Unidirectional mannitol synthesis of Acinetobacter baumannii MtlD is facilitated by the helix-loop-helix-mediated dimer formation.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix­loop­helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

Improving diagnosis of early complications (<1 week) through continuous vital sign monitoring following oncological gastrointestinal surgical procedures.

BACKGROUND: Patients undergoing major oncological abdominal surgery are prone to postoperative complications, making early recognition crucial. Clinical deterioration is often preceded by changes in vital signs, which are typically measured thrice a day by a nurse. However, intermittent measurements may delay recognizing clinical deterioration. Continuous vital parameter monitoring may lead to earlier recognition and management of complications and reduce nursing workload. OBJECTIVE: To compare vital parameter measurements between ward nurses and a wireless continuous monitoring system (Sensium® wireless patch) and assess whether this patch can detect clinical deterioration earlier in patients with complications in the first postoperative week. METHODS: Vital parameters (heart rate, respiratory rate, and temperature) were collected in patients undergoing an oncological resection of the liver, colorectal, or pancreas. Sensium® patch measurements were compared to nurses' measurements to assess the percentages of discordant measurements. In patients with complications in the first postoperative week, time discrepancies between nurses and Sensium® patch measurements were identified in cases of clinical deterioration (respiratory rate ≥15/min, heart rate ≥100/min, and temperature ≥38°C). RESULTS: Among 227 patients, 22% of the patients experienced complications. Nurse and Sensium® measurements were discrepant in 586/2272 measurements (26%). In 506/586 discrepancies (86%), this was due to the respiratory rate (difference ≥4/min). Compared to nurses, the Sensium® patch detected an elevated respiratory rate 14 h earlier and heart rate 2 h earlier within complications in the first postoperative week. For temperature, no difference was observed. CONCLUSION: Continuous monitoring with the Sensium® wireless patch holds promise for earlier recognition of complications in patients who underwent major oncological abdominal surgery.

Membrane-anchored substrate binding proteins are deployed in secondary TAXI transporters.

Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na+-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria.

Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components-the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.

Barriers and facilitators for treatment-seeking in adults with a depressive or anxiety disorder in a Western-European health care setting: a qualitative study.

BACKGROUND: Previous research on barriers and facilitators regarding treatment-seeking of adults with depressive and anxiety disorders has been primarily conducted in the Anglosphere. This study aims to gain insight into treatment-seeking behaviour of adults with depressive and anxiety disorders in a European healthcare system. METHODS: In-depth semi-structured interviews were conducted with 24 participants, aged ≥18 years and diagnosed with an anxiety disorder and/or depressive disorder according to DSM-IV. Participants were purposively sampled from an outpatient department for mental health care in the Netherlands. The seven steps of framework analysis were used to identify relevant themes emerging from the interviews. RESULTS: Data analysis suggested an interplay between individual aspects, personal social system, healthcare system and sociocultural context influences. Amongst the most relevant themes were mental health illiteracy, stigma, a negative attitude toward professional help, the influence of significant others and general practitioner, and waiting time. Financial barriers were not of relevance. CONCLUSIONS: Even in a country with a well-developed mental health care system and in absence of financial barriers, there are many barriers to treatment-seeking in adult patients with depressive and anxiety disorders. National campaigns to increase awareness and decrease stigma in the general population, and to empower the social environment might reduce the treatment gap.

Propagative Oscillations in Codirectional Polariton Waveguide Couplers.

We report on novel exciton-polariton routing devices created to study and purposely guide light-matter particles in their condensate phase. In a codirectional coupling device, two waveguides are connected by a partially etched section that facilitates tunable coupling of the adjacent channels. This evanescent coupling of the two macroscopic wave functions in each waveguide reveals itself in real space oscillations of the condensate. This Josephson-like oscillation has only been observed in coupled polariton traps so far. Here, we report on a similar coupling behavior in a controllable, propagative waveguide-based design. By controlling the gap width, channel length, or propagation energy, the exit port of the polariton flow can be chosen. This codirectional polariton device is a passive and scalable coupler element that can serve in compact, next generation logic architectures.

Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters.

OBJECTIVES: To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. METHODS: Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT-PCR. RESULTS: Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. CONCLUSIONS: TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.

Demonstration of a polariton step potential by local variation of light-matter coupling in a van-der-Waals heterostructure.

The large oscillator strength of excitons in transition metal dichalcogenide layers facilitates the formation of exciton-polariton resonances for monolayers and van-der-Waals heterostructures embedded in optical microcavities. Here, we show, that locally changing the number of layers in a WSe2/hBN/WSe2 van-der-Waals heterostructure embedded in a monolithic, high-quality-factor cavity gives rise to a local variation of the coupling strength. This effect yields a polaritonic stair case potential, which we demonstrate at room temperature. Our result paves the way towards engineering local polaritonic potentials at length scales down to atomically sharp interfaces, based on purely modifying its real part contribution via the coherent light-matter coupling strength g.

Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP.

ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular.

The chloramphenicol/H+ antiporter CraA of Acinetobacter baumannii AYE reveals a broad substrate specificity.

OBJECTIVES: To identify major facilitator superfamily (MFS)-type chloramphenicol transporters of Acinetobacter baumannii AYE, to characterize its substrate specificity and identify CraA substrate and H+ binding sites. METHODS: Five ORFs predicted to encode chloramphenicol transporters were heterologously expressed in Escherichia coli and their substrate specificity was determined by drug susceptibility assays on solid agar medium. CraA transport properties were determined via whole cell fluorescence experiments using ethidium and dequalinium. ACMA quenching was used to characterize the H+/drug antiport process in everted membrane vesicles. The function of CraA in A. baumannii was determined by drug susceptibility assay using A. baumannii ATCC 19606 ΔcraA. RESULTS: CraA, ABAYE0913 and CmlA5 are functionally active when overproduced in E. coli. ABAYE0913 conferred resistance to florfenicol and benzalkonium, CmlA5 conferred resistance to chloramphenicol and thiamphenicol, and craA expression resulted in resistance to chloramphenicol, thiamphenicol, florfenicol, ethidium, dequalinium, chlorhexidine, benzalkonium, mitomycin C and TPP+. Cell expressing craA_E38A showed no resistance to all tested drugs, implying that Glu-38 is involved in the binding of drugs and/or protons. Functional assays indicated that substitution of Asp-46 to Ala resulted in severe susceptibility to cationic drugs, chloramphenicol and thiamphenicol. In contrast, Glu-338 is important for the recognition of chloramphenicol, florfenicol, chlorhexidine and dequalinium. CONCLUSIONS: This study suggests that CraA has a broad substrate specificity, similar to that of E. coli MdfA. However, due to the presence of three charged residues in the transmembrane region conferring different susceptibility profiles upon substitution to Ala, we postulate that CraA has a different substrate recognition mode compared with MdfA.

Identification of the novel class D ß-lactamase OXA-679 involved in carbapenem resistance in Acinetobacter calcoaceticus.

OBJECTIVES: The aim of this study was to characterize the Acinetobacter calcoaceticus clinical isolate AC_2117 with the novel carbapenem-hydrolysing class D ß-lactamase (CHDL) OXA-679. METHODS: Identification of the species and ß-lactamases was verified by genome sequencing (PacBio) and phylogenetic analyses. Antibiotic susceptibility of AC_2117 and transformants harbouring cloned blaOXA-679 was evaluated using antibiotic gradient strips and microbroth dilution. OXA-679 was purified heterologously and kinetic parameters were determined using spectrometry or isothermal titration calorimetry. The impact of OXA-679 production during imipenem therapy was evaluated in the Galleria mellonella infection model. RESULTS: Sequencing of the complete genome of the clinical A. calcoaceticus isolate AC_2117 identified a novel CHDL, termed OXA-679. This enzyme shared sequence similarity of 71% to each of the families OXA-143 and OXA-24/40. Phylogenetic analyses revealed that OXA-679 represents a member of a new OXA family. Cloning and expression of blaOXA-679 as well as measurement of kinetic parameters revealed the effective hydrolysis of carbapenems which resulted in reduced susceptibility to carbapenems in Escherichia coli and A. calcoaceticus, and high-level carbapenem resistance in Acinetobacter baumannii. Infection of larvae of G. mellonella with a sublethal dose of blaOXA-679-expressing A. baumannii could not be cured by high-dose imipenem therapy, indicating carbapenem resistance in vivo. CONCLUSIONS: We identified blaOXA-679 in a clinical A. calcoaceticus isolate that represents a member of the new OXA-679 family and that conferred high-level carbapenem resistance in vitro and in vivo.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives.

The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein- and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.

Assessing Costs Using the Treatment Inventory Cost in Psychiatric Patients (TIC-P), TIC-P Mini and TIC-P Midi.

BACKGROUND: The Treatment Inventory Cost in Psychiatric patients (TIC-P) instrument is designed to measure societal costs in patients with psychiatric disorders and to be applied in economic evaluations. Efforts have been made to minimize respondents' burden by reducing the number of questions and meanwhile retaining the comprehensiveness of the instrument. Previously, a TIC-P Mini version and a TIC-P Midi version were developed and tested in a predominantly inpatient patient population. AIMS OF THE STUDY: The aims of this study are to examine the comprehensiveness of the abridged questionnaires in estimating the societal costs for patients with anxiety or depressive disorders and to assess the impact of productivity costs on the total costs. METHODS: The comprehensiveness of the abridged versions of the TIC-P was assessed in four populations: a group of primary care patients with anxiety disorders (n=175) and three groups of patients with major depressive disorders in various outpatient settings (n=140; n=125; and n=79). Comprehensiveness was measured using the proportion of total health care costs and productivity costs covered by the abridged versions compared to the full-length TIC-P. Costs were calculated according to the guidelines for costing studies using the Dutch costing manual. RESULTS: Our results showed that the TIC-P Mini covered 26%-64% of health care costs and the TIC-P Midi captured 54%-79% of health care costs. Health care costs in these populations were predominantly dispersed over primary care, outpatient hospital care, outpatient specialist care and inpatient hospital care. The TIC-P Midi and TIC-P Mini captured 22% and 0% of primary care costs respectively. In contrast, inpatient hospital care costs and outpatient specialist mental health care costs were almost fully included in the abridged versions. Costs due to lost productivity as measured by the full-length TIC-P were substantial, representing 38% to 92% of total costs. DISCUSSION: A reduction of the number of items resulted in a substantial loss in the ability to measure health care costs compared to the full-length TIC-P, because these outpatient populations consumed health care from a variety of health care providers. Two limitations of the study need to be stressed. Firstly, the number of patients in each of the four studies was relatively small. However, results were consistent over the four studies despite the small number of patients. Secondly, we did not take costs of medication into account. IMPLICATIONS FOR HEALTH POLICIES: In developing mental health policy, it is important to include considerations on cost-effectiveness. Increasing the evidence on instruments to measure costs from a societal perspective may support policymakers to adopt a broader perspective. IMPLICATIONS FOR FURTHER RESEARCH: The TIC-P Mini is not suitable to capture health care costs in outpatients with anxiety or depressive disorders. The comprehensiveness of TIC-P Midi compared to the full-length TIC-P varied. The TIC-P Midi should therefore be revised in order to better capture costs in all patient groups.

Evolution of Temporal Coherence in Confined Exciton-Polariton Condensates.

We study the influence of spatial confinement on the second-order temporal coherence of the emission from a semiconductor microcavity in the strong coupling regime. The confinement, provided by etched micropillars, has a favorable impact on the temporal coherence of solid state quasicondensates that evolve in our device above threshold. By fitting the experimental data with a microscopic quantum theory based on a quantum jump approach, we scrutinize the influence of pump power and confinement and find that phonon-mediated transitions are enhanced in the case of a confined structure, in which the modes split into a discrete set. By increasing the pump power beyond the condensation threshold, temporal coherence significantly improves in devices with increased spatial confinement, as revealed in the transition from thermal to coherent statistics of the emitted light.

Photon-Number-Resolved Measurement of an Exciton-Polariton Condensate.

We measure the full photon-number distribution emitted from a Bose condensate of microcavity exciton polaritons confined in a micropillar cavity. The statistics are acquired by means of a photon-number-resolving transition edge sensor. We directly observe that the photon-number distribution evolves with the nonresonant optical excitation power from geometric to quasi-Poissonian statistics, which is canonical for a transition from a thermal to a coherent state. Moreover, the photon-number distribution allows one to evaluate the higher-order photon correlations, shedding further light on the coherence formation and phase transition of the polariton condensate. The experimental data are analyzed in terms of thermal-coherent states, which gives direct access to the thermal and coherent fraction from the measured distributions. These results pave the way for a full understanding of the contribution of interactions in light-matter condensates in the coherence buildup at threshold.

Experimental Verification of the Very Strong Coupling Regime in a GaAs Quantum Well Microcavity.

The dipole coupling strength g between cavity photons and quantum well excitons determines the regime of light matter coupling in quantum well microcavities. In the strong coupling regime, a reversible energy transfer between exciton and cavity photon takes place, which leads to the formation of hybrid polaritonic resonances. If the coupling is further increased, a hybridization of different single exciton states emerges, which is referred to as the very strong coupling regime. In semiconductor quantum wells such a regime is predicted to manifest as a photon-mediated electron-hole coupling leading to different excitonic wave functions for the two polaritonic branches when the ratio of the coupling strength to exciton binding energy g/E_{B} approaches unity. Here, we verify experimentally the existence of this regime in magneto-optical measurements on a microcavity characterized by g/E_{B}≈0.64, showing that the average electron-hole separation of the upper polariton is significantly increased compared to the bare quantum well exciton Bohr radius. This yields a diamagnetic shift around 0 detuning that exceeds the shift of the lower polariton by 1 order of magnitude and the bare quantum well exciton diamagnetic shift by a factor of 2. The lower polariton exhibits a diamagnetic shift smaller than expected from the coupling of a rigid exciton to the cavity mode, which suggests more tightly bound electron-hole pairs than in the bare quantum well.

Long-Term and Short-Term Antidepressant Use in General Practice: Data from a Large Cohort in the Netherlands.

BACKGROUND: Antidepressant use is highly prevalent. Research has mainly focused on efficacy during short periods of use for depression and anxiety. There is a relative paucity of data regarding the frequency of long-term use. METHODS: To determine the prevalence and possible increase of long-term use of antidepressants over recent years, we analyzed routine general practice care data in a large cohort of patients (n = 156,620) in and around Amsterdam, The Netherlands. Additionally, predictors of long-term use were studied. RESULTS: Prevalence of long-term use of antidepressants is substantial, and such use appears to be increasing: 30.3% of use was long-term over the period 1995-2005 compared to 43.7% for the period 2005-2015. Higher age, a registered diagnosis of anxiety or depression, and the use of SSRIs or SNRIs were associated with long-term use in multivariate analysis. In addition, specific antidepressants were differentially associated with long-term use. CONCLUSIONS: Long-term antidepressant use is substantial and appears to be on the rise. Awareness of this phenomenon should be increased, such use should be prevented when possible, and reasons for long-term use need to be examined.

Utility scores for different health states related to depression: individual participant data analysis.

OBJECTIVES: Depression is associated with considerable impairments in health-related quality-of-life. However, the relationship between different health states related to depression severity and utility scores is unclear. The aim of this study was to evaluate whether utility scores are different for various health states related to depression severity. METHODS: We gathered individual participant data from ten randomized controlled trials evaluating depression treatments. The UK EQ-5D and SF-6D tariffs were used to generate utility scores. We defined five health states that were proposed from American Psychiatric Association and National Institute for Clinical Excellence guidelines: remission, minor depression, mild depression, moderate depression, and severe depression. We performed multilevel linear regression analysis. RESULTS: We included 1629 participants in the analyses. The average EQ-5D utility scores for the five health states were 0.70 (95% CI 0.67-0.73) for remission, 0.62 (95% CI 0.58-0.65) for minor depression, 0.57 (95% CI 0.54-0.61) for mild depression, 0.52 (95%CI 0.49-0.56) for moderate depression, and 0.39 (95% CI 0.35-0.43) for severe depression. In comparison with the EQ-5D, the utility scores based on the SF-6D were similar for remission (EQ-5D = 0.70 vs. SF-6D = 0.69), but higher for severe depression (EQ-5D = 0.39 vs. SF-6D = 0.55). CONCLUSIONS: We observed statistically significant differences in utility scores between depression health states. Individuals with less severe depressive symptoms had on average statistically significant higher utility scores than individuals suffering from more severe depressive symptomatology. In the present study, EQ-5D had a larger range of values as compared to SF-6D.

Maintenance of Clonogenic KIT(+) Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells.

BACKGROUND & AIMS: Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. METHODS: We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. RESULTS: Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. CONCLUSIONS: Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.

Optimizing working space in laparoscopy: CT-measurement of the effect of neuromuscular blockade and its reversal in a porcine model.

OBJECTIVE: The objective of this paper was to determine the effect of neuromuscular blockade (NMB) on working space in a porcine laparoscopy model. BACKGROUND: Conflicting results on the effect of NMB on laparoscopic working space are found in literature. Almost all studies are limited by absence of objective assessment of working space or use surrogate outcomes. METHODS: In a standardized porcine laparoscopy model, laparoscopic working-space dimensions with and without NMB were investigated in 16 animals using computed tomography at intra-abdominal pressures of 0, 5, 10, and 15 mmHg during multiple runs of abdominal insufflation. RESULTS: No statistically significant effect of NMB on abdominal dimensions and laparoscopic working-space volume was found during CO2 pneumoperitoneum. In contrast, the effect of pre-stretching of the abdominal wall by a previous abdominal insufflation was found to be significant. CONCLUSIONS: This experimental study confirms the results from several clinical studies that NMB does not influence laparoscopic working space. Studies dealing with working space during laparoscopy should take note of pre-stretching bias.