Axial Skeletal Malformations in Genetically Modified Xenopus laevis and Xenopus tropicalis.

Skeletal malformations in captive-bred, adult Xenopus spp., have not previously been reported. Here we describe 10 sexually mature, genetically modified laboratory frogs (6 Xenopus laevis and 4 Xenopus tropicalis) with axial skeletal abnormalities. The young adult frogs were described by veterinary staff as presenting with "hunchbacks," but were otherwise considered to be in good health. All affected frogs were genetically engineered using various techniques: transcription activator-like effector nucleases (TALEN) editing using thyroid hormone receptor α TALEN mRNA, restriction enzyme-mediated integration methods involving insertion of the inducible transgene pCAR/TRDN, or via I-SceI meganuclease transgenesis using either pDRTREdpTR-HS4 or pDPCrtTA-TREG-HS4 plasmid sequences. Radiographic findings (6 frogs) and gross necropsy (10 frogs) revealed vertebral column malformations and sacroiliac deformities that resulted in moderate to severe kyphosis and kyphoscoliosis. These findings were confirmed and additional skeletal abnormalities were identified using computed tomography to create a 3D reconstruction of 4 frogs. Additional findings visible on the 3D reconstructions included incomplete vertebral segmentation, malformed transverse processes, and a short and/or curved urostyle. Histopathologic findings included misshapen intervertebral joints with nonconforming articular surfaces, narrowed joint cavities, flattened or irregularly-formed articular cartilage, irregular maturation lines and nonpolarized chondrocytes, excess fibrocartilage, and evidence of irregular bone resorption and growth. While the specific etiology of the vertebral skeletal abnormalities remains unclear, possibilities include: 1) egg/oocyte physical manipulation (dejellying, microinjection, fertilization, etc.), 2) induction and expression of the transgenes, 3) inactivation (knockout) of existing genes by insertional mutagenesis, or 4) a combination of the above. Furthermore, the possibility of undetected changes in the macro or microenvironment, or a feature of the genetic background of the affected frogs cannot be ruled out.

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

Histologic evaluation of activation of acute inflammatory response in a mouse model following ultrasound-mediated blood-brain barrier using different acoustic pressures and microbubble doses.

Rationale: Localized blood-brain barrier (BBB) opening can be achieved with minimal to no tissue damage by applying pulsed focused ultrasound alongside a low microbubble (MB) dose. However, relatively little is known regarding how varying treatment parameters affect the degree of neuroinflammation following BBB opening. The goal of this study was to evaluate the activation of an inflammatory response following BBB opening as a function of applied acoustic pressure using two different microbubble doses. Methods: Mice were treated with 650 kHz ultrasound using varying acoustic peak negative pressures (PNPs) using two different MB doses, and activation of an inflammatory response, in terms of microglial and astrocyte activation, was assessed one hour following BBB opening using immunohistochemical staining. Harmonic and subharmonic acoustic emissions (AEs) were monitored for all treatments with a passive cavitation detector, and contrast-enhanced magnetic resonance imaging (CE-MRI) was performed following BBB opening to quantify the degree of opening. Hematoxylin and eosin-stained slides were assessed for the presence of microhemorrhage and edema. Results: For each MB dose, BBB opening was achieved with minimal activation of microglia and astrocytes using a PNP of 0.15 MPa. Higher PNPs were associated with increased activation, with greater increases associated with the use of the higher MB dose. Additionally, glial activation was still observed in the absence of histopathological findings. We found that CE-MRI was most strongly correlated with the degree of activation. While acoustic emissions were not predictive of microglial or astrocyte activation, subharmonic AEs were strongly associated with marked and severe histopathological findings. Conclusions: Our study demonstrated that there were mild histologic changes and activation of the acute inflammatory response using PNPs ranging from 0.15 MPa to 0.20 MPa, independent of MB dose. However, when higher PNPs of 0.25 MPa or above were applied, the same applied PNP resulted in more severe and widespread histological findings and activation of the acute inflammatory response when using the higher MB dose. The potential activation of the inflammatory response following ultrasound-mediated BBB opening should be considered when treating patients to maximize therapeutic benefit.

Histologic safety of transcranial focused ultrasound neuromodulation and magnetic resonance acoustic radiation force imaging in rhesus macaques and sheep.

BACKGROUND: Neuromodulation by transcranial focused ultrasound (FUS) offers the potential to non-invasively treat specific brain regions, with treatment location verified by magnetic resonance acoustic radiation force imaging (MR-ARFI). OBJECTIVE: To investigate the safety of these methods prior to widespread clinical use, we report histologic findings in two large animal models following FUS neuromodulation and MR-ARFI. METHODS: Two rhesus macaques and thirteen Dorset sheep were studied. FUS neuromodulation was targeted to the primary visual cortex in rhesus macaques and to subcortical locations, verified by MR-ARFI, in eleven sheep. Both rhesus macaques and five sheep received a single FUS session, whereas six sheep received repeated sessions three to six days apart. The remaining two control sheep did not receive ultrasound but otherwise underwent the same anesthetic and MRI procedures as the eleven experimental sheep. Hematoxylin and eosin-stained sections of brain tissue (harvested zero to eleven days following FUS) were evaluated for tissue damage at FUS and control locations as well as tissue within the path of the FUS beam. TUNEL staining was used to evaluate for the presence of apoptosis in sheep receiving high dose FUS. RESULTS: No FUS-related pre-mortem histologic findings were observed in the rhesus macaques or in any of the examined sheep. Extravascular red blood cells (RBCs) were present within the meninges of all sheep, regardless of treatment group. Similarly, small aggregates of perivascular RBCs were rarely noted in non-target regions of neural parenchyma of FUS-treated (8/11) and untreated (2/2) sheep. However, no concurrent histologic abnormalities were observed, consistent with RBC extravasation occurring as post-mortem artifact following brain extraction. Sheep within the high dose FUS group were TUNEL-negative at the targeted site of FUS. CONCLUSIONS: The absence of FUS-related histologic findings suggests that the neuromodulation and MR-ARFI protocols evaluated do not cause tissue damage.

Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice.

Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive lean weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness, they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role in sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute to chronic cachexia development, pathology and potential reversal.

A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection.

The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.

Microbiota-accessible carbohydrates suppress Clostridium difficile infection in a murine model.

Clostridium difficile is an opportunistic diarrhoeal pathogen, and C. difficile infection (CDI) represents a major health care concern, causing an estimated 15,000 deaths per year in the United States alone 1 . Several enteric pathogens, including C. difficile, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut 2 . Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota-accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant effect on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that C. difficile burdens are suppressed through the addition of either a diet containing a complex mixture of MACs or a simplified diet containing inulin as the sole MAC source. We show that switches between these dietary conditions are coincident with changes to microbiota membership, its metabolic output and C. difficile-mediated inflammation. Together, our data demonstrate the outgrowth of MAC-utilizing taxa and the associated end products of MAC metabolism, namely, the short-chain fatty acids acetate, propionate and butyrate, are associated with decreased C. difficile fitness despite increased C. difficile toxin expression in the gut. Our findings, when placed into the context of the known fibre deficiencies of a human Western diet, provide rationale for pursuing MAC-centric dietary strategies as an alternate line of investigation for mitigating CDI.

Acute and Chronic Outcomes of Gas-Bubble Disease in a Colony of African Clawed Frogs (Xenopus laevis).

Gas-bubble disease occurs in aquatic species that are exposed to water that is supersaturated with gases. In February 2007, municipal water supersaturated with gas was inadvertently pumped into the vivarium's aquatic housing systems and affected approximately 450 adult female Xenopus laevis. The inflow of supersaturated water was stopped immediately, the holding tanks aggressively aerated, and all experimental manipulations and feeding ceased. Within the first 6 h after the event, morbidity approached 90%, and mortality reached 3.5%. Acutely affected frogs showed clinical signs of gas-bubble disease: buoyancy problems, micro- and macroscopic bubbles in the foot webbing, hyperemia in foot webbing and leg skin, and loss of the mucous slime coat. All of the frogs that died or were euthanized had areas of mesenteric infarction, which resulted in intestinal epithelial necrosis and degeneration of the muscular tunic. Over the subsequent 2 wk, as gas saturation levels returned to normal, the clinical symptoms resolved completely in the remaining frogs. However, 3 mo later, 85% of them failed to lay eggs or produce oocytes, and the remaining 15% produced oocytes of low number and poor quality, yielding cytosolic extracts with poor to no enzymatic activity. Histology of the egg mass from a single 2- to 3-y-old frog at 3 mo after disease resolution revealed irregularly shaped oocytes, few large mature oocytes, and numerous small, degenerating oocytes. At 6 mo after the incident, the remaining frogs continued to fail to produce eggs of sufficient quantity or quality after hormonal priming. The researchers consequently opted to cull the remainder of the colony and repopulate with new frogs.

Endoluminal ultrasound applicators for MR-guided thermal ablation of pancreatic tumors: Preliminary design and evaluation in a porcine pancreas model.

PURPOSE: Endoluminal ultrasound may serve as a minimally invasive option for delivering thermal ablation to pancreatic tumors adjacent to the stomach or duodenum. The objective of this study was to explore the basic feasibility of this treatment strategy through the design, characterization, and evaluation of proof-of-concept endoluminal ultrasound applicators capable of placement in the gastrointestinal (GI) lumen for volumetric pancreas ablation under MR guidance. METHODS: Two variants of the endoluminal applicator, each containing a distinct array of two independently powered transducers (10 × 10 mm 3.2 MHz planar; or 8 × 10 × 20 mm radius of curvature 3.3 MHz curvilinear geometries) at the distal end of a meter long flexible catheter assembly, were designed and fabricated. Transducers and circulatory water flow for acoustic coupling and luminal cooling were contained by a low-profile polyester balloon covering the transducer assembly fixture. Each applicator incorporated miniature spiral MR coils and mechanical features (guiding tips and hinges) to facilitate tracking and insertion through the GI tract under MRI guidance. Acoustic characterization of each device was performed using radiation force balance and hydrophone measurements. Device delivery into the upper GI tract, adjacent to the pancreas, and heating characteristics for treatment of pancreatic tissue were evaluated in MR-guided ex vivo and in vivo porcine experiments. MR guidance was utilized for anatomical target identification, tracking/positioning of the applicator, and MR temperature imaging (MRTI) for PRF-based multislice thermometry, implemented in the real-time RTHawk software environment. RESULTS: Force balance and hydrophone measurements indicated efficiencies of 48.8% and 47.8% and -3 dB intensity beam-widths of 3.2 and 1.2 mm for the planar and curvilinear transducers, respectively. Ex vivo studies on whole-porcine carcasses revealed capabilities of producing ablative temperature rise (ΔT > 15 °C) contours in pancreatic tissue 4-40 mm long and 4-28 mm wide for the planar transducer applicator (1-13 min sonication duration, ∼4 W/cm(2) applied acoustic intensity). Curvilinear transducers produced more selective heating, with a narrower ΔT > 15 °C contour length and width of up to 1-24 mm and 2-7 mm, respectively (1-7 min sonication duration, ∼4 W/cm(2) applied acoustic intensity). Active tracking of the miniature spiral coils was achieved using a Hadamard encoding tracking sequence, enabling real-time determination of each coil's coordinates and automated prescription of imaging planes for thermometry. In vivo MRTI-guided heating trials in three pigs demonstrated capability of ∼20 °C temperature elevation in pancreatic tissue at 2 cm depths from the applicator, with 5-7 W/cm(2) applied intensity and 6-16 min sonication duration. Dimensions of thermal lesions in the pancreas ranged from 12 to 28 mm, 3 to 10 mm, and 5 to 10 mm in length, width, and depth, respectively, as verified through histological analysis of tissue sections. Multiple-baseline reconstruction and respiratory-gated acquisition were demonstrated to be effective strategies in suppressing motion artifacts for clear evolution of temperature profiles during MRTI in the in vivo studies. CONCLUSIONS: This study demonstrates the technical feasibility of generating volumetric ablation in pancreatic tissue using endoluminal ultrasound applicators positioned in the stomach lumen. MR guidance facilitates target identification, device tracking/positioning, and treatment monitoring through real-time multislice PRF-based thermometry.

Biochemical and Hematologic Reference Intervals for Aged Xenopus laevis in a Research Colony.

Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs.

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

Helicobacter pylori Activates and Expands Lgr5(+) Stem Cells Through Direct Colonization of the Gastric Glands.

BACKGROUND & AIMS: Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. METHODS: We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. RESULTS: H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. CONCLUSIONS: H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.

Gut microbiota-produced succinate promotes C. difficile infection after antibiotic treatment or motility disturbance.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea. The mechanisms underlying C. difficile expansion after microbiota disturbance are just emerging. We assessed the gene expression profile of C. difficile within the intestine of gnotobiotic mice to identify genes regulated in response to either dietary or microbiota compositional changes. In the presence of the gut symbiont Bacteroides thetaiotaomicron, C. difficile induces a pathway that metabolizes the microbiota fermentation end-product succinate to butyrate. The low concentration of succinate present in the microbiota of conventional mice is transiently elevated upon antibiotic treatment or chemically induced intestinal motility disturbance, and C. difficile exploits this succinate spike to expand in the perturbed intestine. A C. difficile mutant compromised in succinate utilization is at a competitive disadvantage during these perturbations. Understanding the metabolic mechanisms involved in microbiota-C. difficile interactions may help to identify approaches for the treatment and prevention of C. difficile-associated diseases.

m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.

N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.

Role of disease-associated tolerance in infectious superspreaders.

Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens.

Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.

Loss of anterior gradient 2 (Agr2) expression results in hyperplasia and defective lineage maturation in the murine stomach.

Recent studies of epithelial tissues have revealed the presence of tissue-specific stem cells that are able to establish multiple cell lineages within an organ. The stem cells give rise to progenitors that replicate before differentiating into specific cell lineages. The mechanism by which homeostasis is established between proliferating stem or progenitor cells and terminally differentiated cells is unclear. This study demonstrates that Agr2 expression by mucous neck cells in the stomach promotes the differentiation of multiple cell lineages while also inhibiting the proliferation of stem or progenitor cells. When Agr2 expression is absent, gastric mucous neck cells increased in number as does the number of proliferating cells. Agr2 expression loss also resulted in the decline of terminally differentiated cells, which was supplanted by cells that exhibited nuclear SOX9 labeling. Sox9 expression has been associated with progenitor and stem cells. Similar effects of the Agr2 null on cell proliferation in the intestine were also observed. Agr2 consequently serves to maintain the balance between proliferating and differentiated epithelial cells.

Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1.

Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent interferon-ß production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.

A novel clinically translatable fluorescent nanoparticle for targeted molecular imaging of tumors in living subjects.

The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin α(v)ß(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future.

Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality.

Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.