Objective: To investigate the dynamic amplitude of low-frequency fluctuations (dALFFs) in patients with Parkinson's disease (PD) and healthy controls (HCs) and further explore whether dALFF can be used to test the feasibility of differentiating PD from HCs.Methods: Twenty-eight patients with PD and 28 demographically matched HCs underwent resting-state functional magnetic resonance imaging (rs-fMRI) scans and neuropsychological tests. A dynamic method was used to calculate the dALFFs of rs-fMRI data obtained from all subjects. The dALFF alterations were compared between the PD and HC groups, and the correlations between dALFF variability and disease duration/neuropsychological tests were further calculated. Then, the statistical differences in dALFF between both groups were selected as classification features to help distinguish patients with PD from HCs through a linear support vector machine (SVM) classifier. The classifier performance was assessed using a permutation test .Results: Significantly increased dALFF was detected in the left precuneus in patients with PD compared to HCs, and dALFF variability in this region was positively correlated with disease duration. Our results show that 80.36% (p < 0.001) subjects were correctly classified based on the SVM classifier by using the leave-one-out cross-validation method.Conclusion: Patients with PD exhibited abnormal dynamic brain activity in the left precuneus, and the dALFF variability could distinguish PD from HCs with high accuracy. Our results showed novel insights into the pathophysiological mechanisms of PD. Parkinson's disease (PD) is a common neurodegenerative disorder characterized by progressive impairment of motor function and widespread non-motor symptoms, which affects patients' quality of life and is, hence, a significant social burden –3. At prIn recent years, advanced neuroimaging techniques have allowed us to noninvasively explore the nature of the human brain in an efficient manner –10. RestPrevious studies on aberrant static ALFF in PD are inconsistent as they report both lower and higher local brain activity in various cerebral areas. However, a static state analysis of ALFF ignores the dynamic characteristics of brain activity during the whole scanning period. Evidences from rs-fMRI that employ a sliding window approach have effectively detected dynamic functional connectivity features with higher sensitivity than the static state method , 19, 20.To date, no studies have evaluated the combined effects of dynamic ALFF and supervised machine-learning approaches on PD. Based on previous findings that a static ALFF has greater heterogeneity in PD and the proven property of dynamic methods, we hypothesized that dynamic intrinsic local spontaneous neuronal activity will show greater variability in subjects with PD than HCs, and that it may be another powerful index of rs-fMRI in exploring the underlying mechanisms of PD. We aimed to identify the cerebral regions that displayed abnormal dynamic local neuronal activity based on the voxel level of the whole brain. Furthermore, we applied SVM to observe whether the aberrant dALFF could be used as a feature to distinguish PD from HCs. This study may improve our understanding of the potential pathophysiological mechanism of PD. Moreover, we hope that this research can contribute to the clinical diagnosis of PD at an individual level.This study was carried out in accordance with the tenets of the Helsinki Declaration and approved by the local ethics committee of Xuzhou Affiliated Hospital, Xuzhou Medical University. Written informed consent was obtained from all subjects before participation in the study.The project used a convenience sample of 31 hospitalized patients with PD who met the UK Bank diagnostic criteria for PD . All patAll participants were scanned in a 3.0 Tesla MRI scanner  with an eight-channel head coil. During the scan, comfortable foam pads were used to stabilize the head of each subject to minimize head motion, and all subjects wore earplugs to reduce the noise from the MRI machine. Then, an echo-planar imaging sequence was employed to acquire resting BOLD images. The parameters of the protocol are as follows: time of repetition, 2,000 ms; time of echo, 30 ms; field of view, 220 mm × 220 mm; slice thickness, 3 mm; slice gap, 1 mm; voxel size, 3.4 mm × 3.4 mm × 4.0 mm; number of slices, 36; flip angle, 90°; and total volume of each subject, 185.http://www.rfmri.org/dpabi)  Brain Imaging (DPABI) (g/dpabi) . The firg/dpabi) –33.The analysis of dynamic amplitude of low-frequency fluctuations (dALFF) was carried out using Temporal Dynamic Analysis (TDA) toolkits based on DPABI . Before t-test was used to observe intergroup differences in age and MoCA/MMSE scores. Sex-based group difference was evaluated using the chi-square test. A general linear mode (GLM) with age, sex, and mean FD as covariates was used to compare the difference of dALFF/ALFF between the PD and HC groups. Multiple comparisons were corrected using Gaussian Random-Field (GRF) method .Two-sample p < 0.05). All statistical analyses were performed using SPSS version 16 .Partial correlation analysis was calculated between dALFF variability and disease duration/MoCA/MMSE/UPDRS/H-Y with age and sex as covariates  method was applied to estimate the generalization ability of our classifier. To verify the ability of the validation strategy, we also made a classification comparison by introducing 10-fold cross-validations. Then, the total accuracy, sensitivity, and specificity were obtained to assess classifier performance.p-value was obtained after the total permutation was accomplished.A permutation test was used to evaluate the statistical significance of this classification accuracy . The perp = 0.652), sex (p = 0.789), and MMSE (p = 0.831) between the PD and HC groups. However, the MoCA score of the PD group was significantly lower than that of the HCs (p < 0.05).The details of age, sex, and MoCA/MMSE scores are listed in p < 0.001). In addition, we found that the CV of dALFF was positively correlated with disease duration  (The intergroup differences in dALFF are shown in = 0.800) , and no = 0.800) . There wp < 0.001, non-parametric permutation approach). The receiver operating characteristic (ROC) curve of the classifier was 0.82. A 10-fold validation was also employed in our study to verify the reliability of the classification method, which generated a classifier accuracy of 71.43%.Classification results are shown in Upon literature review, we observed that only few studies employed a TDA method to explore the neural-activity characteristics of PD. The present study showed the following findings: (1) the dALFF of patients with PD compared to HCs was notably different in the left precuneus; (2) a significant correlation between CV of dALFF in the left precuneus and the course of the disease was found in PD; (3) dALFF in the left precuneus showed high accuracy in distinguishing between patients with PD and HCs.p < 0.001). Dynamic algorithm showed the capability to characterize neural activity of the human brain by identifying specific function signatures.Prior studies have noted the importance of cerebral static local neural activities in PD , 14, 15.18F]fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) proved that the precuneus was the most remarkable area of hypometabolism in the posterior cortical region . Further, to compare the performance of SVM using LOOCV, a nested 10-fold cross-validation method was used to assess the classifier's performance; the accuracy was 71.43%. These findings showed that SVM could achieve better classification capability with LOOCV, and the results also provided evidence that patients with PD could be distinguished from HCs at the individual level when using dALFF variation in the left precuneus. These results support the hypothesis that the dALFF could identify individual PD patients.The imaging diagnosis of PD remains a challenge even now, as a confirmed diagnosis in most patients is still made depending on the clinical symptoms . An asseOur study has some limitations. First, all patients were on medication. Although patients underwent fMRI scanning while they were off medication, the effects of the long-term treatment could not be completely ruled out. Second, the classification power based on the 28 PD patients was still not strong enough, and we just used SVM in the same sample to testify the classification accuracy. Third, the patients did not undergo comprehensive cognitive scales testing, which could have prevented a more accurate detection of cognitive performance. Future research should include a larger sample size and another independent test sample should be recruited for testifying classification accuracy.To our best knowledge, this is the first study to attempt to investigate the dynamic spontaneous neural activities in patients with PD. Our results provided evidence that dynamic analysis was more sensitive to detect alteration of brain activity than a static method. In addition, the CV of dALFF was found to be correlated with the course of the disease, which may ultimately contribute to identifying PD at the individual level. Thus, our results provide novel insights on the pathophysiological mechanisms of PD.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethical committee of Xuzhou Affiliated Hospital. The patients/participants provided their written informed consent to participate in this study.CZ and BD contributed equally to this work for the conception/design of the study, the acquisition, analysis, interpretation of data, drafting of the manuscript, final approval of the version to be published, and agreement to be accountable for all aspects of the research. JW, CH, YR, HZ, and MS were responsible for data analysis, drafting of the manuscript, final approval of the version to be published, and agreement to be accountable for all aspects of the research. QX, NC, and KL were responsible for revision of the manuscript, final approval of the version to be published, and agreement to be accountable for all aspects of the work. KX was responsible for design of the study, revision of the work, final approval of the version to be published, and agreement to be accountable for all aspects of the work.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.


Calumma and a new species, Calumma benovskyi sp. nov., is erected for it. Since all species of this genus are endemic to Madagascar, this fossil uniquely demonstrates the existence of Calumma on continental Africa in the past. Our results challenge the long-held view that chameleons originated on Madagascar and dispersed over water to Africa, and provide a strong evidence of an African origin for some Malagasy lineages. The Oligocene–early Miocene dispersal to Madagascar, using oceanic currents that favoured eastward dispersal at that time, is a highly supported scenario matching the suggested dispersal of lemurs to this island. This is consistent with a previously suggested hypothesis based on molecular data.We here present the first detailed study of the specimen KNM-RU 18340 from Rusinga Island (Kenya), the only known complete early Miocene chameleon skull, using micro-CT. This specimen represents one of the oldest chameleon fossils ever recovered. For the first time, the skull bone internal surfaces, their sutures, and elements contained inside the rocky matrix are observed. Our morphological comparisons and phylogenetic analyses place this specimen confidently in the genus  About half of the accepted species of chameleons occur in Madagascar. This island has therefore been suggested to be a centre of diversity of the clade from where it likely radiated via oceanic dispersal3. Later, in contrast, the molecular phylogeny of Tolley et al.4 suggested that the family most probably originated in Africa, with two separate oceanic dispersal events to Madagascar during the Palaeocene and the Oligocene, when prevailing oceanic currents would have favoured eastward dispersal. The fossil record of these animals, the only form of direct evidence regarding the early evolution and palaeobiogeography of these animals, is unfortunately scant. Thus, a key element for resolving this conundrum is lacking.The Chamaeleonidae is an unusual family of lizards including extant species from Africa, Madagascar, the Middle East, southern India, Sri Lanka, and the Mediterranean region of Europe. It is a highly characteristic and morphologically specialized clade of acrodont iguanians. This clade is mainly composed of arboreal forms, but includes a ground-dwelling desert form –the Namaqua chameleon4, but the oldest known fossil record of crown members only dates back to the early Miocene (MN3)6. Chamaeleonid fossils are mostly reported from the Miocene of Europe12 and are also known from Africa14 and potentially India15. Further African fossils have been documented from early Pliocene deposits17. The vast majority of the fossil record is represented by isolated elements, mostly jaw fragments, which renders taxonomical identification to the genus or species level often difficult or impossible. Only two notable exceptions exist - Chamaeleo intermedius, based on a natural calcite cast described by Hillenius13 from the middle Miocene of Fort Ternan in Kenya; and Ch. andrusovi from the early  Miocene of Europe, based on well-documented isolated cranial elements described by Čerňanský6.Molecular data suggest a Cretaceous origin18 . The skull is not fully freed from the rock, as matrix fills the entire internal region and covers many bone elements  is famous for its numerous fossil mammalsThe aims of this paper are: 1) to use high-resolution X-ray microcomputed tomography (μCT) to virtually prepare all skeletal elements of KNM-RU 18340; 2) to discover potentially hidden bones embedded in the sediment not visible from the outside; and 3) on the basis the obtained new anatomical data to evaluate the possible taxonomic position of KNM-RU 18340 and shed light on scenarios for the origin and diversification of chameleons.21Squamata Oppel, 181122Chamaeleonidae Gray, 1825Calumma Gray, 186523Calumma benovskyi sp. nov.Calumma - born abroad, he reached the island by sailing across the ocean.After Count Móric Beňovský . He was born in Slovakia  and died at the age of 39 in Madagascar (5. 23. 1786). He is known as an important traveler and adventurer - the first European to sail in the North Pacific Ocean (seven years before James Cook) and the first person who explored Saint Lawrence Island. He traveled to Madagascar, where local tribal chiefs elected him as a “king” (ampansacabe) of Madagascar in 1776. His story parallels the history of KNM-RU 18340 : skull, mandible and three cervical vertebrae in connection Figs. .Figure 2Rusinga Island , site R107.Early Miocene, base of the Hiwegi Formation.Calumma distinguishable from other species by the posterior corners of the frontal, forming well-developed posterolateral processes. Besides this feature, this taxon is characterized by the unique combination of the following characters: (1) nasal-frontal contact present; (2) prefrontal fenestra present and connected to external naris; (3) prefrontal inclined laterally in dorsal view; (4) contact between prefrontal and postorbitofrontal, excluding the frontal from the orbit; (5) orbital margin medially concave rather than straight in dorsal view; (6) single line of protuberances on the external surface of jugal; (7) hour-glass shaped parietal, bearing sculpture arrangement roughly resembling the Greek letter Ψ (psi); (8) well-developed and rather regularly distributed sculpture formed by protuberances on the dorsal surface of the frontal; (9) frontal completely pierced by the parapineal foramen; (10) posterodorsal process of postorbitofrontal reaching above the orbit, inducing an horizontal upper temporal fenestra, dorsally open, and not visible in lateral view; (11) posterodorsal process of squamosal more or less horizontal rather than vertical; and (12) the maximum width of the skull in dorsal view located at the level of the frontal-parietal contact rather than posterior to it.A species of Calumma 25. Is is also present in Rhampholeon and Rieppeleon, but not in Bradypodion, Chamaeleo, Kinyongia, Trioceros and Brookesia. The premaxilla-frontal contact is absent in the outgroup and character optimization in Mesquite supports this character state as being the condition at the basal node of the Chamaeleonidae clade. Character optimization in Mesquite shows that the contact of these two bones evolved independently in Rieppeleon, Rhampholeon and some members of Calumma. Among Calumma, the absence of the contact is at the basal node of the clade. So its presence can be regarded as derived among of this clade.The contact of the premaxilla with the frontal. This is present in Bradypodion and Brookesia often have a single nasal24.The paired nasals do not contact one another and are separated by a strut of bone formed by the premaxilla and the frontal. The majority of the chameleons have paired nasals, whereas et al.14). This character state can be observed in some extant chameleons, such as Calumma nasutum, Ca. fallax, Ca. vatosoa, Ca. guibei , but also in Rhampholeon, Archaius and Trioceros melleri26. This connection is absent in e.g., Brookesia, Rieppeleon, Nadzikambia, Bradypodion, Chamaeleo and Furcifer. As for Calumma, character optimization in Mesquite supports absence of the connection as being the condition at the basal node of the clade, whereas its presence is derived.The prefrontal fenestra is connected to the external naris . However, the suborbital process in lateral view is covered by the maxilla in Archaius and many Furcifer species 24.The suborbital process of the jugal in lateral aspect; only a dorsal narrow portion of this process is exposed dorsal to maxilla. This is present in Calumma (except for Ca. parsonii), Brookesia, Rieppeleon and Bradypodion, whereas the contact is usually present in members of the genera Chamaeleo, Nadzikambia, Archaius, Kinyongia, and some members of Furcifer  and Trioceros24. The character optimization in Mesquite supports the absence of jugal - squamosal contact as being the condition at the basal node of the clade formed by all chameleons. Character optimization in Mesquite shows that presence of this contact is the condition at the basal node of the clade [Chamaeleo + Trioceros + Kinyongia + Furcifer + Calumma], whereas the contact of these bones in Archaius evolved independently. The absence of this contact in members of Calumma is regarded as a reversal.The absence of jugal - squamosal contact. This contact is absent in Calumma globifer, Ca. ambreense and Ca. brevicorne 27, Chamaeleo, Trioceros and it can vary among Furcifer - present in F. pardalis and F. lateralis, but not in F. campani and F. bifidus. Conversely, these two elements do not meet in many small chamaeleonids like Brookesia, Rhampholeon and Rieppeleon24, but also in Bradypodion thamnobates, Bra. setaroi and Nadzikambia mlanjensis17. The character optimization in Mesquite supports the prefrontal - postorbitofrontal contact as being the condition at the basal node of the clade formed by [Chamaeleo + Trioceros + Kinyongia + Furcifer + Calumma]. Its absence in some members of this clade can be regarded as a reversal.The exclusion of the frontal fom the orbital margin, due to the prefrontal-postorbitofrontal contact. The prefrontal and postorbitofrontal meet in many chameleons, such as Calumma: while the margin is concave in Ca. nasutum, Ca. fallax, Ca. guibei and Ca. juliae, it is straight in Ca. globifer, Ca. ambreense, Ca. parsonii, Ca. brevicorne and Ca. boettgeri. Besides Calumma, a concave margin occurs in Rhampholeon and some Furcifer . It is straight in Brookesia, Palleon, Rieppeleon, Archaius, Bradypodion, Trioceros, Kinyongia, Chamaeleo and Nadzikambia30.The orbital margin, formed by the prefrontal and postorbitofrontal, is medially concave rather than straight in dorsal view. This varies among members of Calumma, an ornamentation formed by protuberances in the central region of the frontal is present in Ca. globifer, Ca. ambreense, Ca. parsonii, Ca. brevicorne and Ca. lefona, but not in Ca. nasutum or Ca. beottgeri30. Except for Calumma, this can be observed in Brookesia, Rieppeleon and Bradypodion29, but also in some large males of Trioceros jacksonii. Character optimization in Mesquite evaluated its presence in members of Calumma in two equally parsimonious ways: as the condition at the basal node in this lineage with an additional reversal in Ca. nasutum and Ca. boettgeri, or as representing a derived condition in Ca. globifer + Ca. parsonii and Ca. brevicorne.The sculpture formed by protuberances is well-developed and rather regularly distributed on the dorsal surface of the frontal. Among Calumma . Athough a flat parietal is present even in Bradypodion, Rhampholeon and Rieppeleon, it gradually narrows posteriorly, being triangular rather than hour-glass shaped. The posterior portion is usually laterally compressed, forming a crest, in Archaius, Chamaeleo, Furcifer, Nadzikambia, Kinyongia and Trioceros. Brookesia species and Palleon can be distinguished by a presence of the supratemporal processes of the parietal29. Character optimization in Mesquite evaluated the change of laterally compressed parietal in two equally parsimonious ways: the laterally compresed parietal as being the condition at the basal node of the [Chamaeleo + Trioceros + Kinyongia + Furcifer + Calumma] lineage, showing its additional reversal in Calumma, or the lateral compresion as representing an independent derived character in Chamaeleo, Furcifer and Trioceros. Character optimization in Mesquite evaluated the change of additional widening of the posterior portion of the parietal (hour-glass shape) as well as the absence of a dorsally elevated casque as being the conditions at the basal node of the Calumma clade.The hour-glass shaped parietal (mid-constriction present) with a triangular wide posterior portion, which does not form a posteriorly narrowing terminus . The parietal is flat and does not form a dorsally elevated casque. This can be observed in members of the extant Calumma, this is present in Ca. globifer, Ca. parsonii, Ca. ambreense and Ca. brevicorne, but not in Ca. nasutum, Ca. gallus, Ca. boetgeri, Ca. uetzi, Ca. lefona, Ca. juliae, Ca. guibei and Ca. gehringi30. The upper temporal fenestra is well visible laterally in many chamaeleonid genera, e.g. Brookesia, Rieppeleon, Rhampholeon, Bradypodion, Furcifer and Chamaeleo29. The polarity of this character in Mesquite is unresolved. However, the upper temporal fenestra being horizontal, dorsally open, not visible in lateral view appears to be consistently present in the lineage [Ca. parsonii + Ca. globifer + Ca. ambreense] sister to all other species of Calumma4.The posterodorsal process of the postorbitofrontal reaches above the orbit and thus, the upper temporal fenestra is horizontal, dorsally open, being not visible in lateral view. Among Brookesia and Rieppeleon24.The presence of a well-developed supratemporal. The supratemporal is absent in Calumma (except for Ca. parsonii), Rhampholeon, Bradypodion and Brookesia, but present in Chamaeleo, Kinyongia and Trioceros24. Character optimization in Mesquite evaluated this change in two equally parsimonious ways: as the condition at the basal node in the [Chamaeleo + Trioceros + Kinyongia + Furcifer + Calumma] lineage with an additional reversal in Calumma + Furcifer, or as representing independent derivations of the presence of lacrimal in Chamaeleo and Kinyongia + Trioceros. As for the outgroup, the lacrimal is absent in Uromastyx, but present in Leiolepis24. The presence of lacrimal can be regarded as a plesiomorphic state among lizards31.The absence of a lacrimal. The lacrimal is absent in 24 noted that the vomer in chamaeleons may be represented by: (a) a single fused bone; (b) an element divided anteriorly but fused posteriorly; or (c) two separate bones. Most extant chamaeleons possess a vomer that is only partly divided or fused into a single element. Paired vomers are present e.g., in Archaius.The partial fusion of the vomers (at least in the posterior portion). Rieppel and CrumlyRhampoleon spectrum, Furcifer pardalis and Chamaeleo calyptratus, but wide in Calumma globifer.The broad anteriormost region of the palate formed by the vomers and the anterior portions of the vomerine processes of the palatine. It is rather narrow in e.g., 24.The dentary tooth row ends far anterior to the dorsal process of the coronoid24.The presence of an angularUromastyx) and character optimization in Mesquite supports wide skull as being the condition at the basal node of the clade formed by all chameleons. Character optimization in Mesquite shows that narrow, laterally compressed skull is the condition at the basal node of the clade [Chamaeleo + Trioceros + Kinyongia + Furcifer + Calumma], whereas the condition in Calumma is regarded as a reversal.The wide skull (its width is more than 50% of the maximum anteroposterior length). It is present in the outgroup , whereas Ca. globifer  and Ca. parsonii  are sister to this clade . According to Hillenius33, the skull of Ca. nasutum is very similar to that of Rhampholeon. As was already stated by Rieppel and Crumly24, Ca. nasutum lacks numerous apomorphies, possibly due to its small size.In analysis 1, a New Technology (NT) search in TNT produced a single most parsimonious tree. The position of KNM-RU 18340 within ade Fig. . RemarkaCalumma , as sister to Ca. brevicorne.In analysis 2, a traditional heuristic Traditional search in T.N.T. produced a single parsimonious tree. The topology of the examined taxa is identical to that recovered from the NT search Fig. ; KNM-RU et al.4 was used to constrain the ingroup relationships. The analysis produces a single tree, in which KNM-RU 18340 is again nested within Calumma, as sister to Ca. nasutum , despite the different topologies within the Chamaeleoninae, specimen KNM-RU 18340 is consistently recovered as a member of the ade Fig. .In analyChamaeleo (Ch. namaquensis and Ch. dilepis) and two Calumma species (Ca. brevicorne and Ca. globifer) providing further support for the phylogenetic analysis which unambiguously places the fossil within the Calumma group  - they claimed (erroneously as we know now) the complete separation of external nares from the prefrontal fenestra in KNM-RU 18340.Rieppel Thanks to micro-computed tomography (μCT), we were able to identify all skull bones and observe their internal sides as well as their sutures. Moreover, the micro-CT revealed elements that are hidden by the rocky matrix and could not be studied before, such as an almost complete braincase with stapes, the palate region, a ceratohyal and the first three cervical vertebrae Fig. .Calumma. Overall the morphology of the fossil skull most closely resembles that seen in the extant Ca. brevicorne , the presence of the prefrontal-postorbitofrontal contact over the orbit and the maximum width of the skull being located at the level of the frontal-parietal contact in dorsal view . But there are also some differences, e.g. the absence of the premaxilla-frontal contact, the separation of the external naris from the prefrontal fenestra, and the straight orbital margin in dorsal view in the extant species (see above).The morphological comparisons are in agreement with the results of the phylogenetic analyses that specimen KNM-RU 18340 can be distinguished from all extant genera, except from see Fig. , especiaCalumma benovskyi sp. nov. There is also one unique character: well defined and laterally expanded triangular posterolateral processes of the frontal. Although in some species of Calumma 26 the posterolateral corners of the frontal expand laterally, they do not form a well-defined triangular processes as present in the early Miocene Ca. benovskyi sp. nov. The processes are not developed in Brookesia and Palleon, but are well-expanded laterally in the outgroup taxon Uromastyx.The preserved elements of KNM-RU 18340 possess a unique combination of character states (see Diagnosis), and a new taxon name is therefore erected: Calumma lineage on continental Africa. The extant species of this genus 34, which harbours an exceptional biodiversity. The separation of this island from Africa occurred already in the early Cretaceous35. Although the presence of a Malagasy lineage in continental Africa during the early Miocene might appear as a surprise, similar patterns have been observed for Madagascar’s endemic terrestrial extant mammals. Based on phylogenetic analyses, tenrecs37, euplerid carnivores38 and nesomyine rodents36 appear to be monophyletic taxa, whose sister group is found in Africa. They each result from a single colonization event and subsequent radiation within the island39. Likewise, recent palaeontological discoveries suggest that two lemur lineages likely dispersed from Africa to Madagascar across the Mozambique Channel independently, and thus have an African origin40. The dispersal window for these four endemic clades is estimated to span from the Oligocene to the early Miocene40. It is also worth noting that some other squamate lineages such as pythons and varanids did not manage to follow this dispersal route.Our results show that KNM-RU 18340 represents the first evidence of a member of the et al.3 suggested that chameleons originated on Madagascar and dispersed over water at least three times to Africa, and once each to the Seychelles, to the Comoros archipelago (where they occupy two islands) and to Reunion. In contrast, Tolley et al.4 suggested that the family originated in Africa, with two separate oceanic dispersal events to Madagascar during the Palaeocene (Brookesiinae lineage) and the Oligocene, when prevailing oceanic currents would have favoured eastward dispersal. The early Miocene Calumma benovskyi sp. nov. supports an African continental origin for Malagasy chamaeleonine lineages, at least for the Calumma clade. The oceanic dispersal using currents that favoured eastward dispersal appears to be the most probable scenario  —to cross such distances43. Rafting has been suggested for anole lizards migrating around the Caribbean44 or for mabuyid skink Trachylepis atlantica on the island of Fernando de Noronha (Brazil), where the ancestors of this species are believed to have rafted from Africa, across the Atlantic, during the last 9 million years45; Trachylepis is otherwise distributed in Africa and Madagascar46.Raxworthy rio Fig. . The exiCalumma is also supported by the distribution of members forming a clade [Chamaeleo + Trioceros + Kinyongia + Furcifer + Calumma]; where only the latter two taxa are found in Madagascar4. According to Tolley et al.4, Calumma and Furcifer are sister taxa and the split of these two lineages occured in the Eocene. But within these genera, species-level divergence occured during the Oligocene and Miocene. However, if the split is older than the documented early Miocene occurrence of the extinct continental species Ca. benovskyi sp. nov., this might suggest that the last common ancestor of these two lineages most likely lived on the continent. This then suggests a continental origin for both, Calumma and Furcifer.The African origin of Calumma benovskyi sp. nov. represents the oldest known member of this genus and shares some characters with the basal node of this clade, based on our current knowledge we can assume that Ca. beniovskyi most likely does not form an ancestral lineage for the Calumma clade. It is recovered as sister to Ca. brevicorne rather than in a basal position to all Calumma. Moreover, molecular data suggest that the ancestral lineage should be older than the early Miocene4; yet, this remains to be verified with fossil data of an older age. For now the diversity and morphological disparity of the Oligocene and early Miocene Calumma in Africa remains unknown. However, the assignment of KNM-RU 18340 to the genus Calumma and its presence in the early Miocene of Kenya demonstrates that the Calumma lineage was present in Africa at a time when oceanic currents supported eastward dispersal42. This is consistent with the previously suggested hypothesis of Tolley et al.4 based on molecular data.Although the Miocene Calumma from Madagascar to Africa through oceanic dispersal, as previously suggested by Raxworthy et al.3. According to several authors , the prevailing oceanic currents at that time support dispersal from Africa to Madagascar, and not the other way around. According to McCall47, some areas of the Mozambique Channel were dry land during the middle Eocene - early Miocene. Even if animals used several intervening islands in a stepping-stone chain, eastward currents would have hindered their westward dispersal. Moreover, chameleons are relatively poor swimmers rendering the westward route scenario even less probable. Ali and Huber41 also pointed out that shortly after the early Miocene, the currents between Africa and Madagascar turned in the opposite direction , like in present-day surface-water circulation. From the middle Miocene onwards, currents would thus have hindered journey to Madagascar for any non-volant and non-swimming taxa, and could have supported the development of insular endemism for terrestrial animals there.The above scenario challenges a dispersal of the genus 35. But such a bridge would have enabled any taxon, even large animals , to disperse. This scenario would neither explain the quasi absence of large-bodied terrestrial mammals, nor the limited number of mammal families that live on the island today.Hence, the “out of Madagascar” dispersal scenario would have required the existence of fully terrestrial land bridge between the late Oligocene and the early Miocene. Of note is that the separation of Africa and Madagascar occurred already during the MesozoicThe skull KNM-RU 18340 (field number RU 1916'87) is housed at the National Museums of Kenya, Nairobi. The specimen comes from the early Miocene locality Rusinga Island and is remarkably complete and three-dimensionally well-preserved. Its maximum anteroposterior length  is 32.1 mm. The specimen has not been fully prepared from the rock and this matrix still fills the whole internal region and hides many bony elements.We µCT scanned this skull at the University of Witswatersrand (South Africa), using a Nikon Metrology XTH 225/320 LC Xray microtomograph, with an acceleration tension of 100 kV and an intensity of 50 µA. As much as 2000 radiographs were acquired with an exposure time of 4 s and an averaging of 1. The resolution was 18.83 µm. The micro-CT data were analyzed using VG Studio Max 3.2 and Avizo 8.1. The right side of the skull is in better condition and more completely preserved than the left side. For this reason, we focused our reconstruction efforts on the right side, and supplemented certain areas with elements from the left side, when better preserved. For the reconstruction of the whole skull in dorsal view, missing areas were restored by mirror imaging.24 , in addition to the extinct taxon represented by the Kenyan material described here. Uromastyx was used as the outgroup. The principal goal of this analysis is to understand the relationship of the Kenyan early Miocene taxon among Chamaeleonidae. The data matrix was analysed using maximum parsimony as an optimality criterion in the program TNT and the NT (New Technology) search (with ratchet) with 1000 iterations48. All characters were treated as unordered and were equally weighted. Support was estimated through Bremer support indices49. Mesquite v.2.7550 was used to visualize all trees. In the third analysis (see above), the molecular phylogeny of Tolley et al.4 was used to constrain the ingroup relationships. The command used was: . KNM-RU 18340 was the only taxon excluded from the constraint tree, leaving it free to float.A morphological data matrix  performed on the Procrustes residuals. We constructed a neighbor-joining tree on the PC axes that jointly explained 99.6% of the variance to evaluate phenotypic proximity between the fossil and the extant genera. All analyses were performed in R  using the libraries RMORPH53, MASS54, APE55 and ADE456.We ran a 3D geometric morphometric analysis on 27 species of chameleons in addition to the fossil. Extant species covered all genera with most genera represented by two or three species. Specimens were micro CT-scanned and surfaces were cleaned in Geomagic Studio and imported in the ‘Landmark’ software50 was used for optimizations and ancestral state determinations using parsimony (see Supplementary Data et al.4. KNM-RU 18340 is excluded here due to its uncertain topology within Calumma. In contrast, Ca. boettgeri is added to increase taxon sampling in regards of Calumma.Mesquite v.2.75Supplementary Information.Supplementary Information2.


Objective: The prognosis of status epilepticus (SE) is highly related to the underlying etiology. Inflammation of the central nervous system (CNS), including infection and autoimmune encephalitis, is one of the treatable conditions causing SE. The initial presentation of infectious and autoimmune CNS disorders can be quite similar, which may be difficult to differentiate at the beginning. However, treatment for these entities can be quite different. In this study, we aim to identify the differences in clinical features among patients with infectious and autoimmune SE, which could help the clinicians to select initial investigation and ensuing therapies that may improve overall outcomes.Methods: This was a retrospective study that included 501 patients with SE within a period of 10.5-years. Patients with inflammatory etiology were collected and separated into infectious and autoimmune SE. The symptoms at onset, SE semiology, status epilepticus severity score, and END-IT score at admission, treatment for SE, and outcome  on discharge and last follow-up were recorded. Data on the first cerebrospinal fluid, electroencephalography, and magnetic resonance imaging were also collected.Results: Forty-six (9.2%) of the 501 patients had SE with inflammatory etiology. Twenty-five (5%) patients were autoimmune SE and 21 (4.2%) were infectious SE. Patients with autoimmune SE have younger age and female predominance. As for clinical presentations, psychosis, non-convulsive SE, and super refractory SE were more common in patients with autoimmune SE. Nevertheless, the prognosis showed no difference between the two groups.Conclusion: The different initial clinical presentations and patient characteristics may provide some clues about the underlying etiology of SE. When inflammatory etiology is suspected in patients with SE, younger age, female sex, psychosis, non-convulsive SE, and super refractory SE are clinical features that suggest an autoimmune etiology. Status epilepticus (SE) is a neurological emergency associated with significant morbidity and mortality that usually requires admission to an intensive care unit –3. The gBrain inflammation can also cause SE , 11, incOnly two studies have specifically addressed the differences between infectious and autoimmune etiology. Spatola et al. were the first to report that patients with an infectious etiology were older in age and had a more severe clinical presentation at first encounter . SubsequWe retrospectively reviewed the medical records of all patients with SE admitted to the Neurological Intensive Care Unit at Kaohsiung Chang Gung Memorial Hospital between January 2006 and July 2016. This study was approved by the Chang Gung Medical Foundation Institutional Review Board.SE was defined as 5 min or more of continuous clinical and/or electrographic seizure activity or recurrent seizure activity without recovery (returning to baseline) between seizures . RefractInflammatory SE was defined as SE due to acute inflammation of the brain parenchyma, with or without the involvement of the meninges , and furSubacute onset (rapid progression of fewer than 3 months) of working memory deficits (short-term memory loss), altered mental status, or psychiatric symptoms.New focal CNS findingsSeizures not explained by a previously known seizure disorderCSF pleocytosis (white blood cell count of more than five cells per mm3)MRI features suggestive of encephalitisAt least one of the followings:Reasonable exclusion of alternative causesAutoimmune SE was defined as suggested by previous experts' consensus :SubacutSE patients who had positive neuronal surface auto-antibodies testing  in serum or CSF were also considered as autoimmune SE.Infectious SE was diagnosed if microbiologic studies demonstrated an infectious agent. Those without evidence of microbiologic studies would have to fulfill one of the underlying criteria : (1) fevClinical information was recorded using a standardized evaluation form, including the symptoms at onset, SE semiology and classification, status epilepticus severity score (STESS)  and the U-test. p < 0.05 was considered as statistically significant.Statistical analyses were performed using the IBM SPSS Statistics for Windows . To compare demographic data between infectious and autoimmune groups, categorical variables were assessed using Chi-square or Fisher exact tests, and continuous variables were compared using the Mann-Whitney n = 21) and those with an unknown etiology (n = 8) were also excluded (During the 10.5-year study period (January 2006–June 2016), 501 patients with SE were reviewed, of whom 46 (9.2%) had an inflammatory etiology, including 25 females (54.3%) and 21 males (45.7%). Of the excluded patients, 237 had cerebrovascular disease, 77 had metabolic disturbances, 43 had head trauma, 39 had intracranial tumors, 11 had AED withdrawal, 11 had alcohol-related SE, three had neurodegenerative diseases, two had mitochondrial diseases, and three had medically refractory epilepsy. Of the three patients with medically refractory epilepsy, two had Dravet syndrome and one had focal cortical dysplasia. Patients without CSF data , and more of the patients with autoimmune SE were female compared to those with infectious SE . The initial presentation of both groups was similar, including the STESS and END-IT score at admission, onset symptoms, and latency of seizures after the initial symptoms. Psychosis was the presenting symptom only in the autoimmune SE group  and non-convulsive SE was more prevalent among the patients with autoimmune SE compared to those with infectious SE . Refractory SE occurred more commonly in the autoimmune SE than in the infectious SE group, but the difference was not statistically significant . Super refractory SE was more common in the autoimmune SE group than in the infectious SE group . The number of AEDs used was similar between both groups, but the use of general anesthesia was more common in the autoimmune SE group than in the infectious SE group . However, the duration of admission or ICU stay, mRS score at discharge, and mortality rate during admission were similar between the two groups. The sensitivity and specificity for STESS to predict the outcome at discharge were 70.6 and 44.8%, respectively, compared to 68.8 and 45.5% at last follow-up. The sensitivity and specificity for the END-IT score to predict the outcome at discharge were 9.4 and 100.0%, respectively, compared to 21.4 and 100.0% at last follow-up.The clinical features of infectious and autoimmune SE are compared in p = 0.014), higher median white blood cell count , higher percentage of neutrophilic predominance , and higher percentage of low CSF/blood glucose ratio  compared to the patients with autoimmune SE, who had a higher percentage of lymphocytic predominance . There was no significant difference in IgG index between the two groups. The autoimmune SE group tended to have a higher rate of background slowing activity in the first EEG, but the difference between autoimmune and infection was not statistically different . The presence of sporadic epileptiform discharge, periodic discharge, or electrographic seizure was similar among the two groups in the first EEG study. With regards to the first MRI findings, an abnormal FLAIR signal was observed in 11 patients with autoimmune SE and eight patients with infectious SE. A restricted diffusion signal on DWI was found in 14 patients with autoimmune SE and 10 patients with infectious SE. However, there were no significant differences in abnormalities in the FLAIR and DWI signals between the two groups. Detailed descriptions of the locations of the abnormal signals on FLAIR and DWI are presented in The results of CSF and EEG are presented in Inflammatory SE is a previously under-recognized subgroup of SE. In the current study, 9.2% of all cases of SE were related to an inflammatory etiology, which is in accordance with previous studies (range from 6 to 12.8%) , 20. InfAmong all patients with SE, infection accounted for 4.2% and autoimmune accounted for 5%. This suggests that autoimmune SE is as common as infectious SE , 29, andThe onset symptoms of autoimmune SE can be various. Alteration in mental status is the cardinal symptom , but proCSF studies are an important tool to identify the cause of SE, however, such studies can be challenging clinically. Neutrophilic predominant pleocytosis usually points toward a bacterial infection or the early stage of viral encephalitis, especially in the first 24 to 48 hours , 24. IntMRI can aid in the search for the etiology of SE, but with limitations. Limbic encephalitis may present as an increased FLAIR/T2 signal or abnormal DWI in the medial temporal lobes –38, and EEG is routinely used to evaluate patients with seizures or disturbed consciousness. Slow background activity was more dominant in autoimmune patients compared with other etiologies of seizure , althougWe found that general anesthesia was more commonly used in the patients with autoimmune SE. This is in accordance with previous studies in which patients with autoimmune SE were less responsive to AEDs , 30, 41.The functional outcomes were similar in both infectious and autoimmune groups with a similar mRS score at discharge and similar mortality rate during admission. However, most of our patients had a poor outcome at discharge or last follow-up . Our study showed that the predictive values of STESS and END-IT scores were not in the same direction. That is, STESS was more sensitive but END-IT was more specific in terms of predicting the outcomes at discharge. More studies may be needed to compare the use of these two scores. In addition to functional impairments, a recent study reported that patients also had substantial impairments in their quality of life after SE . Our patThe limitations of this study are that it was conducted at a single hospital and that the design was retrospective. In addition, the study was started before the availability of recent autoimmune encephalitis screening tests and immunotherapies, which may have affected the outcomes.In conclusion, we observed that patients with autoimmune SE had a younger age at onset, female predominance, and often presented with psychosis, super-refractory SE and non-convulsive SE. The initial clinical investigations including EEG and MRI only provided limited information about the underlying etiology. CSF tests were helpful in diagnosing bacterial infectious-related SE but had difficulty in differentiating viral encephalitis and autoimmune SE. Since these two etiologies have different treatment strategies and the presenting symptoms are quite similar , 29, it This study is approved by the Chang Gung Medical Foundation Institutional Review Board (IRB No.: 103-3665B and 201800677B0).All authors have read and approved the final manuscript. C-HL contributed to clinical data analysis and draft of the manuscript. Y-TL, C-JH, and F-YS had contributions to clinical data acquisition and analysis. M-HT had substantial contributions to the conception and design of the study, data analysis, critical revision, and final approval of the manuscript.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.


A composite measure of 8 indicatorsof AL ,waist-to-hip ratio, total cholesterol, glycated hemoglobin (hemoglobin A1C),triglycerides and C-reactive protein) was calculated.t = 1.55, p = .126). Aprincipal component factor analysis was conducted on the 8 AL indicators; a2-factor solution explained 57% of the variance. Group differences in thetwo AL factors were analyzed using MANOVA. BMI and waist-to-hip ratios werelower, but blood pressure and triglycerides were higher in the US group andwere mediated by time in the US. Greater acculturation stress wassignificantly related to increased waist-to-hip ratio .there were no significant group differences in AL between Mexican and Mexicanimmigrant women ( findings suggest some measures of AL increased with time in the US, andacculturation stress may be a significant factor. Althoughmigration from Mexico has slowed greatly in the past few years  , Mexico still has a greater percentage of its citizens living abroad(mostly in the US) than any other country in the world  . Moreover, more than one-third of these mexicanos en elextranjero (Mexicans living abroad) have resided in the US for 15 yearsor more, many with precarious legal status  .The US and Mexico are neighbors sharing the longest and most active migrationcorridor in the world. More than 11.7 million Mexican immigrants currently reside inthe United States (-) data, the risk for cardiovascular disease among immigrant Latinos isrelatively low at the time of arrival but increases greatly with length of residencein the US  . Mexican-American women in particular have one of the world’s highestrates (44%) of metabolic syndrome (MS), a disorder characterized by central obesity, insulin resistance/hyperinsulinemia, hypertension, anddyslipidemia  , all of which increase the risk for atherosclerotic disease.Consistent with the Hispanic Paradox -5 da , one factor that may contribute to MS among Mexican immigrant women isperceived stress  . Acculturation-related factors such as family separation, culturalconflicts, low socioeconomic status, language barriers, racism and discriminationand low perceived control over employment may contribute to chronic stress and maypredispose Mexican immigrant women to the development of MS. Recently, the threat ofdeportation is a major stressor for many families where one or more members areamong los sin papeles (the undocumented). To date, there is littleknown regarding patterns of AL accumulation and the impact of chronic stress on ALamong Mexican immigrant women  .Using a framework of allostatic load (AL) 8 , on . In contrast, acculturation stress is defined as a negative reactionto intercultural contact or the cultural adaptation process. Mexican immigrants tothe US may experience acculturation stress when seeking housing, work, or educationor because of racial/ethnic discrimination and loss of social support  . These conflicts are frequently encountered by new immigrants;however, if migration does not result in a substantially higher quality of life andfinancial security, acculturation stress may become a chronic state  . This is particularly true among the undocumented. Acculturationstress has long been associated with poor mental health in a number of studies amongMexican immigrants (-) . Consistent with the model of (AL), recent studies suggest thatcumulative exposures to high levels of chronic psychological stressors may lead to avariety of physiologic conditions as well.In its simplest form, acculturation can be conceptualized as a normative process thatoccurs when a person from one culture is exposed to another culture  . AL is the collective term used to refer to damage incurred by thebody as it adapts to such psychosocial or physical stressors. It has been positedthat AL may negatively affect the body through a variety of biochemical mechanisms . Constant exposure to frequent stress may lead to unexplained surgesin blood pressure, the overproduction of stress-related hormones resulting ingradual degradation of the immune system, and “overloading” of the body’scompensatory mechanisms. Over time, these physiological responses to chronic stresscan manifest through myocardial infarction, stroke, hypertension, type 2 diabetesmellitus, and certain types of cancers.The biobehavioral process of AL   provide . Systolic and diastolic blood pressures, total cholesterol,triglycerides, fasting glucose, glycated hemoglobin (hemoglobin A1C), body massindex (BMI), waist circumference and waist-to-hip ratio serve are examples of someof the biomarkers used to assess AL. Individuals with a high AL are particularlyprone to develop truncal weight gain, insulin insensitivity and othercharacteristics of MS. Although the terms AL and MS are sometimes usedinterchangeably, the data suggest that they are distinct concepts  ; MS can best be understood as one manifestation of AL.AL is generally measured through a composite index of signs and symptoms ofcumulative strain on various organs and tissues, with a concentration on thecardiovascular system  . The authors controlled for a number of demographic, socioeconomic,and health input covariates but did not measure perceived stress directly.Similarly, in a study of adults in Texas, foreign-born Mexicans had lower AL scoresthan US-born Mexican Americans, and acculturation measures did not account for thedifference  . Chronic work-related, financial, and caregiving stressors wereassociated with elevated ALs in a separate study, but immigrant women were not partof the sample  . To date, few studies have focused on the impact of perceived stressassociated with acculturation as a factor in AL among Mexican immigrant women.There is a limited body of knowledge regarding AL among Mexicans and Mexicanimmigrants. In an early study of AL among Mexicans, Mexican immigrants had lower ALscores upon arrival than US-born Mexican Americans, non-Hispanic whites, andnon-Hispanic blacks, and this advantage lessened with duration of residence in theUS This study compared Mexican women from the indigenous state of Oaxaca who immigratedto the US with a matched group of women born and residing in Oaxaca using measuresof perceived stress and a range of biomarkers of AL, including body mass index(BMI), waist-to-hip ratio, systolic and diastolic blood pressure, and finger stickcollections of dried blood spots (DBSs) for C-reactive protein, HgbA1C, totalcholesterol and triglycerides. Four hypotheses were proposed: 1) Levels of AL amongimmigrant Mexican women in the US will be higher than women in Mexico; 2) Length oftime living in the US will predict levels of AL; 3) Perceived stress scores will begreater among women living in the US; and 4) Acculturation stress, but not perceivedstress, will predict levels of AL among women in the US.A matched case-control design was used in this pilot study. Approval for the studywas granted by the Institutional Review Board of Rutgers University, RutgersBiomedical and Health Sciences (Pro 20140000012) and the Vice-Rector of theUniversidad de la Sierra Sur campus of the State University of Oaxaca in Miahuatlán,Mexico.promotoras /trainedcommunity health workers first sought out women who immigrated from the specificcommunity in Oaxaca through personal contacts and announcements placed in churchbulletins. Later, in Mexico, a matched sample of women was recruited through thelocal Health Center/Centro de Salud by a local female physician who was wellregarded by the women in the community. Participants were matched by age. Many ofthe women from the Oaxaca (control) sample were considered blood relatives  of the women in the US (case) sample; the use of relativesas controls helped to assure the groups had significant overlap and helped to limitconfounding by genetic factors thought to be related to MS and elevated AL . The 1:2 sampling ratio was chosen based on published recommendationsfor case-control studies  . Likewise, it has been noted that when the number of cases in a studyis small, the ratio of controls to cases can be raised to improve the ability tofind significant differences  . The sample size of 59 women is consistent with the number of subjectsrequired for pilot studies using a comparative design to calculate effect sizes toestimate power and sample size needed for a larger study  . Sample sizes of 15-25 per group were recommended for small to mediumstandardized effect sizes  .Data collection took place in two locations: 1) an urban community of approximately56,000 residents in central New Jersey, where 40-50% of the full-time residents ofthe community are immigrant families from southern Mexican states, particularlyOaxaca; and 2) a small indigenous community of approximately 14,000 residents in thewestern Sierra Sur region of Oaxaca, which is the home to a large percentage ofthese immigrants. Participants were recruited in both the US and Mexico usingpurposive sampling methods. In the US, Subjects were all premenopausal adult women born in the state of Oaxaca, between18-45 years of age, nonpregnant, and able to understand Spanish and/or English.Menopausal and postmenopausal women were excluded because of the greater prevalenceof MS in this population. suggested that many indigenous Mexican women are comfortable speakingin Spanish but much less comfortable reading and writing in Spanish. Participantsprovided demographic information regarding age, number of years of education,marital status, residence of spouse (US or Mexico), number of pregnancies and livingchildren, place of birth and when applicable, the number of years living in the US.Spanish language versions of the questionnaires were used for both groups; bilingualresearch assistants (Mexico) and promotoras (US) assistedparticipants by reading items to them when necessary. All participants completed a14-item Spanish language version of the Perceived Stress Scale (PSS)  . This version  measured the degree to which social situations were appraised asstressful. It is widely used and has utility in predicting biomarkers of stress.Individual scores on the PSS can range from 0 to 56 with higher scores indicatinghigher perceived stress. Prior to use, the literacy level of the PSS and theHispanic Women’s Social Stressor Scale (HWSSS)  were assessed using the Fernandez-Huerta method  , and both were found to be at a 7 th grade level. The PSShas been tested among adults in Mexico with a reported internal consistency amongwomen of α = .78  . The HWSSS, a 41-item scale used to assess social stressorsexperienced by Mexican and Mexican-American women, was chosen for this study fromamong other scales as a proxy measure of acculturation stress. This decision wasmade in consultation with a group of community promotoras ; themajority of acculturation stress scales reviewed focused on the experiences of newimmigrants (particularly English language proficiency issues). In contrast, theHWSSS appeared to more adequately address the stressors of immigrant women who hadbeen living in the US for a decade or more. The 41 items are based, in part, onitems derived from the Immigrant version of the Hispanic Stress Inventory (HSI), awell-established tool to measure acculturation stress  . Individual scores on the HWSSS can range from 0 to 123, with higherscores indicating higher perceived stress. In preliminary testing, the authorsidentified six subscales of the HWSSS ; Cronbach’s alpha coefficients ranged from .73-.94 .Participants were enrolled using verbal consent since the lead author’s previousexperience working in the immigrant Mexican community  . The packets were then mailed to the laboratory from collection sitesin both the US and Mexico. Elution and analyses of the DBS eluates were carried outin the laboratory using a standardized process  . Participants in the US were compensated with a $10 gift card to apopular “big-box” store. Following consultation with local leaders, participants inMexico were given a bag of groceries containing rice, beans and tuna fish.The aforementioned measures of AL were assessed. DBSs were selected because they area minimally invasive method of incorporating biomarkers into population-based healthresearch in a binational study. SurgiLance Pink SLN 300, 2.88 mm 21-gauge high-flowlancets were used to obtain the finger-stick specimens. After discarding the firstdrop of blood, a drop was placed in each of the five circles on a Whatman 903™Protein Saver Card. In keeping with the protocol established by the BiomolecularLaboratory at the University of Rochester, School of Nursing, the samples were givenadequate time to dry and then stored in low gas-permeability plastic bags with adesiccant packet added to reduce humidity. When the specimens were sufficiently dry,the blood spots were a dark brownish color and no bright red areas were visible , a measure of allostatic load (AL) was designed to summarize levels ofphysiological activity across a range of regulatory systems, including systolic anddiastolic blood pressure, BMI and waist-to-hip ratio, total cholesterol,triglycerides and HgbA1c. For each of the 8 indicators of AL, participants wereclassified into quartiles based on the distribution of scores of the entire cohort.AL was the sum of the number of indicators where the participant was in the highestrisk quartile. In this scoring system, AL scores could range from 0-8. p = .001 with 4 degrees of freedom. One participant had aMahalanobis distance that exceeded the criterion and was excluded from the MANOVAanalyses. Levene’s test was used to test for homogeneity of variance.Following the methods of Seeman et al. 34 , a , more recent research has suggested that a two-factor model mightexplain more of the variance in AL  . More recently, it has been suggested that model invariance acrosssubpopulations does not preclude the possibility that the measurement of AL maydiffer importantly by the studied sample  . Therefore, the one-factor and two-factor models need to be examinedin this study to determine which model is a better fit to the data in thesample.While the original AL model was based on a single factor 8 , moDescriptive statistics were computed for all variables. Next, the eight components ofAL were examined by factor analysis to determine whether a single-factor or atwo-factor model was a better fit to the data. . For hypothesis 3, independent t-tests were used to test for groupdifferences (US and Mexico) in perceived stress (PSS). For hypothesis 4, Pearson’s rwas used to examine the bivariate correlations between the PSS and AL and the HWSSSand AL. Analyses were conducted using SPSS Version 23, and the significancethreshold for all analyses was set at p =.05.For hypothesis 1, the AL scores for the two groups (US and Mexico) were compared byan independent t–test. Multivariate analysis of variance (MANOVA) was used toidentify the specific indicators that led to group differences in AL and to buildsuccessive models to identify covariates that explained the differences. Wilk’slambda was used to assess whether the MANOVAs were significant. Hypothesis 2 wastested using Pearson’s r to identify the specific AL indicators that were associatedwith length of time living in the US. Mediational analysis was then conducted withthe indicator regressed on years in the US and with PSS scores as a mediatorp values werep = .88 and p = .28, respectively). Cronbach’salpha for the PSS was .71 and .95 for the HWSSS. The means and standard deviationsof the study variables (n = 59) are presented in A total of 19 immigrant women were enrolled in the study in the US. These cases werethen matched with 40 women of the same age and indigenous background (Tacuate) whoresided in the home community in Mexico. The ages of women in both countries weresimilar . Ninety percent ofthe women in the US were married, as were 75% of the women in the Mexico sample.Women living in the US had a mean 3.05 children (SD = 1.08), while Mexican women hada mean 2.57 children (SD = 1.70). Forty-two percent of women in the US finished theequivalent of elementary school in Mexico, while another 42% listed middle school astheir highest level of education. While a similar percentage of women in Mexicoattended only elementary school (40%), another 27.5% went on to graduate from highschool, and 17.5% attended or graduated from college. As a community thatexperiences high levels of emigration, 87.5% of the women in Oaxaca reported havingfriends or relatives who had immigrated to the US, and 17% reported that theirspouse was currently living in the US. All of the women in the US lived with theirspouses. The mean time in the US was 16.37 years .Scores on both the PSS and the HWSSS were normally distributed  explained 35% ofthe variance and consisted of systolic and diastolic blood pressure, totalcholesterol and triglycerides. The second component (metabolic) explained anadditional 22% and consisted of BMI, waist-to-hip ratio, hemoglobin A1C andC-reactive protein.Group Differences in AL (Hypothesis 1). The AL scores for the twogroups (US and Mexico) were first compared by an independent t-test. The AL scorefor the US group  was notsignificantly different than the AL score of the Mexican group , t (57) = 1.55, p = .13.However, the US women had lower BMI and waist-to-hip ratios but significantly highersystolic and diastolic blood pressure and triglycerides (TG) than the women inMexico. The result demonstrates that the combined AL score did not differ betweengroups. Separate MANOVAs were then conducted for each factor (cardiovascular andmetabolic). The first MANOVA examining the cardiovascular indicators of AL found astatistically significant difference between the groups, F = 7.719, p < .001; Wilk’s Λ = 0.623, partial η 2 =.377. Levene’s test was not significant, indicating homogeneity of variance. Asshown in F  = 11.50, p< .001; Wilk’s Λ = 0.526, partial η 2 = .474. Levene’s test wassignificant for the indicators of BMI and waist-to-hip ( p <.05)indicating a lack of homogeneity of variance. Examining the ANOVAs for eachindicator revealed significant group differences for 2 of the indicators. As shownin U = 1.84, z = 2.77, p = .006), andwaist-to-hip, showed significant group differences.The second MANOVA found a statistically significant difference between groups in themetabolic indicators of AL, Length of time in the US and AL ( Hypothesis 2 ).The MANOVAs were repeated with the covariates of age and education levels. The samegroup differences were found, and the same 5 indicators were different betweengroups. Given the hypothesis that acculturation stress is related to AL, a covariatewas added to the 2 MANOVAs. The group differences in the cardiovascular factor wereno longer significant when the covariate of years in the US was added  = 0.74, p = .566; Wilk’s Λ = 0.945,partial η 2 = .055). Levene’s test was not significant, indicatinghomogeneity of variance. In addition, there were no group differences in theindicators of DBP, SBP, and TG when accounting for years of residence in the US.This finding suggests that time in the US is associated with specific indicators ofAL, specifically systolic and diastolic blood pressure and triglycerides. To examinethe effect of time in the US on indicators of AL, a mediational analysis wasconducted with systolic blood pressure regressed on years in the US and withPerceived Stress Scale (PSS) scores as a mediator (36) . The overallmodel was significant, F  = 10.27, p <.001, R2 = .32, Time in the US had a significant direct effect on SBP  with no indirect effect through PSS. Similar patternswere found for both diastolic blood pressure and total triglycerides. The conclusionis that time in the US is directly and proportionately associated with increasedSBP, DBP and TG and PSS scores do not mediate the associations.Group differences in perceived stress scores (Hypotheses 3). Theperceived stress scores for the two groups  were compared by anindependent t-test. Perceived stress scores were higher among women in Mexico  than the US , but not significantly so( t (57) =-1.52, p =.13). This finding suggeststhat there were no differences in perceived stress between Mexican and Mexicanimmigrant women in the study.Relationships among perceived stress, acculturative stress and AL(Hypothesis 4). In the US group, acculturation stress scores (HWSSS)were not correlated with total AL scores but were significantly correlated with theAL indicator waist-to-hip ratio . In the US group, PSS was not significantly correlated with total AL scores orany of the 8 indicators of AL. Among women in the US, total scores of the PSS werenot significantly correlated with total scores of the HWSSS , supporting the assertion that the instrumentsaddressed two distinct phenomena, perceived stress (PSS) and acculturative stress(HWSSS). The small sample size of women in the US ( n = 19)precluded the use of regression analyses. Nonetheless, these findings suggest thatthere are unique acculturation-related stressors that may play a significant role inpromoting truncal weight gain among this group of Mexican immigrant women. Specificitems on the HWSSS that caused considerable stress for the US women included livingwith relatives; being ignored or getting poor service at stores or offices becausethey were Hispanic; not having enough money to pay for necessities such as food fortheir families or shoes for their children; feeling lonely and isolated and missingthe help and support of their family in Mexico. , which linked AL among Mexican immigrants with increased time spent inthe US. This relationship was particularly true in this study with regard tosystolic and diastolic blood pressure and triglycerides. Given that the participantsin the study were young to middle-aged women, the prevalence of hypertension in theUS group was somewhat unexpected, although the results of other studies indicatethat hypertension is likely underdiagnosed and untreated among immigrant Latinos .Our findings support previous studies ,18 ,(-) , an additional factor that may parallel this process among Mexicanwomen is the nutrition transition. The nutrition transition is defined as a broadshift in dietary habits and physical activity that coincides with economic,demographic, and epidemiological changes  . For the past twenty years, Mexico has been in Stage 4 of thenutrition transition, which is a phase characterized by weight gain and an increasein nutrition-related noncommunicable diseases. In contrast, the US is shifting intoStage 5, where the focus is on weight loss/weight management and behavioral changes.As a result, it is quite possible that Mexican immigrants who arrive in the US inthe near future may be heavier than their counterparts in the US, a finding thatchallenges the so-called Hispanic Paradox. Further analyses of dietary intake amongboth groups of women may help to confirm these findings.Despite the higher scores for blood pressure and triglycerides, we did not find asignificant difference in AL between the two groups of women. This was likely due tothe unexpectedly higher BMI and waist-to-hip ratios among women in the Mexicangroup. Although acculturation stress has traditionally been thought to contribute toweight gain among immigrant women (-) . However, the presence of a significant association between a specificsource of stress, namely, acculturation, and waist-to-hip ratio, providespreliminary support for the AL model among Mexican immigrant women.Although the number of years spent in the US was associated with increases inselected indicators of AL, the length of time more closely approximatesacculturation, not acculturation stress. Empirical evidence suggests that it is notthe acculturation process itself but the stress of adapting to life in a new countrythat has the greatest impact on the physical and emotional health of Latinoimmigrants. Perceived stress scores were slightly higher among women living inMexico than in the US. This finding is not totally unexpected; the very fact thatsuch a high percentage of Oaxacans immigrate to the US suggests that life isfinancially and emotionally difficult for those who stay behind mexicana en el extranjero for more than a decade, asituation that is becoming more common. Long-term immigrants suffer from a uniqueset of challenges that have not been well addressed in the literature  . The two groups of women were well matched regarding age andindigenous background. This study sets the stage for a larger investigation into theeffects of acculturation stress on AL in Mexican immigrant women, which couldpotentially inform a multipronged intervention to lower AL in this group.This binational pilot study took a novel approach to assess the effect ofacculturation stress on AL among Mexican women from the same community living in theUS and Mexico. In contrast to previous studies that simply linked acculturation toAL using a proxy measure of the number of years living in the US, our study used twodistinct measures to quantify the relationship between stress and AL. In contrast toprevious studies, our US sample contained a significant percentage of women who hadlived as a  . The unique reciprocal relationship between the two binationalcommunities also posed some interesting challenges, including the issue of returnmigrants to Mexico. Following the recession in the late 2000s, a significantpercentage of Mexican immigrants from the US returned to Mexico, convinced theycould make a better living in their homeland. The Mexican community we visited wasno exception. When immigrants return to rural Mexico, they often create a demand forlifestyles they adopted in the US  . These preferences include building fast food restaurants and agreater reliance on automobiles as a means of transportation. These changes affectthe community at large and may add to the obesogenic environment that characterizesStage 4 of the nutrition transition in Mexico. On a related note, we did notdirectly measure physical activity and nutritional intake in this study; these twovariables would add greatly to our understanding of the role that stress plays in ALand should be included in future studies. In this study, we limited our choice ofbiomarkers to measures that could easily be collected in a community setting. Thisdecision precluded us from assessing neuroendocrine markers such as 24-hour urinarynorepinephrine or epinephrine. In addition, the laboratory we used was not able toaccommodate DBS analysis for inflammatory biomarkers such as interleukin6 (IL-6) andtumor necrosis factor alpha (TNF-α). Focus groups may be needed to explore thepotential for collecting additional biomarkers. Finally, it can be argued that thesample size (N = 59) was too small to conduct the two principal component factoranalyses. There is no consensus in the factor analysis literature concerning aminimum sample size. While older references suggest there should be at least 10cases per variable, with a total of at least 100 cases  , other more recent sources recommend limiting the number of variablesand factors to assure moderate to high levels of communality  . Following the more recent guidelines, we restricted the number ofvariables to 8 and the number of factors in our final model to 2. In our study,communalities ranged from .43 to .75, placing it just below the .6 to .8 averagerecommended as a high range  . It has been suggested that small sample sizes may not be a problemwhen the data are highly reliable and communality levels are high  . Accordingly, we had highly reliable measures of biomarkers , and ourcommunality levels were moderate to high. Lastly, we achieved convergence usingPromax rotation as a solution. Often, samples that are too small will fail toconverge  . For these reasons, we argue that principal component factor analysiswas appropriate in our study.There are a number of limitations to the present study. The use of purposive samplinglimits the generalizability of the findings. A significant number of the women inthe US sample did not work outside the home and had not experienced instances ofwork-place discrimination; many lived in close proximity to each other, which mayhave offered some protection for them against acculturation stress, or the so-called“barrio advantage” In summary, this study supports evidence of a link between length of residence in theUS and acculturation stress in some indicators of AL among Mexican immigrant women.To attenuate the declines in health status among Mexican immigrants postulated bythe Hispanic Paradox, further research will be needed to clarify the roles played byacculturation and acculturation stress in the genesis of allostatic load.