Data on outcome of endovascular treatment in patients with acute ischaemicstroke due to large vessel occlusion suffering from intravenousthrombolysis-associated intracranial haemorrhage prior to mechanicalthrombectomy remain scarce. Addressing this subject, we report ourmulticentre experience.A retrospective analysis of consecutive acute ischaemic stroke patientstreated with mechanical thrombectomy due to large vessel occlusion despitethe pre-interventional occurrence of intravenous thrombolysis-associatedintracranial haemorrhage was performed at five tertiary care centres betweenJanuary 2010–September 2020. Baseline demographics, aetiology of stroke andintracranial haemorrhage, angiographic outcome assessed by the Thrombolysisin Cerebral Infarction score and clinical outcome evaluated by the modifiedRankin Scale at 90 days were recorded.In total, six patients were included in the study. Five individualsdemonstrated cerebral intraparenchymal haemorrhage on pre-interventionalimaging; in one patient additional subdural haematoma was observed and onepatient suffered from isolated subarachnoid haemorrhage. All patients exceptone were treated by the ‘drip-and-ship’ paradigm. Successful reperfusion wasachieved in 4/6 (67%) individuals. In 5/6 (83%) patients, thepre-interventional intracranial haemorrhage had aggravated inpost-interventional computed tomography with space-occupying effect.Overall, five patients had died during the hospital stay. The clinicaloutcome of the survivor was modified Rankin Scale=4 at 90 daysfollow-up.Mechanical thrombectomy in patients with intravenous thrombolysis-associatedintracranial haemorrhage is technically feasible. The clinical outcome ofthis subgroup of stroke patients, however, appears to be devastating withhigh mortality and only carefully selected patients might benefit fromendovascular treatment. As a result, various randomised controlled studies arecurrently being conducted to determine if MT without IVT is equally effective.Recently, the randomised DIRECT-MT trial in China indicated that MT was noninferiorto the combined treatment. However, the ongoing SKIP trial in Japan could not establish that skippingIVT was noninferior to the combined approach but was at least associated with alower risk of intracranial haemorrhage (ICH).Mechanical thrombectomy (MT) in combination with intravenous thrombolysis (IVT) isthe standard treatment for patients suffering from acute ischaemic stroke (AIS) dueto intracranial large vessel occlusion (LVO) in the anterior circulation.6 Nevertheless, in primary strokecentres (PSCs), the early initiation of bridging therapy remains the only treatmentfor patients presenting with AIS before admitting these patients to a MT-capablecomprehensive stroke centre (CSC). With increasing numbers of patients treated underthe ‘drip and ship’ paradigm, the occurrence of an IVT-associated ICH prior to theendovascular procedure is becoming more likely. Studies analysing the benefit of MT in this subgroup are limited aspre-interventional ICH remains an exclusion criterion for endovascular therapy inclinical trials. Therefore, we aimed to report our multicentre experience with MT in patientswith AIS due to LVO suffering from IVT-associated ICH.Symptomatic intracerebral haemorrhage is the main intracranial complication of IVTwith rates reported up to 8.8% according to the European Cooperative Acute StrokeStudy II (ECASS II) trial and associated with high mortality rates.We conducted a retrospective study of AIS patients undergoing MT at five tertiarycare centres in Germany between January 2010–September 2020.All patients included in the study were treated with MT due to LVO despite theoccurrence of an ICH after initiation of IVT. Inclusion criteria were missingevidence of ICH on baseline imaging, application of IVT and execution of additionalimaging prior to the intervention using multi-detector or flat-detector computed tomography (CT) with detection of anewly delimited ICH. Extent or location of ICH  were not exclusion criteria. Space-occupying effect of a parenchymalhaemorrhage was defined as any mass effect on adjacent brain structures such as deepgrey matter and gyri with narrowing of sulci, midline shift or brain herniation.Early ischaemic damage was evaluated using Alberta Stroke Program Early CT Score(ASPECTS) on first imaging for the anterior and posterior circulation. Inpost-interventional ASPECTS areas of intraparenchymal haemorrhage in the affectedterritory were included in the assessment. Large vessel occlusion was defined as anyocclusion in cerebral arteries including distal internal carotid artery (ICA),middle cerebral artery , distal vertebral artery, basilarartery (BA) and posterior cerebral artery (P1 segment). Patient demographics,medical history, technical features, angiographic and clinical outcome were noted.The aetiology of the occlusion was based on the Trial of ORG 10172 in Acute StrokeTreatment (TOAST) classification. In addition, the underlying aetiology for ICH and localization of the ICH (in/outside the LVOaffected territory) was reviewed. Any progression in size with consecutive increaseof perifocal oedema was defined as aggravation of the ICH.All patients received IVT and were treated according to the widely accepted selectioncriteria with a weight-based infusion of alteplase at 0.9 mg/kg over 60 min with amaximum dose of 90 mg. Ten per cent of the total treatment dose was given as a bolusover 1 min. There were no limitations on procedural characteristics including theuse of different thrombectomy techniques, which were left to the attendingneuroradiologist’s discretion. Endovascular treatment was performed with approved MTdevices using stent-retrievers, large-bore aspiration catheters or a combination ofboth.Complete reperfusion was defined as the Thrombolysis In Cerebral Infarction (TICI)scale score of three. Successful reperfusion was defined as TICI≥2b. Clinicalefficacy outcome was the rate of functional independence measured by the modifiedRankin Scale (mRS) and defined as 0–2 at discharge and 90 days. National Institutesof Health Stroke Scale (NIHSS) and mRS grades were assessed by a consultantneurologist. Baseline NIHSS was collected at patients’ admission at the CSC.According to the guidelines of the respective local ethics committees, ethicalapproval was given when necessary for this anonymous retrospective study, which wasconducted in accordance with the Declaration of Helsinki. A patient’s consent fortreatment was obtained according to the individual institutional guidelines. Due tothe retrospective nature of the study, additional informed consent was deemedunnecessary.In total, six patients from five tertiary stroke centres were treated with MT due toLVO suffering from IVT-associated ICH. Procedural characteristics per case are shownin Large vessel occlusion of the BA was detected in 3/6 (50%) patients, two patientssuffered from MCA M1 and one patient from distal ICA LVO, respectively. Five out ofsix (83%) individuals demonstrated cerebral intraparenchymal haemorrhage onpre-interventional CT. Of those, 3/5 (60%) patients suffered from space-occupyinghaematoma. In one patient, additional subdural haematoma was observed, and onepatient suffered from isolated subarachnoid haemorrhage (SAH). Five out of six (83%)ICH events were localised in the LVO affected territory .Cardioembolic cause was the most common aetiology for the LVO and found in 3/6 (50%)patients, followed by large artery atherosclerosis . Stroke aetiologyremained unknown in two patients. The underlying aetiology for the ICH remainedundetermined in all patients except for one, for whom further work-up revealed vitalcancer, which might have increased the bleeding propensity. Four out of six patientshad previous medication with antiplatelet agents.Median baseline NIHSS at CSC admission was 22 and median baseline ASPECTS on firstimaging was eight. The initial NIHSS at the primary stroke centre was not documentedin 5/6 patients. The median interval between (a) onset and IVT was 80 min (IQR53–165 min), (b) IVT and groin puncture was 107 min (IQR 94–193 minutes) and (c)onset and groin puncture was 240 min (IQR 190–340 min), respectively. The rate ofpre-treatment functional independence (mRS≤2) was 50% (3/6).The median time interval from groin puncture to final reperfusion was 70 min (IQR61–146 min) and the median number of thrombectomy manoeuvres was three (range1–8). Successful reperfusion was achieved in 4/6 (67%) patients. None of the sixpatients were reperfused completely. The median ASPECTS in post-interventionalCT was six. In the majority of cases , the IVT-associated ICH hadaggravated in post-interventional imaging with space-occupying oedema.Procedure-related, minor SAH had occurred in 1/6 (17%) patients. In one patient,intracranial stenting was performed (Case 6). The patient presented with anocclusion of the proximal BA at a PSC  and afteOverall, five patients had died during hospital stay. The survivor initiallypresented with a non-space occupying ICH and achieved a complete resorption ofthe haemorrhage at discharge; at 90 days, the clinical outcome was moderate anmRS=4 .We provide the first report of a series of AIS patients with LVO suffering fromIVT-associated ICH prior to MT. Our study revealed several findings: (a) theprocedure is technically feasible with an adequate rate of successful reperfusion,(b) the mortality rate of these subgroup of patients is high (83%), and (c) theunderlying cause for the ICH remains undetermined in most cases.As the presence of an intracranial haemorrhage currently represents an exclusioncriterion for MT, the decision-making was done individually. In three of theaffected individuals, the haemorrhage was not space-occupying .One patient showed haemorrhage outside the affected territory (Case 1). One patientsuffered from a basilar artery occlusion and clinical outcome is known to bedevastating if occlusion will not be recanalised (Case 5). In another patient (Case3), it was the decision of the operator and the neurologist as the patient was inher 80s, but functionally independent prior to the stroke. The underlying cause for the ICH was probable cerebral amyloid angiopathy andthe patient was released with an excellent neurological outcome (mRS=0). Incomparison with our study, the patient had lower NIHSS at admission (NIHSS=7) andwas reperfused completely. In addition, the IVT-associated ICH appeared not to bespace-occupying.So far, there is one case report about a successful MT in a 75-year-old patient withMCA occlusion and pre-interventional IVT-associated ICH. and might be due to several factors. First, the median age of the includedpatients was 80 years and all patients had arterial hypertension with increasedlikelihood of difficult vascular access resulting in longer procedure times, lowerrecanalization rates and poorer outcome. Second, half of the occluded vessels were localised in the posteriorcirculation. A recent study reported that futile recanalization occurred morefrequently in BA occlusions, and predictors of futile recanalization included age,stroke severity, manoeuvre count and intracranial stenting.The rate of successful reperfusion in our study was lower compared to the current literature It remains unclear whether the devastating clinical outcome in our study wascaused by the LVO or the ICH itself. Since the ICHs were space-occupying in themajority of cases, it might have been attributed most likely to the neurologicalaggravation. However, it has to be mentioned that two of the patients were notreperfused successfully (TICI=2a each), which is also accompanied with poor clinical outcome. In contrast to the deceased, the survivor in our cohort suffered from an M1occlusion and a small parenchymal haemorrhage, which had resorbed completely atdischarge. Although two other patients also showed non-space-occupying ICHs onpre-interventional CT, the clinical outcome was poor due to underlying BA occlusionand an aggravation of the ICHs during the hospital stay. Although the presentednumber of patients is low, it is conceivable that the extent of baseline ICH andexpansion after MT might influence patients’ outcome.In our study, the mortality rate was high with 83% compared to patients with LVOreceiving IVT in the anterior circulation with rates up to 15% in the interventionalarm (MT and IVT) of the HERMES meta-analysis. On the other hand, high BP levels around reperfusion therapy carry anincreased risk of ICH. In the status of vessel occlusion, low BP levels may lead to hypoperfusion ofischaemic tissue resulting in greater infarction. In the unlikely event of both, ICH and simultaneous cerebral LVO, the optimaltarget BP remains indeterminable, since both variations provoke poor neurologicaloutcome.In this context, blood pressure (BP) control might be one important factor affectingthe outcome. On the one hand, moderately elevated BP is associated with goodcollateral circulation in AIS patients. The study showed that there was approximately a doubling of the odds of ICHwith the presence of a visible acute cerebral ischaemic lesion on pretreatment brain imaging. Some of these baseline factors are also reflected in our cohort regardinghigh median age, high median baseline NIHSS, a median ASPECTS=8 on baseline imaging,previous antiplatelet agents and comorbidities. From this point of view, it might bereasonable in this subgroup of patients, and more particularly in patients treatedby the ‘drip and ship’ paradigm, to enforce a flat detector CT pre-interventionallyin case of deterioration of the patients’ status.In a meta-analysis risk factors for ICH in AIS patients treated with IVT wereidentified including higher age, higher stroke severity and higher glucose level.The underlying cause for the IVT-related ICH was undetermined in most cases. Thismight be due to the fact, that in most patients advanced imaging includingpost-interventional magnet resonance imaging has not been executed.In patients with LVO based on an intracranial atherosclerotic disease the treatmentof the underlying stenosis is challenging. In our study, one individual sufferedfrom a slight pre-interventional SAH and an acute high-grade stenosis of the BA. Atthis point, the risk of progressive bleeding due to the necessity of potentantiplatelet medication must be weighed against re-occlusion of the vessel. Thepatient in our study was treated with intracranial stenting and peri-interventionaladministration of tirofiban, which led to a massive intracranial haemorrhage. Inthese specific cases, alternatively a percutaneous transluminal balloon angioplastymight be a reasonable treatment option with waiving of glycoprotein IIB/IIIAinhibitors administration.The main limitation of our study is the retrospective nature including inherentselection bias and the usage of different thrombectomy equipment and techniques. Thelack of a control group is a further limitation. As highlighted previously, thepresented number of patients is too low to draw definitive conclusions, especiallyas half of the patients suffered from BA occlusion, which by itself is accompaniedwith a poorer outcome compared to anterior circulation strokes and might serve as apossible confounder. However, this multicentre study includes the largest number ofendovascularly treated LVOs following IVT-associated ICHs in the literature sofar.Mechanical thrombectomy in patients with IVT-associated ICH is technically feasible.The clinical outcome of these patients appears to be devastating with high mortalityand only carefully selected patients might benefit from endovascular treatment.However, until we have data from larger trials, it will always be an individualdecision but, finally, it should be the goal to not leave individuals behind thatmight benefit from endovascular treatment in this particular situation.


Epigenetic changes of lung adenocarcinoma (LUAD) have been reported to be a relevant factor in tumorigenesis and cancer progression. However, the molecular mechanisms responsible for DNA methylation patterns in the tumor immune-infiltrating microenvironment and in cancer immunotherapy remain unclear.We conducted a global analysis of the DNA methylation modification pattern (DMP) and immune cell-infiltrating characteristics of LUAD patients based on 21 DNA methylation regulators. A DNA methylation score (DMS) system was constructed to quantify the DMP model in each patient and estimate their potential benefit from immunotherapy.Two DNA methylation modification patterns able to distinctly characterize the immune microenvironment characterization were identified among 513 LUAD samples. A lower DMS, characterized by increased CTLA-4/PD-1/L1 gene expression, greater methylation modifications, and tumor mutation burden, characterized a noninflamed phenotype with worse survival. A higher DMS, characterized by decreased methylation modification, a greater stromal-relevant response, and immune hyperactivation, characterized an inflamed phenotype with better prognosis. Moreover, a lower DMS indicated an increased mutation load and exhibited a poor immunotherapeutic response in the anti-CTLA-4/PD-1/PD-L1 cohort.Evaluating the DNA methylation modification pattern of LUAD patients could enhance our understanding of the features of tumor microenvironment characterization and may promote more favorable immunotherapy strategies. S-adenyl methionine to the C5 position of a cytosine residue to form 5-methylcytosine at CpG sites  is the most common form of lung cancer and is responsible for the cancer deaths worldwide . HoweverIn this study, we collected and integrated the clinical information and genomic data of 513 patients from The Cancer Genome Atlas (TCGA) LUAD cohort and comprehensively evaluated the TME characteristics represented by distinct patterns of DNA methylation modifications. We finally identified two independent DNA methylation patterns (DMPs) by unsupervised consensus clustering the expression of 21 DNA methylation regulator-related genes, which we defined as methylation regulators. The immune cell-infiltrating properties of two DMPs were highly consistent with immune-noninflamed or immune-inflamed phenotype, respectively. We then constructed a DNA methylation score (DMS) system to investigate the efficacy of DNA methylation modification patterns in individual patients and estimated their immunotherapeutic value in several clinical trials.The workflow of this study is presented in  were extracted for DNA methylation modification pattern identification . The clup-values <0.05 were considered statistically significant for GSVA analysis. Gene ontology (GO) and pathway annotation for DNA methylation pattern-related genes were performed using the R package “clusterProfiler” with a cutoff value of p < 0.05.We performed Gene-Set Variation Analysis (GSVA) enrichment analysis with “GSVA” R packages to study the differed biological pathway between different DNA methylation modification patterns in cancer samples . The Halhttps://cibersort.stanford.edu/) using the gene expression profile of LUAD cancer  was implemented to determine variations in immune leukocyte subtype abundance between different DMP clusters using the R package “GSEAbase.” Subsequently, the abundances of 22 immune cell types for each tumor specimen were further identified by estimating relative subsets of RNA transcripts , where i stood for the relevant gene expression of the selected set  to identify the DNA methylation modification-related differentially expressed genes (DEGs) across distinct DMP phenotypes. Univariate Cox model analysis was performed to calculate their association with overall survival and to extract prognostic DEGs to construct a scoring system. We then conducted principal component analysis (PCA) using the identified DEG prognostic genes, and PCA 1–2 components were selected to act as signature scores to construct DNA methylation-relevant gene signature, which we defined as the DMS. This method mainly focused on evaluating the score for each patient in the dataset with the largest group of well-correlated (or anticorrelated) genes. We constructed the DMS using an algorithm similar to the GGI: DMS = Σ, which is a publicly available dataset containing corresponding clinical pathology information . The immIn this study, 21 DNA methylation regulators were collected from the published data. To determine the pattern of DNA methylation modification mediated by 21 regulators, the TCGA-LUAD cohort and their corresponding clinical data were prepared for analysis  via univariate Cox model analysis , demonstrating the crosstalk between the DMS and genetic mutation evaluation  phenotype, which corresponded to a worser survival prognosis. In contrast, the DMP-B–clustered individuals presented with hypomethylation was characterized by the activation of abundant immune effector/pathways and the presence of multiple immune cell infiltrations in the TME, which exhibited an immune-inflamed phenomenon, corresponding to significant survival benefits. Analysis of the tumor immunophenotype in patients with solid tumors has also widely supported the view that the immune environment plays a central role in the pathogenesis of tumorigenesis and metastasis and harbored important clinical implications for effective immunotherapy outcomes , 31–33. To further elucidate the characteristics of transcriptome traits in DMP, the DEGs of distinct DNA methylation modification patterns were obtained and selected as the DNA methylation signature genes, which were concurrently found to be significantly associated with the cell cycle and DNA replication biological behaviors. Based on the mRNA expression levels of these DEGs, three DNA methylation gene clusters with different survival prognosis and immune-infiltrating features were identified, indicating there were indeed different clinical immune phenotypes associated with LUAD , 41. In Consistent with the previous analysis, the DMS, which was negatively correlated with TMB, was found to be markedly enriched in the immune activation and stromal-relevant pathways, underlining the pivotal role of stromal immune activation in resistance to immune checkpoint therapeutics , 44. OurHowever, there were some limitations in this study. Firstly, we used only 21 DNA methylase-related regulators to construct the model system; newer methylation regulators should be included in further analyses. Second, additional clinical data are needed to validate the efficacy of the immunotherapy and to evaluate the accuracy of the DMP and DMS system.We identified two DNA methylation modification patterns in LUAD patients based on 21 methylase-related regulators and constructed a methylation profile score model for individual patients. The TME disparity between the distinct DMPs highlighted the potential complexity and heterogeneity of LUAD formation, casting light on multiple processes fueling tumor evolution, immune infiltration, and drug resistance. The systematic analysis of the immune and/or clinical characteristics in the DMP and DMS will contribute to enhance a deeper understanding of the tumor immune-infiltrating microenvironment and will promote the development of effectively targeted immunotherapies.The original contributions presented in the study are included in the article/DY designed the experiment, analyzed the data, drafted the manuscript, and coordinated the study. ZW revised the paper. YW, FC, LY, and SZ performed the table and figure processing. RT and JL supervised the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Panicum miliaceum) in Mesopotamia, identified using phytoliths in dung-rich sediments from Khani Masi, a mid-second millennium BCE site located in northern Iraq. Taphonomic factors associated with the region’s agro-pastoral systems have likely made millet challenging to recognize using conventional macrobotanical analyses, and millet may therefore have been more widespread and cultivated much earlier in Mesopotamia than is currently recognized. The evidence for pastoral-related multi-cropping in Bronze Age Mesopotamia provides an antecedent to first millennium BCE agricultural intensification and ties Mesopotamia into our rapidly evolving understanding of early Eurasian food globalization.Multi-cropping was vital for provisioning large population centers across ancient Eurasia. In Southwest Asia, multi-cropping, in which grain, fodder, or forage could be reliably cultivated during dry summer months, only became possible with the translocation of summer grains, like millet, from Africa and East Asia. Despite some textual sources suggesting millet cultivation as early as the third millennium BCE, the absence of robust archaeobotanical evidence for millet in semi-arid Mesopotamia (ancient Iraq) has led most archaeologists to conclude that millet was only grown in the region after the mid-first millennium BCE introduction of massive, state-sponsored irrigation systems. Here, we present the earliest micro-botanical evidence of the summer grain broomcorn millet ( Similar to the contemporary world, this form of agricultural production was vital for provisioning large urban centers and financing imperial ambitions across ancient Eurasia4. For millennia, grain production in western Eurasia was limited to winter cereals, primarily wheat and barley, both of which were locally domesticated and adapted to Southwest Asia’s Mediterranean climate, with cool, wet winters and hot, dry summers. Summer grains and their wild relatives are not native to the region and thus summer cultivation only became possible with the translocation of millets and other East Asian and African domesticates.Multi-cropping, defined here as the seasonally sequential production of multiple crops on the same land in the same year, is an agricultural technique aimed at diversifying and intensifying economic and subsistence-based food, fodder, and forage production6. Their short life cycle, drought tolerance, minimal maintenance, high returns, and protein-rich grains make millets versatile, nutritious, and labor-effective food sources for both people and livestock9. Two of the East Asian millets, broomcorn (Panicum miliaceum) and foxtail  millet, were likely transported to western Eurasia both across the continent through Central Asia and along maritime trade routes , in particular, would eventually become one of the most important cereal grains in ancient Eurasia4.Millets represent a variety of fast-growing, small-seeded summer grains, initially domesticated in both Africa and northern China. Millets require summer rainfall (May–October > 120 mm) or irrigation14. Although domesticated millet is found throughout Central and South Asia and as far west as eastern Europe, cultivation of the crop is thought to be mainly restricted to areas with sufficient summer precipitation  is a very minor cultivar in Iraq16 and rarely grows ferally even in perennially damp places17. As a result, most archaeologists believe millet was only introduced to Mesopotamia (ancient Iraq) and other areas that lack summer precipitation with the construction of massive, state-sponsored irrigation systems during the mid-first millennium BCE, which would have made multi-cropping possible and worthwhile18. In contrast, textual evidence suggests millet may have been cultivated in Mesopotamia as early as the third millennium BCE, possibly being introduced via maritime routes from the Indus Valley19 or overland via expanding Bronze Age trade networks22.By the mid-second millennium BCE, long-distance exchange networks connected all of Eurasia marking the near completion of the “Trans-Eurasian Exchange” in which East Asian domesticates arrive in Southwest Asia and Europe and wheat and barley reach East Asia26. The Akkadian term for millet (duḫnu/tuḫnu) is first explicitly mentioned in mid-second millennium BCE cuneiform texts, found at the Mesopotamian sites of Nippur and Nuzi27. Texts from Nuzi suggest that millet was planted in conjunction with sesame: “plant sesame and millet! there is one homer of sesame and millet which is already planted,” or gifted as grain along with barley: “PN [personal name] gave to PN2, PN3, and PN4, four homers of barley and two homers of millet as…for the properties27.” Some scholars link duḫnu/tuḫnu to the Akkadian word arsikku (Sumerian: ar zig), possibly pushing references to millet back into the third millennium BCE28. Old Akkadian (third millennium BCE) texts also refer generically to both “early grain”  and “late grain” , which was sown in spring and harvested in late summer and thus may refer to either millet or sesame30. These are the only known summer-sown “grains,” and certainly all the technology needed for their cultivation would have been available long before the second millennium BCE31.Mesopotamian textual specialists have long argued that millet was an important crop in the region as early as the third millennium BCE32, and sesame, also a summer crop, is a known cultivar in the region from the third millennium BCE onwards34. However, millet is rarely mentioned in discussions of early Mesopotamian agriculture35 and is generally excluded from models of Bronze Age Mesopotamian food production37.Textual evidence contrasts sharply with archaeological perspectives that largely ignore the possibility or implications of millet multi-cropping in Mesopotamia prior to the first millennium BCE. Some archaeologists do not doubt that millet was also present in semi-arid Mesopotamia during the second millennium BCE because it is contemporaneously present in adjacent areas with more temperate climates38 and, until recently, its general scarcity in adjacent regions. The earliest unequivocal archaeobotanical evidence for broomcorn millet in Mesopotamia is from c. 700 BCE when millet grains are found in large numbers at Nimrud and Fort Shalmenesar  and it was never used prior to the large-scale Neo-Assyrian imperial intensification systems, even as a diversification or risk reduction strategy . Likewise, Rosenzweig18 credits the Neo-Assyrians and their agricultural maximization policies with the introduction of millet and other non-local crops to northern Mesopotamia.The contradiction between textual and archaeological perspectives is due to the nearly complete absence of archaeobotanical evidence for millet in Iraq in all periodssar Fig. 39. CitinPanicum miliaceum may be present in impressions of millet grains on ceramics from the site of Jemdet Nasr (ancient Kish) in southern Iraq dating to 3000 BCE43, but the interpretation of botanical impressions has been argued to be unreliable44. A few charred Panicum grains were reportedly found inside a small jar from the same site45, and a single grain of P. miliaceum was identified from a secure Late Bronze Age (14th–13th cent. BCE) oven context at Gurga Chiya in the Shahrizor plain in northeastern Iraq46. In northern Syria, isolated grains of P. miliaceum are also reported in second millennium BCE contexts at Tell Sheikh Hamad48 and Tell Mozan in northeast Syria49; although the Tell Mozan finds are not mentioned in the final report50. The rarity of millets in archaeobotanical data from Bronze Age Mesopotamia has led archaeologists to interpret these finds as exotic imports, intrusive grains, or very minor cultivars, and thus millet has played almost no role in our interpretations of agro-pastoral production in the region.However, archaeobotanical remains are not entirely absent prior to the first millennium BCE. Earlier evidence for 54. At this time, Khani Masi appears to have close cultural and economic ties to Kassite Babylonia, centered in southern Mesopotamia. Excavations have revealed a sequence of major construction episodes, with the earliest phases dated to around 1450 BCE and subsequent building phases during the fourteenth and thirteenth centuries BCE. Settlement at SRP 46 ended following the abandonment of an extensive baked mudbrick building complex around 1100 BCE, and there is no evidence that this part of the site was ever reoccupied51.This study presents new data from the site of Khani Masi, located along the Upper Diyala/Sirwan River, a tributary of the Tigris River, in the Kurdistan Region of Iraq Fig. a. Khani 55, and regional paleoclimate data suggest that a similar climate system prevailed during the second millennium BCE  , is replete with perennial, spring-fed streams, supplied by groundwater originating in the Zagros Mountains to the northeast. This well-watered plain has a rich history of human occupation dating back to the Neolithic period58, and thus it is reasonable to conclude that irrigation was likely practiced for many millennia.The region in which Khani Masi is located has a typical Mesopotamian steppe climate (Irano-Turanian vegetation), with cool, wet winters and hot, dry summersBCE Fig. b56. Toda62), but have rarely been studied in detail in Mesopotamia64. Abundant ceramics and AMS carbon-14 samples securely date the deposit to the mid-late second millennium BCE , we encountered phytoliths in anatomical connection, or multicellular structures , composed of interdigitating phytolith morphotypes69 similar to Panicum miliaceum in ten samples , while samples 67, 73, and 75 yielded only a single interdigitating multicellular structure each. Providing context to these four samples, samples 59, 73, and 75 are from burned animal dung-rich deposits while sample 67 is from an outdoor surface (Supplementary Text and Table 54. Phytolith morphotype results showed that riparian vegetation like sedges (Cyperaceae) were rare (< 2.1%)  vegetation like millet, represent 30% of the short cell assemblage in sample 59 (the most of any sample at Khani Masi) and between 9.3 and 10.4% in samples 67, 73, and 75  from other domesticated millets74, P. miliaceum and other domesticated millets share several features with their wild relatives and other weedy Panicoideae (C4) grasses76. For example, the Panicum species, Panicum bisulcatum (Japanese panicgrass) and Panicum repens (torpedo grass), are morphometrically very similar to P. miliaceum and can only be distinguished from broomcorn millet based on a single criterion78. P. bisulcatum is not native to Iraq, but P. repens is native and present in riparian areas80. In fact, five of the nine genera representing millets and their wild relatives81 are present in modern Iraq   Tzvelev, the debated progenitor, feral relative, or weedy companion of domesticated P. miliaceum78. Panicum ruderale is not native to Iraq and thus its presence would also indicate human translocation.Using five criteria, Panicum miliaceum)  for a total of 90 individually measured phytoliths. Note that partial or broken morphotypes at the edges of the multicellular structures were not measured. 28 of the 30 interdigitating multicellular structures meet five criteria and 19 meet all six criteria , and the process height to body ratio is greater than 1 (criteria 6). 13 multicellular structures have distinctly finger-type endings, and 14 have either both ending types or intermediate appearances (criteria 3). The overall averages for both long cell (elongate) ending lengths  and the ratio of process height to ending length  are within one standard deviation of the values reported by Lu et al.77 for P. miliaceum. All measurements of both the individual and multicellular interdigitating phytoliths exceed the minimum required sample size for ensuring means are within 5% of actual population means at a 90% confidence level (Supplementary Table 88.This study identified 30 phytolith multicellular structures (silica skeletons), with characteristics consistent with the inflorescence bracts of broomcorn millet (m) Figs. . Multiceinterdigitating inflorescence phytoliths, only Paniucm repens (torpedo grass) shares four of the same diagnostic criteria with Panicum miliaceum. P. repens only produces short, ‘wavy-type’ endings, whereas P. miliaceum mostly produces ‘finger-type’ endings (criteria 3)78. Nineteen interdigitating multicellular structures have finger or both finger- and wavy-type endings making them potentially distinct from P. repens. However, cautiously using the stricter criteria of exclusively ‘finger-type’ endings, eleven multicellular structures are securely identified as P. miliaceum  is not well studied from a phytolith perspective, although it does not appear to resemble the strict criteria for P. miliaceum90. Panicum turgidum is also adapted to and moderately present only in the sandy desert regions in the extreme southeast of Iraq (Saharo-Sindian), and is not expected to grow in the moister Khani Masi region91. Future research should focus on generating accessible phytolith references for the wild grasses of Iraq and the greater Mesopotamian region.It is possible that the Panicum miliaceum) is present at the mid-second millennium BCE site Khani Masi located along the Upper Diyala/Sirwan River in northern Iraq. This result represents the first phytolith identification of P. miliaceum from ancient Iraq and aligns with both contemporary textual sources and some regional macrobotanical evidence that suggests millet was present in Mesopotamia at this time.Our results demonstrate that broomcorn millet arid in terms of precipitation, it contains an abundance of perennial water sources that could support summer cereal cultivation without major investments in irrigation. As further articulated below, our results suggest that the absence of evidence for millet in previous investigations is due to the particularly strong taphonomic bias against millet grain preservation as well as to the low archaeological visibility of pastoral lifeways. Thus, millet cultivation was likely far more widespread in the second millennium BCE than is currently recognized. Furthermore, the availability of millet as an alternative food source for people or animals requires the reassessment of Bronze Age models of urban provisioning, resilience, and human-environment interactions.95 meaning it had been practiced for millennia by the second millennium BCE. That sesame is cultivated in the region from the third millennium BCE onwards further emphasizes that Mesopotamian communities were familiar with summer crops and were versed in small-scale summer cultivation. Thus, while the local seasonal climate may be unfavorable for precipitation-based summer cultivation, river discharge rates during April–June suggest that preexisting irrigation infrastructure could easily have been used to irrigate millet sown as a cover crop in river flood control areas or in fallow fields96  has a long history of irrigation and a myriad of perennial water sources, particularly in the ecologically diverse Zagros foothills zone, and the cultivation of millet in the region should be unsurprising. Irrigation technology was developed in the region during the sixth–fifth millennium BCEsee Fig. c. SummerTwo alternative scenarios, although unlikely, could explain the consumption of millet by animals at Khani Masi without local cultivation: (a) the feral growth of the domesticated grain near perennial water sources or (b) the long-distance transport of unhulled grains from their natural growing range.Panicum miliaceum around nearby perennial water sources is unlikely but cannot be completely ruled out. Feral growth requires that broomcorn millet was introduced into the region before the midden was formed at Khani Masi, during late third–early second millennium BCE. If this was the case, the implication is that domesticated Broomcorn millet was still known and actively used for animal forage at this time, centuries earlier than previously accepted.First, the feral growth of domesticated 52. Yet, it is unlikely that sufficient amounts of unhulled millet grains would have been transported hundreds of kilometers from the Taurus-Zagros mountains or from maritime ports in lowland Mesopotamia only to be used as fodder for local sheep-goats . For millet, macrobotanical finds are remarkably rare in all periods, even in periods when millet is intensively cultivated38. Millet finds from Southwest Asia seem to be restricted to a single grain for an entire site (e.g.107) or large caches recovered under exceptional preservation conditions such as roof storage collapse from catastrophic fires ; or in jars with tar (Nimrud39).In Southwest Asia, preservation conditions for macrobotanicals can be poor, especially in shallower sites, and the ubiquities of even the major crops, wheat and barley, can sometimes be too low for meaningful statistical analysis (e.g.Sesamum indicum L.), another small-grained, oil-rich summer plant translocated into Mesopotamia. Like millet, sesame seeds are also textually attested but exceedingly rare in the archaeobotanical record29. Their small size and high oil content make carbonized sesame seeds extremely fragile and prone to disintegrate during the recovery process. Further, their relatively small quantities and processing in off-site areas make them less likely to enter the archaeological record in the first place108. Consequently, no sesame grains are attested in Mesopotamia for the nearly 1000 years between the earliest grains recovered ca. 2300 BCE  and those dating to the late second millennium BCE109. Where data are published, sesame finds, too, are restricted to very few grains or large caches29. However, like millet, recent proteomic, residue, and microbotanical approaches are demonstrating that sesame and other exotic plants were more widespread in second millennium BCE Southwest Asia than previously thought111.In many ways, the archaeobotanical record for millet mirrors that of sesame . In contrast to sesame, millet phytoliths at Khani Masi were primarily recovered from burned and discarded dung-rich sediment that suggests introduction via animal dung116. Third, because dung in ancient settlements is often used as fuel or burned to reduce the volume of dung accumulating in animal penning areas, any fragile millet grains that survive digestion would be subsequently destroyed through burning. Finally, discarded organic rich dung and ashes often decay after deposition (diagenesis) further destroying organic macrobotanical evidence114.Dung-associated plant material is subjected to additional destructive processes that decrease the likelihood of grain identification from macrobotanicals. First, dung is most likely to enter the archaeological record through fuel use and animal penning . Second, unlike wild seeds, which are abundant in ruminant animal dung, domesticated cereal grains are starchy or oil-rich with thin protective outer coatings and rarely survive sheep and goat digestion77. Microbotanical and geochemical approaches , which can effectively identify animal dung and its contents, have not yet been widely used in the region. This study demonstrates, however, that millet was cultivated in Mesopotamia and that phytolith analyses of dung deposits are likely key for investigating the role of forage and fodder in the advent of regional multi-cropping.The strong taphonomic bias against millet grain preservation means that this grain has been below our ability to resolve using traditional macrobotanical methods118), acknowledging that local pastoral systems and their specific practices, level of mobility, and integration into agricultural systems vary widely. Millet grown as a forage crop would be directly consumed by animals, not harvested. The pastoral origins of multi-cropping in Mesopotamia complement multiple botanical and isotopic studies from across Central and South Asia that also suggest millet was adopted slowly, through bottom-up, pastoral initiatives123. Millet’s low investment, high return qualities made it especially well-suited to the needs of the semi-mobile pastoralists who transported it across Central Asia’s ecologically diverse landscapes124. It is fitting then that this new crop may have been first adopted by pastoralists living in the environmentally complex Mesopotamian-Zagros interface.The recovery of millet from animal dung—consumed as a forage crop—suggests that the initial practice of multi-cropping in Mesopotamia is likely associated with small-scale pastoral diversification strategies—not imperial agricultural mandates. Pastoral, here, is defined broadly as the husbandry of sheep-goats  socio-political and economic systems127. In both cases, the possible pastoral origins of multi-cropping highlight the influence of steppe region pastoral practice on the political and land use histories of Southwest Asia. Many Southwest Asian crops and animals were first domesticated in the Zagros foothills (“hilly flanks”)129, and this study suggests that the Zagros foothills may have continued to be a regional center of agro-pastoral innovation for Mesopotamia during the Bronze Age.Pastoral practice outside of institutional spheres has been a topic of intense debate in Mesopotamian archaeology because it is not well documented in Mesopotamia’s archaeological or textual records36 attribute the “decoupling” of urban site size (and population) with climate trends after 2000 BCE and urban size with sustaining area after 1200 BCE to changes in labor organization, taxation, and integration into long-distance trade networks. However, like most models of Mesopotamian economies, they have not yet explicitly considered the impact of new crops37. However, this decoupling of demographic and environmental variables coincides with the arrival of new crops with properties optimally suited to diversifying and strengthening the resilience of Mesopotamia’s agro-pastoral production systems. Even a low-level or opportunistic cultivation of millet, for human or animal consumption, may have had a significant impact on urban provisioning and thus resilience capacity130. Future studies could further investigate the origins of multi-cropping by investigating isotopic δ13C enrichment from low-level millet (C4) consumption and by deploying microscopic methods that acknowledge the taphonomic biases against millet grain preservation. Perhaps uncoincidentally, evidence of millet cultivation is nearly as rare as studies using isotopic131 and phytolith approaches54 with potentially critical impacts on our understanding of Mesopotamia’s social, political, and economic systems.Beyond the Zagros Region, the adoption of millet likely had far-reaching impacts on Mesopotamia’s social, political, and economic systems beginning in the second millennium BCE. The verified presence of millet in mid-second millennium BCE Mesopotamia sheds new light on historical events and trajectories of the region and requires a reassessment of models of urban provisioning, resilience, and human–environment interactions. For example, Lawrence et al.Panicum miliaceum) in Mesopotamia (ancient Iraq) and suggest that the origins of multi-cropping (summer cultivation) begin in the second millennium BCE. This finding aligns with ongoing investigations of early food globalization across Eurasia, a conversation in which Mesopotamia has been notably absent. As in other regions, the initial use of millet in Mesopotamia was likely as a foraging crop. Agro-pastoralists in the Zagros-Mesopotamian interface may have grown millet opportunistically at low levels for centuries as a diversification strategy132 before it was considered food suitable for human consumption or economically advantageous93 to the political economies within the first millennium BCE Neo-Assyrian Empire. Strong taphonomic bias against millet grain preservation provides an explanation for why its recovery has been so rare despite its known presence in textual sources. Micro-remain analysis offers a promising path forward for exploring the processes and practice of multi-cropping in Mesopotamia. In fact, this study highlights that micro-remain analyses have the potential to fundamentally transform our understanding of daily life, the formation of states and empires, and human-environment relationships in one of the most prominent and strategic nodes of ancient Eurasian and African networks.Here we provide the earliest microbotanical evidence of broomcorn millet (2) separated by a 0.5 m baulk were excavated in area Y82 at Khani Masi (SRP46) by the Sirwan Regional Project (SRP). Charcoal samples for 14C dating were collected during excavation and analyzed at the University of Arizona AMS Laboratory. 14C date ranges were calibrated using OxCal v4.4.4  were collected in plastic bags directly from the freshly cleaned baulk section, and sampling tools were cleaned with acetone between every sample. Sample locations were tagged, photographed, and geolocated using an Emlid RS + RTK GNSS system.Two adjacent trenches  when possible67.Phytoliths were extracted using the Katz et al.82  using the morphometric criteria defined bySupplementary Information.


We study the vulnerabilityof single-molecule nanowires againsta temporary disconnection of the junction. To this end, we comparethe room and low-temperature junction formation trajectories alongthe opening and closing of gold–4,4′-bipyridine–goldsingle-molecule nanowires. In the low-temperature measurements, thecross-correlations between the opening and subsequent closing conductancetraces demonstrate a strong structural memory effect: around halfof the molecular opening traces exhibit similar, statistically dependentmolecular features as the junction is closed again. This means thatthe junction stays rigid and the molecule remains protruding fromone electrode even after the rupture of the junction, and therefore,the same single-molecule junction can be reestablished if the electrodesare closed again. In the room-temperature measurements, however, weakopening–closing correlations are found, indicating a significantrearrangement of the junction after the rupture and the related lossof structural memory effects. Upon further pulling, the moleculeslides to the apex and only the linkers couple to the electrodes,yielding a decreased junction conductance (LowG configuration). Here,we study the stability of these molecular arrangements investigatingthe two possible scenarios in T = 4.2 K).BP molecules canattach to gold electrodes in two different bindinggeometries, resulting in double-step molecular plateaus.6 the room-temperature opening traces yield a double-peakfeature −1 is the conductance quantum unit. Onthe other hand, the low-temperature measurements exhibit one dominantpeak at the LowG conductance and only a shoulder is observed aroundthe HighG conductance . The coexistence of the LowG peak in theopening and closing histogram indicates that the LowG configurationcan be recovered as the junction is closed again in a low-temperatureenvironment.The low-temperatureclosing histogram  confirm that molecular plateaus extendto longer displacement during the closing of the junction. Furthermore,these two-dimensional histograms reveal that at low temperature, asignificant number of traces are measured without molecular signatures;i.e., tunneling traces are mixed with molecular ones in both the openingand the closing direction. In contrast, the room-temperature measurementsdo not indicate significant number of tunneling traces in either direction,thus almost all opening and closing conductance traces exhibit molecularplateaus.The two-dimensionalconductance histograms .The above analysis shows that≈45% of the low-temperaturemolecular opening traces exhibit molecular plateaus along the closingprocess as well. In the following, we further analyze this subsetof the traces  is likely tobe reproduced on the subsequent closing trace. This reproducibilityis restricted to the molecular conductance range, and to the opening–closing trace pairs. Note that the strong correlations vanish abovethe HighG conductance range (see the vanishing correlations abovethe conductances of the enframed molecular region in r and the closing trace in the r + 1 opening/closingcycle (see the absence of strong correlations around the diagonalin At this point, it is important to emphasizethat opening/closing2D conductance histograms and average conductance traces 4A convey


Surgical resection is currently the only potentially curative option for patients with pancreatic adenocarcinoma (PDAC). However, 80% of patients are ineligible for surgery due to the presence of invasive disease or distant metastases at the time of diagnosis. Treatment strategies geared towards reclassifying these patients as surgical candidates by reducing metastatic burden represents a promising approach to improving long-term survival. We investiaged the efficacy of a photodynamic therapy (PDT) based treatment regimen at preventing the spread and reducing the burden of metastases in animal models of PDAC. We demonstrate that PDT in combination with the first-line chemotherapeutic nab-paclitaxel dramatically inhibits (up to 100%) the eventual development of metastases in models of early stage PDAC, and completely eliminates metastasis in 55% of animals with already established distant disease in late-stage models. Our findings suggest that PDT-based treatment regimens may enable reclassification of patients with previously inoperable disease as surgical candidates.Despite substantial drug development efforts, pancreatic adenocarcinoma (PDAC) remains a difficult disease to treat, and surgical resection is the only potentially curative option. Unfortunately, 80% of patients are ineligible for surgery due to the presence of invasive disease and/or distant metastases at the time of diagnosis. Treatment strategies geared towards reclassifying these patients as surgical candidates by reducing metastatic burden represents the most promising approach to improve long-term survival. We describe a photodynamic therapy (PDT) based approach that, in combination with the first-line chemotherapeutic nab-paclitaxel, effectively addresses distant metastases in three separate orthotopic PDAC models in immunodeficient mice. In addition to effectively controlling local tumor growth, PDT plus nab-paclitaxel primes the tumor to elicit systemic effects and reduce or abrogate metastases. This combination dramatically inhibits (up to 100%) the eventual development of metastases in models of early stage PDAC, and completely eliminates metastasis in 55% of animals with already established distant disease in late-stage models. Our findings suggest that this light activation process initiates local biological and/or physiological changes within the tumor microenvironment that can be leveraged to treat both localized and distant disease, and potentially reclassify patients with previously inoperable disease as surgical candidates. Pancreatic ductal adenocarcinoma (PDAC) continues to be a devastating disease, with a 5-year survival rate of 10% . DespiteHere, we present a verteporfin–photodynamic therapy (PDT) based approach that, when combined with nab-paclitaxel (NabP), effectively inhibits metastatic colonization and eliminates established distant metastases in early- and late-stage models of PDAC, respectively. Our findings suggest a neoadjuvant approach by which select patients with metastatic disease may qualify for surgical resection. In addition, this approach may prevent metastatic colonization in patients with earlier-stage disease. Given that the presence of confirmed metastases currently precludes surgical intervention, the anti-metastatic effects of combined PDT and NabP may offer a potentially curative surgical opportunity for this patient population.Unfortunately, due to the lack of screening technologies and asymptomatic nature of early-stage PDAC, approximately 60% of patients present with disease that has already metastasized . Only 10Photodynamic therapy utilizes the photochemical properties of small molecule photosensitizers to convert energy from visible or infrared light into locally cytotoxic reactive molecular species. Extensive work by our group and others has consistently demonstrated that PDT can increase intratumoral concentrations of chemotherapy ,9 and seAlthough PDT is delivered locally to primary tumors, it is known to induce a strong systemic response, and preclinical studies of PDT vaccines and tumor rechallenge models have consistently demonstrated the formation of an anti-tumor memory following treatment . PDT-basIn this study, we aim to separately investigate these possibilities using three mouse models of pancreatic cancer, each with a well-defined metastatic status at the time of treatment. We demonstrate that PDT in combination with nab-paclitaxel and gemcitabine (Gem) effectively inhibits the formation of metastases in an early-stage model of PDAC, and clears established distant metastatic colonies in a substantial fraction of animals in a late-stage model. Given its mild and well-characterized side effects and the desperate need for innovative therapies, this PDT-based treatment strategy represents a promising and rapidly translatable approach that may increase the proportion of patients eligible for surgical resection.AsPC-1 (metastatic cells derived from ascites) and MIA PaCa-2 (primary tumor derived) human pancreatic cancer cells were purchased from ATCC , cultured as per the manufacturer’s instructions, and confirmed negative for mycoplasma contamination. Visudyne  photosensitizer was a kind gift from QLT Inc . Nab-paclitaxel, comprising 90% human albumin and 10% active ingredient (paclitaxel), was obtained from Abraxis BioScience . Gemcitabine HCl was purchased from Sandoz Inc. .6 AsPC-1 or MIA PaCa-2 cells suspended in 50 µL of 50% Matrigel  were injected into the body of the pancreas. The incision was closed with 4–0 monofilament suture. Animals were monitored over the course of the experiment per study protocols. At the study endpoint, animals were sacrificed, and tumor tissue excised to record weight. Liver, iliac lymph nodes, and lungs were harvested to evaluate metastatic burden by qRT-PCR.All animal study protocols were approved by the Institutional Animal Care and Use Committee at the Massachusetts General Hospital. Orthotopic pancreatic tumors were initiated in 4–6 week-old male Swiss nude mice . The animal was anesthetized with ketamine/xylazine, and on its left side a 1 cm long incision was made laterally over the spleen. The pancreas was exposed by gently pulling the spleen out of the incision, and 1 × 10Treatment efficacy was evaluated based on tumor weight. For experiments in which treatment was initiated on day 9 post implantation, tumors were approximately 30–50 mg (3–4 mm diameter), and for those initiated on day 24, tumors were approximately 300 mg (~8 mm diameter). All treatments were administered via tail vein injection. For groups receiving PDT, one hour following the injection of Visudyne (0.25 mg/kg), the pancreas was exteriorized as during implantation. The tumor was illuminated with 690 nm laser light . All mice treated with PDT received only a single dose of PDT. Following PDT, the incision was closed with 4–0 sutures. For animals receiving chemotherapy, each cycle consisted of nab-paclitaxel (25 mg paclitaxel/kg per cycle) and/or Gemcitabine (100 mg Gemcitabine/kg per cycle). Chemotherapy was administered starting day 9 (30 min post-PDT if appropriate), and again every 4 days for a total of 4 cycles. Untreated control animals received phosphate buffered saline at the appropriate time point.A quantitative RT-PCR-based method (qRT-PCR) was used to detect human cancer cells in excised mouse samples of liver, iliac lymph nodes (containing surrounding muscle), and lungs. Tissue was immediately harvested from the sacrificed animal at the end of the experiment and snap frozen in liquid nitrogen. Frozen tissues were pulverized, and RNA was extracted using RNeasy Protect Mini Kits  according to the manufacturer’s instructions. Samples were reverse transcribed into cDNA, which served as PCR templates. Relative amount of human and mouse GAPDH genes were quantified using the cycle threshold obtained from qRT-PCR, and number of human cells per gram of tissue was calculated from a set of standard curves.All statistical analyses were carried out using appropriate tools in Graphpad Prism 8. Unless otherwise specified, tumor weights were plotted as mean with standard deviation error bars and analyzed by Kruskal–Wallis ANOVA with Dunn multiple comparisons. Metastases were plotted as medians and analyzed with Kruskal–Wallis ANOVA with Dunn multiple comparisons. Fisher’s exact test was used to analyze datasets comparing the presence or absence of metastases in mice following each treatment. Sample sizes are indicated in figure captions.p = 0.08), control of metastatic spread, as 30% of mice treated with this combination had detectable metastases. Similar results have been previously reported, wherein the addition of Gem to a NabP regimen did not result in any additional survival benefit in subcutaneous PDAC tumors [The MIA PaCa-2 (MP2) cell line is derived from a primary pancreatic tumor, and generally exhibits less aggressive phenotypes compared to ascites-derived AsPC-1 cells, including increased sensitivity to gemcitabine  and a slC tumors . This trGiven its ability to completely inhibit the metastatic potential of PDAC in our model of early-stage disease, we aimed to evaluate whether PDT + NabP had similar efficacy in more aggressive models of PDAC. Mice bearing AsPC-1 tumors were treated with either PDT, Gem, NabP, Gem + NabP, or PDT + NabP, on day 9 post-implantation A, at whiAt the day 24 endpoint, PDT- and Gem-treated mice did not exhibit a significant reduction in primary tumor size compared to untreated animals, whereas all NabP-based treatments resulted in significant and statistically similar control of primary tumors C. Gem + Given the efficacy of PDT + NabP in early-stage models of PDAC, we hypothesized that this combination may reduce metastatic burden in animals that already have established distant metastases. Since the majority of PDAC patients are diagnosed with stage IV disease, we simulated this scenario by initiating treatment on day 24 post-implantation of AsPC-1 cells A, when 9p = 0.17) in AsPC-1 mice  or medium term (60 days) experiments. However, the proportion of animals that developed metastases after 60 days decreased from 83% to 0% in MIA PaCa-2 mice F, and 83C-1 mice G when PDC-1 mice G. These Consistent with the most common anatomical sites of metastases observed clinically , tissue Several prior studies have reported “abscopal” effects of PDT in reducing metastases in immunocompetent models of cancer ,24. Of pWe hypothesize that these mechanisms are at least partially responsible for the significant cytoreduction of distant disease in the T-cell deficient animal model described here. In fact, the immunocompromised mice likely underestimate both the local and systemic immune-mediated effects of PDT + NabP against this immune-privileged disease. These exciting findings point to the inherent ability of light activated neoadjuvant priming to alter the tumor microenvironment in ways that have dramatic effects on distant disease, possibly via the release of diffusible cytokines or activation of the innate immune system. Additional studies investigating these mechanisms in immunocompetent mouse models are warranted and may inform rationally designed PDT and immune checkpoint combination therapies. Furthermore, an in-depth characterization of similar metastatic models using cell lines with a range of chemotherapy/PDT sensitivities and metastatic potentials may lead to a deeper understanding of the impact of PDT and chemotherapy on metastasis control.Although modern neoadjuvant chemotherapy regimens enable ~70% of borderline resectable patients to undergo resection , less thWe demonstrate that PDT combined with NabP completely eliminates metastases in over 50% of animals that have established, distant disease at the time of treatment in an immunocompromised mouse model. Two early phase clinical trials investigating PDT for patients with inoperable, locally advanced PDAC suggest that PDT does indeed have the potential to help downstage disease to a point where it is amenable for resection. In one case, a patient received a relatively low dose (20J) of Visudyne-based PDT without follow up chemotherapy, and was subsequently determined to have resectable disease and underwent an R0 Whipple’s pancreaticoduodenectomy . SimilarWhile PDT + NabP results in uniform reduction in primary tumor size in the late-stage model, 45% of animals still have a substantial metastatic burden comparable to those treated with NabP only . This diPancreatic cancer continues to be a debilitating disease, in part due to the advanced stage of disease at which most patients are diagnosed. Unfortunately, approximately 60% of patients present with metastasized disease and are ineligible for potentially curative resection. Our findings indicate that by capturing the priming effect of PDT combined with front line chemotherapy, established distant metastases are eliminated in a significant fraction of animals, and this combination controls primary tumor growth for at least 7 weeks post treatment. Given recent promising reports of PDT in early-stage clinical trials and the limited treatment options available for these patients, we believe that our current exciting findings suggest that PDT has the potential to complement the current standard of care and provide this vulnerable patient population with an expanded set of therapeutic options. We anticipate that in immunocompetent subjects, the effect of PDP would be further enhanced by the additional benefit of immune activation, leading to a more robust response.


The aim of the study was to analyze unrelated samples from the Rewa male population of Central India by targeting Y-short tandem repeats (STR) and then compare the results to previously published Y-STR haplotype data.A total of 181 unrelated male subjects from the Rewa population were genotyped for seventeen Y-STRs  by using an AmpFlSTR® Yfiler™ multiplex kit . The allele frequencies and forensic parameters were evaluated.A total of 111 distinct Y-STR alleles with corresponding frequencies ranging from 0.006 to 0.829 were identified. The gene diversity values ranged from 0.3092 at DYS437 to 0.8188 at DYS385b. The studied population showed a high level of haplotype diversity (0.9985) and discrimination capacity (0.927). A haplotype analysis was also conducted. Among the 181 unrelated male samples, 165 haplotypes and 153 unique haplotypes were found. Additionally, Rst (genetic distance) values were calculated using the analysis of molecular variance (AMOVA) for the studied population and for other 18 populations described in the literature. The Rst provides a convenient parameter for estimating the level of genetic differentiation from the microsatellite data. Based on these Rst values and using the multidimensional scaling plot, a neighbor-joining tree was constructed.The high values of haplotype diversity and discrimination capacity indicate a great potential for distinguishing between male individuals in the studied population. The present population data are expected to find their use in forensic caseworks and population genetics. According to the 2011 India Census, the total population size of Rewa is about 2.5 million; among them, there are 1.3 million males and 1.2 million females. The population density is 377 people per square kilometre area. Rewa was known as a princely state during the time of British Empire; today, its population is composed of various groups like tribes, casts, and communities living under various environmental conditions like forests, waterfalls, rivers, lush greenery, monuments, and forts Forensic laboratories across the globe are conducting Y-short tandem repeat (STR) typing for their routine casework analysis as well as population genetics studies , 2. Y-STSo far, only a limited number of genetic studies on Y-STRs among the Central Indian population have been reported. Hence, there is a need to conduct more studies on this population –15.The aim of the study was to analyze unrelated samples from the Rewa male population of Central India by targeting Y-short tandem repeats and then compare the results to previously published Y-STR haplotype data.Prior to conducting this study, ethical clearance had been obtained from the Ethical Committee of the Raksha Shakti University . A total number of 181 unrelated male individuals’ blood samples were collected randomly for the purpose of this study. All the samples belong to the population of Rewa, a state in Central India . PeriphePhenolchloroform extraction was performed to extract genomic DNA from whole blood samples (n=181). The extracted DNA samples were quantified using a Quantifiler® Duo DNA Quantification Kit  in accordance with the manufacturer’s protocol. Quantified DNAs were normalized to the concentration of 1.0 ng/μl and then taken for PCR amplification.DNA samples were subjected to the amplification by using seventeen Y-STR loci . PCR conditions were set as per the manufacturer’s instructions. The total volume of the PCR reaction mixture comprised 25 μl of reagents . The amplification was run on a GeneAmp PCR System 9700 Thermal Cycler  using an AmpFLSTR Yfiler™ PCR kit (Thermo Fisher Scientific). Positive and negative controls were also run throughout the system.The PCR products were size separated with the help of capillary electrophoresis using an ABI 3100 Genetic Analyzer  and size-characterized with the GeneScan 500 LIZ internal lane size standard (Thermo Fisher Scientific) as per the manufacturer’s recommended protocol. The GeneMapper ID-X Software Version 1.4 (Applied Biosystems) was used to determine the amplified fragments’ sizes. All alleles’ designations were based on a comparison with the allelic ladders provided by the AmpFLSTR Yfiler™ system (Thermo Fisher Scientific). The present study was carried out in accordance with the quality assurance standards recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) .Forensic and population genetic parameters were evaluated for seventeen Y-STRs. The gene diversities (GD) of the analysed 17 Y-STRs were calculated according to Nei and Tajima ; Uttar Pradesh, India [Afridi Pathan]; Punjab, India [Balmiki]; Gujarat, India [Bhil]; Andhra Pradesh, India [Brahmin]; Karnataka, India [Indian]; Madhya Pradesh, India [Gond]; Jharkhand, India [Indian]; Madhya Pradesh, India [Indian]; Tamil Nadu, India [Iyengar]; Madhya Pradesh, India [Kanyakubja Brahmin]; Maharashtra, India [Konkanastha Brahmin]; Tripura, India [Riang]; Tripura, India [Tripuri]; Malaysia [Indian]; Nepal [Newar]; Balochistan, Pakistan [Baloch]; and Singapore [Indian] , 22–28. Due to the absence of recombination process in the Y-STR chromosome markers, they are considered highly effective in studying the genetic diversity of male individuals in a population. In order to understand the origin of modern humans, it is important to conduct genetic studies of various populations. Although such studies have been reported from different regions of Central India, no population studies have been conducted and reported from the Rewa region.The present study was conducted with 181 unrelated male individuals of the Rewa population whose Y-STR markers were determined, processed, and then compared with published data on other populations for the genetic distance or genetic relationships. According to the results, the DC value (0.9985) was higher and these seventeen Y-STRs had more polymorphism and higher systematic efficiency in the Rewa populations; these parameters can be used as a useful forensic marker set. The GD value exceeded 0.5 in fourteen Y-STR loci, which showed a higher level of polymorphism. Along with that, three loci had GD values of less than 0.5; that indicated a lower level of polymorphism, which is less suitable for forensic purposes in this population.The pairwise comparison between the studied population and those reported by others demonstrate that the Rewa people have a close affinity to Madhya Pradesh, India [Gond], followed by Gujarat, India [Bhil] Malaysia [Indian], Punjab, India [Balmiki], Singapore [Indian], and Madhya Pradesh, India [Indian]. The studied population showed a greater genetic affinity with the population of Uttar Pradesh, India [Afridi Pathan]. As observed in the neighbor-joining tree, the Madhya Pradesh, India [Gond], Gujarat, India [Bhil] Malaysia [Indian], Punjab, India [Balmiki], and Madhya Pradesh, India [Indian] cluster belong to the same tree branch. The tree also revealed that in Central India, the populations of the same ethnic origin  belong to different clusters. The authors encourage other researchers to explore more genetic polymorphism in the Rewa population.An attempt has been made to better understand the genetic relationship between Rewa  and other neighbouring populations. To this end, 181 Rewa male individuals were genotyped using an AmpFlSTR® Yfiler™ multiplex kit. This is the first report that provides population-based data on genetic variations and Y-STR polymorphism in the Rewa population using seventeen Y-STR markers. Here, we also report on the 17 Y-STR loci allelic frequencies and haplotype distribution in the Rewa population, which exhibit a significant discrimination capability, which can be applied to forensic casework and population studies.


Clinicians have learnt valuable lessons throughout the COV-SARS-2 pandemic, many of which have produced solutions that we aim to continue to implement within the foreseeable future. Optimising patients’ surgical pathways to reduce the length of stay and complications is an area of particular importance, both for maximal utilisation of available resources and for reduction of the exposure of inpatient and elective patients to an increased risk of infection within healthcare facilities.The aim of this review was to investigate the possible implications of using low-pressure laparoscopic gynaecological surgery versus standard- or high-pressure pneumoperitoneum surgeries. The primary outcome was postoperative pain, with secondary outcomes including duration of surgery, length of inpatient stay and rate of complications.MEDLINE, Embase and Cochrane CENTRAL were searched from inception to December 2020. We searched for published randomised control trials comparing low-pressure laparoscopic surgery (≤8 mmHg) to at least one additional standardised pneumoperitoneum pressure (≥12 mmHg and/or ≥15 mmHg). A total of 203 studies were reviewed, five of which were included in this analysis. Studies comparing low-pressure laparoscopic surgery against gasless abdominal cavities were excluded.2 = 0%). Studies measuring postoperative pain using a numerical rating scale displayed significant pain reduction at all hours measured (p ≤ 0.01). The rate of intraoperative complications was 1% for all groups measured. Cumulative analysis of the duration of surgery did not differ significantly between groups (p = 0.99).The meta-analysis of the results was pooled and calculated within RevMan 5.0 software . Studies using a visual analogue scale (1-10) to compare low versus standard pneumoperitoneum pressures did not display a significant diminution of postoperative pain at ≤ 6 or 24 hours: -0.30  and -0.66 , respectively. Studies additionally demonstrated worse visualisation of the surgical field within the low-pressure group (risk ratio 10.31; 95% CI, 1.29-82.38 IThe pandemic has revealed new issues that must be addressed by clinicians to promote the safety of patients and the efficiency of inpatient stay. This review has paved the way for new possibilities and innovative approaches to address the issue of optimising patient surgical pathways; however, at present, we cannot give a firm justification for the use of low-pressure gynaecological laparoscopy. Reasons for this include the minimal reduction in pain scores between low, standard and high pneumoperitoneum pressures, leading to a mixture of statistically significant results, as well as a reduction in the visualisation of the surgical field and the small population sizes in the reviewed papers. Additional research is required to further explore the potential clinical benefits of gynaecological laparoscopy to ensure its effective ambulatory use within mainstream surgical operations. During the SARS-COV-2 pandemic, hospitals have been under extreme pressure, with their capacity having stretched beyond its limits. The pandemic has notably caused problems with inpatient hospital flow, leading to the stagnation of patients within wards. This poses an increased risk of the development of hospital-acquired infections .Clinicians have learnt valuable lessons throughout the duration of the pandemic, many of which have produced solutions that we aim to continue to implement in the foreseeable future. Optimising patients’ surgical pathways to reduce the length of stay and complications is an area of particular importance both for maximal utilisation of available resources and for the reduction of the exposure of inpatient and elective patients to an increased risk of infection within healthcare facilities .Laparoscopic surgery, particularly within gynaecological procedures, is frequently the most favourable surgical procedure, compared to open surgery. Laparoscopic surgery provides well-known advantages, including earlier patient mobilisation, reduced length of stay and reduced pain, alongside a decreased incidence of surgical site infections ,4.The Royal College of Obstetricians and Gynaecologists (RCOG) recommends that intra-abdominal pressure should be maintained between 12 and 15 mmHg to sustain a suitable surgical field of view, as well as to minimise ventilatory and haemodynamic risks to the patient . HoweverIn recent years, emerging research has tackled this issue by exploring the use of lower pneumoperitoneum pressures (≤8 mmHg) during surgical procedures. This research may be significant in the streamlining of lessons learned from the pandemic. Interestingly, Vlot et al. highlighted in 2013 that 85% of abdominal expansion during porcine laparoscopic surgery was achieved at a pneumoperitoneum pressure of 10 mmHg . A pertiThis systematic review thus delves into the literature available on low-pressure gynaecological laparoscopy versus standard and, in some cases, high pneumoperitoneum pressures, while evaluating both intra- and postoperative outcomes.MethodThe principles and results of this review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines .Search StrategyThe electronic databases MEDLINE, Embase and the Cochrane library were used to search all available studies from inception to December 2020 and to analyse the available material on postoperative pain within low-pressure laparoscopy. The keywords and index terms used included gynaecological laparoscopy, gynaecologic laparoscopy, laparoscopic hysterectomy, low pressure, pneumoperitoneum and insufflation. To increase search accuracy, a population, intervention, comparison and outcome (PICO) model was used, as follows: Patient: women, Intervention: low-pressure gynaecological laparoscopy, Comparison: standard-pressure gynaecological laparoscopy, Outcome: postoperative pain. The details of this search strategy can be found within the appendices under Tables Inclusion and Exclusion CriteriaOnly texts published in the English language were included. All records deemed eligible for assessment had their reference lists scrutinized to identify any additional citations to be included in the analysis that may not otherwise be extracted through the standardised literature search. One additional study was retrieved in this manner.Studies including multiple laparoscopy insufflation pressures were included; however, all records eligible for qualitative and quantitative analysis were required to include at least a comparison between a low-pressure and standard-pressure pneumoperitoneum. Studies that included both benign and malignant operations were included in this review.All studies analysed were required to include outcomes on postoperative pain; however, pain could be measured using different standardised methods. Differences were assessed on the basis of the overall mean pain scores between laparoscopy pressures and times assessed postoperatively. Our primary aim was to measure postoperative pain; secondary outcomes included estimated blood loss, duration of surgery, types of operations and rate of complications. An additional factor measured included the number of surgeons operating. This was deemed to be a relevant factor, as the inclusion of multiple surgeons within one study would aim to reduce operator bias, thus allowing the results to be more generalisable to the surgical population.All records of patients under the age of 18 were excluded. Additionally, studies measuring standard pneumoperitoneum pressures versus gasless abdomens were excluded. Cohort, case-control and case report studies were excluded, as was low-pressure non-gynaecological laparoscopy. Studies excluded based on full-text analysis can be found within the appendices under Table Data ExtractionTwo independent authors screened eligible studies by title, abstract and, when required, full text to see if they met the inclusion criteria for review. Any disagreements were concluded through arbitration with the senior author. For each trial assessed, information was collected regarding the study design, characteristics of participants, interventions used, comparisons made and postoperative outcomes assessed. Low-pressure insufflation pressures were set at ≤8 mmHg, standard insufflation pressures were set between 12 and 15 mmHg, and high insufflation pressures were set at ≥15 mmHg.2 measurements to determine heterogeneity calculated using RevMan5 software (version 5.3)  [For studies measuring postoperative pain on a visual analogue scale, the mean differences, standard deviations and 95% confidence intervals were pooled and calculated. Consistency IEngland) . SubgrouAnalysis of Study QualityThis review assessed five randomised control trials for quality using the risk of bias analysis tool created by the Cochrane Collaboration . AnalysiResultsA total of 203 studies were identified for possible inclusion after duplicates were removed. Of these, 179 studies did not meet the eligibility criteria based on their titles and abstract content. Through further analysis, 19 of the 24 remaining studies were found not to meet the criteria for analysis after full-text assessment. Finally, five studies were selected for discussion. Figure Five studies for a total of 380 patients were included in this review. These studies analysed pain, mainly shoulder tip and abdominal pain, up to 24-48 hours postoperatively. The characteristics of all included studies can be seen in Table Studies Implementing a Visual Analogue ScaleTopcu et al. performed a prospective randomized trial of 150 patients undergoing laparoscopic gynaecological surgery, who were split into three laparoscopic categories: low pressure (8 mmHg), standard pressure (12 mmHg) and high pressure (15 mmHg) . IndicatIn conjunction, Bogani et al. explored the implementation of low-pressure laparoscopy (8 mmHg) versus standard pneumoperitoneum pressure (12 mmHg) in 42 patients undergoing laparoscopic hysterectomies . IndicatTable 2 = 0%).Cumulative analysis of the two studies demonstrated worse visualisation of the surgical field within the low-pressure group  and 91 patients within the standard-pressure group (12 mmHg) and exploring their symptoms up to 48 hours postoperatively, using a numerical analogue scale instead of a visual analogue scale . The maiA 2017 pilot study by Sroussi et al. delved into this topic further by exploring the possibility of performing laparoscopic surgery at even lower pneumoperitoneum pressures (7 mmHg) than in previous literature with the use of the AirSeal® system. Thirty patients underwent gynaecological laparoscopy at pressures of 7 mmHg, and 30 further patients at 15 mmHg, for benign laparoscopic conditions . As in tTable Study Measuring Pain as Secondary OutcomeIn contrast, Kim et al. randomised 46 patients into low-pressure (8 mmHg) and standard insufflation pressure (13 mmHg) groups . IndicatDiscussionWithin this review, we found that all studies but one demonstrated at least a single reduction in postoperative pain by low-pressure laparoscopic surgery (≤8 mmHg) compared to standard-pressure laparoscopy (≥12 mmHg) at the postoperative times measured. However, Bogani et al. favoured follow-up times less than 12 hours postoperatively, in contrast to Topcu et al., who chose times significantly above 12 hours postoperatively, despite using the same mode of measurement ,21. The We observed a reduction in postoperative pain in low-pressure groups in studies using a visual analogue scale, but these results were not significant when particular studies using this analogue scale were combined within a meta-analysis. A significant reduction of pain in low-pressure groups was observed in all-time categories by studies measuring with a numerical analogue scale, but it was not feasible to conduct a meta-analysis to truly evaluate the results. Despite significant results, any reduction in pain scores between groups was minimal, and clinical descriptions of relative pain from severe to moderate, moderate to mild or mild to milder were not evaluated further within included studies, thus making it difficult to assess the results to determine the true reduction in pain between groups based solely on numerical values. Studies additionally displayed little difference in the duration of hospital stay and consumption of analgesics between groups, thus further reducing the amplitude of significance for the interpretation of pain scores alone.The results of our review are in concordance with those of previous reviews by Hua et al. and Raval et al., who also explored the effect of low-pressure laparoscopy cholecystectomies on postoperative pain ,25. In tConfounding variables to which could be attributed the fluctuating nature of reported pain scores are the initial trocar insertion pressure, time taken to deflate the abdomen to lower pressures, the irregular amplitude of intra-abdominal pressures during each procedure once sustained low or standard pneumoperitoneum pressure was achieved and, finally, degree of Trendelenburg tilt. Insufflator systems can control regulatory intra-abdominal pressures and flow rates; however, the rate of response can vary according to manufacture specifications ,27. One  In this review, we observed a low rate of complications and conversion to open surgery within low-pressure groups. However, the studies reviewed performed most surgical procedures for benign conditions; thus, increasing the use of low-pressure procedures for malignant conditions may demonstrate shortfalls in low-pressure, particularly when it is combined with a reduction in visual field. Furthermore, studies within this review contained low population sizes. As gynaecological complications tend to be uncommon in surgical procedures, future studies may warrant larger population sizes to detect any differences in complication rates between pressure groups. Overall, we believe that this review does not contain adequate statistical evidence to convincingly promote the use of low-pressure laparoscopic surgery.LimitationsThis review included five papers, consisting of a total of 380 participants. Although these studies provide novel insights into a previously unexplored area of research, the limited variety of papers exploring low-pressure gynaecological laparoscopy make it difficult to draw sufficient conclusions for application within the medical community. Many studies in this review did not measure pain further than 24 hours postoperatively. Future studies may consider measuring pain for longer than 24 hours to provide consistency in published results, making them suitable for analysis to identify any further benefits of low-pressure laparoscopy.Second, whilst the included studies evaluated pain on a numerical value, the studies did little to evaluate the clinical impact of pain. Albeit the differences in pain scores might have been significant at times, the included studies did little to evaluate the use of additional important outcomes such as prolonged narcotic use, delayed discharge or delayed mobility. Future studies may warrant a greater exploration of these outcomes, as a sole decrease in post-operative pain would not be substantial enough to employ the use of ambulatory low-pressure laparoscopy in routine practice.Furthermore, 60% of the included studies had only one surgical team performing all operations on participants. This may indicate an element of operator bias, as surgeons within the studies may be more experienced than the general cohort in performing low-pressure gynaecological operations, thus reducing the observed surgical failure rates and intra-abdominal complications, such as expected blood loss. A more diverse surgical team within future studies will help provide more consistent results that are more representative of the wider surgical community. If low-pressure laparoscopy were to be incorporated as an ambulatory approach to gynaecological operations, the justifications for this need to be explored much further.Despite these limitations, our study rigorously explored major databases to retrieve all available material and conduct a thorough analysis of this topic. We included a variety of randomised control trials comparing at least two surgical pneumoperitoneum pressures without regard to the date of publication or type of surgeries performed to enhance the validity and generalisability of the results.To conclude, this review cannot justify the current use of low-pressure gynaecological laparoscopy. Meta-analysis of studies using the visual analogue scale did not display a significant reduction in post-operative pain. However, studies using the numerical analogue scale did display significant results in all times measured, but a meta-analysis could not be conducted. Studies displayed worse visualisation of the abdominal cavity. The rate of secondary complications, duration of inpatient stay, estimated blood loss and duration of surgery were relatively similar between pressure groups. The studies included in this review were of sound quality and displayed a low risk of bias; thus, we conclude that this review is of a moderate to the high level of evidentiary quality.Following the resolving SARS-COV-2 pandemic, the medical community aims to harness the lessons learned regarding hospital inpatient management and infection control. Optimising surgical pathways can aid in streamlining patients to discharge in a timelier manner. Low-pressure laparoscopy may assist with this in the near future; however, the beneficial qualities needed to substantiate its use in routine gynaecological procedures are not currently proven. Additional research will be needed before employing low-pressure genealogical surgery as an ambulatory modality for surgeons.


Paramuricea clavata  and the red coral Corallium rubrum  have followed different trends in terms of size structure, but a similar progressive reduction in density and biomass. Concurrently, recurrent MHWs were observed in the area during the 2003–2018 study period, which may have hindered populations recovery. The studied octocorals play a unique habitat-forming role in the coralligenous assemblages (i.e. reefs endemic to the Mediterranean Sea home to approximately 10% of its species). Therefore, our results underpin the great risk that recurrent MHWs pose for the long-term integrity and functioning of these emblematic temperate reefs.Understanding the resilience of temperate reefs to climate change requires exploring the recovery capacity of their habitat-forming species from recurrent marine heatwaves (MHWs). Here, we show that, in a Mediterranean highly enforced marine protected area established more than 40 years ago, habitat-forming octocoral populations that were first affected by a severe MHW in 2003 have not recovered after 15 years. Contrarily, they have followed collapse trajectories that have brought them to the brink of local ecological extinction. Since 2003, impacted populations of the red gorgonian  In particular, marine heatwaves (MHWs), which are discrete prolonged anomalously warm water events, are becoming more frequent and severe, causing deleterious impacts  across the oceans –3. AmongParamuricea clavata and Corallium rubrum) have been among the most affected organisms, providing excellent biological models to explore the resistance and recovery capacity. However, while our understanding of their resistance to MHWs is improving , slow growth rates (less than 3 cm per year), low recruitment rates or high post-settlement mortality) and restrained dispersal capacity )Click here for additional data file.