Chewing improves visuospatial performance through locus coeruleus (LC) activation. The effects of bilateral and unilateral mastication were investigated in subjects showing different degrees of asymmetry in masseter electromyographic (EMG) activity during clenching and in pupil size at rest (anisocoria), which is a proxy of LC imbalance. Correlations between performance changes and asymmetry values were found in males, but not in females. Among males, subjects with low asymmetry values  were more sensitive than those with high asymmetry values  to bilateral and unilateral chewing on the side with higher EMG activity (hypertonic). The opposite was true for hypotonic side chewing. BAL subjects were sensitive to unilateral chewing on both sides, while in IMB subjects, hypertonic side chewing did not influence performance in either males or females. Bilateral chewing elicited larger effects in BAL subjects than in IMB subjects, exceeding the values predicted from unilateral chewing in both groups. Finally, pupil size and anisocoria changes elicited by chewing were correlated with asymmetry values, independent of sex. Data confirmed the facilitation of visuospatial performance exerted by chewing. Trigeminal asymmetries modulate the chewing effects, making occlusal rebalancing an appropriate strategy to improve performance. Trigeminal nerve stimulation (TNS) has been utilized for clinical purposes, such as the treatment of drug-resistant epilepsy and refractory trigeminal neuralgia, as well as major depression, post-traumatic stress, and attention deficit hyperactivity disorders . TNS actThese chewing effects on cognitive performance might be related to the influence of chewing on alertness ,16 and aWhile chewing enhances cognitive visuospatial performance, a trigeminal imbalance is detrimental. Indeed, in subjects showing an asymmetric development in their masseter electromyographic (EMG) activity during clenching, bite plate wearing improved both visuospatial performance and haptic task-related mydriasis ,29,30,31The stimulating effect of chewing on visuospatial cognition  is biaseThe purpose of the present study was to assess, in the same population of subjects, the effects of bilateral and unilateral chewing on visuospatial performance and pupil size. Specifically, we aimed to study whether the effects of bilateral chewing could be predicted by the sum of the effects elicited by consecutive bouts of unilateral chewing on each side. Moreover, we investigated whether the effects of chewing on visuospatial cognition and pupil size were modulated by the degree of trigeminal imbalance, leading to a significant correlation between cognitive visuospatial improvement and EMG asymmetry.The experiments were performed on 30 healthy, right-handed subjects , who did not complain of pain or discomfort during habitual masticatory activity. Five subjects  showed mild signs of temporomandibular disorders (TMDs) during the objective clinical evaluation .This study analyzed the modifications in pupil size, anisocoria, and cognitive visuospatial performance elicited under four different experimental conditions, which were tested in three different experimental sessions. Moreover, between the second and third experimental session, subjects could be summited to two additional sessions for (1) manufacturing a bite splint and (2) testing its effectiveness in modifying EMG asymmetry. The flow chart of the whole experimental procedure is illustrated in In experimental session 1 A, as a pThe degree of asymmetry in the average EMG value during clenching  was quantified by the difference in EMG activity between the hypertonic and hypotonic sides divided by the corresponding mean: (EMG hypertonic − EMG hypotonic)/((EMG hypertonic + EMG hypotonic)/2).The participants showing masseter EMG asymmetry during clenching higher than 20%  were selected for an occlusal rebalancing procedure. We refer to these 22 participants as imbalanced (IMB) subjects. Twelve of them  showed higher EMG activity on the right side, while ten showed higher activity on the left side . Four of them  displayed mild signs of TMDs. The remaining 8 subjects with EMG asymmetry less than 20% were all males, and only one of them showed signs of TMDs. We refer to these participants as balanced (BAL) subjects.(a)Bilateral recording of pupil size at rest;(b)Performance assessment in a cognitive visual search task, based on the Spinnler–Tognoni numeric matrices test , in the Following this preliminary assessment, pupil size and performance-related variables were studied either at rest or under bilateral chewing conditions. Whatever the condition analyzed, the initial T0 baseline measurements consisted of:These measurements took about five minutes and were followed either by a two minute period of hard pellet bilateral chewing  , occurring approximately 7 min after T0 and followed by a further 30-minute period of rest (T2).From 2 to 10 days later, in experimental session 2 A, subjecp = 0.001). An example of this reduction in trigeminal asymmetry is shown in the insets of About 18 days after experimental session 2, the IMB subjects underwent the bite splint-manufacturing session A. AccordBaseline (T0) measurements ;Unilateral chewing on the right side (1 min);T1 measurements ;T2 measurements .Experimental session 3 C began fAfter one hour of rest, the entire T0-T1-T2 sequence was repeated, inviting the subjects to chew a new pellet with the same characteristics as the previous pellet on the left side.(1)Bilateral pupil size at rest;(2)Difference in pupil size (anisocoria)—this parameter could be calculated either as left–right or hypertonic–hypotonic side differences;(3)Performance index (PI: target numbers retrieved in 15 s/15), this metric has been described in detail elsewhere ,22).The analysis was based on the following parameters, evaluated at T0, T1, and T2:Subjects sequentially scanned three (10 × 10) numerical matrices, retrieving and ticking with a pencil as many of the (target) numbers indicated above each matrix as possible . To prev2). The two electrodes of each couple were placed on the incisura sigmoidea and on the submental triangle. Biphasic  current pulses  were delivered at the frequencies of 0.618 Hz (incisura sigmoidea) and 40 Hz  by two independent IACER stimulators , as described elsewhere [The left and right trigeminal mandibular nerves were activated by two separate couples of electrodes  were performed using a corneal topographer-pupillographer , which maintained constant artificial lighting of 40 lux over the eye and captured pictures of the eye surface. The iris image was acquired by the camera with an acquisition time of 33 msec. Pupil images and size measurements were displayed on the computer screen. The haptic task was based on a tangram, a puzzle of triangular, square, and parallelogram-shaped forms. Subjects were seated with their heads restrained by the pupillometer, with the dental arches apart. During the task, each subject received the parallelogram piece from the experimenter, which was to be repositioned in its original place using the dominant hand under tactile control. Left and right pupil measurements were collected during separate task repetitions at rest when the subject began to explore the puzzle surface. During this procedure, vision was occluded.The pellet for chewing  had a cylindrical shape (1.0 × 1.0 × 1.5 cm), a reticular structure, and was made of silicon rubber . Its hardness (60 Shore OO) was unmodified by chewing. It was gray in color, sugar-free, odor-free, and tasteless.Masseter EMG activity was recorded using Duotrode Ag/AgCl electrodes , placed as previously described ,38. EMG The distribution of the data was assessed using the Komolgorov–Smirnov test. When the data did not comply with a spherical distribution, the Huynh–Feldt correction was applied to the ANOVA output. The comparison between the changes in performance and pupil-related parameters elicited by the different chewing patterns at T1 and T2 with respect to T0 was performed using a 4 Conditions  × 2 Times  repeated measures ANOVA. To assess the significance of the chewing-induced modifications on cognitive visuospatial performance and pupil size-related parameters, a 3 Times  repeated measures ANOVA was applied for each condition. These analyses were applied to the whole population, as well as separately to males and females.The changes observed in pupil size, anisocoria, and PI at T1 and T2 with respect to T0 were separately correlated with EMG asymmetry and anisocoria by linear regression analysis. Statistics were obtained for the whole population, as well as separately for males and females.Finally, to verify the degree of coupling between changes in pupil size parameters and performance, differences between time points were evaluated  separately for each chewing condition  and submitted to linear regression analysis. Comparisons of slopes and correlation coefficients were also performed ,40,41. The significance level was set at 0.05 for PI. Since pupil size-related variables  could be interdependent, Bonferroni’s correction was applied and the significance level was set at 0.05/3, i.e., to 0.017. IBM SPSS Statistics for Windows version 20.0  was used for the analysis.p = 0.001; EMG asymmetry: 25.3 ± 21.10%, p = 0.028). This finding was attributable to the absence of females showing symmetric EMG activity during clenching (BAL subjects) and the positive correlation between anisocoria and EMG activity. Indeed, when pupil size and EMG asymmetries were expressed as left–right difference, a positive correlation was observed between these two parameters, with the larger pupil size being located on the side with higher EMG activity. Although the data from the whole population could be well fitted by a single regression line , a separate analysis of males . When anisocoria and EMG asymmetry values were evaluated as hypertonic–hypotonic side difference (p = 0.001) but not in females . Average baseline anisocoria and EMG asymmetry values  corresponded to 0.32 ± 0.29 mm and 34.6 ± 23.5%, respectively. Average anisocoria (0.49 ± 0.29 mm) and EMG asymmetry values (43.8 ± 22.7%) observed in females were significantly higher than those observed in males  repeated measures ANOVA, which was separately applied for each condition. The results of this analysis are summarized in p < 0.0005). In this group, PI values at T1 (1.68 ± 0.37 Nos./s) were significantly larger than the T0  values.While simple repetitions of the measurement did not affect the PI, PI values increased significantly at T1 for all chewing patterns, although the changes elicited by chewing on the hypertonic side were very small. However, the effect persisted at T2 only for bilateral chewing. No between-gender differences were found under the conditions of bilateral chewing and chewing on the hypotonic side. Following chewing on the hypertonic side, a significant time effect was observed only for males  = 15.67, p = 0.030) observed for anisocoria in the rest condition was not present after Bonferroni’s correction, the post hoc comparison indicated a significant difference at T1 with respect to T0. This increase in anisocoria values at T1 was exclusively due to females showing a significant time effect  = 9.971, p = 0.001), with the T1 values (0.53 ± 0.28 mm) being significantly higher with respect to both the T0  and T2  values. At variance, neither a significant time effect  = 1.228, p = 0.308) nor significant differences between the T0 (0.11 ± 0.19 mm), T1 (0.15 ± 0.22 mm), and T2 (0.06 ± 0.24 mm) time points could be observed in the male population. Bilateral chewing elicited a significant drop in anisocoria values both at T1 and T2 with respect to T0, and the same occurred following unilateral chewing on the hypotonic side, but in this instance, anisocoria values at T2 and T0 did not differ. At variance, chewing on the hypertonic side significantly increased anisocoria values at both T1 and T2 , hypertonic side pupil  = 4.75, p = 0.008), hypotonic side pupil  = 22.47, p < 0.0005), and anisocoria  = 32.20, p < 0.0005), which are decomposed in Changes in performance and pupil size parameters at the different times and under different conditions were analyzed using 4 Conditions × 3 Times repeated measures ANOVA. Significant condition × time effects were observed for PI  = 8.86, At T1, bilateral chewing had opposite effects on the size of the hypertonic (reduction) and hypotonic (increase) side pupils, while unilateral chewing always increased pupil size bilaterally, although to a larger extent on the ipsilateral side. Contralateral pupil size changes elicited by unilateral chewing were not significantly different from those observed in the rest condition. Pupil size changes at T2 were not significantly different from those at T0 and they were not significantly different from those observed in the rest condition. Finally, the decreases in anisocoria values elicited at T1 by bilateral and unilateral chewing on the hypotonic side were significantly larger than those in the rest condition, while this was not the case for the increase in anisocoria values elicited by unilateral chewing on the hypertonic side. At T2, only the anisocoria changes elicited by bilateral chewing were significantly larger with respect to the rest condition.p < 0.0005). Similar findings were obtained in the different gender and asymmetry  groups. A strong negative correlation was also observed between PI and anisocoria changes , with similar results observed in males and females.An attempt was made to verify whether PI changes between time points could be predicted by the corresponding pupil size and anisocoria changes. This analysis focused on those changes in pupil parameters that were significantly different from the spontaneous fluctuations observed in the rest condition . Moreovep < 0.0005). This correlation, however, was entirely due to the 8 BAL male subjects , since it was not observed among the IMB subjects , irrespective of their gender. It was also observed when only males (BAL and IMB) were taken into account .Under the hypertonic side chewing condition, a correlation was observed between PI and hypertonic side pupil changes . While males and females showed the same trend for the hypertonic side pupil, the correlation observed for the hypotonic side pupil was exclusively due to the females , while the males did not show any trend between these parameters . When BAL and IMB subjects were taken into account, this correlation was only observed among IMB subjects. Under the bilateral chewing condition, PI changes were also negatively correlated with anisocoria changes in IMB subjects . This correlation was mainly due to the female group , with males showing a similar but non-significant slope . Under the bilateral chewing condition, a loose correlation was observed between PI and pupil size changes . This held true for both males and females.In the present study, unilateral chewing lasted for one minute, whatever the side, while bilateral chewing consisted of two chewing bouts of one minute (one on the hypertonic side and one on the hypotonic side). For this reason, we investigated whether the effects of bilateral chewing on PI could be predicted by summating those of unilateral chewing. The changes induced by bilateral chewing at T1 with respect to T0  were significantly higher than those obtained by summing the effects of unilateral chewing  but not to anisocoria baseline values . A more careful analysis revealed that females did not show a correlation of the experimental-predicted difference in PI change with EMG asymmetry  and anisocoria , while males did show these correlations . The difference between R values of males and females was significant for EMG asymmetry . Within the whole population, the difference between the experimental and predicted values of PI change was loosely correlated with EMG asymmetry . The same held true for the hypotonic side pupil, the size of which was increased under all chewing conditions .The size of the hypertonic side pupil increased following both conditions of unilateral chewing, while it decreased following bilateral chewing; consequently, the predicted values (0.14 ± 0.06 mm) were significantly higher than the experimental ones  observed between BAL  and IMB  subjects. p = 0.006). This finding was supported by the significant difference in PI change (p = 0.003). The same results could also be observed for pupil asymmetry . As shown in p = 0.023) and anisocoria . When chewing on the hypertonic side was considered, changes in PI at T1 were significantly and negatively correlated with EMG asymmetry  but not for IMB subjects . Indeed, PI changes at T1 in BAL males (0.142 ± 0.043 Nos./s) were larger than those in IMB males  and females . Interestingly, among males, the T1 and T0 PI values were significantly different for BAL subjects , leading to the significant correlation between the T2–T1 PI difference with EMG asymmetry and anisocoria  with respect to IMB males . The T2–T1 PI difference in BAL males was also significantly different than that of IMB females  and anisocoria  values within the whole population. As shown in Finally, when chewing occurred on the hypotonic side, changes in PI at T1 were not significantly correlated with baseline EMG asymmetry , with the T1 PI values larger than T0 values in both BAL  and IMB  subjects. The T1–T0 PI difference (0.249 ± 0.144 Nos./s) was significant also among females , all of whom being IMB subjects.Among males, PI changes in BAL subjects (0.133 ± 0.036 Nos./s) were smaller than those in IMB subjects , with similar but insignificant trends observed in the two gender groups. A similar result was observed for the hypotonic side pupil, but in this instance, pupil size changes and anisocoria values showed a positive correlation .The spontaneous fluctuations in pupil size and anisocoria observed in the rest condition at T1 with respect to T0 were not correlated with baseline values of EMG asymmetry and anisocoria, whatever population was considered. This finding reinforced the results of the regression analysis performed on the pupil size and anisocoria changes elicited by the different chewing conditions. Only changes significantly larger than those elicited in the rest condition were analyzed. Pupil size changes elicited by bilateral chewing on each side were not correlated with baseline values of EMG asymmetry within the whole population, nor among males and females. A loose correlation was found between the changes on the hypertonic side and baseline values of anisocoria , with similar negative trends in both males and females.Ipsilateral pupil size changes elicited by hypertonic side chewing were not correlated with EMG asymmetry, whatever population was considered . A loose correlation was observed with baseline values of anisocoria . Separate analyses of males and females indicated similar trends in the two groups, which reached the significance level in males  but not in females .As shown in p < 0.0005), as well as among males  and females .p = 0.013). Similar but non-significant trends were observed among males  and females . A strong correlation between these anisocoria changes and the corresponding baseline values was observed within the whole population , with males  and females  showing similar trends. These data are illustrated in Significant anisocoria changes were elicited at T1 by bilateral chewing and by chewing on the hypotonic side. p = 0.003), with both males  and females  showing similar trends. Anisocoria changes were negatively correlated with baseline anisocoria values within the whole population , with similar trends among males  and females . These data are summarized in Finally, anisocoria changes elicited by chewing on the hypotonic side were also correlated with baseline values of EMG asymmetry  but not anisocoria values . As shown in p = 0.562; anisocoria: R = 0.149, p = 0.595). The differences in R values between males and females were significant for both EMG asymmetry  and anisocoria .To assess whether the relative efficacy of the chewing patterns in modulating performance and pupil size was affected by the baseline values of EMG and pupil size asymmetries, we calculated the differences in performance and pupil size changes at T1 under the different chewing  conditions. When the whole population was considered, the hypotonic–hypertonic side difference in PI change was significantly correlated with baseline EMG asymmetry  but not with anisocoria values . While females did not show any correlation of this relative performance indicator with the two measures of asymmetry , strong correlations were observed among males for both EMG asymmetry and anisocoria  or anisocoria baseline values . However, analysis of the two gender groups revealed strong correlations among males with both EMG asymmetry and anisocoria (p = 0.926) or anisocoria . Finally, when the bilateral–hypertonic side chewing difference in PI change was considered within the whole population, this parameter was not correlated with either EMG asymmetry  ,47,48, wThe results of the present study indicated that the effects on performance in a cognitive visual search task elicited by two sequential bouts of chewing on the left and right sides cannot be predicted by assuming a simple additive effect of the two chewing periods . The difference between the predicted and experimental values was significant whatever the gender and level of trigeminal balance/imbalance. The observed difference could be due to the presence of a build-up of excitation that progressively increased the stimulating effect of chewing on cognitive visuospatial performance. Further studies are required to verify this hypothesis. A well-documented example of brainstem build-up phenomenon is the velocity storage mechanism , which pThe present study showed that, in males, trigeminal asymmetries and the related anisocoria values accounted for a sizable fraction of the variability observed in the chewing-related changes in PI, as well as in the relative efficacy of the different chewing patterns in enhancing visuospatial attention. Also, the decay of the effect of bilateral chewing over time (from T1 to T2) was related to the asymmetry indices . These cAt least among males, however, the stimulating effect of trigeminal sensorimotor signals elicited by chewing was modulated by the level of trigeminal/LC asymmetry, which made the hypertonic side of IMB subjects somehow refractory to chewing-related activation . In addiThe refractoriness of the hypertonic side to chewing stimulation could be due to too high LC activity on that side. It is well known that cognitive performance/task-related mydriasis are related to the basal level of LC activity ,4; when The higher sensitivity of BAL subjects to bilateral chewing can be attributed to the fact that their visuospatial performance was affected by chewing on either side and they were more sensitive to the time-cumulative effects of chewing stimulation. Why the hypotonic side in IMB subjects is more sensitive to unilateral chewing than in BAL subjects needs to be explained. It is possible that, in IMB individuals, the persistence of a low resting level of LC activity on this side leads to an enhancement of its excitability following stimulation of trigeminal afferents. Alternatively, we must consider that chewing on the hypotonic side reduces anisocoria in IMB subjects, which might induce enhanced performance in these subjects. Indeed, as proposed by Tramonti Fantozzi and colleagues ,30, the Overall, these findings suggest that, in males, the level of trigeminal asymmetry determines the degree of LC imbalance, which finely modulates the effectiveness of chewing-related trigeminal signals in facilitating cognitive visuospatial improvements in a side-dependent manner.The present investigation showed that, at variance with unilateral chewing, which increased pupil size on both sides , bilaterThe difference observed between the hypertonic and hypotonic side pupils during bilateral chewing could arise from the interaction of two opposing factors. Prolonged chewing activity might reduce the level of stress in subjects ,61, reduThe pupil size changes, elicited by chewing on the hypotonic side in the ipsilateral pupil, were tuned by EMG asymmetry (and anisocoria) so that the larger post-chewing increases in pupil size occurred in the subjects showing the largest asymmetry values. Also, this finding was consistent with a higher sensitivity of the LC neurons on the hypotonic side to trigeminal stimulation in IMB subjects, leading to a slightly higher LC discharge and pupil size soon after the chewing activity. Assuming that this larger pupil size is indicative of higher excitability of LC neurons during the matrices test and, consequently, of larger task-related mydriasis, this would explain the higher sensitivity of PI to hypotonic side chewing in IMB subjects. This higher sensitivity of the LC on the hypotonic side in IMB subjects would also explain the larger drop in anisocoria values of these subjects, which may further enhance chewing-elicited visuospatial improvement. Finally, the present experiments showed that a drop in anisocoria values occurred also following bilateral chewing, which was larger in subjects showing the highest asymmetry values. This might have improved the sensitivity of IMB subjects to bilateral chewing. The neurophysiological mechanisms responsible for these changes in neuronal excitability following chewing  are unknown. Following bilateral chewing, no increase in pupil size at rest was observed, suggesting that no relevant changes occurred in the tonic LC activity of both sides. However, larger task-related mydriasis was induced following chewing activity , indicatIn conclusion, the present study showed that BAL subjects benefitted from the stimulating effects of unilateral chewing irrespective of chewing side and were more sensitive than IMB subjects to bilateral chewing. Moreover, the effects of bilateral chewing could not be predicted by summing those elicited by unilateral chewing on both sides, suggesting that prolonged chewing activity leads to a build-up of excitation within the central structures that mediates the stimulating effect of chewing on performance of a visual attention task. BAL subjects were more sensitive than IMB subjects to the stimulating effects of bilateral chewing, likely due to the effectiveness of both sides in activating ARAS during chewing and a higher ability to recruit a build-up of excitation within brainstem structures. Finally, in males, the changes in visuospatial performance elicited under all chewing conditions, as well as their relative efficacy, were tuned by the degree of trigeminal asymmetry and anisocoria. The present investigation explores the influence of chewing on visuospatial attention and its interaction with the presence of basal trigeminal asymmetry, which are coupled to an apparent imbalance of the central noradrenergic system. There are two possible translational applications arising from this study and previous ones targeting the same topic ,30,32,34The second aspect may concern the field of rehabilitation. There is growing evidence that neurodegenerative diseases may be triggered by neurodegenerative processes occurring in the LC ,74. In pFurther studies are needed to verify the suitability of chewing and occlusal rebalancing within these two promising fields of application. We must acknowledge that this study had several limitations. First, the sample size was rather small. This limited the possibly of detecting significant correlations within the individual male and female populations. Moreover, all of the females were IMB subjects, thus making it impossible to draw final conclusions about the lack of correlations between pupil size/performance changes and asymmetry indices within this gender group.


Prolagus sardus , was an iconic lagomorph species that populated Sardinia and Corsica and became extinct during the Holocene. There is a certain scientific debate on the phylogenetic assignment of the extinct genus Prolagus to the family Ochotonidae (one of the only two extant families of the order Lagomorpha) or to a separated family Prolagidae, or to the subfamily Prolaginae within the family Ochotonidae. In this study, we successfully reconstructed a portion of the mitogenome of a Sardinian pika dated to the Neolithic period and recovered from the Cabaddaris cave, an archaeological site in Sardinia. Our calibrated phylogeny may support the hypothesis that the genus Prolagus is an independent sister group to the family Ochotonidae that diverged from the Ochotona genus lineage about 30 million years ago. These results may contribute to refine the phylogenetic interpretation of the morphological peculiarities of the Prolagus genus already described by palaeontological studies.Palaeogenomics is contributing to refine our understanding of many major evolutionary events at an unprecedented resolution, with relevant impacts in several fields, including phylogenetics of extinct species. Few extant and extinct animal species from Mediterranean regions have been characterised at the DNA level thus far. The Sardinian pika,  In particular, mitochondrial DNA (mtDNA) represents a useful tool to understand phylogeny and population dynamics due to its abundance in ancient remains with respect to the nuclear genome, high mutation rate and nearly neutral mode of evolution12.Studies on ancient DNA (aDNA) of extinct animal species have made possible to dig into the past and complement classical palaeontological approaches to reconstruct evolutionary trajectories at an unprecedent level16. An interesting case study is based on one Sardinian extinct species. Palaeogenomic data related to the enigmatic extinct Sardinian dhole, Cynotherium sardous Studiati, 1857, demonstrated that this unique canid species, that populated the island till the end of the Late Pleistocene, represents a separate taxon from all other living canids and became genetically isolated from other mainland canid lineages approximately 500–300 kya (Middle Pleistocene)4.A few extinct animal species from Mediterranean regions have been characterised at the DNA level thus far, providing information on the unique evolutionary history of isolated and endemic speciesProlagus sardus 19. The genus Prolagus Pomel, 1853, which belongs to the order Lagomorpha, is documented by several species which lived during the Neogene and the Quaternary21. Fossils pertaining to this genus have been identified in many sites of Europe, North Africa, and Anatolia. The evolutionary history and the anatomy of Prolagus have been discussed by several authors who traditionally considered this genus as a peculiar member of the family Ochotonidae Thomas, 189729, that currently includes only the living pikas, all classified within the genus Ochotona Link, 1795. A few other authors, however, have proposed that all extinct Prolagus species belong to the separate family Prolagidae Gureev, 1964, or alternatively to a subfamily of Ochotonidae, Prolaginae28. The lack of the third lower molar (M3) is one of the main anatomic elements that differentiates Prolagus species from the genus Ochotona30. Prolagus is thought to have been anagenetically derived from the monospecific genus Piezodus Viret, 1929, a taxon from the Oligocene-Lower Miocene of Europe, which differs from Prolagus for the presence of rooted cheek teeth and metaflexids, and the lack of protoconulid in the third lower premolar32. Prolagus species are known to be present in Mediterranean insular environments as documented by the taxa retrieved in the Gargano palaeo-archipelago (Late Miocene?—earliest Pliocene) and in the Sardinia-Corsica Block (Late Pliocene—Holocene). In Corsica and Sardinia, the genus Prolagus is represented by an anagenetic lineage that includes three endemic taxa: Prolagus aff. figaro (early Late Pliocene of Capo Mannu D1), Prolagus figaro López-Martínez, 1975 (latest Pliocene?/earliest Pleistocene—late Early Pleistocene) and Prolagus sardus  (Middle Pleistocene—Holocene)36.Another enigmatic and iconic extinct species that populated Sardinia and Corsica is the Sardinian pika Prolagus sardus are certainly the most abundant44. It is likely that P. sardus was a prey of several carnivores (canids and mustelids) and prey birds of the Quaternary insular palaeo-ecosystem of Corsica and Sardinia. In particular, it seems possible that the endemic canid Cynotherium sardous was specialised to hunt P. sardus46. Burnt remains collected in several archaeological sites documented that P. sardus was regularly hunted and eaten by the first communities of modern humans who colonised the Sardinia-Corsica Block39. This lagomorph was certainly present in Sardinia until the Iron Age  and in Corsica possibly until the Roman era (between 2343 BP and the sixth century CE according to the site of Castellu)48. The extinction of P. sardus is however probably linked to the introduction of new predators and ecological competitors in the insular ecosystem. The transmission of pathogens by introduced species such as rats and hares cannot be excluded as potential alternative events or co-events that led to the extinction of this species39.Among the fossil remains of Quaternary vertebrates of Sardinia, those of P. sardus derived from a radiocarbon-dated bone specimen collected in a Sardinian archaeological site. Phylogenetic reconstructions based on these DNA sequences may suggest that the extinct Prolagus genus could be better placed in an independent sister group to the family Ochotonidae.In this study, we report the retrieval of sequences from the mitochondrial genome of 3, the absence of M3, and the presence of three lobes in M2, clearly allowed to assign the bone to P. sardus and to exclude all extinct and living Sardinian lagomorphs.The analysed bone (specimen n. PR6) was an incomplete left hemimandible belonging to a mature Sardinian pika collected at Cabaddaris cave  method indicated a calibrated range from 7575 to 7431 cal BP with a probability of 95.4% , indicating poor conservation of aDNA of this specimen.The most sensitive and cutting-edge protocols for aDNA analysis were applied and the efficiency of the analysis was evaluated in each step. More than 32 million paired-end reads were generated from specimen PR6, but only an extremely low fraction of all obtained sequences (0.008%) mapped against the O. curzoniae mitochondrial reference genomes reported typical patterns of degraded DNA, such as short length of the fragments (40 bp on average) and increasing frequency of C > T transitions at the most extreme positions of every read , O. princeps (NC_005358.1) and Oryctolagus cuniculus (NC_001913.1) mitochondrial genomes, with 2721, 2608 and 2535 unique mapped reads (after filtering for duplicates), respectively.Sequences that mapped to the P. sardus, if referred to the modern mitochondrial reference sequence of the ochotonid O. curzoniae . The distribution of the obtained sequences along this reference mitochondrial genome is shown in Supplementary material Fig. A total of 3393 bp was obtained with clear ancient DNA damage patterns, corresponding to a 19.8% of the complete mitochondrial genome of O. curzoniae reference mtDNA sequence produced a total of 51 consensus regions, of which 12 were longer than 100 bp . The mitochondrial genome of the rodent species Cricetulus griseus was used as outgroup , as well as the basal node of 63% clustered the root of the Ochotona group.Phylogenetic trees were constructed using the obtained sequence information from oup Fig. . Maximumade Fig. . In the P. sardus indicated that it diverged from the Ochotona lineage about 30 Ma 55 and that, therefore, what we retrieved could be considered as derived from the true ancient mtDNA of P. sardus.The authenticity of the sequence data was evidenced by clean negative controls and DNA-damage patterns, which cast no doubts about the ancient origin of the retrieved sequence information. The low fraction of aDNA assigned to Ochotona curzoniae, O. princeps and Oryctolagus cuniculus represent those with the highest fraction of hits, we recovered ~ 20% of the mitochondrial genome of P. sardus. Although the retrieved sequence was just about one fifth of the total expected mitochondrial genome, the ancient sequence data enabled the first molecular phylogenetic placement of this extinct species, as also obtained for other species in previous studies that used partial mtDNA sequence information12.In general, by mapping reads against several lagomorph species of which Ochotona genus58. These two families diverged about 51.4 Ma61. However, there is no commonly accepted consensus of the scientific community regarding the intraordinal distinction of the order Lagomorpha when the extinct groups are considered66. Conversely, several extinct families of Lagomorpha have been described, including †Paleolagidae Dice, 1929, †Mytonolagidae Burke, 1941, †Desmatolagidae Burke, 1941, †Prolagidae Gureev, 1962, †Mimolagidae Erbajeva, 1986, and †Strenulagidae Averianov and Lopatin, 200562. In addition, various intraordinal classifications of the order Lagomorpha were proposed by other authors67. The family Prolagidae groups pika-like lagomorphs reported from the Oligocene-lowermost Miocene to the Holocene fossil records of the peri-Mediterranean area.Only two extant families are recognised in the order Lagomorpha: Leporidae, which includes the rabbits, the hares, and the jackrabbits; Ochotonidae, which includes the pikas, all grouped in the monophyletic P. sardus sequences were performed with a dataset of corresponding 12S, 16S and D-loop sequences of mtDNA from modern species of Lagomorpha from different continents, using the mitochondrial genome of a rodent species as outgroup. As expected, the phylogenetic analysis supported the nodes of the two extant families with strong values for both Leporidae and Ochotonidae. In addition, within the Ochotonidae family, fine phylogenetic relationships were in line with previous studies of this group of mammals based on cytochrome b and ND4 mtDNA genes70, suggesting that partial mtDNA sequence used in this study did not miss phylogenetic microstructures and relationships within the order.Phylogenetic analyses that we carried out with the obtained P. sardus in a sister clade to the Ochotonidae family. The Prolagus-Ochotona clade was recovered with good statistical support in both analyses . In general, the node values increased with the addition of the D-loop sequence in the alignment, for all divergence nodes while this value decreased for the node of the P. sardus lineage. This might be mainly attributable to the different mutation ratios present in this hypervariable region72.The phylogenetic trees obtained with and without the D-loop region were consistent in supporting the position of P. sardus diverged from Ochotona spp. about 30 Ma (CI: 15–59 Ma). Therefore, our molecular phylogenetic analyses support an independent evolutionary scenario for the Oligocene-Holocene Piezodus-Prolagus lineage as previously suggested by palaeontological data. Furthermore, the available data support the hypothesis to include the genus Prolagus in the independent family Prolagidae. Our results can therefore suggest that a new taxon including Prolagus may better explain the molecular diversity found and, in turn, the morphological diversity described by palaeontological studies, in line to what was suggested by some authors using only palaeontological information28.Bayesian molecular dating implemented in BEAST highlighted that the P. sardus, paving the way for its correct phylogenetic assignment. Further studies are needed to obtain a complete mtDNA sequence of this extinct species able to confirm the inferred molecular dating. More P. sardus specimens should be also investigated to retrieve aDNA from more individuals and understand the intra-specific diversity of this species. It could be also interesting to evaluate the evolutionary trajectory of this species over time by analysing aDNA of other specimens dated over a wide time window and retrieved both in Sardinia and Corsica.This study provides the first molecular systematic characterisation of Prolagus sardus (PR6) excavated from the archaeological site of the Cabaddaris cave  in Sardinia  was used in this study  were well preserved. No remains of other lagomorphs were found in the Cabaddaris cave in association with this mandible and the human artifacts. The sample was provided by the Soprintendenza Archeologia, Belle Arti e Paesaggio per le Province di Sassari e Nuoro. The Soprintendenza represents the territorial branches of the Government, responsible for the management and entrustment of cultural heritage in Italy.One mandible of udy Fig. . The samhttps://www.morphosource.org/concern/media/000494910).Biomolecular  and physical-chemical  analyses, provide fundamental information and data, however, they often implicate destructive or micro-destructive approaches. To preserve precious ancient remains, it is important to apply, when possible, appropriate methods of digital acquisition, with the aim to allow future analyses or to leave the possibility of having access to a reproduction of the remain for museum purposes. Thus, microphotogrammetry was used to obtain a 3D graphical model of the mandible before DNA extraction and radiocarbon dating Fig. . We perfProlagus sardus, we weighed the jaw with a precision laboratory balance (0.9 g). The sample was sent to the Centre of Dating and Diagnostic, Department of Mathematics and Physics (CEDAD) laboratory, University of Salento  for radiocarbon dating, that was obtained with the CEDAD Accelerator Mass Spectrometry (AMS), following the methods previously described74. Reference samples of known oxalic acid concentration provided by the National Institute of Standard and Technology (NIST) were used as a quality control of the results. Radiocarbon dating was then calibrated in calendar age using OxCal Ver. 3.10 software based on INTCAL20 atmospheric data75.To generate radiocarbon dating for the remain of 78. In fact, disposable coverall suits, double pair of gloves, boots, face masks and plastic face shields were worn during the handling of samples. The worktop and instruments were regularly cleaned after each experiment with DNA Exitus Plus solution  or ~ 5% commercial NaClO and exposed for 30 min to ultraviolet radiation at 254 nm. Moreover, DNA-free reagents were used, and negative controls were processed for each batch of samples. In addition, PCR reactions and post-PCR procedures were carried out in a physically isolated area.The PR6 sample, represented by an almost complete left jaw Fig. , was pro81. One extraction negative control was included in the process. Briefly, the digestion buffer was composed by 0.45 M EDTA, 0.25 mg/mL proteinase K, 0.05% Tween, the binding buffer was the PB Buffer  and silica columns were the ones from High Pure Viral Nucleic Acid Large Volume kit . Two washing steps with PE Buffer (Qiagen) were performed and finally the DNA was eluted in 50 µL of EB Buffer (Qiagen) and quantified with Qubit instrument  and High Sensitivity Assay dsDNA kit (Thermo Fisher Scientific).The teeth, represented by one incisor and the cheek teeth, all still encased into the alveoli, were sampled and pooled together. Due to the small size of the teeth, it was not possible to sample only the cementum part. They were decontaminated by spraying 1.5–2.0% sodium hypochlorite on the surface of the teeth, and then a clean paper towel, rinsed with DNA-free water, was used to remove the bleach. They were subsequently air-dried for a minimum of 20 min under ultraviolet (UV) light (254 nm wavelength) at a distance of ~ 25 cm from the bulb. Then, they were crushed in a mortar and ~ 100 mg of powder was recovered. The DNA extraction was performed using a modified version of a silica-based column protocol, by adding a pre-digestion step82 with home-made baits obtained from long range PCR of Ochotona princeps DNA but the enrichment of ancient mtDNA was unsuccessful (data not shown). Then we proceeded with a shotgun approach. A total of 20 µL of the eluted material was converted in single strand libraries83 along with a library as negative control. The optimal number of PCR cycles for indexing was determined by Real-Time amplification on ABI 7500 PCR System (Thermo Fisher Scientific). Indexing amplification was performed by splitting 48 µL of each library in two 50 µL reactions for 18 cycles, using a dual indexing approach and AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific). The amplified libraries were purified using 0.9X AMPure SPRI-beads  and quantified on the Bioanalyzer  using the DNA1000 kit (Agilent Technologies). The indexed libraries from these and other ancient samples were then pooled in equimolar amounts and sequenced on a lane of an Illumina HiSeq X platform run  for 75 × 2 paired-end sequencing, generating more than 32 million paired-end reads.We first attempted a multiplexed mtDNA sequence capture protocol84 to evaluate the performance of the sequencing process. Sequences were processed with Paleomix85 by aligning paired-end reads to the complete versions of seven mitochondrial genomes of different species: Sciurus vulgaris (accession number: NC_002369.1), Lepus capensis (GU937113), Mus musculus (NC_005089.1), Ochotona curzoniae (NC_011029.1), O. princeps (NC_005358.1), Oryctolagus cuniculus (AJ001588.1) and Homo sapiens (NC_007092.1). Briefly, adapters were trimmed with lenient parameters using AdapterRemoval . To improve mappings of ancient reads at the ends of the circular reference genomes, we generated elongated versions  of the mitogenomes using the CircularGenerator tool implemented in EAGER86. The collapsed reads were initially aligned to the elongated references using BWA backtrack algorithm, with minimum quality of reads to be aligned ≥ 20 and seed region use disabled87. Preliminary BAM files were then realigned to the standard version of the reference mitogenomes using the CircularMapper tool from EAGER. Duplicates were removed with PCRDuplicates on Paleomix85. The authenticity of data was finally estimated by computing frequencies of C > T along the 5’ and 3’ ends of the reads with mapDamage288 and by observing the number of reads aligning to the human reference genome. Sequences with a minimum depth of 3X were subsequently manually inspected with Integrative Genomics Viewer (IGV)89 and used for further analyses. Reconstruction of the P. sardus mtDNA contigs was obtained with GenomeVX90 using O. curzoniae mtDNA reference sequence .Raw data were initially investigated with FastQCProlagus sardus sequences obtained in this study were aligned with 18 previously published 12S, 16S and D-loop regions of mtDNA from modern Lagomorpha species of different continents and one Rodentia species (Cricetulus griseus) as outgroup. The list of species and the relative information are shown in Supplementary material Table 91, with the tool ClustalW. Phylogenetic analyses were carried out on phylogenetically informative regions of the mtDNA genome  that have been previously established for the two lagomorph families, Leporidae and Ochotonidae92. Two phylogenetic analyses were performed using the following sequence information: (i) a contig obtained joining three partial ribosomal sequences  for a total of 531 bp of P. sardus mtDNA sequence: (ii) to this contig, a portion of the D-loop sequence of 200 bp was added to obtain another combined contig of 731 bp of the P. sardus mitochondrial genome sequence. Maximum likelihood trees were obtained using RAxML-NG93, providing, for each dataset, a starting Neighbour-joining (NJ) tree based on the Jukes and Cantor (JC) model substitution matrix94, with 1000 bootstraps, as suggested by the tool “Models”, implemented in MEGA X91. The most suitable model (JC) was chosen since it showed the best probability with and without outgroup .P. sardus and other Lagomorpha taxa was obtained with BEAST 2.595 using alignments of partial sequences of D-loop, 16S and 12S mtDNA regions already used for the phylogenetic analyses. Bayesian estimation analyses were performed including species of the two families of Lagomorpha order (Leporidae and Ochotonidae) and one outgroup of the order Rodentia . BEAST 2.5 was run by assuming a combination of parameters including a JC69, Gamma4 substitution model, a relaxed uncorrelated lognormal molecular clock, and a Yule process of speciation96. The following three calibration points were defined considering a Log normal distribution: (i) the divergence  between Ochotonidae and Leporidae families 61; (ii) the divergence node of Ochotona group 61; (iii) the node between Romerolagus diazi and the Lepus genus 97. Analyses were based on two independent Markov Chain Monte Carlo (MCMC) chains, each consisting of 10,000,000 chains. Data were collected every 1,000 points, and the burn-in was set to 10%. The chain convergence and effective sampling size (ESS) values higher than 200 were evaluated with Tracer v. 1.5 (Available: http://tree.bio.ed.ac.uk/software/tracer). TreeAnnotator v. 2.6.7  and Figtree v. 1.4.4  were used to annotate and illustrate the final trees.Bayesian estimation of the divergence time between 99. Branch lengths were calculated using the maximum likelihood (ML) method and the Jukes-Cantor substitution model was applied100. A Log-normal distribution calibration type setting, with two calibration constraints  was applied with the same timing used for BEAST 2.5 analyses. Tao et al.99 method was used to set minimum and maximum time boundaries on nodes for which calibration densities were provided and confidence intervals were computed with the same method100. Bars around each node represent 95% confidence intervals. A discrete Gamma distribution was used to model evolutionary rate differences among taxa. The geological time scale followed Gradstein et al.101An independent clock analysis was carried out by applying the RelTime method available in the software MEGA XSupplementary Information.


Dental calculus is becoming a crucial material in thestudy ofpast populations with increasing interest in its proteomic and genomiccontent. Here, we suggest further development of a protocol for analysisof ancient proteins and a combined approach for subsequent ancientDNA extraction. We tested the protocol on recent teeth, and the optimizedprotocol was applied to ancient tooth to limit the destruction ofcalculus as it is a precious and irreplaceable source of dietary,microbiological, and ecological information in the archeological context.Finally, the applicability of the protocol was demonstrated on samplesof the ancient calculus. Itcarries various particles that enter the oral cavity, allowing usto study the composition of the diet,et Yip23). Thus, the layers and segments of the calculus have avariable composition of bacteria, food residues, host biomolecules,or environmental particles. In this way, the dental calculus representssamples with high heterogeneity.The process of the deposition of dental plaque depends on manyvariables that are not yet fully understood. Many factors such aspH, oral microbiome, amount, or even chance determine whether a certainpiece of debris is caught and later preserved in a calcified plaque–calculus protocol publishedby Wiśniewski et al.40—usuallymodified for smaller volumes and ancient samples by Jeong et al.38 and Cappellini et al.41 , or gel-aidedsample preparation (GASP).10 In this study, we performeda comparison of several protocols for protein extraction using recentteeth to limit the destruction of ancient material and proposed acombined protocol that allows protein and DNA isolation from the sametooth or calculus material, adopting a different approach to the currentprotocol for unified decalcification35 tooffer an alternative for processing of ancient calculus samples. Theproposed protocol was applied to ancient teeth and calculus.Most studies use EDTA overnightfor up to 7 days for demineralization.We used teeth from recent patientsobtained within the past decade by a dentist to test different extractionconditions on nonancient specimens and two individuals buried in a hospital cemetery dated between the13th and 16th centuries in Náměstí Svobody, Znojmo,Czech Republic.43The origin and dating of the analyzed samplesare summarized in Recent tooth samples were submerged in 3% hydrogenperoxide forsterilization and stored at room temperature prior to the experiment.Prior to protein extraction, all tooth samples were cleaned with 1%bleach and ultrapure water, frozen at −80 °C for 48 h,and pulverized in an oscillatory mill (Retsch MM 301), see Table S1) were treated with 100 μL of HCl—5and 10% in MQ water (v/v), citric acid (CA)—5 and 10% in MQwater (w/v), solution of sodium calcium edetate, and SDT buffer , as shown in g), and the supernatants were collected.For the selectionof the best protein extraction approach, ∼20 mg samples ofpooled powder from recent teeth REC1 and REC2 (see Table S2). In this case, the SDT buffer extractionprocedure described above was applied.For evaluationof the performance of the selected extraction method in ancient samples,medieval tooth and dental calculi were used  performed using an UltiMate 3000 RSLCnano systemconnected to an Orbitrap Fusion Lumos Tribrid spectrometer (ThermoFisher Scientific). Peptides from the ancient tooth and dental calculuswere analyzed on a nanoElute system online coupled with a timsTOFPro spectrometer (Bruker). See the Supporting Information file for full details regarding the analyses anddata evaluation, including the custom database described in Table S3.Protein extracts were subjectedto filter-aided sample preparation as described elsewhere.Table S7). 100 μL of SDT buffer was addedto the powder in the samples of groups A and B , 25 μL of proteinaseK solution , and 450 μL of EDTA . For comparison, samples  with an elution volume of 60 μL.Concentration was measured with a Qubit dsDNA HS Assay Kit in Qubit2.0 with 10 μL of isolate, and the samples with the highestand lowest input  were measured on a FragmentAnalyzer using a DNF-474 High-Sensitivity NGS Fragment Analysis Kit.Lysis was performed overnight at 56 °C and 900 rpm, and 450μL of the supernatant was used for DNA isolation by silica columnsaccording to the protocol by Yang et al.48 andfurther sequenced by Sanger sequencing, resulting sequences were runthrough mtDNAprofiler49 and MITOMASTER.50 Results were compared to the whole mitogenomepreviously obtained by Illumina sequencing after target enrichmentaccording to the protocol by Senovska et al.51 The whole mitogenome was also evaluated by DamageProfiler52 to confirm the authenticity and typical ancientDNA damage.The extracted aDNA and the blanks wereamplified using primersfor HVI (IFb-16128 and IR-16348) and HVII (IIFa-45 and IIR-287) ofmtDNA, as done in Nilsson et al.,Initially, we sought for thematerial to be as homogeneous as possiblein a sufficient amount, which allowed us to do reasonable testingand subsequent optimization of the sample preparation methods. Weselected a homogenized pooled sample of whole recent teeth based onthe assumption that if the extraction protocol shows satisfactoryefficiency in protein isolation from highly mineralized samples, lessmineralized calculus should not pose a problem. Our initial propositionwas supported by the fact that ancient DNA is often extracted fromdental calculus by similar methods used for bone and tooth samples,and thus parallel DNA extraction could be involved in our optimizedprotocol.A sample of a pulverized tooth provides enough materialfor experimentreplication under various conditions and is more frequent even inthe historical context.Next, the optimized protocol was appliedto ancient samples, earlymedieval tooth, and finally dental calculus, the targeted sample type.40On the basis of reported studiesand our experience, we selected several extraction solutions to testthe efficiency of the protein extraction step using samples of pulverizedrecent teeth. We applied solutions of citric acid (5 and 10%), hydrochloricacid (5 and 10%), sodium calcium edetate, which is used for partialtooth demineralization in dentistry, and SDT buffer used for proteinlysis during FASP sample preparation of protein samples.Table S4). Full results are listed in Datasheet 1. On average, we identified 608 nonbacterialproteins  compared to less than 100using acidic treatments. Treatment with edetate allowed for the identificationof 302 proteins , providing better resultsthan acidic extraction but still substantially lower than SDT extraction.Taking into account the results, we continued with the applicationof a SDT buffer for protein extraction in subsequent experiments.Under the given conditions, the highestnumber of proteins identified by LC–MS/MS analysis was obtainedfrom samples treated with SDT buffer, under elevated temperature (Table S5). Full results arelisted in Datasheet 2.Next, we applied a selected protocolthat combined SDT protein extraction with FASP to an early medievaltooth (ZH539T) as an example of an ancient tooth. After pulverization,we prepared four samples of tooth powder of different weights , each weight in three replicates  is probably a result of native degradation duringthe time since burial.25 Full resultsare listed in Datasheet 3.After method optimization usingtooth samples, we applied this protocol to three samples of ancientdental calculus . We identified 113–893proteins based on at least 2 unique peptides .The calculus of individual NSZ 151 (NSZ151C) iscomposed of a greatnumber of human proteins, which might suggest the breakdown of oralor even lung tissues that could be connected to pathological processes,including human hemoglobin  not presentin the other studied calculus samples. In contrast, in sample NSZ120Cwe found abundant microbial taxa, and the early medieval sample ZH576Cmostly contained various proteins from Selenomonas sputigena, Actinomycesdentalis, and other Actinomyces spp., Porphyromonas gingivalis, or Fretibacterium fastidiosum (NSZ120C). We identifiedhuman proteins demonstrating an active immune reaction, such as complementC3 and C4 (NSZ120C) or hemoglobin (NSZ151C) that could result frombleeding caused by periodontal disease.Several proteins were assigned to oral bacteria suchas Bos sp., consumption of milk and possibly other bovine products. Samplefrom NSZ 151 also contained other animal proteins that would implycontact or possibly consumption of, e.g., Oryctolaguscuniculus or Sus scrofa.Dietary proteins belongto one of the most informative in the contextof ancient dental calculus analysis, and thus they are often the primaryfocus of studies. Unfortunately, proteins related to milk consumptionare frequent laboratory contaminants. However, in sample ZH576C, wefound a large amount of bovine milk proteins  that were not present or were very minor in blanks.It suggests, together with other abundant proteins of 54 However, both sites studied here are only hundreds of years oldand are well preserved, therefore deamidation levels of analyzed samplesare far lower compared to, e.g., Pleistocene Mylodon.26 We include modifications of the reportedpeptides in Table S6. Deamidation is quitevariable, it depends on many factors, and it has been proposed tobe used rather as an indicator of protein preservation.56 We suggest, e.g., additional targeted DNA analysis of organismsof interest to confirm the presence of taxa identified by proteomicanalyses, for example, by metabarcoding and targeted PCR as done bySawafuji et al.57 This should be possiblefrom a single piece of material, e.g., if performed by the unifiedprotocol presented here, or as done by Fagernäs et al.35We also evaluated protein modifications as deamidation analysiswas suggested to be a reliable tool for confirmation of the authenticityof ancient proteins and was used primarily for collagen, especiallyin case of very ancient samples that are thousands or even millionsof years old.To maximizethe use of the sample material, we tested subsequent DNA extraction.The powder samples after protein extraction were treated with DNAlysis buffer, and the DNA content was analyzed.Table S7 and Figure S1. The Qubit dsDNAHS Assay Kit only measures double-stranded DNA; however, some of theDNA after SDT treatment will be single-stranded and therefore undetectablethis way. The average yield after SDT incubation was 258.09 pg ofDNA from 1 mg of powder without EDTA wash (sample group A), 199.15pg/mg with EDTA wash (group B), and 411.35 pg/mg (group D) withoutSDT incubation, excluding samples with the lowest input of 1 mg. Thus,we can still recover about 63% of the aDNA from samples after proteinextraction compared to the standard extraction of the aDNA from untreatedsamples. This is comparable to the 57% aDNA yield obtained using theunified decalcification protocol by Fagernäs et al.35 where the supernatant was used for DNA extractionand the pellet for protein extraction, i.e., in the opposite way toour approach. The DNA yield in our protocol is higher without thewashing step between SDT and EDTA treatment, which was used to unifyfurther analysis with samples without proteomic extraction . Our protocol is time-efficient, with both extractionsfinished in 24 h compared to standard approaches of multiple daysof demineralization, thus saving time, energy, and laboratory as wellas personnel capacities.The concentrationof aDNA extracts from ancient tooth powder (ZH539T) is summarizedin Figure S2). Therefore, we suggesthigher input when possible, even if aDNA analysis is feasible forinput of about 1 mg of tooth powder, depending on the preservation.The amount needed for analysis might be lower for more concentratedmaterials such as dental calculus.The results of Sanger sequencing wereconsistent with previouslydetermined haplogroup W4; however, samples with the lowest input amounts(4A–4D) produced very weak and damaged products that were harderto sequence with more apparent aDNA damage such as cytosine deamination(see Table S8, and theancient DNA damage patternis shown in Figure S3. The aDNA in extractsis identical; only yield and quality vary between the extraction groups.SNPs found in the whole mitogenomeof ZH 539a are shown in 35 a pellet of debris remains after protein and aDNA extractions, possiblyallowing further analysis of microremains by light microscopy, potentiallyallowing three diverse analyses of a single precious material.Similar to Fagernäs et al.,This study describes a new method for parallelprotein and DNAextraction that can be used on both recent and ancient teeth and dentalcalculus, enabling the most efficient use of very complex and preciousmaterial such as dental calculus. A significant advantage of thisapproach is that DNA and proteins are extracted from an identicalpiece of material, allowing for comparison of the obtained data andavoiding redundant sampling and material destruction as well as potentiallyallowing for further microscopic analysis of microremains. Our protocolis time-efficient: both aDNA and protein extractions can be performedwithin 24 h.However, as the yield of DNA extraction in thecombined protocolis about two-thirds of the yield of the standard protocol yield, itis not recommended for a purely genomically oriented experiment design.The proteins identified in the samples were consistent with theexpected results from teeth and dental calculus, including proteinsof human, dietary, and microbiome origin, indicating the applicabilityof the procedure to ancient dental material.


In addition, the highest rate of birth asphyxia was found in group 2 (5.9%), which was significantly higher than that in Groups 1 (1.8%) and 3 (3.3%) (p = 0.007). (4) Conclusions: Pregnant women with abnormal 50 g GCTs without a diagnosis of GDM had undesirable maternal and neonatal outcomes, as well as those who had GDM, suggesting that healthcare providers should closely monitor them throughout pregnancy and the postpartum period.(1) Background: An abnormal 50 g glucose challenge test (50 g GCT) during pregnancy, even without a diagnosis of gestational diabetes mellitus (GDM), may result in undesirable obstetric and neonatal outcomes. This study sought to evaluate the outcomes in pregnant women with abnormal 50 g GCT in secondary care hospitals in Thailand. (2) Methods: A total of 1129 cases of pregnant women with abnormal 50 g GCT results who delivered between January 2018 and December 2020 at Thasala, Sichon, and Thungsong hospitals were retrospectively reviewed and divided into three groups: abnormal 50 g GCT and normal 100 g oral OGTT , abnormal 50 g GCT and one abnormal 100 g OGTT value , and GDM . (3) Results: Cesarean section rates in group 3 (61.9%) were statistically higher than those in groups 1 (43.6%) and 2 (49.4%) ( If the results exceed the screening threshold, then they undergo a 100 g oral glucose tolerance test (OGTT) for GDM diagnosis. [An abnormal test is associated with a higher risk of gestational diabetes mellitus (GDM), independently of whether it resolves or not after delivery. It is usually diagnosed after 20 weeks of gestation . There iagnosis. , which uagnosis. ,7. GDM iagnosis.  or the 7The prevalence of GDM has increased dramatically. The highest prevalence of GDM (24.2%) has been found in Southeast Asia . In AsiaIn Thailand, there are no guidelines on standard care for pregnant women with abnormal 50 g GCTs. Furthermore, there is still a lack of study data regarding the outcomes in pregnant women with abnormal 50 g GCTs who do not have diagnoses of GDM. There is still a lack of study data regarding the outcomes in pregnant women with abnormal 50 g GCTs without diagnoses of GDM. There are different contexts and guidelines for GDM management among institutions. In Thailand, previous studies conducted in tertiary hospitals in urban areas have reported the effects and risk factors of abnormal GDM screening ,20, but This retrospective study was carried out at three community hospitals  located in Nakhon Si Thammarat Province, Thailand. At these hospitals, approximately 120–250 pregnant women per month use antenatal care and delivery services, and there are 1–3 obstetricians, 2–3 midwives in the antenatal care unit, and 8–10 midwives in the labor room. Sichon, Thasala, and Thungsong Hospitals are located in Sichon District , Thasala District , and Thungsong District , respectively .e is the acceptable sampling error (0.05). The confidence level was maintained at 95%. The sample size from the three hospitals was 1100. We determined the prevention of missing data based on a sample size of 5%. Therefore, the final sample size was 1155 participants. However, only 1129 complete medication records were included in this study.Pregnant women with abnormal 50 g GCTs who had attended antenatal clinics and delivered between January 2018 and December 2020 at Thasala, Sichon, and Thungsong Hospitals were enrolled in this study. The sample size was determined using the single-proportion population formula:The inclusion criteria were women with singleton pregnancies, who had undergone GDM screening by 50 g GCT at 24–28 weeks of gestation, and who had attended and delivered at Sichon, Thasala, and Thungsong Hospitals between January 2018 and December 2020. The exclusion criteria were pregestational diabetes, gestational hypertension, preeclampsia, chronic hypertension, and incomplete medical data records. Finally, a stratified random sampling method was used for data collection. In this study, the pregnant women were classified into three groups depending on the number of abnormal 100 g OGTT values: group 1 consisted of pregnant women who had abnormal 50 g GCTs and normal 100 g OGTTs; group 2, pregnant women with abnormal 50 g GCTs and one abnormal 100 g OGTT, who had a GCT value ≥140 mg/dL and were positive for a single 100 g OGTT; and group 3, pregnant women with GDM who had been diagnosed with a GCT value ≥140 mg/dL and who had two or more positive 100 g OGTT values. The diagnosis of GDM was based on the Carpenter–Coustan criteria, and was based on the 100 g OGTT. GDM was defined as having any two values at or above thresholds of 95 mg/dL, 180 mg/dL, 155 mg/dL, and 140 mg/dL for fasting, 1 h, 2 h, and 3 h plasma glucose levels, respectively .2; family history of diabetes; presence of hypertension; previous GDM; and a history of fetal macrosomia, stillbirth, or fetal anomaly) [Retrospective data were extracted from the HOSxP program and medical records . There were three parts, including (1) the baseline maternal characteristics, such as age, religion, past pregnancy history, risk factors for GDM  , gravidianomaly) , Apgar sanomaly) ), heart This study was reviewed and approved by the Ethics Committee on Human Research at Walailak University, Thailand. This study was performed in accordance with the Declaration of Helsinki . The requirement for written consent documentation was waived due to the unidentifiable personal information in the secondary data.p-value < 0.05.The data were analyzed using SPSS for Microsoft Windows . Descriptive statistics were used to analyze the demographic data and maternal and neonatal outcomes. Continuous variables are presented as medians and interquartile ranges (IQRs), and categorical variables are presented as frequencies and percentages. The Kolmogorov–Smirnov test was used to check the normality of the continuous data. Regarding the continuous variables, the Kruskal–Wallis and Mann–Whitney U tests were applied to compare the median of data among more than two groups and two groups, respectively. For categorical variables, the chi-square test was used to examine bivariate associations between demographic data and maternal and neonatal outcomes among the groups. Statistical significance was set at a 2 at Thasala Hospital were higher than those at Sichon and Thungsong Hospitals. Of these, 397 (35.2%) were pregnant women with normal 100 g OGTTs; 425 (37.6%) were pregnant women with one abnormal 100 g OGTT value; and 307 (27.2%) had been diagnosed with GDM.The most common maternal outcome was cesarean section , followed by postpartum hemorrhage  and shoulder dystocia . The rates of postpartum hemorrhage and shoulder dystocia at Thasala Hospital were higher than those at Sichon and Thungsong Hospitals. The most common neonatal outcome was hypoglycemia , followed by LGA  and respiratory distress syndrome . The rates of macrosomia and LGA were higher at Thasala Hospitals than at Sichon and Thungsong Hospitals. In addition, the rates of respiratory distress syndrome at Sichon Hospital were higher than those at Thasala and Thungsong Hospitals .p < 0.001). The median pre-pregnancy BMI, systolic blood pressure, and previous GDM in group 3 were significantly higher than those in groups 1 and 2  (2 than groups 1 and 2 (p < 0.001). There was no statistically significant difference between the groups regarding family history of DM, history of macrosomia, or rate of total weight gain greater than the recommendation , was significantly higher than that in groups 1  and 2  ( < 0.05) . Group 3endation .p < 0.001). Nonetheless, there were no statistically significant differences in the rates of shoulder dystocia or postpartum hemorrhage among the groups. Regarding the neonatal outcomes, the highest rate of birth asphyxia was found in group 2 (5.9%), which was statistically higher than that in groups 1 (1.8%) and 3 (3.3%) (p = 0.007). The rates of neonatal intensive care unit admission and hypoglycemia in group 3 were significantly higher than those in groups 1 and 2. The median neonatal birth weight, preterm birth, macrosomia, LGA, and rates of respiratory distress syndrome were comparable between the groups.2) [This study was designed to compare the maternal and neonatal outcomes among pregnant women who had an abnormal 50 g GCTs with normal 100 g OGTT, pregnant women with abnormal 50 g GCTs and one abnormal 100 g OGTT value, and pregnant women with GDM. The majority of the pregnant women in secondary care hospitals in Nakhon Si Thammarat province in southern Thailand were positive for 50 g GCT and negative for 100 g OGTT, aged more than 25 years, and multigravida, and had family histories of DM, GDM risks of more than one factor, total weight gain within the recommendation, and vaginal deliveries. Similarly to previous studies in large tertiary hospitals in Bangkok, Thailand, the results showed that the majority of pregnant women with abnormal GCT were aged ≥25 years and multigravida ,19,20. A2) . Some st2) . In the 2) . However2) , which iRegarding maternal outcomes, the results showed that the most common maternal outcome was cesarean delivery (50.8%). The cesarean section rate in this study was comparable to the rates of cesarean delivery in Thailand in the previous report . The ratWith regard to neonatal outcomes, the most important finding in this study was the prevalence of birth asphyxia in pregnant women who had abnormal 50 g GCTs and one abnormal 100 g OGTT value, which was statistically higher than that in the abnormal 50 g GCT, normal 100 g OGTT, and GDM groups. In this study, women with abnormal 50 g GCTs and one abnormal 100 g OGTT value had a 3.48 times increased risk of delivering infants with birth asphyxia compared to those with abnormal 50 g GCTs only . This is inconsistent with previous research that found the frequency of birth asphyxia to be significantly higher in the infants of mothers with prior diabetes (22%) and GDM (20%) than those with non-diabetic mothers (6%) . These rThe other important findings of this study were the comparable rates of neonatal birth weight, preterm birth, macrosomia, LGA, and rates of respiratory distress syndrome among pregnant women with GDM and pregnant women with abnormal 50 g GCTs and without GDM. Pregnant women with abnormal 50 g GCTs without diagnoses of GDM may also deliver LGA babies with macrosomia. Hyperglycemia can lead to fetal exposure to hyperglycemia during pregnancy. Hyperglycemia leads to an increase in fat and protein stores in the fetus. Most LGA babies have maternal hyperglycemia ,28,34. Lp < 0.001). These findings, therefore, confirmed the previous results that indicated that neonatal hypoglycemia was more common in pregnancies with GDM [The rates of neonatal intensive care unit admission and hypoglycemia in the GDM group were statistically higher than in the group of pregnant women who had abnormal 50 g GCTs without a diagnosis of GDM (with GDM . Similarwith GDM . Neonatawith GDM . FurtherThe study’s limitations are as follows. The retrospective study design of this study might have resulted in selection bias, as participant selection was carried out by convenience sampling. The results were also subjected to confounding, as other risk factors that involve obstetric and neonatal outcomes might be present, but not measured. However, our study’s strengths were its large sample size and the fact that comprehensive data involving maternal and neonatal outcomes were gathered and analyzed. Additional studies should be performed prospectively to determine the maternal and neonatal outcomes of pregnant women with abnormal 50 g GCTs without diagnoses of GDM. Further studies to identify the risk factors for undesirable outcomes among these pregnant women are also suggested.This study investigated the impact of an abnormal 50 g GCT during pregnancy without a diagnosis of GDM on maternal and neonatal outcomes in secondary care hospitals. The highest rate of birth asphyxia was found in pregnant women who had abnormal 50 g GCTs and one abnormal 100 g OGTT value, which was statistically higher than those in the abnormal 50 g GCT/normal 100 g OGTT and GDM groups. In addition, the mean neonatal birth weight and rates of preterm birth, macrosomia, LGA, and respiratory distress syndrome were not significantly different between pregnant women with abnormal 50 g GCTs without diagnoses of GDM and those with GDM. Therefore, this study suggests that healthcare providers should consider pregnant women with abnormal 50 g GCT tests without diagnoses of GDM as a high-risk group which should be closely monitored throughout pregnancy, until the postpartum period.


This study aimed to explore the clinical features and prognosis of cardiac metastatic tumors. In addition, whether continuing antitumor therapy after the development of cardiac metastases can benefit patients and the response of cardiac metastases were investigated.A retrospective analysis was conducted on patients with malignancies who were admitted to Fujian Cancer Hospital and Fujian Provincial Hospital from January 2007 to September 2022, and the follow-up period ended in March 2023. Clinical data were gathered, treatment efficacy was evaluated, and survival analysis was performed.P < 0.001). The responses of cardiac metastases corresponded to the responses of the primary tumors. Patients with a cardiac response had a median survival time of 55.60 months, which exceeded the 13.40 months observed for those without a cardiac response. However, there was no significant difference (P = 0.375).After the patients developed cardiac metastasis, the overall 30-day, 3-month, 6-month, and 12-month survival rates were 85.00%, 59.00%, 51.00% and 38.00%, respectively. With continued treatment, the average survival time was 27.33 months , which exceeded the 6.6 months  observed for patients who withdrew from treatment (In conclusion, continuing antitumor therapy after the development of cardiac metastases can significantly prolong patient survival. Cardiac metastases and primary tumors respond consistently to antitumor treatment. The risk of death due to heart failure in cancer patients with cardiac metastases needs to be further investigated. Cardiac tumors, whether benign or malignant, are relatively rare among oncological diseases, with an overall prevalence of no more than 0.33% . CardiacA retrospective analysis was conducted on patients with malignancies who were admitted to Fujian Cancer Hospital and Fujian Provincial Hospital from January 2007 to September 2022, and the follow-up period ended in March 2023. Patients with malignancies were diagnosed through detailed medical history, complete physical examination and pathologic results. Cardiac metastases were confirmed by imaging examinations , and all tumors had metastasized to the heart and/or invaded the pericardium from external sources. Both patients who continued treatment and those who withdrew from treatment were included. This study obtained only clinical information  and was carried out in accordance with the standards of the Declaration of Helsinki. Informed consent was obtained from all patients or their legal guardians. This study was approved by the Ethics Committee of Fujian Cancer Hospital and the Ethics Committee of Fujian Provincial Hospital.Efficacy evaluation of antitumor treatments for primary tumors and metastases was performed. For solid tumors, it was based on Response Evaluation Criteria In Solid Tumors (RECIST version 1.1) ; for lymAll statistical analyses were performed using SPSS v27.0. Continuous variables are presented as the mean ± SD when normally distributed and as the median (interquartile range) otherwise. Descriptive parameters such as frequencies and percentages were calculated for categorical data. For normally distributed continuous variables, between-group differences were performed with independent-sample t tests; for nonnormally distributed data, Wilcoxon rank-sum tests were used. The 1-, 3-, 6-, and 12-month survival outcomes were examined, and comparisons were evaluated by Kaplan‒Meier curves and log-rank statistics. The survival of the following groups was compared: continued treatment versus discontinued treatment and cardiac response versus no cardiac response. All survival data were right-censored. All tests were 2-sided, and an alpha value of 0.05 was used to define statistical significance.A total of 41 patients with metastatic cardiac malignancies were included in the current study. As shown in Tables 2. Most cardiac metastases were single lesions, while another 3 patients had more than 3 cardiac metastases. The most common location of the cardiac metastases was the right ventricle (16 cases), followed by the left ventricle (14 cases), pericardium (13 cases), right atrium (5 cases), left atrium (4 cases), and interventricular septum (4 cases); in the other 5 cases, the locations were difficult to distinguish. Ten patients had both ventricular and pericardial involvement.A large plurality of primary malignancies was lymphoma (10 of 41), followed by lung cancer (7 of 41), esophageal cancer (4 of 41), cervical cancer (3 of 41), renal cancer (3 of 41), melanoma (2 of 41), colorectal cancer (2 of 41), osteosarcoma (2 of 41), soft tissue tumors (2 of 41), and other unclassified primary tumors (2 of 41). The average length of the metastatic cardiac tumors was 3.84 ± 2.00 cm, and the average cross-sectional area was 12.86 ± 11.36 cmSeven patients had a history of heart disease. Ten patients had hypertension, and 5 patients had diabetes. A small number of patients had a history of smoking or drinking. A history of cardiovascular disease and cardiovascular risk factors did not seem to be associated with the development of cardiac metastases. After cardiac metastases developed, 17 patients had electrocardiographic abnormalities, 15 patients had hydropericardium, and only 12 patients had cardiac symptoms such as chest distress and tachypnea. Echocardiography showed that 25 patients had more than one instance of valvular regression. However, no decrease in ejection fraction was observed. The details are shown in Tables P > 0.05). There was also no significant change in LVEF (P > 0.05) among the 8 patients, but three patients displayed E/e’ > 12 after cardiac metastasis, which suggested the potential possibility of diastolic insufficiency.Eight patients had available serial echocardiography reports. As shown in Table P < 0.001) between patients with or without continued treatment, and patients who continued treatment had a longer survival time  based on the treatment response of cardiac metastasis  of patients were observed to have lymphoma as the primary tumor. The second most common type of primary tumor was lung cancer 7 of 41), followed by esophageal cancer, cervical cancer, renal cancer, melanoma, colorectal cancer, osteosarcoma, soft tissue tumors and other unclassified primary tumors. As lymphoma is a non-solid tumor of lymph-hematopoietic origin, it has been reported that the heart seems to be more often involved in non‐Hodgkin's lymphomas, and the pericardium is more often infiltrated in Hodgkin's lymphoma , 19. Thu of 41, fEchocardiography is a common method for assessing cardiac function. Echocardiographic imaging may show a thickened myocardium, an abnormal myocardial structure and abnormal contractility after cardiac metastasis , 20. AltProlonging the survival of oncology patients has always been a common goal. The occurrence of cardiac metastasis indicated that the tumor was in the terminal stage. The 2022 ESC guidelines on cardio-oncology hold the view that systemic chemotherapy is needed for the treatment of cardiac metastases, but a lack of evidence to support its benefits and not much explanation is given in the guidelines . To the Of the patients who had efficacy evaluations, when primary tumors appeared to recede, there were corresponding remissions or stabilization of the cardiac metastases and other metastases. When the primary tumors stabilize or progress, cardiac metastases and other metastases also appear to stabilize or progress. Thus, it was tentatively concluded that when cardiac metastases responded to a therapeutic regimen, their responses were accompanied by primary tumor responses. Among patients with cardiac metastases, there were 5 in whom the efficacy of treatment was evaluated as CR; 4 of these patients had lymphoma. The primary tumors were also evaluated as achieving CR or PR. For lymphoma patients, active chemotherapy after cardiac metastasis can still achieve good results. Therefore, after cardiac metastasis occurs, systemic therapy to address the primary tumors remains important, and a cardiac response can lead to favorable curative effects and prolong the survival period.Theoretically, myocardial replacement with tumor cells may eventually cause heart failure . The deaIn conclusion, continuing antitumor therapy after the development of cardiac metastases can significantly prolong patient survival and can benefit patients. Cardiac metastases and primary tumors respond consistently to antitumor therapy. The risk of death due to heart failure in cancer patients with cardiac metastases needs to be further investigated.The strengths of this study include the first to show that continuing antitumor therapy after the development of cardiac metastases can benefit patients, and effective cardiac metastasis responses are accompanied by primary tumor responses. However, there are some limitations to the present study. First, cardiac metastatic cancer is rare, and the number of samples collected in this study was small, which was a major limitation; therefore, this study focused more on clinical characteristics and prognosis rather than on the analysis of risk factors. Our next study will include multicenter data to expand the sample size for more in-depth research. Second, this study initially found that continuing antitumor therapy after the development of cardiac metastasis could prolong the survival of patients, but we could not determine which specific regimen was superior due to the limited samples at present. Future studies will explore the impact of treatment option selection on the prognosis of patients with cardiac metastatic cancer based on expanded samples.