These include nine individuals from the “Crusaders’ pit” in Sidon, a mass burial in South Lebanon identified from the archaeology as the grave of Crusaders killed during a battle in the 13th century CE. We show that all of the Crusaders’ pit individuals were males; some were Western Europeans from diverse origins, some were locals , and two individuals were a mixture of European and Near Eastern ancestries, providing direct evidence that the Crusaders admixed with the local population. However, these mixtures appear to have had limited genetic consequences since signals of admixture with Europeans are not significant in any Lebanese group today—in particular, Lebanese Christians are today genetically similar to local people who lived during the Roman period which preceded the Crusades by more than four centuries.During the medieval period, hundreds of thousands of Europeans migrated to the Near East to take part in the Crusades, and many of them settled in the newly established Christian states along the Eastern Mediterranean coast. Here, we present a genetic snapshot of these events and their aftermath by sequencing the whole genomes of 13 individuals who lived in what is today known as Lebanon between the 3 Human migrations, which often accompanied historical battles and invasions, have profoundly reshaped the genetic diversity of local populations in many regions. The Mongols, around Genghis Khan’s time, spread their male lineages throughout Asia from the Pacific to the Caspian Sea,th century CE.Ancient DNA (aDNA) from medieval Crusaders who traveled to the Near East and from local people who were their contemporaries and interacted with them can potentially resolve the discrepancy between the historical records reporting admixed descendants of Crusaders in the Near East and the genetics of modern populations not displaying such a signal. aDNA from this period can also resolve uncertainties in the demographic processes that accompanied the Crusades, providing insight into questions such as what was the provenance of the Crusaders’ armies and the extent to which they were from genetically and geographically diverse groups. How genetically similar are modern Near Easterners to the medieval populations and what genetic changes occurred after the Crusades? These questions can potentially be answered today in great detail using ancient genomics; however, obtaining aDNA from Crusaders is challenging and hindered by two major factors: the hot and humid climate in the Near East negatively impacts the survival of aDNAhs37d5 reference sequence, removed duplicates, removed bases from the end of the reads until the frequency of nucleotide misincorporation dropped to below 5%, and randomly sampled a single sequence with a minimum base quality of ≥20 to represent each SNP. We found that seven samples from the Crusaders’ pit had less than 2% of endogenous DNA from the first exploratory sequencing runs and thus we excluded them from additional sequencing and from further analyses. The remaining nine samples from the Crusaders’ pit and all five samples from the Roman period had 2% to 58% endogenous DNA with post-mortem damage patterns typical of ancient DNA . This is expected to be negative when A is European because of the excess of WHG ancestry in Europeans compared with the Roman period Lebanese, but should not be significantly different from zero when A is a Near Easterner. As expected, we found a significant contrast between Europeans and Near Easterners from their genetic affinity to WHG , who contributed ancestry to all Europeans but not to Near Easterners.y to WHG , confirmf4 is always positive when A is any Near Eastern population  Following a battle between Crusaders/Europeans and a Muslim army/Near Easterners, the dead from both sides were buried in the same pit. (2) Alternatively, the Crusaders recruited local Near Easterners into their army and thus the burial was for individuals who were all fighting with the Crusaders. The historical records explain that the attacks on the Crusaders were led by Muslim armies mostly recruited from Syria, Iraq, Egypt, Turkey, and Bedouin tribes in the region. But we find the statistic pulation , suggestqpAdmqpAdm inferences by testing, similarly to SI-41 and SI-53, the European individuals from the Crusaders’ pit and, as expected, we found that most of their ancestry was derived from Europeans in contrast to SI-41 and SI-53  who we could not associate with any specific group. We consequently wanted to explore the possibility that these individuals’ particular pattern on the PCA might be due to their genomes being admixed. Therefore, we first ran an ADMXITUREnd SI-53 . Individnd SI-53  show thaNext, we analyzed the variations on the Y chromosomesf4 which quantifies the genetic changes related to West Eurasians that occurred in Lebanon between the Bronze Age and the Roman period. We found there was an increase in Eurasian hunter-gatherer and Steppe population ancestry in Lebanon after the Bronze Age  and found that the statistic is not significantly different from zero for any Ancient West Eurasian tested, thus indicating that there were no significant genetic changes between the Roman period and the medieval period in Lebanon  and f4; there were no significant genetic differences between medieval Lebanese and present-day Lebanese Christians  and f3, which test whether the Lebanese groups descended from a mixture between medieval Lebanese and another population. We found that Lebanese Christians cannot be modeled in this way (f3-statistics when A was an African or a Central/East Asian population (rarecoal-toolsOur data suggest that admixture occurred in Lebanon during the Crusaders’ time; therefore, we sought to investigate the consequences of this admixture for the genomes of the Lebanese by tracking the genetic changes that occurred in Lebanon before and after the Crusades. We started by using the SGDP set to compute the statistic onze Age A, which  Lebanon B. We theristians C, but weristians D. This gthis way A, but Lepulation B, indicaRemarkably, the significant genetic changes in the Lebanese following the Crusades appear to have been not from the Crusaders and were not in the Lebanese Christians, but were mostly restricted to the Lebanese Muslims. We analyzed admixture LDIn this report, we have presented new whole-genome sequence data from ancient individuals who lived during times when major historical events were unfolding in the Near East. Our samples from the Crusaders period are evidence of a remarkable genetic diversity that coexisted in this region—Europeans, Near Easterners, and their mixed descendants—but this heterogeneity was transient in the genomic history of the Near East, since, with the exception of some Y chromosome lineages, present-day populations derive most of their ancestry from local people who preceded the Crusades. Ancient DNA from the warm Near East is still problematic to retrieve, and in addition our samples from the Crusaders’ pit were crudely buried and partly burned, but our study shows that recovering such aDNA is feasible and that the historical and genetic insights from it are exceptional and not possible from modern DNA alone.The authors declare no competing interests.


The prevalence of perioperative surgical complications is a worldwide issue: In many cases, these events are preventable. Audio-video recording during laparoscopic surgery provides useful information for the purposes of education and event analyses, and may have an impact on the focus of the surgeons operating. The aim of the present study was to investigate how audio-video recording in the operating room during laparoscopic surgery affects the focus of the surgeon and his/her assistant.A group of laparoscopic procedures where video recording only was performed was compared to a group where both audio and video recordings were made. All laparoscopic procedures were performed at Lindesberg Hospital, Sweden, during the period August to September 2017. The primary outcome was conversation not relevant to the ongoing procedure. Secondary outcomes were intra- and postoperative adverse events or complications, operation time and number of times the assistant was corrected by the surgeon.p = 0.002). No differences in perioperative adverse event or complication rates were seen.The study included 41 procedures, 20 in the video only group and 21 in the audio-video group. The material comprised laparoscopic cholecystectomies, totally extraperitoneal inguinal hernia repairs and bariatric surgical procedures. Irrelevant conversation time fell from 4.2% of surgical time to 1.4% when both audio and video recordings were made .Available at FOU Sweden (ID: 232771) and retrospectively at Clinical  Despite increased awareness of perioperative safety, the prevalence of intraoperative adverse events and postoperative complications remains an important issue, with perioperative complications being estimated to result in one million deaths each year. It is said that up to one half of all these are potentially preventable , 2. SeveRoutine video recording of surgical procedures has been reported to have a positive impact on the behavior of personnel and may improve surgical outcome –10. FurtThe aim of this study was to evaluate whether the introduction of simultaneous audio and video recording during laparoscopic surgery has an impact on the focus of the surgeon and his/her assistant. We also considered the effect on complication and adverse event rates of these procedures.From August 29 until September 28, 2017, all laparoscopic surgical procedures performed at Lindesberg Hospital, Sweden, were consecutively included in the study. Due to the lack of previous data to base a Power calculation on, the study was planned as a pilot study aiming to include 20 operations in each of two groups – one with and one without audio recording. During the first three weeks of the study period, only standard video recording of the laparoscopic procedure was performed. Only the operation as seen from the laparoscope was recorded. During the second 3-week period, the surgeon and the assistant surgeon each had a microphone and both video (of the operation) and audio recordings were made during the procedure. This resulted in one period with video but no audio recording (control group) and one group with both video and audio recording (intervention group). No surgical procedure was excluded.Data collection and recordings were performed by a single external observer. The observer was present in the operation room but not interfering with the operation team. The operation team consisted of two surgeons , two operation room nurses (one sterile and one non-sterile assistant), and two anesthesia nurses. A stopwatch was used to note the time when the surgeon and assistant surgeon were engaged in conversation considered irrelevant to the procedure. Conversations considered to be of educational purpose were not regarded as irrelevant. Registration of operation time and potential disturbances began following time-out, in accordance with the surgical safety checklist provided by the World Health Organization  , and was conducted in accordance with the standards of the 1964 Helsinki Declaration and its later amendments .During the inclusion period, 41 elective surgical procedures were observed and included in the study, no procedure was excluded. The surgical procedures performed in the control group (video recording only) were: laparoscopic gastric bypass and laparoscopic sleeve gastrectomy for morbid obesity; elective cholecystectomy; and totally extraperitoneal inguinal hernia repair. In the intervention group (audio-video recording) the surgical procedures performed were: laparoscopic gastric bypass; laparoscopic gastric sleeve; and elective cholecystectomy . With adjustment for operation time, the median percentage time spent in irrelevant conversation during surgery dropped from 4.2% (IQR 2.1–9.9%) to 1.4% (IQR 0.74–2.4%) when audio-video recordings were made during surgery (p = 0.002). Unadjusted time in irrelevant conversion is visualized in Fig. The time spent in irrelevant conversation was shorter in the intervention group (intervention group 62 s IQR 29–100 s) compared to the control group 160 s (IQR 91–490 s), 00 s compp = 0.59). Details of these events are listed in Table Only one patient suffered an intraoperative complication . An intraoperative adverse event occurred during six procedures in the control group 30%), and during eight procedures (38%) in the intervention group . No postoperative complication occurred in the control group.p = 0.53). The median number of external interferences during the procedure was 10 (IQR 4.3–15) in the control group and 11 (IQR 6.5–16) in the intervention group (p = 0.30).The median number of corrections of the assistant was two (IQR 0.3–2.0) in the control group and one (IQR 1.0–2.0) in the intervention group (Audio recording reduced irrelevant conversation time during surgery. Similar results have been seen in association with endoscopy , but to Apart from reducing irrelevant conversation during surgical procedures, routine audio-video recording may be positive in other ways such as the creation of a database which may be used in the analysis of intraoperative events or near events; information that surgeons can learn from . RecordiThe major obstacles to routine audio recording in the operating room are mainly practical, ethical and perhaps legal. Audio-video recording in the working environment may affect the quality perceived, as well as the privacy of all personnel present and that of the patient. In addition, knowing that you are being recorded may add stress to the surgeon, in particular during complex maneuvers. The use of the recordings at a later stage must therefore be judicious. When used in surgical training and event analysis, it is possible for the material to be used to track errors of an individual with subsequent criticism or reprimand . FurtherGiven the low intraoperative and postoperative complication rates in modern minimally invasive bariatric surgery , 25, lapThere are several potential benefits to be gained from audio-video recording during laparoscopic surgery, and the use of microphones is reported to be safe . SurgeonAudio-video recording during laparoscopic abdominal surgery reduces irrelevant conversation time and may improve intraoperative safety and surgical outcome.


Human chromatin remodeling enzyme INO80 can be recruited to the p53REs of p21 promoter and negatively regulates p21. As one of the key subunits of the INO80 complex, YY1 has also been confirmed to bind to the p53RE sites of p21 promoter. Importantly, YY1 was recently reported to be bound and stabilized by BCCIP (BRCA2 and CDKN1A-interacting protein). Therefore, we hypothesized that the YY1/BCCIP complex plays an important role in regulating the transactivation of p21. Here we present evidence that the YY1/BCCIP complex coordinatively regulates p53RE-mediated p21 transactivation. We first confirmed the cross-interaction between YY1, BCCIP, and p53, suggesting an intrinsic link between three proteins in the regulation of p21 transcription. In dual luciferase assays, YY1 inhibited p53RE-mediated luciferase activity, whereas BCCIP revealed the opposite effect. More interestingly, the region 146–270 amino acids of YY1, which bound to BCCIP, increased p53-mediated luciferase activity, indicating the complexity of the YY1/BCCIP complex in co-regulating p21 transcription. Further in-depth research confirmed the co-occupancy of YY1/BCCIP with p53 at the p53RE-proximal region of p21. Lentiviral-mediated knockdown of BCCIP inhibited the recruitment of p53 and YY1 at the p53RE proximal region of p21; however, this phenomenon was reversed by expressing exogenous YY1, suggesting the collaborative regulation of YY1/BCCIP complex in p53RE-mediated p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.Transactivation of  TP53 gene encodes the p53 protein that plays critical role in tumor prevention by taking control of a wide variety of cellular responses and the expression of multiple genes that regulates stress signal pathways [The pathways . In cancpathways . Howeverpathways ,5. Thesepathways .p21Waf1/Cip1, p21) is a universal inhibitor of cyclin kinases that controls the cell cycle by regulating the cyclin-dependent kinases [p21 is also one of the most studied downstream target genes of p53. Two highly conserved p53 responsive elements (p53REs) in the p21 promoter region can be recognized and bound by activated p53 to activate p21 gene expression [p21 expression is negatively regulated by the INO80 chromatin remodeling complex through binding to the p53REs in the p21 promoter region [p21 promoter region in a p53-mediated mechanism. As a DNA-binding protein, YY1 contains both transcriptional activation and repression domains, thus showing a bidirectional function in gene transcription regulation [p21 promoter region [It is well known that CDKN1A , not only directly binds to p21 [http://genome.ucsc.edu), the enrichment of YY1 at the BCCIP transcriptional start site (TSS) proximal region in several cancer cells including A549 lung cancer cells, HepG2 human hepatocellular carcinoma cells, and HCT116 human colon cancer cells was found, suggesting the function of YY1 in regulating BCCIP gene transcription. This view is also confirmed by later experiments showing that YY1 and INO80 together transcriptionally regulate BCCIP in HCT16 cells [p21, suggesting the involvement of BCCIP in p21 transcription [p21 in cells is unclear. Therefore, in an effort to address this issue, using a series of biochemical and biological experiment approaches, we present evidence of the cross-talk between YY1, BCCIP, and p53. In addition, we investigated how YY1 and BCCIP coordinate with each other to regulate p53 and its target gene p21.BCCIP, a protein that is characterized based on its interaction with BRCA2 and CDKN1A  and p53-null (p53−/−) HCT116 cells. As shown in To further explore the coordinative roles of BCCIP and YY1 in the regulation of the p53RE-mediated transcription of downstream target genes, multiple p53 responsive elements containing pp53-TA-Luc plasmid (p53RE-Luc) A were us##p < 0.01, columns 4–7 in comparison with column 3). In contrast, the increase in p53RE luciferase activity due to exogenous BCCIPα and BCCIPβ was superimposed by knocking down YY1 , has been shown to induce p53 . In our 0 µM 5FU A. Howeve0 µM 5FU B, lane 40 µM 5FU D. Similap21, six primer sets including p53 binding sites in the p21 locus were used to amplify ChIP DNA . The enrichment of three proteins at −2.3 kb upstream of the p21 locus in 5FU-treated cells was more obvious. Although not very strong, we can see the recruitment of p53 at −1.8 kb and p53/YY1/BCCIP at −0.9 kb upstream of the p21 TSS. From the above experimental results, the existence of cross-talk between p53, YY1, and BCCIP in the transcriptional regulation of p21 can be speculated. To address this speculation, ChIP assays were further carried out in stably expressing shBCCIP cells with or without 5FU treatment. Select primer sets at the p21 locus including -2.3 kb and -1.5 kb were used to amplify ChIP DNA. As expected, silencing BCCIP prevented the recruitment of both p53 and YY1 at −2.3 kb upstream of the p21 TSS compared to the shNT group  the INO80 complex represses p21 expression by regulating the promoter proximal nucleosome arrangement under normal cellular conditions; ii) in Doxo-treated cells, the INO80 complex rapidly removes accumulated H2A.Z and relieves its inhibition of p21, thereby inducing the transcriptional expression of p21 [In general, tumor suppressor gene  periods . Based oods [p21 ,24. Likeods [p21 . It has p21 gene . Using Rechanism . It is wechanism . Our datcription ,13, suggn of p21 .p21, was surmised. Consistent with this, the luciferase activity of p53RE-Luc, which contains multiple p53REs, was regulated by both YY1 and BCCIP. As previously reported [p53.On the other hand, we recently found that BCCIP not only stabilizes YY1 protein, but also directly binds to YY1 in HCT116 cells. The 146–270 amino acid of YY1 seemed to be the key region for maintaining the interaction between YY1 and BCCIP . Howeverreported , YY1 inhp21 and p53T18P expression levels, suggesting that p53 phosphorylation increases the transcriptional activity of p53 by enhancing p53 stability [p21 promoter, suggesting the coordinative roles of YY1/BCCIP/p53 in regulating p53RE-mediated p21 transcription  and anti-c-Myc agarose, as well as anti-Flag M2 (F3165) mouse monoclonal antibody were purchased from Sigma . Anti-BCCIP (16043-1-AP) and anti-p21 (10355-1-AP) polyclonal antibodies were from Proteintech Group . Anti-YY1 (H414) (sc-1703 or sc-1703X) rabbit polyclonal antibody, anti-Myc  mouse monoclonal antibody, rabbit total IgG (sc-2027), and mouse total IgG (sc-2025) were obtained from Santa Cruz Biotechnology . Anti-p53 mouse monoclonal antibody was provided by Boster Group . The ChIP grade anti-p53 mouse monoclonal antibody was from Abcam . Anti-p53S15P (RLP0205), anti-p53T18P (RLT0212), anti-p53S20P (RLT0206), anti-GADD45 (RLT1832), anti-Bax (RLT0456), and anti-Flag (RLG0004) rabbit polyclonal antibodies, Bcl2 (RLM3041) and anti-HA (RLM3003) mouse monoclonal antibodies were obtained from Ruiying . Anti-GAPDH  polyclonal antibody and anti-BCCIP  mouse polyclonal antibody were raised against bacterially expressed proteins . 5-Fluorouracil (5FU)  was dissolved in dimethyl sulfoxide (DMSO) and prepared at a 500 mM concentration for storage. The final concentration of 5FU in cell culture medium was 250 μM.2.The human HCT116 colon carcinoma cells including p53 wild type (p53+/+) or p53-null (p53−/−) and the human embryonic kidney (HEK) 293T cells were cultured in RPMI 1640 Medium  and Dulbecco’s Modified Eagle’s Medium , respectively, containing 10% fetal bovine serum  and a 1% penicillin–streptomycin mixture , at 37 °C in the presence of 5% COWhole-cell lysate was prepared 48 h after transient transfection plasmids. The method was described previously . Bound pFull-length cDNAs encoding human BCCIP (NM_078468), p53 (NM_001276760), YY1 (NM_003403), and different truncations of YY1 including YY1 (146–270aa and YY1/Δ146–270aa) proteins were cloned with Flag and Myc tags into pcDNA3.1 (−). Tagged YY1, BCCIPα/β, and p53 plasmids (pcDNA3.1 as control) were transiently transfected to HCT116 cells using 4 µL PEI  according to the manufacturer’s recommendations. Cells were harvested and lysed 48 h after transfection. The proteins were analyzed by western blot with specific antibodies.Pp53-TA-Luc plasmid  including multiple p53 response elements (ACGTTTGCCTTGCCTGGACTTGCCTGGCCTTGCCTTGGACATGCCCGGGCTGTC) was obtained from Beyotime Biotechnology . HCT116 cells were co-transfected with Pp53-TA-Luc (0.4 μg), which encodes firefly luciferase, the control plasmid renilla luciferase vector (0.12 ng), which encodes renilla luciferase, and the plasmids expressing YY1 and BCCIP using PEI reagent . Total effector plasmid (1.7 μg) in each transfection was adjusted with empty vectors. pp53-TA-Luc transactivity was estimated 48 h later by measuring firefly and renilla luciferase activities using the Dual-Luciferase reporter assay kit  and by normalizing firefly to Renilla luciferase .Isolation of total RNA and reverse transcription (cDNA synthesis) were done using RNAiso Plus (D9109)  and PrimeScript 1st Strand cDNA Synthesis Kit (Takara) essentially as described . P53 andTo knock down BCCIP or YY1 expression, the lentivirus system was used to express shRNA ,21. AccoAbout 30% cultured HCT116 cells in 24-well plates containing a cover slip  on each well were treated with shBCCIP or shYY1 for 72 h. Cells were then washed, fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.3% TritonX-100 in PBS buffer for 5 min. After incubating cells with p53 (1:50), p21 (1:100), and YY1 (1:100) primary antibodies at 37 °C for an hour, cells were stained with FITC- or TRITC-conjugated secondary antibodies . Cell nuclei were stained by DAPI containing Vectashield . Fluorescence images were observed with an Olympus BX40F microscope .7 HCT116 cells. ChIP DNA was analyzed by quantitative real-time PCR (qPCR). Each experiment was performed 2–3 independent times. ChIP and no antibody signal were normalized to total input. Six primer sets for qPCR on the promoter region of p21 were designed: p21 −2.8kb (−2876bp~−2470bp), 5′-TGATGCTAGGAACATGAGCAA-3′ (forward) and 5′-CCCGAGTAGCTGGGATTACA-3′ (reverse); −2.3kb (−2292bp~−2111bp), 5′-CTGTGGCTCTGATTGGCTTT-3′ (forward) and 5′-CTCCTACCATCCCCTTCCTC-3′ (reverse); −1.8kb (−1876bp~−1788bp), 5′-ACATTCAAGTGCATGGTTGC-3′ (forward) and 5′-CTTCTAGCTCACCACCACCA-3′ (reverse); -1.5kb (−1511bp~−1326bp), 5′-GCTTAGAGTGGGGTCCTGAG-3′ (forward) and 5′-CCTCTAACGCAGCTGACCTC-3′ (reverse); -0.9kb (−982bp~−848bp); 5′-TTGTCATTTTGGAGCCACAG-3′ (forward) and 5′-GGGCTCAGAGAAGTCTGGTG-3′ (reverse); -0.3kb (−293bp~−193bp), 5′-GGGGCTCATTCTAACAGTGC-3′ (forward) and 5′-GACACATTTCCCCACGAAGT-3′ (reverse).ChIP assays were carried out essentially as described using p53, BCCIP, and YY1 antibodies . Each Cht-test.Results are expressed as the means and standard deviation (SD). Comparisons between two groups were made with an unpaired Student’s p21 transactivation. Also, the cross-interaction between YY1, BCCIP, and p53 indicates the intrinsic link between three proteins in the regulation of p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.In summary, our data suggest that the YY1/BCCIP complex coordinatively regulates p53RE-mediated


The prefrontal cortex (PFC) plays a central role in guiding decision making, and its function is altered by alcohol use and an individual’s innate risk for excessive alcohol drinking. The primary goal of this work was to determine how neural activity in the PFC guides the decision to drink. Towards this goal, the within-session changes in neural activity were measured from medial PFC (mPFC) of rats performing a drinking procedure that allowed them to consume or abstain from alcohol in a self-paced manner. Recordings were obtained from rats that either lacked or expressed an innate risk for excessive alcohol intake, Wistar or alcohol-preferring (P) rats, respectively. Wistar rats exhibited patterns of neural activity consistent with the intention to drink or abstain from drinking, whereas these patterns were blunted or absent in P rats. Collectively, these data indicate that neural activity patterns in mPFC associated with the intention to drink alcohol are influenced by innate risk for excessive alcohol drinking. This observation may indicate a lack of control over the decision to drink by this otherwise well-validated supervisory brain region. Aberrant decision-making is both a risk factor for, and the result of, an Alcohol Use Disorder (AUD). Thus, identifying the neural mechanisms regulating drinking decisions may lead to unique prevention and intervention strategies. The current study demonstrates that the intent to drink alcohol is encoded by the activity of neural populations within the medial prefrontal cortex (mPFC). However, the encoding of this intention signal is greatly reduced in a population of rodents with an extensive family history of high alcohol consumption. These data suggest that identifying strategies to restore the contribution of mPFC in the decision to drink alcohol may provide effective targets to treat an AUD, particularly in those with family history of AUDs.Aberrant decision making is both a risk factor for, and the result of, an alcohol use disorder AUD; . TherefoDysfunction of the mPFC has been repeatedly found in populations of subjects that drink alcohol excessively . ExposurThe PFC has also long been known to be involved in the regulation of executive processes required to guide reward-based decision making , and aniThe data presented herein are novel in-depth analyses of previously published data . In thisP rats have been selectively bred for >75 generations for their high drinking phenotype , and areN = 22) were ordered from the Indiana Alcohol Research Center Animal Production Core, and male Wistar rats (N = 21) were ordered from Envigo. All animals were shipped via truck to our vivarium, and were single housed and placed on a 12/12 h dark/light reverse cycle. Animals were ≈70 d of age before testing and had ad libitum access to food and water. All procedures were approved by the Animal Care and Use Committee and conformed to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research  IAP access session animals received access to an unsweetened 10% alcohol (v/v) solution for 2CAP sessions. 2CAP sessions occurred during the dark phase of the light/dark cycle, starting 1–3 h after lights off. The conditioning box configuration is illustrated in N = 3) and P rats (N = 4) with matched 15 d 2CAP alcohol consumption history  were selected for electrophysiological experiments, and were unilaterally implanted with multi-tetrode arrays in the mPFC  were conducted while electrophysiology was recorded using a 96-channel electrophysiological recording system . Animals were then given approximately three water sessions where the sippers contained water. A primary goal of these water sessions was to make a direct connection to studies of brain function in humans, wherein alcohol-associated cues are presented in the absence of access to alcohol . ElectroSpike trains were manually identified and sorted into individual cell clusters based on the features of the wave form in Spike Sort 3D . After cell sorting, duplicate timestamps and interspike intervals <3 ms were removed from spike trains. Only spike trains containing >150 spikes were analyzed.xy coordinates were converted to voltage and fed directly into electrophysiology hardware where they were recorded in parallel to neuronal activity. Raw tracking values were used to plot the location of the animals within the conditioning apparatus.One video camera was used in conjunction with ANY-maze software to track the head location of animals while they performed the task, and another was used to record high-definition video and audio to identify trials where animals ultimately consumed fluid  were created by aligning binned (100 ms) spike trains for each neuron to the onset of each trial. PSTHs were smoothed using a Gaussian function with a standard deviation of 300 ms, and softmax normalized to avoid being biased by high firing rate neurons by dividing the firing rate of each neuron by its maximum variance  was used to quantify the degree to which each neurons activity changed in response to the task stimuli compared to pre-task (baseline) activity as well as chance (surrogate testing); binned (100 ms) spike trains were not transformed or normalized in any way before these analyses. Individual neurons were evaluated for the degree of responsiveness using d´ . SpecifiFollowing d´ analyses, we next used MI , where x was the discretized smoothed spiking rate of the neuron  and y was either the trial type  or the drink outcome . The smoothed spiking rate of the neuron was discretized such that the values across trials were ranked and binned into three states  with equal number of counts . The probability was then calculated by dividing the number of joint state observations by the total number of trials. For instance, in the case of drink outcome encoding, for a given neuron and time bin, we may have observed five joint states in which the neuron had a low firing rate during drinking trials. In this example, pi,t = 5/20 = 0.25. In the case of stimulus encoding, we might have observed seven joint states in which the neuron had a high firing rate during real trials. In this example, pi,t = 7/40 = 0.175. Note that drink encoding only used real trials, so only 20 total observations where performed, whereas trial encoding used both real and null trials, resulting in 40 total observations.We began the MI calculation by aligning the first 10 drinking, the first 10 non-drinking, and 20 null trials relative to the cue onset  or a randomly chosen time point during the ITI . Null trials were constructed from periods of the neural recording that were randomly selected from the ITI such that full null trials did not overlap real trials at any time. At a given time bin For each neuron, time bin, and encoding type , we calculated the MI using i,t(x) and pi,t(y) are found by summing over the other variable:We used base 2 for the logarithm in The MI quantifies how much information one variable provides about the other. In this case, if a neuron tends to fire much more frequently on drinking trials than non-drinking trials, for instance, a large MI value would result. However, if drinking status and neuron firing rate were unrelated, then a small MI value would result. By calculating the MI at each time bin for each neuron, we were able to evaluate encoding dynamically throughout the task.p value for each MI result by randomizing the joint observations 100 times and recalculating the MI for these null surrogates. The randomization procedure preserved the marginal distributions. The p value was then calculated as the proportion of null surrogates with a MI result greater than or equal to the observed value in the real data. In the case where all null MI values were less than the result from the real data, the p value was set to 0.005 = 0.5 × (1/100) due to the resolution associated with using 100 null surrogates.Due to the discrete nature of experimental trials and the fact that MI results cannot be lower than 0, noise tends to bias MI results upwards . To assep values were used to calculate a weight (w) for each MI result via w = -log10(p). These weights were then normalized by dividing each weight by the sum of all the weights and used to calculate the weighted mean . We applied a threshold of p < 0.01 to all such Kolmogorov–Smirnov tests to assess significant ensemble encoding.While the weighting procedure above allowed us to highlight the importance of significant information results, in time bins where few significant information results were observed, an upwards bias in the information results would still be observed. To detect cases where the ensemble of information values was not significantly different from null, we also used a Kolmogorov–Smirnov test to compare the distribution of real MI results to the distribution of MI results from null surrogate data used to calculate the individual neuron p value was then calculated as the proportion of randomized trials with differences greater than or equal to the difference in the real data, accounting for the sign of the difference. In the case where all randomized trial difference values were less than the result from the real data, the p value was set to 0.00005 = 0.5 ×  due to the resolution associated with using 10,000 randomized trials. These p values were then corrected for multiple comparisons across time bins within a given figure using FDR control , we used a bootstrap approach to compare the weighted mean MI between P and Wistar rats at each time bin. We compared the difference between the weighted mean MI values in the real data to the difference weighted mean MI results from 10,000 randomized trials (identity of P and Wistar neurons randomized preserving number of neurons in each group). The  control .PCA was conducted to evaluate the predominant population-level firing rate dynamics. PCA is commonly used as a dimensionality reduction tool that requires minimal assumptions of the data . A singlFor SS analyses, neural population activity was projected onto the first three PCs of PCA space. These analyses allowed us to determine the time course of alterations in the pattern of firing rate. Similar patterns of population activity reside close to each other in 3-dimensional space, and when different are further apart. Differences in distance between 3-dimensional population activity vectors were evaluated on a bin-by-bin basis via Euclidean distance analyses . The meap < 0.05; p > 0.20) were observed in the mean number of drinking  or non-drinking trials . Collectively these data indicate that the behavioral response to the DS was not predictive of a drinking trial.A summary of statistics for each figure is provided in table format . To asseF = 38.03, p < 0.0001; 2 = 3.24; p = 0.20) or water sessions  was compared to a baseline period 2 s immediately before the trial A. Heteroamount of information encoded by trial- and drink-encoding neurons over time, information theoretic statistical approaches were used. The goal of these analyses were to capture the amount of information encoded in each neuron about the trial-associated stimuli  and if the neural firing rates dissociated drinking/non-drinking trials (drink-encoding). Additionally, these analyses focused on drink-encoding that occurred before fluid availability , as this time interval was expected to contain cue-elicited encoding of the intention to drink or abstain. In addition to quantifying the amount of information using MI, these analyses captured different encoding strategies  at each time bin during a trial .To quantify and compare the p < 0.05; p < 0.05; Examples of trial-encoding neurons are plotted in p < 0.05; p < 0.05; Examples of drink-encoding neurons are plotted in p < 0.05; p < 0.05; p < 0.05; p < 0.05; p < 0.05; To determine whether differences in information encoding observed at the single neuron level were maintained at the population level, SS analyses were performed to quantify how neural activity patterns, captured via principle components, evolved throughout a trial. To quantify the evolution of neural trajectories, Euclidean distance to a corresponding time bin of a null trial was computed for drinking, non-drinking, and null trials . Euclidean distance was calculated from a multidimensional space that was defined by the first 3 principle components. Larger values of Euclidean distance correspond to larger differences in neural activity patterns, which indicate that the predominant patterns of neural firing were different for two comparisons A. VideosVideo 1.Data from Wistar rats given alcohol access. The top panel in all videos is identical to data found in 10.1523/ENEURO.0489-18.2019.video.1Video 2.Data from P rats given alcohol access. The top panel in all videos is identical to data found in 10.1523/ENEURO.0489-18.2019.video.2Video 3.Data from Wistar rats given access to water. The top panel in all videos is identical to data found in 10.1523/ENEURO.0489-18.2019.video.3Video 4.Data from P rats given access to water. The top panel in all videos is identical to data found in 10.1523/ENEURO.0489-18.2019.video.4The goal of the current study was to determine if the intent to drink alcohol was encoded by populations of neurons in the rodent mPFC, and if such encoding was influenced by a family history of alcohol drinking. Task-stimuli-evoked changes in neural activity were observed in mPFC of both strains of rats . ContrarP rats are an extremely well-validated rodent model of AUD . One feaSeveral studies have found that P rats display persistent alcohol-seeking behavior in the presence of cues previously associated with alcohol access/consumption compared to other strains . This suIn the current study, the encoding of drinking intent  was diminished in mPFC of P rats compared to Wistar rats. While, these data are the first to provide evidence that mPFC neurons directly encode the intent to consume alcohol, they also indicate this signal is diminished in animals with increased risk of excessive drinking. These data suggest that increased familial risk diminishes the contribution of the mPFC in the decision to seek and drink alcohol. However, there is also substantial evidence for transitions in encoding in subcortical brain regions, such as the striatum. The dorsomedial striatum directly influences alcohol consumption that is still sensitive to devaluation , whereas the dorsolateral striatum modulates alcohol consumption only after prolonged training in which animals have become insensitive to devaluation and display habitual behavioral responding . Thus, oCollectively, the data provided herein indicate differences in the role of the mPFC in alcohol consumption between populations with or without increased familial risk of excessive drinking. This finding is characterized by two primary features observed in P rats. (1) The encoding of the decision to drink is blunted during alcohol drinking. (2) And the encoding of stimuli previously associated with alcohol is enhanced during water sessions. We have also observed that neurons in the mPFC of P rats may be uniquely sensitive to the alcohol-associated context of the 2CAP conditioning chamber (i.e., before task;


In this paper we present a novel no-reference video quality measure, NR-FFM (no-reference frame–freezing measure), designed to estimate quality degradations caused by frame freezing of streamed video. The performance of the measure was evaluated using 40 degraded video sequences from the laboratory for image and video engineering (LIVE) mobile database. Proposed quality measure can be used in different scenarios such as mobile video transmission by itself or in combination with other quality measures. These two types of applications were presented and studied together with considerations on relevant normalization issues. The results showed promising correlation values between the user assigned quality and the estimated quality scores. The most reliable way of doing this is by conducting visual experiments under controlled conditions, in which human observers grade the quality of the multimedia contents under evaluation . UnfortuFR quality measures require the original undistorted or unprocessed signal. NR quality measures require only the processed/degraded signal. RR quality measures need information derived from the original signal .In this paper, we present no-reference video quality measure for frame freezing degradations called NR-FFM (no-reference frame–freezing measure). This type of degradation often occurs during video transmission in different types of TV broadcasting , with low SNR  margin. It will be shown that the proposed measure achieves high correlation with MOS and that it can be used in cases where only this type of degradation is expected or in combination with other degradation types, so that NR-FFM is combined with some other FR or RR objective measure.This paper is organized as follows: A survey that classifies and compares current objective video quality assessment algorithms can be found in . GeneralBecause frame-freezes produce video sequences that may have a total play time longer than reference video sequence , it is unclear how to compare such video sequences by using full or reduced reference quality measures. Also, by matching segments of the degraded sequence to corresponding segments of the reference, usual quality measures would always be perfect .The paper  presentsThere are also other algorithms for the detection of dropped video frames. In one of the state-of-the-art algorithms , the autIn this study, we developed a no-reference measure for frame-freezing degradations (NR-FFM) on the basis of AFR and spatial information of the video content. NR-FFM can be used to quantify frame freezing degradations alone, or in combination with other measures to calculate quality in the presence of different types of degradations. LIVE mobile database  was usedNR-FFM measure based on overall frame duration, number of frame freezings, and spatial information of the tested video sequence;NR-FFM obtains best correlation among other tested frame freezing objective measures in LIVE mobile dataset and similar correlation in VQEG HD5 dataset as other tested measures;NR-FFM can be combined with other objective measures designed for other degradation types, tested on LIVE mobile dataset with five degradation types, and combined with RVQM and STRRED as reduced reference measures.Compared to the existing measures, contributions in this paper are:H.264 compression (four test videos per reference);Wireless channel packet-loss (four test videos per reference);Frame-freezes (four test videos per reference);Rate adaptation (three test videos per reference);Temporal dynamics (five test videos per reference).To develop and test the proposed measure NR-FFM, we used sequences from the LIVE Mobile Video Quality Database   for longer frame freezing in the case of the stored streaming scenario (degradation types 1–3) with the same affected frame rate;DMOS score was higher (worse) in the case of live streaming scenario (1 × 120 + 1 × 60 frame freeze) where frames were ‘lost’, compared with the case of stored video and 2 × 120 frame freezes; however, it was not clear by how much, as the former had a freeze at the end lasting for 60 frames;DMOS score was different for different video sequences and the same degree of AFR, which means that the final DMOS score depended also upon video content—a good example would be sequence number 11, with a very low DMOS score, as this sequence had low spatial and temporal information, so the long frame freeze did not have a higher impact on the subjective score.Several conclusions can be drawn from the graph  values. By default, Sobel operator should be calculated for both horizontal and vertical edges. However, we later calculated SI for only horizontal (later described as SIH), only vertical (later described as SIV), and both horizontal and vertical edges :To be able to further analyze video content, we used spatial and temporal activity indicators, the spatial information (SI), and the temporal information (TI) for all degraded video sequences. SI and TI are defined as follows :(1)SI=maResults of TI versus SI are presented in Firstly, we tried to find if there was a relationship between spatial information (SI), temporal information (TI), and DMOS scores, of all the 40 degraded sequences in the LIVE dataset. A genetic algorithm (GA)  was usedn is number of freezes in one sequence j, NFD is normalized frame duration of one freezing , SI(j) and TI(j) are spatial and temporal information of the whole sequence j (according to the Equation (1)), respectively, and coefficients α, β, and γ were optimized using a GA to maximize Spearman’s correlation. Coefficient α takes into account impact of the duration of one frame freeze as well as overall frame freeze duration on final video quality. It should be expected that 0 < α < 1. α < 1 means that one freezing event with longer duration will have a better (lower) objective grade than few shorter freezing events , which is in accordance with α > 0 means that a longer frame freeze will have a higher impact  on video quality than a shorter frame freeze. Coefficients β and γ show the impact of spatial and temporal information on video quality. It would be expected that β and γ were higher than 0, meaning that higher spatial or temporal activity will have a higher influence on video quality.We then hypothesized the following form for the reference frame freezing measure (NR-FFM) for degraded video sequence j :α and β over the non-overlapping test set. We divided 10 original sequences in 7 or 8 for training  and 3 or 2 for testing . This gave (10 over 7) = 120 combinations or (10 over 8) = 45 combinations. Each combination was run 10 times and best (highest) Spearman’s correlation was taken as being the correct model. Afterwards, the model was tested on the remaining part of the frame-freezing dataset. The model was trained to have the highest possible Spearman’s correlation. Running GA 10 times , using all combinations, we obtained the best results with mean γ near 0. Because of that, we decided to calculate NR-FFM using SI information only. By discarding TI from Equation (2), proposed NR-FFM should then have a following form:We used LIVE mobile database frame freezing degradation sequences (40 video sequences) to train the proposed model from Equation (2). We calculated GA over the training set and tested calculated parameters x and y:x is results from different objective measures and y is results from subjective tests), whereas x and y. Spearman’s correlation assesses how well an arbitrary monotonic function can describe the relationship between two variables without making any assumptions about the frequency distribution of the variables 1 and Q2 1 and Q3 fitting functions. Q2 had a somewhat lower correlation and Q4 function gave the lowest correlation (as could be expected from linear fitting function).From 1, Q2, Q3, and Q4.When the entire mobile dataset was used for training, we obtained Kendall’s, Spearman’s, and Pearson’s correlation according to When comparing Spearman’s and Pearson’s correlations from To further compare proposed NR-FFM measure, we also used DMOS tablet scores (20 video sequences with frame freezing degradations) with proposed NR-FFM from Equation (7). It has to be noted that these scores were obtained from the same video sequences as in the mobile dataset, however, they were shown on the tablet display.1, Q2, Q3, and Q4, respectively. 1–b5, as defined in Equation (6), for this case.If we used the NR-FFM measure defined in Equation (7), we obtained Kendall’s correlation of 0.6000; Spearman’s correlation of 0.8030; and Pearson’s correlation of 0.8357, 0.8353, 0.8358, and 0.8253 for Q1, Q2, Q3, and Q4 as fitting functions), also for this case.Another solution to determine NR-FFM measure coefficients would be to train it on a tablet dataset (with 20 video sequences) and test it on a mobile dataset (with 40 video sequences). Training was performed equally as previously described for the overall mobile dataset. Parameters α and β in NR-FFM, Equation (4), were in this case determined to be 0.6356 and 0.1098, respectively. To be able to combine the proposed NR measure with other objective measures, it has to be rescaled and to have the same regression as the targeted objective measure. One way of achieving this is by using the nonlinear regression model proposed in Equations (8) and (9), where free parameter δ was calculated using GA to give the highest possible Spearman’s correlation with DMOS. Rescaling was done using the maximum and minimum grade from the objective measure tested in advance for other degradation types, as well as from the proposed NR measure. We implemented proposed NR measure from Equation (4) on two reduced video quality measures, RVQM , and STRRVQM measure is based on 3D  steerable wavelet transform (Riesz transform ) and modSTRRED  is another measure with variable range of the reference information . It combines spatial RRED index  (SRRED) In the case of implementing the proposed measure of frame freezing degradations in RVQM, rescaling was done according toAt the end, combined measures could be calculated as follows:In Equation (10), STRRED combined was rescaled to start from 1 , otherwise the combined measure would give 0 even if only one part of the measure gave a perfect grade (zero).As in the previous case (only frame-freezing degradations), we divided the dataset in seven or eight  for training and the rest for testing . This gave overall (multiplied by four degradation levels per degradation type and five degradation types) 140 or 160 video sequences for training. The rest of the non-overlapping video sequences were used for testing . Results for Kendall’s, Spearman’s, and Pearson’s correlation between RVQM, STRRED, and DMOS are presented in If the entire dataset was used for training in Equations (8) and (9), δ was calculated using GA running 10 times ); δ1 = 3.2023 and δ2 = 3.8115. In this case, rescaled measures can be explicitly written as in following equations:1, Q2, and Q3 have similar correlations, whereas Q4 produces a much lower correlation. This means that the linear fitting function Q4 cannot be used in this case. It has to be also noted that in this database, only one part from Equation (10) has an influence on the final objective metric (frame-freezing degradation or any other degradation type), whereas the other part shows perfect quality (with score 1).Pearson’s correlation was calculated after nonlinear regression using Equation (6). In these tables, results for VQM measure  that incH (from Equations (1) and (2)) as spatial information values.In this subsection we will compare newly proposed NR-FFM measure with several existing measures, named “Borer” (no reference objective measure) , “FDF” , FDF (no-reference), Quanhuyn (no-reference), and FDF-RR (reduced reference). In the LIVE mobile dataset, NR-FFM gave the best correlation results between all tested measures . In the The main difference between LIVE mobile frame freezing degradations and VQEG-HD5 frame freezing degradations were the type and overall duration of the freezing occurrences. In the LIVE mobile dataset, there were online degradations , which resulted in frame drops and offline degradations , where no frame was actually lost, only delayed. Also, in the LIVE mobile dataset, overall freezing duration was generally the same—in offline frame freezing 8 × 1 s long, 4 × 2 s long, and 2 × 4 s long. Only online freezing had one 4 s long freeze and one 2 s long freeze. In the VQEG-HD5 dataset, all freezing frames were online degradations, resulting in frame drops. Also, higher packet loss ratio (PLR) resulted in longer frame freezes and, consequently, more lost frames. This meant that overall frame freezing duration was different for different degradation types (in the VQEG-HD5 dataset this was due to the different PLR ratio). Probably because of this, the NR measures FDF and Borer had lower correlation in the LIVE mobile dataset compared to the VQEG-HD5 dataset. Also, motion intensity or temporal information (or any other measure that would take differences between consecutive frames into account) did not have a higher value in offline frame freezing compared to the original video sequence (and that degradation was present only in the LIVE mobile dataset). NR-FFM measure had the best correlation in the LIVE mobile dataset compared to the other tested measures, probably also because it was calculated only on the basis of frame freezing duration and spatial information. NR-FFM measure also had different values for the equal overall frame freezing, but with different numbers of occurrences (see Equation (7)). Nonetheless, in the VQEG-HD5 dataset, NR-FFM had similar correlation to Borer and FDF measures without pretrained values on the VQEG-HD5 dataset.When comparing online degradation  and offline degradation  types in the LIVE mobile dataset , offlineNR-FFM measure was also tested using both the LIVE mobile (40 video sequences) and LIVE tablet study (20 video sequences) and was trained/tested using another dataset, showing similar fitting coefficients and correlations in both cases (trained on the LIVE mobile and tested on the LIVE tablet and vice versa).Furthermore, we combined the proposed NR-FFM measure with some existing reduced-reference measures (RVQM and STRRED) and tested it using all video sequences from the LIVE mobile/tablet dataset. Results have shown that it is possible to obtain similar correlation for the overall combined objective measure using all five degradation types in the LIVE mobile dataset .In this paper we proposed a new NR-FFM measure for frame freezing degradations. It can be used only for this degradation type or in combination with other degradation types, not lowering their overall correlation .Future research may be based on temporal information (or motion intensity) implementation in the proposed NR-FFM measure to obtain higher correlation with subjective grades for video sequences with both offline and online frame freezing. Alternatively, different formulae maybe defined for spatial and temporal information instead of the proposed formula in Equation (7). A larger dataset should also then be developed to be able to calculate correlations with higher confidence.


Archaeological evidence indicates that domestic pigs arrived in Europe, alongside farmers from the Near East ∼8,500 y ago, yet mitochondrial genomes of modern European pigs are derived from European wild boars. To address this conundrum, we obtained mitochondrial and nuclear data from modern and ancient Near Eastern and European pigs. Our analyses indicate that, aside from a coat color gene, most Near Eastern ancestry in the genomes of European domestic pigs disappeared over 3,000 y as a result of interbreeding with local wild boars. This implies that pigs were not domesticated independently in Europe, yet the first 2,500 y of human-mediated selection applied by Near Eastern Neolithic farmers played little role in the development of modern European pigs. Archaeological evidence indicates that pig domestication had begun by ∼10,500 y before the present (BP) in the Near East, and mitochondrial DNA (mtDNA) suggests that pigs arrived in Europe alongside farmers ∼8,500 y BP. A few thousand years after the introduction of Near Eastern pigs into Europe, however, their characteristic mtDNA signature disappeared and was replaced by haplotypes associated with European wild boars. This turnover could be accounted for by substantial gene flow from local European wild boars, although it is also possible that European wild boars were domesticated independently without any genetic contribution from the Near East. To test these hypotheses, we obtained mtDNA sequences from 2,099 modern and ancient pig samples and 63 nuclear ancient genomes from Near Eastern and European pigs. Our analyses revealed that European domestic pigs dating from 7,100 to 6,000 y BP possessed both Near Eastern and European nuclear ancestry, while later pigs possessed no more than 4% Near Eastern ancestry, indicating that gene flow from European wild boars resulted in a near-complete disappearance of Near East ancestry. In addition, we demonstrate that a variant at a locus encoding black coat color likely originated in the Near East and persisted in European pigs. Altogether, our results indicate that while pigs were not independently domesticated in Europe, the vast majority of human-mediated selection over the past 5,000 y focused on the genomic fraction derived from the European wild boars, and not on the fraction that was selected by early Neolithic farmers over the first 2,500 y of the domestication process. The emergence of agricultural societies in the Near East at least 12,500 y before the present (BP) was followed by the westward dispersal of farmers into Europe beginning ∼8,500 y BP 1. This NeSus scrofa present in Europe before the arrival of Near Eastern farmers can be classified as domestic, indicating that European hunter-gatherers did not independently domesticate local wild boars.Traditional methods for distinguishing between wild and domestic pigs rely primarily on archaeological context and size differences  or are bAlthough the phenotype associated with Near Eastern domestic pigs does not appear to vary considerably following their introduction to Europe –20, therOne possible mechanism to account for the apparent discontinuity between genotype and phenotype is gene flow from local European wild boars into the introduced domestic population. Domestic pigs have likely always interacted and interbred with wild populations, and this process has been suggested wherever domestic animals have arrived , and the gene flow from wild boars did not substantially affect the phenotype of the domestic population, it is possible that modern domestic pigs retain a sufficient, yet undetected, fraction of Near Eastern ancestry that underlies domestic traits . This scn = 230] and next-generation sequencing (NGS) data [n = 327]) and nuclear data, including 2 high-coverage (>10-fold), 7 medium-coverage (1- to 10-fold), and 54 low-coverage (<1-fold) genomes from an assessment of >500 archeological pig remains (Dataset S1). Our dataset (including publicly available sequences) spans the past 14,000 y and includes a total of 2,099 samples from the Near East and Europe, including samples from contexts that precede and follow the origins of pig domestication.Here, we assessed whether modern domestic pig genomes retain a Near Eastern component that is essential for maintaining their domestic characteristics, and characterized the extent, speed, and mechanisms by which pigs acquired European wild boars ancestry. To do so, we obtained mitochondrial , and 1 from the Near East, including haplogroups mt-Y1 and mt-ArmT . These results substantiate previous findings that mt-Y1 and mt-ArmT are indigenous to the Near East, although mt-Y2, previously thought to be found exclusively in the Near East (SI Appendix). In addition, the mt-Y1 signature, originally restricted to the Near East (SI Appendix).Our mtDNA analysis revealed 2 broad groups: 1 from Western and Eastern Europe, including mt-Italian, mt-A, mt-C, and mt-Y2 haplogroups . The persistence of the mt-Y1 signature within pigs on islands mimics the patterns seen in isolated island populations of both sheep and humans. For instance, sheep in Orkney and St. Kilda , in Crete , in Sardinia  , near Nat. Kilda , were noSI Appendix, Fig. S10). An ADMIXTURE analysis of 38 wild boars nuclear genomes, including an ancient wild boars from Aşıklı Höyük  reveals that modern wild individuals from Greece possess 33 to 38% Near Eastern nuclear ancestry, while those from Italy possess only 6 to 10% . It is also possible, however, that a portion of the Anatolian ancestry found in Italian wild boars is the result of admixture from domestic pigs derived from the Near East into wild populations, instances of which have previously been shown to have occurred in northern Germany . In fact, when modern European domestic pigs are treated as a single population, our haplotype-based analyses [GLOBETROTTER (SI Appendix), and most of this Near Eastern signal is derived from a few modern breeds from Italy, Hungary, and Spain that possessed 1.7 to 6.4% Near Eastern nuclear ancestry . It is therefore likely that the limited Near Eastern ancestral component detected in these samples was acquired through gene flow with local wild boars , and maintained as a result of a lack of admixture with introduced Chinese pigs.Additional ADMIXTURE analyses, including 111 genomes, clearly demonstrate that most modern domestic pigs (77 of 85) do not possess significant levels of Near Eastern ancestry . In Europe, 4 ancient high/medium-coverage domestic pigs did possess Near Eastern nuclear ancestry (Dataset S1), and also had substantially more Near Eastern ancestry than any of the ancient or modern European wild boar , as well as significant D-statistics of the form D  . These results indicate that European wild boars were being incorporated into domestic populations relatively soon after the latter were introduced from the Near East.We further assessed the degree of Near Eastern ancestry in archaeological pigs. Our ADMIXTURE analysis indicates that Bronze Age domestic pigs from western Iran  and Armenia  did not possess any European ancestry, and were exclusively derived from ancient Near Eastern wild boar (ancestry B. Specifild boar B. This iSI Appendix) to be confidently projected onto a principal component analysis (PCA) alongside both modern and (high- and medium-coverage) ancient genomes. We analyzed these data together with those of Asian wild and domestic pigs. In this analysis, principal component 1 (PC1) separated European and Asian pigs, while PC2 separated Near Eastern and European pigs . After removing Asian pigs, PC1 separated modern European domestic pigs from all other samples, while PC2 separated European from Near Eastern pigs .To obtain a more precise temporal and geographic resolution of the disappearance of Near Eastern genomic signatures in Europe, we performed additional analyses of 54 low-coverage ancient genomes (<1-fold) that possessed sufficient data . We also identified 3 samples from Buran-Kaya, Crimea  that also cluster close to Near Eastern wild boars, although they each possess the mt-Y2 haplotype and so are thought to be local wild boars (Dataset S1).The PCA revealed 2 groups of ancient European pigs  C. The fiDataset S1).The second group of ancient European samples was closer to wild and modern domestic pigs from Europe and included samples that are mostly younger in age than the first group. This second group consisted of 18 domestic samples from overall more recent archaeological sites dating from 7,100 to 900 y BP, including the following: Herxheim, Germany ; Oulens, France ; Bozdia, Poland ; Durrington Walls, England ; Utrecht, The Netherlands ; Basel, Switzerland ; Coppergate, England ; Undir Junkariusfløtti, Faroe Islands ; and Ciechrz, Poland (∼900 y BP: AA139). This group also comprised 7 ancient samples that could not be identified as either wild or domestic, including the following: la Grotte du Taï, France ; Santa Maria in Selva, Italy (Late Neolithic: AA629); and El Portalón, Spain . Lastly, 2 ancient wild boars, 1 from Birsmatten-Basisgrotte, Switzerland  and 1 from Siniarzewo, Poland  were also found to fall closer to modern European wild boars. All of these samples possessed a European mtDNA signature , Danube , and Rhin  river systems in Germany, Romania, Macedonia, and Serbia possessed substantially more Near Eastern ancestry than is present in European wild boar B and C. SI Appendix, Fig. S19). For admixture values ≥5%, the probability of observing only 8 genomes with Near Eastern ancestry is <1% . This indicates that ADMIXTURE should detect significantly more pigs with Near Eastern ancestry if the genome of every modern domestic pig possessed a Near Eastern component ≥5%. Additionally, our simulations indicate that the GLOBETROTTER , which is less than what is present in modern Italian and Balkan wild boar. If a degree of Near Eastern ancestry was essential for the maintenance of the domestic phenotype in Europe, we would therefore predict that the underlying causative variants are present in no more than ∼4% of the genome.To assess the threshold above which we could confidently identify Near Eastern ancestry in our ancient data, we simulated genomes with predefined Near Eastern ancestry proportions and analyzed the data using ADMIXTURE . We thenETROTTER  analysisSI Appendix). Our results show that a large majority of domestic pig haplotypes within these sweep regions share a closer genetic affinity to European wild boars than to Near Eastern wild boar . In fact, we did not identify a single region that was closer to Near Eastern wild boars . This suggests that the majority of human-mediated selection that took place after the arrival of pigs in Europe most likely did not target haplotypes of Near Eastern origin. We could not, however, distinguish between European and Near Eastern ancestry in ∼10 sweep regions. Given the bias toward modern European wild boar haplotypes in our dataset, it is possible that our analysis did not possess sufficient power to identify Near Eastern ancestry in those ∼10 regions. Doing so will require additional sequencing of modern and ancient Near Eastern pigs.To further explore this possibility, we investigated whether regions of modern domestic pig genomes reported to be subjected to positive selection  were morMC1R) gene. This gene has been shown to harbor functional mutations (linked to the loss of camouflage coat color) that are highly correlated with domestic status (SI Appendix). Our analyses of previously published and novel modern and ancient MC1R sequence data (269 domestic pigs and 46 wild boar) demonstrate that a specific nonsynonymously derived mutation [D124N . The only wild boar that possessed 1 copy of the derived allele originated from a population in The Netherlands that is known to have recently interbred with domestic pigs (Dataset S1), we identified 64 of 76 animals with at least 1 copy of the derived allele (the remaining 12 were homozygous for the wild type). Altogether, this suggests that while the ancestral allele at this locus cannot be used to unequivocally distinguish wild and domestic pigs, the derived allele is highly indicative of domestic status.To further assess the potential relevance of Near Eastern ancestry to the genetic and phenotypic makeup of early and modern domestic pigs, we investigated the Melanocortin 1 Receptor .The earliest pigs that possessed the derived allele were found at Neolithic Ulucak Höyük in western Anatolia . The earliest European pigs that possess the derived allele are from Neolithic sites in Bulgaria , Romania , and Germany . Further phylogenetic analysis of the ∼100-kb region surrounding the SI Appendix, Fig. S9). This finding indicates that we do not possess the resolution to infer whether the D124N mutation (now fixed in many domestic breeds) first arose in the Near East or in Europe. Although we cannot definitively identify the geographic origin of the D124N mutation using phylogenetic analysis, the fact that it occurred in Anatolia before the arrival of domestic pigs into Europe, and that it likely arose only once, strongly suggests that this trait originated in Anatolia and was present in the first pigs that were transported into Europe.This result suggests that the D124N mutation found in Near Eastern and European pigs arose just once and was maintained, despite substantial gene flow with European wild boars. Interestingly, the nearest clade to this monophyletic cluster consisted of 2 haplotypes found in modern wild boar with European ancestry (The Netherlands) and Near Eastern ancestry , the Near Eastern genomic proportion of domestic pigs in Europe had dropped to <50%, and the Near Eastern fraction is now 0 to 4% in modern European domestic pigs. This near-complete genomic replacement and gradual disappearance of Near Eastern ancestry occurred over 3 millennia in continental Europe and was the result of hybridization between Near Eastern domestic pigs and European wild boars. This further implies that European domestic pigs did not originate from an independent domestication process, but rather from the continuous management of herds that were interbred  with local wild boar. In Mediterranean regions, including Sardinia , CorsicaMC1R gene) that was transferred from the Near East into Europe by early farmers, where it resisted introgression from wild boar. This finding suggests that other regions of the genome that govern domestic phenotypes  may also have retained their Near Eastern ancestry, but our analyses indicate that these regions make up no more than 4% of the genome. In fact, we show that the vast majority of human-mediated selection over the past 5,000 y focused instead on the genomic fraction derived from the European wild boars, and not on genomic variants that were selected by Near Eastern Neolithic farmers during the first 2,500 y of the domestication process.The introgression from European wild boars eroded the proportion of Near Eastern ancestry in European pigs to levels that are potentially below our detection threshold. As predicted by a model in which European pigs were not independently domesticated, we found the existence of a genetic variant leading to black coat color (within the Previous coalsecent simulations have shown that a genomic replacement of this magnitude, as a result of introgression from a local population into an invading population is expected, so long as the incoming population is relatively small and strong barriers to interbreeding do not exist . The degOverall, our results suggest that domestication narratives are not as straightforward as a simple dispersal of fully domesticated plants and animals out of the area of initial domestication. Instead, domestication is a protracted process, a significant proportion of which takes place through continual admixture and human-mediated selection. These perspectives underscore the temporally dynamic nature of the relationship between humans and domestic taxa, and our increasing ability to monitor this process by analyzing ancient genomic data within the context of metrical, isotopic, and other analyses.Supplementary FileSupplementary FileSupplementary File


Alterations in the corticostriatal system have been implicated in numerous substance use disorders, including alcohol use disorder (AUD). Adaptations in this neural system are associated with enhanced drug-seeking behaviors following exposure to cues predicting drug availability. Therefore, understanding how potential treatments alter neural activity in this system could lead to more refined and effective approaches for AUD. Local field potentials (LFPs) were acquired simultaneously in the prefrontal cortex (PFC) and nucleus accumbens (NA) of both alcohol preferring (P) and Wistar rats engaged in a Pavlovian conditioning paradigm wherein a light cue signaled the availability of ethanol (EtOH). On test days, the catechol-o-methyl-transferase (COMT) inhibitor tolcapone was administered prior to conditioning. Stimulus-evoked voltage changes were observed following the presentation of the EtOH cue in both strains and were most pronounced in the PFC of P rats. Phase analyses of LFPs in the θ band (5–11 Hz) revealed that PFC-NA synchrony was reduced in P rats relative to Wistars but was robustly increased during drinking. Presentation of the cue resulted in a larger phase reset in the PFC of P rats but not Wistars, an effect that was attenuated by tolcapone. Additionally, tolcapone reduced cued EtOH intake in P rat but not Wistars. These results suggest a link between corticostriatal synchrony and genetic risk for excessive drinking. Moreover, inhibition of COMT within these systems may result in reduced attribution of salience to reward paired stimuli via modulation of stimulus-evoked changes to cortical oscillations in genetically susceptible populations. Alcoholism is highly heritable and genetic vulnerability is associated with increased likelihood of alcohol use-related problems. Presentation of environmental stimuli paired with alcohol are capable of inducing craving and relapse in alcohol-dependent individuals. The work described here using a rodent model of cued ethanol (EtOH) availability suggests that altered corticostriatal activity may be associated with genetic vulnerability to abuse alcohol. Additionally, our data suggest that inhibition of the catechol-o-methyl-transferase (COMT) enzyme activity may reduce the influence that alcohol-conditioned stimuli (CSs) elicit on behavior, resulting in a reduction of cued alcohol seeking in genetically susceptible individuals.Environmental stimuli associated with drugs of abuse acquire motivational properties  capable Theta oscillations  have been shown to facilitate plasticity , as wellAlcohol preferring (P) rats are a validated preclinical model of AUD . P rats The catechol-o-methyl-transferase (COMT) enzyme metabolizes catecholamine’s including DA in cortical regions . Using tN = 10, 37 datasets; Indiana University) and Wistar  rats. All animals arrived from both respective outside vendors within the same week to ensure habituation and rearing conditions were identical for both strains. Animals were single housed, weighed ∼250–300 g at the start of experiment, maintained on a reverse light dark cycle and supplied with food and water ad libitum. All animals initially received four weeks of intermittent two bottle access for 20% EtOH to pre-expose animals to EtOH  or vehicle  as described previously , 2018.x-, y-coordinates in separate voltage traces. These voltage traces were then used to compute the velocity of the animal’s movement  fed through silica tubing and then secured to custom-made head caps via epoxy. Wires were externally referenced to a stainless-steel skull screw over the cerebellum. Probes were implanted over the mPFC  and NA x¯t=HxThen the analytic signal is projected on the unit circle (3)zt=ζtζThe following widely used measure of the strength of phase locking between these two signals was calculated:  (4)γ=1N∑strength . To explTo investigate the impact of CS presentation on the phase of the signals, we analyzed the phase delay. Phase delay was calculated by first computing the averaged time difference between peaks of LFP phases for 1 s before CS on for each brain region . Then, the time difference between the first peak right after the CS and the last peak right before the CS was computed. Phase delay was derived by computing the difference between the first value and second value. Positive values indicate that the CS causes a delay of peak while negative values suggest that the CS causes the advance of the peak.To measure the CS-evoked precision of phases across trials in the θ band, ITPC was calculated. Drinking trials were analyzed for 200 ms before, and 1 s after the CS. ITPC was calculated per After calculating ITPC for each trial, averages across trials were taken in the θ band (5–11 Hz) for each dataset. Statistics were performed on the dataset-averaged time series.https://www.r-project.org/). Unless specified otherwise, all comparisons were first subjected to ANOVA testing or mixed-design ANOVA (a mixture of between subject factor and within subject factors) followed by post hoc tests for all multiple comparison procedures.All analyses were performed in MATLAB (MathWorks) and R  = 4.56, p = 5.70 × 10−5; t test, t(36) = 9.71 × 10−1, p > 0.05; F = 15.5, p = 8.21 × 10−5) and non-drinking trials  = 1972, p = 9.21 × 10−6), trial × strain interaction  = 2.90 × 101, p = 7.46 × 10−8; Consistent with our previous findings , behavioF = 1.39, p = 1.05 × 10−5; F = 2.78 × 102, p < 1.00 × 10−16; main effect of time F = 3.15, p < 1.00 × 10−16; On trials that animals drank, a robust change in voltage of the mPFC and NA was observed following the presentation of the CS in both strains of animals . The chaF = 6.75, p = 4.6 × 10−6; P rat, trial × time interaction, F = 6.82, p = 4.1 × 10−6). The increase in mPFC-NA synchrony was more robust in P rats  = 1.39 × 101, p = 1.26 × 10−3), indicating that, overall, synchrony differed for trials in which animals chose to drink versus trials where animals abstained.Phase synchrony during the US but not before the CS was observed A–C. To dF = 2.42, p = 2.97 × 10−3; F = 4.00, p > 0.05) or epoch  = 2.63 × 10−1, p > 0.05) were observed. Additionally, no two-way or three-way interactions were detected (ps > 0.05). Collectively, these data indicate that, on non-drinking trials, the observed synchrony is influenced by trial number but not strain or epoch.Synchrony was next analyzed for drinking and non-drinking trials separately. A mixed-design ANOVA on non-drinking trials revealed a significant main effect of trial number  = 4.64, p = 3.97 × 10−2), epoch  = 1.50 × 102, p = 5.45 × 10−6), and trial number  = 4.61, p = 8.72 × 10−8) were observed. Post hoc tests indicated that phase synchrony for Wistars was significantly higher overall compared to P rats  and immediately after the sipper was removed from the chamber . Additionally there were significant strain × epoch  = 1.10 × 101, p = 8.69 × 10−5), and strain × trial  = 2.89, p = 3.52 × 10−4) interactions, indicating the pattern of phase synchrony across trials and epochs was different across rat strain. Therefore, drinking trials were analyzed separately for Wistar and P rats to determine differences in synchrony across each epoch and trial.On drinking trials, significant main effects of strain  = 5.18, p = 3.48 × 10−8), but neither a main effect of epoch  = 1.76 × 10−1, p > 0.05) nor an epoch × trial interaction  = 1.33, p > 0.05) were observed  = 1.40, p > 0.05). However, synchrony differed by epoch  = 2.62 × 101, p = 1.82 × 10−7; Post hoc tests indicated that synchrony during drinking (US) was significantly higher than synchrony before CS presentation  and after drinking . There was no difference in synchrony before CS on and after drinking . In summary, consistent with wavelet coherence analyses, increases in θ band synchrony were observed when alcohol was available during drinking trials  = 4.37, p = 4.54 × 10−2) but not brain region  = 3.48, p > 0.05) or trial number  = 5.97 × 10−1, p > 0.05). Both two-way and three-way interactions were not significant (ps > 0.05). Next, the difference of the phase delays was assessed for both strains in each brain region separately. In the mPFC, the phase delay of P rats was significantly longer compared to Wistars . During non-drinking trials, a mixed-design ANOVA was employed to investigate the change in the phase of the signals due to CS presentation. There was no main effect of strain  = 1.36 × 10−1, p > 0.05), brain region  = 1.51 × 10−1, p > 0.05) or trial number  = 1.39, p > 0.05; ps > 0.05). Collectively these analyses indicate that the phase resets are most robust in P rats in the mPFC on drinking trials.To determine how the presentation of the CS influenced oscillatory dynamics, changes in θ phase over time in the mPFC and NA were assessed . To deteF = 3.02, p = 5.50 × 10−6) and P rats  = 3.73, p = 0.1 × 10−6). CS-evoked changes in ITPC were assessed by comparing ITPC values 200 ms before stimulus onset to values 1 s after CS presentation  = 1.47 × 101, p = 9.01 × 10−4) but no effect of strain  = 7.23 × 10−1, p > 0.05). There was also a significant strain × treatment interaction  = 4.39, p = 4.79 × 10−2). Post hoc test indicated that tolcapone decreased intake of EtOH . In particular, tolcapone decreased intake in P rats  but not Wistars  = 6.19, p = 2.28 × 10−2) was observed, but no main effect of strain  = 1.14, p > 0.05) or strain × treatment interaction  = 8.39 × 10−1, p > 0.05) were observed. Post hoc testing indicated that treatment with tolcapone resulted in fewer drinking trials compared to treatment with saline . To investigate the effects of tolcapone on mPFC-NA synchrony (γ), we performed a four-way ANOVA with treatment , strain, epoch, and drinking status  as factors. There were significant main effects of treatment  = 3.16 × 101, p = 2.27 × 10−8), strain  = 1.72 × 102, p < 1.0 × 10−16), and drinking status  = 5.24, p = 2.21 × 10−2), but not epoch  = 1.47, p > 0.05). There was also a significant treatment × strain interaction  = 2.83 × 101, p = 1.17 × 10−7), while all other two-way, three-way, or four-way interactions were not significant (ps > 0.05). Post hoc tests indicated that administration of tolcapone resulted in reduced mPFC-NA synchrony  but not P rats . Consistent with non-treatment sessions, overall mPFC-NA synchrony in Wistars was higher compared to P rats . Moreover, mPFC-NA synchrony during drinking trials was significantly higher than during non-drinking trials .In the following analyses, data are collapsed across trial number due to the few drinking trials observed following tolcapone treatment in P rats. There was no effect of tolcapone on CS-evoked changes in voltage  in eitheF = 7.31, p = 6.96 × 10−3). Post hoc testing indicated that the phase delays of P rats were significantly shorter compared to Wistars  = 3.07 × 10−1, p > 0.05), drinking  = 1.37, p > 0.05) or brain region  = 2.30 × 10−1, p > 0.05). However, there was a significant treatment × strain interaction  = 4.24, p = 3.97 × 10−2). Post hoc testing indicated that tolcapone reduced the phase delay in the mPFC of Prats during drinking trials . Similar to non-treatment sessions, a main effect of strain was observed  = 1.08 × 101, p = 1.08 × 10−3) with post hoc test indicating a shorter phase delay in P rats, relative to Wistars .Phase-response curves were computed to assess CS-induced phase resets following treatment and were collapsed across trials. A four-way ANOVA  was performed. There was a significant main effect of strain  = 1.09, p = 0.37), whereas a main effect of time was observed following tolcapone administration  = 3.00, p = 1.04 × 10−4; F = 2.95, p = 7.41 × 10−5), but not tolcapone  = 1.30, p = 0.178).To further determine how changes CS-evoked synchrony were influenced by treatment, ITPC was assessed following saline and tolcapone injections on drinking trials. In Wistars, no main effect of time was observed following saline administration (repeated-measures ANOVA, These experiments investigated the neural mechanisms by which drug paired stimuli elicit reward seeking in a rodent model of addiction vulnerability. Relative to Wistars, P rats exhibited an increase in mPFC-NA synchrony when drinking and a greater CS-induced phase delay in mPFC. Following tolcapone administration, a reduction in EtOH intake in P but not Wistar rats was observed. While no effect of tolcapone on synchrony in P rats was observed, a tolcapone-mediated suppression of the mPFC phase delay was observed in P rats but not Wistars during drinking trials only. These data suggest a novel neural mechanism whereby tolcapone suppresses drinking in genetically susceptible populations. These data also further elucidate the modulatory role of catecholamines in the corticostriatal system on alcohol seeking behaviors.Theta oscillations in the mPFC are hypothesized to play a critical role in encoding reward value. MPFC θ phase locking was shown to be present during both reinforced and non-reinforced licks in a high-value context while phase locking was notably weaker during both reinforced and non-reinforced licks in a low-value context, suggesting that the strength of mPFC θ activity corresponds to reward value . StrongeIn the present experiment, P rats exhibited lower mPFC-NA θ synchrony than Wistars overall D,E. Low Alcohol is hypothesized to disrupt the structure of oscillations by making them asynchronous . AlcoholA wealth of literature supports the hypothesis that PFC-NA connections are important for reward seeking, especially in the presence of CSs . In the A robust θ phase reset was observed in mPFC following cue presentation B2. PhaseAlcohol associated cues elicit increases in the frontal BOLD response  and the Strain differences in tolcapone’s ability to reduce the phase reset may be associated with differences in basal DA tone and subsequent differences in cue-evoked DA transmission. Alcohol-paired cues elicit increases in DA concentrations in both clinical  and precThe results obtained here highlight the heterogeneity of factors which may contribute to excessive drinking phenotypes. Whereas alterations in corticostriatal synchrony may be associated with excessive drinking, catecholamine activity in the PFC appears to more strongly influence attribution of motivational salience to drug paired stimuli in addiction vulnerable organisms. These results highlight the role that catecholamines play on shaping how motivating stimuli are processed by PFC. Furthermore, these results suggest that targeting the catecholamine system in PFC may provide an effective way to blunt the incentive motivational properties of drug-associated stimuli. In this way, novel treatments that target this system might be effective in preventing craving and relapse.