Excess body weight is thought to increase the risk of aggressive prostate cancer (PCa), although the biological mechanism is currently unclear. Body fatness is positively associated with a diminished cellular response to insulin and biomarkers of insulin signalling have been positively associated with PCa risk. We carried out a two-pronged systematic review of (a) the effect of reducing body fatness on insulin biomarker levels and (b) the effect of insulin biomarkers on PCa risk, to determine whether a reduction in body fatness could reduce PCa risk via effects on the insulin signalling pathway. We identified seven eligible randomised controlled trials of interventions designed to reduce body fatness which measured insulin biomarkers as an outcome, and six eligible prospective observational studies of insulin biomarkers and PCa risk. We found some evidence that a reduction in body fatness improved insulin sensitivity although our confidence in this evidence was low based on GRADE . We were unable to reach any conclusions on the effect of insulin sensitivity on PCa risk from the few studies included in our systematic review. A reduction in body fatness may reduce PCa risk via insulin signalling, but more high-quality evidence is needed before any conclusions can be reached regarding PCa. Prostate cancer (PCa) is the leading cause of morbidity and mortality in men worldwide . In 20182 increment; 95% confidence interval (CI) 1.01–1.08). In the same study, the relative risk was stronger for advanced (RR 1.12 per 5 kg/m2 increment 95% CI 1.01–1.23) PCa and weaker and inverse for localised PCa  compared with controls [Excess body weight, resulting from imbalances in diet and physical activity, has been implicated as a risk factor for the incidence and progression of several cancers including PCa ,5. In 20controls .With the increasing worldwide prevalence of overweight and obesity in men , a betteInsulin is a hormone made and secreted by the pancreas that plays a key role in the regulation of blood glucose levels. Insulin binds to receptors on cell membranes, initiating a cascade of reactions that activate specific signalling pathways within the cell. This results in cellular uptake of glucose, promoting cell growth and inhibiting apoptosis .Body fatness is positively associated with increasing blood insulin levels ,15,16, cEvidence suggests that insulin and biomarkers of higher circulating insulin, including C-peptide, are positively associated with PCa risk . A diagnWhilst there is evidence that excess body fatness is a risk factor for aggressive PCa , it is nOur objective was to systematically review and synthesise evidence from studies investigating whether changing body fatness might impact on PCa risk via the insulin signalling pathway; in doing so, we investigated the insulin signalling pathway as a potential mechanistic link between body fatness and PCa incidence or progression.Relevant studies which contributed evidence for mechanistic links were those that reported an intermediate phenotype  as either an outcome or an exposure. Defined using PICO/PECO , we separately identified studies which assessed: (1) the effects of dietary interventions (I) to reduce body fatness in adult males (P) on insulin outcomes (O) compared to controls (C); and (2) whether insulin signalling (I/E) in adult males (P) impacted on PCa outcomes (O) compared to controls (C). Our interest was primarily in humans so we prioritised studies in humans and a priori decided to only review animal studies where the former were absent.This systematic review and meta-analysis followed the WCRF International/University of Bristol methodological framework . The revWe included experimental studies examining the effects of dietary intake interventions to change body fatness on the insulin signalling pathway. We excluded studies where the only intervention had a physical activity component, as physical activity is likely to have an independent effect on insulin signalling. Randomised controlled trials (RCTs) with human adult participants were eligible for inclusion, with no restriction on race or nationality. Since only males can develop prostate cancer, we only included studies with male subjects analysed separately. Studies which included a high proportion of participants with a diagnosis of type 1 or 2 DM at baseline were excluded, although we included population-based studies which did not exclude participants on the basis of diabetes.Our exposure of interest was a change in body fatness measured by indirect methods including anthropometric biomarkers , crude weight and skinfold thickness) and bioelectrical impedance analysis (BIA), or direct methods including densitometry, computed X-ray tomography (CT), magnetic resonance imaging (MRI), and dual-energy X-ray absorptiometry (DXA).Outcomes of interest were serum or plasma insulin or biomarkers and surrogate indices of the insulin signalling pathway and insulin resistance , homeostatic model assessment for insulin sensitivity (HOMA-S), glycated hemoglobin (HbA1c) and quantitative insulin sensitivity check index (QUICKI)).We included studies examining the association between the insulin signalling pathway and PCa outcomes. Only RCTs or prospective observational studies were eligible. To reduce the possibility of the results being affected by reverse causation we only included studies in which exposure was measured at least two years (or with a study mean/median of at least 5 years) before outcomes were counted. Cross-sectional and retrospective study designs were excluded. Commentaries, editorials and conference proceedings and studies published only as protocols were excluded.Studies of human adult males (aged 18 years or over) with no restriction on race or nationality were eligible for inclusion. Men with a PCa diagnosis at baseline were excluded, with the exception of studies examining cancer progression as the outcome. Within such studies, men undergoing androgen deprivation therapy (ADT) for PCa were excluded. ADT increases the risk of insulin resistance and diabetes in this population ; by exclExposures of interest were serum or plasma insulin or biomarkers and surrogate indices of the insulin signalling pathway and insulin resistance .Our outcomes of interest were PCa incidence, measures of progression  and PCa-specific mortality. We included any stage of cancer but investigated incident cancer and cancer progression separately. Among the included studies, a broad range of PCa definitions was reported. For the assessment of the insulin–PCa association, a set of 7 broader categories was adopted instead ; Clinicaltrials.gov ; PROSPERO  was also conducted in May 2020 to source additional papers not returned in the search. Results from the literature searches were imported into Endnote X9, where duplicates were identified and removed using the Endnote function.We carried out two separate searches to identify studies examining: (i) effects of changes to body fatness on insulin signalling; and (ii) associations between biomarkers of insulin signalling and PCa outcomes. Searches were conducted using the Cochrane Database of Systematic Reviews ; Embase Ovid (1980 to July 2020); and BIOSIS (1969 to July 2020). The search strategy comprised MeSH terms, text words and keywords. Full search terms that were implemented in MEDLINE, EMBASE and BIOSIS are shown in the We used a sequential approach to the identification and selection of studies, in terms of both the source of the studies and the types of studies  independently screened titles and abstracts of studies for possible inclusion against the inclusion criteria. If a title or abstract met the eligibility criteria, or eligibility could not be determined, a full-text version of the article was obtained and independently screened by two of three reviewers . Discrepancies between reviewers were resolved via discussion until a consensus was reached.p value, odds ratio, 95% confidence intervals). For studies of body fatness–insulin sensitivity association, we also extracted details of the intervention and the control and baseline adiposity and, within group pre–post-intervention mean differences in insulin biomarker levels; where this was not reported we calculated this ourselves as outlined in the Following the screening process, data from the eligible primary studies were independently extracted by two of three reviewers  using a predefined data extraction form. Disagreements from this process were resolved through discussion. Data extracted for all study types included publication details , study characteristics , intervention or exposure , outcomes of interest and results , we performed meta-analysis of results from included studies to estimate a summary measure of effect. Both fixed-effect models and random-effects models were applied to compute pooled standardised mean difference (SMD) and relative risk (RR), with the intention of focusing on the random-effects results unless there was evidence that the model was unsuitable .2). Small study effects were assessed visually using funnel plots and tested statistically using an Egger test [p values, allowing comparison of the direction of effect and strength of evidence across studies even when there is some heterogeneity in the exposure and outcome [Results were reported and graphically displayed using forest plots where we had at least 2 studies which measured the same exposure and outcome, and which provided data in a format that allowed them to be combined. Heterogeneity in effect size between studies was assessed by estimating the between-study variance in effect sizes  or PCa that had spread to more distant organs (distant metastases). High-grade PCa was defined as having a Gleason score of greater than or equal to 7 and low-grade PCa as a score less than 7. Risk of Bias of Included StudiesWe included two main types of study designs within the review-RCTs and observational studies and we assessed risk of bias using selected up-to-date tools for either RCTs or non-randomised studies of exposures. We used the revised Cochrane risk-of-bias tool (RoB 2) to evaluate the risk of bias in RCTs . The tooFor non-randomised studies of exposures, we used a preliminary version of the Risk of Bias in Non-randomised studies of Exposures (ROBINS-E) tool. Similar to ROBINS-I, bias is assessed across 7 domains, bias due to: confounding; measurement of exposure; selection of participants into the study; post-exposure interventions; missing data; measurement of outcomes; and selection of the reported result. Judgements of risk of bias are categorised as ‘low risk’, ‘low risk except for concerns of uncontrolled confounding’, ‘some concerns’, ‘high risk’ or ‘very high risk’. Results assessed to be at high’ or ‘very high’ risk of bias were excluded from syntheses.In our inclusion criteria, we prespecified age and BMI as important confounders of the insulin–prostate cancer association and did not include studies unless they had adjusted for these confounders. Other important confounders for the insulin–PCa association were ethnicity, family history of PCa, history of cancer diagnosis (aside from nonmelanoma skin cancer), height and insulin-like growth factor (IGF) and these were used to assess risk of bias due to confounding.Risk-of-bias assessments were performed independently by two reviewers (OD and SL), with any discrepancies resolved by discussion until a consensus was reached. An overall risk-of-bias judgement was assigned to each included study and assessments are presented in summary tables for each study.The certainty in the evidence from in the included studies was rated using the GRADE framework , informeOur searches identified 15,478 potentially eligible studies of body fatness and insulin signalling. After removing duplicates and screening studies for eligibility, we identified seven eligible studies ,36,37,38We identified 3152 potentially eligible studies on insulin sensitivity and prostate cancer but only six of these studies  met our Six of the seven studies measured fasting circulating glucose levels as the outcome, five measured fasting circulating insulin, two measured HOMA-IR, two measured both circulating glucose and insulin following a glucose tolerance test, one study measured glucose disposal rate and another one study measured circulating C-peptide.A further 11 potential studies of interventions to reduce body fatness with biomarkers of insulin as outcomes were identified by our searches, but were subsequently excluded  because Risk of BiasFor our body fatness–insulin association studies, we present one risk-of-bias assessment per study, because each outcome measure was based on a blood sample collected in the same manner and processed in the same laboratory. For the study by Alves et al. , we assep = 0.05. Ross et al. [All studies included in this systematic review achieved their aim of reducing body fatness except the study by Alves et al. . All thos et al.  was the Grade AssessmentThe GRADE assessment of certainty in the evidence was downgraded by two points from high certainty to low certainty due to: the risk of bias in individual studies  (1 level); imprecision due to the small number of studies (0.5 level) and small number of included individuals in each study; and (0.5 level) reporting bias because these biomarkers were secondary outcomes within the RCTs we included and it is possible that other RCTs have not published data on these outcomes. We did not downgrade due to indirectness of evidence because all studies were conducted in adult men, who were recruited from the general population. We also did not downgrade the evidence due to heterogeneity as there was no evidence of this after excluding one study which did not observe a reduction in body fatness following their intervention .There were six prospective observational studies ,42,43,44We excluded nine studies which did not adjust their insulin biomarker-PCa results by BMI . The reaRisk of BiasWe assessed risk of bias in each study separately for each insulin biomarker exposure and each separate outcome  . For eveThe results for the association between insulin and PCa risk from the studies included in our review are given in C-PeptideIn a small study (139 cases) published in 2010 by Lai et al. , high C-Fasting Glucose and Glucose ToleranceThree studies ,41,44 inDarbinian et al.  investigHOMA-IR, Insulin and Molar RatioIn a small study by Albanes et al. , the autGRADE AssessmentWe downgraded the evidence on insulin signalling and prostate cancer to very low certainty due to: risk of bias, which was at least moderate for all studies; heterogeneity between the studies; and imprecision (1 level for each).We found very few studies (n = 7) which investigated the effect of a reduction in body fatness in males on biomarkers of insulin sensitivity. Of the eligible studies we identified, one did not observe a reduction in body fatness despite having implemented a calorie-restricted dietary intervention . This saWe excluded several potentially eligible studies either because they did not carry out randomisation of intervention, they did not present their analysis separately for males, or because they were performed in men who already had impaired insulin sensitivity. The studies which were included in our review were assessed as having at least some concerns in relation to risk of bias.It was difficult to draw conclusions on the effect of insulin sensitivity on PCa risk from the few studies we were able to include in our systematic review. With the exception of fasting glucose, there were only one or two studies investigating each biomarker and the evidence was heterogeneous. We excluded several studies either because they did not allow at least a two-year lag between exposure measurement and PCa or did not adjust their insulin biomarker-PCa association by BMI. A lag period is important to avoid reverse causation particularly in relation to PCa risk which has a long latency period. The hypothesis we were investigating in this review was whether body fatness affected PCa risk via insulin signalling. If we had included studies which did not adjust by BMI, we would be addressing the question of whether BMI was associated with PCa risk since BMI is so strongly correlated with biomarkers of insulin.We used methodology that we have previously developed with WCRF to systematically investigate the evidence for specific mechanisms between exposures and cancer outcomes . We carrHowever, we found that the overall certainty of the evidence was low for the effect of reducing body fatness on biomarkers of insulin sensitivity in men and very low for the effect of insulin sensitivity on PCa risk. Despite there being a wealth of evidence on the association between body fatness and insulin signalling , there was very little evidence which met our inclusion criteria, or which directly addressed the question of whether reducing body fatness in adult men could impact this mechanistic pathway. Two important reasons for investigating mechanisms between exposures and cancer is to determine whether the association is causal or not, and to identify potential targets for intervention. The approach we have taken is more cost effective and will provide answers more quickly than a trial of interventions to reduce body fatness with cancer risk as the outcome. However, it does depend on evidence relating to the intermediate phenotype being available.Our review has highlighted that insulin sensitivity is a potential mechanistic pathway via which body fatness could impact on PCa risk and has suggested that reducing body fatness may improve insulin sensitivity. However, the evidence linking insulin sensitivity to prostate cancer risk is inconclusive due to a lack of high-quality studies investigating this. Therefore, much more research is needed in this area before any firm conclusions on this mechanistic pathway can be drawn.


Anthropogenic environmental changes are altering ecological and evolutionary processes of ecosystems. The possibility that ecosystems can respond abruptly to gradual environmental change when critical thresholds are crossed (i.e. tipping points) and shift to an alternative stable state is a growing concern. Here I show that fast environmental change can trigger regime shifts before environmental stress exceeds a tipping point in evolving ecological systems. The difference in the time scales of coupled ecological and evolutionary processes makes ecosystems sensitive not only to the magnitude of environmental changes, but also to the rate at which changes are imposed. Fast evolutionary change mediated by high trait variation can reduce the sensitivity of ecosystems to the rate of environmental change and prevent the occurrence of rate-induced regime shifts. This suggests that management measures to prevent rate-induced regime shifts should focus on mitigating the effects of environmental change and protecting phenotypic diversity in ecosystems. These cEcosystem responses to a changing environment are driven by the individual responses of the organisms that are part of it. These responses are in turn driven by phenotypic traits that determine organismal sensitivity to environmental stress, which underlies the response capacity of ecosystems to stress . PhenotyIn this study, I investigate how the interaction between ecological, evolutionary and environmental stress dynamics influences the occurrence of regime shifts in ecosystems with ASSs. Using a general framework to investigate eco-evolutionary dynamics of communities under environmental stress, I incorporate trait evolution in three ecological model systems in which the presence of ASSs has been documented. Subsequently, I examine (1) whether and how trait evolution alters system stability (i.e. asymptotic behaviour), and (2) how ecological, evolutionary and stress dynamics interact and affect the risk of regime shifts (i.e. transient dynamics). Finally, I summarize the findings across the three model systems in the section ‘General patterns in eco-evolutionary systems with ASSs under stress'.. 2I investigate how ecological, evolutionary and environmental stress dynamics influence regime shifts in ecological systems with ASSs in three different model systems corresponding to different ecological scales: a population subjected to an Allee effect; a predator–prey interaction with stage-structured prey; and a shallow lake ecosystem. To do so, I incorporate trait evolution to these systems using standard quantitative genetics techniques  and anal (a)per capita growth rate (i.e. fitness) is correlated with population size. Although this effect can influence a wide range of densities, the most commonly described type of Allee effect is that in which per capita growth rate decreases with decreasing density )In the three ecological systems investigated here, the sensitivity to the rate, and not just the magnitude, of environmental change is caused by the different time scales at which ecological and evolutionary dynamics occur. This kind of regime shifts, also known as rate-induced critical transitions (or rate-tipping), was hypothesized to be a general property of dynamical systems that have processes operating on different time scales (fast–slow systems) . In lineEcological theory has considered regime shifts in which ecosystems move from one stable state to another. As a consequence, after a regime shift, the ecosystem persists in the alternative stable state if environmental stress remains at this level . Here I A large body of ecological theory has investigated how evolutionary changes induced by natural selection can rescue populations experiencing environmental stress from extinction ,37. HerePerhaps one of the biggest challenges will be to empirically study how eco-evolutionary feedbacks make ecosystems sensitive to the rate of environmental change. A first attempt may use an approach combining models and data. For instance, a recent study, using a model whose parameters were estimated from data, showed that behavioural feedbacks may cause rate-induced regime shifts in coral reefs . Howevereco and TPcecoevo in the results). Therefore, if evolution in antagonistic species shifts the tipping point to a lower environmental stress level, rate-induced regime shifts may occur before stress levels exceed the tipping point of the ecological system without evolution. Predicting the effects of co-occurring evolutionary processes of multiple interacting species on ecosystem tipping points and their associated dynamics will be more challenging, especially when evolutionary rates greatly differ due to, for instance, generation time differences among the species. Further research is therefore needed to gain insights into the responses to environmental change of ecosystems with coevolving species to inform management strategies.In this study, I consider the evolution of the species that is abundant in the ecological state present at low levels of environmental stress. In this case, the tipping point that marks the transition to the ASS is shifted to a higher level of environmental stress. Perhaps, the evolution of antagonistic species  may shift the tipping points to a lower stress level. As in the cases studied here, I expect rate-induced regime shifts to occur in evolving ecological systems when environmental stress increases to levels in between the tipping point set by the absence of evolution and the tipping point set by the presence of evolution (transition region between TPcSimultaneous ecological and evolutionary responses to environmental changes are common in ecosystems . It willClick here for additional data file.


Salmo salar) is presented and discussed as a case study from information provided by two major Scottish salmon producers. The percent of total marine sites (n = 80) included in this case study, that reported with suspected or clinical anemia covering the period mid-late summer 2017 to 2019, was between 1 and 13%. The findings from this case study suggest that anemia experienced in most cases was regenerative and most likely linked to blood loss from the gills.Anemia in salmonid aquaculture is a recognized blood disorder resulting from the reduction of hemoglobin concentration and/or erythrocyte count. Because of sub-optimal oxygen supply to the tissues, as a negative impact of anemia fish will experience reduced growth and poor health. This health challenge may be linked with several factors including anthropogenic changes in the marine environment, infectious etiology , nutritional deficiencies, or hemorrhaging. From the mid-late summer of 2017 to 2019, Scottish salmon farming companies began to report the occurrence of anemic events in open-net marine sites. At that time, the industry had little understanding of the pathogenesis and possible mechanisms of anemia and limited the ability to formulate effective mitigation strategies. Clinical examination of fish raised suspicion of anemia and this was confirmed by generating a packed cell volume value by centrifugation of a microhematocrit tube of whole anticoagulated blood. Company health team members, including vets and biologists, reported discoloration of gills and local hemorrhages. This paper reviews various commercially significant cases and lesser-known cases of anemia in cultured salmonid species induced by various biological factors. The current methods available to assess hematology are addressed and some future methods that could be adopted in modern day fish farming are identified. An account of the most recent anemic event in Scottish farmed Atlantic salmon ( •Anemia outbreaks identified in 1–13% of Scottish Atlantic salmon farms surveyed mid-late summer 2017 to 2019.•In most cases, anemia was regenerative and likely linked to blood loss via the gills.•Anemia is most likely associated with microhemorrhages identified on the gills and liver.•Routine hematology required for fish health monitoring and the development of automated analyzers is required. The greus spp.)  and amoeerurans) . These perurans) .An emerging health challenge for farmed Atlantic salmon has been the occurrence of anemia during the marine growing phase, although there is very limited information regarding the etiology. In general, anemia can be associated with many of the most commercially significant infectious and non-infectious diseases and is relatively common in various species of intensely farmed livestock e.g., ruminants , sheep aAnemia is defined as a reduction in the number of red blood cells and/or the hemoglobin (Hb) concentration of the blood below a normal level  and has This review will address (i) an overview of commercially significant and lesser-known health challenges where anemia has been identified in salmonid aquaculture (ii) current and novel methods for anemia assessment (iii) a case study of anemia in Scottish salmon aquaculture.2Multiple causative factors can contribute to clinical anemia in fish e.g., viral, bacterial, and parasitic infections, toxins, nutritional deficiencies, and blood loss . This coAnemia in salmonid farming has been associated with some of the most commercially significant bacterial and viral infections. Infectious salmon anemia (ISA) is a viral infection of Atlantic salmon caused by an orthomyxovirus . The disRenibacterium salmoninarum  caused mainly by oninarum , cold waoninarum , infectioninarum  in salmooninarum . Other loninarum  and viraoninarum . HoweverOncorhynchus tshawytscha) farms in British Columbia, Canada. Fish farmers termed the condition ‘marine anemia’ due to the pale appearance of the gills accompanied by enlargement of the spleen and kidney and fluid in the body cavity   across multiple chinook salmon  . Kent an factors . Thus, i3Hematology is a valuable tool to highlight subtle changes to an individual's health through identifying deviations in the complete blood cell count (CBC). A CBC typically includes the quantification of multiple cellular blood components: RBC, white blood cells (WBC), WBC differential, platelets (thrombocytes in lower vertebrates), hematocrit (Hct), hemoglobin (Hb), and the RBC indices including: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).Fish hematology has gathered momentum for monitoring health in various aquatic sectors  and is considered a useful diagnostic method due to its non-destructive nature and the capacity for increasing sample size compared to the gold standard, histopathology . AdoptinManual methods for analyzing fish blood parameters tend to be normal practice due to the nature of the blood, which has a high ratio of nucleated RBCs to WBCs, making it challenging for automated analyzers to correctly quantify and categorize cell types . In veteA primary concern in misinterpretation of total WBC counts and the ratio of different classes of WBCs is the time between when a blood sample is taken and when it is analyzed. 4One of the main limitations of using hematology in salmon aquaculture is the lack of well-defined and robust reference values for cultured Atlantic salmon. These shortcomings make it difficult to interpret blood parameters and indices between normal and unhealthy individuals under different farming scenarios. Automated hematology analyzers enable large volumes of samples to be processed for a range of domestic and farmed animals. Fish blood is not currently an available pre-installed option on commercial bench-top hematology analyzers due to nucleation of RBC and morphological similarities of WBC and therefore manual methods are preferred . DevelopTo help overcome the issues with blood degradation during transportation and the technical complexity of automated hematology analyzers, cell image analysis software may be an alternative solution for the automation of blood diagnosis. Image analysis has been developed over many years for various cell biology applications but has been typically used to process fluorescent and phase contrast images and limited in the use of light microscopy images . Digitaln = 8) that were identified as clinically anemic from corresponding Hct values of <10% (unpublished data). These biomarkers will help to investigate the cause of anemia such as inflammation, hemorrhaging, or malnutrition , gross examination of gills and fish condition . The results indicated a wide geographical distribution of sub-clinical and clinical cases of anemia throughout the Scottish marine regions of Clyde, Argyll, West Highlands, and Outer Hebrides (n = 80) in Company A and Company B during mid-late summer 2017 to 2019, it is estimated that between 1 and 13% were affected by anemia annually  completed questionnaires  that covered the initial outbreaks in mid-late summer 2017 until 2019 (excludes any subsequent years). The total number of samples during this period for Company A and Company B included Hct , blood smears , proliferative gill disease (PGD), or complex gill disease (CGD). AGD can have significant health and welfare implications and cause economic loss with reports of up to 82% mortalities . PGD is Tenacibaculum maritimus, Piscichlamydia salmones, Candidatus Clavochlamydia salmonis, N. perurans, Loma salmonae, Ichthyobodo spp., Trichodina sp.) and non-infectious agents (phytoplankton and zooplankton) that could be present on the gills  suggest that in most cases it is regenerative anemia, resulting from loss of RBC (hemorrhage) or destruction of RBC (hemolysis). Examination of blood smears revealed no obvious evidence of hemolysis as there was no intracellular hemosiderin within neutrophils or monocytes. A small number of histological results (n = 5) did not show hemolytic anemia due to the lack of melanomacrophage centers with high hemosiderin content (Observations made by Company A based on blood smears (n = 695) and Hct values  to enable accurate interpretation of blood parameters as has been established for many species in veterinary medicine. Due to the complex nature of intensive farming of salmonids, in addition to the multiple factors that influence hematological values, it is an arduous task to establish ‘normal’ reference ranges using traditional manual methods for a CBC. To enable the development of a representative reference dataset, it would be beneficial to develop automated systems to handle large sample volumes for rapid screening, whether that be from laboratory-based hematology analyzers or handheld devices for use on-site. For a holistic approach to health management and complementary methods for hematology examination, the use of clinical chemistry, with endpoints such as ferritin, bilirubin, albumin, and globulin, would aid in the identification of the mechanisms and provide another non-invasive method for health monitoring in aquaculture.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.None.


Three meta-analysis reviews were included and they all concluded that the use of fresh and frozen sperms for ICSI from patients with NOA showed comparable fertilisation and pregnancy rates.: The use of frozen testicular sperm from men with NOA results in fertilisation and clinical pregnancy rates similar to those of fresh sperm. This may encourage fertility centres to use frozen testicular sperm samples, as this policy has certain advantages that would help with organising their workflow.Abbreviations: CPR: clinical pregnancy rate; 2PN%: two pronuclei % fertilisation rate; ICSI: intracytoplasmic sperm injection; NOA: non-obstructive azoospermia; OA, obstructive azoospermia; SCO: Sertoli cell-only syndrome; (micro-)TESE:  testicular sperm extraction Azoospermia is defined by a complete absence of sperm in the ejaculate after centrifugation of two semen specimens and affects about 1–2% of males and 10–15% of the infertile males [Obstructive azoospermia (OA) is the absence of spermatozoa in the ejaculate secondary to a transport failure between the testis and urethra . While tin situ in patients with azoospermia [An isolated diagnostic testicular biopsy should rarely be indicated, as it will not provide definitive proof of whether sperm will be found during sperm retrieval, particularly in Sertoli cell-only syndrome (SCO) and maturation arrest cases . Histopaospermia .A suitable treatment for NOA is microsurgical testicular sperm extraction (micro-TESE) followed by intracytoplasmic sperm injection (ICSI). A testicular biopsy can be performed on the day of oocyte retrieval and fresh sperm can be used to fertilise the oocytes .For those cases with anticipated difficult sperm retrieval, it is better to start testicular sperm retrieval at least 8 h before ovum retrieval to avoid post-maturity oocyte damage . HoweverSome authors suggest repeating the sperm retrieval in such cases due to poorer ICSI results when frozen–thawed testicular sperm samples are used compared with fresh sperm samples, as they are convinced that during the process of freezing and thawing spermatozoa are subjected to a series of drastic changes in their environment. Phase transitions of the lipids in the plasma membrane may impair the function of membrane proteins that are needed for ion metabolism and structural integrity. Freezing may also lead to extracellular ice nucleation producing osmotic changes with efflux of water from the cells, with loss of stability of the lipid bilayer. Further consequences may also include denaturation of proteins and structural deformation of the cell organelles ,14.The aim of the present systematic review was to review the debate about the routine use of cryopreserved testicular sperm in ICSI in patients with NOA, as some authors suggest repeating the sperm retrieval in such cases due to poorer ICSI results when frozen–thawed testicular sperm samples are used compared with fresh sperm samples.A systematic literature review was performed in August 2020 using the Medical Literature Analysis and Retrieval System Online (MEDLINE), Web of Science databases, and the Excerpta Medica dataBASE (EMBASE). Review articles, congress abstracts and editorials were excluded. Search terms included ‘non-obstructive azoospermia’ in combination with the term ‘cryopreservation’ OR in combination with the term ‘intracytoplasmic sperm injection’. The search was limited to the English literature. References cited in selected articles and in review articles retrieved in the search were used to identify other studies and articles that were not included in the initial searches. We included the articles that provided the highest level of evidence. The systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  Figure .Figure 1n = 908). Non-relevant records were excluded based on full-text evaluation (n = 255). Three meta-analysis reviews were included and they all concluded that the use of fresh and frozen sperm samples in ICSI in patients with NOA showed comparable fertilisation and pregnancy rates.In all, 1189 publications were screened and 26 of them were included in this systematic review based on our inclusion criteria and were considered eligible for this study. Duplicate studies and abstracts were excluded. Only full-text articles in the English language were included. Non-relevant records were excluded based on title and abstract , while repeating the sperm retrieval procedure cannot be done before 3  or 6 monSo to settle the debate about the success rates of fresh vs frozen testicular sperm use, three meta-analysis reviews were conducted. They all concluded that fresh and frozen sperms in ICSI from patients with NOA showed comparable fertilisation and pregnancy rates.et al. [The first meta-analysis review article by Nicopoullos et al.  in 2004 et al. [In the second meta-analysis review article by Ohlander et al.  in 2014 et al. [In the third meta-analysis review article by Yu et al.  in 2018 et al. .Table 2.Surgical retrieval of rare sperms can be achieved in clinical practice. Conventional cryopreservation cannot help those patients due to its technical limitations. More complicated technologies have been developed over the years to manage this situation. A number of devices have been suggested to cryopreserve rare spermatozoa like empty zona, Cryolock, Cell Sleepers, Petri dishes, the novel sperm vitrification device (SpermVD) and other biological or non-biological devices. None of these have achieved widespread use due to technical requirements and cost concerns .Use of frozen testicular sperm from men with NOA results in fertilisation rates similar to that of fresh sperm samples. In addition, the CPR was found to be similar when comparing fresh vs frozen sperm use in that group of patients. This may encourage fertility centres to use frozen testicular sperm samples, as this policy has certain advantages that would help organising the workflow.Significant progress has been made over the past few years in our understanding of male infertility. This understanding, as well as rapid technological progress, has played a great role in managing males with azoospermia. In view of these findings, sperm cryopreservation has to be considered in every surgical sperm retrieval operation to guarantee the best use of those valuable sperms. Patients with azoospermia need to be reassured that frozen–thawed viable spermatozoa are as good as the fresh ones, and they should be adequately counselled before any surgical procedure (


The mural thrombus in the ascending aorta is rare, most of which are associated with aneurysm or atherosclerotic lesions, with high risks of causing catastrophic thrombotic events. A mural thrombus in the non-aneurysmal and non-atherosclerotic ascending aorta is exceptionally uncommon.We reported a large mural thrombus in normal ascending aorta of an asymptomatic patient. Preoperative imaging confirmed the presence of the sessile thrombus located at the left anterior wall of ascending aorta. Given that it had the potential to cause fatal thrombotic complications, surgical removal and segment of ascending aorta replacement were executed. The patient had an uneventful recovery and discharged 14 days after surgery.Anticoagulant is the therapeutic cornerstone of ascending aortic thrombus, but surgery should be performed aggressively when the thrombus is large or floating to avoid severe embolic complications or recurrence. The mural thrombus in the ascending aorta(AA) is rare, most of which are associated with aneurysm or atherosclerotic lesions, with high risks of causing catastrophic thrombotic events, such as ischemic stroke, acute myocardial infarction and peripheral arterial embolism. A mural thrombus in the non-aneurysmal and non-atherosclerotic AA is exceptionally uncommon. Herein, we report an asymptomatic large mural thrombus in normal AA which was successfully removed by surgery.A 59-year-old man complaining about right lower limb pain and swelling was transferred to our institution for an ascending aortic mass which had been found incidentally in local hospital. Extremity ultrasound confirmed deep vein thrombosis in his right lower limb, which was responsible for his suffering. He had a past history of massive pulmonary embolisms, which were reduced obviously after 5-month course of oral anticoagulant. Except smoking, he had no other risk factors like hypertension, diabetes mellitus and dyslipidemia and he took no medication at that time. The thoracic contrast-enhanced computed tomography(CT) showed a low-density defect with no enhancement in the left anterior wall of AAFig. A and theGiven that the large thrombus in the AA with high risks of fatal thrombotic complications, surgical removal was executed. The surgery was performed through a median sternotomy and then cardiopulmonary bypass was established by cannulation of the right axillary artery and right atrium. After AA cross clamped, it was incised under cardiac arrest and a sessile mass located on the left anterior wall was identified Fig. A. The maMural thrombi in the non-aneurysmal and non-atherosclerotic AA are extremely rare. Although some of those have been reported without significant signs of aortic atherosclerosis on preoperative imaging, they have been found attached at atherosclerotic lesions after aortotomy , which wAs for diagnostic tools, contrast-enhanced CT should be considered as the first choice because it can clearly show whether the aorta has dissection, hematoma or atherosclerosis, and whether the mass displays enhancement. Moreover, the size and location of the mass can be visually seen on reconstructive images. In our case, since the asymptomatic patient was incidentally detected a sessile mass in his non-aneurysmal and non-atherosclerotic AA, the possibility of ascending aortic tumor(e.g. sarcoma) should be taken into account. Thus, we applied PET to exclude the malignancy and evaluate the necessity of surgery. Besides, although transesophageal echocardiography is effective for evaluating the ascending aortic thrombus, this invasive procedure is very uncomfortable for patients and related to possible complications such as gastrointestinal perforation and aspiration pneumonia , which iAt present, there is no corresponding guidelines or consensus on the treatment of the ascending aortic thrombus. There are two main treatment approaches: surgery and conservative therapy with anticoagulant. Endovascular treatment and thrombus aspiration have been also applied in recent years. Anticoagulant should be regarded as the therapeutic cornerstone whether surgery or not, especially appropriate for the patients with high risks of surgical intervention. Some researchers have reported good outcomes of anticoagulant therapy alone . HoweverWe presented a large and sessile mural thrombus in the normal AA of an asymptomatic patient. Ascending aortic thrombus should be taken into consideration when meet with a relatively young patient showing clinical manifestations of arterial embolism. Although anticoagulant therapy is generally recommended, we advocate that surgery should be performed aggressively when the thrombus is large or floating to avoid severe embolic complications or recurrence.


Autoimmune rheumatic diseases have a major impact on public health as one of the most common morbidities, and many of these disorders involve both local and systemic manifestations with severe consequences for patient health and quality of life. However, treatment options for many of these diseases remain inadequate for a substantial portion of patients, and progress in developing novel therapeutics has been slow. This lack of progress can be largely attributed to an insufficient understanding of the complex mechanisms driving pathogenesis. Recently, the emergence of single-cell RNA sequencing (scRNAseq) has offered a powerful new tool for interrogating rheumatic diseases, with the potential to assess biological heterogeneity and individual cell function in rheumatic diseases. In this review, we discuss the major insights gained from current scRNAseq interrogations of human rheumatic diseases. We highlight novel cell populations and key molecular signatures uncovered, and also raise a number of hypotheses for follow-up study that may be of interest to the field. We also provide an outlook into two emerging single-cell technologies  that have yet to be utilized in the field of rheumatic diseases, but which offer immense potential in expanding our understanding of immune and stromal cell behavior. We hope that scRNAseq may serve as a wellspring for the generation and interrogation of novel hypotheses regarding autoreactive lymphocytes and tissue infiltration patterns, and help uncover novel avenues for therapeutic development. Autoimmune rheumatic diseases have a major impact on public health as one of the most common morbidities, affecting some 3% of the general population . Many ofHowever, the development of new therapeutics for improved treatment has been painstakingly slow, with no treatment having been approved for primary Sjogren’s syndrome, and very few for systemic lupus erythematosus, leading to major difficulties in prescribing more precise clinical guidelines tailored for individual patients , 4. ThisSince its advent from relatively low-throughput applicability, single-cell RNA sequencing (scRNAseq) has evolved into a powerful tool for assessing biological heterogeneity and individual cell function in complex environments at extremely high throughput , 6. UnliIn this review, we first summarize major findings obtained from current scRNAseq studies in rheumatoid arthritis, systemic lupus erythematosus, and primary Sjogren’s syndrome. We also highlight a number of provocative questions raised by the results obtained from these studies, and which may be of relevance towards furthering current knowledge in the field. We further discuss the potential value for integrating other forms of single-cell omics data into these mapping studies and beyond, to preview some forms of potential knowledge to come from future studies.Rheumatoid arthritis (RA) is an autoimmune disease characterized by erosion of the protective collagen layer in joints across the body under inflammatory stress . In a siA number of efforts have been made to generate a cellular atlas of rheumatoid arthritis in both peripheral circulation and in afflicted joints. Two early single-cell mapping studies using a homebrew microfluidic system  or the pHLA-DRA. These results raise an interesting possibility that differentiation of synovial FLS towards a pro-inflammatory state may occur as an early event during RA pathogenesis, with this conversion potentially contributing to the recruitment of immune cells to synovial tissue. The potential of synovial FLS derived from RA patients to act as additional antigen-presenting cells to activate T cells has been previously demonstrated in several reports  has already been initiated to target this FLS in patients refractory to methotrexate, and results from these studies may yield additional insights into their function  in a cohort with minimal treatment intervention . Interesnologies , 21. It At the same time, the study also noted a substantial increase in the expression of chemokines from the CCL family in both dendritic cells and macrophages of ACPA- patients compared to ACPA+ counterparts. If these increases are indeed independent of current patient disease severity, this result may suggest an alternative, more innate-centric model for disease progression in ACPA- patients, compared to a more lymphocyte-driven reaction in ACPA+ counterparts. Such a divergence in mechanism may partly explain clinically observed differences in disease progression . FurtherOther recent studies have focused on a specific population of cells to investigate them in greater detail. For instance, a study focused on the macrophage compartment in RA synovial tissue emphasized the existence of CD206+MerTK+ macrophages with a unique regulatory signature in RA patients experiencing clinical remission, and correlated with remission maintenance . This povia antibody-mediated damage (Systemic lupus erythematosus (SLE) is a severe autoimmune disorder typically involving systemic manifestations capable of causing major, and oftentimes chronic, organ and tissue damage , 28. Thed damage , 31. As In order to map this systemic disease, large cohort studies have been conducted to characterize the immune populations present in peripheral circulation of SLE patients. The first of these studies focused on identifying populations of immune cells especially responsive to interferon stimulation . These aWhile a large number of interferon genes exist, there has long been evidence suggesting that IFNα may be the one most strongly correlated with disease activity in SLE, and treatment with IFNα may provoke SLE-like side effects in some patients , 36. IntAt the same time, additional explorations of other biological pathways and processes that are highly correlated with progression towards ISGhi signatures may provide additional insights into the mechanisms by which these cell populations exert pathological functions. In particular, it is unclear if continued expression and stimulus of interferon receptors is required for the maintenance of these ISGhi cell states, or if they represent a more stable end-state. Trajectory analyses that reconstruct the differentiation hierarchies between cell states may be particularly useful in this context for identifying potential novel forks in differentiation caused by the elevated levels of interferon observed. The inclusion of additional data from currently unpublished efforts to study signatures in patients with acute flares and with other unique pathological features may help to further clarify this possibility.Beyond the peripheral transcriptome, other studies have focused on evaluating regional differences brought about by SLE pathology in the kidneys and skin. By isolating immune cells from renal biopsies, one study was able to capture a complex landscape of renal-infiltrating immune cells broadly similar to the peripheral atlas, including several populations of T and B cells defined by an ISG signature . Part ofBLHLE41, consistent with the hypothesis that the function and transcriptome of residential immune cells may also be significantly altered as a result of interferon signaling in SLE. However, substantial numbers of T and B cells have also been demonstrated to be present in the kidneys under physiological conditions  (Primary Sjogren’s syndrome (pSS) is characterized by excessive dryness at mucosal membranes due to insufficient fluid production from secretory glands  . Histolo2962895) . FurtherThus far, one preliminary scRNAseq study has been performed examining the peripheral blood of patients with pSS as compared with healthy controls . The autThe establishment of a clonal relationship between peripheral and gland-infiltrating T cell populations may be especially important in the context of pSS due to the complexity of the peripheral signature of pSS. A number of large-cohort studies using bulk RNAseq of peripheral blood have found remarkably few differences between the pSS patients and healthy controls, with most of the variation being concentrated in the interferon signaling pathways –53. As sAtlas-focused mapping studies have been conducted in each of the three rheumatic diseases reviewed above, and have helped provide a broad contour of the general immune cell landscape in each of these diseases, in both circulation and in afflicted tissues. These studies have also made some inroads into identifying some potentially pathogenic and protective cell types that have been previously underappreciated. A summary of the primary findings reached thus far is included as via monoclonal antibodies or other means, may serve to effectively restrain disease activity in SLE. scRNAseq studies in SLE have thus emphasized a direction for translational research with the establishment of a dominant signaling pathway and the characterization of unique cell subtypes. For instance, if additional mappings are performed to gauge therapeutic response in SLE patients to a particular treatment, it may be possible to utilize these signatures as a key reference point. Assessment of these signatures might also help as a leading indicator to predict relapse or remission. Importantly however, these assessments will require validation in much larger cohorts of patients than those typically sequenced in scRNAseq studies in order to reach sufficient power. As such, developing other assays that can capture the nuanced insights obtained from scRNAseq data will be necessary to further translational research into SLE.In the case of SLE, which has the largest number of patients sequenced thus far, interferon signaling appears to play the biggest role in differentiating single-cell profiles during both active disease and remission. Multiple immune cell types appear to have skewed differentiation, and this skew extends to tissue infiltrating immune cells. The pervasiveness of this signature raises hope that disruption of the interferon signaling axis, whether In the context of RA, less is known about the full spectrum of immune cells involved and the essential signaling mechanisms involved. While several sequencing studies of the synovium in RA have been performed, these studies have largely emphasized more local alterations in fibroblast and myeloid signatures in joints. In contrast to SLE, these alterations are not necessarily reflected in peripheral circulation. However, from the clinical correlation analyses reported thus far, it appears that the presence of some of these populations is positively associated with increases in systemic scores of disease activity and inflammation . It is currently unclear how these local changes in one joint may propagate to others, or otherwise influence RA-involved conditions in other tissues. Extensive future investigations are necessary to gain a clearer picture of the relevant molecular mechanisms and immune response patterns in RA, and how these mechanisms might enable autoimmune reactions across the body.The landscape of gland-infiltrating immune cells in pSS is similarly underexplored. As reviewed above, only a single scRNAseq study of circulating immune cells in pSS has been performed thus far, identifying a potential increase in CD4 CTLs. However, the relationship between circulating phenotypes and glandular damage in pSS remains unclear. Although there have been some indications of interferon signaling playing an important role in pSS based on transcriptome differences relative to healthy controls, it is currently unclear if strong ISG signatures can be found in both circulating and gland-infiltrating immune cell populations on a single-cell level in a manner akin to SLE. Detailed mappings of both peripheral blood and infiltrated glands in pSS patients will be necessary to clarify these and other possibilities.Besides the information already obtained from atlas studies, scRNAseq also has immense potential for uncovering additional biological insights by being integrated into other forms of omics information. In recent years, other sequencing-based omics technologies have become increasingly mature and are now available through established protocols. In the following section, we highlight single-cell immune repertoire sequencing and spatial transcriptomics as two technologies with particular potential to be applied in assessing rheumatic diseases and answer key questions regarding their pathological progression.Current scRNAseq studies of rheumatic diseases have elucidated a significant number of distinctive T and B cell populations as reviewed above. However, classification of these T and B cells has been driven by transcriptome-level information on expression profiles, and not based on their antigenic specificity, thus neglecting a critical component of T and B cell identity. Antigen specificity of T and B cells is determined by the sequence of unique recognition receptors  encoded as specific sequences generated by genetic recombination , 57. UnfFortunately, new approaches for library construction have made it increasingly possible to capture full-length sequences, including in the TCR and BCR regions. In plate-based workflows, utilization of unique enzymes and/or amplification sequences  generate full-length information and capture entire TCR and BCR sequences , 59. In This advance has opened up new possibilities, as the importance of autoreactive T and B cells clones in mediating rheumatic diseases has long been appreciated. Early studies in animal models raised the hypothesis of there being ‘forbidden clones’ driving autoimmune diseases , and subHowever, it is also important to note that autoreactive T and B cell clones will only represent a small portion of the total immune repertoire of any individual at a given time, particularly in circulation. Furthermore, positive serum detection of autoantibodies has been reported to precede clinical disease in SLE, SS, and RA, and a high percentage of people who do not develop rheumatic diseases may also have circulating autoantibodies . In the Given the fact that patients with rheumatic diseases typically maintain significant circulating titers of autoantibodies, one might assume that autoreactive clones may tend to occupy a higher percentage of class-switched memory and plasma cells which can directly secrete large amounts of antibody. However, several studies that have inspected the proportion of ANA-reactive B cells by stage in SLE patients have found only a surprisingly small increase in circulating ANA-reactive plasma cells compared to controls , 69. SimAlternatively, it is possible that a more precise definition of B cell status derived from scRNAseq, when analyzed together with clonal identity, might help identify subtypes of plasma cells highly enriched for autoreactive clones. Such enrichment may also be more prominent when analyzing a more specific context, such as during clinical remission or abrupt flares. Furthermore, it is critical to remember that B cells can undergo somatic hypermutation and affinity maturation, wherein B cells can reshape their BCR sequence to increase their affinity for a given antigen in germinal centers under T cell help . As suchSince T cells lack the ability to undergo repeated rounds of affinity maturation and class-switch recombination, the TCR sequence of any given clone can be expected to stay stable, allowing for easier analysis. However, due to technical difficulties with generating tetramer libraries against possible autoantigens, even less is known about the behavior and frequencies of autoreactive T cell clones in rheumatic diseases at this time. In the context of pSS, and cloned T cells lines recognizing SSA have been generated , and autAs such, further work will be necessary to distinguish the identities of autoreactive and bystander memory clones. A summary of how inclusion of repertoire information may help guide analysis is depicted in The recent advent of spatially-aware transcriptomics has made it possible to gain a concurrent understanding of the tissue localization and transcriptomes of individual cells . While pFor instance, SLE is known to cause systemic manifestations in an extraordinarily wide range of tissues spanning the entire body, and current studies of skin and renal manifestations. Single-cell mapping studies have further demonstrated that significant numbers of ISGhi immune cells may enter into tissue sites. However, current analyses of these infiltrating immune cells have been limited to transcriptome information without spatial location. A spatial mapping might be able to resolve the context in which these immune foci form, as well as the contents of each foci. One question of particular interest is whether all of these foci are indeed dependent on interferon signaling, and which communication signals from surrounding stromal tissue may serve to help/hinder their formation. For instance, immune foci have been found to be preferentially localized near certain types of cells, but not others, in the context of tumors , 84, butAs reviewed above, histology-guided analyses in RA have already helped to identify differences in spatial distribution between Thy1+ and CD55+ FLS, and have found preferential accumulation of immune cells near Thy1+ FLS. A preliminary spatial transcriptomic study of RA synovium has also been conducted, and has suggested co-localization between CD8+ T effector memory cells and class-switched B cells, and segregation between T cells and plasma cells . UnfortuFurthermore, spatial analyses may also help to compare newly-infiltrating immune cells with long-lived residential immune cells that can persist in many tissues. For example, the skin under normal conditions is expected to contain large numbers of Langerhans cells and memory T cells. These cells provide an important barrier function as a first line of defense against external pathogens, and have been conditioned to survive and function in the dermal environment. These residential cells can thus be expected to express unique transcriptome profiles that enable local adaptation, and can more readily respond to local insults by virtue of their localization . TraditiThe application of scRNAseq into human rheumatic disease holds great promise for answering foundational questions regarding how autoimmune reactions are instigated and maintained in complex environments, as well as identifying novel targets for therapeutic intervention. Current efforts to map the atlas of immune cell types involved in each disease have already identified a number cell types and pathways as being of notable interest, particularly in the context of SLE. These mapping studies have also identified notable modifications in regional, tissue-resident populations of both immune and non-immune cells as a result of disease progression that point to larger-scale remodeling caused by immune cell activity. If recently emerging technologies for repertoire and spatial mapping can be integrated into scRNAseq studies, a much more complete picture of auotreactive lymphocytes and tissue infiltration patterns can also be obtained. Combination of these assays with other emerging single-cell technologies in each sample, such as ATAC-seq  and protYW and LZ generated the central concepts for the manuscript. ZZ and LC collected and assessed the primary literature in the field and drafted initial figures. ZZ, LC, JL, and GC wrote the initial manuscript. YW, GC, and LZ extensively revised the manuscript. All authors contributed to the article and approved the submitted version.This work was funded by support from the National Natural Science Foundation of China , and from the Chongqing International Institute for Immunology (2020YJC06 to LZ).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.


The lack of standardized language assessment tools in Russian impedes clinical work, evidence-based practice, and research in Russian-speaking clinical populations. To address this gap in assessment of neurogenic language disorders, we developed and standardized a new comprehensive assessment instrument–the Russian Aphasia Test (RAT). The principal novelty of the RAT is that each subtest corresponds to a specific level of linguistic processing  in different domains: auditory comprehension, repetition, and oral production. In designing the test, we took into consideration various (psycho)linguistic factors known to influence language performance, as well as specific properties of Russian. The current paper describes the development of the RAT and reports its psychometric properties. A tablet-based version of the RAT was administered to 85 patients with different types and severity of aphasia and to 106 age-matched neurologically healthy controls. We established cutoff values for each subtest indicating deficit in a given task and cutoff values for aphasia based on the Receiver Operating Characteristic curve analysis of the composite score. The RAT showed very high sensitivity (> .93) and specificity (> .96), substantiating its validity for determining presence of aphasia. The test’s high construct validity was evidenced by strong correlations between subtests measuring similar linguistic processes. The concurrent validity of the test was also strong as demonstrated by a high correlation with an existing aphasia battery. Overall high internal, inter-rater, and test-retest reliability were obtained. The RAT is the first comprehensive aphasia language battery in Russian with properly established psychometric properties. It is sensitive to a wide range of language deficits in aphasia and can reliably characterize individual profiles of language impairments. Notably, the RAT is the first comprehensive aphasia test in any language to be fully automatized for administration on a tablet, maximizing further standardization of presentation and scoring procedures. Single word comprehension subtests were also correlated. Interestingly, it was specifically verb comprehension that was found to be related to sentence and discourse level comprehension, supporting the idea of the role of the verb as a central sentence element. This correlation emerged even though most of the trials in the Sentence Comprehension subtest had syntactic but not semantic distractors. Accordingly, it is in line with the hypothesis that the verb’s grammatical properties  are processed during comprehension and production of isolated verbs , linkingRepetition subtests remained highly correlated; it should be noted that specifically Nonword Repetition correlated strongly with Sentence Repetition likely indicating critical involvement of the phonological buffer in both tasks. As expected, Object and Action Naming subtests were related. Both naming subtests were also related to Nonword and Word Repetition subtests, but again it was specifically Action Naming that was related to the Sentence Repetition subtest and the Discourse Production subtest. Overall, the prominent relationships observed between performance on subtests targeting verb processing and sentence/discourse level tasks highlights the importance of assessing verb comprehension and action naming in aphasia. Future studies are needed to explore these differential association patterns in greater detail.We estimated internal reliability of subtest scores using Cronbach’s alpha  calculatp > .05). Overall, these results demonstrate that with the provided scoring instructions, the RAT can be reliably scored by trained clinicians.Inter-rater reliability for repetition and production subtests was calculated based on 20 evaluations of PWA responses . This observation in combination with good to excellent internal reliability estimates for all comprehension subtests implies that the below-normal scores on these subtests can still be confidently interpreted as such. Still, given that particularly single word comprehension subtests demonstrated poor test-reliability reliability, we would caution future test users against interpreting them in isolation or in contrast scores, especially for individual cases.Test-retest reliability was calculated based on 20 additional individuals with chronic aphasia who were evaluated two times each on two separate occasions  in language functioning and provide guidance to clinicians on how to interpret observed changes in the scores in a meaningful way. Regression-based approaches for determining clinically significant changes in the scores should also be explored in future work .Another limitation of the current normative dataset is that we only collected data from individuals with brain damage who were diagnosed with aphasia. Future inquiries need to include persons with brain damage but without language impairments, so that sensitivity of the test specifically to language deficits rather than to general cognitive sequalae of brain injury  is ascertained. While the selective relationships observed between different RAT subtests demonstrate that subtests are differentially sensitive to specific language deficits and underscore the test’s construct validity, data from a non-aphasic brain injury group would further raise the diagnostic value of the RAT.Finally, the RAT currently does not assess reading and writing abilities. We hope that in the near future we will be able to develop and standardize these subtests as well.The RAT is the first comprehensive aphasia language battery in Russian, sensitive to a range of deficits and with properly established validity, inter-rater and test-retest reliability according to contemporary psychometric standards. It provides a multidimensional characterization of impaired and spared aspects of language functioning at different linguistic levels in different domains: auditory comprehension, repetition, and oral production. By using a ROC-curve analysis Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Although these TRexpansions could not be classified definitely as pathogenic, as there are no availableguidelines to facilitate pathogenicity interpretation, these expansions were in the size rangeof disease-causing expansions seen in other conditions. Furthermore, to the best of theauthors’ knowledge, this is the first time TR expansion has been reported in Lennox-Gastautsyndrome. This finding raises the question: is this due to TR expansions being rare inLennox-Gastaut syndrome or simply because TR expansions are rarely looked for inLennox-Gastaut syndrome patients? As only six patients with Lennox-Gastaut syndrome wereincluded in this study, of which two had TR expansions, it is possible that the latter istrue, but we need to acknowledge that the sample size overall is small. Nevertheless, thisintriguing finding raises the question of how important TR might be for other developmentaland epileptic encephalopathies or other forms of human genetic disease? How much of ourunderstanding of the pathogenicity and strategies for potential treatments are being missed bythe focus on protein-coding genes in general? This possibility is further underlined bywell-known examples of causal TR expansions in other human diseases such as Huntington’sdisease and amyotrophic lateral sclerosis,Perhaps, most intriguing was the identification of TR expansions in the genes DiscoInteracting Protein 2 Homolog B (The research performed by Qaiser et al.


Malignant cells often demonstrate a proliferative advantage when compared to non-malignant cells. However, the rapid growth and metabolism required for survival can also highlight vulnerabilities specific to these malignant cells. One such vulnerability exhibited by cancer is an increased demand for amino acids (AAs), which often results in a dependency on exogenous sources of AAs or requires upregulation of de novo synthesis. These metabolic alterations can be exploited by therapy, which aims to improve treatment outcome and decrease relapse and reoccurrence. One clinically utilised strategy targeting AA dependency is the use of asparaginase in the treatment of acute lymphoblastic leukaemia (ALL), which results in a depletion of exogenous asparagine and subsequent cancer cell death. Examples of other successful strategies include the exploitation of arginine deiminase and methioninase, nutrient restriction of methionine and the inhibition of glutaminase. In this review, we summarise these treatment strategies into three promising avenues: AA restriction, enzymatic depletion and inhibition of metabolism. This review provides an insight into the complexity of metabolism in cancer, whilst highlighting these three current research avenues that have support in both preclinical and clinical settings. Cancer is a disease at the cellular level that results from oncogenic mutations that lead to altered cellular metabolism, promoting tumorigenesis  and acute myeloid leukaemia (AML). Kelly et al.  demonstrAnother example supported by recent evidence is the enzymatic depletion of the essential AA, methionine. Cancer cell proliferation appears dependent on exogenous methionine and is known as methionine dependence or the Hoffman effect Hoffman . HoffmanClostridium sporogenes and successfully inhibited the growth of Walker carcinosarcoma 256 implanted in male Wistar rats (Kreis and Hession 2) and melanoma (3755 vs 858 mm2) cancer models  , which was reflected in an increased survival. Despite the promising preclinical data, human studies have not shown consistent clinical improvement. However, many human studies are limited by factors including low patient numbers, poor adherence, lack of randomisation and the absence of a control group. These factors are controlled in preclinical studies utilising animal models, which may be the reason that preclinical success was not replicated in the human trials.A common example of dietary cancer therapy is the ketogenic diet, providing a fat-rich, low carbohydrate diet. The rationale is to reduce circulating glucose levels and induce ketosis. This promotes energy starvation in malignant cells that are unable to utilise ketone bodies, whilst non-malignant cells adapt Klement . The ketIncreasing evidence has highlighted that BCAAs, leucine, isoleucine and valine, are required for malignant growth and act as a source of energy in many cancers (Ananieva and Wilkinson It is apparent that BCAA metabolism in cancer is complex. The long-term dietary restriction of all 3 BCAAs is required for maple syrup urine disease treatment and has demonstrated a successful clinical outcome for an otherwise fatal disease (Blackburn et al. BCAT gene demonstrating reduced proliferation of cancer cells both in vitro and in vivo (Hattori et al. isocitrate dehydrogenase 1 (Tönjes et al. A number of pharmacological inhibitors have been extensively researched to inhibit the function of key enzymes involved in AA metabolism, thus disrupting cancer progression. The BCAT enzyme has been identified in having a central role in the pathogenesis of a number of cancers (Hattori et al. Not all studies have confirmed that the anti-proliferative effects of gabapentin on tumour cells are dependent of BCAT1 inhibition. Grankvist et al.  demonstrIn the 1950s, a dependence on an abundant supply of exogenous glutamine was noted in mammalian cell lines Eagle . GlutamiThere are a number of approaches to target glutamine metabolism including blocking cellular uptake of glutamine, depletion of glutamine in the blood and inhibition of enzymes involved in glutamine synthesis and catabolism, all of which provide potential avenues for cancer therapy (Lukey et al. Phase 1 clinical trials have demonstrated the success of telaglenastat as a well-tolerated monotherapy in patients with multiple myeloma and lymphoma through the successful inhibition of glutaminase in tumours (Vogl et al. The extensive mutational heterogeneity of cancer highlights a growing need for personalised medicine. Therapies currently exist to target the many pathways associated with tumour progression. Yet many patients suffer due to the toxicity of these treatments, in addition to cancer cell adaptations which results in chemoresistance. Research needs to identify novel therapeutic avenues to tackle the resistant forms of these cancers or provide a less aggressive alternative to current therapies in patients not deemed fit to treat. Amino acids are required for a number of vital processes and many malignant cells exhibit genetic abnormalities that compromise their ability to obtain or utilise an adequate supply of the AAs required for tumorigenesis. Nutrient restriction, enzymatic depletion and enzymatic inhibition of AA metabolism have all demonstrated success in preclinical and clinical models, supporting these therapies as viable strategies for cancer treatment Table . These a


We argue that the reconstruction of prehistoric networks offers a timely opportunity to test the interplay between social connectivity and culture and ultimately helps to disentangle evolutionary mechanisms in the archaeological record.Culture is increasingly being framed as a driver of human phenotypes and behaviour. Yet very little is known about variations in the patterns of past social interactions between humans in cultural evolution. The archaeological record, combined with modern evolutionary and analytical approaches, provides a unique opportunity to investigate broad-scale patterns of cultural change. Prompted by evidence that a population's social connectivity influences cultural variability, in this article, we revisit traditional approaches used to infer cultural evolutionary processes from the archaeological data. We then propose that frameworks considering multi-scalar interactions  over time and space have the potential to advance knowledge in cultural evolutionary theory. We describe how social network analysis can be applied to analyse diachronic structural changes and test cultural transmission hypotheses using the archaeological record (here specifically from the Marine Isotope Stage 3 This article is part of a discussion meeting issue ‘The emergence of collective knowledge and cumulative culture in animals, humans and machines’. The backbone of this argument is that the selection of inheritable social phenotypes would not have been possible without variations in individuals' social strategies. It is the transmission of social/cultural characters, in which individuals copy or learn from others, that produces evolutionary changes . BecauseMultiple evidence has shown that individual social decisions (i.e. with whom and how frequently to interact) affect population-level outcomes, such as cultural transmission –6. This ca 11.7 ka onwards), its application may also be feasible to older periods . We believe this approach provides new avenues to study cultural evolution in human prehistory.In this manuscript, we revisit the main approaches used to infer evolutionary processes from archaeological data. This allows us to propose state-of-the-art methodologies to estimate past human interaction patterns. Specifically, we argue that we can gain important insights into cultural transmission mechanisms by implementing modelling approaches to formally analyse prehistoric hunter–gatherer socio-spatial networks . The network analysis methodology considers distinct levels of interactions . The latter enables integrating empirical approaches and structural comparisons, thus providing a deeper understanding of cultural transmission processes. Because our methodological approach relies on the reconstruction of socio-spatial networks, its application is restricted to periods of human prehistory in which the empirical evidence  holds sufficient resolution to trace diachronic changes in both cultural behaviour and social connectivity. While the examples discussed throughout this manuscript come from the Late Glacial and Holocene hunter–gatherers (Marine Isotope Stage (MIS) 1, . 2Prehistoric hunter–gatherers were organized into a fluid multi-level social structure . This me−2 if the mobile people depended upon terrestrial animals or if they exploited aquatic resources, respectively. Another example comes from a study comparing the structure of 1189 social groups in 339 extant hunter–gatherers: they showed an organization in hierarchical, self-similar networks of predictable group size ). 5When studying cultural evolution, it is important to remember that the stone age archaeological record suggests that some technological innovations appeared as punctuations after long static periods, with abrupt cultural changes that varied across time and space. The process was multifaceted and depended, among others, on environmental fluctuations, social factors (such as demography and migratory events) and learning opportunities –73. In tThis explicit network-based approach can be applied to any macrophenomenon in which patterns of cultural change are linked to demographic variation. Classical examples include the emergence of innovations in small populations  and the . 6We generally expect cultural transmission to be dependent on social structure, but the degree to which cultural specialization, loss and/or diversification are a consequence of prehistoric social networks is still poorly understood. Considering the macroscale of prehistoric archaeological proxies, modes of cultural transmission are mostly restricted to horizontal and oblique ones. Vertical transmission would happen within families, but such a resolution is rarely detectable in the archaeological record. In a simplified scheme of cultural transmission modes, we could assume that horizontal and oblique transmissions expose individuals to more social learning opportunities than vertical transmission does. This would have consequences for cultural changes in the short term (a few generations) and long term . If culture is hypothetically dependent only on vertical transmission, cultural changes should be slow, because individuals have access to a limited cultural pool. The contrary would be expected of individuals who learn from non-related conspecifics . Therefoneed to investigate whether: (iv) cultural loss happens in small and disconnected networks; and (v) cultural diversity is higher in mildly connected networks—as structural property seems to maximize both information transmission [We may expect to observe in the temporal snapshots of the prehistoric data: (i) larger social networks during periods of relatively high population density; (ii) stronger links within regional groups; and (iii), changes in density and length covered by links according to resource distribution and availability . These smission  and cultsmission ,76. Thessmission . These psmission ). Here, Therefore, in order to reconstruct prehistoric networks, it is necessary to consider the levels of social interactions  and the archaeological proxy. For example, it has long been considered that personal ornaments (such as shells and fossil molluscs) could be traced to recreate networks that were socially motivated , and tha. 7Our understanding of human cultural evolution would certainly benefit from the reconstruction of prehistoric socio-spatial networks. An increasingly large number of studies analyse the archaeological record from an evolutionary perspective, yet the effect of social connectivity on cultural transmission is still largely unknown. We highlighted that the temporal and spatial scales of social interactions are relevant to evaluate the multi-scalar nature of human–environmental interactions and cultural changes. SNA could help to decipher human cultural responses to changing environments. An obvious concern is whether the archaeological record is robust enough to formally reconstruct and analyse prehistoric hunter–gatherer social networks. Among the reasons to believe that the application of network analysis is timely, is the access to higher-quality archaeological data and state-of-the-art methodologies. The latter enables us to assess the reliability of these networks. By applying SNA, researchers are able to uncover relevant patterns of social interactions, and consequently obtain a better picture of the selective mechanisms underlying human cultural evolution.