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Background and Aims: Liver fibrosis staging is challenging in patients with obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Liver biopsies are invasive, whereas noninvasive tests such as vibration-controlled transient elastography (VCTE) can be inaccurate in patients with obesity. We hypothesized that EUS-guided shear wave elastography (EUS-SWE) is more accurate for liver fibrosis staging in patients with MASLD and obesity; the aim of this pilot study was to test this hypothesis and establish optimal fibrosis stage cutoffs for EUS-SWE., Methods: This was a multicenter, cross-sectional study from prospectively collected data. Consecutive patients who underwent EUS-SWE with subsequent liver biopsy were included. EUS-SWE was compared with Fibrosis-4 Index (FIB-4) and VCTE. Area under the receiver-operating characteristic (AUROC) curve analysis was performed, and 90% sensitivity and specific cutoffs were calculated to determine optimal cutoffs., Results: Sixty-two patients were included. Mean body mass index was 40.74 kg/m 2 . EUS-SWE was superior to FIB-4 in discriminating significant fibrosis (F2; AUROC, .87 vs .61; P < .0048) and advanced fibrosis (F3; AUROC, .93 vs .63; P < .0001), but not cirrhosis (F4; AUROC, .95 vs .81; P = .099). EUS-SWE was superior to VCTE in predicting advanced fibrosis and cirrhosis (P = .0067 and P = .0022, respectively). The 90% sensitivity cutoffs for EUS-SWE were 7.50, 8.48, and 11.30 for F2, F3, and F4, and the 90% specificity cutoffs were 9.82, 10.20, and 14.60., Conclusions: In this pilot study, EUS-SWE was superior to FIB-4 and VCTE for liver fibrosis staging in patients with MASLD and obesity. (Clinical trial registration number: NCT05728697.)., Competing Interests: Disclosure The authors disclosed the following financial relationships: M. Ryou: consultant for Olympus, Fujifilm, Cook, Boston Scientific, GI Windows Surgical, and EnteraSense. P. Jirapinyo: consultant/advisory board member/research support, Apollo Endosurgery and Fractyl; consultant/research support, Boston Scientific; consultant, ERBE and Spatz Medical; and research support, GI Dynamics and USGI Medical. C.C. Thompson: consultant/research support, Apollo Endosurgery, Boston Scientific, GI Dynamics, Olympus/Spiration, Fujifilm, Lumendi, and EndoQuest Robotics; general partner, BlueFlame Healthcare Venture Fund; consultant, Covidien/Medtronic and Xenter; founder/consultant/board member, Enterasense Ltd, EnVision Endoscopy, and GI Windows; research support, ERBE; consultant/advisory board, Fractyl; and consultant/advisory board member/research support, USGI Medical. All other authors disclosed no financial relationships., (Copyright © 2024 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Endoscopic liver "palpation" can be performed by indenting the liver surface under EUS. Indentation depth is measured with the use of sonographic calipers. We hypothesized that fibrotic livers are more difficult to indent, and that indentation can accurately predict liver fibrosis staging. We compared EUS-guided liver palpation and conventional screening modalities in patients with suspected metabolic dysfunction-associated steatotic liver disease., Methods: This was a cross-sectional pilot study. Consecutive patients at 3 hospitals from 2021 to 2023 underwent EUS-guided palpation with liver biopsy. Liver palpation was compared with fibrosis-4 index (FIB-4), aspartate transaminase to platelet ratio index (APRI), nonalcoholic fatty liver disease fibrosis score (NFS), and transient elastography in predicting fibrosis staging on histology. Area under the receiver operating characteristic curve analysis was performed., Results: Seventy-three patients were included. Mean age was 49.1 years, and 71.2% were female. Mean body mass index was 41.1 kg/m. 2 Indentation depth was negatively correlated with fibrosis stage (Kruskal-Willis test, P < .0001). EUS palpation demonstrated c-statistics of 0.79 and 0.95 in discriminating advanced fibrosis and cirrhosis, respectively. EUS liver palpation was superior to NFS in predicting advanced fibrosis (P = .0057) and superior to APRI and NFS in predicting cirrhosis (P = .0099 and P = .045, respectively). EUS palpation was not significantly different from FIB-4. EUS palpation was superior to transient elastography in predicting cirrhosis (P = .045). When optimal cutoffs were used, indentation measurement ≤3.5 mm yielded 100% predictive value for ruling in advanced fibrosis, and ≥4.0 mm yielded 100% predictive value for ruling out cirrhosis., Conclusions: EUS liver palpation is a novel, accurate, and easy-to-use screening tool for advanced fibrosis and cirrhosis in patients with metabolic dysfunction-associated steatotic liver disease., Competing Interests: Disclosure The following authors disclosed financial relationships: C. C. Thompson: Consultant, Apollo Endosurgery and Covidien/Medtronic; Venture Fund General Partner, BlueFlame Healthcare; Consultant/Research Support, Boston Scientific; Founder/Consultant/Board Member, Enterasense Ltd; Founder/Consultant/Board Member, EnVision Endoscopy; Research Support, ERBE; Consultant/Advisory Board, Fractyl; Consultant/Research Support, GI Dynamics; Founder/Consultant/Board Member, GI Windows; Consultant/Research Support, Olympus/Spiration; Consultant/Advisory Board Member/Research Support, USGI Medical; Consultant/Research Support, Fujifilm, Consultant/Research Support, Lumendi; Consultant, Xenter; Consultant/Research Support, Endoquest Robotics. K. Chang: Consultant for Olympus and Fujifilm. M. Ryou: Consultant for Olympus, Fujifilm, Cook, Boston Scientific, GI Windows Surgical, and EnteraSense. P. Jirapinyo: Consultant/Advisory Board Member/Research Support, Apollo Endosurgery; Consultant/Research Support, Boston Scientific; Consultant, ERBE; Consultant/Advisory board Member/Research Support, Fractyl; Research Support, GI Dynamics; Consultant, Spatz Medical; Research Support, USGI Medical. The other authors disclosed no financial relationships., (Copyright © 2024 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Eyed needles are among the most iconic of Paleolithic artifacts, traditionally seen as rare indicators of prehistoric clothing, particularly tailoring. However, recent finds across Africa and Eurasia show that other technologies like bone awls also facilitated the creation of fitted garments. Nonetheless, the advent of delicate eyed needles suggests a demand for more refined, efficient sewing. This refinement may signify two major developments: the emergence of underwear in layered garment assemblages, and/or a transition in adornment from body modification to decorating clothes, as humans covered themselves more completely for thermal protection. Archaeological evidence for underwear is limited, but the Upper Paleolithic saw an increase in personal ornaments, some sewn onto clothing. Eyed needles may mark a pivotal shift as clothes acquired the social functions of dress, decoupling clothing from climate and ensuring its enduring presence.

Shifts in projectile technology potentially document human evolutionary milestones, such as adaptations for different environments and settlement dynamics. A relatively direct proxy for projectile technology is projectile impact marks (PIM) on archaeological bones. Increasing awareness and publication of experimental data sets have recently led to more identifications of PIM in various contexts, but diagnosing PIM from other types of bone-surface modifications, quantifying them, and inferring point size and material from the bone lesions need more substantiation. Here, we focus on PIM created by osseous projectiles, asking whether these could be effectively identified and separated from lithic-tipped weapons. We further discuss the basic question raised by recent PIM research in zooarchaeology: why PIM evidence is so rare in archaeofaunal assemblages (compared to other human-induced marks), even when they are explicitly sought. We present the experimental results of shooting two ungulate carcasses with bone and antler points, replicating those used in the early Upper Paleolithic of western Eurasia. Half of our hits resulted in PIM, confirming that this modification may have been originally abundant. However, we found that the probability of a skeletal element to be modified with PIM negatively correlates with its preservation potential, and that much of the produced bone damage would not be identifiable in a typical Paleolithic faunal assemblage. This quantification problem still leaves room for an insightful qualitative study of PIM. We complement previous research in presenting several diagnostic marks that retain preservation potential and may be used to suggest osseous, rather than lithic, projectile technology., Supplementary Information: The online version contains supplementary material available at 10.1007/s12520-024-01944-3., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2024.)

The earliest European carvings, made of mammoth ivory, depict animals, humans, and anthropomorphs. They are found at Early Aurignacian sites of the Swabian Jura in Germany. Despite the wide geographical spread of the Aurignacian across Europe, these carvings have no contemporaneous counterparts. Here, we document a small, intriguing object, that sheds light on this uniqueness. Found at the Grotte des Gorges (Jura, France), in a layer sandwiched between Aurignacian contexts and dated to c. 36.2 ka, the object bears traces of anthropogenic modifications indicating intentional carving. Microtomographic, microscopic, three-dimensional roughness and residues analyses reveal the carving is a fragment of a large ammonite, which was modified to represent a caniformia head decorated with notches and probably transported for long time in a container stained with ochre. While achieving Swabian Jura-like miniaturization, the Grotte des Gorges specimen displays original features, indicating the craftsman emulated ivory carvings while introducing significant technical, thematic, and stylistic innovations. This finding suggests a low degree of cultural connectivity between Early Aurignacian hunter-gatherer groups in the production of their symbolic material culture. The pattern conforms to the existence of cultural boundaries limiting the transmission of symbolic practices while leaving space for the emergence of original regional expressions., (© 2023. Springer Nature Limited.)

Puncture alignments are found on Palaeolithic carvings, pendants, and other fully shaped osseous artifacts. These marks were interpreted as abstract decorations, system of notations, and features present on human and animal depictions. Here, we create an experimental framework for the analysis and interpretation of human-made punctures and apply it to a highly intriguing, punctured bone fragment found at Canyars, an Early Upper Palaeolithic coastal site from Catalonia, Spain. Changes of tool and variation in the arrangement and orientation of punctures are consistent with the interpretation of this object as the earliest-known leather work punch board recording six episodes of hide pricking, one of which was to produce a linear seam. Our results indicate that Aurignacian hunters-gatherers used this technology to produce leather works and probably tailored clothes well before the introduction of bone eyed needles in Europe 15,000 years later.

Fully shaped, morphologically standardized bone tools are generally considered reliable indicators of the emergence of modern behavior. We report the discovery of 23 double-beveled bone tools from ~ 80,000-60,000-year-old archaeological layers at Sibudu Cave in KwaZulu-Natal, South Africa. We analyzed the texture of use-wear on the archaeological bone tools, and on bone tool replicas experimentally used in debarking trees, processing rabbit pelts with and without an ochre compound, digging in sediment in and outside a cave, and on ethnographic artefacts. Debarking trees and digging in humus-rich soil produce use-wear patterns closely matching those observed on most Sibudu tools. This tool type is associated with three different Middle Stone Age cultural traditions at Sibudu that span 20,000 years, yet they are absent at contemporaneous sites. Our results support a scenario in which some southern African early modern human groups developed and locally maintained specific, highly standardized cultural traits while sharing others at a sub-continental scale. We demonstrate that technological and texture analyses are effective means by which to infer past behaviors and assess the significance of prehistoric cultural innovations., (© 2022. The Author(s).)

Personal ornaments have become a key cultural proxy to investigate cognitive evolution, modern human dispersal, and population dynamics. Here, we reassess personal ornaments found at Zhoukoudian Upper Cave and compare them with those from other Late Paleolithic Northern Chinese sites. We reappraise the information provided by Pei Wen Chung on Upper Cave personal ornaments lost during World War II and analyze casts of 17 of them, along with two unpublished objects displayed at the Zhoukoudian Site Museum and three original perforated teeth rediscovered at the Zhoukoudian Site Museum. We apply archeozoological, technological and use-wear analyses to document variation in ornamental practices and their change throughout the site stratigraphy. Badger, fox, red deer, sika deer, marten, and tiger teeth as well as carp bone, bird bone, Anadara shell, limestone beads, and perforated pebble appear to have been the preferred objects used as ornaments by Upper Cave visitors. Multivariate analysis of technological data highlights a correspondence between cultural layers and perforation techniques, with radial incising being typical of layer L2 and bidirectional incising of L4. The three rediscovered badger canines display features suggesting they were sewed on clothing rather than suspended from necklaces or bracelets. Elemental scanning electron microscopy coupled with energy dispersive X-ray spectromety and mineralogical (μ-Raman) analyses of red residues adhering to the rediscovered teeth indicate these objects were originally coated with ochre and identify variations that match differences in technology. The two ornaments exhibited at the Zhoukoudian Site Museum are ancient teeth that were recently perforated and should be excluded from the Upper Cave assemblage. A seriation of Late Paleolithic ornaments found at Northern Chinese sites identifies a clear-cut difference in preferred ornament types between western and eastern sites, interpreted as reflecting two long-lasting traditions in garment symbolic codes., Competing Interests: Conflict of interest The authors declare no conflicting interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Activities attested since at least 2.6 Myr, such as stone knapping, marrow extraction, and woodworking may have allowed early hominins to recognize the technological potential of discarded skeletal remains and equipped them with a transferable skillset fit for the marginal modification and utilization of bone flakes. Identifying precisely when and where expedient bone tools were used in prehistory nonetheless remains a challenging task owing to the multiple natural and anthropogenic processes that can mimic deliberately knapped bones. Here, we compare a large sample of the faunal remains from Lingjing, a 115 ka-old site from China which has yielded important hominin remains and rich faunal and lithic assemblages, with bone fragments produced by experimentally fracturing Equus caballus long bones. Our results provide a set of qualitative and quantitative criteria that can help zooarchaeologists and bone technologists distinguish faunal remains with intentional flake removal scars from those resulting from carcass processing activities. Experimental data shows marrow extraction seldom generates diaphyseal fragments bearing more than six flake scars arranged contiguously or in interspersed series. Long bone fragments presenting such characteristics can, therefore, be interpreted as being purposefully knapped to be used as expediency tools. The identification, based on the above experimental criteria, of 56 bone tools in the Lingjing faunal assemblage is consistent with the smaller size of the lithics found in the same layer. The continuity gradient observed in the size of lithics and knapped bones suggests the latter were used for tasks in which the former were less or not effective., Competing Interests: The authors have declared that no competing interests exist.

Background: There is significant variability in weight loss after bariatric surgery. We hypothesize that part of this variability may be predetermined by genetic differences associated with metabolic homeostasis. MicroRNA (miRNA) are short pieces of RNA that regulate gene expression and are readily detectable in serum. They are implicated in numerous metabolic processes, including weight homeostasis. In this pilot study, we briefly review the role of miRNA, and assess the feasibility of using them in the clinical setting of obesity treatment., Objectives: To evaluate the feasibility of using miRNA to predict weight loss after bariatric surgery., Setting: Academic medical center., Methods: Serum was collected from patients at the initial bariatric surgery consultation. Weight loss data were collected 6 to 12 months postoperatively. Individuals experiencing the least and the greatest amount of percentage of excess weight lost at 6 months were analyzed to assess for genetic differences in miRNA expression., Results: The median percentage of excess weight lost was 51% (range, 34%-63%) for those who lost the least and 87% (range, 82%-111%) for those who lost the most weight. Groups were similar in age, sex, diabetic status, and type of surgery. In total, of the 119 miRNA detected in the serum of the patients, 6 demonstrated potential for discriminating between the high and low weight loss groups. These miRNA have previously been implicated in regulation of fatty acid biosynthesis, adipocyte proliferation, type 2 diabetes, and obesity., Conclusions: In this pilot study, we demonstrated the feasibility of identifying genetic differences between high and low weight loss groups by identifying distinct serum miRNA. In the near future, these biomarkers could facilitate informed decisions about surgery. In addition, these miRNA could open new genetic pathways that describe the pathophysiology of obesity, and provide targets for future treatment., (Copyright © 2020 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

The recent identification of cave paintings dated to 42-40 ka BP in Borneo and Sulawesi highlights the antiquity of painted representations in this region. However, no instances of three-dimensional portable art, well attested in Europe since at least 40 ka BP, were documented thus far in East Asia prior to the Neolithic. Here, we report the discovery of an exceptionally well-preserved miniature carving of a standing bird from the site of Lingjing, Henan, China. Microscopic and microtomographic analyses of the figurine and the study of bone fragments from the same context reveal the object was made of bone blackened by heating and carefully carved with four techniques that left diagnostic traces on the entire surface of the object. Critical analysis of the site's research history and stratigraphy, the cultural remains associated with the figurine and those recovered from the other archeological layers, as well as twenty-eight radiometric ages obtained on associated archeological items, including one provided by a bone fragment worked with the same technique recorded on the object, suggest a Late Paleolithic origin for the carving, with a probable age estimated to 13,500 years old. The carving, which predates previously known comparable instances from this region by 8,500 years, demonstrates that three-dimensional avian representations were part of East Asian Late Pleistocene cultural repertoires and identifies technological and stylistic peculiarities distinguishing this newly discovered art tradition from previous and contemporary examples found in Western Europe and Siberia., Competing Interests: The authors have declared that no competing interests exist.

Objectives: The purpose of this study was to specify the number of male filicide-suicide committed in the province of Quebec between 1997 and 2012, and to evaluate whether there has been an increase in the number of male filicide-suicide according to the period studied and the type of motivation to commit the crime., Method: The data cover all officially registered male filicides committed in the province of Quebec from 1997 to 2012, against youths under 18 years old. A total of 50 cases were divided by five years spans starting in 1997 and analyzed using the Grille d'analyse multidimensionnelle de l'homicide intrafamilial., Results: The results show that among the 50 male filicides committed between 1997 and 2012, 13 of these were followed by the aggressor's suicide. Also, the likelihood of suicide committed as a result of filicide is higher among individuals who committed filicide between 2007 and 2012 and who were motivated by marital separation, compared to filicides committed between 1997 and 2001 motivated by another reason. In particular, the majority of perpetrators of filicide committed between 2007 and 2012, motivated by marital separation, committed suicide as a result of the act of committing, compared to individuals who were motivated by another reason for the same period., Conclusions: On one hand, the present study demonstrates the importance of considering self-destruction and, more specifically, the suicide of these individuals. On the other hand, our study emphasizes the importance of considering the type of motivation to commit filicide, including spousal separation, which is an element of understanding specific to male filicide-suicide., (Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.)

When, how, and following which paths hominins created the innovations that allowed them to colonize regions of the planet that were not suited to their thermal physiology is still a matter of inquiry. In this paper, we elaborate a theoretical framework to investigate the origin and diversification of bone needles, summarize the evidence for their emergence, create a large database of their morphometric and stylistic characters, and present results of the study of an exceptionally well-preserved collection of needles from Shuidonggou Locality 12 (SDG12), a site located in the Ningxia Hui Autonomous Region, Northern China, dated to ca. 11.2 ka BP. Bone needles are reported from 271 sites and 355 archaeological layers. Revision of the evidence shows they represent an original cultural innovation that emerged in Eurasia between 45-40 ka BP. Size differences between the earliest known specimens, found in Siberia and China, indicate needles may have been invented independently in these two regions. Needles from Eastern Europe may represent either an independent invention or a geographic extension of earlier Siberian and Caucasian sewing traditions. In Western Europe, needles appear during the Solutrean. The wider size range characteristic of Magdalenian specimens supports the idea that needles of different sizes were used in a variety of tasks. In China, the robust sub-circular needles found at sites dated between 35-25 ka BP are followed, between 26-23 ka BP, by small flat needles, which may represent an innovation associated with the microblades/microcores toolkit. At SDG12, technological, functional, and morphometric analyses of finished needles and manufacturing by-products identify two previously undetected reduction sequences for the production of needles of different size and, probably, function. The bone needles found at Paleoindian sites are the smallest and reflect a never previously achieved mastery in the production of such tools., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Most Chinese lithic industries dated between 300,000 and 40,000 are characterized by the absence of Levallois debitage, the persistence of core-and-flake knapping, the rarity of prepared cores, their reduction with direct hard hammer percussion, and the rarity of retouched flakes. Here we report the discovery of seven bone soft hammers at the early hominin Lingjing site (Xuchang County, Henan) dated to 125,000-105,000. These artefacts represent the first instance of the use of bone as raw material to modify stone tools found at an East Asian early Late Pleistocene site. Three types of soft hammers are identified. The first consists of large bone flakes resulting from butchery of large herbivores that were utilized as such for expedient stone tools retouching or resharpening. The second involved the fracture of weathered bone from medium size herbivores to obtain elongated splinters shaped by percussion into sub-rectangular artefacts. Traces observed on these objects indicate intensive and possibly recurrent utilization, which implies their curation over time. The last consists of antler, occasionally used. Lingjing bone tools complement what we know about archaic hominin cultural adaptations in East Asia and highlight behavioural consistencies that could not be inferred from other cultural proxies. This discovery provides a new dimension to the debate surrounding the existence of the Middle Palaeolithic in the region. The attribution of East Asian sites to the Middle Palaeolithic assumes that cultural traits such as the Levallois method represent evolutionary hallmarks applicable to regions of the world different from those in which they were originally found. Here, we promote an approach that consists in identifying, possibly from different categories of material culture, the original features of each regional cultural trajectory and understanding the behavioural and cognitive implications they may have had for past hominin populations.

How and when did hominins move from the numerical cognition that we share with the rest of the animal world to number symbols? Objects with sequential markings have been used to store and retrieve numerical information since the beginning of the European Upper Palaeolithic (42 ka). An increase in the number of markings and complexity of coding is observed towards the end of this period. The application of new analytical techniques to a 44-42 ka old notched baboon fibula from Border Cave, South Africa, shows that notches were added to this bone at different times, suggesting that devices to store numerical information were in use before the Upper Palaeolithic. Analysis of a set of incisions on a 72-60 ka old hyena femur from the Les Pradelles Mousterian site, France, indicates, by comparison with markings produced by modern subjects under similar constraints, that the incisions on the Les Pradelles bone may have been produced to record, in a single session, homologous units of numerical information. This finding supports the view that numerical notations were in use among archaic hominins. Based on these findings, a testable five-stage scenario is proposed to establish how prehistoric cultures have moved from number sense to the use of number symbols.This article is part of a discussion meeting issue 'The origins of numerical abilities'., (© 2017 The Author(s).)

Background: As clinical outcome data are increasingly tied to hospital reimbursement, balancing quality care with training of surgical residents has become critical. We used the ACS-NSQIP database to determine impact of resident participation in laparoscopic gastric bypass on 30-day morbidity and mortality., Methods: We queried the ACS-NSQIP database from 1/2005 to 12/2012 for laparoscopic gastric bypass, dividing cases between those with or without resident involvement. Univariate and multivariate analyses of intraoperative and postoperative outcomes were assessed. A sub-analysis was performed to address whether different resident training levels affected outcomes., Results: A total of 43,477 laparoscopic gastric bypass cases were available for analysis; 22,189 had resident involvement (resident = R), and 21,288 did not (no resident = NR). Preoperative characteristics were similar between groups. On multivariate analysis, procedures with resident assistance had increased risk of the following complications: superficial site infection (R = 2.1 vs. 1.5 %, p < 0.001), renal failure (R = 0.4 vs. NR = 0.3 %, p = 0.002), urinary tract infection (R = 1.1 vs. 0.9 %, p = 0.027), and sepsis (R = 0.8 vs. NR = 0.6 %, p = 0.019). Increased operative time in the resident group (29 min, p < 0.0001) demonstrated direct linear association with resident trainee level. There was no statistical difference in the incidences of the following: pulmonary embolism, deep venous thrombosis, deep surgical site infection, organ space infection, pneumonia, unplanned intubation, mechanical ventilation >48 h, septic shock, cardiac arrest, return to the operating room, or mortality., Conclusion: Resident participation in laparoscopic gastric bypass was associated with statistically significant, but clinically insignificant increase in incidence of superficial site infection, renal failure, readmission rate, and length of stay. Therefore, although resident participation in laparoscopic gastric bypass is associated with significantly increased operative time, it does not lead to increased mortality and has no clinically significant effect on morbidity.

Graft versus host disease (GVHD) is a phenomenon that occurs after allogeneic bone marrow transplants. Gastrointestinal (GI) manifestations of acute GVHD are common, but severe GI GVHD complications, such as bowel perforation, occur rarely and necessitate surgical intervention. To our knowledge, there are no recorded cases of colonic perforation resulting from GVHD with negative cultures for infectious agents such as cytomegalovirus. We present a case of large bowel perforation due to GVHD.

A series of aryl thiotetrazolylacetanilides were synthesized and found to be potent inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases. The incorporation of an alkynyl fragment on the aniline provided inhibitors with excellent cellular activity and extensive SAR led to the identification of one inhibitor having good oral bioavailability in rats.

Tipranavir is a novel, non-peptidic protease inhibitor, which possesses broad antiviral activity against multiple protease inhibitor-resistant HIV-1. Resistance to this inhibitor however has not yet been well described. HIV was passaged for 9 months in culture in the presence of tipranavir to select HIV with a drug-resistant phenotype. Characterization of the selected variants revealed that the first mutations to be selected were L33F and I84V in the viral protease, mutations which together conferred less than two-fold resistance to tipranavir. At the end of the selection experiments, viruses harbouring 10 mutations in the protease (L10F, I13V, V32I, L33F, M36I, K45I, I54V, A71V, V82L, I84V) as well as a mutation in the CA/SP1 gag cleavage site were selected and showed 87-fold decreased susceptibility to tipranavir. In vitro, tipranavir-resistant viruses had a reduced replicative capacity which could not be improved by the introduction of the CA/SP1 cleavage site mutation. Tipranavir resistant viruses showed cross-resistance to other currently approved protease inhibitors with the exception of saquinavir. These results demonstrate that the tipranavir resistance phenotype is associated with complex genotypic changes in the protease. Resistance necessitates the sequential accumulation of multiple mutations.

A series of novel 8-substituted dipyridodiazepinone-based inhibitors were investigated for their antiviral activity against wild type human immunodeficiency virus (HIV-1) and the clinically prevalent K103N/Y181C mutant virus. Our efforts have resulted in a series of benzoic acid analogues that are potent inhibitors of HIV-1 replication against a panel of HIV-1 strains resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Furthermore, the combination of good antiviral potency, a broad spectrum of activity, and an excellent pharmacokinetic profile provides strong justification for the further development of compound (7) as a potential treatment for wild type and NNRTI-resistant HIV-1 infection.

The guanine nucleotide exchange factor (GEF) Son-of-sevenless (Sos) encodes a complex multidomain protein best known for its role in activating the small GTPase RAS in response to receptor tyrosine kinase (RTK) stimulation. Much less well understood is SOS's role in modulating RAC activity via a separate GEF domain. In the course of a genetic modifier screen designed to investigate the complexities of RTK/RAS signal transduction, a complementation group of 11 alleles was isolated and mapped to the Sos locus. Molecular characterization of these alleles indicates that they specifically affect individual domains of the protein. One of these alleles, SosM98, which contains a single amino acid substitution in the RacGEF motif, functions as a dominant negative in vivo to downregulate RTK signaling. These alleles provide new tools for future investigations of SOS-mediated activation of both RAS and RAC and how these dual roles are coordinated and coregulated during development., (2004 Wiley-Liss, Inc.)

A series of 8-heteroarylthiomethyldipyridodiazepinone derivatives were prepared and evaluated for their antiviral profile against wild type virus and the important K103N/Y181C mutant as an indicator for broad activity. 2,6-Dimethylpyridine derivative 16 was found to have a good pharmacokinetic profile in spite of poor metabolic stability in rat liver microsomes.

The retinal determination (RD) gene network encodes a group of transcription factors and cofactors necessary for eye development. Transcriptional and posttranslational regulation of RD family members is achieved through interactions within the network and with extracellular signaling pathways, including epidermal growth factor receptor/RAS/mitogen-activated protein kinase (MAPK), transforming growth factor beta/DPP, Wingless, Hedgehog, and Notch. Here we present the results of structure-function analyses that reveal novel aspects of Eyes absent (EYA) function and regulation. We find that the conserved C-terminal EYA domain negatively regulates EYA transactivation potential, and that GROUCHO-SINE OCULIS (SO) interactions provide another mechanism for negative regulation of EYA-SO target genes. We have mapped the transactivation potential of EYA to an internal proline-, serine-, and threonine-rich region that includes the EYA domain 2 (ED2) and two MAPK phosphorylation consensus sites and demonstrate that activation of the RAS/MAPK pathway potentiates transcriptional output of EYA and the EYA-SO complex in certain contexts. Drosophila S2 cell two-hybrid assays were used to describe a novel homotypic interaction that is mediated by EYA's N terminus. Our data suggest that EYA requires homo- and heterotypic interactions and RAS/MAPK signaling responsiveness to ensure context-appropriate RD gene network activity.

Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by approximately 10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target.

Mice infected with murine acquired immunodeficiency syndrome (MAIDS) virus developed lymphoadenopathy and profound immunodeficiency. Concomitantly the expression of endogenous mammary tumor virus (MTV) mRNA increased significantly, especially for the 1.7-kb 3' open reading frame (ORF) mRNA encoding MTV superantigen. B cell lines that are established from MAIDS mice and exhibit superantigen activity also express a high level of 1.7-kb endogenous MTV and mRNA. Infection of a B cell tumor line in vitro with retrovirus containing the cloned MAIDS virus gene induced superantigen activity and this cell line also expressed the 1.7-kb superantigen coding MTV 3' ORF mRNA. These results strongly suggest a link between MAIDS virus infection and the induction of endogenous superantigen activity. This may play an important role in the pathogenesis of the MAIDS virus.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring -1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.

One hope to maintain the benefits of antiviral therapy against the human immunodeficiency virus type 1 (HIV-1), despite the development of resistance, is the possibility that resistant variants will show decreased viral fitness. To study this possibility, HIV-1 variants showing high-level resistance (up to 1,500-fold) to the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS have been characterized. Active-site mutations V32I and I84V/A were consistently observed in the protease of highly resistant viruses, along with up to six other mutations. In vitro studies with recombinant mutant proteases demonstrated that these mutations resulted in up to 10(4)-fold increases in the Ki values toward BILA 1906 BS and BILA 2185 BS and a concomitant 2,200-fold decrease in catalytic efficiency of the enzymes toward a synthetic substrate. When introduced into viral molecular clones, the protease mutations impaired polyprotein processing, consistent with a decrease in enzyme activity in virions. Despite these observations, however, most mutations had little effect on viral replication except when the active-site mutations V32I and I84V/A were coexpressed in the protease. The latter combinations not only conferred a significant growth reduction of viral clones on peripheral blood mononuclear cells but also caused the complete disappearance of mutated clones when cocultured with wild-type virus on T-cell lines. Furthermore, the double nucleotide mutation I84A rapidly reverted to I84V upon drug removal, confirming its impact on viral fitness. Therefore, high-level resistance to protease inhibitors can be associated with impaired viral fitness, suggesting that antiviral therapies with such inhibitors may maintain some clinical benefits.

Protease inhibitors are potent antiviral agents against human immunodeficiency virus type 1. As with reverse transcriptase inhibitors, however, resistance to protease inhibitors can develop and is attributed to the appearance of mutations in the protease gene. With the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS, 350- to 1,500-fold-resistant variants have been selected in vitro and were found not only to contain mutations in the protease gene but also to contain mutations in Gag precursor p1/p6 and/or NC (p7)/p1 cleavage sites. Mutations in cleavage sites give rise to better peptide substrates for the protease in vitro and to improved processing of p15 precursors in drug-resistant clones. Importantly, removal of cleavage site mutations in resistant clones leads to a decrease or even an absence of viral growth, confirming their role in viral fitness. Therefore, these second-locus mutations indicate that cleavage of p15 is a rate-limiting step in polyprotein processing in highly resistant viruses. The functional constraint of p15 processing also suggests that additional selective pressure could further compromise viral fitness and maintain the benefits of antiviral therapies.

The T-cell receptor repertoire was analyzed in C57BL/6 mice upon infection with helper-free stocks of the pathogenic murine AIDS (MAIDS) defective virus in order to demonstrate if, as previously reported, this virus encodes a superantigen. A polyclonal T-cell stimulation involving T cells expressing multiple V beta subsets occurred within the first week of infection, while late in the disease we could note only a 50% deletion of V beta 5 CD8+ cells. Transfection of the MAIDS virus genomic DNA into fibroblasts and B cells expressing major histocompatibility complex class II molecules failed to show any stimulation of cells expressing the specific V beta (V beta 5) previously reported to respond to MAIDS virus-infected cells. In addition, mice lacking V beta 5 cells did not show any significant decrease in susceptibility to the disease compared with mice expressing V beta 5 and bred on the same genetic background. Our in vivo and in vitro results fail to demonstrate a role for a superantigen encoded by the MAIDS defective viral genome in the pathogenesis of MAIDS.

Excavation at the Ecuadorian highland site of La Florida in suburban Quito revealed six deep shaft tombs yielding high-status individuals (n = 9) as well as apparent sacrifices and other low-status individuals (n = 23). Determination of sex and age at death of the recovered skeletal remains resulted in a sample of 32 individuals aged from approximately 7 to 50 years of age. The sample of 18 individuals over the age of 18 years included 14 females and 4 males. Temporally, the remains are assigned to the Chaupicruz Phase (circa 100 to 450 AD) of the Regional Developmental Period. In this study, we analyze stable isotopes of carbon and nitrogen from human bone in order to compare the diets of the high- and low-status individuals. Stable carbon isotope analyses were carried out on preserved protein and biological apatite (bioapatite), and stable nitrogen isotope analyses were carried out on preserved protein. There is a statistically significant difference in delta 13C between the two groups for both protein and mineral sources of carbon with evidence for the greater consumption of maize in the high-status group. There is no significant difference in delta 15N between the two groups, nor is there a significant difference in the spacing between protein and mineral delta 13C values between the two groups. Ethnohistorical evidence for the 16th century AD provides the expectation that the only dietary difference was the higher consumption of animal protein by the elite. There is no evidence for this based on the bone chemistry data from La Florida.(ABSTRACT TRUNCATED AT 250 WORDS)

CD4 is a cell surface molecule expressed mostly on cells of the T-cell lineage. Studies have shown that this molecule plays an important role in positive and negative selection of T cells in the thymus. It is not surprising therefore, that in T-cell ontogeny, CD4 starts to be expressed on thymocyte subpopulations about to undergo these selection processes. The human CD4 molecule was expressed in mouse thymus ontogeny using a promoter, MMTVD, which targets expression as early as day 14 of ontogeny, prior to expression of endogenous TCR, CD4 and CD8. Thymic ontogeny is delayed in foetal MMTVD-CD4 mice. Human CD4-expressing thymuses show a twofold reduction in cellularity at days 17 and 18 of ontogeny compared with non-transgenic control littermate thymuses, and paradoxically, MMTVD-CD4 thymuses contain more cells in the S and G2/M stages of the cell cycle than control thymuses do. At the cell surface marker level, MMTVD-CD4 thymocytes show a delay in surface expression of CD3, murine CD4 and murine CD8, along with persistent expression of IL2R alpha compared with foetal non-transgenic littermates. Biochemical studies show that, although MMTVD-CD4 thymocytes do not express surface CD3, cytoplasmic CD3 epsilon proteins as well as TCR beta incomplete and complete transcripts are present in foetal day-17 thymocytes. Low levels of surface CD3/TCR expression, however, could partly be due to the low levels of zeta mRNA and proteins detected in these cells. These results suggest that CD4 is not expressed until a certain stage of differentiation not only because it is not yet required for selection processes, but because it can lead to a reversible deregulation of thymocyte development.

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to be derived from the progenitor double-positive T cells. These results suggest the existence of similar and common factors in CD4+ CD8- and CD4- CD8+ T cells and support a model of differentiation of CD4+ CD8+ T cells through common signal(s) involved in turning off the expression of the CD4 or CD8 gene.

Human thymic dendritic cells (DC) have previously been shown to be intimately associated with thymocytes in situ and in culture. We report that thymic DC express LFA-3 and ICAM-1 adhesion molecules and may spontaneously associate with autologous thymocytes within mitogen-independent clusters. Moreover, the accessory activity of isolated human thymic DC was investigated in Con A-stimulation assays. By proliferation experiments, measured as [3H]TdR incorporation, we demonstrated that irradiated thymic DC strongly increase the mitogen-induced activation of autologous PBL as well as of unfractionated thymocytes. More interestingly, in coculture assays performed with purified thymocyte subsets, we have found that thymic DC greatly enhance the Con A proliferation of CD1- CD3bright thymocytes whereas the accessory activity toward the CD1+ CD3- thymocytes was very weak. Inhibition experiments demonstrated that the DC accessory activity is inhibited by anti-DR-related and anti-IL-2R mAb. However, blocking assays with anti-CD11b, anti-CD11c, anti-LFA-3, and anti-ICAM1 mAb showed that the accessory function obtained is similar to that with untreated cultures. We conclude that isolated human thymic DC may present potent DR- and IL-2-dependent accessory activity mainly directed toward the CD1- CD3bright thymocyte subpopulation, suggesting that thymic DC may be involved in the in vivo proliferation of mature thymocytes.

Genetic linkage analysis requires the identification and documentation of large families with many affected members present, preferably in more than one generation. The IMAGE Project has been establishing a population-based Alzheimer disease (AD) registry in the Saguenay - Lac-Saint-Jean region of the Province of Quebec. The population of this region has a well-documented ancestry, with reliable genealogical records (since 1842) computerized by SOREP. We have recently begun to investigate the pedigrees of selected probands (definite, probable and possible) from the IMAGE registry in order to identify informative pedigrees for genetic linkage analysis. Interviews were carried out with close relatives of the probands (at least one informant per sibship) to identify secondary AD cases. The questionnaires used pertain to the accuracy of genealogical records, to family medical history and to a retrospective diagnosis of AD for people with cognitive deficits. By these means, we have documented a large extended pedigree in which a total of 15 individuals with cognitive deficits were ascertained over three generations. Of these cases, 7 are still living and there is autopsy confirmation in another one. Computer simulations using the program SIMLINK revealed that this is a potentially informative family for linkage analysis. Horizontal extension of the pedigree to second cousins of the proband is now being carried out. This will render the family IMAGE/1 even more informative in genetic linkage analysis studies.