Adaptation to volumetric compression drives hepatoblastoma cells to an apoptosis-resistant and invasive phenotype.

Liver cancer involves tumor cells rapidly growing within a packed tissue environment. Patient tumor tissues reveal densely packed and deformed cells, especially at tumor boundaries, indicative of physical crowding and compression. It is not well understood how these physical signals modulate tumor evolution and therapeutic susceptibility. Here we investigate the impact of volumetric compression on liver cancer (HepG2) behavior. We find that conditioning cells under a highly compressed state leads to major transcriptional reprogramming, notably the loss of hepatic markers, the epithelial-to-mesenchymal transition (EMT)-like changes, and altered calcium signaling-related gene expression, over the course of several days. Biophysically, compressed cells exhibit increased Rac1-mediated cell spreading and cell-extracellular matrix interactions, cytoskeletal reorganization, increased YAP and ß-catenin nuclear translocation, and dysfunction in cytoplasmic and mitochondrial calcium signaling. Furthermore, compressed cells are resistant to chemotherapeutics and desensitized to apoptosis signaling. Apoptosis sensitivity can be rescued by stimulated calcium signaling. Our study demonstrates that volumetric compression is a key microenvironmental factor that drives tumor evolution in multiple pathological directions and highlights potential countermeasures to re-sensitize therapy-resistant cells. Significance statement: Compression can arise as cancer cells grow and navigate within the dense solid tumor microenvironment. It is unclear how compression mediates critical programs that drive tumor progression and therapeutic complications. Here, we take an integrative approach in investigating the impact of compression on liver cancer. We identify and characterize compressed subdomains within patient tumor tissues. Furthermore, using in vitro systems, we induce volumetric compression (primarily via osmotic pressure but also via mechanical force) on liver cancer cells and demonstrate significant molecular and biophysical changes in cell states, including in function, cytoskeletal signaling, proliferation, invasion, and chemoresistance. Importantly, our results show that compressed cells have impaired calcium signaling and acquire resistance to apoptosis, which can be countered via calcium mobilization.

HIV self-testing intervention experiences and kit usability: results from a qualitative study among men who have sex with men in the SELPHI (Self-Testing Public Health Intervention) randomized controlled trial in England and Wales.

OBJECTIVES: SELPHI (HIV Self-Testing Public Health Intervention) is the largest randomized controlled trial (RCT) of HIV self-testing (HIVST) in a high-income setting to date, and has recruited 10 000 men who have sex with men (cis- and transgender) and transgender women who have sex with men. This qualitative substudy aimed to explore how those utilizing self-tests experience HIVST and the implications for further intervention development and scale-up. This is the first qualitative study in Europe investigating experiences of HIVST among intervention users, and the first globally examining the experience of using blood-based HIVST. METHODS: Thirty-seven cisgender MSM SELPHI participants from across England and Wales were purposively recruited to the substudy, in which semi-structured interviews were used to explore testing history, HIVST experiences and intervention preferences. Interviews were audio-recorded, transcribed and analysed through a framework analysis. RESULTS: Men accessed the intervention because HIVST reduced barriers related to convenience, stigma and privacy concerns. Emotional responses had direct links to acceptability. Supportive intervention components increased engagement with testing and addressed supportive concerns. HIVST facilitated more frequent testing, with the potential to reduce sexually transmitted infection (STI) screening frequency. Substudy participants with an HIV-positive result (n = 2) linked to care promptly and reported very high acceptability. Minor adverse outcomes (n = 2; relationship discord and fainting) did not reduce acceptability. Ease of use difficulties were with the lancet and the test processing stage. CONCLUSIONS: Intervention components shaped acceptability, particularly in relation to overcoming a perceived lack of support. The intervention was broadly acceptable and usable; participants expressed an unexpected degree of enthusiasm for HIVST, including those with HIV-positive results and individuals with minor adverse outcomes.

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials.

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-3H] leucine or [methyl-3H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum.

In vitro adherence of Pseudomonas aeruginosa to four intraocular lenses.

PURPOSE: To compare the relative degrees of adherence of a clinical strain of Pseudomonas aeruginosa to the optic material of four intraocular lenses (IOLs). SETTING: Center for Applied and Environmental Microbiology, Georgia State University, Atlanta, Georgia, USA. METHODS: Intraocular lens optics made of poly(methyl methacrylate) (PMMA), AcrySof-acrylic, and silicone were included in this study. The IOLs were incubated in a minimal medium with cells of P. aeruginosa for 2 hours and 18 hours. Cells in the 2 hour experiment were prelabeled with 3H-leucine; those in the 18 hour experiments were postlabeled. After rinsing the IOLs to remove loosely adherent cells, we determined the number of cells adhered to coded lenses from calibration curves of disintegrations per minute versus cells per square millimeter. Additional lenses were incubated with P. aeruginosa and examined with scanning electron microscopy. RESULTS: The adherence of P. aeruginosa in order of increasing magnitude was AcrySof-acrylic < PMMA < silicone 1 < silicone 2. The differences between all groups were statistically significant. The scanning electron microscopy observations were in general agreement with the radiolabel studies. CONCLUSIONS: The AcrySof-acrylic IOL was less susceptible to primary adherence and 18 hour biofilm formation by P. aeruginosa than the PMMA and silicone IOLs, indicating that this material reduced pseudomonad adherence and the risk of endophthalmitis following cataract surgery.

In vitro evaluation of the efficacy of a silver-coated catheter.

Bacteria commonly associated with nosocomial urinary tract infections were examined in vitro for their relative adherence to latex, 100% silicone-, hydrogel-coated latex-, and hydrogel/silver-coated latex urinary catheters. Degrees of adherence within 2 h were determined with cells radiolabeled with leucine. Adherence was greatest and equivalent on silicone and latex catheters. Adherence of four strains of Escherichia coli to the hydrogel/silver-coated catheter was decreased by 50% to 99% in comparison with the other catheters. Repeat testing with strains of E. coli and Pseudomonas aeruginosa with over 50 catheters demonstrated a consistency in the inhibition. The hydrophilic coating of the catheter appeared to be primary in the decreased adherence phenomenon followed by a secondary biocidal effect of the silver ion.

Effect of adhered bacteria on the binding of Acanthamoeba to hydrogel lenses.

OBJECTIVES: To determine the effect of Pseudomonas aeruginosa and Staphylococcus epidermidis on the binding of Acanthamoeba species to hydrogel lenses. METHODS: Cells of amebae and bacteria were incubated with different types of hydrogel lenses. Densities of amebae that were bound to the lenses after rinsing were determined from direct counts with a cell detachment procedure and from scintillation counts of cells, which were radiolabeled with tritiated leucine. RESULTS: With both methods, amebae showed significantly increased binding to hydrogel lenses with attached P aeruginosa. The numbers of amebae that were retained on lenses with attached S epidermidis were not significantly different from those that were retained on lenses without bacteria. The binding of amebae to unworn hydrogel lenses, in contrast to the irreversible adherence of P aeruginosa, was tenuous. CONCLUSIONS: The binding of Acanthamoeba species to unworn hydrogel lenses was tenuous and appeared to be related to water content, surface tensions, and ionic charge. The presence of adhered P aeruginosa on the hydrogel lenses facilitated the binding of Acanthamoeba species. The cocontamination of lens systems with bacteria (eg, P aeruginosa) may be a prime factor in the development of amebic keratitis.

Effect of Staphylococcus epidermidis on hydrogel contact lens retention on the rabbit eye.

A slime-producing isolate of Staphylococcus epidermidis attached to FDA Group II hydrogel contact lenses persisted on rabbit eyes for up to 14 days, but except for minor redness of the eye no other effect was observed. Eye flora of eight representative New Zealand White rabbits included four different species of Staphylococcus including S. epidermidis and one species of Micrococcus, none of which produced overtly obvious biofilms. The slime-producing strain of S. epidermidis adhered more effectively to lenses than a non-slime-producing strain, and lenses challenged with the slime-producing strain remained on the rabbit eye for longer time periods than those with a non-slime-producing strain. Bacteria associated with the contact lens may affect the retention of the lens on the rabbit cornea during experimental studies.

Binding of Acanthamoeba to hydrogel contact lenses.

The numbers of Acanthamoeba binding to new hydrogel contact lenses of different polymer and water content were determined with two quantitative methods, a radiolabeled-cell method and a detaching-fluid method. Numbers of amoebae retained on nonionic lenses increased with increasing water content of the lenses. With both nonionic and ionic lenses numbers of associated amoebae decreased with successive rinsing steps. The retentions of amoebae on unworn hydrogel lenses, in contrast to the irreversible adhesion of bacteria, were tenuous and appeared to be effected mainly by surface tension, surface charge and water content.

Effects of polyhexamethylene biguanide and chlorhexidine on four species of Acanthamoeba in vitro.

We determined the relative minimal inhibitory and minimal amoebicidal concentrations of chlorhexidine digluconate and polyhexamethylene biguanide for four species of Acanthamoeba. The amoebae were grown in peptone-glucose-yeast extract broth for 72 h in tissue culture flasks. Either washed trophozoites (approximately 10(5)) or cysts (approximately 10(5)) were incubated in the enrichment broth in 96 well microtiter trays. Antimicrobial concentrations of the biguanides were determined from microscopic examinations of methylene blue uptake and from subcultures. In general, killing was time dependent. Minimal amoebicidal concentrations at 24 h ranged from 50 to 100 mg/ml and to as low as 25 mg/ml by 72 h. Trophozoites were killed more rapidly than cysts. Both biguanides had similar levels of activity. A synergistic combination of chlorhexidine and polyhexamethylene biguanide (total concentration 25 mg/ml) was most evident for A. castellanii and A. polyphaga. Cysts of A. culbertsoni and A. hatchetti stained more rapidly after exposure to the combination of biguanides than to the single biguanides, but there were no statistically significant differences in the final numbers of dead or stained cysts after exposure to the combination or to the single biguanides.

Adherence of organisms to silver-coated surfaces.

Pure silver-, silver oxide- and silver chloride-treated surfaces in comparison to polypropylene inhibited both growth and adherence from saline of Serratia marcescens, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans. These same organisms demonstrated enhanced adherence to an Ion-Beam-Assisted-Deposited silver surface followed by loss of viability. This type of surface in contrast to the other silver surfaces did not produce zones of inhibition in agar diffusion tests.

Resistance of adhered bacteria to rigid gas permeable contact lens solutions.

We examined eight conditioning or disinfecting solutions recommended for use with rigid gas permeable (RGP) or hard contact lenses for their efficacy against planktonic and attached cells of bacteria and Candida albicans. Most solutions, particularly those containing phenylmercuric nitrate and polyaminopropyl biguanide, gave marked inhibition (99.99% reduction within 4 hours) of planktonic cells of bacteria. Planktonic cells of Serratia marcescens and C. albicans survived in certain solutions containing chlorhexidine and benzalkonium chloride. In contrast, cells of all test microorganisms adhered to wells of polyethylene contact lens cases showed various degrees of survival after 4, 6, and 12 hours of exposure to most contact lens solutions. Drying of the lens case with adhered cells for 10 hours prior to addition of the lens solution usually reduced the incidence of recovery. Studies demonstrated that biofilms developed in cases with solutions of low and high efficacy. The enhanced resistance of adhered cells requires the periodic disinfection or replacement of lens cases used with RGP lenses.

Effects of silver on adherence of bacteria to urinary catheters: in vitro studies.

Strains of Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis, and Klebsiella pneumoniae, mostly from complicated urinary tract infections, showed reduced adherence to silver-treated silicone or latex catheters as compared with latex or silicone catheters. The relative degrees of cell adherence to catheters at 2 h or 18 h, as indicated by radiolabeled cell assays, were in general agreement with growth rate-reduction assays and scanning-electron-microscopy data. For strains of E. coli, the correlation between cell hydrophobicity and degree of adherence to catheters was not significant. Antibiotic resistance (tetracycline, sulfathiazine, neomycin, kanamycin) and silver resistance were not associated. The radiolabel adherence procedure provided a quantitative method for evaluating the relative antimicrobial efficacy of silver-treated catheters.