The aims of this study were to assess cognitions relating to hypoglycaemia in adults with type 1 diabetes and impaired awareness of hypoglycaemia before and after the multimodal HypoCOMPaSS intervention, and to determine cognitive predictors of incomplete response (one or more severe hypoglycaemic episodes over 24 months).This analysis included 91 adults with type 1 diabetes and impaired awareness of hypoglycaemia who completed the Attitudes to Awareness of Hypoglycaemia (A2A) questionnaire before, 24 weeks and 24 months after the intervention, which comprised a short psycho-educational programme with optimisation of insulin therapy and glucose monitoring.p=0.001). The attitudinal barrier ‘hyperglycaemia avoidance prioritised’  decreased from baseline to 24 weeks, and this decrease was maintained at 24 months (mean±SD=5.3±0.3 vs 4.3±0.3 vs 4.0±0.3). The decrease in ‘asymptomatic hypoglycaemia normalised’ from baseline  was significant at 24 weeks (1.5±0.3 vs 0.8±0.2). Predictors of incomplete hypoglycaemia response (one or more further episodes of severe hypoglycaemia) were higher baseline rates of severe hypoglycaemia, higher baseline scores for ‘asymptomatic hypoglycaemia normalised’, reduced change in ‘asymptomatic hypoglycaemia normalised’ scores at 24 weeks, and lower baseline ‘hypoglycaemia concern minimised’ scores .The age and diabetes duration of the participants were 48±12 and 29±12 years, respectively (mean±SD). At baseline, 91% reported one or more severe hypoglycaemic episodes over the preceding 12 months; this decreased to <20% at 24 weeks and after 24 months (Participation in the HypoCOMPaSS RCT was associated with improvements in hypoglycaemia-associated cognitions, with ‘hyperglycaemia avoidance prioritised’ most prevalent. Incomplete prevention of subsequent severe hypoglycaemia episodes was associated with persistence of the cognition ‘asymptomatic hypoglycaemia normalised’. Understanding and addressing cognitive barriers to hypoglycaemia avoidance is important in individuals prone to severe hypoglycaemia episodes.www.isrctn.org: ISRCTN52164803 and https://eudract.ema.europa.eu: EudraCT2009-015396-27.The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-022-05847-7. Hypoglycaemia remains a common side effect of the insulin treatment required by people with type 1 diabetes , and has1c [Structured education in flexible insulin therapy and use of diabetes technologies (insulin pumps and glucose sensors) may reduce severe hypoglycaemia episodes, and education may improve IAH without increasing HbA1c , 19–24. 1c , 26. How1c . Emergin1c , 24. Qua1c , 28. Add1c . Attitud1c . The que1c  in a sec1c .1c of 5 mmol/mol (0.5%) [In the HypoCOMPaSS RCT, participants with type 1 diabetes and IAH were allocated to insulin pump therapy  or multiple daily insulin injections (MDI) for insulin delivery. All participants optimised glucose monitoring using finger-prick self-monitoring of blood glucose (SMBG) alone or with added real-time continuous glucose monitoring (RT-CGM) . Prior tl (0.5%) , 33.The aims of the present study were to: (1) assess the three A2A attitudinal barriers before and at 24 weeks and 24 months after HypoCOMPaSS RCT participation; and (2) investigate whether A2A attitudinal barriers at baseline, and/or changes from baseline to 24 weeks, were associated with an incomplete response to the intervention, defined as reporting at least one further severe hypoglycaemic episode in the 24-month follow-up .Details have been published describing the HypoCOMPaSS study design , 24-weekNow; No delay’ (never delay hypoglycaemia treatment); ‘Establish your Extra risks’ (and times when risk is highest); ‘Scan for Subtle Symptoms’ (of hypoglycaemia); be Wary even While asleep (through watchful detection and active prevention of hypoglycaemia while asleep). Participants completed the A2A questionnaire at baseline, 24 weeks and 24 months [During the initial 24-week RCT, everyone received equivalent support/attention from healthcare professionals and attention to therapeutic targets, regardless of the randomised intervention, including four-weekly visits and review of the ‘my hypo compass’ principles, returning to usual care at 24 weeks. The ‘my hypo compass’ programme is a two-hour standardised psycho-educational programme targeting hypoglycaemia avoidance around the four compass points NESW: ‘4 months .n=91). Those who also had complete A2A data at 24 weeks and 24 months (n=54) were included in the analysis of the impact of HypoCOMPaSS on A2A attitudinal barriers over time. To study the relationships between the three A2A attitudinal barriers at baseline or their change over 24 weeks and incomplete response to the intervention (defined by at least one severe hypoglycaemia episode over the 24 months’ follow-up in participants reporting one or more severe hypoglycaemia episode over the 12 months prior to randomisation), 60 participants were included . Comparisons of ‘incomplete’ responses vs ‘complete’ responses in all participants were planned before study commencement, with formalisation of definitions preceding any data analysis [For the present study, the baseline analysis included all participants with complete A2A data prior to randomisation  and the Medicines and Healthcare Products Regulatory Agency (17136/0246/001-0001). The trial was registered (ISRCTN52164803 Eudract number 2009-015396-27). Oversight was provided by an independently chaired Trial Steering Committee and a Data Monitoring and Ethics Committee. Participants gave written informed consent.Episodes of severe hypoglycaemia over the 12 months prior to randomisation were reported, and recorded within the groupings 0; 1; 2; 3 or 4; 5–10; 11–15; 16–20; 21–30; 31–50; >50. Following intervention, episodes were recorded prospectively by participants, and the data were collected by researchers every 24 weeks and expressed within the same frequency groupings as at baseline. To report, annualised rates of severe hypoglycaemia episodes (episodes/person-year) at each time point, means for each grouping were calculated , and the total number during the 24-week period was multiplied by two for each participant.IAH was assessed using the Gold score . This asPart 1 of the 19-item A2A questionnaire  asks parAt the end of the study, participants were classified into three groups: (1) complete responders ; (2) incomplete responders ; and (3) indeterminate .n) and frequencies (%). The χ2 test  was used to compare categorical variables, and unpaired Student’s t tests were used to compare continuous variables between the 54 participants with complete A2A data at 24 weeks and 24 months and those with missing A2A data at 24 weeks and/or 24 months (n=37). Repeated-measures ANOVA was used to examine the impact of the HypoCOMPaSS intervention on each of the three attitudinal barriers (n=54) and the rate of severe hypoglycaemia (n=55) (within the 83 people with one or more severe hypoglycaemic episodes at baseline or within the 64 complete or incomplete responders over the 24-month follow-up period), with a post hoc analysis using Sidak’s correction where indicated. Unpaired t tests were used to compare the three A2A factor scores at baseline (n=64) and the change in scores from baseline to 24 weeks (n=62) between complete and incomplete responders. Effect sizes were measured using Cohen’s d for data compared by Student’s t test, using 0.2, 0.5 and 0.8 to indicate small, medium and large effects [2p) for ANOVA data, using 0.01, 0.06 and 0.14 for small, medium and large effects [Analyses were performed using IBM SPSS 24 for Windows. Descriptive statistics are presented as mean±SD or absolute numbers  had complete data for part 1 of the A2A questionnaire and 91 (95%) had complete data for part 2 of the A2A questionnaire at baseline. Table 2p=0.342, p<0.001 for all pairwise comparisons; Table p<0.001). Of the 64 participants for whom data were available, 40 (62.5%) had a complete response over 24 months, with the remaining 24 (37.5%) being incomplete responders. Participants with complete vs missing A2A data at 24 weeks and 24 months  and response to the intervention over the 2-year follow-up . The proportion of participants with missing A2A data at 24 weeks and/or 24 months was higher among incomplete responders (50% vs 15%).Following the study intervention, the severe hypoglycaemia rate was reduced, with the annualised rate remaining significantly lower than baseline at all follow-ups , with 74% responding ‘a lot’ or ‘extremely’ to the question; likewise motivation to regain awareness was high (3.2±0.8), with 83% responding ‘a lot’ or ‘extremely’, while belief in their ability to regain awareness was lower (1.8±1.0), with 71% responding ‘somewhat’ or ‘slightly’. In part 2 of the questionnaire, the highest concordance rates were for item 8 (‘good’ diabetes management consists mostly in avoiding hyperglycaemia) and item 16 (having hyperglycaemia necessarily causes high levels of frustration and/or anxiety), with 30% and 42%, respectively, responding ‘very true’  in ‘hyperglycaemia avoidance prioritised’ at 24 weeks (p=0.001) and 24 months (p=0.004). There was also a significant reduction (η2p=0.113) in ‘asymptomatic hypoglycaemia normalised’ at 24 weeks (p=0.039), which appeared sustained but was no longer statistically significant at 24 months . There was no significant change in ‘hypoglycaemia concern minimised’ .Table Participants with a complete or incomplete response to the HypoCOMPaSS intervention did not differ in baseline values for any attitudinal barrier or the change in those values from baseline to 24 weeks (ESM Table p>0.05), with Nagelkerke R2 values of up to 0.445. Higher baseline rates of severe hypoglycaemia episodes predicted an incomplete response to the intervention . Higher baseline values for ‘asymptomatic hypoglycaemia normalised’ (p=0.028) and reduced change at 24 weeks (p=0.040) also predicted an incomplete response (model B). Conversely, lower baseline values for ‘hypoglycaemia concern minimised’ predicted an incomplete response . Although participants at Cambridge had a higher probability of an incomplete response compared with the reference site (Sheffield) , the overall effect of clinical site was not significant .Table This study measured cognitions relating to hypoglycaemia over a 24-month period in adults with type 1 diabetes and IAH, examining the potential of the HypoCOMPaSS intervention (brief psycho-education and optimisation of insulin treatment with glucose monitoring to reduce hypoglycaemia) to modify attitudinal barriers to hypoglycaemia avoidance, and investigate whether such cognitions, and/or changes in them from baseline to 24 weeks, predict incomplete response to the programme, defined by at least one severe hypoglycaemia episode over the 24-month follow-up period. The predominant attitudinal barrier to hypoglycaemia avoidance in this cohort was ‘hyperglycaemia avoidance prioritised’. This cognition was reduced significantly 24 weeks after the multimodal HypoCOMPaSS intervention, with the change sustained over 24 months. The belief that asymptomatic hypoglycaemia is ‘normal’ was also reduced, being significantly lower after 24 weeks. Stronger endorsement of this cognition at baseline and a reduced change after 24 weeks were associated with an incomplete response during follow-up, as was a lower baseline score for ‘hypoglycaemia concern minimised’.Participants were recruited to the HypoCOMPaSS RCT due to their well-established problematic hypoglycaemia. At baseline, 74% of participants reported high levels of concern about this, and 83% were highly motivated to regain hypoglycaemia awareness. This contrasts with the qualitative study from which the A2A questionnaire was developed, in which only 24% of people with well-established problematic hypoglycaemia expressed a high level of concern . This isEndorsement of the barrier ‘hyperglycaemia avoidance prioritised’ fell significantly during the study. A recently published RCT of psycho-educational interventions successfully targeting otherwise treatment-resistant problematic hypoglycaemia in type 1 diabetes also reported changes in cognitions relating to hypoglycaemia , 38. The1c at 24 months, with improved hypoglycaemia awareness and reduction of severe hypoglycaemia episodes [The reduced over-prioritisation of hyperglycaemia after the HypoCOMPaSS intervention detected by the A2A questionnaire was mirrored by significantly reduced ‘worry’ regarding high glucose levels, attenuated ‘low blood glucose’ preference, less ‘avoidance of glucose extremes’, and a lower drive to take ‘immediate action’ for high glucose in participants , as repoepisodes .The ‘unacceptability’ of any glucose reading <4 mmol/l was emphasised within the ‘my hypo compass’ intervention, which focused on individualised reappraisal of existing knowledge and changing behaviours in light of this reappraisal. Being watchful (W) and acting now (N) without delay were key objectives. In addition, protocol-driven insulin dose reductions were negotiated with participants to address glucose levels under 4 mmol/l. Although the score for ‘asymptomatic hypoglycaemia normalised’ was lower at 24 weeks, the absence of a significant difference from baseline at 24 months suggests that ongoing review of this attitudinal barrier is required in those at continued high risk, with consideration of the need for ‘top-up’ psycho-educational input. The association between further severe hypoglycaemia post-intervention and a higher endorsement of the barrier ‘asymptomatic hypoglycaemia normalised’ at baseline and in participants with the least change in this cognition 24 weeks after intervention provides further support for the need for ongoing monitoring using the A2A questionnaire. The lack of association between continued experience of severe hypoglycaemia episodes and the change in scores for ‘hyperglycaemia avoidance prioritised’ is compatible with the hypothesis that it is lack of change in the less prevalent lower-scoring factor ‘asymptomatic hypoglycaemia normalised’ that is associated with continued experience of severe hypoglycaemia episodes in our cohort. People in this minority group may benefit from more intensive cognitive and psychological support.‘Hypoglycaemia concern minimised’ was the predominant attitudinal barrier to hypoglycaemia avoidance in only 6% of participants. This was not affected by the HypoCOMPaSS intervention. The association between a lower tendency to minimise these concerns at baseline and ongoing severe hypoglycaemia episodes during the follow-up period is consistent with our previous findings that those continuing to experience severe hypoglycaemia episodes had the highest fear of hypoglycaemia at baseline and at study completion (24 months) . This suThis study has limitations. As the RCT was designed to compare insulin delivery and glucose self-monitoring modalities, with all participants receiving the psycho-educational intervention and intensified support from healthcare professionals, the impact of ‘my hypo compass’ as a stand-alone intervention has not been determined. Causality cannot be attributed to the described associations, given the lack of a control arm without provision of the ‘my hypo compass’ intervention. However, the highly comparable biomedical and person-reported outcomes between the intervention arms, and the a priori plan to evaluate pre/post outcomes in the whole cohort, alongside an incomplete vs complete responder analysis in all participants, provide at least partial justification for our conclusions. The finding that another psycho-educational intervention targeting cognitions has recently been shown to change A2A scores , 40, whiThe current study also has strengths. It specifically recruited adults with type 1 diabetes and IAH. It provided equivalent psycho-education, support/attention from healthcare professionals and therapeutic targets for all participants regardless of randomised technological intervention, with detailed follow-up 18 months after return to standard clinical care. The ‘my hypo compass’ intervention can be delivered by a single trained facilitator in a one-to-one session or a 2-hour group session, with one telephone follow-up/consolidation session 4 weeks later. The multimodal HypoCOMPaSS RCT has demonstrated the feasibility and utility of implementing this in parallel with medical optimisation of conventional self-management supported by diabetes technology. The current study adds to the growing interest in providing tools that help not only to assess hypoglycaemia risk in an individual or group  but alsoIn conclusion, this study provides further evidence for the importance of cognitions in hypoglycaemia risk. It shows that ‘hyperglycaemia avoidance prioritised’ is a predominant attitudinal barrier to avoiding hypoglycaemia among some adults with type 1 diabetes, established IAH and severe hypoglycaemia episodes, and that it is possible to address this by a practical intervention. It also suggests that resistance to change of other attitudinal barriers may underpin the failure of such interventions to achieve complete success (i.e. no further severe hypoglycaemia episodes). Formal assessment of the cognitive barriers limiting successful hypoglycaemia avoidance using the A2A questionnaire may enable a deeper understanding of individualised needs, drivers and concerns in those with IAH . This stESM(PDF 181 kb)


Peroxisomes are organelles that play essential roles in many metabolic processes, but also play roles in innate immunity, signal transduction, aging and cancer. One of the main functions of peroxisomes is the processing of very-long chain fatty acids into metabolites that can be directed to the mitochondria. One key family of enzymes in this process are the peroxisomal acyl-CoA oxidases , the expression of which has been shown to be dysregulated in some cancers. Very little is however known about the expression of this family of oxidases in non-small cell lung cancer (NSCLC). ACOX2 has however been suggested to be elevated at the mRNA level in over 10% of NSCLC, and in the present study using both standard and bioinformatics approaches we show that expression of ACOX2 is significantly altered in NSCLC. ACOX2 mRNA expression is linked to a number of mutated genes, and associations between ACOX2 expression and tumour mutational burden and immune cell infiltration were explored. Links between ACOX2 expression and candidate therapies for oncogenic driver mutations such as KRAS were also identified. Furthermore, levels of acyl-CoA oxidases and other associated peroxisomal genes were explored to identify further links between the peroxisomal pathway and NSCLC. The results of this biomarker driven study suggest that ACOX2 may have potential clinical utility in the diagnosis, prognosis and stratification of patients into various therapeutically targetable options.The online version contains supplementary material available at 10.1186/s12890-022-02115-7. Lung cancer remains the most common diagnosed malignancy in the world and the most recent Globocan analysis indicates that there will be an estimated 19.3 million new cancer cases worldwide, of which 11.4% will be lung cancer and at 18% the leading cause of cancer death (with an estimated 1.8 million deaths)  and the Since their discovery in 1954 , peroxisEvidence linking peroxisomes to the biology of NSCLC would stem strongly from the earlier studies demonstrating the roles of peroxisome proliferator-activated receptors (PPARs) in lung cancer tumorigenesis . MoreoveOne of the main functions of peroxisomes is the processing of very-long chain fatty acids (VLCFAs) via either α- or β- oxidation into metabolites that can be directed to the mitochondria . PeroxisAs such these diverse roles indicate that peroxisomes by their nature play important roles in a large number of globally important human disease including obesity, cancer and age-related disorders . In the This pathway has not been studied in such detail in NSCLC, although evidence is beginning to emerge linking the β-oxidation of peroxisomal fatty acids to NSCLC. In the analysis of the peroxisomal pathway in NSCLC by Zhang et al. , 38 diffAs ACOX2 was suggested to be elevated at the mRNA level in over 10% of NSCLC , we undeTwenty-two surgically resected chemotherapy naïve fresh-frozen tumour specimens were used in this study, taken from surgical resections of at St. James’s Hospital, Dublin during the period 2011–2016. Immediately following resection all samples were evaluated by a pathologist and tumour tissue along with matched normal tissue were dissected for downstream analysis. In total, 11 adenocarcinomas and 11 squamous cell carcinomas were utilized in this study and a summary of the histopathological and clinicopathological data is presented in Table A total of 204 surgically resected NSCLC tumour specimens from the period 1999–2007 were included in this study. All surgically resected tumour specimens and control specimens were fixed with 10% formalin and embedded in paraffin (FFPE). The Union for International Cancer Control Tumour-Node-Metastasis (TNM) Classification of Malignant Tumours 8th edition was used to stage the tumours , 27 and A Beecher Manual Tissue Arrayer (Model MTA-1) was used to generate of a tissue microarray (TMA) containing quadruplicate cores (0.6 mm) of the FFPE embedded samples and a 4 µm sections were subsequently used for immunohistochemistry (IHC) of ACOX2.IHC was performed on TMA sections by utilizing a standard protocol to deparaffinise, rehydrated and wash the slides. Subsequently, ULTRA cell conditioning (ULTRA CC1), pH9.1, was used for heat induced epitope retrieval (HIER). For ACOX2 antibody staining, slides were incubated with rabbit polyclonal primary antibody HPA038280  diluted in ventana antibody diluent (Ref: 251–018) (1:50) for 32 min at ambient temperature and stained using the OptiView™ DAB IHC detection kit on a Ventana BenchMark XT processor.Following IHC, staining was independently assessed by two pathologists blinded to the clinical, pathological and follow-up data. Staining intensity was designated as either 0, 1 + , 2 + or 3 + and each tumour section was given a H-score between 0 and 300 = 3(% at 3 +) + 2(% at 2 +) + 1(% at 1 +). No samples were observed with a H-Score of 300.Tumors with high ACOX2 expression were designated as those with an average H score above the median value and low expression below the median. Kaplan–Meier analyses were constructed using Prism 5.01 (GraphPad).Total RNA was isolated and converted to cDNA using our previously described methodology –32. BrieqPCRs for ACOX2 (relative quantification method) were subsequently conducted on these samples using an Illumina Eco qPCR and 2 × SYBR Green qPCR Master Mix  using the manufacturers protocol in a 2-step qPCR program with histone H3 as the internal control using the following primers:ACOX2 FWD: 5’– CCAAGTGGACATGGCAAGAA – 3’ACOX2 REV: 5’ – GTGACTTCTGAGCCCACTGGA – 3’Histone H3 FWD: 5’ – GGTAAAGCACCCAGGAAGCA – 3’.Histone H3 REV: 5’ – CCTCCAGTAGAGGGCGCAC– 3’.The following cycling parameters were used:An initial Polymerase activation of 95 °C for 2 min followed by 35 cycles of 95 °C 15 s and annealing/amplification 61 °C for 1 min. A melting curve analysis was conducted at the end of each PCR using 95 °C 15 s, 55 °C 15 s and a final 95 °C for 15 s. The final concentration of primer in each reaction was 200 nM, and the.Data was analysed using either the default in-built Eco software or imported into EcoStudy (Illumina) for analysis using the ΔΔCq method .To determine if acyl-CoA oxidases show significantly altered expression in NSCLC a systematic analysis was carried out using Lung Cancer Explorer (LCE)  to conduThe STRING database  was inteValidations of altered ACOX2 expression were examined in the TCGA NSCLC cohorts using LCE. Meta—analyses of additional gene expression datasets were also conducted using LCE . Tumour—Altered protein expression in the TCGA-LUAD was validated using UALCAN  to interTo examine for prognostic value, five separate analyses were conducted using either KM-Plot (Microarray and RNA-Seq) , LCE 3434, UALCAAll analyses use the Cox proportional hazards model.To examine whether the altered expressions of acyl-CoA oxidases (ACOX1-3) were correlated with CNVs, the datasets for TCGA-LUAD and TCGA-LUSC were analysed through cBioPortal , 42.To identify mutated genes from the associated TCGA datasets that result in changes of expression of USO1 in NSCLC an analysis was conducted using the MuTarget platform with ACOX2 set as the target gene, and with mutation prevalence set at 2% . All tarCorrelations between DNA methylation and altered ACOX2 expression were examined initially using UALCAN , and subTo interrogate how altered expression of ACOX2 expression was associated with tumour infiltrating immune cells (TIICs) in NCSLC we used TIMER  or TIMERThe relative expressions of ACOX2 in NSCLC cell lines and associated pharmacological profiles for 24 anti-cancer drugs were downloaded from the Cancer Cell Line Encyclopaedia (CCLE) database . The halA further analysis for drugs potentially capable of targeting NSCLC based on ACOX2 expression was conducted using the DepMap PRISM repurposing Primary Screen .All data are expressed as mean ± SEM unless stated otherwise. Statistical analysis was performed with Prism 5.01  using either paired two-tailed Student’s t-test or the Mann–Whitney two-tailed t-test. Correlations were conducted using the Spearman non-parametric correlation coefficient. Differences were considered significant when p < 0.05. All other analyses through web based servers used the default settings in each instance.–4) and LUSC  respectively. The other acyl-CoA oxidases were found to have significantly decreased expression of ACOX1 mRNA and ACOX3 mRNA in the LUSC samples only  and the results show that ACOX2 protein levels are significantly reduced in tumour samples compared to normal . The resulting network of first neighbours is presented in Additional file -3. The rAn initial analysis of the prognostic value of ACOX2 was conducted using KM-Plot . OverallUsing LCE, we conducted a systematic analysis of ACOX2 mRNA for OS benefit. Overall, ACOX2 mRNA was found to have OS benefit in LUAD (p = 0.00049) but not in LUSC  to determine whether mutations within these key genes were correlated with altered ACOX2 expression levels and the results are presented in Table  to deterFinally, using muTarget , we thenAs ACOX2 is significantly downregulated in both LUAD and LUSC using UALCAN we next examined whether methylation may be an important element in the downregulation of this gene. While significant methylation is presented for both LUAD Fig. A and LUSTo assess the potential impact of decreased ACOX2 expression on tumour immunity, an analysis of tumour-infiltrating immune cells in NSCLC was conducted using TIMER . FollowiWe re-assessed the effects of ACOX2 on immune infiltrates using TIMER2 , which pFinally, the ESTIMATE package  was used−07).To investigate the relationship between ACOX2 we used TIMER2.0 to assess correlations between its expression and the expression of important markers of T cell exhaustion , 56. The–21). The signature for genes associated with MMR showed no correlations in LUAD, but again had a very significant negative correlation in LUSC (3.2 × 10–19).Tumour mutational burden (TMB) is widely considered to be a biomarker for predicting potential patient response to immune checkpoint inhibitor therapy , 58. UsiWhilst this represents a proxy for assessing the effect of ACOX2 expression on TMB, we subsequently reassessed the TCGA datasets for whether ACOX2 expression is associated with TMB using functions in UCSCXenaShiny  and alsoTaken together, these results suggest that TMB may be associated with ACOX2 expression particularly in LUAD.To examine any associations between ACOX2 expression and drug sensitivity, we examined the effects of ACOX2 expression against the pharmacological profiles for 24 anticancer drugs from the CCLE database . Using pMetabolic pathway reprogramming is now considered to be a hallmark of cancer , and is Loss of ACOX2 expression due to mutation has been shown to have a functional role in cardiac cancer , with asEpigenetics plays a role in the dysregulation of many genes in cancer . One of One emerging area in cancer where the peroxisomal pathway plays important roles is in the regulation of cellular immune responses . Indeed,It has also been suggested that peroxisomes play a role in the Warburg effect , wherebyIt has been suggested that it may be possible to target peroxisomes by inducing “pexophagy” or the autophagocytic degradation of peroxisomes . HoweverHowever, since the vast majority of patients with NSCLC display decreased ACOX2 at both the mRNA and protein level Fig. , it may Nevertheless, it may be also possible to potentially restore levels of ACOX2 in patients where decreased ACOX2 is expressed through (PPAR)-α/γ dual activators such as SN158, which was shown to significantly upregulate ACOX2 mRNA , althougOverall, the results presented in this study raise interesting possibilities with respect to the potential use of ACOX2 in the diagnosis/prognosis of patients with NSCLC. Moreover, the results suggest that ACOX2 levels could potentially be useful to stratify patients into subsets that may respond to checkpoint inhibitor therapy. In addition, expression of ACOX2 can also potentially be used to stratify patients that may respond to various drugs including Tanespimycin, Crizotinib and MEK inhibitors. However, further studies will be required to elucidate the exact roles of these genes in the pathogenesis of LUAD and LUSC respectively, and also to further delineate the potential for use of ACOX2 expression to stratify or link patient responses to MEK inhibitors and potential subsets of patients with actionable mutations such as BRAF, KRAS, RASA1 and NF-1.Additional file 1. Analysis of TCGA-LUAD and –LUSC datasets for alterations in acyl-CoA peroxidases at the mRNA and protein level.Additional file 2: Figure S1. Protein-Protein Interaction Networks associated with acyl-CoA oxidases. Functional enrichment analysis of acyl-CoA oxidases was carried out on the STRING database  generating forest plots summarizing hazard ratios for OS in (A) LUAD and (B) LUSC specific datasets.


Studies of color perception have led to mechanistic models of how cone-opponent signals from retinal ganglion cells are integrated to generate color appearance. But it is unknown how this hypothesized integration occurs in the brain. Here we show that cone-opponent signals transmitted from retina to primary visual cortex (V1) are integrated through highly organized circuits within V1 to implement the color opponent interactions required for color appearance. Combining intrinsic signal optical imaging (ISI) and 2-photon calcium imaging (2PCI) at single cell resolution, we demonstrate cone-opponent functional domains (COFDs) that combine L/M cone-opponent and S/L + M cone-opponent signals following the rules predicted from psychophysical studies of color perception. These give rise to an orderly organization of hue preferences of the neurons within the COFDs and the generation of hue “pinwheels”. Thus, spatially organized neural circuits mediate an orderly transition from cone-opponency to color appearance that begins in V1. Psychophysical studies predict that retinal cone-opponent mechanisms interact in specific combinations to generate color perception. Here, the authors show that the predicted interactions are implemented by organized circuits in primary visual cortex. Among these extraspectral colors is “true red”, a color that human observers report as being neither purplish nor orangish2. The percept of true red cannot be generated with monochromatic light, and true red is not a component of the rainbow.Color vision has intrigued scientists for centuries, largely because it is readily apparent that the colors we perceive are an abstraction from physical reality. For example, the most distant wavelengths of visible light, “red” and “blue” are linked together perceptually through a continuum of extraspectral colors that can only be generated by mixing long and short wavelengths7 ; L-OFF center with M-ON surround (L−M + ); M-ON center with L-OFF surround (M + L−); and M-OFF center with L-ON surround (M−L + ). The fifth type of P cell is S-OFF and (L + M)-ON (S−(L + M) + )12. “Blue-ON” bipolar cells have S-ON and (L + M)-OFF responses (S + (L + M)−) but little or no spatial opponency. Neurons in the LGN have the same cone-opponent properties as their RGC inputs and have laminar spatial distributions consistent with the projections of corresponding RGC types8.Accordingly, color vision has played a central role in efforts to understand the neural mechanisms that generate perception. Rigorous quantitative psychophysical studies that preceded methods for measuring neuronal activity or deciphering neural connectivity led to a Three-stage model of color perception7 Fig. . It was 16. It is, therefore, apparent that V1 is the locus for mechanisms that begin to generate color appearance, but comparisons of the distributions of neuronal color preferences in V1 versus higher visual areas indicate that further processing in V2, V4, and inferotemporal cortex is likely to be required to establish the populations of neurons that encode for color perception20.Importantly, the cone-opponent mechanisms that account for the ability to discriminate colors do not account for color appearance and color perception. Instead, color appearance involves further interactions between the cone-opponent mechanisms − and S−(L + M)+ mechanisms Fig. . To reco31, as well as “unique colors”33. Specifically, the appearance of each of the four “unique colors”  requires a particular mixture of the possible combinations between L/M opponent and S + or S- mechanisms, which are L + S+, M + S−, L + S−, and M + S+, respectively. The colors used to illustrate the L-, M-, and S-cones  Stage 2 mechanisms and therefore allow assessment of responses that would require integrated inputs. Assessment of preferred DKL colors with ISI-generated hue-phase maps revealed that interactions at the intersections of COFD pairs form hue “pinwheel” or “linear” structures. Both ISI hue-phase maps and 2PCI of color preferences of individual neurons reveal that the functional architecture and microarchitecture within intersecting COFDs is as expected from relatively linear combinations of cone-opponent signals  methods to map the locations in V1 that are responsive to the ON and OFF phases of cone-isolating stimuli across large swaths of cortical territory. These maps reveal cone-opponent functional domains (COFDs) where neurons are preferentially activated by the ON or OFF phases of cone-isolating stimuli. Results show that L-ON (L+) and M-OFF (M−) responsive regions are always spatially overlapping, as are L-OFF (L−) and M-ON (M+) regions, to generate L + M− and M + L− domains. The L + M− and M + L− domains occur as adjacent non-overlapping pairs. S+ and S− domains are also organized as non-overlapping pairs. Importantly, the axes joining the L + M−/M + L− pairs are often organized roughly orthogonally  cone-opponent mechanisms. To explicitly investigate possible interactions between cone-opponent mechanisms and whether they occur functionally to generate neurons with preferred colors reflecting those interactions we: (1) developed and validated ISI methods to reveal cone-opponent functional domains (COFDs) across large V1 regions  quans Figs. ; (3) usens Figs.  and hue-ns Figs. . Togethens Figs. .25. Here we present data collected from seven adult male and female macaque monkeys (M. fascicularis)  subjected to 2PCI; data presented here include responses to visual stimuli and analyses not described in the previous publication.We conducted ISI and 2PCI to map the functional architecture and measure the visual responses of neurons in superficial layers of V137, therefore, the orientations, spatial frequencies (SFs), and spatial phases were identical between each set. Fluorescence changes were converted to inferred spike-rate time sequences that were then used to calculate spike-triggered averages (STA) of stimuli as linear estimates of receptive fields , and plotted the ON/OFF dominance map in red/blue, based on the locations of neurons. Maps from all imaging regions are presented in the main and supplementary Figures.  at 0.017 Hz, 0.08 Hz or 0.1 Hz  “blobs” and “interblobs” Hz Fig. . SubsequV1 Figs. . . In contrast, comparisons between S-cone and either L- or M-cone maps generate pixels relatively evenly distributed across all four quadrants, as predicted. In particular, for the LS comparison, 42.66% of pixels fall into the opposite-sign quadrants (2nd and 4th quadrants) and 57.34% into the same-sign quadrants . And for the MS comparison, 56.48% of pixels fall into the opposite-sign quadrants and 43.52% into the same-sign quadrants . Similar results are observed for 3 additional animals/imaging regions , each pixel within the COFD mask was assigned three values corresponding to the modulation (including magnitude and sign) by each of the three cone-isolating stimuli at that pixel. Pixel-by-pixel comparisons were then made across each of the three possible comparisons of cone types . Typical results are illustrated in Fig. In addition to interactions between cone-opponent mechanisms within a 2-dimensional isoluminant plane  cone-opponent mechanisms predictable from the COFD maps, as expected from the mixing of cone-opponent inputs? Do the individual neurons within the COFDs prefer colors that would be predicted from the mixing of the cone-opponent signals within the COFDs? Answering these questions is central to assessing whether interactions across the COFDs implement the Stage 3 mechanisms.34. We chose the DKL isoluminant plane because there is a close correspondence between DKL hues and the cone-opponent signals13, allowing straightforward predictions of hue preferences expected from linear integration of cone-opponent inputs. The 12 DKL hues were presented as drifting gratings for 2PCI or temporally modulated full-field stimuli for ISI, and the responses of neurons within the COFDs were imaged. We generated both ISI hue-phase maps , the hue-phase maps generated using DKL stimuli were composed of ON/OFF pairs with strong signals surrounded by regions with weak or no phasic modulation Fig. . We usedaps Fig. . The resres Fig. , but othres Fig. , but werres Fig. .Fig. 6ThWe next assessed whether individual neurons within the COFDs prefer colors that would be predicted from the mixing of the cone-opponent signals within the COFDs. Figure n = 2589 neurons from 5 imaging regions). We then generated a mask for each neuron to identify its corresponding ISI pixels, and the normalized pixel values from L, M, and S-COFD maps (see above) for each neuron were averaged to yield the magnitudes and signs modulated by L-, M-, and S-cone-isolating stimuli. Similar to Fig. P < 0.0001, Fisher’s exact test, two-sided); and neurons with pixels in the M + L− quadrant are significantly more likely to prefer hues around 180°  than around 0° . For the S-cone versus L and M-cone comparisons  includes modulation not only in the isoluminant plane, as we have explored here, but also along the achromatic axisDs Figs. , and theConsistent with 2PCI results indicating the mixing of cone-opponent signals to generate intermediate hue-selectivities of individual neurons within COFDs (above), we also observed gradual shifts in ISI hue-phase maps Fig.  generate25 , or than neurons with significant S-cone OFF STAs . Similarly, neurons with significant S-cone OFF STAs were more likely to prefer the DKL hue at 270° than neurons without significant S-cone STAs , or than neurons with significant S-cone ON STAs .There is also a clear separation of a set of neurons near the middle of the diamond plots with relatively strong S-cone STAs Fig. . Note thThe relatively weaker influence of the S-cone relative to the L/M opponent signals during interactions between the COFDs can be seen not only in the hue tuning of the individual neurons (see above) but also in the tuning of ISI pixels within the different COFDs to the DKL stimuli. To further quantify the relationships between COFDs and DKL hue responses, the COFD masks were used to identify pixels in each COFD ON or OFF region Fig. , as wellTo more directly assess the predictions from the mixing of COFDs to generate color-opponent signals, we also generated DKL hue tuning curves for the pixels at the intersections corresponding to the regions where neurons would be expected to be shifted toward each of the red (L + ∩ S+), yellow (L + ∩ S−), blue (M + ∩ S+), and green (M + ∩ S−) color appearance mechanisms Fig.  to combi27  or for ON or OFF domains of particular cones . Alignment of COFD maps to postmortem CO histology shows that they have similar periodicity and are often overlapping, but there are also clear regions that do not overlap  only for the comparisons based on phase-unassigned COFDs , as maximal statistical significance for four animals is P = 0.0625. The achromatic ON and OFF domains were also significantly biased toward blob regions for all three animals in which achromatic phase maps were generated.Previous studies have suggested that cytochrome-oxidase blobs in layer 2/3 are specialized for the processing of color informationlap Fig. . The bia10, we were particularly interested in whether S + domains might be more strongly biased toward high CO intensity regions than other COFDs. Comparisons between cone types and phases within animals revealed consistent significant differences only for the S + domains, which were significantly more biased toward higher CO intensities than S− for all four animals . S + was also significantly higher than both L + and M− in three of four animals. Other comparisons were significant for only one or two out of the four animals. It is noteworthy that the densest S + LGN terminals and CO staining are found deep within layer 2/3 (layer 3B)52, so there might be a closer correspondence between S + responses and CO deeper in cortex.Because a previous study has shown that S + (L + M)− LGN afferents preferentially terminate in the CO blobs25; neurons preferring green were observed to be at locations of higher CO staining intensity than those preferring red.We also quantified the relationships between CO staining and the preferred directions in DKL color space of neurons imaged with 2PCI Fig. . The hig23. We have generated ISI maps using the same methods applied in those studies  and directly compared those maps to COFDs imaged in the same animals. We find that the color domains and COFDs are closely related  within V1In summary, we have demonstrated that: (1) L-, M-, S-COFDs, and achromatic ON/OFF domains are a prominent feature of the functional organization of primate V1 that can be readily revealed with ISI imaging; (2) 2PCI-assayed neurons within ON/OFF domains of L, M, S and achromatic have the same ON/OFF sign of that domain type; (3) both qualitative and quantitative assessment of the spatial intersections of COFDs shows that they follow the mixing rules predicted from the Three-stage model; (4) DKL hue-phase maps show that the hue preferences at intersections between COFDs correspond to hues predicted from functional mixing and generate hue pinwheel or linear structures; (5) 2PCI of DKL hue preferences at the intersections of COFDs shows that functional mixing occurs at the level of individual neurons; and (6) COFDs are biased toward regions of high CO intensity. We conclude that the neural substrates that underlie the mixing required for a transition from cone-opponent (Stage 2) mechanisms to color-opponent (Stage 3) mechanisms are highly organized and implemented by specific connections between neurons in the geniculate input layers  and the neurons that we have assayed in more superficial V1.13. The “unique colors”  are not necessarily expected to be preferred by larger numbers of neurons than other hues, but instead reflect perceptual transitions within the color-opponent mechanisms, where, for example, “red” appears neither “bluish” or “yellowish”3.  Consistent with these expectations, our observations and analyses indicate that the mixing of L/M and S/(L + M) opponent signals within COFDs gives rise to shifts in preferred hues that are in the directions expected to generate color-opponent mechanisms. But the contribution of the S/(L + M) mechanism (S-cone contributions) is likely much less than expected to fully account for color appearance. Careful calculations based on psychophysical measures assign just a 2.55-fold stronger influence to the L/M mechanism3, while the distribution of color preferences we have measured from neurons in upper layer 2/3 of V1 appear to reflect a much stronger relative influence of the L/M mechanism  that receive direct S/(L + M) signals10 and can project directly to V2 might also have more dominant S-cone influences that could contribute to stronger S-cone influences in higher visual areas.The most prominent feature of Stage 3 in the Three-stage model is the specific mixing between cone-opponent mechanisms that are predicted to give rise to each of the four color-opponent mechanisms Fig. . This miism Fig. . It is l48 and direction maps50. Hue-direction pinwheels were surrounded by regions of iso-hue direction, again reminiscent of iso-orientation and iso-direction domains. Hue-direction pinwheels were often found at the intersections of L/M opponent and S-cone COFDs and, in the most striking cases, aligned with the intersections of orthogonal L + M−/M + L− and S+/S− COFD pairs  and may share common principles with those underlying emergence of orientation and direction maps.We were surprised to find that ISI mapping of the locations preferring DKL hues modulated in different directions (hue-direction maps) revealed “pinwheel” structures Fig.  reminisc28. The COFDs that we observe, as well as their relationships to CO staining, argue against this interpretation and provide an explanation for why different electrode penetrations might sometimes be biased to sample neurons with different color opponency. There may, in fact, be color columns that extend across cortical layers, but they likely correspond to the more closely spaced COFDs with different cone inputs rather than to separate blobs (see further below). Future experiments using ISI imaging of COFDs to guide subsequent electrode penetrations  that have both dendritic and axonal processes that arborize selectively in both layers 4Cβ and 4 A/3B58. These neurons could directly integrate L/M opponent with S+ or S− LGN inputs and provide their mixed signals to neurons in layers 4Cβ and 4 A/3B. Such a mechanism could account for Blue-Yellow opponent neurons that have been reported in layer 4C28. While our observations clearly point to these circuits as the neural substrates that mediate the mixing of cone-opponent mechanisms to generate the rudiments of color opponency, further experiments will be required to more precisely identify the micro-circuitry involved.We have observed a functional organization (COFDs) in the most superficial layers of V1 that involves systematic mixing of L/M opponent with S+ and S− signals. The fact that L/M opponent LGN inputs terminate in a separate layer (layer 4Cβ) from S + (layer 3B) and S− inputs (layer 4 A) indicates that mixing of these cone-opponent systems can only occur through neural substrates that span across these layers and also suggest that there are likely to be some laminar differences in cone responses and color tuning. The most prominent substrate for potential mixing is the axons of layer 4Cβ spiny stellate neurons that extend to and densely arborize in layers 4 A and 3B, likely mixing L/M opponent with S-cone signals23. While the overall organization of color patches was similar to CO blobs with substantial overlap, there was clearly not a one-to-one correspondence. Those observations clarified that when single-unit recordings are related to blobs, blobs are an indirect and imperfect proxy for color functional architecture. Other studies combining single-unit recordings with postmortem CO histology provided the first evidence for a columnar organization for color preference and suggested that separate blobs might process “red-green” (L/M) versus “blue-yellow” (S/L + M) opponency28. But the first ISI maps of V1 locations preferring different colors suggested color transitions on a finer spatial scale than would be expected from separate L/M and S/(L + M) blobs24. This was confirmed when 2PCI was used to definitively identify functional microarchitecture for color preferences at the level of single neurons26. Neurons preferring similar colors are grouped together within a functional color microarchitecture, and neurons preferring different colors are spaced more closely than would be compatible with their separation by blobs.The V1 functional organization and micro-organization related to cone-opponency and color preferences that we have reported here build on a long history of prior studies using varied technologies and sometimes leading to apparently contradictory results. However, in light of the higher resolution and denser sampling afforded by 2PCI technology, sources of the apparent discrepancies are now much clearer. Before the development of ISI, studies used CO blobs as an anatomical landmark for linking the functional properties of sparsely sampled neurons to possible functional architecture. Notably, Livingstone and Hubel first reported a prevalence of neurons in and around CO blobs that were color-selective but not orientation selective, suggesting that color tuning would likely have some sort of functional organization.  than 0° (“pink”). Here we also report that all of the regions within COFDs (generated with full-field stimuli) are significantly biased toward high CO activity (“blobs”) and that S-ON regions are significantly more biased than S-OFF COFDs. Altogether, these results point to an organization in which the blobs are a reliable marker of locations receiving direct S-ON input from the LGN52 and are part of a somewhat larger, overlapping system of COFDs. The COFDs mediate an orderly mixing of the S-ON input to blobs with L + /M− and M + /L− input from layer 4 Cβ and S-OFF input from layer 4 A, that follows the stage 3 mixing rules to generate a broader range of color preferences arranged in hue pinwheels.Using CIE colors, Garg et al.M. fascicularis) under anesthesia in this study. They are referred as A1–A7. Details and allocation of these animals can be found in Supplementary Table All procedures involving live animals were conducted in accordance with the guidelines of the NIH and were approved by the Institutional Animal Care and Use Committee (IACUC) at The Salk Institute for Biological Studies. We recorded from seven adult macaque monkeys , physiological recordings (nonrecovery), and euthanasia in this study are described below.For animals used for 2PCI , two sequential surgeries were performed. First, a recovery surgery was conducted for the injection of AAVs to express the genetically encoded calcium sensor, GCaMP6f (see below). Then after 10–12 days of recovery to allow for GCaMP expression, a second nonrecovery surgery was performed, and an imaging chamber was implanted for ISI and 2PCI. For other animals used in this study, only the nonrecovery surgery was performed.2) and sterile conditions. Animals were intubated (tracheostomy was performed during nonrecovery surgery) and mechanically ventilated. EKG, SpO2, end-tidal CO2, and body temperature were all monitored throughout the procedure and during all recordings. Lactated Ringer’s with 5% dextrose, antibiotics , and dexamethasone  were supplied.Before all surgeries, animals were anesthetized with ketamine (10 mg/kg) and pretreated with atropine sulfate (0.02 mg/kg) intramuscularly. During surgeries, animals were under general anesthesia  was pressure injected via fine glass micropipettes at 1–3 depths in each of 6–12 locations in V1 . Injections were spaced about 1 mm apart to yield nearly continuous labeling. Following the injections, a thin piece of silicone artificial dura was placed between the cortical surface, and the natural dura, and these were glued in place with Vetbond . The bone was replaced and cemented in place. Animals were recovered. Buprenorphine was administered for 3 days . Antibiotics  and dexamethasone  were administered daily to prevent infection and reduce brain swelling.During the first surgery, following a craniotomy over V1, a 1:1 mixture of AAV5-TRE3-GCaMP6f and AAV5-thy1s-tTaIn the nonrecovery surgery, a head-post was implanted to stabilize the animal. A large imaging chamber (20 mm inner diameter) was cemented onto the skull over V1 and V2. A coverslip insert was screwed into the chamber to reduce brain movement. Wires were implanted through the small hole in the skull and connected to an amplifier to monitor EEG. Anesthesia was gradually transitioned from isoflurane to sufentanil citrate , and vecuronium bromide (0.05–0.2 mg/kg/hr i.v.) was administered to paralyze the animal before recording, and EEG was subsequently monitored to continue along with other vital signs to assess anesthetic depth. Anesthesia dosage was adjusted at a level that insured slow-wave EEG activity. Eyes were dilated with 1% atropine sulfate and 0.5% proparacaine hydrochloride ophthalmic solution and fitted with contact lenses of appropriate curvatures to focus on a screen 57 cm from the eyes. First, we used ISI to obtain functional maps  to target the regions of interest  during 2PCI  or extracellular electrophysiology recording . The animals were humanely euthanized by anesthetic overdose followed by perfusion at the end of the procedure (see “Histology and Image Alignment”).Surface blood vessel images were acquired under 540 nm illumination. Images of reflectance change  were acquired under 630 nm illumination. Imaging was performed using a GigE camera  and custom Matlab software.42. . For creating ocular dominance, orientation, and isoluminant color maps, we utilized the first ISI paradigm. Images were acquired during the pre-stimulus and stimulus periods  at 10 Hz. For cone-opponent and DKL hue-phase maps, we utilized the second ISI paradigm, images were acquired at 10 Hz during stimulus presentation, which consists of a 2 sec pre-stimulus period and a 10–20 min stimulus period.In this study, we conducted two ISI paradigms known as standard episodic stimulation combined with blockwise data acquisition and continuous-periodic stimulation combined with continuous data acquisitionMicroscopy for two-photon calcium imaging (2PCI) was performed using a customized setup incorporating a Sutter movable objective microscope  with a resonant scanner  and data acquisition controlled by a customized version of Scanbox . GCaMP6f was excited by a Ti:sapphire laser  at 920 nm. Of three animals used for 2PCI, two (A3 and A4) were single-plane continuously scanned at 15.49 Hz ; one (A1) was double-plane continuously scanned with an electrically tunable lens  at 30.98 Hz . An area of about 1168 × 568 µm (A3 and A4) or 1100 × 725 µm (A1) was imaged with a ×16, 0.8 NA objective lens . The back aperture of the objective was not overfilled due to the technical limitations of the microscope.60. Briefly, signals were sampled at 30 kHz and filtered separately to an action potential band (0.3–10 kHz) and a local field potential band , then streamed to a computer under the control of SpikeGLX (http://billkarsh.github.io/SpikeGLX) software. The probe was held and lowered into the cortex using an oil hydraulic manipulator , which was attached to the imaging chamber in order to ensure perpendicular penetration to the brain surface through a small hole in the coverslip. The locations of the electrode penetration were within cone-opponent functional domains (COFDs) revealed by ISI, and the probe was slowly lowered 2.5–3.1 mm below the brain surface. The brain surface at the location of the penetration was covered with 2% agarose and then with silicone elastomer  to prevent drying. Data were recorded at least 30 min later after the electrode was stable and visual fields were subsequently mapped.Extracellular electrophysiological data were collected using Neuropixels Phase 3 probes with 384 recording channelshttp://psychtoolbox.org/) for MATLAB  on a CRT monitor  with a refresh rate of 100 Hz. For all experiments, the monitor was positioned 57 cm in front of the animal’s eyes. Prior to each experiment, the luminance of each of the red, green, and blue phosphors was linearized. Then a white point and mean luminance were chosen  as the background for spectra measurement of each phosphor with a spectroradiometer . Then we utilized the spectra of each phosphor to calculate gun gains for cone-isolating stimuli  and DKL hue stimuli , and we used the same white point and mean luminance as background, interstimulus interval and blank condition during cone-isolating stimuli, DKL hue stimuli and full contrast achromatic grating stimuli  presentation. Another white point  was chosen as the background and blank condition for physical equal luminance CIE  hue stimuli25 .Visual stimuli were generated with the Psychophysics Toolbox Version 3  were presented binocularly to locate orientation columns and monocularly to obtain an ocular dominance (OD) map. Four CIE hues  were chosen to construct Red/Green and Blue/Yellow isoluminant square-wave drifting gratings  to obtain Red/Green and Blue/Yellow isoluminant color maps. In order to increase the signal-to-noise ratio, each stimulus condition was presented in a randomized order with 20–30 repeats. For each trial, the stimulus was displayed for 4 sec with a 2 sec pre-stimulus period and 8 sec interstimulus interval. Continuous-periodic full-field cone-isolating Fig. , achromaFor 2PCI, only episodic stimuli (drifting and Hartley flashed sine-wave gratings) were used, and all stimuli were presented monocularly. First, the dominant eye was selected based on the ISI ocular dominance map of the imaging area; the non-dominant eye was occluded. All stimuli were given through the dominant eye. Next, the receptive field of the imaging area was mapped by showing a series of drifting gratings, and the stimulus size (1.3–1.5° in diameter) was chosen to be as small as possible while still covering the receptive fields of all neurons within the imaging region.Drifting gratings  were presented for 3–4 sec with a 3 to 4 sec interstimulus interval. Achromatic drifting gratings were presented as sine-wave gratings at 8 directions (0 to 315° in increments of 45°), 5 or 6 spatial frequencies , and a single temporal frequency of 5 cycles/sec. Each condition was repeated five times in random order. DKL and CIE hue drifting gratings were presented as square-wave gratings with 0.5 spatial duty cycle, 8 directions (same as achromatic gratings), 4 spatial frequencies , and a single temporal frequency of 5 cycles/sec. Each condition was repeated 3 times in random order.37 consisted of 120 blank stimuli and 5640 unique gratings with different combinations of orientation, spatial frequency (range: 0.2–6.0 cycles/degree), and spatial phase   set0°) Fig. . The stiFor extracellular electrophysiology recording, a uniform patch was temporally modulated along two axes in the DKL isoluminant plane at 0.33 Hz with 0.5 temporal duty cycle (see “DKL Hues” below). The patch was presented monocularly to measure the ON/OFF responses to verify the ON/OFF phases  of the cone-opponent and DKL hue-phase maps.38, we first calculated a 3 × 3 transformation matrix (M) by taking the dot product of the monitor spectral power distribution (r(λ), g(λ), b(λ). λ was sampled in the range nm every 2 nm) of the 10° cone fundamentals (l(λ), m(λ), s(λ)) published by Stockman and Sharpe61.To calculate cone-isolating directions62 with equal increments from 0 to 330°  was the same background we used for measuring phosphor spectra . The same cone fundamentals used for cone-isolating stimuli were used for DKL hue calculation. Twelve hues were selected from the DKL isoluminant plane30° Fig. . For A1,25; more details of the stimuli can be found in the supplementary materials of that paper. Briefly, twelve hues were selected from the HSL plane  with an equal increment from 0 to 330°. Then the CIE-1931 xyY coordinates  to 10 ± 0.1 cd/m2. Because of this adjustment and the gamut of the CRT monitor, the twelve hues were not in the same plane of the HSL color space, but they had the same luminance (Y). The CIE xy coordinates of each hue remained the same between different experiments. For A3 and A4, we presented the CIE hues. One cycle of the CIE hue spatial square-wave gratings consists of a CIE hue and the background color  with 0.5 duty cycle.The CIE hue stimuli were identical to those described in a previous publicationAnimals were euthanized by a lethal dose of pentobarbital sodium , and transcardial perfusion was performed using 0.9% saline, followed by 4% paraformaldehyde (PFA), 10% sucrose with PFA, and 20% sucrose with PFA, and the brain was extracted. The recording region was then blocked, flattened, and sunk in 30% sucrose and sectioned (50 µm) tangentially using a freezing microtome. Tissue was stained for cytochrome-oxidase (CO), and sections were imaged. Both CO and 2PCI images were aligned manually with the ISI blood vessel map in Adobe Photoshop. Regions from ISI images corresponding to 2PCI images were interpolated in Adobe Photoshop using ‘Bicubic Automatic’ method.R/R) of each pixel for each stimulus condition was first calculated using the following formula:R is the average response of a pixel during the stimulus period, and R0 is the average response of a pixel during the pre-stimulus period. The ∆R/R image of each condition was then filtered using a fourth-order Butterworth bandpass filter . Next, we computed a t-map44, and based on the ∆R/R images of two conditions to reveal the OD columns and orientation columns. The orientation polar map  of the image, and then filtered using the contrast-limited adaptive histogram equalization (CLAHE) method provided by MATLAB. Hemodynamic delay in the phase map was not compensated42; instead, we took advantage of the results from 2PCI or electrophysiology recording to identify the sign of ON/OFF phases in the ISI phase maps , then contours were extracted from filtered images to generate COFD contour maps. Contours corresponding to the highest 15% and lowest 15% of values correspond are used to illustrate ON or OFF regions Figs. , b, 9b.To plot bivariate histograms, COFD masks were generated by selecting contour level values that best corresponded to the domain sizes of each phase map. We manually removed some regions  to make sure the masks are specific to regions of interest. A blood vessel mask and area mask were also added to exclude pixels within blood vessel regions or other areas outside V1 . Next, COFD phase maps and DKL hue-phase maps were filtered with a two-dimensional Gaussian kernel  and normalized to [−1 to 1]. Then pixels on COFD maps were selected based on the combined L-, M-, S-cone COFD masks to plot bivariate histograms to show spatial relationships between different COFD maps Fig. . SimilarGvM) as described by the following equation64:θ is the DKL hue direction in the range of . The mean pixel value of each DKL hue-phase map was calculated based on each L-, M-, and S-cone COFD mask Fig.  or basedTo plot the spatial relationship between CO intensity and COFD maps, blood vessel holes in CO images were first filled using ‘Content-Aware’ method in Adobe Photoshop. Then CO images were filtered with a 2-dimensional Gaussian kernel  and normalized to [0 to 1] with a higher pixel value means higher CO intensity. Pixels within COFD regions  and non-COFD regions were selected (pixels within blood vessels were excluded) to plot the histogram Fig. . If ther65 (https://scanbox.org/). For two-plane bidirectional scanning, raw fluorescence signals were interpolated before spike estimation. Thus, interpolated signals and spikes had 30.98 Hz sampling rate for both planes. Once signals and spikes were extracted, they were analyzed using customized software written in MATLAB and The Julia Language . The change in fluorescence (∆F/F) was computed by the following equation, where F is the average fluorescence during stimulus presentation and P < 0.05 to either two-way ANOVA across all stimulus conditions (including blank stimuli), or two-tailed Welch’s t-test between the strongest response and response to the blank stimulus.Regions of interest (neuron cell bodies) were manually segmented using Adobe Photoshop. Raw fluorescence signals were extracted, and the spike rate was estimated by Scanbox67 to average the responses of eight direction to get one direction vector. Each condition was repeated five times in the experiment, so there were five direction vectors for each neuron. Similarly, five orientation vectors were obtained by averaging two directions of motion at each orientation. Then we projected the five vectors onto their final mean vector, thus the two-dimensional distribution of 5 vectors was transformed to a one-dimensional distribution. Note that each of those five vectors depended on how the conditions combined as one repeat, therefore the condition combination affected the distribution of five vectors. To reduce the distribution bias introduced by low sampling, we randomly shuffled direction or orientation conditions 100 times and calculated the vectors to produce 500 data points demonstrating the distribution of the direction or orientation response magnitude of a neuron. We did the same procedures for the blank condition and calculated the distribution of the blank response magnitude of the same neuron. Then we analyzed the two distributions using ROC analysis68 and calculated the probability of the area under a ROC curve  as a parameter to indicate the significance of direction or orientation selectivity. If AUC > 0.7, we considered the neuron to be direction or orientation selective. The preferred direction or orientation is the angle of the final mean vector from the circular variance calculation  share the same Hartley basis function, which is radially symmetric, therefore, c, which is the same for all Hartley stimulus set. Note that this constantc is canceled when calculating magnitude change and does not affect the normalized cone weights calculation neither (see below). The estimated kernel α) multiples with its unit vector  of the maximal absolute value in its best STA, and used the sign to plot the ON (positive) and OFF (negative) maps Figs. .To calculate the normalized cone weights in Fig. ded Fig. , the S-cded Fig. .71. Therefore, we utilized the LFP gamma band power to identify the ON/OFF phases of the COFD phase map. The LPF band  was bandpass filtered (1–100 Hz), and 60 Hz line noise was removed. Then, LPF power spectrums were estimated by Multi-Taper methods from 0–300 ms following and −300 to 0 ms before stimulus onset, which corresponds to the response  within COFD regions were compared with the median (y) within non-COFD regions to test whether (x-y) has zero median against the alternative hypothesis that the median is not zero using Wilcoxon signed rank test (two-sided). For comparison across animals, we applied Sign test (right-tailed) to see whether the difference between the median of CO intensity within all COFDs and non-COFD is significant across animals. The same Wilcoxon signed rank test (two-sided) was also performed on achromatic ON/OFF regions versus non-COFD regions. For animals in which we identified the ON/OFF sign of ISI phase map, we compared the CO intensity distribution in ON regions versus OFF regions within and between each cone type in each animal. The L-, M-, and S-COFD masks were the same as described above. We performed Wilcoxon rank sum test (two-sided) to check whether two distributions have equal medians.To statically compare the CO intensity distribution within COFD regions versus non-COFD regions, COFD and non-COFD regions were selected using the masks described above. For within animal comparisons, each COFD region was treated as an independent sample, which was represented as the median of the CO intensity within that COFD region. For each animal, the CO intensity median values (n = 20 bins) corresponding to overlaps in the same-sign and in the opposite-sign quadrants (cf. Figure To test the significance of overlaps between different COFDs measured with ISI, normalized distributions of pixel densities from overlaps of COFDs for each combination of two cone types (e.g., Fig. Further information on research design is available in the Supplementary InformationReporting Summary


Primate cerebral cortex is highly convoluted with much of the cortical surface buried in sulcal folds. The origins of cortical folding and its functional relevance have been a major focus of systems and cognitive neuroscience, especially when considering stereotyped patterns of cortical folding that are shared across individuals within a primate species and across multiple species. However, foundational questions regarding organizing principles shared across species remain unanswered. Taking a cross-species comparative approach with a careful consideration of historical observations, we investigate cortical folding relative to primary visual cortex (area V1). We identify two macroanatomical structures—the retrocalcarine and external calcarine sulci—in 24 humans and 6 macaque monkeys. We show that within species, these sulci are identifiable in all individuals, fall on a similar part of the V1 retinotopic map, and thus, serve as anatomical landmarks predictive of functional organization. Yet, across species, the underlying eccentricity representations corresponding to these macroanatomical structures differ strikingly across humans and macaques. Thus, the correspondence between retinotopic representation and cortical folding for an evolutionarily old structure like V1 is species-specific and suggests potential differences in developmental and experiential constraints across primates.The online version contains supplementary material available at 10.1007/s00429-021-02427-0. A major goal in systems and cognitive neuroscience is to understand the evolution of the human cerebral cortex . Common approaches for functional mapping of the human brain such as inflating or flattening a digitally reconstructed cortical surface provide utility by visualizing areas buried within sulci, which comprise the majority of cortex. However, the inflation and flattening process invariably distorts important features of the cortex. For example, while the rCaS is easily identifiable in post-mortem brains Fig.  as well n = 24) and macaques (n = 6). We focused on eccentricity representations as classic electrophysiology studies have related eccentricity representations to the rCaS in macaques  using fMRI in human participants  were randomly selected from the 181 participants comprising the HCP 7 T Retinotopy Dataset  participated in this study. Four monkeys were selected from  and eccentricity (annulus) mapping were conducted in separate experiments each consisting of 8–12 runs with an equal split in the direction of rotation. Each polar angle run consisted of eight cycles lasting 40 s each. Each eccentricity run consisted of seven cycles lasting 40 s each with 10 s of blank, as well as black backgrounds in between. These additional blank periods were inserted to temporally separate responses to the foveal and most peripheral positions. The monkeys were rewarded with juice for maintaining a central fixation within a 2° window. Gaze direction was monitored using an infrared eye tracker . See Arcaro and Livingstone . Macaque functional data were collected in a 3T Siemens TimTrio scanner with an AC88 gradient insert using 4-channel surface coils (custom made by Azma Mareyam at the Martinos Imaging Center). Macaque anatomical data were collected in a 3T Siemens Skyra scanner using a 15-channel transmit/receive knee coil.T1-weighted (T1w) and T2-weighted (T2w) structural scans at 0.7-mm isotropic resolution were acquired at 3T and used as the anatomical substrate for the retinotopy data. See Benson et al.  for fullWhole-brain scans were collected using a T2*-sensitive gradient echo planar pulse sequence at a resolution of 1.6 mm isotropic and 1 s TR . See Benson et al.  for fullA whole-brain structural volume was acquired, while the animals were anesthetized with a combination of Ketamine (4 mg/kg) and Dexdomitor (0.02 mg/kg). Monkeys were scanned using a T1w magnetization-prepared rapid gradient echo (MPRAGE) sequence; 0.5 × 0.5 × 0.5 resolution; FOV = 128 mm; 256 × 256 matrix; TR = 2700 ms; TE = 3.35 ms; TI = 859 ms; flip angle = 9°). 3 whole-brain anatomical images were collected in each animal.Whole-brain scans were collected using a T2*-sensitive gradient echo planar pulse sequence at a resolution of 1 mm isotropic and 2 s TR . To enhance contrast , SUMA, FreeSurfer  then passed into FreeSurfer’s autosegmentation pipeline. If poor segmentations were detected, the white matter mask and control points were edited, and the surface reconstruction was rerun until corrected. To fix segmentation errors, average anatomical volumes were manually edited to improve the grey/white matter contrasts and to remove surrounding non-brain structures . See Arcaro and Livingstone  for morehttp://cvnlab.net/analyzePRF).The data were processed using the HCP pipelines as previously published  to increase the signal-to-noise ratio (SNR), while preserving spatial specificity. Data were registered using a two-step linear then non-linear alignment approach  to a standard anatomical template for all monkeys. Fourier analysis was used to identify spatially selective voxels from polar angle and eccentricity experiments. Results from this analysis approach were originally reported in Arcaro and Livingstone  and are “Traced from before backwards, or from within outwards, the line of this sulcus presents a strongly marked, but irregular, upward convexity.On making successive transverse sections of this cerebrum from before backwards —is posteriorly bounded by the retrocalcarine sulcus and anteriorly by the variable occipitopolar sulcus. On the lateral surface, the boundary of the striate area is delineated by the gyrus descendens.” (pg. 16).In more modern work, Duvernoy  defines It should also be noted that Duvernoy denotes different labels for the bifurcated branches, or rami, of the retrocalcarine sulcus as the inferior  and superior (pg. 214) retrocalcarine sulci.Likewise, in the most recent atlas to define the rCaS in the human brain, Petrides  also defGuided by these definitions, we defined the rCaS in humans and macaques as the two bifurcated components of the posterior CaS positioned on the medial side of the occipital pole.“When it is recalled that the sulcus occipitalis superior occupies a position (within the area striata) on the lateral surface analogous to that of the retrocalcarine  on the mesial surface it will be apparent that Cunningham’s term “external calcarine” is not inappropriate as a designation for the former furrow. Moreover, the gradual slipping over of the area striata lateralis on to the mesial surface in the human brain must imply that the homologue of that caudal part of the lateral stria-bearing cortex, which is folded to form the posterior part of the superior occipital  sulcus in the Apes, will in the human brain form the walls of the caudal part of the sulcus retrocalcarinus .” (pgs. 134–135).The external calcarine sulcus (eCaS) is very prominent on the lateral surface of the occipital lobe in macaque  appears to be the most suitable as it is a part of the calcarine complex, and like the posterior or retrocalcarine, is axial to the striate area.” (pg. 8).Connolly  also wro“Beginning just above the lower end of the lunate sulcus, the main constituent of the lateral calcarine (lc) extends obliquely backward to the occipital pole. Just above it is its detached dorsal ramus.” (pg. 27).As reflected in the above quotation, Connolly referred to the eCaS as the lateral calcarine in his atlas across species Fig. E. He alsFinally, in the most recent atlas to define the eCaS, Petrides  refers tFollowing these classic and modern definitions of the eCaS, we defined the eCaS in all 48 hemispheres in humans  and 12 hemispheres in macaques relative to the rCaS.In humans, rCaS and eCaS were manually defined by a neuroanatomist (KSW) in each individual based on the classic and modern definitions described above. In macaques, rCaS and eCaS were also identified by a neuroanatomist (KSW) guided by the work described in the previous section, as well as additional modern studies  and normalized to the total surface area of V1 in each participant .For rCaS and eCaS, cortical surface area was measured along the pial and (smoothed) white matter surface segmentations using AFNI’s SurfMeasures. Surface area was measured both in raw units  within V1 and the foveal confluence was estimated along pial surface segmentations using AFNI’s SurfDist. Because the vertical meridian boundary between V1 and V2 is difficult to measure within the fovea using fMRI, the foveal confluence was defined as a curved line through the most foveal measurements linking the upper and lower vertical meridian boundaries identified within the surrounding parafoveal regions. Cortical distance from the fovea was defined as the minimum Euclidean distance along the cortical surface between each V1 surface node and the foveal confluence line. Treating the foveal confluence as a single point defined at the midpoint along this line yielded qualitatively similar results for subsequent analyses and did not change interpretation of the data. We focused on eccentricity representations as classic electrophysiology studies have related eccentricity representations to the rCaS in macaques , d̂ = cortical distance, and a and b are free parameters. The constant term (− 1) allows for fitting foveal (< 1°) measurements. The curve was fit using MATLAB’s Curve Fit Tool, minimizing the sum of the squared errors between the actual eccentricity values and the eccentricity values predicted by the curve.For each participant, eccentricity measurements within V1 were plotted as a function of cortical distance from the foveal confluence. This produced a scatter plot in which each data point represents a single surface node Fig. . An expoTo further illustrate the relationship of the rCaS and eCaS to the cortical magnification of V1 in humans and macaques, we computed a 2D histogram using bins of 0.5° eccentricity and 1 mm cortical distances for all datapoints in V1. We then used MATLAB’s contour function to identify isolines positioned at 99, 75, 50, and 25% of the maximum bin for data within the rCaS and eCaS separately.The average eccentricity representation within both rCaS and eCaS was calculated for each participant. The group mean and standard error across participants were calculated. Polar angle and eccentricity measurements for each surface node were converted to Cartesian space (Matlab’s pol2cart) and visualized in scatter plots for humans and macaques separately.We evaluated visual field coverage and areal size differences across species using two-way ANOVAs with species (humans/macaques) and hemisphere (right/left) as factors.The retrocalcarine (rCaS) and external calcarine (eCaS) sulci were identified bilaterally in 24 humans and 6 macaques. In both species, the rCaS and eCaS were localized to the medial and lateral surfaces of occipital cortex, respectively. In both macaques and humans, the rCaS was located on the most posterior portion of the medial surface with the long axis oriented along the inferior-superior dimension. The macaque eCaS was located in the ventral half of the operculum of the occipital lobe with the long axis oriented along the posterior–anterior axis. The human eCaS was identified as one or a series of smaller sulci located toward the occipital pole positioned dorsally to the occipito-polar sulci Petrides . To exam2 ± 39.01; average macaque: 187.91 mm2 ± 12.07) was larger than the eCaS  and comprised a larger portion of V1’s total surface area . Notably, the size of the eCaS relative to V1 was substantially larger in macaques than humans  = 158.36, p < 0.001; no significant effect of hemisphere and no interaction, p > 0.05). The size of the rCaS relative to V1 was also larger in macaques than humans  = 8.38, p < 0.006; no significant effect of hemisphere and no interaction, p > 0.05).In macaques, both the rCaS and eCaS fell within V1 in each individual. In humans, the rCaS fell within V1 in each individual, but the eCaS fell outside of V1 for several participants (9 of 48 hemispheres). In both species, the surface area of the rCaS (average human: 279.68 mm° (± 0.03) in humans and 2.45° (± 0.12) in macaques  = 551.34, p < 0.0001; no significant effect of hemisphere and no interaction, p > 0.05). Across participants, the mean eccentricity representation of the rCaS was 2.73° (± 0.11) in humans and 7.34° (± 0.06) in macaques  = 403.5, p < 0.0001; no significant effect of hemisphere and no interaction, p > 0.05). Differences in visual field coverage between humans and macaques were apparent by visualizing the range of eccentricity representations within the eCaS, within the rCaS, and across the rest of V1 as a function of cortical distance from the foveal confluence  = 725.37, p < 0.0001; no effect of hemisphere or interaction, p > 0.05; Fig. F = 10.82, p < 0.002; no significant effect of hemisphere or interaction, p > 0.05; Fig. Fs < 1.67, ps > 0.201).In both species, the rCaS and eCaS were localized to specific parts of the eccentricity map in V1 Figs. , 6 and 7within species, but not across species.Finally, visual field coverage with respect to polar angle also differed between species for both sulci Fig. , right. “Above all, the striking fact that in Man almost the entire striate area is found buried in a long and deep calcarine fissure wholly, or almost wholly, located on the medial face of the hemisphere, whereas in the lower Primates not less than half of the striate area is spread over the lateral face of the occipital lobe, requires a satisfactory phylogenetical explanation.” Polyak , Pg. 467Consistent with the classic quotation from Polyak  above, mMorphologically, our results identify a vertical, or bifurcated, portion of the calcarine sulcus on the medial surface toward the occipital pole in human and non-human primates Huxley , b. DespTo our knowledge, this is the first study to clearly show that the same macroanatomical structure (rCaS) can be coupled with different retinotopic representations between species Figs. , 6 and 7Our findings support an interpretation that nearly all of the V1 map is shifted medially in human occipital cortex relative to macaques Figs. D and 5 aIt is likely that underlying changes in microarchitecture and connectivity accompany shifts in cortical localization. Co-occurring with V1’s displacement is an increase in myelination on the medial surface of the occipital lobe in humans compared to macaques and chimpanzees  and external calcarine (eCaS) sulci and retinotopic representations within V1 in macaques and humans using anatomical and functional MRI. We find a consistent sulcal–functional relationship across individuals separately within each species, but a different sulcal–functional relationship among individuals between species. Specifically, the rCaS represents eccentricities 2°–5° in humans, but 7°–10° in macaques. These results indicate that the same macroanatomical structure can be coupled with different functional representations between species. Likely inter-related phylogenetic and anatomical mechanisms contribute to the fact that the same macroanatomical structure can be coupled with different functional representations between species. Future studies examining whether sulcal–functional mappings also diverge in other cortical regions will help us better understand the development and evolution of the cerebral cortex across species.Supplementary file1 (PDF 668 KB)Below is the link to the electronic supplementary material.