may alter cardiovascular physiology and affect disease progression during stress. Multiple post-translational modifications (PTMs) can modify proteins and alter their stability or conformation to regulate their interactions and functional activities. Prominent examples include acetylation, phosphorylation, palmitoylation and glycosylation, together with a substantive array of oxidative PTMs (oxPTMs). Collectively, these modifications orchestrate homeostatic responses of cells; however, these mechanisms of control may be dysregulated and contribute to disease pathogenesis, including those of the cardiovascular system.Changes in the abundance of reactive oxygen species (ROS), reactive nitrogen species (RNS) or cellular reducing biomolecules are altered during health and disease. Proteins sense these perturbations in cellular redox, with ROS or RNS reacting with amino acids to introduce oxPTMs, which can couple to a functional change that may alter physiology . SignifiS-sulfhydration), S-nitrosylation, S-sulfenylation or S-glutathionylation, following reactions with hydrogen sulfide, reactive derivatives of nitric oxide , hydrogen peroxide or derivatives of glutathione , respectively . D. DS-nitrin vitro E. C141 w do this . It is pS-nitrosated proteins generically transition to form a disulfide bond . O. O68]. Oide bond ; hence, S-nitrosation or disulfide bond formation, namely the scenarios mentioned above that activate sEH, a decrease in cell proliferation and migration may be expected. However, treatment with a diol restored blood flow in sEH null mice to levels observed in WT mice during hind limb ischemia [S-nitrosation or disulfide formation may be beneficial. While it is clear that sEH plays a role in angiogenesis, further research is required to elucidate how sEH is regulated to elicit its opposing angiogenic effects in different tissues and the precise roles redox regulation play in specific cells or scenarios.Decreasing sEH activity, either pharmacologically or genetically, has illustrated a broad role for this hydrolase and its EET substrates in vascular homeostasis. EETs are angiogenic, anti-inflammatory, and suppress platelet aggregation ,47,70,71ischemia . Similarischemia . TherefoThe anti-inflammatory actions of EETs also contribute to vascular homeostasis. 11–12 EET decreased the number of adherent and rolling mononuclear cells in mice following TNF-injections . SimilarCa) to cause arterial dilation [2+-induced intermediate-conductance (IKCa) and small-conductance (SKCa) potassium channels that may also participate in cell hyperpolarization [Ca and ATP-sensitive potassium (KATP) channels to induce hyperpolarization and vasodilation [2-OA, covalently adducted to sEH at C521 to inhibit it, increased EETs/DHETs, thereby lowering blood pressure in angiotensin II-induced hypertensive mice. This study clearly demonstrates a role for the redox control of sEH in blood pressure regulation and the pathogenesis of hypertension. It is plausible that other mechanisms of inhibitory sEH oxidation, such as tyrosine nitration, may also couple to vasodilation. On the other hand, oxidative modifications that activate the enzyme are anticipated to have the opposite effect and induce vasoconstriction instead due to the increased hydrolysis of vasodilatory EETs. Interestingly, the vasopressor angiotensin II not only increases cellular H2O2 [EETs are vasodilators that induce vascular smooth muscle cell (VSMC) hyperpolarization and the activation of large-conductance calcium activated potassium channels (Kdilation ,84. Somerization , which srization . EETs carization . EETs redilation . Based odilation ,81,87,88lar H2O2 ,90 but alar H2O2 .Ca channels [2 also demonstrated that EETs relax the coronary vasculature, whereby the electrophilic lipid induced a prominent and sustained vasodilation in ex vivo perfused rat hearts. This coronary vasodilation was due to 15d-PGJ2 adducting to C521 and inhibiting sEH, a mechanism that contributed to coronary hypoxic vasodilation [Myocardial sEH has been significantly investigated in terms of cardiac pathophysiology, with relatively few studies investigating its role in physiological functions during health. However, similar to the dilation of the systemic vasculature, EETs can also relax the coronary vasculature by activating Kchannels . Our wordilation .+ channels [2+ channels [ATP channel openings [In myocytes, 5,6-EET or 11,12-EET increased cell shortening and intracellular calcium concentrations . Other schannels , activatchannels , as wellopenings . Despiteopenings . FurtherThe role of myocardial sEH in cardioprotection has been widely studied. Hearts were protected from cardiac hypertrophy as well as myocardial ischemia and reperfusion injury when the hydrolase was absent in sEH null mice or was pharmacologically inhibited in WTs ,88,96. C2-OA to hearts prior to ischemia and reperfusion protected them from loss of contractile function [Administering 10-NOfunction , which i2O2 [Interestingly, following angiotensin II-induced cardiac hypertrophy or ischemia and reperfusion injury, sEH activity is increased ,98. This2O2 ,100,101.2O2 .It notable that the Oximouse compendium shows that a number of cysteine residues in sEH, both in the human and mouse proteome, are susceptible to oxidation, and these are altered during aging in various different tissues . The OxiFor a protein to be considered truly redox regulated, the oxPTMs need to be able to be reversed as this enables dynamic, two-way control. In terms of the oxPTM in sEH discussed above, one wonders about their reversibility and if they are enzymatically reversed. In the case the intra-protein disulfide in sEH, we provided evidence that thioredoxin reduces the disulfide back to the basal reduced state . S-nitroInterestingly, the studies describing covalent adduction of endogenous electrophilic lipids offers some hope for the generation of a novel class of sEH inhibitors which have advantages over classical inhibitors . The sitPreviously, antioxidants have been tested as a therapy for cardiovascular diseases in an attempt to combat excessive oxidant production. However, despite multiple clinical trials assessing their efficacy, the outcomes have been disappointing, with no cardiovascular protection observed, and in some instances they were even harmful ,111. HowThis review has primarily focused on the hydrolase domain of sEH. However, as mentioned above, sEH also contains an N-terminal lipid phosphatase domain ,114 whicIt is now abundantly evident that many proteins are regulated by oxPTMs that couple changes in cellular redox status to functional responses of proteins and the cells they are found in. Over the last 20 years or so, we have increasingly changed our view of the role of oxidants in cells and tissues. Although they have important roles in dysregulation and disease pathogenesis, there is a plethora of evidence for their regulatory roles during health and adaptive changes that limit disease progression. It is evident that the redox regulation of cardiac and vascular sEH is highly intricate, and much remains to be understood. Importantly, redox control of the hydrolase contributes both to protective as well as maladaptive pathways, and this likely depends upon the specific ROS source and their concentration and locality to sEH, as shown in