Immobilization rapidly selects for chemoresistant ovarian cancer cells with enhanced ability to enter dormancy

Around 20–30% of ovarian cancer patients exhibit chemoresistance, but there are currently no methods to predict whether a patient will respond to chemotherapy. Here, we discovered that chemoresistant ovarian cancer cells exhibit enhanced survival in a quiescent state upon experiencing the stress of physical confinement. When immobilized in stiff silica gels, most ovarian cancer cells die within days, but surviving cells exhibit hallmarks of single-cell dormancy. Upon extraction from gels, the cells resume proliferation but demonstrate enhanced viability upon reimmobilization, indicating that initial immobilization selects for cells with a higher propensity to enter dormancy. RNA-seq analysis of the extracted cells shows they have signaling responses similar to cells surviving cisplatin treatment, and in comparison to chemoresistant patient cohorts, they share differentially expressed genes that are associated with platinum-resistance pathways. Furthermore, these extracted cells demonstrate greater resistance to cisplatin and paclitaxel, despite being proliferative. In contrast, serum starvation and hypoxia could not effectively select for chemoresistant cells upon removal of the environmental stress. These findings demonstrate that ovarian cancer chemoresistance and the ability to enter dormancy are linked, and immobilization rapidly distinguishes chemoresistant cells. This platform could be suitable for mechanistic studies, drug development, or as a clinical diagnostic tool.     

Cloning of genomic DNA of Lactococcus lactis that restores phage sensitivity to an unusual bacteriophage sk1-resistant mutant

An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of Lactococcus lactis C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (beta-1,4-N-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of Lactobacillus and Streptococcus and also revealed DNA homologous to noncoding sequences of temperate phage of L. lactis, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to sun and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism.     

Linkage of <Emphasis Type="Italic">Patr-AL</Emphasis> to <Emphasis Type="Italic">Patr-A</Emphasis> and-<Emphasis Type="Italic">B</Emphasis> in the major histocompatibility complex of the common chimpanzee (<Emphasis Type="Italic">Pan troglodytes</Emphasis>)

Patr-AL is a recently described gene found only in the common chimpanzee, but closely related in structure to the highly polymorphic Patr-A and HLA-A genes of the chimpanzee and human MHCs, respectively. Unlike Patr-A and HLA-A, the Patr-AL gene has little polymorphism and is not fixed in the chimpanzee genome. To determine whether Patr-AL is located in the MHC or elsewhere, we compared segregation of the Patr-AL gene with segregation of Patr-A and - B alleles in chimpanzee families. The results demonstrate that Patr-AL is an MHC class I gene present on different MHC haplotypes as defined by their combination of Patr-A and B alleles.     

Molecular dynamics simulations of the first steps of the reaction catalyzed by HIV-1 protease

The mechanism of the first steps of the reaction catalyzed by HIV-1 protease was studied through molecular dynamics simulations. The potential energy surface in the active site was generated using the approximate valence bond method. The approximate valence bond (AVB) method was parameterized based on density functional calculations. The surrounding protein and explicit water environment was modeled with conventional, classical force field. The calculations were performed based on HIV-1 protease complexed with the MVT-101 inhibitor that was modified to a model substrate. The protonation state of the catalytic aspartates was determined theoretically. Possible reaction mechanisms involving the lytic water molecule are accounted for in this study. The modeled steps include the dissociation of the lytic water molecule and proton transfer onto Asp-125, the nucleophilic attack followed by a proton transfer onto peptide nitrogen. The simulations show that in the active site most preferable energetically are structures consisting of ionized or polarized molecular fragments that are not accounted for in conventional molecular dynamics. The mobility of the lytic water molecule, the dynamics of the hydrogen bond network, and the conformation of the aspartates in the active center were analyzed.     

The glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension

Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Psychometric properties and construct validity of the Weight-Related Eating Questionnaire in a diverse population

This study evaluates the 16‐item, four‐factor Weight‐Related Eating Questionnaire (WREQ), which assesses theory‐based aspects of eating behavior, across diverse, nonclinical subgroups. A total of 621 men and women aged 18–81 years (34.3 ± 16.4) with a mean BMI of 25.7 ± 6.1 kg/m² (range 15.5–74.1 kg/m²) were recruited from general education classes at the University of Hawai'i, Manoa and an online survey panel of Hawai'i residents to complete a web‐based survey. Participants were predominantly white (23%), Asian/Asian‐mix (42%), or Native Hawaiian/Pacific Islander (18%). The WREQ's factor structure was successfully replicated by confirmatory factor analysis (CFA) for the entire sample and by weight status, gender, age, and race with strong internal consistency. Four‐week test‐retest reliability (n = 31) for the subscales was excellent with interclass correlations of 0.849–0.932. Tests of population invariance confirmed the generalizability of the WREQ across all subgroups having provided no evidence that the factor structure, factor loadings, or indicator intercepts varied significantly between the groups. Multivariate regression analyses showed that emotional eating was independently associated with BMI (β = 0.272, P < 0.001) as well as moderate‐ and long‐term weight change rates (weight gain) in young adults (β = 0.152, P = 0.042) and adults (β = 0.217, P = 0.001). Compensatory restraint was negatively associated with weight gain in adults (β = ?0.133, P = 0.039). Routine restraint and emotional eating were highest among dieters. All associations remained significant after accounting for gender, age, and race. The hypothesized WREQ measurement model demonstrated very good construct validity, confirming the unbiased generalizability of the WREQ measure across sex, age, race, and BMI subgroups, and excellent criterion‐related validity with respect to current BMI, weight change, and weight control status.     

Epigenetic mechanism underlying the development of polycystic ovary syndrome (PCOS)-like phenotypes in prenatally androgenized rhesus monkeys

The pathogenesis of polycystic ovary syndrome (PCOS) is poorly understood. PCOS-like phenotypes are produced by prenatal androgenization (PA) of female rhesus monkeys. We hypothesize that perturbation of the epigenome, through altered DNA methylation, is one of the mechanisms whereby PA reprograms monkeys to develop PCOS. Infant and adult visceral adipose tissues (VAT) harvested from 15 PA and 10 control monkeys were studied. Bisulfite treated samples were subjected to genome-wide CpG methylation analysis, designed to simultaneously measure methylation levels at 27,578 CpG sites. Analysis was carried out using Bayesian Classification with Singular Value Decomposition (BCSVD), testing all probes simultaneously in a single test. Stringent criteria were then applied to filter out invalid probes due to sequence dissimilarities between human probes and monkey DNA, and then mapped to the rhesus genome. This yielded differentially methylated loci between PA and control monkeys, 163 in infant VAT, and 325 in adult VAT (BCSVD P<0.05). Among these two sets of genes, we identified several significant pathways, including the antiproliferative role of TOB in T cell signaling and transforming growth factor-β (TGF-β) signaling. Our results suggest PA may modify DNA methylation patterns in both infant and adult VAT. This pilot study suggests that excess fetal androgen exposure in female nonhuman primates may predispose to PCOS via alteration of the epigenome, providing a novel avenue to understand PCOS in humans.