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Purpose:The effectiveness of immune checkpoint inhibitors (ICI) is limited in pancreatic ductal adenocarcinoma (PDAC). We conducted a phase I study to evaluate the safety of ICI with stereotactic body radiation therapy (SBRT) in patients with metastatic PDAC.Patients and Methods:Patients enrolled must have received at least one line of prior systemic chemotherapy for metastatic disease. Cohorts A1 and A2 received durvalumab every 2 weeks plus either 8 Gy in one fraction of SBRT on day 1 or 25 Gy in five fractions on day −3 to +1. Cohorts B1 and B2 received durvalumab plus tremelimumab every 4 weeks and either 8 Gy in one fraction of SBRT on day 1 or 25 Gy in five fractions on day −3 to +1. ICIs were continued until unacceptable toxicity or disease progression. The primary objective was the safety and feasibility of treatment. Objective response was assessed in lesions not subjected to SBRT.Results:Fifty-nine patients were enrolled and 39 were evaluable for efficacy. No dose-limiting toxicities were seen. The most common adverse event was lymphopenia. Two patients achieved a partial response (one confirmed and the other unconfirmed). The overall response rate was 5.1%. Median PFS and OS was 1.7 months [95% confidence intervals (CI), 0.8–2.0 months] and 3.3 months (95% CI, 1.2–6.6 months) in cohort A1; 2.5 months (95% CI, 0.1–3.7 months) and 9.0 months (95% CI, 0.5–18.4 months) in A2; 0.9 months (95% CI, 0.7–2.1 months) and 2.1 months (95% CI, 1.1–4.3 months) in B1; and 2.3 months (95% CI, 1.9–3.4 months) and 4.2 months (95% CI, 2.9–9.3 months) in B2.Conclusions:The combination of ICI and SBRT has an acceptable safety profile and demonstrates a modest treatment benefit in patients with metastatic PDAC.

Supplemental table S1 and S2

Supplemental figure S1: Representative immunochemistry staining of MHC-1 from tumor biopsied samples of partial response (top), stable disease (middle), and progressive disease (bottom), respectively. Scale bar: 20 X.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays., iScience, 25 (8), ISSN:2589-0042

Connexins are a family of transmembrane proteins that regulate diverse cellular functions. Originally characterized for their ability to mediate direct intercellular communication through the formation of highly regulated membrane channels, their functions have been extended to the exchange of molecules with the extracellular environment, and the ability to modulate numerous channel-independent effects on processes such as motility and survival. Notably, connexins have been implicated in cancer biology for their context-dependent roles that can both promote or suppress cancer cell function. Moreover, connexins are able to mediate many aspects of cellular metabolism including the intercellular coupling of nutrients and signaling molecules. During cancer progression, changes to substrate utilization occur to support energy production and biomass accumulation. This results in metabolic plasticity that promotes cell survival and proliferation, and can impact therapeutic resistance. Significant progress has been made in our understanding of connexin and cancer biology, however, delineating the roles these multi-faceted proteins play in metabolic adaptation of cancer cells is just beginning. Glucose represents a major carbon substrate for energy production, nucleotide synthesis, carbohydrate modifications and generation of biosynthetic intermediates. While cancer cells often exhibit a dependence on glycolytic metabolism for survival, cellular reprogramming of metabolic pathways is common when blood perfusion is limited in growing tumors. These metabolic changes drive aggressive phenotypes through the acquisition of functional traits. Connections between glucose metabolism and connexin function in cancer cells and the surrounding stroma are now apparent, however much remains to be discovered regarding these relationships. This review discusses the existing evidence in this area and highlights directions for continued investigation.

The development and advancement of aptamer technology has opened a new realm of possibilities for unlocking the biocomplexity available within proteomics. With ultra-high-throughput and multiplexing, alongside remarkable specificity and sensitivity, aptamers could represent a powerful tool in disease-specific research, such as supporting the discovery and validation of clinically relevant biomarkers. One of the fundamental challenges underlying past and current proteomic technology has been the difficulty of translating proteomic datasets into standards of practice. Aptamers provide the capacity to generate single panels that span over 7000 different proteins from a singular sample. However, as a recent technology, they also present unique challenges, as the field of translational aptamer-based proteomics still lacks a standardizing methodology for analyzing these large datasets and the novel considerations that must be made in response to the differentiation amongst current proteomic platforms and aptamers. We address these analytical considerations with respect to surveying initial data, deploying proper statistical methodologies to identify differential protein expressions, and applying datasets to discover multimarker and pathway-level findings. Additionally, we present aptamer datasets within the multi-omics landscape by exploring the intersectionality of aptamer-based proteomics amongst genomics, transcriptomics, and metabolomics, alongside pre-existing proteomic platforms. Understanding the broader applications of aptamer datasets will substantially enhance current efforts to generate translatable findings for the clinic.

Summary Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss., Graphical Abstract, Highlights • Dynamic intestinal crypt remodeling induced by short-term Notch inhibition • Intestinal stem cells are maintained but exhibit a transient loss of function • Paneth cells are rapidly lost by apoptotic cell death • A regenerative response replenishes Paneth cells to repair the crypt, Samuelson and colleagues demonstrate that intestinal stem cell (ISC) niche disruption by short-term Notch inhibition induces dynamic crypt cell remodeling. Surprisingly, Paneth cells were rapidly lost by apoptosis. Although ISCs were maintained, they exhibited a transient loss of function. A subsequent crypt regenerative response was observed with activation of multiple stem/progenitor cells to replenish the Paneth cell pool and return to homeostasis.

3046 Background: Extracellular Vesicles (EV) are of broad interest as carriers of molecular signatures of tumor progression and cancer treatment response. EVs, which contain nucleic acids, lipids, and proteins, are released from cells for waste excretion and communication. Numerous proteins and markers are expressed within and on the surface of EVs, but classification markers for murine EV subsets are lacking. To identify tumor and dendritic cell- derived EV markers for preclinical models of breast cancer, we investigated surface marker repertoires of EVs produced by the murine breast cancer and dendritic cell lines, 4T1 and DC2.4. Methods: Cells were cultured in serum free media for 2 days. EVs were harvested and isolated by ultrafiltration followed by size exclusion chromatography. EV particle size and concentration were estimated by nanoparticle tracking analysis and microBCA. To identify highly expressed EV markers, a mouse EV multiplex flow cytometry assay was performed using detection antibodies, CD9, CD63, and CD81, with sets of >35 barcoded capture beads, representing more than 100 specific capture: detection combinations. EV marker expression was analyzed using the FCMPASS/MPAPASS software (nano.ccr.cancer.gov). > 250 beads were assessed for each capture- and detection- antibody combination for each EV type and dilution tested; mean fluorescent intensity was determined; and pairwise comparisons between test and control sample sets were evaluated by t-tests. Results: Breast cancer (4T1)-derived EVs but not dendritic cell (DC2.4)-derived EVs were strongly detected with CD326 (EpCAM) and CD49b (integrin alpha5, VLA-2) capture beads, using each of the three tetraspanin antibodies. Both types of EVs were detected with anti-CD9 and anti-CD81 when captured by anti-CD44 and anti-CD49e (integrin beta1, VLA-5) beads. DC2.4 EVs were distinctively identified by CD11b capture. CD63 capture and detection antibodies robustly recognized EVs from 4T1 but provided minimal recognition of DC2.4 EVs. Mouse serum EVs from non-tumor bearing mice, showed minimal or no detectable CD326 or CD11b. Conclusions: Multiparametric MPAPASS-processed EV repertoire analysis of EVs from murine breast cancer and dendritic cell lines identified CD9, CD81, CD44, and CD49e as common epitopes among both types of evaluated EVs. CD326, CD49b, and CD63 distinguished 4T1 from DC2.4 EVs, and CD11b distinctively identified the DC2.4 EVs. The absence of detected CD326+ and CD11b+ in the serum of non-tumor bearing mice indicates the potential of these two markers for detection of specific tumor and antigen presenting cell EV subsets in serum from mice bearing CD326+ tumors such as 4T1. These results establish a foundation for further tests of detection and tracking of tumor-specific CD326+ EVs as "liquid biopsies" in blood samples as correlates to tumor progression and/or response to treatment.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPA

PURPOSE: The effectiveness of immune checkpoint inhibitors (ICIs) is limited in pancreatic ductal adenocarcinoma (PDAC). We conducted a phase I study to evaluate the safety of ICI with stereotactic body radiation therapy (SBRT) in patients with metastatic PDAC. PATIENTS AND METHODS: Patients enrolled must have received at least one line of prior systemic chemotherapy for metastatic disease. Cohort A1 and A2 received durvalumab every 2 weeks plus either 8Gy in one fraction of SBRT on day 1 or 25Gy in 5 fractions on day −3 to +1. Cohort B1 and B2 received durvalumab plus tremelimumab every 4 weeks and either 8Gy in one fraction of SBRT on day 1 or 25Gy in 5 fractions on day −3 to +1. ICIs were continued until unacceptable toxicity or disease progression. The primary objective was the safety and feasibility of treatment. Objective response was assessed in lesions not subjected to SBRT. RESULTS: Fifty-nine patients were enrolled and 39 were evaluable for efficacy. No dose limiting toxicities were seen. The most common adverse event was lymphopenia. Two patients achieved a partial response (one confirmed and the other unconfirmed). The overall response rate was 5.1%. Median PFS and OS was 1.7 months (95% CI 0.8–2.0 months) and 3.3 months (95% CI 1.2–6.6 months) in cohort A1; 2.5 months (95% CI 0.1–3.7 months) and 9.0 months (95% CI 0.5–18.4 months) in A2; 0.9 months (95% CI 0.7–2.1 months) and 2.1 months (95% CI 1.1–4.3 months) in B1; and 2.3 months (95% CI 1.9–3.4 months) and 4.2 months (95% CI 2.9–9.3 months) in B2. CONCLUSION: The combination of ICI and SBRT has an acceptable safety profile and demonstrates a modest treatment benefit in patients with metastatic PDAC.

The use of flow cytometry to analyze small particles has been implemented for several decades. More recently small particle analysis has become increasingly utilized owing to the increased sensitivity of conventional and commercially available flow cytometers along with growing interest in other small particles, such as extracellular vesicles. Despite an increase in small particle flow cytometry utilization, the lack of standardization in data reporting has resulted in a growing body of literature regarding extracellular vesicles (EVs) that cannot be easily interpreted, validated, or reproduced. Methods for fluorescence and light scatter standardization are well-established and the reagents to perform these analyses are commercially available. Here we outline FCM(PASS) as software package for performing fluorescence and light scatter calibration of small particles while generating standard reports conforming to the MIFlowCyt-EV standard reporting framework. This unit shall cover the workflow of implementing calibration using FCM(PASS) as follows: acquisition of fluorescence and light scatter calibration materials, cataloguing the reference materials for use in the software, creating cytometer databases and datasets to associate calibration data and fcs files, importing fcs files for calibration, inputting fluorescence calibration parameter, inputting light scatter calibration parameter, and applying the calibration to fcs files.

A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis.

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts. CA extern

Background & Aims: Loss of leucine-rich repeat-containing G-protein–coupled receptor 5–positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. Methods: Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator–ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. Results: Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter–sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10–Notch–dependent manner. Conclusions: Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model. Keywords: Defensin, Paneth Cell, Intestinal Stem Cells, Regeneration, Enteroid, Notch, Chemotherapy

Gene trap mutagenesis is a powerful tool to create loss-of-function mutations in mice and other model organisms. Modifications of traditional gene trap cassettes, including addition of conditional features in the form of Flip-excision (FlEx) arrays enabling directional gene trap cassette inversions by Cre and Flpe site-specific recombinases, greatly enhanced their experimental potential. By taking advantage of these conditional gene trap cassettes, we developed a generic strategy for generating conditional mutations and validated this strategy in mice carrying a multipurpose allele of the Prdm16 transcription factor gene. We demonstrate that the gene trap insertion creates a null mutation replicating the Pierre Robin sequence-type cleft palate phenotype of other Prdm16 mutant mice. Consecutive breeding to Flpe and Emx1IREScre deleter mice spatially restricted Prdm16 loss to regions of the forebrain expressing the homeobox gene Emx1 demonstrating the utility of the technology for the analysis of tissue-specific gene functions.

TO THE EDITOR Defects in melanocytes have been implicated in the etiology of a variety of human skin diseases and disorders (Lin and Fisher, 2007; Fistarol and Itin, 2010; Rees, 2011). There is long-standing interest in studying the development and dysfunction of human melanocytes, but there has not been a reliable and accessible system to study early events in human melanocyte differentiation. An in vitro system that reliably and efficiently produces normal human melanocytes from embryonic stage cells would allow us to better dissect the physiological and pathological development of melanocytes. Recent advances in stem cell biology have led to the establishment of human induced pluripotent stem cell (hiPSC) techniques that enable researchers to reprogram somatic cells to the pluripotent state (Takahashi et al., 2007). Differentiation of human and mouse pluripotent stem cells (PSCs) toward the melanocyte lineage has been reported (Yamane et al., 1999; Pla et al., 2005; Fang et al., 2006; Nissan et al., 2011; Ohta et al., 2011; Yang et al., 2011), but existing protocols have shortcomings that may limit their research and clinical applications. For example, the use of embryonic stem cells could lead to allogeneic immunoincompatibility of differentiated melanocytes and transplant recipients. In addition, the use of hiPSCs generated by integrative reprogramming strategies raises concerns about reactivation of retained transgenes, some of which are oncogenes. In addition, the current methods for melanocyte differentiation from hiPSCs require optimization in order to reproducibly generate high-purity melanocytes from multiple hiPSC lines. We have established a strategy to produce human melanocytes in vitro for use as a platform for pigment cell research and the development of cell-based therapies. We first derived transgene-free hiPSCs from two distinct types of skin cells: human primary melanocytes (HMs) and human dermal fibroblasts (HDF51) (Figure 1a and Supplementary Figure S1a online). We used a nonintegrative reprogramming approach mediated by Sendai virus–based vectors independently encoding POU5F1, SOX2, KLF4, and MYC (Fusaki et al., 2009; Macarthur et al., 2012). As shown in Figure 1b and Supplementary Figure S1b online, biomarkers of cellular pluripotency, including endogenous OCT4/POU5F1, NANOG, Tra-1-81, and UEA-I (Wang et al., 2011), were positive in HMi-506, HMi-503, and HDF51i-509 hiPSCs. Cells were also shown to be pluripotent using a gene expression diagnostic test (PluriTest; Muller et al., 2011), by differentiation into cells that express biomarkers relevant to all three germ layers in vitro (Figure 1c and Supplementary Figure S1c, S1d and S1e online) and by generation of teratomas (Supplementary Figure S1d online). Figure 1 Generation and differentiation of transgene-free human induced pluripotent stem cell (hiPSCs). (a) HMi-506 cells generated from human primary melanocyte (HM) cells using a Sendai virus–based reprogramming system were cocultured with mouse embryonic ... We newly developed two differentiation protocols based on previously reported methods. One protocol involves an aggregation-in-suspension step, whereas the other does not (Supplementary Figure S2 online). Both protocols generated cells displaying typical melanocyte morphology and pigmentation (Figure 1d) from hiPSCs after 30 days of directed differentiation, suggesting that the aggregation-in-suspension step is dispensable. The melanin granules that accumulated at the dendritic tips of differentiated cells were intensely stained by Fontana–Masson staining, indicating that the pigmentation of these cells was due to melanogenesis (Supplementary Figure S3 online). In addition, MITF (microphthalmia-associated transcription factor), a marker for melanocyte progenitors, was expressed in more than 90% of the differentiated derivatives after 30 days (Figure 1e and Supplementary Figure S4 online), which appears to be a higher differentiation efficiency than other reported protocols (Nissan et al., 2011; Ohta et al., 2011). As expected, MITF was not detected in the undifferentiated hiPSCs, and was present in the primary melanocytes (Figure 1e). Notably, our protocols resulted in similarly high levels of melanocyte differentiation for all four independent hiPSC lines examined, highlighting their reproducibility. Other melanocytic biomarkers including TYR (tyrosinase), MLANA (melan-A), TYRP1 (tyrosinase-related protein 1), PMEL (premelanosome protein), PAX3 (paired box 3), and SOX10 (SRY-box 10) were highly expressed in the differentiated derivatives (similar to primary melanocytes, Figure 2a and b). The melanin content and cell signaling involved in melanin production in the differentiated derivatives was increased by treatment with α-melanocyte-stimulating hormone (α-MSH) in a dose-dependent manner (Figure 2c and d and Supplementary Figure S5 online). These findings indicate that the differentiated derivatives possess molecular features of bona fide melanocytes and accurately mimic their ability to respond to α-MSH, which is the factor that activates melanogenesis and enhances skin pigmentation during the tanning response (Thody, 1999). Figure 2 Molecular and functional characterization of the melanocyte-like differentiated cells. (a) Heat map and dendrogram of melanocytic biomarkers showing that these transcripts were preferentially expressed in human primary melanocyte (HM) cells and HMi-506_Mel ... Genome-wide gene expression profiling and unsupervised hierarchical clustering revealed that the melanocytes (HMi-506_Mel Diff_1 and HMi-506_Mel Diff_2) differentiated from the HMi-506 cells were closely clustered with HMs and were distinct from all undifferentiated hiPSC samples (Figure 2e). As genetic abnormalities may occur in hiPSC genomes during the reprogramming and differentiation processes, we tested the genomic stability of the cells by comparing the differentiated derivatives with the parental primary melanocytes using high-resolution single-nucleotide polymorphism (SNP) genotyping and copy number variation analysis. As shown in Figure 2f, the HMi-506_Mel Diff derivatives and parental cells showed highly similar genotyping profiles, showing that the cellular genome remained stable during reprogramming and differentiation. Similar to human melanocytes in vivo, the differentiated derivatives in semiautologous skin reconstructs were located at the dermis–epidermis interface and interspersed with keratinocytes (Supplementary Figure S6a, S6b, S6c and S6d online), indicating their ability to integrate with the skin tissue of transplant recipients. Similar to the autologous dermal fibroblasts used for generating transgene-free hiPSCs, the differentiated derivatives stimulated limited proliferation of peripheral blood mononuclear cells that were isolated from the blood of the same individual in a mixed lymphocyte reaction assay (Supplementary Figure S6e online). These results attest to the clinical advantages of melanocytes differentiated from hiPSCs using the reprogramming and differentiation approaches described here. In this study, we have demonstrated that genetically stable melanocytes can be efficiently differentiated from transgene-free hiPSCs generated from two different types of cutaneous cells. This differentiation protocol takes less time than previously reported melanocytic differentiation protocols, and we showed that it is equally effective for multiple independent hiPSC lines. We performed a thorough investigation of the differentiated cells, including genome-wide gene expression analysis and SNP genotyping in addition to functional assays. Our approach can serve as an unlimited source of custom human melanocytes that can be used for novel approaches for modeling human skin disease (e.g., melanoma and vitiligo) and to provide material for transplantation.

The epithelium lining the gastrointestinal tract serves many functions, including aiding in digestion, nutrient absorption, barrier function, and immunity. In the small intestine, the intestinal epithelium is comprised of repeating crypt-villus units which undergo constant renewal to maintain intestinal homeostasis and tissue integrity (1). Multipotent intestinal stem cells (ISCs) located in the base of the crypt are responsible for constant renewal and rapid replenishment of cells lining the crypt-villus axis. ISCs give rise to rapidly proliferating transit-amplifying (TA) cell population which, as they exit crypt, differentiate into the four major mature cell types: enteroendocrine cells, goblet cells, and Paneth cells belong to the secretory lineage, while enterocytes comprise the absorptive lineage. Although it is agreed upon that each crypt contains ISCs, the exact identity of the stem cell population has been debated for many years (2,3).

Context:Few population-based studies have examined the incidence of meniscal injuries, and limited information is available on the influence of patient's demographic and occupational factors.Objective:To examine the incidence of meniscal injuries and the influence of demographic and occupational factors among active-duty US service members between 1998 and 2006.Design:Cohort study.Setting:Using the International Classification of Diseases (9th revision) codes 836.0 (medial meniscus), 836.1 (lateral meniscus), and 836.2 (meniscus unspecified), we extracted injury data from the Defense Medical Surveillance System to identify all acute meniscal injuries among active-duty military personnel.Patients or Other Participants:Active-duty military personnel serving in all branches of military service during the study period.Main Outcome Measure(s):Incidence rate (IR) per 1000 person-years at risk and crude and adjusted rates by strata for age, sex, race, rank, and service.Results:During the study period, 100201 acute meniscal injuries and 12115606 person-years at risk for injury were documented. The overall IR was 8.27 (95% confidence interval [CI] = 8.22, 8.32) per 1000 person-years. Main effects were noted for all demographic and occupational variables (P< .001), indicating that age, sex, race, rank, and service were associated with the incidence of meniscal injuries. Men were almost 20% more likely to experience an acute meniscal injury than were women (incidence rate ratio = 1.18, 95% CI = 1.15, 1.20). The rate of meniscal injury increased with age; those older than 40 years of age experienced injuries more than 4 times as often as those under 20 years of age (incidence rate ratio = 4.25,95% CI = 4.08, 4.42).Conclusions:The incidence of meniscal injury was sub-stantially higher in this study than in previously reported studies. Male sex, increasing age, and service in the Army or Marine Corps were factors associated with meniscal injuries.

OBJECTIVE: Recent epidemiologic studies found that 20% of subjects with the diagnosis of panic disorder had attempted suicide. This study sought to determine the prevalence of suicidal ideation and suicide attempts among patients with panic disorder and whether the presence of comorbid borderline personality disorder influenced the prevalence of suicidal thoughts and behavior. METHOD: At two outpatient clinics, experienced clinicians conducted retrospective reviews of data from the intake interviews and charts of 59 patients with panic disorder and comorbid borderline personality disorder and 234 patients with panic disorder with or without axis II disorders other than borderline personality disorder. RESULTS: Suicide attempts were reported by 2% of the patients with panic disorder, compared to 25% of the patients with both panic disorder and borderline personality disorder. In addition, 2% of the patients with panic disorder, compared to 27% of the patients with panic disorder and borderline personality disorder, reported suicidal ideation that was judged to be of clinical significance. CONCLUSIONS: The rate of suicidal ideation and suicide attempts for psychiatric outpatients with panic disorder was discrepant with the findings of the earlier studies. The increased suicide risk in this group of patients was associated with borderline personality disorder, increased substance abuse, and affective instability. While 61% of the panic disorder patients and 78% of the patients with both panic disorder and borderline personality disorder reported thinking about death, this must be distinguished from actual suicidal ideation and clinical risk of suicide. Language: en

In search of a more thorough understanding of Posttraumatic Stress Disorder (PTSD), theories of etiology have emerged from virtually every theoretical persuasion. Many provide a framework useful for understanding certain facets of the disorder. However, based on current knowledge, a model must be comprehensive enough to encompass the constellation of symptoms that comprise the disorder, the differential severity of symptoms, the presence of PTSD in some individuals but not others experiencing similar trauma, and recent empirical research bearing on these factors. In this paper, we review etiological models of PTSD and propose a new model based on a recent conceptualization of the process and origins of anxiety and panic. This model includes consideration of the role of biological and psychological vulnerabilities, negative life events, alarms (fear reactions), perceptions of control, social support, and coping strategies. Empirical research supporting the inclusion of these components is presented. Finally, etiological and phenomenological similarities between anxiety disorders, specifically panic disorder and PTSD, are discussed.

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.

The frequency of sexual imagery outside of sexual behavior was assessed. In addition, an attempt was made to determine the viability of a distinction between internally generated sexual thoughts (fantasy) and externally provoked sexual thoughts (urge). After a 3-day practice period, 49 male and 47 female heterosexual undergraduates self-monitored the frequency of fantasies, urges, and masturbatory fantasies for 7 consecutive days. The most significant finding was that men reported a significantly greater frequency of sexual urges and masturbatory fantasies than women. No gender differences emerged with respect to fantasies. These findings suggest that a distinction between internally and externally generated sexual imagery is viable and may be useful in future studies examining the frequency, functions, and determinants of sexual imagery.

Analysis of statewide survey data revealed that 22.5% of individuals with mental retardation who were served by the Oklahoma mental retardation system were receiving antipsychotic medication. Anxiolytic medications were prescribed for 9.3%, and antidepressants were prescribed for 5.9%. Higher levels of medication use were found in institutional settings and Intermediate Care Facility settings.

This research represents an initial attempt to extend Lang's (1979) bioinformational processing view of emotion to the realm of male sexuality-particularly psychogenic impotence. Sexually functional and sexually dysfunctional men received training in which they were shaped to incorporate physiological responses during imagery. Preceding and following this training, heart rate, penile tumescence, frontalis myotonia, and skin resistance were monitored while subjects imagined scenes differing in thematic content (fear, erotic, and neutral) and propositional structure (stimulus and/or response cues). In general, the results failed to replicate previous findings supporting this model. During fear scenes, only myotonia followed the predicted-inverted-V pattern in which an increase in responding occurred while listening to the scene, a greater response while imagining the scene, and a decrement in response once imagery ceased. Similar responding did not occur on heart rate, skin resistance, or tumescence. With the exception of myotonia, subjects failed to differentiate between the stimulus and the response cues embedded in scripts. Results are discussed in the context of Lang's (1979) model as it may apply to sexual dysfunction.