Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS 1b is widespread in Providencia spp. and encoded on the chromosome. T3SS 1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS 1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS 1a or T3SS 1b was seen in the calf infection model. However, T3SS 1b was required for the rapid killing of Drosophila melanogaster . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Machine learning for rhabdomyosarcoma histopathology.

Correctly diagnosing a rare childhood cancer such as sarcoma can be critical to assigning the correct treatment regimen. With a finite number of pathologists worldwide specializing in pediatric/young adult sarcoma histopathology, access to expert differential diagnosis early in case assessment is limited for many global regions. The lack of highly-trained sarcoma pathologists is especially pronounced in low to middle-income countries, where pathology expertise may be limited despite a similar rate of sarcoma incidence. To address this issue in part, we developed a deep learning convolutional neural network (CNN)-based differential diagnosis system to act as a pre-pathologist screening tool that quantifies diagnosis likelihood amongst trained soft-tissue sarcoma subtypes based on whole histopathology tissue slides. The CNN model is trained on a cohort of 424 centrally-reviewed histopathology tissue slides of alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma and clear-cell sarcoma tumors, all initially diagnosed at the originating institution and subsequently validated by central review. This CNN model was able to accurately classify the withheld testing cohort with resulting receiver operating characteristic (ROC) area under curve (AUC) values above 0.889 for all tested sarcoma subtypes. We subsequently used the CNN model to classify an externally-sourced cohort of human alveolar and embryonal rhabdomyosarcoma samples and a cohort of 318 histopathology tissue sections from genetically engineered mouse models of rhabdomyosarcoma. Finally, we investigated the overall robustness of the trained CNN model with respect to histopathological variations such as anaplasia, and classification outcomes on histopathology slides from untrained disease models. Overall positive results from our validation studies coupled with the limited worldwide availability of sarcoma pathology expertise suggests the potential of machine learning to assist local pathologists in quickly narrowing the differential diagnosis of sarcoma subtype in children, adolescents, and young adults.

Mental health diagnoses and early removal of long-acting reversible contraception.

BACKGROUND: Having a mental health diagnosis is associated with contraceptive non-adherence and user-related contraceptive failures of short-acting methods. There is a lack of research on the relationship between mental health diagnoses and early discontinuation of highly effective long-acting reversible (LARC) methods such as the intrauterine device (IUD) and subdermal implant (SDI). METHODS: Using a Primary Care and Obstetrics and Gynecology Patient Data Registry, we conducted a cross-sectional analysis of the relationship between any mental health diagnosis (any anxiety disorder or depression) and early LARC removal (<1 year post-insertion) among 385 reproductive-aged (14-50 years) women in 2008-16. Adjusted logistic regression was used to calculate odds ratios and 95% confidence intervals. RESULTS: Almost 10% (n = 37) of the sample had an early LARC removal, of which 62.2% were hormonal IUD and 37.8% were SDI. Women with a mental health diagnosis had a higher prevalence of early LARC removal (13.6% vs. 8.0%, p = =.090). Although non-significant, there was a trend in adjusted analyses indicating twice the odds of early removal for women with a mental health diagnosis versus no diagnosis (OR = =2.04, 95% CI = =0.97-4.27). LIMITATIONS: This study is limited by a small sample size and availability of variables from a reportable medical record database. Pregnancy intentions and side effects of method use could not be accounted for which may have impacted timing of removal. CONCLUSIONS: Understanding why women choose early LARC removal can inform counseling to help women make informed choices about their contraceptive method that meets their reproductive needs.

Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo.

BACKGROUND: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and Acinetobacter baumannii are active in vitro and in vivo. METHODS: PPMOs were evaluated in vitro using minimum inhibitory concentration (MIC) and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment. RESULTS: MICs of PPMOs ranged from 0.1 to 64 µM (approximately 0.6-38 µg/mL). The most effective PPMO tested was (RXR)4-AcpP, which is targeted to acpP. (RXR)4-AcpP reduced viability of A. lwoffii and A. baumannii by >10(3) colony-forming units/mL at 5-8 times MIC. Mice treated with ≥0.25 mg/kg of (RXR)4-AcpP survived longer and had less inflammation and bacterial lung burden than mice treated with a scrambled-sequence PPMO or phosphate-buffered saline. Treatment could be delayed after infection and still increase survival. CONCLUSIONS: PPMOs targeted to essential genes of A. lwoffii and A. baumannii were bactericidal and had MICs in a clinically relevant range. (RXR)4-AcpP increased survival of mice infected with A. lwoffii or A. baumannii, even when initial treatment was delayed after infection. PPMOs could be a viable therapeutic approach in dealing with multidrug-resistant Acinetobacter species.

Clinical Evaluation of the Articulinx Intercarpometacarpal Cushion for the First CMC Joint: A Feasibility Study.

Background The Intercarpometacarpal Cushion (ICMC; Articulinx, Cupertino, CA, USA) is an implantable spacer designed as a less invasive surgical treatment for osteoarthritis (OA) of the first carpometacarpal joint (CMC-1). Description of Technique Following local anesthesia and exposure of the joint capsule the ICMC, attached to a needle and suture tethers, is guided into the joint space under fluoroscopic visualization through a dorsal approach. The needle is pulled through the thenar eminence to the opposite side of the hand and, once proper device placement is confirmed, cut free and the joint capsule closed. Patients and Methods Eight female patients (median age 56 years; range, 42-83) were treated and followed for 6 to 24 months. Safety of the implant procedure was evaluated intraoperatively. Pain, joint function, and strength were evaluated at 6 weeks, 3, 6, 12 and 24 months with a Visual Analog Scale (VAS) for pain, the QuickDASH inventory, Canadian Occupational Performance Measure (COPM), and pinch and grip strength measurements. Results At 2 years (n = 6), mean VAS pain scores decreased from 6.3 (± 1.5) to 2.2 (± 1.1) (p < 0.001), mean QuickDASH scores improved from 47 (± 15) to 31 (± 11) (p < 0.10), mean COPM performance scores improved from 5.0 (± 1.2) to 5.5 ( ± 1.3) (p = NS). Mean pinch and grip strength measurements also improved compared with baseline. No serious adverse events occurred. Two device removals occurred, associated with a traumatic event and Stage IV OA with device displacement, at 6 and 9 months respectively. Conclusion The ICMC can be implanted safely. Effectiveness needs to be confirmed in future studies.

Protective effects of aspirin and vitamin E (alpha-tocopherol) against copper- and cadmium-induced toxicity.

A 24-h exposure to copper (400 microM, 600 microM) or cadmium (5 microM, 10 microM) significantly reduces the viability of COS-7 cells. A 2-h preincubation with vitamin E does not protect COS-7 cells from copper-induced toxicity, but does protect against cadmium-induced toxicity. Preincubation with aspirin protects cells from both copper- and cadmium-induced toxicity. A combination of aspirin and vitamin E (10 microM and 25 microM, respectively) increases cell viability in copper-exposed cells in a clearly additive manner, while in cadmium-exposed cells the effects are slightly additive. These results indicate that aspirin and vitamin E can protect cells from metal-induced toxicity. Differences in the protective effects of aspirin and vitamin E on copper versus cadmium-induced toxicity may be due to alternative mechanisms of metal toxicity or antioxidant activity.

PathMaster: content-based cell image retrieval using automated feature extraction.

OBJECTIVE: Currently, when cytopathology images are archived, they are typically stored with a limited text-based description of their content. Such a description inherently fails to quantify the properties of an image and refers to an extremely small fraction of its information content. This paper describes a method for automatically indexing images of individual cells and their associated diagnoses by computationally derived cell descriptors. This methodology may serve to better index data contained in digital image databases, thereby enabling cytologists and pathologists to cross-reference cells of unknown etiology or nature. DESIGN: The indexing method, implemented in a program called PathMaster, uses a series of computer-based feature extraction routines. Descriptors of individual cell characteristics generated by these routines are employed as indexes of cell morphology, texture, color, and spatial orientation. MEASUREMENTS: The indexing fidelity of the program was tested after populating its database with images of 152 lymphocytes/lymphoma cells captured from lymph node touch preparations stained with hematoxylin and eosin. Images of "unknown" lymphoid cells, previously unprocessed, were then submitted for feature extraction and diagnostic cross-referencing analysis. RESULTS: PathMaster listed the correct diagnosis as its first differential in 94 percent of recognition trials. In the remaining 6 percent of trials, PathMaster listed the correct diagnosis within the first three "differentials." CONCLUSION: PathMaster is a pilot cell image indexing program/search engine that creates an indexed reference of images. Use of such a reference may provide assistance in the diagnostic/prognostic process by furnishing a prioritized list of possible identifications for a cell of uncertain etiology.

Involvement of a pertussis toxin-sensitive G protein in the mitogenic signaling pathways of sphingosine 1-phosphate.

Sphingosine 1-phosphate, a sphingolipid metabolite, was previously reported to increase DNA synthesis in quiescent Swiss 3T3 fibroblasts and to induce transient increases in intracellular free calcium (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). In the present study, pretreatment of Swiss 3T3 fibroblasts with pertussis toxin reduced sphingosine 1-phosphate-induced DNA synthesis. Sphingosine 1-phosphate decreased cellular cAMP levels and also caused a drastic decrease in isoproterenol- and forskolin-stimulated cAMP accumulation. Pertussis toxin treatment prevented the inhibitory effect of sphingosine 1-phosphate on cAMP accumulation, suggesting that a pertussis toxin-sensitive Gi or Gi-like protein may be involved in sphingosine 1-phosphate-mediated inhibition of cAMP accumulation. Mitogenic concentrations of sphingosine 1-phosphate stimulated production of inositol phosphates which was inhibited by pertussis toxin, while the response to bradykinin was not affected. Furthermore, calcium release induced by sphingosine 1-phosphate, but not by bradykinin, was also attenuated by pertussis toxin treatment. However, sphingosine 1-phosphate-induced phosphatidic acid accumulation was unaffected by pertussis toxin. The increase in specific DNA binding activity of activator protein-1, which was induced by treatment of quiescent Swiss 3T3 fibroblasts with sphingosine 1-phosphate, was also inhibited by pertussis toxin. These results suggest that some of the sphingosine 1-phosphate-induced signaling pathways are mediated by G proteins that are substrates for pertussis toxin.

Stereospecificity of sphingosine-induced intracellular calcium mobilization and cellular proliferation.

Sphingosine is a positive regulator of cell growth in Swiss 3T3 fibroblasts (Zhang, H., Buckley, N. E., Gibson, K., and Spiegel, S. (1990) J. Biol. Chem. 265, 76-81). The present study investigated the stereospecificity of sphingosine-induced cell proliferation and its mitogenic signal transduction mechanisms. D-(+)-erythro Stereoisomers (cis and trans) stimulated DNA synthesis, whereas neither L-(-)-threo-sphingosine (cis or trans) nor DL-threo-dihydrosphingosine had any effect. Previously, we have shown that sphingosine-1-phosphate may mediate the mitogenic effect of sphingosine (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). However, no major differences were found in the formation of D-(+)-erythro- and L-(-)-threo- sphingosine-1-phosphate derived from the respective sphingosine isomers in intact cells. Thus, the stereospecificity of the response to sphingosine may reside at the level of specific intracellular targets for sphingosine-1-phosphate. Sphingosine-1-phosphate triggers dual signal transduction pathways of activation of phospholipase D leading to increases in the levels of phosphatidic acid and mobilization of calcium from internal stores. Both D-(+)-erythro- and L-(-)-threo-sphingosine isomers induced similar increases in phosphatidic acid concomitant with identical decreases in phosphatidylcholine levels. In contrast, only the D-(+)-erythro-stereoisomers (cis and trans) were effective in releasing calcium from intracellular stores. Our results suggest that the formation of phosphatidic acid is not sufficient to mediate sphingosine-stimulated DNA synthesis. However, the stereospecificity of the sphingosine-induced mobilization of calcium from internal stores seems to correlate with the induction of DNA synthesis by sphingosine stereoisomers.

Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway.

Sphingosine-1-phosphate, a metabolite of sphingolipids which has previously been shown to stimulate DNA synthesis and cell division in quiescent cultures of Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167), induced a transient increase in intracellular free calcium independent of extracellular calcium. The increase in calcium was completely abolished when intracellular calcium pools were depleted with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The dose-response for calcium release induced by sphingosine-1-phosphate correlated closely with the concentration required for stimulation of DNA synthesis. The magnitude of the calcium response decreased with successive challenges, although sphingosine-1-phosphate did not attenuate the responses to either bradykinin or ionomycin. Conversely, prior stimulation of the cells with bradykinin had no effect on the sphingosine-1-phosphate-induced calcium signal. Although sphingosine-1-phosphate increased inositol (1,4,5)-trisphosphate levels, complete inhibition of inositol phosphate formation by pretreatment with 12-O-tetradecanoylphorbol-13-acetate did not block sphingosine-1-phosphate-mediated calcium responses. Moreover, in permeabilized cells, heparin, an inositol (1,4,5)-trisphosphate antagonist, blocked Ca2+ release induced by inositol (1,4,5)-trisphosphate, but did not significantly alter the Ca2+ release induced by sphingosine-1-phosphate. Sphingosine-1-phosphate did not stimulate the release of arachidonic acid, another signaling molecule known to elevate [Ca2+]i without inositol lipid turnover or calcium influx. Our data suggest that sphingosine-1-phosphate mobilizes Ca2+ from internal stores primarily through a mechanism independent of inositol lipid hydrolysis and arachidonic acid release and that sphingolipid metabolism may be important in calcium homeostasis.

Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts.

Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-1-phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling pathways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-1-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-1-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-1-phosphate.

Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation.

Sphingosine and sphingosine-1-phosphate, metabolites of membrane sphingolipids, have recently been shown to stimulate release of calcium from internal sources and to increase proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in sphingosine-1-phosphate levels which precede the increase in the potent mitogen, phosphatidic acid. Sphingosine-1-phosphate itself induces a more rapid increase in phosphatidic acid, thus suggesting that it may mediate the effects of sphingosine on phosphatidic acid accumulation. The concentration dependence for the formation of phosphatidic acid induced by sphingosine-1-phosphate correlates with its effect on DNA synthesis. Similar to sphingosine, sphingosine-1-phosphate also stimulates the activity of phospholipase D, although a significant effect is observed at a much lower concentration. However, in contrast to previous reports with sphingosine, sphingosine-1-phosphate does not inhibit the phosphatidic acid phosphohydrolase activity in cell homogenates. Thus, in addition to its effect on mobilization of calcium, sphingosine-1-phosphate can increase the level of phosphatidic acid, most likely via activation of phospholipase D. We suggest that sphingosine-1-phosphate mediates the effect of sphingosine on phosphatidic acid accumulation in Swiss 3T3 fibroblasts and may regulate cellular proliferation by affecting multiple transmembrane signaling pathways.

Measuring morphine's capacity to establish a place preference.

A series of experiments are described providing an assessment of the procedures of conditioned place preference (CPP) testing involving an automated system having 12 separate chambers. Experiment 1 provides data to demonstrate (a) that in these chambers no initial preferences for one side over the other exists among rats, (b) that this neutrality of sides is not affected by session lengths between 15 and 60 min, and (c) that the optimal session length for tests in these chambers is on the order of 30 min. Experiment 2 demonstrates the stability of control groups' scores across a number of conditioning and testing sessions. Experiments 3 and 4 provide data to demonstrate (a) that a positive CPP can be established in our chambers using injections of morphine, (b) that a regimen of dosing with unequal numbers of days of putative and alternate conditioning is a reliable and conservative test of the opioid's ability to establish a CPP, and (c) that although the activity of rats decreases across a session, the general activity of rats before and after conditioning procedures is the same. Experiment 5 replicates the procedures employed by Scoles and Siegel (25) and demonstrates that the tendency for rats to explore novel environments is strong, and care must be taken to provide an opportunity for rats to pair different experiences with each side of the chamber in order for a CPP to emerge.

PCP and conditioned place preferences.

Phencyclidine (PCP), in doses of 0.25, 0.35, and 0.45 mg/kg, was administered systemically to male Sprague-Dawley rats in order to determine if a positive conditioned place preference (CPP) could be achieved. Other subjects received systemic injections of morphine, 4.0 mg/kg, as a standard for comparison. At testing, rats receiving 0.45 mg/kg PCP showed a positive CPP compared to controls, as did rats receiving morphine. Previous research had shown that larger doses of PCP and prolonged times after PCP administration produced aversion as indexed by CPP testing. The narrow dose range and short time span in which PCP's positively reinforcing properties are apt to emerge may be related to PCP's psychotomimetic potential and to its ability to sustain its own intake even though aversive effects are often manifest.

Ethanol with small doses of morphine establishes a conditioned place preference.

Conditioned place preference (CPP) testing is a way of indexing the reinforcing efficacy of drugs among rats. CPP testing involves using an alley with two distinctive sides. Typically, rats have drug experiences on one side and placebo experiences on the other. At testing, without drugs, their preference for side is tabulated. Rats' (6 groups of 12 each) place preferences were assessed before and after they were placed, once a day for 9 days, in the putative side of conditioning, and on 3 interspersed days, in the other side. During putative conditioning, one group received saline prior to being placed in both sides (a control group). Two groups had either morphine (2.0 mg/kg) or ethanol (0.5 g/kg) with the putative side of conditioning and saline with the other side. Three groups received morphine plus ethanol before being placed in the putative side of conditioning and either saline, morphine, or ethanol in the other side. At testing, rats that received morphine plus ethanol on side of putative conditioning showed a strong CPP whereas others did not. Results are compatible with the idea that ethanol's reinforcing effect is enhanced when there is a surfeit of opioidergic activity.