Meliponini: Hypotrigonina) is described from Southeast Asia. Ebaiotrigona Engel & Nguyen, gen. nov., is based on the type species Lisotrigonacarpenteri Engel, recorded from Vietnam, Thailand, Laos, Cambodia, and southern China. The species was previously considered an enigmatic member of Lisotrigona Moure, but is removed to a new genus based on its unique male terminalia that differs considerably from that of Lisotrigona and instead shares resemblances with Austroplebeia Moure, and the presence of yellow maculation . It is possible that Ebaiotrigona is the extant sister group of Austroplebeia, but this requires confirmation by future phylogenetic analyses. Based on available biological observations, Ebaiotrigonacarpenteri could not be confirmed as lachryphagous as is well documented from the tear-drinking species of Lisotrigona and Pariotrigona Moure.A new genus of minute stingless bees ( Meliponini), several species stand out for their Lilliputian sizes, typically with body sizes under 4.1 mm. Concomitant with these minute proportions is the further reduction of the wing venation beyond that of most meliponines, lacking closed cells in the hind wing, lacking defined submarginal cells, and the disappearance of 2Rs+M or 3M without a bend apically .Among the considerable diversity of stingless bees  ; Division of Invertebrate Zoölogy, American Museum of Natural History, New York (AMNH); and Insect Ecology Department, Institute of Ecology & Biological Resources (IEBR), Hanoi, Vietnam. Morphological terminology follows that of Material of Taxon classificationAnimaliaHymenopteraApidaeEngel & Nguyengen. nov.C798E650-7253-50E4-B47E-6C63F1E040F3http://zoobank.org/99CCDC03-E122-46B9-81F6-D8C11E432DA2Lisotrigonacarpenteri Engel, 2000.Lisotrigona and the smallest individuals of Austroplebeia. The presence of yellow maculation on the face and mesosoma of the worker differentiates this caste from those of Lisotrigona  and have minute plumose facial setae covering the entire face, including the upper frons . While yellow maculation is shared between workers of Ebaiotrigona and Austroplebeia, the latter has a more complete wing venation , with 1Cu, 2Cu, and 3Cu present, at least as nebulous if not tubular veins, and thereby a more completely delimited subdiscoidal  cell. Additionally, Austroplebeias.str. has 6 distal hamuli , and Anteplebeina has 1M and 1cu-a confluent (versus 1M distad 1cu-a in Lisotrigona and Austroplebeias.str.). Perhaps the most dramatic differences between Ebaiotrigona and other minute stingless bees are in the male terminalia. The male terminalia of Ebaiotrigona . Setae generally pale to white; those of body fine, short, and simple, face with minutely plumose setae except on upper frons with only minute simple setae , scape with fine minute simple setae but without erect short bristles (erect short bristles present in Lisotrigona); mesoscutum with numerous erect, fine, short, simple setae; disc of mesoscutellum with similar setae to those of mesoscutum except twice as long or longer, particularly along posterior margin.⚲: Minute, total length ca 3.95–4.15 mm, forewing length ca 2.96–3.10 mm; integument generally smooth and polished, some places with widely scattered minute punctures on head and mesosoma, with distinct pale yellow maculation on face, specifically undersurface of scape, supraclypeal area, and clypeus; pronotal lobe; and sometimes as small triangle on lower parocular area, as thin line on lateral margin of mesoscutum bordering tegula and as apicolateral spots on mesoscutellar apical margin ; labrum transverse, simple, apical margin rounded; mandible with apical margin slightly oblique, largely edentate except for two small teeth on upper margin of margin , teeth well defined, acute, and incised, interdental spaces distinct but not broadly incised; labial palpomeres I and II with a few, scattered, elongate, apically arched to slightly wavy setae, such setae more clustered apically and sparse elsewhere (similar to that of Lisotrigona).Head as broad as mesosoma, slightly broader than long, with face narrower than compound eye length; vertex gently rounded, not produced or ridged; preoccipital area rounded; scape shorter than antennal-ocellar distance, not reaching median ocellus; ocelli near top of vertex; flagellomere I trapezoidal, longer than flagellomere II; flagellomere II about as long as flagellomere III, each slightly broader than long; middle flagellomeres about as long as or slightly longer than broad; intertorular distance slightly shorter than torulocular distance; upper torular tangent below facial midlength; gena rounded, narrower than compound eye in profile; supraäntennal area with triangular medial elevation bordered laterally by converging furrows forming distinct scapal basins; frontal carina absent, indicated on supraäntennal triangle by weakly demarcated ridge, otherwise a narrowly polished line to median ocellus; malar area nearly linear, subequal to or shorter than 0.5× flagellar diameter ; 1M distad 1cu-a ; submarginal slightly acute; 1M weakly arched; 2M not angled apically ; r-rs slightly longer than 3Rs. Hind wing with 5 distal hamuli; no closed cells and veins posterior to R absent except proximal arch of M+Cu.Metatibia approximately triangular, approximately 2.8–3.0× as long as greatest width; retromargin gently curved with subangulate distal superior angle, retromarginal setae and superior prolateral surface setae simple; prolateral surface shallowly concave apically, with corbicula occupying slightly less than apical half; retrolateral surface with broad keirotrichiate zone and narrow superior subglabrous zone, without defined clivulus except proximally; keirotrichiate zone approximately 4× as broad as superior glabrate zone, keirotrichiate zone extending to apical margin, with keirotrichiate zone narrowing and superior glabrate zone broadening in apical-most portion of metatibia; inferior penicillum and rastellar comb present, each composed of fine soft setae. Metabasitarsus with proventral margin straight, retrodorsal margin generally paralleling proventral margin, apical margin weakly convex to comparatively straight, distal superior angle not projecting; retrolateral surface without basal sericeous area.Metasoma subtriangular, about as wide as mesosoma, with metasomal terga smooth and shining except apical margins more matte and faintly and minutely imbricate, all terga almost glabrous, except by minute, erect, simple setae apically forming a narrow band on apical margin, such setae progressively longer, more abundant, and more spread on apical-most terga; sterna largely smooth and glabrous, with long to elongate, fine, erect, simple setae apically.Ebaiotrigona truly lack facial maculation, but this will require confirmation through extensive future sampling of males throughout the range of the species], except pronotal lobe consistently yellow. Scape shorter and broader than that of worker  and the generic name Trigona Jurine. The gender of the name is feminine.The new generic name is a combination of the Ancient Greek adjective Ebaiotrigonacarpenteri (Engel), new combination.Currently, the genus includes only the type species, Ebaiotrigona.The following modifications to the key of Taxon classificationAnimaliaHymenopteraApidae(Engel)comb. nov.575C2D28-D4B0-5DC4-A161-410B5976B64BLisotrigonacarpenteriAMNH). Engel, 2000: 232. Holotype ⚲  1.04 mm, 1.19 mm, respectively; compound eye length, upper and lower interorbital distances 0.92 mm, 0.53 mm, 0.45 mm, respectively.Compound strongly converging below, inner orbits only feebly concave above; compound eye length greater than upper interocular distance; antennae arising below lower third of face so that distance from lower clypeal margin to lower margin of antennal torulus is about one-half distance from upper margin of torulus to lower margin of median ocellus; clypeus about twice as broad as long, lower margin scarcely below lower ocular tangent, upper margin separated from antennal torulus by about one-half torular diameter; head surface in vicinity of antennal toruli depressed, not visible in profile; interocellar distance over twice ocellocular distance, almost 3× ocellar diameter; ocelli on submit of vertex, head surface declivitous immediately behind lateral ocelli with no ridge or carina; genal area much narrower than compound eye in profile; malar area linear ; mandibles comparatively long, conspicuously crossing one another, mandibular apex slender, apical margin oblique with lower apical angle pointed, upper portion of apical margin faintly sinuous and simulating a faint preapical bump (scarcely large enough to denote as a “tooth”) Fig. ; antennaIntegument of head and mesosoma black except basal one-third of scape , mandible, labrum, clypeus, spot between antennae, mesoscutellum, metanotum, propodeum, legs except coxae and femora, and all metasomal segments, brown or suffused with brown ranging to black ; pronotal lobe markedly yellow; protibia, meso- and metatibial apices, and tarsi light yellowish brown; tegulae yellowish brown; wings clear, veins light brown; stigma translucent, light brownish.Integument shining, smooth, head and mesosoma with well separated, fine, small punctures, punctures on lateral part of mesoscutum denser; punctures on mesoscutellum much stronger and denser than those on mesoscutum, separated by 2–3× a puncture width; mesepisternum centrally with coarse punctures separated by 2–3× a puncture width, otherwise integument between punctures smooth; basal area of propodeum shining, minutely tessellate, glabrous; posterior surface smooth, shining. Metasomal terga smooth except minute punctures on narrow posterior margins.Pubescence mostly exceptionally short, mostly yellowish white, setae of lower half of face white and conspicuously plumose, other setae simple or nearly so. Posterior part of mesoscutellum with zone of upcurved setae, longest on body except for some on apical margin of clypeus and mandible, about 2.5× as long as median ocellar diameter, otherwise long setae (nearly 2× ocellar diameter) sparse on apical margin of clypeus, vertex, coxae, and trochanters, rather numerous on mandibular lower margin. Setae of mesoscutum short, some setae at apicolateral corner longer, about as long as ocellar diameter. Metasomal terga I–IV glabrous except for minute erect setae on posterior margins.AMNH) is of the darker morph.Workers exhibit noticeable variation in overall coloration, and can loosely fall into a lighter and dark morph, although there are individuals who seemingly intergrade and so these morphs are not discrete Figs , 5–7. Ge20°21.35'N, 105°39.47'E], 22 June 2021, coll. Tuấn Anh Trương et al.  (IEBR); 18⚲⚲, Yên Hân [Community], Chợ Mới [District], Bắc Kạn [Province], 27 June 2021, coll. Tuấn Anh Trương et al.  (IEBR); 53⚲⚲, Nam Cường [Community], Chợ Đồn [District], Bắc Kạn [Province], coll. Tuấn Anh Trương et al. ; 21⚲⚲, Lân Nghè, Hữu Liên Natural Reserve, Hữu Liên [District], Lạng Sơn [Province], 21°33'48.6"N, 106°24'36.4"E, ca 289 m, 11 June 2018, coll. Liên Thị Phương Nguyễn et al. (IEBR).Vietnam: 1♂, 67⚲⚲, Tân Thành [Village], Yên Thịnh [Community], Yên Thủy [District], Hòa Bình [Province] , Hà Tĩnh [Province], Pariotrigona . In Pariotrigona the basal bulb is smaller and somewhat transverse, and is abruptly narrowed to the thin elongate process. In Lisotrigona, the penis valves are comparatively short relative to the overall size of the genitalia, with a broad basal bulb and tapering to a comparatively short process that is scarcely longer than the bulb. While the Asian and Australian minute species lack a spatha, Pariotrigona is an outlier in that the spatha is present as a large membranous structure. The form of each of these genera is seemingly apomorphic when compared to putative outgroups among related members of the Hypotrigona group of genera . A further elaboration of the nests and biology of E.carpenteri will be of considerable significance for understanding the distribution and occurrence of lachyrphagy in Asiatic Meliponini. If true, lachryphagy is restricted to Lisotrigona and Pariotrigona, then it may be that this is a biological synapomorphy for these two genera, with Ebaiotrigona more closely allied to Austroplebeia. Ideally, once the biology has been more fully elucidated for E.carpenteri, an analysis of morphological and molecular data can be undertaken for the Hypotrigona group (Table The nests and the biology of up Table  to explo


In the context of nuclear medicine and theranostics, integrin-related research and development was, for most of the time, focused predominantly on 'RGD peptides' and the subtype αvβ3-integrin. However, there are no less than 24 known integrins, and peptides without the RGD sequence as well as non-peptidic ligands play an equally important role as selective integrin ligands. On the other hand, multimerization is a well-established method to increase the avidity of binding structures, but multimeric radiopharmaceuticals have not made their way into clinics yet. In this review, we describe how these aspects have been interwoven in the framework of the German Research Foundation's multi-group interdisciplinary funding scheme CRC 824, yielding a series of potent PET imaging agents for selective imaging of various integrin subtypes.The gallium-68 chelator TRAP was utilized to elaborate symmetrical trimers of various peptidic and non-peptidic integrin ligands. Preclinical data suggested a high potential of the resulting Ga-68-tracers for PET-imaging of the integrins α5β1, αvβ8, αvβ6, and αvβ3. For the first three, we provide some additional immunohistochemistry data in human cancers, which suggest several future clinical applications. Finally, application of αvβ3- and αvβ6-integrin tracers in pancreatic carcinoma patients revealed that unlike αvβ3-targeted PET, αvβ6-integrin PET is not characterized by off-target uptake and thus, enables a substantially improved imaging of this type of cancer.Novel radiopharmaceuticals targeting a number of different integrins, above all, αvβ6, have proven their clinical potential and will play an increasingly important role in future theranostics. Multimerization is a venerable concept, and its theoretical foundations have been established decades ago . There i18F-Galacto-RGD  . These are widely referred to as 'RGD peptides,' and a clear distinction between the different compounds is rarely made.A look on the wealth of pertinent literature reveals that the terms "integrin," "αvβ3," "RGD" and "(neo-)angiogenesis" are often closely associated. Frequently, they are even used in a synonymous manner , which mBy 1994, Cheresh and coworkers found that αvβ3-integrin plays a major role in angiogenesis, i.e., the sprouting of new vessels from existing ones  . This prThis notion is somewhat problematic, not because it is incorrect, but because it does not picture reality in its entirety. First, there are also integrin-independent pathways that regulate angiogenesis, such as vascular endothelial growth factor receptor 2 (VEGFR2) signaling . Second,18F]FDG, quietly disregarding the question whether a tracer uptake might actually be related to angiogenesis or not     type, it is hard to imagine that a greater variety of conjugates has been generated for any other small-molecule targeting motif. The same might apply to multimers thereof . The imp68 Ga-chelator TRAP ], which was originally developed at Charles University in Prague in the late 2000's   , which, NMe)K] . Trimeriarcinoma . Figure C lesion . A compay stages  undersco68 Ga-labeled trimeric αvβ6-integrin-targeted PET radiopharmaceutical shows excellent performance for imaging of pancreatic carcinoma in a clinical setting. Hence, we believe that multimeric probes in molecular imaging are no longer a future vision but, from now on, should be considered clinical reality [The functional diversity of the 24 different integrins bears a huge, largely untapped potential for novel theranostic approaches, particularly in the field of nuclear medicine. In this review, we outlined some lines of thought on how this hidden treasure could be lifted in the future, driven by novel, selective ligands, and optimized radiopharmaceuticals. We described that multimers of integrin ligands often display superior performance at the preclinical stage and furthermore demonstrated that a


Ki-67 is a marker of cell proliferation, used as an important diagnostic marker in the pathologic differentiation of various lesions. It is also relevant for developing targeted molecular therapies. We carried out a systematic review to assess the Ki-67 labeling index (LI) in odontogenic cysts and tumors. Databases were searched, including PubMed (MEDLINE), Scopus, CINHAL, PsycoInfo, the Cochrane Library, and Proquest. The meta-analysis was carried out based on the data of 608 lesions. When a 5% cut-off point was set, ki-67 LI of all benign odontogenic tumors dropped below this point. All the malignant tumors demonstrated an LI of over 15.3%; a significantly higher Ki-67 LI in malignant odontogenic lesions (17.59±2.80) was observed. Among benign tumors, the largest and the smallest Ki-67 LIs were seen in ameloblastoma (4.39±0.47) and adenomatoid odontogenic tumor (0.91±1.71). The mean values of Ki-67 LI in tumors and cysts were 4.23 (0.38) and 1.04 (0.07), respectively. Among odontogenic cysts, the highest Ki-67 LI was found in odontogenic keratocyst (OKC) (3.58±0.51), and the lowest in the radicular cyst (1.29±0.62%). Ki-67 LIs in all odontogenic cysts were <3%, except for OKC. This controversial lesion seems to have a profile more similar to a tumor, and a treatment plan similar to tumors might be suggested. We found that odontogenic lesions have diverse proliferative activities that help differentiate between various lesions and suggest therapeutic plans. OKCs have a different origin, progression course, and biological behavior from other odontogenic cysts, e.g., dentigerous or radicular cysts.2 Relatively high mitotic activity and increased epithelial cell turnover rates have been proposed to account for the growth of OKCs.3 In 2005, OKC was classified as a benign odontogenic tumor in the WHO classification; however, it is still debated whether OKC is a benign tumor or an odontogenic cyst.4Odontogenic lesions are one of the most common pathologic entities in the jaw region. Odontogenic cysts arise from different types of odontogenic epithelial cell types. The most common odontogenic cyst is the dentigerous cyst, which accounts for 20% of all the mandibular epithelial cysts.5 Unicystic ameloblastoma displays a lining of focal or fully ameloblastic epithelium upon histopathological examination.6Ameloblastoma, the most prevalent clinically significant odontogenic tumor, comprises 1% of all jaw cysts or tumors, and 10‒45.2% of all odontogenic tumors. Ameloblastoma is a benign tumor with a tendency for local invasion and a high recurrence rate; it is divided into solid/multicystic, unicystic, and peripheral subtypes.8Ameloblastic carcinoma is a malignant epithelial odontogenic tumor that might develop within an existing ameloblastoma.10 Ki-67 is involved in all the active stages of the cell cycle , but is not present in the resting G0 phase.11 The most common standard for evaluating the tumor for Ki-67 expression is to calculate the percentage of positive nuclear staining with an anti-Ki-67 antibody, which is described as the Ki-67 labeling index (LI).12 Ki-67 expression has been used in the diagnosis of many different human tumors, e.g., breast cancer, meningioma, prostate cancer, non-Hodgkin lymphoma, soft tissue sarcoma, etc.13Abnormal cell proliferation is an essential feature of tumorigenesis. Ki-67 is a 319–358 kDa protein, considered a reliable marker for the proportion of proliferating cells and to predict the lesion’s behavior.14 In the maxillofacial region, Ki-67 has been used for distinguishing between adenoid cystic carcinoma (ACC) and polymorphous low-grade adenocarcinoma (PLGA), two histopathologically similar lesions, but with different biological behavior and clinical outcome. Based on previous studies, the mean Ki-67 LI for PLGA and ACC has been reported to be 1.24% and 3.71%, respectively.15 The Ki-67 LI has also been used to distinguish between two other lesions, basal cell adenocarcinoma (BCAC) and basal cell adenoma. The mean Ki-67 LI values in BCAC and BCA were 4.4% and 2.1%, respectively. The authors suggested a Ki-67 LI cut-off point of 5% and observed that 91% of BCAC LI values were higher than this cut-off point. Accordingly, a high Ki67 LI value could be helpful in BCAC diagnosis.16Ki-67 is a diagnostic molecular marker involved in the growth of cancer cells. It is often assayed during the histopathological examination and has also been used for developing therapeutic methods.14 This study aimed to systematically review the role of Ki-67 LI in odontogenic cysts and tumors. To the best of our knowledge, this is the first review assessing Ki-67 LI in odontogenic lesions.Ki-67 is a diagnostic and prognostic marker based on many statistical and laboratory findings and is, therefore, a promising candidate to differentiate between various lesions and suggest the best treatment approach.This systematic review was carried out based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines for reporting systematic reviews.PubMed (MEDLINE), Scopus, CINHAL, PsycoInfo, the Cochrane Library, and Proquest were used as the search databases.The key words were selected based on Medical Subject Heading (MeSH) terms. The studies were retrieved by searching for the following keywords:“odontogenesis”, “odontogenic cyst”, “keratocyst”, “odontogenic keratocyst”, “radicular cyst”, “ki67”, “malignant ameloblastoma”, “ameloblastic sarcoma”, “ameloblastic fibrosarcoma”, “odontogenic carcinoma”, “odontogenic malignancy”, “CEOT”, “KCOT”, “MIB-1 antigen”, “MIB-1 protein”, “antigen MIB 1”, “protein MIB 1”, “Ki67 antigen”, “dentigerous cyst”, “radicular cyst”, “follicular cyst”, “periapical cyst”, “calcifying odontogenic cyst”, “residual cyst”, “glandular odontogenic cyst”, “dental tissue neoplasm”, “odontogenic tumor”, “odontogenic tumor”, “odontogenic adenomatoid tumor”, “odontogenic adenomatoid tumor”, “odontogenic neoplasm”, “odontogenic cancer”, “odontoma”, ”ondontome”, “squamous odontogenic tumor”, “meloblastic sarcoma”, “ameloblastic fibrosarcoma”, “odontogenic sarcoma”, “malignant odontogenic tumor”, “benign odontogenic myxoma”, “ameloblastic fibroma”, “squamous odontogenic tumour” and “calcifying epithelial odontogenic tumor” from 1990 to 2019 (August).The inclusion criteria were as follows:Studies on the Ki-67 expression levels in odontogenic cysts and tumors published from 1990 to 2019 (August). Articles published in the English language with full-text availability. Ki-67 values expressed as a percentage or LI evaluated by counting  ×100 Ki-67 LI reported as mean ± SD The following studies were excluded: non-human research, reviews, letters, editorials, expert opinions, articles written in a language other than English, studies that reported ranges instead of means ± SD, studies in which the value was not expressed as a percentage, articles of poor quality, studies that did not include qualitative staining and immunohistochemistry analysis, duplicated articles (risk of bias).Using the CASP  quality checklist, the included articles were independently assessed by two specialists in oral and maxillofacial pathology (M.K. and M.V.), and low-quality studies were excluded.Two investigators (M.K. and M.J.) independently extracted the articles’ information and summarized them in the standardized extraction form. Extracted data comprised: first author’s name, year of publication, study type, sample size, mean values and standard deviations of Ki-67 expression, lesion type (tumor/cyst), and the lesion name. Endnote X5 Resource Management Software was used to organize titles and abstracts of the articles and identify duplicates.2 were used to determine heterogeneity. A probability value of I2 value >50% and P value<0.10 for Cochran’s Q indicated significant heterogeneity. Based on heterogeneity analysis, fixed or random-effects models were used. Subgroup analysis was performed based on the type of lesion, the lesion’s name, and the sample size. Statistical significance was set at P<0.05.The Comprehensive Meta-Analysis  software, version 2.0, was used for data analysis. Mean and standard deviation values of ki-67 expression for each study were extracted from the selected articles. Q statistics and IThe initial search retrieved 1458 articles. Of 1458 papers, 859 studies remained to be assessed after the duplicates were removed. A total of 449 articles were excluded after reviewing the titles and abstracts, and the remaining 150 full-text articles were separately reviewed for quality by two investigators. 17 the mean score of Ki-67 was reported instead of Ki-67 LI or percentage. In the study by Otero et al,18 the mean ± SD was not reported separately for each lesion, and only a broad comparison was made. Also, in an article by Sathi et al,19 the results of immunohistochemical staining were not reported as mean ± SD, and therefore, it was removed from the study. Finally, 18 studies were included in this meta-analysis. At this stage, 132 studies were removed because the inclusion and exclusion criteria were not met. A few studies were excluded because of the method they used to report the immunohistochemical results. For example, in one study by Ramadoss et al,P<0.001) showed statistically significant publication bias.An estimation of potential publication bias was carried out using the funnel plot. The asymmetry of the plot suggested publication bias . Egger l31 The results revealed that the mean of Ki-67 expression in tumors was as follows: pooled mean=4.23, SD=0.38 (P<0.001). Considering the significant heterogeneity , the random-effects model was used to calculate pooled LI mean at 95% CI . Considering the significant heterogeneity , the random-effects model was used to calculate pooled LI mean at 95% CI  were higher than ameloblastoma (4.2 ± 3.6%). The findings of Bello et al25 provide additional support for the statistically significant difference in Ki-67 LI levels between ameloblastoma and ameloblastic carcinoma.Although Ki-67 has increasingly been studied as a prognostic indicator in recent years, this marker’s more critical diagnostic role was emphasized in the present study. Simple histopathological techniques are not always able to differentiate between these challenging lesions. Random-effects model analysis was carried out for this purpose. Significantly higher Ki-67 LI levels were noted in ameloblastic carcinoma (17.59 ± 2.80%). The mean value obtained was significantly different from that in benign odontogenic lesions, consistent with previous studies. As observed by Yanamoto et al,38 reported higher Ki-67 expression in the malignant component of carcinoma tissue in pleomorphic adenoma than the benign component. The results were obtained from 29 cases of salivary gland tumors and the Ki-67 LI range for benign tumors was 0.5–6%, but the range was 3.6%–50% for malignant tumors. Therefore, the estimation of the potential proliferation rate of salivary gland tumors and identifying a subgroup of patients with invasive tumors, are useful in diagnostic pathology. This approach can differentiate between benign and malignant lesions and predict the prognosis of salivary gland tumors. In another study by Tadbir et al39 on Ki-67 expression in salivary gland tumors, the Ki-67 LI levels in malignant tumors (10.74 ± 10.8%) were higher than benign tumors, such as pleomorphic adenoma (0.78±0.2%).The same crucial diagnostic question has been explored in studies carried out on lesions in other anatomical areas, and the results are comparable to our findings. Faur et alP< 0.001) compared with odontogenic cysts . In a similar study, Piattelli et al23 found that Ki-67 expression in unicystic ameloblastoma was higher than in dentigerous cysts.We also found that Ki-67 LI levels were higher in odontogenic tumors , odontoma (2.96 ± 1.77%), and AOT (0.91 ± 1.71%) in descending order. Accordingly, the lowest Ki-67 expression among odontogenic tumors was seen in AOT. These results will be helpful in the pathologic diagnosis and differentiation of these tumors.After ameloblastic carcinoma, the highest Ki-67 expression was seen in ameloblastoma (4.39 ± 0.47%), followed by unicystic ameloblastoma (4.12 ± 0.85%). A study by Olimid et alAs shown in Among odontogenic cysts, the highest Ki-67 LI expression was seen in OKC (3.58 ± 0.51%), and the lowest was observed in radicular cysts (0.98 ± 0.47%). These findings might be helpful in the histopathological analysis of cysts, especially when inflammation is present.40 Tsuneki et al34 reported that the expression level of Ki-67 in OKC was higher than that in dentigerous and radicular cysts. However, in contrast to our findings, Živković et al37 found that Ki-67 expression in OKC was lower than both dentigerous and radicular cysts; this finding might be explained because the evaluation only concerned the suprabasal layer. On the other hand, in a study by Kuroyanagi et al40 the expression level of Ki-67 was assessed in both the basal and suprabasal layers, and they observed higher Ki-67 expression in OKC compared with other odontogenic cysts. In another study by Pan et al,41 on OKC’s epithelial lining, Ki-67 immunopositive cells were found in both the basal and suprabasal layers.The order of Ki-67 LI expression in odontogenic cysts was as follows: OKC (3.58±0.51%), COC (2.43±0.65%), dentigerous cysts (1.29±0.62%), and radicular cysts (0.98 ± 0.47%) in descending order. These findings suggest that the assessment of Ki-67 LI in OKC during pathological diagnosis might be useful for diagnostic and therapeutic purposes.42 reported higher Ki-67 expression in OKC compared with inflammatory and developmental cysts.In the present systematic review, covering 118 cases of OKC, 92 cases of radicular cysts, 33 cases of calcified odontogenic cysts (COC), and 48 cases of dentigerous cysts, we found the highest Ki-67 LI expression in OKC and the lowest expression in radicular cysts . Therefo29According to the data summarized in 2 studies with statistical values that were <50%, we used the fixed-effects model and the Mantel-Hansel method, but in other studies where the values were >50% or the P-value was <0.05, the random-effects model was the most reliable option to calculate the overall effect size. The included studies’ heterogeneity might be caused by different methodological techniques, imperfect reporting of cases, absence of true case verification, or other uncertain reasons. Despite these limitations, we believe that crucial diagnostic information can be extracted from the meta‐analysis since the results were based on the best available evidence.In the present study, the search strategy was limited to articles published in English. Articles that might contain data with potential high quality, written in other languages, were not included because of difficulties with accurate medical translation. In IIn this systematic review, we found that when a cut-off point of 5% was set, ki-67 LI of all benign odontogenic tumors dropped below this point. All malignant tumors demonstrated an LI of >15.3%; a significantly higher Ki-67 LI in malignant odontogenic lesions (17.59±2.80) was observed. Among odontogenic tumors, the maximum and the minimum Ki-67 LIs were found in ameloblastoma (4.39±0.47) and AOT (0.91±1.71). Among odontogenic cysts, the highest Ki-67 LI was seen in OKC (3.58±0.51), with the lowest for radicular cysts (1.29±0.62). Ki-67 LI in all odontogenic cysts was <3%, except for OKC. OKC might be more similar to a tumor, and a treatment plan similar to that for tumors might be suggested. Therefore, odontogenic lesions have diverse proliferative activities that help differentiate between various lesions and suggest therapeutic plans.MJ: acquisition of data, drafting the work, revising the paper, and approving final draft. FPA: analysis and interpretation of data, drafting the work, revising the paper, and approving final draft. MVS: acquisition of data, revising the paper, and approving final draft. MK: concept and design of the work, interpretation of data, drafting the work, revising the paper, and approving final draft. MRH: interpretation of data for the work, drafting the work, revising the paper, and approving final draft. NV: acquisition a of data, revising the paper, and approving final draft.This paper is based on a thesis submitted for DDS degree (NO. 62900) and was financially supported by the Research Council of Tabriz University of Medical Sciences.The authors declare no conflict(s) of interest related to the publication of this work.The research protocol was approved by the Ethics Committee of Tabriz University of Medical Sciences (IR.TBZMED.VCR.REC. 1398.331).


Amorphophallus xiei intercropped with maize (intercropping condition) or grown in an adjacent open site (monoculture condition). Both intercropping leaves and monoculture leaves exhibited very fast induction responses. The times taken to achieve 90% maximum net photosynthetic rate in intercropping leaves and monoculture leaves were 198.3 ± 27.4 s and 223.7 ± 20.5 s during the photosynthetic induction, respectively. During an 8-min simulated sunfleck, the proportion of excess excited energy dissipated through the xanthophyll cycle-dependent pathway (ΦNPQ) and dissipated through constitutive thermal dissipation and the fluorescence  pathway increased quickly to its maximum, and then plateaued slowly to a steady state in both intercropping and monoculture leaves. When the illumination was gradually increased within photosystem II (PSII), ΦNPQ increased quicker and to a higher level in monoculture leaves than in intercropping leaves. Relative to their monoculture counterparts, intercropping leaves exhibited a significantly lower accumulation of oxygen free radicals, a significantly higher content of chlorophyll, and a similar content of malondialdehyde. Although monoculture leaves exhibited a larger mass-based pool size of xanthophyll cycle [V (violaxanthin) + A (antheraxanthin) + Z (zeaxanthin)] than intercropping leaves, intercropping leaves had a higher ratio of (Z + A)/(V + Z + A) than monoculture leaves. intercropping leaves had markedly higher glutathione content and ascorbate-peroxidase activity than their monoculture counterparts. Similar activities of catalase, peroxidase, dehydroascorbate reductase, and monodehydroascorbate were found in both systems. Only superoxide dismutase activity and ascorbate content were lower in the intercropping leaves than in their monoculture counterparts. Overall, the xanthophyll cycle-dependent energy dissipation and the enzymatic antioxidant defense system are important for protecting plants from photooxidation in an intercropping system with intense sunflecks.Photosynthetic and photoprotective responses to simulated sunflecks were examined in the shade-demanding crop  Crop production is a complex light-driven system. Around 90% of the dry matter accumulated in crops comes from photosynthetic production , and thuZea mays) with soybean (Glycine max) can reduce the incidence of leaf blight and rust and reduce the density of weeds , Yunnan Province, China. The annual average precipitation and temperature at the study site is 1654.6 mm and 19.6°C, respectively. The rainy season is from May to October, when the total precipitation accounts for 90% of annual precipitation. The dry season is from November to April.A. xiei was first found in the understory of a tropical forest in the southwest of Yunnan Province, China, more than 14 years ago , 120 kg P2O5 ha–1 (superphosphate), 100 kg K2O ha–1 (potassium chloride), and 20,000 kg organic fertilizer ha–1. Besides the N fertilizer, the remaining was used as a base fertilizer. Thirty percent of N was used as a base fertilizer, and 70% of N as topdressing that was applied 50 days after seedling transplantation. All plots were watered by an automatic irrigation system. The crops were well protected from diseases, pests, and weeds. Field measurements were conducted in the middle of August when A. xiei was growing vigorously. Leaf discs was punched, submerged into liquid nitrogen, and transported to the lab for biochemical analysis.The variety of maize used in the present experiment is Yunrui 8, a variety widely cultivated in Yunnan Province, China, and commonly used in intercropping system with –2).The tagged leaves were cut down, and the area of these leaves was determined using a leaf area meter . Afterward, the leaves were oven dried to constant weight. Specific leaf area (SLA) was calculated as the ratio of leaf area to leaf dry weight (g cm2-response curves were completed between 09:30 and 11:30 using a portable photosynthesis apparatus (LI-6400XT). The tagged leaves were enclosed in a chamber where light was controlled at 800 μmol m–2 s–1 by a built-in LED light source, temperature at 25°C by setting block temperature, and CO2 concentration at 380 μmol mol–1 by a CO2 injection system. During the measurements of light-response curves, temperature and CO2 concentration in the chamber was retained at 25°C and 380 μmol mol–1, respectively, but light intensity was sequentially reduced in the following order: 1,500, 1,000, 800, 600, 400, 300, 200, 150, 100, 80, 60, 40, 20, and 0 μmol m–2 s–1. To complete the measurements, an automatic measurement program of light-response curves was conducted. The relationship between net photosynthetic assimilation (Pn) and photon flux density (PFD) was fitted to a non-rectangular hyperbola (Pn (PFD) = Pmax-c × eb*PFD–, where Pmax is the maximum net photosynthetic assimilation under saturating light. The light saturating point (LSP) was estimated from ln (0.1 × a/c) × (−1/b). The apparent quantum yield (AQY) and light compensation point (LCP) were estimated as the slope and x-intercept of the linear region of the light response curve, respectively.Light-response curves and COyperbola : Pn (PFDPn and intercellular CO2 concentration (Ci) was evaluated by a CO2 response curve. The tagged leaves were enclosed in a chamber where temperature, PFD, and CO2 concentration were adjusted to 25°C, 800 μmol m–2 s–1, and 400μmol mol–1, respectively, and the leaf was fully induced by illumination for more than 15 min. Afterward, an automatic program of CO2 response curves was conducted to measure the change in gas exchange rate with varied CO2 concentration. The CO2 concentration in the chamber was varied in the following order: 400, 300, 200, 150, 100, 50, 400, 600, 800, 1,000, and 1,200 μmol mol–1. CO2 response curves were obtained by fitting the data to a non-rectangular hyperbola. Carboxylation efficiency (CE) was estimated by the slope of the linear region of the CO2 response curve.The relationship between –2 s–1. When the readings were completely stable, gas exchange rate was then automatically recorded at 30-s intervals. Afterward, PPFD was increased by one step to a high light level of 1,500 μmol m–2 s–1, and gas exchange rate was automatically recorded at 30-s intervals until stable readings were achieved. The process of photosynthetic induction was completed through an automatic program of time-lamp responses. The temporal change in Pnet during photosynthetic induction was fitted to a non-rectangular hyperbola  were calculated from the photosynthetic induction curve.where a fluorescence yields (F0 and Fm) were measured in the fully dark-adapted leaves, and the maximum efficiency of PSII photochemistry (Fv/Fm) was calculated as (Fm–F0)/Fm. Minimum, variable, maximum, and steady-state fluorescence intensity  were recorded in measurements of light response curves and photosynthetic induction curves. Maximum efficiency of PSII photochemistry in the light (Fv′/Fm′) was calculated as (Fm′–F0′)/Fm′, actual efficiency of PSII photochemistry in the light (ΔF/Fm′) as (Fm′–Fs)/Fm′, electron transport rate (ETR) as ΔF/Fm′ × PFD × 0.84 × 0.5, non-photochemical quenching (NPQ) as (Fm–Fm′)/Fm′, and photochemical quenching (qP) as (Fm′–Fs)/(Fm′–F0′).At predawn, minimum and maximum chlorophyll (Chl) f,d = Fs/Fm, (2) ΦPSII = (Fm′–Fs)/Fm′, and (3) ΦNPQ = Fs/Fm′–Fs/Fm. Φf, d is the sum of the fractions of light energy absorbed by PSII that is lost by either constitutive thermal dissipation or via fluorescence, ΦPSII corresponds to the fraction of absorbed light energy that is consumed by photosynthetic electron transport, and ΦNPQ is the fraction of absorbed light energy that is dissipated thermally via light-dependent non-photochemical quenching processes. The allocation of absorbed light energy by PSII was analyzed in the fully light-induced leaves that were used to measure light response curves, and in the thoroughly dark-adapted leaves that were used to measure photosynthetic induction curves.The fate of absorbed light energy by PSII was estimated by the method described by 440, obtained from pure pigments.At midday on a clear day in August, leaf discs (2 cm in diameter) were punched from the tagged plants, and were then immediately submerged in liquid nitrogen. The samplings were taken to the lab for biochemical analysis. Carotenoid and Chl content was determined according to the method described by g for 10 min using an analytical method based on the reduction of ferric ion to ferrous ion. Reduced glutathione was assessed by dithio-bis-nitrobenzoic acid (DTNB) colorimetry at an absorbance of 412 nm as described by 2-EDTA, followed by centrifugation at 12,000 g for 20 min. Afterward, 0.5 mL phosphate buffer  and 0.5 mL DTNB (4 mM) were added into 1 mL supernatant, respectively.Reduced ascorbate was determined as described by g) were crushed into fine powder with a mortar and pestle under liquid nitrogen. Homogenates for antioxidant enzyme analyses were prepared according to the method described by 4-EDTA, and 200 μL enzyme extract. The reaction mixtures were illuminated for 15 min at a light intensity of 4,000 lx. One unit of SOD activity was defined as the amount of the enzyme required to induce 50% inhibition of nitroblue tetrazolium, as monitored at 560 nm.Leaves harvested at midday on a clear day were immediately frozen in liquid nitrogen. Frozen leaves , which decomposes Ht 240 nm . Each 3-2O2, and 200 μL enzyme extract. One unit of POD activity was expressed as μmol guaiacol oxidation min–1 g–1 fresh weight leaves.Peroxidase (POD) activity was assayed by monitoring the increase in absorbance at 470 nm caused by guaiacol oxidation. Each 3-mL reaction mixture contained 50 mM phosphate buffer (pH 7.0), 16 mM guaiacol, 10 mM H2, 0.3 mM ascorbic acid, 60 μM H2O2, and 200 μL enzyme extract. Enzyme activity was determined by monitoring the decrease in absorbance at 290 nm. One unit of APX activity was expressed as μmol ascorbate oxidized min–1 g–1 fresh weight leaves.Ascorbate peroxidase (APX) activity was analyzed according to the method described by –1 g–1 fresh weight leaves.Glutathione reductase (GR) activity was determined by monitoring the disappearance of NADPH at 340 nm. Each 3-mL reaction mixture contained 100 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, 0.2 mM NADPH, 0.5 mM oxidized glutathione, and 200 μL enzyme extract. One unit of GR activity was expressed as μmol NADPH oxidation min–1 g–1 fresh weight leaves.Monodehydroascorbate (MDHAR) activity was measured by monitoring decreases in absorbance at 340 nm, which mirrored the oxidation of NADPH. Each 3-mL reaction mixture contained 25 mM potassium phosphate (pH 7.8), 0.2 mM EDTA, 0.1 mM ascorbate, 0.5 U of ascorbate oxidase, 0.1 mM NADPH, and 100 μL enzyme extract. One unit of MDHAR activity was expressed as μmol NADH min–1 g–1 fresh weight leaves.Dehydroascorbate reductase (DHAR) activity was measured by monitoring the increase in absorbance at 265 nm due to the formation of ascorbate. Each 3-mL reaction mixture contained 100 mM potassium phosphate (pH 7.0), 50 mM reduced glutathione, 5 mM dehydroascorbate acid, and 100 μL enzyme extract. One unit of DHAR activity was expressed as μmol reduced ascorbate min2–) were measured according to the method of 2. Fresh leaf samples (1.0 g) were ground with liquid nitrogen, homogenized in 5 mL of 50 mM phosphate buffer (pH 7.8), and centrifuged at 12,000 g for 10 min at 4°C. The resulting supernatant was then re-centrifuged at 18,000 g for 20 min at 4°C. Reaction mixtures consisting of 1 mL supernatant and 1 mL of 1 mM hydroxylamine hydrochloride (using 50 mM phosphate buffer [pH 7.8] as solvent) were incubated at 25°C for 1 h. After addition of 1 mL of 17 mM aniline sulfonic acid and 1 mL of 7 mM α-naphthylamine, the reaction was performed at 25°C for 20 min. Absorbance was recorded at 530 nm using sodium nitrite as a standard.Superoxide ions , and the variables were given as the mean ± SD. Data were subjected to analyses of variance based on a completely randomized experimental design, and A. xiei were intercropped with two rows of maize. The row and plant distance for A. xiei under the intercropping system was the same as in the monoculture system. In the intercropping system, the distance between rows of A. xiei and rows of maize was 45 cm, and the row and plant distance for maize was 50 and 30 cm, respectively. The design of the plane graph is shown as in A. xiei growth light environment. The light transmission in the intercropping was measured to be around 45%FL  in intercropping leaves and monoculture leaves was 16.97 μmol m–2 s–1 and 9.46 μmol m–2 s–1, respectively, the former being significantly higher than the latter , light compensation point (LCP), light saturation point (LSP) (Fv′/Fm′), actual efficiency of PSII photochemistry in the light (ΔF/Fm′) and electron transport rate (ETR) were recorded in the intercropping leaves than in the monoculture leaves; however, a reverse trend was observed for non-photochemical quenching (NPQ)  . Under ing (NPQ) .–2 s–1, the time course of photosynthetic induction in both intercropping leaves and monoculture leaves resembled a hyperbolic response in Pnet. The hyperbolic response was characterized by a rapid increase in Pnet to 50–90%  than that of understory leaves (PSII) to decrease rapidly to the minimum value and increase slowly thereafter to the steady-state but the proportion of excess excited energy dissipated through xanthophyll cycle-dependent pathway (ΦNPQ) and the proportion of excess excited energy dissipated through constitutive thermal dissipation and fluorescence  to increase fast to the maximum value and then decline slowly to the steady-state  and electron transport rates (ETR) increased rapidly at first and then increased slowly to the steady-state  decreased rapidly at first and then dropped slowly to the steady-state  as reviewed by A. xiei intercropping with maize was also very similar to that inhabited by understory plants, and in such light environments, sunflecks provide a significant light resource. Thus, for the shade-demanding crop A. xiei, a rapid photosynthetic response to sunflecks might be of great importance to its carbon balance, allowing it to maximize the use of light energy from sunflecks and maintain a positive carbon balance, and is thus highly advantageous to its final yield under shade condition of the intercropping system.In view of the relative importance of sunflecks for daily carbon gain, understory plants are expected to effectively use fluctuating light. The effective use of fluctuating light requires rapid photosynthetic induction, because the photosynthetic induction state determines how rapid the leaf assimilates COntensity . Furtherntensity . Howeverand T90% . Despiteunflecks  when comunflecks . Indeed,in 4 min , which i2–, H2O2, and OH–, can be produced  has been documented as an important mechanism of protecting PSI and PSII in plants exposed to abiotic stress . CEF-defardation . The CEFardation . Previoul stress . There wl stress , indicatl stress . Therefol stress .F/Fm′ declined to near zero at the start of the simulated sunfleck  . At middHaematococcus pluvialis grown under high light condition (β-car is an important auxiliary pigment in plant cells. High content of β-car could dissipate excess excitation energy and scavenge reactive oxygen species, protecting photosynthetic apparatus from damage . Mutantsondition . Thus, β2O2, and plays a critical role in keeping ROS under control in plants (2O2 and of O2– radicals. In the present study, oxygen free radicals were more likely to accumulate in the monoculture leaves than in the intercropping leaves (During normal metabolism, ROS are generated as a side product in the photosynthetic transport of electrons, such as photosynthesis and respiration. Once the balance between the production of ROS and the quenching activity of antioxidants becomes upset, plants will be subjected to oxidative stress, often resulting in oxidative damage . Under cn plants . In thisg leaves . On the g leaves  could heA. xiei demonstrate a very fast response to sunflecks, and this can help them maintain a high positive carbon gain, because of their high rates of photosynthesis compared with the monoculture counterparts; this is thus highly advantageous to its final yield under shade conditions of the intercropping system. The intercropping leaves of A. xiei apparently retain robust photoprotective mechanisms such as xanthophyll cycle-dependent energy dissipation and the enzymatic antioxidant defense system, which are comparable to those of their monoculture counterparts. Taking the low-light environment inhabited by the intercropping leaves of A. xiei into account, these large biochemical investments in photoprotection might represent a wasteful use of resources. However, these investments could efficiently protect the leaves from the injury from intense sunflecks, alleviate photoinhibition, and thus might be associated with the conservative behavior linked to the origin of this species as a shade-tolerant plant.The intercropping leaves of The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.JZ wrote the manuscript and made a statistical analysis of the experimental data. JC and SX designed the experiment and provided ideas for writing manuscript. SS and LZ were responsible for the measurement of steady-state gas exchange and photosynthetic induction during simulated sunflecks. All authors agreed to the publication of this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects, we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species, we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic analyses, we propose a comprehensive classification of three main clades and seven subclades that are intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid phenotype is primarily determined by the presence/absence of single-copy genes. Although wild populations of Cannabis are still unknown, increased sampling of landraces and wild/feral populations across its native geographic range is likely to uncover additional cannabinoid oxidocyclase sequence variants. Significance statementCannabis genome sequencing efforts have revealed extensive cannabinoid oxidocyclase gene variation. However, phylogenetic relationships and evolution of these genes remain unclear. Our comprehensive analysis of currently available data reveals that these genes comprise three main clades and seven subclades that originated through Cannabis-specific gene duplication and divergence. Our new conceptual and evolutionary framework serves as a reference for future description and functional analyses of cannabinoid oxidocyclases.The plant Cannabis sativa L. (henceforth Cannabis) is an ancient yet controversial crop. Cannabis cultivars are commonly divided into “Fiber-type” (or hemp) cultivars that are used for the production of fiber and/or seed oil and “drug-type” (or marijuana) cultivars that are used for recreational, ritual, or medicinal purposes  and cannabidiol (CBD), but more than 120 others have been identified in Cannabis  or cannabigerol   of THC and intermediate content (2–4%) of CBD while drug-type cultivars typically have high content (14–40%) of THC and low content (<1%) of CBD . Howevertype IV) . These ltype IV) .Cannabis plant, cannabinoids are synthesized in multicellular epidermal glands (glandular trichomes) that are most abundant on the bracts of female inflorescences. The cannabinoid biosynthetic pathway has been largely elucidated, and for many steps in the pathway, the corresponding enzymes have been isolated and characterized , CBDA synthase (CBDAS), and CBC synthase (CBCAS) are highly similar in their biochemical properties and sequence characteristics . The amiEschscholzia californica involved in alkaloid biosynthesis and part of the larger oxygen-dependent FAD-linked oxidoreductase family (PF02913) -like gene family (PF08031) . This faPF02913) . Like otPF02913) . In accoactivity . CBCAS iactivity . Howeveractivity  and consentified , it stilT and BD, respectively)  . Howeverctively) . Thus, tTHCAS gene comprises a 1638 bp intronless open reading frame that is found in all drug-type cultivars (THCAS gene but accumulates the cannabinoid precursor CBGA instead of THCA (chemotype IV). This is probably due to a single amino acid mutation leading to a defective (BT0) variant (THCAS locus (CBCAS (see below). Thus, it remains unclear if THCAS occurs in multiple copies and, consequently, if copy number variation could be a target for the breeding of cultivars.The genes encoding THCAS, CBDAS, and CBCAS have been identified . The THCultivars . For thiultivars . It shou variant . Recentl variant . Gene coAS locus . But ampCBDAS gene comprises a 1635 bp intronless open reading frame that is found in all CBDA-producing cultivars , Medicago truncatula (Fabaceae), and Arabidopsis thaliana (Brassicaceae) were included as outgroups, allowing us to place our results within a broader phylogenetic perspective and in the context of the existing BBE-like gene family classification , Medicago truncatula (Fabaceae), and Arabidopsis thaliana (Brassicaceae) were included as outgroups  as well as nearly defective (BT0) coding sequences . Type 1/4 gene sequences from drug-type cultivars such as “Purple Kush” and “Jamaican Lion” as well as fiber-type cultivars such as “Finola” and “Carmen”. It includes the functionally characterized CBCAS , “mutateequences , and “inequences . Subcladequences  online.Subclade A3 comprises at least one full-length coding sequence from cultivar “Finola” and at least two nonfunctional (pseudo)gene copies from drug-type cultivars “Purple Kush” and “Jamaican Lion.” These sequences can be characterized by a duplication of the 3rd codon  and 13 unique nonsynonymous substitutions  online. Subclade A4 comprises three nonfunctional (pseudo)gene sequences from whole-genome assemblies of cultivars “Purple Kush,” “Finola,” and “Jamaican Lion.” They share four nonsense mutations and 14 unique aa substitutions and can be divided into two types  online.CBDAS sequences from chemotype III cultivars such as “Finola,” “Carmen,” and “CBDRx” (CBDAS1 and a defective sequence BD01 coding sequence (D), weakly functional (BDW), and defective (BD01 and BD02) coding sequences gene sequences from THCA-producing cultivars such as “Purple Kush,” “Jamaican Lion,” “Skunk#1,” “Chocolope,” and “Northern Light”; including “mutated CBDAS2 and CBDAS3 from a chemotype III “domestic” cultivar from Japan described as having no CBDA synthase activity by Clade C comprises full-length coding as well as nonfunctional (pseudo)gene sequences from cultivars “Purple Kush,” “Finola,” “CBDRx,” and “Jamaican Lion” as well as Cannabis cultivars “CBDRx,” “Jamaican Lion,” (mother) “Finola,” “Purple Kush,” and a putatively wild Cannabis plant from Jilong, Tibet. Based on nucleotide alignments and protein comparisons, we found that all cannabinoid oxidocyclase genes occur in two main syntenic clusters, together with other BBE-like genes. The first main syntenic cluster comprises a tandemly repeated array of genes from clade C in the genome assemblies of cultivars “Finola,” “CBDRx,” and “Jamaican Lion” genes, and a nonfunctional (pseudo)gene from the A4 subclade. It is present in cultivars “Jamaican Lion,” “Purple Kush,” and the putatively wild plant from Jilong, Tibet. The second variant comprises a single copy of a type 1 CBDAS and can be found in cultivars “Jamaican Lion” and “CBDRx.” The third variant comprises only a type 2 CBDAS and can be found in cultivar “Finola.” We found no nucleotide-level alignments between these variants except for the context around the group 5.2 BBE-like in the second and third variants gene copies and the second of which is only partially assembled. In the “CBDRx” genome, it comprises one full-length CBCAS coding sequence and five nonfunctional (pseudo)gene copies. Interestingly, it also includes a nonfunctional (pseudo)gene from subclade A3 but given the lack of additional A3 copies within the array, this appears to be a relatively recent insertion. In the genome assemblies of cultivars “Finola” and “Purple Kush,” subclade A2_CBCAS gene copies appear in several unplaced scaffolds probably representing the same array  and the receptor-like protein, the Patellin protein, the TWINKLE DNA primase-helicase, and the caseinolytic protease listed above  within the BBE-like gene family (Rhododendron dauricum daurichromenic acid synthase (RdDCAS), another plant cannabinoid oxidocyclase  and moracin C oxidase (MaMO) genes that are responsible for the production of the medicinal compound chalcomoracin  comprising a total of seven (sub)clades . THCAS amutation  online. mutation  but baseTHCAS . In case sequences fall outside any of our described clades, new (sub)clades can be defined in accordance with our system.Our clade-based classification is intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. For example, based on our analyses we were able to confirm that sequences variously named “Fiber-type,” “inactive,” or “obscure” THCAS  can be cS  and C; clade B was apparently lost. Block 2 retained ancestral genes representing clades A  and B; clade C was apparently lost. Support for this hypothetical tandem duplication event can be found in the BBE-like gene tree where the two corresponding group 5.2 BBE-like genes also comprise a Cannabis-specific duplication clades may include a variable number of pseudogenes . Type 2 on the other hand can be found in chemotype I drug-type cultivars “Purple Kush”, “LA confidential,” and on the THCAS-containing haplotype of chemotype II cultivar “Jamaican Lion” (mother). Similarly, the full-length subclade A3 gene that is closely linked to CBDAS in the genome of chemotype III fiber-type cultivar “Finola” is sister to the subclade A3 pseudogenes closely linked to THCAS in drug-type cultivars “Purple Kush,” “Jamaican Lion,” and the plant from Jilong , Humulus japonicus cultivar “Cascade” , Parasponia andersonii , Trema orientalis, Morus notabilis , Medicago truncatula , and Arabidopsis thaliana   and moracin C oxidase (MaMO) in Morus notabilis (Rhododendron dauricum (accession BAZ95780.1) and berberine bridge enzyme from Eschscholzia californica (accession AAC39358.1)   online ( N = 29) . Some CaQ8GTB6.1 . We indiotabilis . In addi39358.1) . SequencC. sativa for homologs of characterized cannabinoid oxidocyclase sequences  using BLASTP and BLASTN implemented in Geneious Prime 2019 . This resulted in 13 cannabinoid-related genes from cultivar “CBDRx” (GCF_900626175.1), 26 from “Jamaican Lion” (mother: GCA_012923435.1) (N = 11), “Chemdog91” , “Jamaican Lion” (father) , “LA confidential” , and “Pineapple Banana Bubble Kush”  (We mined available near chromosome-level genome assemblies of 23435.1) , 15 from23435.1) , and 8 f23435.1)  We simil N = 11)  online ( N = 11) . When neCannabis and Humulus were used as outgroups.In addition, we compiled homologous nucleotide sequences available from ncbi databases the majority of which came from published studies  online . Based oMultiple sequence alignment was performed with MAFFT v7.450 with automatic selection of appropriate algorithm, a gap open penalty of 1.26, and an offset value 0.123. For aligning the protein and nucleotide sequence data sets, we used the BLOSUM62 and 100PAM scoring matrix, respectively. Optimal models of sequence evolution as determined using Modeltest-NG v.0.1.5 on XSEDE via the CIPRES gateway  were WAGTHCAS reference sequence described by THCAS clade, type names were kept in accordance with those from Within the berberine bridge gene family tree, clades were numbered in accordance with earlier classification . Within H. lupulus cultivar “Cascade,” P. andersonii, and T. orientalis based on predicted protein sequences (Nucleotide microsynteny was assessed for the cultivars “Jamaican Lion” (mother), “CBDRx,” “Finola,” “Purple Kush,” and a putatively wild specimen from Jilong, Tibet. Because we found inconsistencies between the different genome assemblies in the ordering and orientation of sequences into scaffolds we considered genomic contigs only. Nucleotide-level alignments were generated by performing gapped extensions of high-scoring segment pairs using Lastz version 1.04.03. To avoid seeding in repetitive sequence we indexed unique words with single alignments only . To reduce runtime we set –notransition and –step = 20. To keep tandem repeats we set –nochain. To filter short and/or dissimilar alignments we set –hspthresh = 100,000, –filter = identity: 95, and –filter = nmatch: 2000. The –rdotplot output was used to generate alignment dotplots in R. For nucleotide level microsyntenic blocks of interest we further assessed microsynteny with genomic sequences from equences .Genome Biology and Evolution online.evab130_Supplementary_DataClick here for additional data file.


Scientific Reports 10.1038/s41598-021-00730-8, published online 02 November 2021Correction to: The original version of this Article contained errors.Firstly, citation numbers after reference 11 (with the exception of citations to References 7 and 10) were inaccurate by two.Additionally, in the Introduction,7,8, the upper part of the planet interior has certainly been affected by the circulation and downwelling of low-pH and oxidizing solutions8–10.”“Although different authors report neutral to alkaline conditions for underground Marsnow reads:7,8, the upper part of the planet interior has certainly been affected by the circulation and downwelling of low-pH and oxidizing solutions9–11.”“Although different authors report neutral to alkaline conditions for underground MarsFurthermore, in the Materials and methods section, the subheading ‘Sample 010,109–1’ now reads ‘Sample 010109-1’, and105.”“Thin sections of sample 010,109–1 were prepared for mineral and textural analysis under an optical microscope and a Scanning Electron Microscope (SEM) coupled with an Electron Dispersive Spectroscopy (EDS) probe, and molecular surface analysis by ToF-SIMSnow reads:107.”“Thin sections of sample 010109-1 were prepared for mineral and textural analysis under an optical microscope and a Scanning Electron Microscope (SEM) coupled with an Electron Dispersive Spectroscopy (EDS) probe, and molecular surface analysis by ToF-SIMSFinally, in the Materials and methods section, under the subheading ‘ToF–SIMS analysis’, exponents were incorrectly captured as citations, and“To prevent this problem, the thin sections of samples 010,109–1 and BH8-24c were cleaned by surface sputtering with a 100 nA 500 eV oxygen ion beam for 3 s.”now reads:“To prevent this problem, the thin sections of sample 010109–1 were cleaned by surface sputtering with a 100 nA 500 eV oxygen ion beam for 3 s.”The original Article has been corrected.


Scientific Reports 10.1038/s41598-018-36060-5, published online 14 March 2019Correction to: The original version of this Article contained an error in the Results and Methods section. The experiments measuring glycogen and glucose content, as well as enzymatic activities, were reported to have been performed on whole fly homogenates, however, these experiments were performed on thorax muscle homogenates.Therefore, in the Results section, the subheading,“Glycogen and glucose contents in whole fly homogenates”now reads,“Glycogen and glucose contents in thorax muscle homogenates”The subheading,“Enzymatic activities in whole fly homogenates”.now reads,“Enzymatic activities in thorax muscle homogenates”.And the text,“PK and CS activities were measured in whole fly homogenates and were analysed with a one-way ANOVA.”now reads,“PK and CS activities were measured in thorax muscle homogenates and were analysed with a one-way ANOVA.”In the Methods, the subheading,“Glycogen and glucose contents in whole flies”now reads,“Glycogen and glucose contents in thorax muscle”The subheading,“Enzymatic activities in whole flies”.now reads,“Enzymatic activities in thorax muscle”.The text,“Following 0, 1, 2, 4, and 10 days of exposure to the HFD or to the SD, the heads and abdomens were dissected from the flies, and the resulting thorax were either directly processed for mitochondrial respiration or frozen in liquid nitrogen and stored at – 80 °C for further measurements of ATP content. On sampling days whole flies were also collected, put on ice, rinsed two times in ice-cold phosphate-buffered saline (PBS), and frozen in liquid nitrogen for measurements of glucose and glycogen contents as well as for pyruvate kinase (PK) and citrate synthase (CS) enzymatic activities.”now reads,“Following 0, 1, 2, 4, and 10 days of exposure to the HFD or to the SD, the heads and abdomens were dissected from the flies, and the resulting thorax were either directly processed for mitochondrial respiration or frozen in liquid nitrogen and stored at −80 °C for further measurements of ATP content, glycogen and glucose content and enzymatic activities.”And the text,“Seven flies were homogenized”.now reads,“Seven thorax were homogenized”.In the original version of this Article, Figure 7 incorrectly refers to whole fly homogenates, rather than thorax muscle homogenates. As a result, the Figure legend,P < 0.05).”“Effects of the high-fat diet (HFD) on (A) Glucose and Glycogen contents of flies, (B) Pyruvate kinase activity, and (C) Citrate synthase activity. Results are means ± s.e.m for each day of the exposure  and have been analyzed using a one-way ANOVA followed by pairwise comparisons of the least-squares means using adjusted P-values (Tukey method). Dissimilar lowercase letters denote changes between days of exposure to a specifc diet, with each diferent letter(s) representing statistically significant differences .”“Effects of the high-fat diet (HFD) on (A) Glucose and Glycogen contents of thorax muscle, (B) Pyruvate kinase activity, and (C) Citrate synthase activity. Results are means ± s.e.m for each day of the exposure  and have been analyzed using a one-way ANOVA followed by pairwise comparisons of the least-squares means using adjusted P-values (Tukey method). Dissimilar lowercase letters denote changes between days of exposure to a specific diet, with each different letter(s) representing statistically significant differences (The original Fig. These changes do not affect the interpretation of the results, or the other sections of the Article, and have been implemented in the HTML and PDF versions of the manuscript.


IKAROS and CTLA4 deficiencies are inborn errors of immunity and show similar clinical phenotypes, including hypogammaglobulinemia and autoimmune diseases (ADs). However, the differences in clinical features and pathogenesis of these are not fully understood. Therefore, we performed systematic literature reviews for IKAROS and CTLA4 deficiencies. The reviews suggested that patients with IKAROS deficiency develop AD earlier than hypogammaglobulinemia. However, no study assessed the detailed changes in clinical manifestations over time; this was likely due to the cross-sectional nature of the studies. Therefore, we conducted a retrospective longitudinal study on IKAROS and CTLA4 deficiencies in our cohort to evaluate the clinical course over time. In patients with IKAROS deficiency, AD and hypogammaglobulinemia often develop in that order, and AD often resolves before the onset of hypogammaglobulinemia; these observations were not found in patients with CTLA4 deficiency. Understanding this difference in the clinical course helps in the clinical management of both. Furthermore, our results suggest B- and T-cell-mediated ADs in patients with IKAROS and CTLA4 deficiencies, respectively. NFKB1, NFKB2, CTLA4, LRBA, IKZF1, STAT3, and PIK3CD (Common variable immunodeficiency (CVID) is the most prevalent disease of inborn errors of immunity (IEI) and is characterized by hypogammaglobulinemia with poor specific antibody production. In addition to increased susceptibility to recurrent bacterial infections, clinical manifestations of CVID include noninfectious complications, such as autoimmune diseases (ADs), enteropathy, lymphoproliferation/granulomatous diseases, and malignancy . AlthougIKZF1 is a key transcription factor of hematopoietic development, particularly early B-cell development. Patients with IKAROS haploinsufficiency (HI) or haploinsufficiency of the dimerization domain (DD) present with progressive B-cell deficiency, whereas IKAROS dominant-negative (DN) results in more severe manifestations involving B-, T-, and myeloid cell defects (collectively termed as IKAROS deficiency)  –6. IKAROiciency) . Additioiciency) , 8. Althiciency) . ConsideSubdividing CVID by identifying its genetic defects has reduced the sample size of the studies. Early studies on CVID, especially cross-sectional studies, that clarified the clinical manifestations and appropriate management practices used large heterogeneous cohorts of 100 to >1,000 patients , 4. AlthThe primary aim of this study was to clarify the limitations of performing a literature review on IKAROS and CTLA4 deficiencies and to emphasize that a longitudinal study could compensate for its limitations. The secondary aim was to provide a possible pathogenesis for complications through a longitudinal study. This study focused on IKAROS and CTLA4 deficiencies from among the many known genetic defects because cohorts for these conditions have been previously described  and the We searched PubMed and Web of Science databases for articles published in English from April 2012 to June 2021 on IKAROS deficiency and from September 2014 to June 2021 on CTLA4 deficiency. The search terms were a “IKAROS haploinsufficiency”, “IKAROS haploinsufficient”, “IKAROS deficiency”, “IKAROS deficient”, “IKAROS mutation”, “IKAROS mutations”, “IKAROS variant”, “IKAROS variants”, “IKZF1 mutation”, “IKZF1 mutations”, “IKZF1 variant”, and “IKZF1 variants” for IKAROS deficiency, and “cytotoxic T-lymphocyte-associated protein 4 haploinsufficiency”, “cytotoxic T-lymphocyte-associated protein 4 haploinsufficient”, “cytotoxic T-lymphocyte-associated protein 4 deficiency”, “cytotoxic T-lymphocyte-associated protein 4 deficient”, “cytotoxic T-lymphocyte-associated protein 4 insufficiency”, “cytotoxic T-lymphocyte-associated protein 4 insufficient”, “CTLA4 haploinsufficiency”, “CTLA4 haploinsufficient”, “CTLA4 deficiency”, “CTLA4 deficient”, “CTLA4 insufficiency”, “CTLA4 insufficient”, “CTLA-4 haploinsufficiency”, “CTLA-4 haploinsufficient”, “CTLA-4 deficiency”, “CTLA-4 deficient”, “CTLA-4 insufficiency”, “CTLA-4 insufficient”, “CTLA4 mutation”, “CTLA4 mutations”, “CTLA4 variant”, and “CTLA4 variants” for CTLA4 deficiency. A search was performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for article identification, screening, eligibility, and inclusion , central nervous system (CNS) involvement, and other symptomatic lymphoproliferation/granulomatous diseases were included in AD as they are inflammatory complications and most likely associated with impaired immunological tolerance and lymphocyte infiltration . AsymptoIn the longitudinal study, AD remission was defined as absent or minimal symptoms, normal laboratory data without treatment, and no relapse until the last follow-up. For patients who underwent hematopoietic stem cell transplantation (HSCT), onset and remission were evaluated for the period before HSCT. Furthermore, if hypogammaglobulinemia and AD simultaneously identified, AD onset was considered to be before that of hypogammaglobulinemia, because serum IgG levels and B-cell counts were further decreased in all available patients.U-test or Wilcoxon signed-rank test for numerical data and chi-square test for non-numerical data. Moreover, 95% confidence intervals (CIs) were calculated using the exact binomial method (Clopper-Pearson method). Differences were considered significant at P values of <0.05. For constructing Kaplan-Meier curves, we defined censoring as loss of follow-up or HSCT.For statistical analyses and construction of Kaplan-Meier curves, GraphPad Prism 8 software (GraphPad) was used. The groups were compared using Mann-Whitney Electrophoresis mobility shift assay (EMSA) and co-immunoprecipitation (Co-IP) assay were performed as previously described . For EMSIntracellular CTLA4 expression was evaluated using flow cytometry. Peripheral blood mononuclear cells stimulated with anti-CD3/CD28 beads were stained with anti-CD4-PC7 (SFCI12T4D11 (T4), Beckman Coulter), fixed, permeabilized, and stained for anti-CTLA4-PE-Cy5  and anti-FOXP3-Alexa Fluor 647 .First, systematic literature reviews of IKAROS and CTLA4 deficiencies were performed, and 122 and 177 articles, respectively, were identified. Of those, 19 and 28 articles were considered eligible for systematic review. Furthermore, 95 and 246 patients were reported in these articles, and, after excluding patients to both groups, 90 and 179 unique patients remained for data analysis, respectively , 12, 21.IKZF1 variants: 66 patients (20 variants) with IKAROS HI, including large deletion and early truncation; 16 patients (6 variants) with IKAROS DD; and 8 patients (two variants) with IKAROS DN  and 0.6 (0.3-1) years, respectively. All developed infections, including Pneumocystis pneumonia, in infancy. One patient developed T-cell ALL at the age of 13 years, and AD was not observed.CTLA4 variants  described the clinical manifestations of substantial cohorts of >10 patients , 11, 23.These systematic literature reviews suggest that patients with IKAROS deficiency develop in the order of AD and hypogammaglobulinemia, whereas those with CTLA4 deficiency do not. Therefore, to evaluate the clinical course over time in detail, we conducted a retrospective longitudinal study on IKAROS and CTLA4 deficiencies in our Japanese cohort.IKZF1 variants bearing 10 distinct variants and 31 patients with CTLA4 variants bearing 16 distinct variants  with several positive autoantibodies, including antinuclear, antidouble-stranded DNA, anticardiolipin and antiribonucleoprotein antibodies. Disease activity in this patient was severe, and he required methylprednisolone pulse and immunoadsorption; however, he subsequently received prednisolone for 7 years without showing SLE-related symptoms. Hypogammaglobulinemia was detected at the same time as the SLE symptoms disappeared. He has been in clinical remission for 3 years without SLE treatment.Patient 11.1 initially presented with enteropathy at the age of 10 years. He had hypogammaglobulinemia and started immunoglobulin replacement therapy when he was 24 years old. He had enteropathy and developed additional complications of type 1 diabetes and CNS involvement in his 30s. Additionally, he developed gastric adenocarcinoma at 34 years, which led to his death from sepsis 2 years after gastrectomy.Patient 16.2 presented with pneumonia and hypogammaglobulinemia at the age of 18 years, he also had alopecia. He developed GLILD and showed CNS involvement after 2 years. Because those complications were refractory to glucocorticoids, immunosuppressant agents, and abatacept, he received HSCT at 21 years of age. Unfortunately, he died of HSCT-related complications 11 months after HSCT.Patient characteristics are presented in Among the 9 and 15 patients with hypogammaglobulinemia in IKAROS and CTLA4 deficiency conditions, respectively, only one patient with CTLA4 deficiency (patient 15.1) achieved remission . The laboratory of SL receives fundings and supports from the Ligue Contre le Cancer-Equipe Labelisée (France), Institut National de la Santé et de la Recherche Médicale (France), the French Foundation of Rare Diseases (France), the Agence Nationale de Recherche ANR (France) , the Institut National du Cancer (France) , the Société Française de Lutte contre les Cancers et Leucémies de l’Enfant et de l’Adolescent, AREMIG (France), and the Fédération Enfants et Santé (France).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Understanding chiral-inducedspin selectivity (CISS), resultingfrom charge transport through helical systems, has recently inspiredmany experimental and theoretical efforts but is still the objectof intense debate. In order to assess the nature of CISS, we proposeto focus on electron-transfer processes occurring at the single-moleculelevel. We design simple magnetic resonance experiments, exploitinga qubit as a highly sensitive and coherent magnetic sensor, to provideclear signatures of the acceptor polarization. Moreover, we show thatinformation could even be obtained from time-resolved electron paramagneticresonance experiments on a randomly oriented solution of molecules.The proposed experiments will unveil the role of chiral linkers inelectron transfer and could also be exploited for quantum computingapplications. Transport experiments were done on self-assembled monolayersof chiral (χ) molecules or on individual molecules addressedby atomic force microscopy.8 Additional studies revealed polarization also invery different contexts, in which no steady-state current flows throughchiral molecules  processes through a chiral bridge linking adonor and an acceptor , coupled to the acceptor in a D-χ-A-Q setup, as alocal probe of this polarization transfer and time-resolved electronparamagnetic resonance (TR-EPR) as the experimental tool. By actingas an external and local sensor, the qubit gives direct access tothe acceptor polarization, without influencing the ET process. Thisprovides a unique means to assess the nature of CISS at the single-moleculelevel, much more directly compared to previous setups,D-χ-A System. We consider the followingexperimental scenario, schematically shown in +-χ-A–. In the case of a linear bridge linkingdonor and acceptor (top inset), this is still a two-electron singletstate  A singlet state, p = p ≤ 1 and ≠0). Here p = −2Tr[ρpSzA] is the final polarizationof the acceptor. This state (representedin p = 1) could result from spin selective ET through a chiralbridge, as found in measurements on photosystem-I33 and in other experiments,41 where charge polarization wasinduced by application of an electric field, thus making these situationssomewhat similar to ET.(2) A polarized state, represented by the density matrixρU⟩ polarization,i.e., ⟨ψU | SzD | ψU⟩ = ⟨ψU | SzA|ψU⟩ = 0.(3) A non-Boltzmann but nonpolarized(correlated) state |ψU⟩ 1c resultd in ref . The mosThe three possible ET outputs are clearlydiscriminated by theexperiments proposed below.Detecting Polarization Usinga Qubit Sensor. Todetect spin imbalance in the D+-χ-A– unit, we propose the use of a qubit, i.e., a paramagnetic S = 1/2 center showing long coherence. Molecular spin qubitsare very promising sensors43 thanks tothe very long coherence times they can reach if properly chemicallyengineered.58 In addition, the capability to link these qubits to other unitsmake them ideal candidates for the proposed architecture. As a modelexample, we consider a qubit based on the VO2+ unit, whichhas already demonstrated remarkable coherence even at room temperature.50 Interestingly, a setup consisting of a chainof three radicals was studied in refs ( in refs −64.Jij = [gi ·gj – 3(gi · rij)(gj · rij)]μB2/rij3, and in thefollowing we consider for simplicity a linear 3-spin chain , these conditions are easily fulfilled with 6 Å ≲ rAQ ≲ 11 Å and workingin Q-band. We thus fix rAQ = 8 Åin the following simulations.After ET, the whole D-χ-A-Q system is described bythe followingspin Hamiltonian:hain see 2a, with H̃, R̃, and K̃ are superoperators associated withthe system Hamiltonian  and with phenomenological incoherent mechanisms. These are(i) relaxation, (ii) dephasing, and (iii) recombination  for each of the three spins of T1 ≈ 2 μs, T2 ≈ 0.5 μs, and TR ≈ 10 μs.50 The precise value of these parameters yields only a broadening ofthe peaks in the field-dependent spectrum and a damping in the timeevolution, as shown by detailed simulations in the Supporting Information. However, as far as these characteristictimes are above a few hundred nanoseconds, they practically do notaffect our conclusions. The recorded signal then corresponds to ⟨Sy⟩ = Tr[∑iSyiρ(t)]. In order to unambiguously unveil the nature of CISS, we consideroriented D-χ-A-Q molecules, with the static field parallel tothe chiral axis, z .Two different experiments exploiting the qubitas a sensor of theacceptor polarization are proposed. The first consists of TR-EPR measurementsrecorded immediately after ET. To simulate TR-EPR spectra, we computethe time evolution of the system density matrix by integrating theLiouville equation ρ̇ = le, refs  and 65),U⟩,modify the radical-pair spectrum(black vs blue curves in the gray-shaded area) but not the qubit response(right part of the spectrum), which is affected only by ⟨SzA⟩. The qubit absorptionpeak close to ∼1.24 T is split by the interaction with theacceptor. If the latter is completely polarized , a single peak appears, correspondingto the |↑↓↓⟩ → |↑↓↑⟩transition.77 Conversely, an unpolarizedDA state induces the additional excitation corresponding to |↑⟩A with approximately the same intensity . Note that in thepresent simulation, ⟨Sy⟩ shows a weaktime dependence (see the Supporting Information), thus making the choice of the time window of integration not crucial.The three states (1–3) give distinct TR-EPR signals,asshown in the time-integrated spectra of I =1/2 (such as 19F) coupled to A by (isotropic) hyperfineinteraction I, split by hyperfine interactionwith A. Again, the probe is only sensitive to ⟨SzA⟩ and weakly perturbs the system,thus giving direct access to the acceptor polarization.78Similar information can be obtained by broadband NMR spectroscopy,as reported in Supporting Information), thus making the relativeintensity of the two peaks a measure of spin polarization. Remarkably,both for EPR and NMR experiments, this feature is not hampered byperforming the experiment at high temperature, which only inducesan overall attenuation of the signal (see simulation in the Supporting Information). Moreover, opposite polarization(arising in model 2 by changing the enantiomer) yields inversion ofthe intensity of the two peaks, thus providing direct proof of theoccurrence of CISS, in contrast to other polarization mechanisms suchas chemically induced dynamic electron polarization (CIDEP).69Partial polarization leads to intermediatesituations . This also emerges from the short-time spectra,as a function of B (panel c), and from the time dependence,reported in panel d for B corresponding to the firstpronounced peak. The different order of maxima and minima as a functionof B can be understood by considering the form ofthe initial state along different directions. An illustrative diagramof the  eigenstates is shown in the insetof JDA. An initial T0⟩ = (|↑↓⟩ + |↓↑⟩)/√2.Hence, the EPR signal shows emission lines for 2–1 and 3–1transitions and absorptions for 2–4 and 3–4. Conversely,an initial ρp state  is strongly anisotropic. For a generic orientationof the molecule with respect to the external field (which definesthe quantization axis) such a state has sizable components on states|↑↑⟩ and |↓↓⟩. If these components are larger than that on |T0⟩, we get emission lines for 4–2 and 4–3transitions and absorption for 1–2 and 1–3. This situation(opposite to that of the singlet) dominates on the spherical average(see the Supporting Information for details).Then, the order of maxima and minima is determined by the sign ofthe spin–spin interaction (fixed by considering a dipolar coupling).p, this measurement does not probethe acceptor polarization but distinguishes a factorized state ρp from a singlet. As shown in the Supporting Information, unpolarized states |ψU⟩ give different patterns, intermediatebetween p, thus requiring, in general,a preliminary characterization of thesystem Hamiltonian to clearly reveal CISS.We finally note that, because the spectrum in panel b isthe samefor any choice of Identificationof a Suitable D-χ-A-Q. Theidentification of a suitable D-χ-A-Q requires the optimizationof many factors, including ET efficiency and stability of the helicoidalstructure in solution, but appears within reach. Considering the individualbuilding blocks, D–A dyads providing long-lived radical pairsare available in the literature.73 The most commonlyused acceptors are C60 or derivatives of naphthalenediimide,whereas pyrene, oligophenylene-vinylene, and tetrathiafulvalene aresuitable donors. Linkers employed in these dyads are usually shortand strongly conjugated and thus significantly more conductive thanfoldamers based on polypeptides commonly used to detect CISS in transportmeasurements.2 Interestingly, the strong dipolemoment of α helices has been shown to enhance intramolecularET. Long distance ET, up to 5 nm, has been instead observed in D-χ-Aunits based on helically folded oligoamide of 8-amino-2-quinolinecarboxylicacid.74 However, both forms of handednessare present and interconversion is relatively fast in solution. Nevertheless,racemization can be hampered in more rigid helicoidal scaffolds suchas helicene. Condensation of a D–A unit with a radical as aqubit has been recently achieved.75 In our case,where pulses separately addressing the photogenerated radical andthe permanent qubit are required, transition metal-based qubits withlong coherence58 are preferred because of their g value differingfrom 2, as discussed above.4+S = 1 complex.76 This is a crucial step toward the physical implementationof quantum computers. A CISS-based approach would be more costly interms of chemical engineering, but it could reveal tremendous potential.Indeed, the photoexcitation step could be replaced by electricallyinduced CISS-ET and combined with electric read-out.In conclusion, we have proposedsimple magnetic resonance experimentsexploiting a qubit as a probe of the acceptor polarization in electron-transferprocesses through a chiral bridge. These experiments will ultimatelyunravel the nature of chiral-induced spin selectivity at the single-moleculelevel. We finally note that, by applying the proposed sequence forpolarization transfer, the CISS effect could be exploited for initializationand read-out of the qubit state, an alternative to optical initializationrecently achieved in a Cr


Palliative care knowledge and health literacy are frequently underestimated in American adults; for example, as measured by the Newest Vital Sign , 79.2% (n = 247) of participants within a Geriatrics Clinic sample displayed “adequate” functional health literacy, while 11.8% (n = 37) scored within the “possibly limited” range, and 9.0% (n = 28) scored within the “highly limited” range. There was additionally a significant association between health literacy and age  within this sample. The Palliative Care Knowledge Scale  was administered to participants, and higher scores indicated a greater knowledge of palliative care. This construct is particularly important to measure, as racial/ethnic disparities exist within this domain; for example, African Americans may have lower overall knowledge of palliative care services and advance care planning than non-Hispanic Whites . In the current study, knowledge of palliative care was measured using the PaCKS , and scores represented the widest possible range of 0 to 13 . There was a significant correlation between age and PaCKS score , as palliative care knowledge increased with age. Females scored significantly higher  than males , t(309) = 3.18, p < .001. There was no main effect of race on palliative care knowledge, and post-hoc analysis using Tukey HSD did not demonstrate significant differences between groups.