APP genetic causes of AD , older individuals with Down syndrome (DS) showing the pathology of Alzheimer’s disease (AD) and individuals with sporadic (early and late onset) AD . The aim of this was to elucidate important group differences and to provide mechanistic insights related to clinical and neuropathological phenotypes. Since lipid and cholesterol metabolism is implicated in AD as well as vascular disease, we additionally aimed to explore the role of APOE genotype in CAA severity and subtypes. Plaque formation was greater in DS and missense APP mutations than in APPdup, sEOAD and sLOAD cases. Conversely, CAA was more severe in APPdup and missense APP mutations, and in DS, compared to sEOAD and sLOAD. When stratified by CAA subtype from 1 to 4, there were no differences in plaque scores between the groups, though in patients with APPdup, APP mutations and sEOAD, types 2 and 3 CAA were more common than type 1. Conversely, in DS, sLOAD and controls, type 1 CAA was more common than types 2 and 3. APOE ε4 allele frequency was greater in sEOAD and sLOAD compared to APPdup, missense APP mutations, DS and controls, and varied between each of the CAA phenotypes with APOE ε4 homozygosity being more commonly associated with type 3 CAA than types 1 and 2 CAA in sLOAD and sEOAD. The differing patterns in CAA within individuals of each group could be a reflection of variations in the efficiency of perivascular drainage, this being less effective in types 2 and 3 CAA leading to a greater burden of CAA in parenchymal arteries and capillaries. Alternatively, as suggested by immunostaining using carboxy-terminal specific antibodies, it may relate to the relative tissue burdens of the two major forms of Aβ, with higher levels of Aβ40 promoting a more ‘aggressive’ form of CAA, and higher levels of Aβ42(3) favouring a greater plaque burden. Possession of APOE ε4 allele, especially ε4 homozygosity, favours development of CAA generally, and as type 3 particularly, in sEOAD and sLOAD.In this study, we have compared the severity of amyloid plaque formation and cerebral amyloid angiopathy (CAA), and the subtype pattern of CAA pathology itself, between The online version of this article (10.1007/s00401-018-1866-3) contains supplementary material, which is available to authorized users. APP) or presenilin (PSEN) genes.Alzheimer’s disease (AD) is a neurodegenerative disorder characterised clinically by a progressive loss of memory and cognition, accompanied by functional impairments of orientation and praxis. Pathologically, the major changes involve a deposition of amyloid β protein (Aβ) in brain parenchyma (as amyloid plaques) and hyperphosphorylated tau within neurones (as neurofibrillary tangles). Additionally, most cases display deposits of Aβ within blood vessel walls—a change known as cerebral amyloid angiopathy (CAA). While more than 90% cases of AD are without obvious genetic cause, and termed ‘sporadic’, the remainder is associated with mutational events involving either the Amyloid Precursor Protein  result in increased catabolic breakdown of APP by β- and/or γ-secretase into Aβ, and confer a pathological picture similar to that seen in sporadic AD. On the other hand, mutations lying in the juxtamembrane region, such as APP692 or APP693, are more associated with CAA than plaques, and often manifest clinically as acute stroke. There are also rare French [APP locus, resulting in APP overproduction. In most of these families, the duplication has been validated only in living patients and confirmed cases with brain donation are scarce. An APP duplication has also been reported in a Spanish patient with apparently sporadic AD and severe CAA [APP locus. In both APPdup and DS individuals, it is presumed that the early deposition of Aβ plaques and CAA stems from an overexpression of APP and the consequent degradation of an excessive production of APP. In addition, recent work suggests that a mutation in the 3′untranslated region of APP also result in APP overexpression and might act as a genetic determinant in some cases of CAA [With respect to the transmembrane protein e French , 37, Dute French , Finnishe French , Japanese French , Swedishe French  and Brite French  familiesvere CAA , but othvere CAA . It has vere CAA , 25. In s of CAA .PSEN-1) mutations located beyond codon 200 compared to those where the mutation lies before codon 200 [PSEN-1 mutations are associated with an unusual morphological form of amyloid plaques, known as ‘cotton wool’ plaques, and often present clinically with a spastic paresis [Although all cases of AD are defined pathologically by the presence of numerous plaques and tangles, and usually CAA, throughout the cerebral cortex and hippocampus, the morphological appearance of these changes, especially plaques and CAA, can vary according to the underlying genetic background. For example, a much more severe CAA is seen in patients with presenilin-1  is that of progressive dementia frequently accompanied by seizures and intracerebral haemorrhage (ICH) [APP copy number. Nevertheless, CAA can present in sporadic and familial AD in various phenotypic histological forms, involving differing combinations of leptomeningeal and parenchymal arterial pathology, sometimes extending into the capillary bed [The cardinal clinical presentation in ge (ICH) , 38, 41,ge (ICH) , 14. CAAge (ICH)  where thlary bed , 46.APPdup [APPdup, this is a more poorly defined feature of individuals with DS, suggesting the presence of a mechanism(s) that acts protectively [APPdup in the ~ 270 other genes located on chromosome 21 that are also triplicated.We have recently reviewed epidemiological data on stroke (including haemorrhagic stroke) related to CAA to make comparisons between DS and APPdup . Althougectively . This miAPPdup, and other APP mutations has never been undertaken as far as we are aware, and consequently the degree and nature of tissue differences in CAA, ICH, and Aβ deposition between these disorders remain unclear. Therefore, in this analysis, we aim to compare the severity of amyloid plaque formation and CAA, and the subtype pattern of CAA pathology itself, and the relationship between CAA severity and CAA phenotype, in APP genetic causes of AD , DS and sporadic (early and late onset) AD . The observations might help to elucidate important differences between the patient groups, and thus provide mechanistic insights related to clinical and neuropathological phenotypes. Since lipid and cholesterol metabolism is implicated in AD as well as vascular disease, we additionally aimed to explore the role of APOE genotype in CAA severity and subtypes.However, a direct comparison of vascular amyloidosis at pathological level between DS, APP gene through Prof Annie Laquerrière at University of Rouen. These were from two separate families. Three patients (patients #1–3) were members of the same family  and 2 with a point mutation at codon 692 (Flemish mutation). Four of these were obtained through MBB, eight from IOPPN Brain Bank (courtesy of C Troakes), and four from Queen Square Brain Bank (QSBB) (courtesy of L Parsons). Additionally, brains were obtained from 34 patients with clinical diagnosis of sEOAD , 34 with sLOAD  and 30 elderly controls  through MBB  with genetically proven duplications in vely, in ). SecondNone of the individuals in any of the pathological groups had been known to have suffered from stroke or ICH.40 and Aβ42(3) monoclonal antibodies  was performed on 30 cases, selected to be representative of each CAA phenotype within each pathological group, as described previously [APPdup cases (case #1–3), six with DS , five with missense APP mutations (#49–52 and 54), six with sEOAD , seven with sLOAD  and three controls .Paraffin sections were cut at 6 µm from formalin fixed blocks of frontal lobe (BA8/9), temporal lobe (BA21/22) with posterior hippocampus, occipital lobe BA17/18) and cerebellar hemisphere. Following titration to determine optimal immunostaining, antibodies were identically employed in a standard immunohistochemical protocol . Section/18 and c40 (BA27) and Aβ42(3) (BC05) specific changes.Sections were examined microscopically for the appearance, severity and topographical distribution of immunostaining within brain parenchyma (as plaques) and cerebral vessels (as CAA). A five-point scoring system, similar to that employed by Olichney et al. , was empPlaquesGrade 0—no Aβ plaques in parenchyma.Grade 1—A few Aβ plaques in parenchyma occupying each low power (× 10 microscope objective) field.Grade 2—A moderate number of Aβ plaques in parenchyma occupying each low power (× 10 microscope objective) field.Grade 3—Many dispersed Aβ plaques in parenchyma occupying each low power (× 10 microscope objective) field.Grade 4—Very many densely packed Aβ plaques in parenchyma occupying each low power (× 10 microscope objective) field.CAAGrade 0—No CAA in blood vessel walls in leptomeninges or brain parenchyma.Grade 1—Occasional blood vessels with CAA in leptomeninges and/or within brain parenchyma, usually not occupying the full thickness of the wall.Grade 2—A moderate number of blood vessels with CAA in leptomeninges or brain parenchyma in leptomeninges or within brain parenchyma, some occupying the full thickness of the wall.Grade 3—Many blood vessels with CAA in leptomeninges or brain parenchyma, most occupying the full thickness of the wall.Grade 4—Most or all blood vessels with severe CAA in leptomeninges or within brain parenchyma, occupying the full thickness of the wall.Representative images for each plaque and CAA grade are presented in Supplementary Fig. 1.Plaque and CAA scores were separately summated across all four brain regions examined to provide a ‘global’ plaque and CAA score for each patient.CAA subtype, based on examination of frontal, temporal and occipital cortex in sections immunostained for Aβ, was assigned to all cases as previously described . Type 1 APOE genotype was determined [APOE genotyping was possible for all 4 APPdup patients, 25/34 DS individuals, 13/16 missense APP mutation patients, all 34 sEOAD patients, 33/34 sLOAD patients, and all 30 controls. The lack of available frozen tissue prevented APOE genotyping for those remaining individuals.DNA was extracted from frozen cerebellum or frontal cortex by routine methods from which termined . APOE geAPOE alleles and genotypes were performed using chi-square test. Correlations between rating data were made using Spearman rank correlation test. In all instances, significance levels were set at p < 0.05.Rating data were entered into an excel spreadsheet and analysed using Statistical Package for Social Sciences (SPSS) software (version 20.0). Patients were stratified according to genetic subtype for statistical analysis of the effect of each mutation on the underlying Aβ pathology. Comparisons of semi-quantitative scores for intensity of Aβ immunostaining were performed using Kruskal–Wallis test with post hoc Mann–Whitney test where Kruskal–Wallis yielded a significant difference between antibody staining scores. Group comparisons of age at onset, age at death and duration of illness were made using ANOVA with post hoc Tukey test. Comparisons of frequency of APPdup, DS, missense APP mutations, sEOAD and sLOAD groups with respect to mean age at onset of disease . By definition, patients with sLOAD had a significantly later age at onset of illness (p < 0.001) compared to those with APPdup, DS, missense APP mutations and sEOAD, but there were no significant differences in age at onset between any of the early onset AD and DS groups  with patients with sLOAD, and controls, all (by definition) dying at a later age (p < 0.001) than patients with APPdup and missense APP mutations, sEOAD and individuals with DS. Again, there were no significant differences in age at death between any of the early onset AD and DS groups (Table p = 0.017). The controls died at a later age than the patients with sLOAD (p = 0.002)  between patients with APPdup, DS, missense APP mutations, sEOAD and sLOAD. Patients with missense APP mutations had a longer duration of illness than individuals with DS (p < 0.001) and patients with sLOAD (p = 0.042).There were significant differences between p = 0.004), but no significant gender difference was seen with respect to duration of illness. There were no gender differences in age at onset, age at death or duration of illness for each CAA subtype within each patient group.Consistent with general longevity, mean age at onset and mean age at death, were both significantly later in females compared to males in the sLOAD group alone  were at Braak stage 5 and 23 (68%) at Braak stage 6; all were at Thal stage 5. Of the 34 patients with sEOAD, 10 (29%) were at Braak stage 5 and 24 (71%) were at Braak stage 6, and 2 (6%) were at Thal stage 4 and 32 (94%) were at Thal stage 5. Of the 34 patients with sLOAD, 2 (6%) were at Braak stage 4, 6 (18%) were at Braak stage 5 and 26 (76%) were at Braak stage 6, and 6 (18%) were at Thal stage 4 and 28 (82%) were at Thal stage 5. Of the 30 controls, 1 (3%) was Braak stage 0, 13 (43%) were at Braak stage 1 and 16 (54%) were at Braak stage 2, and 9 (30%) were Thal stage 0, 10 (33%) were at Thal stage 1 and 11 (37%) were at Thal stage 2 (see Supplementary Table 1).Based on AT8 immunostaining for tau proteins (Braak stage) and 4G8 immunostaining for Aβ , all 4 patients with As evidenced by 4G8 immunostaining, CAA was severe in all four patients, but did not present with a uniform appearance.In patients #1 Fig. a–c, #3 g–i and #In patient #2, severe CAA was seen across all three cortical regions affecting leptomeningeal Fig. e but manAs seen in 4G8 immunostaining, amyloid deposition in the form of plaques and CAA was widely present in all 34 individuals with DS. CAA was present as type 1 in 15 (44%) cases, type 2 in 9 (26%) cases and type 3 in 10 cases (29%)  in which arteriolar and capillary CAA with perivascular deposition of amyloid (dyshoric angiopathy), but few/no plaques, was predominant within occipital cortex, in addition to severe leptomeningeal CAA , type 2 in 20 patients (59%) and type 3 in 5 patients (15%)), in 30/34 patients with sLOAD (as type 1 in 17 patients (57%), type 2 in 7 patients (23%) and type 3 in 6 patients (20%), but only in 17 controls (57%) (as type 1 in 15 subjects (88%) and type 2 in the other 2 subjects (12%)  than those with sEOAD (27%) but not those with sLOAD (57%). Also, the proportion of individuals with sLOAD (57%), and that of controls (88%), showing type 1 CAA was both significantly greater than that proportion of sEOAD patients with type 1 CAA . Conversely, the proportion of patients with sEOAD with type 2 CAA (59%) was significantly greater than that in those with DS  or sLOAD  or in controls . For type 3 CAA, the proportion of individuals with APPdup (75%) was significantly greater than those with DS , sEOAD  and sLOAD , and was also greater (p < 0.05 by Fisher exact test) than those with missense APP mutations and controls (0% in both).The proportion of patients showing different CAA phenotypes varied between the pathological groups Fig. . The proAPPdup, missense APP mutations, sEOAD and sLOAD, or for mean age at death between any of the CAA phenotypes for APPdup, DS, missense APP mutations, sEOAD, sLOAD and control cases  between CAA phenotypes, with CAA type 1 cases having a significantly longer duration of illness than those with type 2 CAA (p = 0.01)   scores differed significantly across the six groups. The degree of plaque formation  was significantly greater in both DS and missense APP mutations than in sEOAD and sLOAD cases (p < 0.001 in every instance). As would be expected, all 5 pathological (AD) groups had a significantly greater degree of plaque formation than controls (p < 0.001), but the degree of plaque formation was not significantly different between sEOAD and sLOAD cases  Fig. a and CAAp < 0.001) but, in contrast to plaque scores, the degree of CAA was also significantly greater in sEOAD than sLOAD (p = 0.014) , missense APP mutations  and DS  than in sEOAD and sLOAD, and the severity of CAA was greater in APPdup than that in DS (p = 0.014) but not so for missense APP mutations (p = 0.056), with there being no significance difference between the missense APP mutations and DS  groups had a significantly greater degree of CAA than controls (14) Fig. b. On theThere were no significant differences between males and females in either plaque or CAA scores for each pathological group, or for each CAA subtype within each pathological group.χ2 = 13.0, p < 0.001), sEOAD  and sLOAD , but not for patients with APPdup or missense APP mutation, or controls. In both missense APP mutations and sEOAD there was a significantly greater level of CAA as both CAA type 2  and type 3  than as type 1, with no significant differences in CAA severity between type 2 and type 3 cases. In DS, sLOAD and controls, there was a significantly greater level of CAA in type 1 than type 2 and type 3 (p < 0.006 and 0.002), with no significant differences in CAA severity between type 2 and type 3 cases.When stratified by CAA subtype, there were no significant differences in overall plaque scores between each CAA subtype for any of the six groups. However, overall severity of CAA did vary significantly across subtypes for DS  or separately as individual pathological groups . For controls, this correlation failed to reach significance . The number of cases with APPdup was too low to permit this particular analysis. For all groups, CAA severity scores progressively increased with ascending CAA phenotype class  with BC05. On the other hand, all plaques detected by 4G8 also appeared to be detected by BC05, but only a subset (of cored plaques) was detected by BA27  with scores for rating of plaque density with 4G8 antibody being significantly greater than that for BC05 antibody (p = 0.019) and BA27 antibody (p < 0.001), and that for BC05 also being significantly greater than BA27 (p < 0.001). Similarly, the degree of immunostaining for CAA differed significantly between the three antibodies  with scores for rating of CAA density with 4G8 and BA27 antibodies being significantly greater than that for BC05 antibody . No significant difference between scores for 4G8 and BA27 antibodies was found (p = 0.582). Despite there being significant differences in the levels of immunostaining for plaques and CAA between the three antibodies, regression analysis showed highly significant correlations between plaque scores for each of the three antibodies (p < 0.001 in every instance), and likewise for CAA scores (p < 0.001 in every instance).Semi-quantitative analysis of the rating data for the level of immunostaining of plaques and CAA by 4G8, BC05 and BA27 antibodies was performed on the 30 cases collectively. The degree of immunostaining of plaques differed significantly between the three antibodies , missense APP patients (p < 0.001) and controls (p < 0.001), as was this in sLOAD patients compared to DS (p < 0.001), missense APP mutations (p < 0.001) and controls (p < 0.001)  Table . There wts Table . None ofAPOE ε2 allele frequency between any of the groups, except that this was significantly higher in controls (p = 0.038) compared to sLOAD patients . Moreover, when stratified according to possession of none, one or two ε4 alleles, the severity of CAA in sLOAD was significantly greater in patients with two APOE ε4 alleles than both those with one or no APOE ε4 alleles (p = 0.013 in both instances) with no significant difference between those with one or no APOE ε4 alleles. No such effect was seen in sEOAD. It was not possible to test for the effects of homozygosity for APOE ε4 allele in DS as there were no such individuals represented in the cohort. Likewise, the effects of possession of APOE ε4 allele could not be tested in APPdup or missense APP mutation due to lack of patient representation.There was no significant effect of possession of at least one copy of APOE ε4 allele, it was not possible to ascertain whether APOE genotype influenced CAA phenotype in patients with APPdup and missense APP mutations , and tending to be greater in type 2 CAA (p = 0.065), compared to type 1 CAA, with no significance between type 2 and type 3 CAA.Due to the low frequency of s Tables , 3. CompAPP, others with missense mutations in APP, patients with sEOAD and sLOAD, older individuals with DS and elderly controls. The main findings to emerge were:APP mutations than in sEOAD and sLOAD cases, while the degree of plaque formation was not significantly different between sEOAD and sLOAD.The degree of plaque formation was greater in both DS and missense APPdup and missense APP mutations, and DS, compared to sEOAD and sLOAD. Again, all five pathological (AD) groups had a significantly greater degree of CAA than controls, and the degree of CAA was also significantly greater in sEOAD than sLOAD, and in APPdup compared to DS.Conversely, the severity of CAA was significantly greater in both APP mutations and sEOAD there was a significantly greater level of CAA as types 2 and 3 CAA compared to type 1. Conversely, in DS, sLOAD and controls there was a significantly greater level of CAA in type 1 CAA than types 2 and 3. In APPdup type 3 was the predominant CAA phenotype.When stratified by CAA subtype, no significant differences in overall plaque scores were seen between each CAA subtype for any of the six groups. However, in both CAA severity scores progressively increased across CAA types 1–4 for all cases combined and for each pathological group individually.APOE ε4 allele frequency was overall significantly higher in sEOAD than in DS, missense APP and controls, as was ε4 allele frequency in sLOAD compared to DS, missense APP mutations and controls. There were no significant differences in APOE ε4 allele frequency between DS, missense APP and controls. None of the APPdup patients bore APOE ε4 allele.All blood vessels stained for CAA by 4G8 appeared to be detected by BA27 but fewer were detected with BC05. Conversely, all plaques detected by 4G8 appeared to be detected by BC05, but only a subset was detected by BA27.APOE ε4 allele frequency varied numerically between each of the CAA phenotypes in APPdup, missense APP mutations, sEOAD, sLOAD, DS and controls, but not significantly so. Nonetheless, APOE ε4 homozygosity was more commonly associated with type 3 CAA than types 1 and 2 CAA, and was associated with a greater severe of CAA overall in sLOAD but not in sEOAD or DS.In the present study, we have compared the extent and pattern of Aβ deposition, both as plaques and as CAA, in patients with duplications in APP mutations were not specifically investigated in this latter study. Nonetheless, like ourselves, these authors found that CAA occurred at significantly higher frequencies in the brains of individuals with DS compared to sLOAD cases and controls, with the DS cohort being 1.2 times more likely to have CAA relative to sLOAD cases, and 4.6 times more likely to have CAA compared to control cases. On the other hand, atherosclerosis and arteriolosclerosis were rare in cases with DS. Such observations of significantly more frequent CAA, and a greater severity of CAA when present, in people with DS relative to sLOAD and control cases are consistent with the hypothesis that such changes are driven, at least partially, by an overexpression of APP. The lack of AD neuropathology and CAA, even at greater than 70 years of age, in rare cases of DS with partial trisomy 21 where APP is not overexpressed [In a recent study, Head and colleagues  comparedxpressed , 35 woulAPP might be translated into enhanced CAA remains unclear, but could involve deficiencies in clearance mechanisms when faced with such an overload of Aβ. In this latter context, it has been postulated that the strong association between age, CAA and AD pathology in the general population is driven, at least partially, by an impaired efficiency of cerebral vessels in later life in expelling extracellular fluid containing soluble forms of Aβ as a consequence of atherosclerosis/arteriosclerosis within such vessels [APP mutations or DS since these would only be anticipated to occur beyond an age at which most would generally survive to. However, Aβ can also be cleared from the brain through several other routes, involving endocytosis by microglia and astrocytes, or enzymatic degradation. It is, therefore, possible that failures in these latter pathways, in conjunction with the overexpression of APP, might foster an inability to expel Aβ and result in severe CAA.Nonetheless, how an overexpression of  vessels . Nonethe vessels  and athe vessels , 22. ParAPP locus, and an overexpression of APP protein, there are clear differences in clinical presentation between APPdup and DS individuals. Stroke and ICH are the main clinical consequences of vascular amyloidosis in APPdup, occurring in at least one-third of all cases, but both are uncommon in individuals with DS, with only a handful of case reports of this in the literature . Indeedure , although designated as CAA type 3, showed a particularly severe phenotype that was reminiscent of type 4 CAA. Consequently, patients with APPdup in the present study had a more severe CAA phenotype than most individuals with DS, despite the similar ages, with only 30% DS individuals showing this type 3 phenotype, and then not to the same degree of severity as in patients with APPdup. It is possible, therefore, that it is the lesser extent of CAA in DS (compared to APPdup) that lowers the risk of CAA-related stroke and haemorrhage in such individuals. Correlations between CAA severity scores and CAA phenotype suggest that the different CAA phenotypes exist on a continuum with type 1 being the least ‘aggressive’ form, and types 2–4 following progressively as Aβ becomes deposited further along the arterial tree reaching into parenchymal arteries and arterioles (type 2) and finally into capillaries (types 3 and 4). Certainly, this would accord with the proposal put forward by Weller et al. [Alternatively, it may be differences in the actual CAA phenotype present that are responsible. Type 1 CAA tended to be more common in DS and sLOAD, and type 2 CAA was more common in missense r et al.  based on40, compared to the longer, more rapidly aggregating form, Aβ42(3). It has been shown that in both AD and DS the Aβ in CAA is composed mostly of Aβ40, whereas in plaques it is Aβ42(3) that predominates [40 and Aβ42(3), with the less aggregation prone Aβ40 travelling further along perivascular drainage channels and ultimately reaching blood vessel walls, compared to the less abundant though more rapidly aggregating Aβ42(3) which coalesces into plaques within the brain parenchyma. In familial AD due to certain missense mutations in APP (for example those at or around codon 717) proteolytic processing of APP elevates levels of Aβ42(3) relative to Aβ40 [42(3) containing plaques being formed as a consequence [40 and Aβ42(3) without affecting levels of production. As mentioned earlier triplication at APP locus will increase production of both Aβ40 and Aβ42(3).APP is processed by proteolytic enzymes known as secretases. Cleavage within the APP domain containing the Aβ sequence by α-secretase precludes its formation, whereas sequential cleavage at the amino- and carboxyl- termini of the Aβ sequence by β- and γ-secretases, respectively, releases Aβ into the extracellular fluid of the brain. Most of this occurs as a more slowly aggregating form, Aβominates , 40, 44. to Aβ40  with excsequence . Other m40 and Aβ42(3) being generated. In APPdup and DS, where excess amounts of Aβ40 (and Aβ42(3)) are produced, this could lead to failure to expel this from the extracellular fluid leading to a massive build up in smaller arteries and capillaries evidenced as the more extensive type 3 CAA. In missense APP mutations such as those at codon 692, the mutated, more highly aggregation prone, form of Aβ40 would promote its excessive deposition in vessel walls, and again result in the severe type 4 CAA (see [APP693 mutations), whereas no excess of Aβ40 is generated in missense mutations in APP occurring around codon 717, and in these cases the less extensive type 1 and 2 CAA predominate. In sEOAD and sLOAD, where normal levels of both Aβ40 and Aβ42(3) are produced, the different CAA phenotypes present might reflect the relative efficiencies in which Aβ40 is cleared through the perivascular drainage channels. Present observations that all blood vessels stained for CAA by 4G8, irrespective of genetic or pathological group, or CAA phenotype, also appeared to be strongly immunoreactive for Aβ40 but less strongly for Aβ42(3). On the other hand, all plaques detected by 4G8 were strongly immunoreactive for Aβ42(3) but only a subset (of cored plaques) appeared to contain Aβ40. Such findings are consistent with our previous studies in familial AD and DS [APPdup might also involve amyloid clearance, but alternative mechanisms could involve a unique oxidative stress profile or immune response in DS [Hence, the different CAA phenotypes seen here in the different forms of AD might in some way reflect the relative proportions of AβCAA  in sLOAD [APOE ε4 allele/E4 isoform could potentiate the development of CAA, as type 3 CAA, in those patients with sLOAD bearing APOE ε4ε4 genotype, a suggestion in keeping with previous studies [APP mutations involving codon 717, would also accord with the relative infrequency of APOE ε4 allele and ε4ε4 homozygosity in such individuals. However, having said that, overall severity of CAA and prevalence of type 3 CAA were equivalent in DS individuals as in patients with sEOAD and sLOAD, despite there being in DS a relative lack of APOE ε4 alleles, and a complete absence of ε4ε4 homozygotes.Interestingly, as others have shown , 40, 46,in sLOAD , 27, posin sLOAD . In thesWhile there were no significant differences in overall plaque scores between sEOAD and sLOAD cases, overall CAA scores were lower in sLOAD than sEOAD. Furthermore, age at death did not vary significantly between CAA phenotypes for these two groups though the proportion of cases with the less severe type 1 CAA was greater in sLOAD than sEOAD, suggesting that advancing age per se may, if anything, lessen the severity of CAA and phenotype present, at least in AD. This observation was not due simply to a shorter duration of disease in sLOAD compared to sEOAD which might potentially have terminated disease progression at an early stage in the later onset cases.40 or shorter sized peptides, or factors which promote its aggregation such as ApoE E4 isoform, or some combination of all of these. Moreover, why there should be a distinction in CAA phenotype profiles between DS and APPdup is puzzling and it is curious why sEOAD should be more strongly associated with type 2 CAA compared to sLOAD (and vice versa for type 1 CAA), but differential possession of APOE ε4 allele does not appear to determine this. Interestingly, previous studies [PSEN-1 mutations, especially in those where the mutation is located after codon 200, in the absence of any APOE ε4 allele modifying effect. Possibly sEOAD shares some genetic or mechanistic risk affinity with such PSEN-1 mutations, which ultimately translate into a similar CAA phenotype. The neuropathological differences between the different forms of AD highlighted in this study require further study to elucidate the underlying mechanisms. The scientific value in knowing what CAA phenotype is present will help to reduce variability of findings, and provide greater consistency of results, when factors relating to promotion of CAA are being investigated.Hence, the factors that determine CAA phenotype are complex and remain unclear, possibly involving differential levels of production or clearance of Aβ studies  have shoSupplementary material 1 (DOCX 4649 kb) Supplementary Fig. 1. Representative examples of the different plaque and CAA scores. Panel a, plaque score = 0, CAA score = 2. Panel b, plaque score = 1, CAA score = 0. Panel c, plaque score = 2, CAA score = 3. Panel d, plaque score = 3, CAA score = 4. Panel e, plaque score = 3, CAA score = 1. Panel f, plaque score = 4, CAA score = 2. Immunoperoxidase-haematoxylinSupplementary material 2 (XLSX 19 kb) Supplementary Table 1. Clinical, pathological and genetic characteristics of the patient and control study groupsBelow is the link to the electronic supplementary material.


For predicting texture suited for South and South East Asia, most of the breeding programs tend to focus on developing rice varieties with intermediate to high amylose content in indica subspecies. However, varieties within the high amylose content class may still be distinguishable by consumers, who are able to distinguish texture that cannot be differentiated by proxy cooking quality indicators.This study explored a suite of assays to capture viscosity, rheometric, and mechanical texture parameters for characterising cooked rice texture in a set of 211 rice accessions from a diversity panel and employed multivariate approaches to classify rice varieties into distinct cooking quality classes. Results suggest that when the amylose content range is narrowed to the intermediate to high classes, parameters determined by rheometry and RVA become diagnostic. Modeled parameters distinguishing cooking quality ideotypes within the same range of amylose classes differ in textural parameters scored by a descriptive sensory panel.Our results reinforced the notion that it is important to define cooking quality classes in indica subtypes based on multidimensional parameters, by going beyond amylose predictions. These predictive cooking models will be handy in capturing cooking and eating quality properties that address consumer preferences in future breeding programs. Policy implications of such findings may lead to changes in criteria used in assessing grain quality in the intermediate to high amylose classes.The online version of this article (10.1186/s12284-018-0245-y) contains supplementary material, which is available to authorized users. In rice varietal improvement programs, the texture of cooked rice is primarily indicated by amylose content (AC) Juliano . This paOne of the best ways to determine the associations of other cooking quality factors with texture is to make AC a constant in studies. An approach is to focus analyses on waxy rice varieties, which have negligible concentrations of amylose. However, the global waxy rice market is small (only 1% of the rice trade); thus the waxy rice approach is not widely used Calpe . The bigpeak viscosity , trough viscosity , final viscosity , breakdown , lift-off , and setback . The pasting curve also shows the pasting temperature  and the peak time  [Fitzgerald et al. G’max) and feature the curves for G’, loss modulus (G”), and tan (δ) [Hsu et al. Information about other cooking quality attributes within an AC class may be obtained by further characterising the behaviour of starch during the cooking and the cooling process. Rapid Visco-Analysis (RVA) is routinely used to determine the pasting behaviour of starch-water suspensions. The pasting curve provides metrics that indicate disintegration and retrogradation of starch, and is based on rheological principles [Zaidul et al. Texture parameters could be classified into three types: geometrical properties, mechanical properties, and properties related to moisture and fat content [Brandt et al. Classification of diverse germplasm within intermediate and high AC classes based on multidimensional data such as those generated by RVA, rheometry, and texture analysis will shed interesting insights about cooking and eating quality. Such insights could then be obtained through dimension reduction and correlation studies applied to multidimensional cooking quality data. Predicted cooking quality classes can also be used to determine grain quality attributes that affect the market prices of rice varieties [e.g., Cuevas et al. In this study, multinomial logistic regression and random forests are the data mining techniques that were employed. The multinomial logistic regression is an extension of the binary logistic regression, a technique used to calculate the probability of membership in one of more than two nominal or unordered categories (outcome variables) based on maximum likelihood estimation [Dixit et al. To the best of our knowledge, these data mining techniques have not been applied to classify rice accessions based on cooking and organoleptic attributes. The objectives of this study, therefore, were to (1) characterise the textural and cooking properties of a collection of rice varieties belonging to the intermediate- and to the high-AC classes; and (2) apply modeling techniques to predict distinct cooking quality ideotypes based on visco-elastic and textural attributes.n = 211) of indica rice accessions was selected based on genetic diversity and their geographic distribution, listed as Additional file A set n = 21 of indi3COOH (1 N) and 30% KI-I2 (2%:0.2%) to form amylose-iodine complex. Absorbance of the sample’s amylose-iodine complex was measured at a wavelength of 620 nm and AC was quantified from a standard curve using varieties of known ACs . The ACs of the varieties used in the standard curve were determined using the reference method of ISO 6647 [International Organization for Standardization Amylose content (AC) was determined based on the colorimetric reaction of the amylose-iodine complex developed using the method of ISO 6647 [International Organization for Standardization − 1, using a DSC model Q100 . Data on thermal transitions were collected and analysed using the Universal Analysis 2000 software. The peak of the endotherm was reported as the GT. Samples were classified as low-GT (below 67 °C), as intermediate-GT (68–73 °C), and as high-GT (GT ≥ 74 °C) [Cuevas et al. The GT of the rice samples were characterised through differential scanning calorimetry (DSC) as previously described [Cuevas et al. Rice flour (3 g) was suspended in reverse osmosis-purified (RO) water (25 g) in a canister and viscosity changes were then measured using a Rapid Visco Analyzer , following the heat (50–95 °C) – hold (95 °C) – cool (95–50 °C) time/temperature profile described in the AACC Method 61–02 [American Association of Cereal Chemists Inc w/v)) and then placed at the centre of a Peltier plate of the Advanced Rheometer 2000 . The rheometer was fitted with a parallel plate geometry (Ø = 40 mm). To minimise evaporation during the test, the sample was covered with a solvent trap sealed with water. During the test, the sample was subjected to heating ramp (35–95 °C) at 4 °C min− 1 ramp rate then to a cooling ramp (95–35 °C) at the same ramp rate. The frequency of oscillation was set at 1 cycle per second (1 Hz). Measurements were performed in triplicate. The TA Advantage Software 2003 (version 4.0.0) was used to record the data (Table G”) at G’max, the tan (δ) at G’max (the ratio of G” to G’ at G’max), and the temperature at G’max were determined based on the G’ and the G” curves. Slopes 1 and 3 (S1 and S3) were measured from the gel point (where tan (δ) = 1) to G’max and G”max. Slope 2 (S2) was measured at G’max to the lowest point of the decreasing G’ (G’trough) while Slope 4 (S4) was measured from G”max to the point before the G” leveled off.For each sample, rice flour was suspended in reverse-osmosis water , adhesiveness , cohesiveness , and springiness  to the time elapsed from starting point to the peak of the first curve (T1), representing sample height recovery after the initial compression) [Lyon et al. − 1. For each cooking replicate, three compression replicates were conducted.For each sample, 25 unbroken milled rice grains were submerged in 1 mL water for 15 min in a test tube, which was then covered to minimise water evaporation. The test tube was heated in a boiling water bath (20 min) and then placed in a water bath (50 °C) until texture profile analysis. Three cooking replications were conducted. Three cooked unbroken rice grains were subjected to a two-cycle compression test using a TA.XT-Plus Texture Analyser equipped with a cylindrical probe . The texture profile resulting from this two-compression test is composed of two sets of one positive and one negative curves, which can be divided into regions that represent downstrokes  and upstrokes   for 1 h at 370 °C. The sample was then cooled to room temperature and made up to volume (20 mL) with deionised water. It was kept overnight to allow for sedimentation. The liberated ammonium in the digest was then allowed to react with a solution containing sodium salicylate (0.94 M) and sodium nitroprusside (0.00026 M), and aqueous sodium hypochlorite  while going through the glass transition lines in a San ++ Segmented Flow Analyser (SFA) system at 16 mL min− 1. Absorbance values of the ammonia-salicylate complex were determined at λ = 660 nm. The % Kjeldahl N values of the samples were determined based on the linear relationship between absorbance and analyte concentration, following the Beer-Lambert Law. This linear relationship was based on a standard curve developed using ammonium sulfate solutions with different concentrations. The crude PC was calculated by multiplying the Kjeldahl N value by 5.95 [Villareal et al. Crude protein content (PC) was measured using a modified protocol based on the automated colorimetric method (AACC Method 46–09) AACC . A test Statistical analyses were carried out in R . Ward’s cluster analysis was used to group the samples into three clusters based on 25 variables . The raw dataset was used in developing a multinomial logistic regression (MLR) model to identify different clusters using Eq. 1:f is the score associated with the sample i assigned to cluster k , βk is the vector of regression coefficients associated with cluster k, and Xi is the vector of explanatory variables describing sample i.where Tests of random forests (RF) were conducted with 500 trees and three variables randomly selected at each split. These random forests generated standardised scores that indicated the importance of each of the nine retained variables (determined by MLR) in classifying samples into the three clusters and also identified the most important variables per cluster. To rank the variables according to importance, the random forest algorithm determined the magnitude of increase in the prediction error  when the out-of-bag data were permuted (or excluded) for one variable while data for all other variables were held constant [Liaw and Wiener v/v) ratio with water in rice cookers . After the rice was cooked to completion, the rice was mixed, ensuring that the grains touching the sides and the bottom were undisturbed. Sub-samples were distributed into glass custard cups (pre-labelled with three-digit codes), sealed with a plastic lid, and then monadically presented for sensory evaluation to a previously trained set of panellists. Along with the sample, a tablespoon and a cup of drinking water were provided. To ensure that the samples were kept warm during the evaluation, the samples were kept in the rice cooker (at the “Warm” setting) and only placed in sample cups once the panellists requested for the samples. A rice breeding line, IR06N155 (harvested in the dry season of 2013 at IRRI’s Long-Term Continuous Cropping Experiment), was used as a standard. It was served to the panellists six times, randomly distributed in different rice tasting sessions.Five samples from each cluster were selected for sensory evaluation using the texture profiling method [Lyon et al. The sensory panelists who participated in sensory evaluation were selected based on their availability and previous training in sensory descriptive profiling. The training phase included a battery of difference tests, sample and method familiarisation, and adjustment of the lexicon based on the panelists’ contexts [Champagne et al. The attributes tested during the rice tasting sessions represented the texture perceived at various stages of eating rice: from when the rice grains are first placed in the mouth up to after swallowing the rice. Panellists evaluate the intensity of each attribute on a 150-mm scale that has been adapted from established 15-point reference scales [Goodwin Jr. et al. r > 0.75 or r < − 0.75, Table max was positively correlated with G” at G’max and negatively correlated with temperature at G’max. Thus, six of these correlated attributes  were removed as redundant from the subsequent analyses and 19 variables were retained, including GT, LO, G’max, and PV.This study characterized cooking quality properties of 211 diverse rice accessions based on 25 cooking quality variables including those routinely tested in grain quality evaluation programs  along with specialized traits to capture viscoelastic properties measured by rheometry  often regarded as one of the most important factors affecting cooked rice quality and it is often viewed in combination with AC because an increase in AC reportedly leads to elevated GT [Juliano et al. Protein content (PC) has been reported to affect cooked rice stickiness [Champagne et al. The TPA provided measurements for HRD, ADH, COH, and SPR. The three clusters had similar values for HRD Table , with raPasting parameters are additional indicators of organoleptic quality, with parameters extensively studied particularly with their relationships with AC, PC, and mechanical texture attributes [reviewed in Champagne et al. max and G’trough while the other two clusters had similar values. Likewise, cluster 1 could clearly be distinguished from cluster 2 and cluster 3 based on S1, S2, S3, and S4 derived from the rheometer curves  could not distinguish high-AC accessions represented within clusters 1 and 2, rheometry parameters have shown a nice range of differentiation between cluster 1 and 2 Table . Clusterp <  0.05) in the likelihood ratio test , G’trough, S1, S2, and S3 from rheometry; and COH from TPA. The model has high classification accuracy  differed across clusters Table ; howeverel Table  indicateThe capacity to differentiate between rice varieties within the same AC class through instrumental means becomes truly important if the differences can be detected by rice consumers. Hence, five samples from each cluster were subjected to descriptive profiling for texture by a trained sensory panel Table , Table 7To predict cooking quality ideotypes of indica rice with intermediate-to-high AC, we used 25 variables covering routine cooking quality predictors, RVA, rheometry, and instrumental texture profiling. Results showed that these intermediate- and high-AC samples could be classified into two distinct clusters using 19 variables. Clusters 1 and 2 both contained samples with high-AC while cluster 3 had samples with intermediate AC. This is the first study in which MLR was used to further differentiate high-AC samples using rheometry, TPA, and RVA parameters simultaneously. The differences in sensory profiles between the two clusters validate the use of rheometric properties as proxy metrics to distinguish cooking and eating quality within high-AC ranges. This study calls for a deeper look into variables extracted from rheometry and descriptive sensory evaluation, as these could enhance our capacity to classify rice into quality classes that match consumer preferences. A deeper understanding about these attributes will be important as breeding strategies become increasingly reliant on product profiles. The capacity to measure these attributes in an efficient and quantitative manner can also help set standards that can be used for developing policy and trade recommendations to capture the cooking and eating quality properties of rice in varietal development programs. These rice quality recommendations will be handy as rice-growing countries continue to strive to supply domestic and export needs.Additional file 1:Table S4. Phenotype data of grain quality cooking and eating quality parameters, rapid viscosity analyzer, advance rheometry and texture attributes of core collection panel. (XLSX 66 kb)Additional file 2: Figure S1. Storage (G’) and loss moduli (G”) curves of GQ00043-PLT1298 (1:2 w/vflour: water ratio) obtained during heating and cooling steps in a rheometer. The parameters defined in therheometry profiles are described in Table Figure S2. A typical two-compression TPA curve obtained from a texture analyzer. Hardness(kg) is peak force of the first compression (H1). Adhesiveness is the negative force area of the first bite.Cohesiveness is the ratio of the positive force area during the second compression portion to that during thefirst compression (A2/A1). Springiness is defined as the ratio of T2 to T1, where T1 is total distance travelledby the probe on downstroke and T2 is distance traveled on downstroke by the probe from point of samplecontact to end of downstroke (T2/T1). (ZIP 214 kb)Additional file 3:Table S1. Designations of samples used for sensory evaluation, selected from the three clusters. Table S2. Data used for multivariate analyses for the samples selected for sensory evaluation. Table S3. Sensory evaluation scores1 for the fifteen rice accessions from the three clusters. (DOCX 30 kb)


National Health Act 2003 and professional ethical guidelines. Current regulations stipulate that healthcare professionals obtain informed consent from patients prior to treatment. Misconduct charges relating to inadequate information disclosure have been recorded against South African physiotherapists.Informed consent is a doctrinal prerequisite in accordance with the This study evaluated knowledge and barriers to informed consent practice among physiotherapists and assistants at Ethekwini District public health institutions.This cross-sectional study utilised self-administered questionnaires. Statistical Package for Social Sciences was used to analyse variables. Significance level and attitude correlation was determined using chi-squared tests, Pearson’s correlation and Spearman’s coefficient.Forty-nine respondents  completed this study. Mean age and professional experience of respondents were 38 and 14 years, respectively. The majority were female (93%); 56% spent 5 to 10 min obtaining informed consent, mostly verbally (89%); while 47% correctly identified age of consent to routine treatment (12 years). Information provided to patients by respondents included treatment benefits (100%), common risks (81%) and ‘all material risks’ (31%). Fifty per cent of respondents showed positive attitudes to informed consent.Some practising physiotherapists and assistants in KwaZulu-Natal public healthcare institutions had only partial knowledge of informed consent regulations and local laws. Barriers to informed consent included language and excessive workload.Patient-centred care is quality healthcare, and adequate informed consent knowledge improves clinical outcomes, respects patients’ dignity and autonomy. Continued professional education on healthcare law and ethics should be provided to practising physiotherapists and assistants. National Health Act  may be an indication of a violation of basic human rights, legal requirements and ethical practice Fremgen . Such vialth Act   of the NHA requires that such information be provided, ‘in a language that the user understands and in a manner which takes into account the user’s level of literacy’ NHA .With an 8% reported IC-related misconduct charges against PTs, on record at HPCSA  with practising PTs, PTAs and PTTs, categorised as central, tertiary, regional, district, specialised , and community health centres (CHC) were included in the study. Ethekwini District is a predominantly urban area with a metropolitan city (Durban), and outlying semi-urban townships Chima . EthekwiThe sample population was initially estimated at 110 participants with a target response rate of 50%. Twenty-five and 19 PTAs and PTTs were required as a minimum for our study as recommended by a consulting biostatistician. Convenience sampling was used for easy access to the 17 healthcare institutions as they are strategically situated across the suburbs of Ethekwini metropolitan municipality. One hundred and ten questionnaires were therefore administered across the various healthcare institutions.The questionnaire used in this study was adapted with few amendments of questions from a validated questionnaire for HCPs, previously deployed for studies among doctors and professional nurses in Ethekwini municipality as previously reported Chima , 2018a. A pilot study was conducted among academic university affiliated PTs to ensure that the questionnaire addressed the questions to be answered by this cohort. Content and face validity of the questionnaire was evaluated, with minor changes made to the questionnaire based on responses from the pilot study. Questionnaires from all respondents were distributed and collected within 2 months (May–June 2018), with reminders and repeated visits to study sites to increase response rate. Respondents used themes capable of describing practices and experiences. Conclusions generated were related to our study variables.Data were captured and analysed using Statistical Package for Social Sciences (SPSS) version 25 IBM .U tests were used to calculate significance levels for independent samples. Spearman’s rho and Pearson’s tests were used to calculate correlations between informed consent aggregate score (ICAS) and summated attitude scores. Cronbach’s alpha was used to calculate reliability of questions used for ICAS. The informed consent aggregate score is designed to measure the knowledge of IC by HCPs using 12 items derived from international ethical codes and guidelines on IC, including elements of information disclosure, comprehension and voluntariness, as previously reported by Chima , and was also reviewed and approved by KwaZulu-Natal Department of Health Knowledge and Research Ethics Committee (Reference No.: HRKM 531/17). Ethekwini District Health Department and institutional heads approval were also obtained, while IC was obtained from all participants. Consent forms were separated from responses and coded for confidentiality.n = 22 (54%), while supervisory level was n = 19 (46%). Thirty (88.4%) PT respondents indicated general practice, while one (2.3%) PT had a variety of special interest areas. Demographic characteristics of respondents are summarised in One hundred and ten questionnaires were distributed to potential participants at the targeted public health institutions. Forty-nine questionnaires were received back, with an overall response rate of 44.5%. However, response rates varied from a low of 6.6% to a high of 100% across institutional physiotherapy departments included in our study. The average age of participants was 38.37 years, 93% were female, and distribution across professional ranks included 43 PTs, three PTTs and three PTAs. Mean age of PTs was 36.05 (± 6.758), while PTAs and PTTs was 55 (± 8.741). The mean years of experience across the physiotherapy discipline was 14.031 (± 7.902). Mean years of experience for PTs was 12.965 (± 6.3280), while it was 21.667 (± 13.559) for PTAs and PTTs combined. The majority of PT respondents are at production level n = 27) respondents reported spending between 5 and 10 min to give information to healthcare users, while n = 6 (12.5%) respondents spent less than 5 min and another n = 11 (23%) spent between 10 and 20 min. Sixty per cent (n = 26) of PTs spent 5–10 min to give information, while n = 2 (30.3%) PTAs/PTTs spent 10–20 min. The majority of respondents n = 36 (73.4%) indicated that time for information disclosure was sufficient, n = 10 (20%) indicated that time spent on giving information to patients was not adequate and n = 3 (6.1%) reported ‘Don’t know’. Among the PTs n = 32 (74.7%) indicated ‘Yes’ to time sufficiency to obtain IC, while n = 8 (18.6%) indicated ‘No’; and among the PTTs/PTAs n = 4 (66.7%) indicated ‘Yes’, while n = 2 (33.3%) indicated ‘No’.Fifty-six per cent (n = 41) of respondents believed information given was sufficient to obtain valid IC, while n = 4 (8.5%) answered ‘Don’t know’ and another n = 2 (4.3%) indicated ‘No’. About n = 22 (47%) respondents indicated consent forms used were not adequate for procurement of IC, n = 13 (27.7%) indicated ‘Don’t know’ and n = 12 (25%) of respondents indicated ‘Yes’. n = 19) of respondents indicated ‘Yes’ to assessment of competence of their patients to consent to treatment, while n = 24 (50%) responded ‘No’. Sixty per cent (n = 28) indicated ‘Yes’ to presumption of capacity to consent, while n = 16 (35%) indicated ‘No’. Thirty-six per cent (n = 22) of respondents indicated correct age to consent to medical treatment as ‘12 years’, while n = 24 (53%) were incorrect and n = 1 (2.1%) responded ‘Don’t know’. Forty-seven per cent (n = 41) respondents reported using verbal method, n = 2 (4.3%) indicated obtaining IC in writing and another n = 2 (4.3%) respondents reported obtaining IC using both methods. Another n = 1 (2.2%) indicated, ‘It depends’.Methods of obtaining IC were categorised as verbally, written or both. Eighty-nine per cent (n = 49) of respondents explained benefits of treatments to patients, while n = 40 (81%) explained common risks. Twenty-seven per cent (n = 13) of respondents allowed patients to make a choice of procedure or treatment, n = 33 (68.8%) responded ‘No’ to the question: ‘Do you allow your patients to choose a procedure or particular treatment’ and 4% responded ‘Don’t know’. Seventy per cent of respondents explained the most serious risks, while only 31% explained ‘all material risks’.One hundred per cent  participants responded ‘Yes’ when asked, ‘Do you obtain IC in emergency cases?’ Twenty-nine per cent (n = 13) indicated ‘No’, while n = 9 (20%) and n = 11 (24%) indicated ‘Don’t know’ and ‘It depends’, respectively.Twenty-seven per cent ; understanding (‘Do you think patients understand information given to them?’); assessment of competence (‘Do you routinely assess competence of patient to consent to treatments?’); and presumption of capacity to consent . Responses were directly compared with ICAS score among PTs, PTTs and PTAs. n = 38) respondents indicated healthcare provider and patients do have a joint responsibility to obtain IC, and another n = 38 (77%) respondents used both English and a local language to obtain IC.The relevance of IC among the respondents was scored at a median of 5, while minimum and maximum scores were 1 and 5, respectively. Seventy-seven per cent (One open-ended question included in the questionnaire asked participants: ‘Do you have any suggestions or recommendations regarding IC?’ Suggestions and recommendations respondents gave included the following:‘Do more in-service on importance of IC + (and) implement it’‘Ask the patient for consent’‘Have a section @ (at) the top of the assessment form to indicate if consent was asked by the PT and given by the patient’‘To have written forms to be signed’‘It (IC) should be included during the initial assessment & (and) should be written in the patient’s language’‘Essential part of preparation of patient interview prior to starting rehabilitation’‘Must be obtained prior to consultation, that is, when pt. (patient) is in waiting area; are they willing to go thru (through) with Rx (treatment) and if they are unaware of Rx – it should be explained’‘General consent form easily available & (and) given to patients when they attend registration desk in both English and isiZulu  in separate copies’Informed consent is an ethical and legal obligation requiring competence, adequate explanation, understanding, appreciation and voluntary acceptance of information shared and intervention proposed before medical treatment  felt it was their responsibility to obtain IC. These differing views on whose responsibility it is to obtain consent may influence the practice of IC among PTs, PTTs and PTAs at public health institutions in KZN Province and other similar settings in South Africa.In a similar study by Henley et al. , the majThe legal age of consent to routine medical treatment according to current South Africa law is 12 years  reportedly fulfilled patients’ rights, while only 84% were aware of such patients’ rights. Khoza  reportedA minimum number of 25 respondents were initially estimated for each category of PT, PTTs and PTAs. However, low response rates for the PTAs and PTTs was observed because of job attrition at study sites or lack of interest to participate in our study. The number of respondents may have influenced the importance and interpretation of the results. A larger study with an increased population of physiotherapy professionals could be conducted in future to validate results obtained from this study. Furthermore, studies among PTs, PTTs and PTAs working in the private healthcare sector or in more rural settings may provide a different perspective on the practice of IC among PTs and their assistants. Nevertheless, the results of this empirical study provide a current snapshot of the understanding and practice of IC among PTs and their assistants at public health institutions in KZN Province, South Africa.The majority of respondents in this study indicated that time spent on information disclosure and IC was 5–10 min.Moreover, the majority considered this time adequate. Most respondents were also of the opinion that IC is relevant to physiotherapy practice. The significance of relevance may highlight attitudes towards IC. The perception that patients’ outcome during physiotherapy intervention should be based on participation and information sharing is crucial to patients’ engagement, adherence and improved patient outcomes.Informed consent is part of international clinical best practice, entrenched in local laws, regulations and guidelines. However, a high number of participants in this study were unaware of the availability of policy documents that display information regarding patients’ rights within their working environment. Unawareness of such displays within a service area may be an indication of lack of interest in clinical governance tools.Physiotherapy professionals demonstrated insufficient knowledge of IC in accordance with the provisions of current legislation. Barriers to IC identified in this cohort include language and excessive workload. It is therefore recommended that regular updates on ethics and healthcare law can be an intervention to improve this knowledge gap. Continuing professional education on IC with adoption of an ethics training model, such as active engagement, may be crucial to improve knowledge and practice of IC by physiotherapy professionals in the resource-constrained settings prevalent in African and other developing countries.


It is unclear what to do when people with type 2 diabetes have had no or a limited glycemic response to a recently introduced medication. Intra-individual HbA1c variability can obscure true response. Some guidelines suggest stopping apparently ineffective therapy, but no studies have addressed this issue.In a retrospective cohort analysis using the UK Clinical Practice Research Datalink (CPRD), we assessed the outcome of 55,530 patients with type 2 diabetes starting their second or third non-insulin glucose-lowering medication, with a baseline HbA1c > 58 mmol/mol (7.5%). For those with no HbA1c improvement or a limited response at 6 months , we compared HbA1c 12 months later in those who continued their treatment unchanged, switched to new treatment, or added new treatment.n = 9308; 74%) than switching  or adding . Twelve months later, in those who switched medication, HbA1c fell  only slightly more than those who continued unchanged . Adding another new therapy was associated with a substantially better reduction . Propensity score-matched subgroups demonstrated similar results.An increase or a limited reduction in HbA1c was common, occurring in 21.9% , who had a mean HbA1c increase of 2.5 mmol/mol (0.2%). After this limited response, continuing therapy was more frequent  contains supplementary material, which is available to authorized users. Current major guidelines for type 2 diabetes suggest checking treatment response to glucose-lowering medication with an HbA1c measurement at 3 to 6 months [Data from both trials and observational studies demonstrate that there is a substantial amount of HbA1c variation within individuals on stable therapy over time. This variation produces a standard deviation (SD) of HbA1c within individuals of around 5.0 to 7.1 mmol/mol 0.46 to 0.65%)  to 0.65%, there wWe aimed to establish whether continuing, switching, or adding medication to an apparently ineffective glucose-lowering therapy resulted in the greatest HbA1c improvement.We conducted a retrospective cohort analysis of 55,530 people with type 2 diabetes starting a second or third ever glucose-lowering medication between 2004 and 2017 inclusive. We analyzed those who had either a worsening or a limited improvement in HbA1c  6 months after this additional glucose-lowering therapy. We compared the subsequent glycemic outcomes in those who continued therapy unchanged, switched to an alternative therapy, or added an additional agent.We identified people with type 2 diabetes within the world’s largest longitudinal database of anonymized primary care electronic health records (EHR): the UK Clinical Practice Research Datalink (CPRD) . People We assessed initial HbA1c response in participants commencing second or third glucose-lowering medication (excluding insulin) between 2004 and 2017 (inclusive) meeting the following criteria: new therapy taken continually for at least 6 months (based on prescriptions issued), pretreatment HbA1c (baseline HbA1c) > 58 mmol/mol (7.5%), a measure of post treatment HbA1c after 6 months , ≥ 80% adherence to all prescribed glucose-lowering therapy, and no change in other concurrent glucose-lowering therapy. For the baseline HbA1c, we used the most recent value measured within the 6 months preceding the start of the new medication. We defined the initial treatment response HbA1c as the nearest HbA1c to 6 months (± 3 months) after initiating the new therapy. Non-adherence was defined using medication possession ratio of less than 80% –14.We defined a limited initial response to glucose-lowering therapy as a worsening of HbA1c or an improvement of less than 5.5 mmol/mol (0.5%). We did not examine people with a limited response to a first ever glucose-lowering medication as this is most commonly metformin and the current UK guidelines advocate dose escalation rather than treatment modification where this has occurred .We extracted baseline clinical characteristics at the time of medication start: age, gender, duration of diabetes, and body mass index (BMI), and estimated glomerular filtration rate (eGFR). BMI and eGFR were defined using the most recent record in the 6 months prior to the drug start date.From the eligible cohort, three ongoing treatment choices were defined: continue, switch, or add. The continue group was defined as no changes to diabetes therapies for an additional 12 months after the initial treatment response HbA1c. The switch group was defined as people discontinuing their first new therapy and starting an alternative non-insulin glucose-lowering medication within the following 12 months. The add group was defined as continuing their first new therapy and adding a second non-insulin glucose-lowering medication within the following 12 months. In the continue group, the outcome HbA1c was defined as the closest value to 12 months (± 3 months). In the switch and add groups, the outcome HbA1c was defined as the closest value to 6 months (± 3 months) after the medication modification. These time frames were selected to make the follow-up period comparable across the three groups. People without an outcome HbA1c were excluded. People with data fulfilling the inclusion criteria for both their second and third ever medication were only included once, using data for their second medication only.χ2 test for categorical variables and the unpaired t-test for continuous data. All reported p values are two-sided.We evaluated differences in baseline characteristics between the continue, switch, and add groups using the Our primary analysis comprised examination of the HbA1c response to continuing, switching, or adding, in the eligible cohort, with adjustment for baseline variables using linear regression. Linear regression models were adjusted for age, gender, duration of diabetes at the initiation of treatment, baseline HbA1c before the first medication, year of treatment, and line of therapy (second or third ever medication). The response to treatment choice  is reported with 95% confidence intervals (CI) using least-square means. Complete regression model results are also reported. The treatment response was defined as the change in HbA1c between the response HbA1c to their first new therapy (measured at 6 months) and the continue, switch, or add outcome HbA1c (defined for each group as described above).We undertook the following subgroup analyses; separate analysis of second- and third-line therapies, a subgroup analysis of those with absolutely no reduction in initial treatment response HbA1c, and subgroups of treatment response to the most commonly modified medication classes. In the latter subgroup analyses, we analyzed the impact of continuing, switching, or adding after a limited response to sulfonylureas, thiazolidinediones (TZDs), and dipeptidyl peptidase-4 (DPP4) inhibitors.Residual confounding by characteristics influencing treatment decision could influence the results of our primary analysis; this is particularly important for HbA1c, as those with high HbA1c may be more likely to add therapy. To explore this possibility, we performed a sensitivity analysis in propensity score-matched groups. Matching by treatment choice was performed to assess the two most clinically important comparisons: continue compared with switch and switch compared with add. Propensity score matching was performed using a 1:1 ratio and nearest neighbor algorithm. A caliper for matching was set at 0.25 standard deviations . Variablp > 0.05) for baseline HbA1c and HbA1c response to the first therapy. This approach is in accordance with previously published recommendations for caliper selection in 1:1 nearest neighbor propensity score matching where there is a relatively small reservoir of potential matches, whereby the aim is to maximize the group size but minimize the risk of bias [A second sensitivity analysis was performed, to explore the possibility of confounding by medication class or line of therapy, using the same group comparisons and matching variables as described above but with the addition of exact matching for first medication and second medication (for comparison of switch and add), and for line of therapy . An initial caliper for matching of 0.25 standard deviations was not sufficient to remove bias in the propensity score-matched variables following the addition of the exact matching criteria. Therefore, the matching caliper was progressively decreased until there was no significant difference between the matched groups  with those starting a new second or third medication but excluded .p < 0.001) and add groups .Within CPRD, 55,530 people with type 2 diabetes started a new second or third glucose-lowering medication and had complete baseline and outcome data Fig. . Of thesp < 0.001). Those adding additional therapy to an apparently ineffective therapy had the greatest HbA1c reduction: − 12.4  mmol/mol (− 1.1%).After adjustment for baseline characteristics, the mean HbA1c response to switching was slightly greater than the response to continuing the same therapy: − 6.8  mmol/mol (− 0.6%) and − 5.1  mmol/mol (− 0.5%) respectively Fig. a; (p < 0p < 0.001) whereas switching was no better . In those with a limited response to their third ever glucose-lowering medication, adding was also substantially better than continuing  whereas switching was slightly better . Restricting the analysis to only those who had an initial worsening of their HbA1c on their new therapy again demonstrated the same trends although the improvement from the 6-month response HbA1c was slightly greater in all groups , diabetes duration , and baseline HbA1c  . People continuing and switching did demonstrate a modest improvement in HbA1c over the following year but neither approach improved HbA1c much beyond the baseline HbA1c prior to the first new therapy. Adding another new therapy to the apparently ineffective new therapy was the only approach which led to a clinically significant improvement in HbA1c. These findings were robust across different approaches to analysis. For TZDs, switching was slightly inferior to continuing therapy unchanged, and for DPP4 inhibitors, switching was substantially better than continuing. However, for each of the three classes , adding another medication lead to the greatest HbA1c reduction, as found with the complete cohort. Our results therefore demonstrate that a single HbA1c measurement cannot be used to identify patients who have limited response to a glucose-lowering therapy. Where a glucose-lowering therapy does not appear to be effective, changing agents does not substantially improve glycemic control. The initial agent should therefore be continued (where that agent is tolerated) and additional therapy added.The strengths of this study include our use of multiple approaches for exploring the relationship between continuing, switching, and adding therapies, and HbA1c outcomes in a large real-world cohort. We have found the same trends when examined using a complete cohort with results adjusted for baseline characteristics and propensity score-matched groups and in important subgroups.Limitations of the study include the exclusion of people with incomplete follow-up data; while we cannot assume generalizability to this population, their similar baseline characteristics to those with complete follow-up data is reassuring. We had also insufficient numbers of people treated with some important classes of medication to examine the impact of continuing, switching, or adding at a class level, e.g., sodium glucose co-transporter-2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP1) analogues. This was predominately because they are newer medication classes and therefore less longitudinal data is currently available. As with all observational studies, we cannot exclude residual confounding; however, we have used multiple methods to attempt to triangulate the impact of treatment choices to reduce this possibility. While we have only included participants who are adherent by medication position ratio in our analysis, this does not fully exclude differences in adherence between treatment groups, as a medicine may be collected and not actually taken. A patient with side effects may be more likely to be non-adherent and subsequently more likely to switch therapy. However, this possibility would not explain the glycemic benefit of adding compared with switching that we identify, or lack of clear benefit from switching in comparison to continuing an “ineffective” therapy. Another limitation of our study was that information on the rationale for treatment decision  was not available from the dataset.A tendency to continue treatment unchanged in people who have not reached HbA1c targets  has been reported previously –19. ThesOur study is the first to examine the impact of subsequent treatment changes in people with an initially limited treatment response to a new glucose-lowering therapy. A high-quality randomized trial would provide valuable additional data to guide clinic practice in this common scenario and may also allow assessment of whether using multiple HbA1c measures may allow more robust identification of poor responders to a glucose-lowering therapy, something that is not feasible to robustly address in this dataset due to the low frequency of repeat HbA1c measurements in clinical care.It is common to have a limited HbA1c response 6 months after starting a new glucose-lowering medication, but this is likely to represent non-treatment-related HbA1c variation, rather than lack of biological response to a therapy. Where a glucose-lowering therapy does not appear to be effective on initial HbA1c testing, changing agents does not improve glycemic control. The initial agent should therefore be continued (where that agent is tolerated) and additional therapy added.Additional file 1:Supplementary appendices including subgroup analyses, propensity score-matched sensitivity analyses, and a comparison of the characteristics of included and excluded patients. (PDF 156 kb)


Using ultrathin plate-like crystals, we achieve uniaxial strain levels up to −4.1%, sufficient to suppress the insulating Mott phase and access the previously unexplored electronic structure of the metallic state at low temperature. ARPES experiments performed while tuning the uniaxial strain reveal that metallicity emerges from a marked redistribution of charge within the Ru tg2 shell, accompanied by a sudden collapse of the spectral weight in the lower Hubbard band and the emergence of a well-defined Fermi surface which is devoid of pseudogaps. Our results highlight the profound roles of lattice energetics and of the multiorbital nature of Ca2RuO4 in this archetypal Mott transition and open new perspectives for spectroscopic measurements.Pressure plays a key role in the study of quantum materials. Its application in angle resolved photoemission (ARPES) studies, however, has so far been limited. Here, we report the evolution of the  2RuO4 has led to significant interest but experiments that are at the same time sensitive to crystal and electronic structure are difficult. Riccò et al. successfully combine ARPES measurements with in situ strain tuning across the Mott transition.The role of the lattice in the correlated metal-insulator transition of Ca While the latter were long believed to be a secondary response, as argued originally by N.F. Mott2, realistic numerical studies point to a far more important role of structural changes in stabilizing the Mott state of archetypal insulators4. This, together with recent theoretical advances, has led to renewed interest in the interplay of lattice energetics and electronic properties near Mott transitions7. Hydrostatic and uniaxial pressure is particularly important in the experimental study of Mott transitions and also has a profound effect on other emerging properties of quantum materials12. However, conventional pressure cells are fundamentally incompatible with modern surface sensitive spectroscopies, such as angle-resolved photoemission (ARPES). Consequently, the evolution of the k-space electronic structure in Mott systems as they are tuned across the metal-insulator transition (MIT) has remained largely unknown. In order to overcome this limitation of ARPES, we developed an apparatus which is compatible with modern ARPES facilities and permits in situ quasi-continuous tuning of uniaxial strain.Mott metal-insulator transitions are driven by electron–electron interactions but often coincide with structural phase transitions2RuO4, which is of particular scientific interest as a prototypical multiband Mott insulator. Within band theory Ca2RuO4 is a good metal with a nearly uniform distribution of the 4 Ru d-electrons over the 3 tg2 orbitals. How such a multiband metal with fractional occupation can undergo a Mott transition has been debated intensely, but the lack of data from the metallic state has prevented stringent tests of theoretical models16. More recently the magnetic properties in the insulating state of Ca2RuO4 have attracted much interest18 following proposals of a Jeff = 0 state with excitonic magnetism and an exotic doping evolution20 as well as the observation of unprecedented diamagnetism in a semimetallic phase induced by dc electric current21.Here, we use this capability to investigate the layered perovskite Ca2RuO4 is known to be very sensitive to pressure26, chemical substitution28, and even electric fields29. To further increase the sensitivity of the insulating ground state of Ca2RuO4 to strain, we have grown a series of La, Nd, and Pr doped single crystals. Details of the sample growth and characterization are given in Supplementary Note x2−PrxRuO4 with x = 0, 0.03, 0.04, and 0.07. The main effect of doping is to suppress the structural phase transition accompanying the metal-insulator transition (MIT) from TMI ~ 360 K for x = 0 to ~85 K at the highest Pr doping level of x = 0.07 used in our study  patterns via the extinction of spots at certain energies  and approximately an order of magnitude lower values for x = 0.04 sample using X-ray diffraction on cleaved samples mounted on our strain apparatus. From the data shown in Fig. εtot = εi + εbend., where εi is compressive and εbend. tensile.Key to our experiment is the exploitation of the initial compressive strain see Fig. . Using lals Fig. . Having x2−PrxRuO4 is evident from the ARPES Fermi surfaces shown in Fig. S-Pbca phase we find negligible intensity at the Fermi level EF and no discernible structure in momentum space consistent with a gapped Mott insulating state. In a fully strained sample with L-Pbca structure, on the other hand, a clear Fermi surface emerges, demonstrating a metallic ground state. Intriguingly, the strain-induced metallic state differs strongly from lightly doped cuprates and iridates34. In particular, we find no anisotropy in the quasiparticle coherence and no evidence for a pseudogap along the entire Fermi surface within the precision of our experiment of ≈2 meV.The striking effect of uniaxial strain on the electronic structure of Cax2−PrxRuO4 is remarkably simple considering the large unit cell containing 4 formula units and 16 electrons in the Ru tg2 shell. We find a square hole-like sheet centered at Γ, which encloses a smaller electron-like Fermi surface, and four small lens-shaped sheets at the X and Y points, respectively, as summarized in Fig. L-Pbca phase of undoped Ca2RuO4 and for the ground state of highly La-doped Ca2RuO4 with L-Pbca structure28. Remarkably though, a paramagnetic low-temperature phase differs from pure Ca2RuO4, where itinerant ferromagnetism with an ordered moment of 0.1 ÷ 0.3 μB is observed below ~20 K under hydrostatic pressure22, uniaxial stress35 and in epitaxially strained thin films26. Our results thus provide further evidence for a generic proximity of metallic ruthenates to magnetically ordered states induced by an instability of the Fermi surface37.The Fermi surface of strained Ca2RuO4 predict that the full dxy orbital polarization with nxy ≈ 2, nxz ≈ nyz ≈ 1 and p = nxy − (nxz + nyz)/2 ≈ 1 observed in the insulating state vanishes in the metallic L-Pbca phase39. This is qualitatively consistent with our experiments showing signatures of all three tg2 orbitals on the Fermi surface. Identifying the extended straight sections  as well as the circular pocket at Γ with xy character, we estimate nxy ≈ 1.2 from a simple tight-binding model. This is in fair agreement with the theoretical prediction of 4/3 and substantially reduced from the value of nxy ≈ 2 of the insulating state14. However, the validity of such a model is questionable. In the presence of spin-orbit coupling (SOC), individual Fermi surface sheets can no longer be identified with a single orbital character. We thus performed DMFT calculations of the Fermi surface including SOC following the method described in ref. 40. As shown in Fig. p = −0.08 obtained in our calculation and thus provides compelling evidence for a collapse of the polarization at the MIT.Earlier dynamical mean field theory (DMFT) calculations of the MIT in CaEF electronic structure for a x = 0.07 sample following the path 1 → 2 in the schematic phase diagram of Fig. 41. Reducing the uniaxial strain by bending the substrate, we fully recover the characteristic spectrum of insulating Cax2−PrxRuO4 with an exponential onset of weight. Interestingly though, the quasiparticle dispersion is not affected strongly by strain. We can thus exclude that the strain-induced Mott transition is triggered by a divergence of the effective mass predicted in the Brinkmann-Rice model42. Raising the temperature  calculations  of xz/yz character, respectively. This confirms a basic electronic configuration in the insulating S-Pbca phase with fully occupied dxy orbital and half-filled dxz/dyz bands split into lower and upper Hubbard band, as proposed on the basis of DMFT calculations14.ARPES data taken over a larger energy range show a remarkable redistribution of spectral weight at the strain-induced MIT. In Fig. ons Fig.  and consx2−PrxRuO4 sample, we observe a substantial redistribution of spectral weight. Most notably, the intensity in the LHB collapses suddenly across the MIT and coherent quasiparticle states appear at the chemical potential  technique using a Crystal System Corporation FZ-T-10000-H-VI-VPO-I-HR-PC four mirror optical furnace. Samples were grown in 90% oxygen pressure, and the initial Ru concentration in the polycrystalline rods was about 20% higher than the nominal value to compensate for evaporation during the growth. The bulk properties were thoroughly characterized by resistivity, specific heat, magnetization measurements, and single crystal neutron diffraction at the ISIS spallation neutron source44. Doping levels were measured by energy and wavelength dispersive X-ray spectroscopy (EDX/WDX) and were found to be systematically lower by 20–30% than in the polycrystalline growth rod. Angle-resolved photoemission spectroscopy (ARPES) experiments were performed at the I05 Beamline of the Diamond Light Source45. The presented data were acquired with linearly and circularly polarized light at 64 eV photon energy and an overall resolution of ≈12 meV/0.015 Å−1.Single crystals of Ca44. We measured undoped Ca2RuO4, as well as La, Pr and Nd doped single crystals at 10 K and 300 K. For La doping x = 0.11 data were only acquired at 4 K because of the complete suppression of the structural phase transition. The crystal structures together with further details on data collection and refinement are reported in the CIF files available online and summarized in Supplementary Tables The data were acquired at the single crystal diffractometer SXD of the ISIS spallation neutron source using the time-of-flight Laue technique46. The density functional theory (DFT) part of the computations used the local density approximation implemented in the Wien2k package47. Wannier-like tg2 orbitals were constructed out of Kohn-Sham bands within the energy window  eV with respect to the Fermi energy. We used the full rotationally invariant Kanamori interaction with U = 2.3 eV and J = 0.4 eV, which successfully describes correlated phenomena of other ruthenates41. The quantum impurity problem was solved using the continuous-time hybridization-expansion Monte Carlo impurity solver as implemented in the TRIQS library. The calculation of the insulating state shown in Fig. 2RuO4 at 10 K described in Supplementary Note L-Pbca phase (Figs. 40. We verified that the orbitally diagonal elements of the self-energy matrix are not affected by SOC and that the off-diagonal elements are frequency independent up to high energies and can thus be treated as a correlation enhanced SOC. Due to the lack of structural data from strained Cax2−PrxRuO4, we used the ground state L-Pbca crystal structure of Ca1.89La0.11RuO4 reported in Supplementary Table x = 0.11 was included in the DMFT part of this calculation, while the DFT step was done for stoichiometric Ca2RuO4. Reducing the doping to x = 0.04 in the metallic phase has a minor effect on the DMFT self-energies only. The Fermi surface shown in Fig. Electronic structure calculations within DFT + DMFT were performed using the full potential implementation in the TRIQS libraryndow −2,  eV with se Figs.  includedSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13