A DNA methylation atlas of normal human cell types.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.

Nucleotide deficiency promotes genomic instability in early stages of cancer development.

Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability.

Oral bacteria accelerate pancreatic cancer development in mice.

OBJECTIVE: Epidemiological studies highlight an association between pancreatic ductal adenocarcinoma (PDAC) and oral carriage of the anaerobic bacterium Porphyromonas gingivalis, a species highly linked to periodontal disease. We analysed the potential for P. gingivalis to promote pancreatic cancer development in an animal model and probed underlying mechanisms. DESIGN: We tracked P. gingivalis bacterial translocation from the oral cavity to the pancreas following administration to mice. To dissect the role of P. gingivalis in PDAC development, we administered bacteria to a genetically engineered mouse PDAC model consisting of inducible acinar cell expression of mutant Kras (Kras +/LSL-G12D; Ptf1a-CreER, iKC mice). These mice were used to study the cooperative effects of Kras mutation and P. gingivalis on the progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. The direct effects of P. gingivalis on acinar cells and PDAC cell lines were studied in vitro. RESULTS: P. gingivalis migrated from the oral cavity to the pancreas in mice and can be detected in human PanIN lesions. Repetitive P. gingivalis administration to wild-type mice induced pancreatic acinar-to-ductal metaplasia (ADM), and altered the composition of the intrapancreatic microbiome. In iKC mice, P. gingivalis accelerated PanIN to PDAC progression. In vitro, P. gingivalis infection induced acinar cell ADM markers SOX9 and CK19, and intracellular bacteria protected PDAC cells from reactive oxygen species-mediated cell death resulting from nutrient stress. CONCLUSION: Taken together, our findings demonstrate a causal role for P. gingivalis in pancreatic cancer development in mice.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.

OBJECTIVE: Cellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development. DESIGN: To uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment. RESULTS: We found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma. CONCLUSIONS: These findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

A single cell atlas of the human liver tumor microenvironment.

Malignant cell growth is fueled by interactions between tumor cells and the stromal cells composing the tumor microenvironment. The human liver is a major site of tumors and metastases, but molecular identities and intercellular interactions of different cell types have not been resolved in these pathologies. Here, we apply single cell RNA-sequencing and spatial analysis of malignant and adjacent non-malignant liver tissues from five patients with cholangiocarcinoma or liver metastases. We find that stromal cells exhibit recurring, patient-independent expression programs, and reconstruct a ligand-receptor map that highlights recurring tumor-stroma interactions. By combining transcriptomics of laser-capture microdissected regions, we reconstruct a zonation atlas of hepatocytes in the non-malignant sites and characterize the spatial distribution of each cell type across the tumor microenvironment. Our analysis provides a resource for understanding human liver malignancies and may expose potential points of interventions.

Rapid Clearing for High Resolution 3D Imaging of Ex Vivo Pancreatic Cancer Spheroids.

The currently accepted imaging methods have been a central hurdle to imaging the finer details of tumor behavior in three-dimensional (3D) ex vivo multicellular culture models. In our search for an improved way of imaging tumor behavior in its physiological-like niche, we developed a simple, efficient, and straightforward procedure using standard reagents and imaging equipment that significantly enhanced 3D imaging up to a ~200-micron depth. We tested its efficacy on pancreatic spheroids, prototypes of high-density tissues that are difficult to image. We found we could both save time with this method and extract information about pancreatic tumor spheroids that previously was difficult to obtain. We were able to discern clear differences in the organization of pancreatic tumor spheroids generated from different origins, suggesting cell-specific, inherent, bottom-up organization with a correlation to the level of malignancy. We also examined the dynamic changes in the spheroids at predetermined time points, providing important information related to tissue morphogenesis and its metabolic state. Lastly, this process enabled us to assess a drug vehicle's potential to penetrate dense tumor tissue by improving our view of the inert particles' diffusion in the 3D spheroid. This clearing method, a simple procedure, can open the door to more accurate imaging and reveal more about cancer behavior.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Efficiency of cell-free and cell-associated virus in mucosal transmission of human immunodeficiency virus type 1 and simian immunodeficiency virus.

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.

Senescent cancer-associated fibroblasts in pancreatic adenocarcinoma restrict CD8+ T cell activation and limit responsiveness to immunotherapy in mice.

Senescent cells within tumors and their stroma exert complex pro- and anti-tumorigenic functions. However, the identities and traits of these cells, and the potential for improving cancer therapy through their targeting, remain poorly characterized. Here, we identify a senescent subset within previously-defined cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinomas (PDAC) and in premalignant lesions in mice and humans. Senescent CAFs isolated from mouse and humans expressed elevated levels of immune-regulatory genes. Depletion of senescent CAFs, either genetically or using the Bcl-2 inhibitor ABT-199 (venetoclax), increased the proportion of activated CD8+ T cells in mouse pancreatic carcinomas, whereas induction of CAF senescence had the opposite effect. Combining ABT-199 with an immune checkpoint therapy regimen significantly reduced mouse tumor burden. These results indicate that senescent CAFs in PDAC stroma limit the numbers of activated cytotoxic CD8+ T cells, and suggest that their targeted elimination through senolytic treatment may enhance immunotherapy.

A fully 3D-printed versatile tumor-on-a-chip allows multi-drug screening and correlation with clinical outcomes for personalized medicine.

Optimal clinical outcomes in cancer treatments could be achieved through the development of reliable, precise ex vivo tumor models that function as drug screening platforms for patient-targeted therapies. Microfluidic tumor-on-chip technology is emerging as a preferred tool since it enables the complex set-ups and recapitulation of the physiologically relevant physical microenvironment of tumors. In order to overcome the common hindrances encountered while using this technology, a fully 3D-printed device was developed that sustains patient-derived multicellular spheroids long enough to conduct multiple drug screening tests. This tool is both cost effective and possesses four necessary characteristics of effective microfluidic devices: transparency, biocompatibility, versatility, and sample accessibility. Compelling correlations which demonstrate a clinical proof of concept were found after testing and comparing different chemotherapies on tumor spheroids, derived from ten patients, to their clinical outcomes. This platform offers a potential solution for personalized medicine by functioning as a predictive drug-performance tool.

Transesophageal endoscopic myotomy for achalasia: recognizing potential pitfalls before clinical application.

BACKGROUND: Laparoscopic Heller esophagomyotomy is the standard of care for achalasia treatment. This procedure, although effective, must be performed with the patient under general anesthesia and is associated with several serious potential complications. The authors aimed to develop a method of performing transesophageal endoscopic esophagomyotomy (TEEM) that would obviate the need for both general anesthesia and external incisions while offering lower intra- and postoperative complications. METHODS: The TEEM procedure was performed on eight pigs. For six of the pigs, the procedure aimed at survival. A mid-esophageal mucosal incision was performed using an endoscope, and a submucosal plane was developed. The lower esophageal sphincter (LES) muscle fibers were clearly visualized and divided. The mucosal incision was closed using fibrin sealant. After 2 weeks of survival, a gastrografin swallow study and necropsy were performed. RESULTS: The TEEM procedure was performed successfully in all eight porcine models. The myotomy included the LES fibers and extended 4 to 6 cm proximally to the esophagus. The proximal gastric muscle was divided up to 1 to 2 cm. No injuries to the abdominal or mediastinal structures occurred. One pig died on postoperative day 1 due to an unrecognized pneumothorax. Two pigs had ischemic ulcers at the myotomy site. The last three pigs had an uneventful recovery. The mucosal incision site healed completely in all the survived pigs, and except for the pig with mediastinal sepsis, all ate heartily and gained weight as expected. CONCLUSION: The TEEM procedure is technically feasible. Due to the morbidity encountered in the first three pigs, the reported technique was modified to include a slimmer endoscope, a shorter tunnel, and a partial-thickness myotomy. These changes together with an understanding of the pitfalls involved in this procedure led to successful results for the next three pigs. Nevertheless, the authors believe that TEEM is not yet ready for prime time. Perfection of the technique and development of dedicated instruments are mandatory before safe translation of this method to human patients.

Trauma care and case fatality during a period of frequent, violent terror attacks and thereafter.

BACKGROUND: From September 1999 through January 2004 during the second Intifada (al-Aqsa), there were frequent terror attacks in Jerusalem. We assessed the effects on case fatality of introducing a specialized, intensified approach to trauma care at the Hebrew University-Hadassah Hospital Shock Trauma Unit (HHSTU) and other level I Israeli trauma units. This approach included close senior supervision of prehospital triage, transport, and all surgical procedures and longer hospital stays despite high patient-staff ratios and low hospital budgets. Care for lower income patients also was subsidized. METHODS: We tracked case fatality rates (CFRs) initially during a period of terror attacks (1999-2003) in 8,127 patients (190 deaths) at HHSTU in subgroups categorized by age, injury circumstances, and injury severity scores (ISSs). Our comparisons were four other Israeli level I trauma centers (n = 2,000 patients), and 51 level I U.S. trauma centers (n = 265,902 patients; 15,237 deaths). Detailed HHSTU follow-up continued to 2010. RESULTS: Five-year HHSTU CFR (2.62 %) was less than half that in 51 U.S. centers (5.73 %). CFR progressively decreased; in contrast to a rising trend in the US for all age groups, injury types, and ISS groupings, including gunshot wounds (GSW). Patients with ISS > 25 accounted for 170 (89 %) of the 190 deaths in HHSTU. Forty-one lives were saved notionally based on U.S. CFRs within this group. However, far more lives were saved from reductions in low CFRs in large numbers of patients with ISS < 25. CFRs in HHSTU and other Israeli trauma units decreased more through the decade to 1.9 % up to 2010. CONCLUSIONS: Sustained reductions in trauma unit CFRs followed introduction of a specialized, intensified approach to trauma care.

Targeting Persistent Biofilm Infections: Reconsidering the Topography of the Infection Site during Model Selection.

The physiology of an organism in the environment reflects its interactions with the diverse physical, chemical, and biological properties of the surface. These principles come into consideration during model selection to study biofilm-host interactions. Biofilms are communities formed by beneficial and pathogenic bacteria, where cells are held together by a structured extracellular matrix. When biofilms are associated with a host, chemical gradients and their origins become highly relevant. Conventional biofilm laboratory models such as multiwall biofilm models and agar plate models poorly mimic these gradients. In contrast, ex vivo models possess the partial capacity to mimic the conditions of tissue-associated biofilm and a biofilm associated with a mineralized surface enriched in inorganic components, such as the human dentin. This review will highlight the progress achieved using these settings for two models of persistent infections: the infection of the lung tissue by Pseudomonas aeruginosa and the infection of the root canal by Enterococcus faecalis. For both models, we conclude that the limitations of the conventional in vitro systems necessitate a complimentary experimentation with clinically relevant ex vivo models during therapeutics development.

Methionine aminopeptidase 2 as a potential target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive metastatic cancer with a very low survival rate. This tumor is hypovascularized and characterized by severe hypoxic regions, yet these regions are not impeded by the oxidative stress in their microenvironment. PDA's high resilience raises the need to find new effective therapeutic targets. This study investigated the suitability of methionine aminopeptidase 2 (MetAp2), a metallopeptidase known to play an important role in tumor progression, as a new target for treating PDA. In our examination of patient-derived PDA tissues, we found that MetAp2 is highly expressed in metastatic regions compared with primary sites. At the cellular level, we found that the basal expression levels of MetAp2 in pancreatic cancer cells were higher than its levels in endothelial cells. Pancreatic cancer cells showed a significant suppression of proliferation in a dose-dependent manner upon exposure to TNP-470, a selective MetAp2 inhibitor. In addition, a significant reduction in glutathione (GSH) levels - known for its importance in alleviating oxidative stress - was detected in all treated cells, suggesting a possible anti-cancer activity mechanism that would be feasible for treating highly hypoxic PDA tumors. Furthermore, in an orthotopic pancreatic cancer murine model, systemic oral treatment with a MetAp2 inhibitor significantly reduced tumors' growth. Taken together, our findings indicate that MetAp2 enhances tumor sensitivity to hypoxia and may provide an effective target for treating hypoxic tumors with high expression levels of MetAp2.

Calcium carbonate mineralization is essential for biofilm formation and lung colonization.

Biofilms are differentiated microbial communities held together by an extracellular matrix. µCT X-ray revealed structured mineralized areas within biofilms of lung pathogens belonging to two distant phyla - the proteobacteria Pseudomonas aeruginosa and the actinobacteria Mycobacterium abscessus. Furthermore, calcium chelation inhibited the assembly of complex bacterial structures for both organisms with little to no effect on cell growth. The molecular mechanisms promoting calcite scaffold formation were surprisingly conserved between the two pathogens as biofilm development was similarly impaired by genetic and biochemical inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. This work offers underexplored druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

Efficient transgene expression from naked DNA delivered into an arterio-venous fistula model for kidney dialysis.

BACKGROUND: Patients with kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which a vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external 'artificial kidney'. In most patients, neo-intimal hyperplasia (NIH) ensues, causing narrowing and subsequent occlusion of the vein, leading to significant morbidity. The cellular events causing venous NIH may serve as ideal targets for molecular-based therapies. However, therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery. MATERIALS AND METHODS: To explore the feasibility of a plasmid-based vascular gene delivery system, we established a rat AVF model that develops NIH. Plasmids encoding for reporter or therapeutic genes were delivered into the blood vessels at the time or after AVF formation. RESULTS: Intra-luminal injection of plasmid into the AVF resulted in extensive and long-term reporter gene expression at the venous limb mainly at the site of NIH formation. Transgene expression was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia and form the NIH lesion. There was no detrimental tissue reaction to plasmid delivery, contrasting with the severe inflammatory response observed after adenovirus infection. Intra-vascular delivery of a plasmid carrying the endothelial nitric oxide synthase gene resulted in sustained production of nitric oxide, previously shown to mitigate NIH formation. CONCLUSIONS: These findings open the possibility of vascular transduction with naked DNA bearing therapeutic genes in areas prone to NIH to ameliorate vein graft pathologies using simple and clinically applicable vector delivery methods.

Posttraumatic stress disorder in hospitalized terrorist bombing attack victims.

BACKGROUND: Posttraumatic stress disorder (PTSD) is a psychiatric disorder that results from exposure to a traumatic event and consists of intrusive and unwanted recollections; avoidance followed by emotional withdrawal; and heightened physiologic arousal. Hospitalized victims of suicide bombing attacks (SBAs) are unique because of the circumstances and severity of their injuries, which could affect the occurrence and delay the recognition of PTSD. Our objectives were to evaluate the prevalence and severity of PTSD among hospitalized SBA victims and to assess variables of physical injury as risk factors for the development of PTSD. METHODS: Forty-six hospitalized SBA victims were evaluated for PTSD using the PTSD symptom scale self-report questionnaire by phone. Demographic and medical data regarding the severity and type of injury and medical treatment were collected from medical files. Injury Severity Score was used to assess severity of physical injury. RESULTS: Twenty-four of 46 (52.2%) hospitalized SBA victims developed PTSD. Presence of blast lung injury was significantly higher in the PTSD group compared with the non-PTSD group (37.5% versus 9.1%, respectively; p < 0.04). There was no significant difference in Injury Severity Score between PTSD and non-PTSD groups. Blast lung injury and intracranial injury were found to be positive predictors of PTSD (odds ratio, 125 and 25, respectively). No correlation was found between the length of stay, length of intensive care unit stay, or severity of physical injuries and the severity of PTSD. CONCLUSIONS: Hospitalized victims of SBA are considerably vulnerable to develop PTSD. Victims should be monitored closely and treated in conjunction with their physical treatment. Blast lung injury and intracranial injury are predictors of PTSD.

A mouse model for sleeve gastrectomy: applications for diabetes research.

INTRODUCTION: Discovery of enhanced glucose tolerance following bariatric surgery has sparked renewed interest in the investigation of unchartered underlying pathways of glucose homeostasis. Delineation of this pathway may ultimately be the first step in the creation of a novel therapy for type II diabetes. Nevertheless, the technical complexity and formidable nature of these surgeries coupled with the fragile nature of small rodents has made the creation of a mouse model to study these effects incredibly challenging. We have created a simplified sleeve gastrectomy mouse model to study the effects of bariatric surgery on glucose tolerance and beta cell proliferation. METHODS: Nineteen mice were randomized to undergo either sleeve gastrectomy (SG) (9) or sham operation (SH) (10). Weight and serum glucose were measured three times weekly and serum insulin measurements and pancreatic harvest were performed at the time of sacrifice. Five mice from each group were sacrificed after one week and the remainder sacrificed after one month. RESULTS: Survival of mice was 100% for both groups. The SG group demonstrated an initial drop in weight and serum glucose as compared to SH, which normalized by one month following surgery. Serum insulin levels and rate of beta cell proliferation were similar in both groups after one week and one month. CONCLUSION: The simplified sleeve gastrectomy is a technically straightforward, low-mortality technique for creating a bariatric mouse model which most faithfully replicates bariatric surgery performed in humans. This model can be a valuable tool to investigate the glucose tolerance and beta cell effects of bariatric surgery.

Pure "cable-tie partial nephrectomy": a porcine model.

BACKGROUND: The standard surgical procedure for polar renal tumors is nephron-sparing surgery, currently performed laparoscopically in most cases. This technically challenging surgery involves clamping of the hilar vessels and warm kidney ischemia. This study tested the feasibility of performing polar nephrectomy using a cable tie without hilar clamping or any other hemostatic procedure. METHODS: General anesthesia and pneumoperitoneum were induced in four farm pigs, and three trocars were inserted intraperitoneally. A kidney pole (either a lower or an upper pole of either the left or right kidney) was exposed and encircled with a plastic cable tie. The pole then was transected sharply with scissors, opening the urinary collecting system and major blood vessels. Neither tissue sealants nor any form of energy was used throughout the cases. The animals were killed 3 weeks later. RESULTS: All the pigs tolerated the surgery well. The renal part of the surgery required less than 12 min, and blood loss was minimal (<30 ml) for all the animals. The postoperative course was benign. Laparotomy performed 3 weeks later showed no peritoneal or retroperitoneal abnormalities. The plastic cable tie remained in the same position covered with a thin layer of mesothelial cells. A thin sector of coagulation necrosis was found under the pressure of the cable. CONCLUSIONS: In a porcine model, polar nephrectomy using a plastic cable tie is a very easy and rapid procedure. Cheap and bloodless, it eliminates the need for renal reconstruction. Hilar dissection and renal ischemia are avoided. "Cable-tie partial nephrectomy" may become a method for removing a tumor-bearing renal pole.

Providing more through less: current methods of retraction in SIMIS and NOTES cholecystectomy.

BACKGROUND: As the field of minimally invasive surgery continues to develop, surgeons are confronted with the challenge of performing conventional laparoscopic surgeries through fewer incisions while maintaining the same degree of safety and surgical efficiency. Most of these methods involve elimination of the ports previously designated for retraction. As a result, minimally invasive surgeons have been forced to develop minimally invasive and ingenious methods for providing adequate retraction for these procedures. Herein we present our experience using endoloops and internal retractors to provide retraction during Single Incision Minimally Invasive Surgery (SIMIS) and Natural Orifice Transluminal Endoscopic Surgery (NOTES) cholecystectomy. We also present a review of the alternative retraction methods currently being employed for these surgeries. METHODS: SIMIS was performed on 20 patients and NOTES was performed on 5 patients at our institution. Endoloops or internal retractors were used to provide retraction for all SIMIS procedures. Internal retractors provided retraction for all NOTES procedures. RESULTS: Successful cholecystectomy was accomplished in all cases. One SIMIS surgery required conversion to standard laparoscopy due to complex anatomy. There were no intraoperative complications. Although adequate retraction was accomplished in all cases, the internal retractors were found to provide superior and more versatile retraction compared to that of endoloops. CONCLUSION: Adequate retraction greatly simplifies SIMIS and NOTES surgery. Endograb internal retractors were easy to use and were found to provide optimal retraction and exposure during these procedures without complications.