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Most modern excavations intensively collect data from flotation, including both light and heavy fractions. While the light fraction (floated) is usually extensively analyzed by archaeobotanists, the heavy fraction or microdebris is often ignored or minimally examined since it requires intensive efforts at the microscopic level to recover and identify the remains. In recent years, a few studies have demonstrated the utility of intensive examination of the microdebris from archaeological sites as a means for investigating behavior on the microscopic level. When collected systematically across surfaces, the analysis of microdebris allows for the identification of different activities and deposits that are often less visible with macroscopic remains. This paper describes the goals and collection methods for microdebris analysis and presents some preliminary analysis of the microdebris from the excavations of the Early Bronze III nonelite residential neighborhood at Tell eṣ-Ṣâfi/Gath, Israel. The results demonstrate that various types of materials are deposited differen- tially between depositional contexts. Some types of deposits yield very little microdebris (e.g., alleyways), while others are characterized by their abundance (e.g., room interiors). Consequently, the systematic collection and analysis of con- textually differentiated microdebris samples from across archaeological surfaces can help guide excavation strategies since it allows for certain deposits to be clearly targeted for intensive examination..

Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.

This study investigates the relationship between performances on the speaking component of the TOEFL iBTTM with performances on academic oral assessment tasks. For the academic tasks, we recorded a...

Background: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). Results: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Conclusions: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.

Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.

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Exosomes are cell-derived extracellular vesicles that have great potential in the field of nano-medicine. However, a fundamental challenge in the engineering of exosomes is the design of biocompatible molecular scaffolds on their surface to enable cell targeting and therapeutic functions. CD63 is a hallmark protein of natural exosomes that is highly enriched on the external surface of the membrane. We have already described the engineering of CD63 as a molecular scaffold in order to introduce cell-targeting feature to the exosome surface. Despite this initial success, the restrictive M-shaped topology of full-length CD63 may hinder specific applications that require N- or C-terminal display of cell-targeting moieties on the outer surface of the exosome. In this study, we describe new and topologically distinct CD63 scaffolds that enable robust and flexible surface engineering of the exosome. In particular, we conducted sequential deletions of the transmembrane helix of CD63 to generate a series of CD63 truncates, each genetically-fused to a fluorescent protein. Molecular and cellular characterization studies showed truncates of CD63 harboring the transmembrane helix 3 (TM3) were correctly targeted and anchored to the exosome membrane and exhibited distinct n-, N-, Ω-, or I-shaped membrane topologies in the exosomal membrane. We further established that these truncates retained robust membrane-anchoring and exosome-targeting activities when stably expressed in the HEK293 cells. Moreover, HEK293 cells produced engineered exosomes in similar quantities to cells that expressed full-length CD63. On the basis of the results of our systematic sequential deletion studies, we propose a model to understand molecular mechanisms that underlie membrane-anchoring and exosome targeting features of CD63. In summary, we have established new and topologically distinct scaffolds based on engineering of CD63 that enable flexible engineering of the exosome surface for applications in disease-targeted drug delivery and therapy.

The discovery of a sacrificed puppy at Tel Miqne-Ekron, a major Philistine settlement in Israel's southern coastal plain, highlights the role of dogs in Iron I Philistia. Though dog sacrifice is described in Hittite religious texts and attested in lands bordering the Aegean during the second–first millennia BCE, evidence for this practice, or even of dog bones, is largely absent from Late Bronze and non-Philistine Iron I (ca. 1550–1000 BCE) Levantine contexts. What distinguishes the Tel Miqne-Ekron puppy interment from later Persian- and Hellenistic-period dog burials, is the placement of its severed head between its hind legs. Microscopic analyses of cut marks on several vertebrae indicate that the iron knife found nearby was likely used in its decapitation. This article examines the Tel Miqne-Ekron puppy burial within its eastern Mediterranean milieu and explores the ritual role of dogs and cynophagy (dog-eating) in second–first millennia society.

Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal β-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.

Analysis of a sacrificed and interred domestic donkey from an Early Bronze Age (EB) IIIB (c. 2800–2600 BCE) domestic residential neighborhood at Tell eṣ-Ṣâfi/Gath, Israel, indicate the presence of bit wear on the Lower Premolar 2 (LPM2). This is the earliest evidence for the use of a bit among early domestic equids, and in particular donkeys, in the Near East. The mesial enamel surfaces on both the right and left LPM2 of the particular donkey in question are slightly worn in a fashion that suggests that a dental bit (metal, bone, wood, etc.) was used to control the animal. Given the secure chronological context of the burial (beneath the floor of an EB IIIB house), it is suggested that this animal provides the earliest evidence for the use of a bit on an early domestic equid from the Near East.

Analysis of a sacrificed and interred domestic donkey from an Early Bronze Age (EB) IIIB (c. 2800-2600 BCE) domestic residential neighborhood at Tell eṣ-Ṣâfi/Gath, Israel, indicate the presence of bit wear on the Lower Premolar 2 (LPM2). This is the earliest evidence for the use of a bit among early domestic equids, and in particular donkeys, in the Near East. The mesial enamel surfaces on both the right and left LPM2 of the particular donkey in question are slightly worn in a fashion that suggests that a dental bit (metal, bone, wood, etc.) was used to control the animal. Given the secure chronological context of the burial (beneath the floor of an EB IIIB house), it is suggested that this animal provides the earliest evidence for the use of a bit on an early domestic equid from the Near East.