DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.

Optimization and Clinical Evaluation of an Automated Commercial Analyte-Specific Reagent Assay for Mycoplasmoides genitalium Macrolide Resistance Detection in Primary Clinical Specimens.

With improvement in laboratory diagnosis of Mycoplasmoides genitalium infection through molecular diagnostics, macrolide resistance determination within M. genitalium-positive patients is necessary. In this study, we report baseline parameters for an analyte-specific reagent (ASR) macrolide resistance real-time reverse transcriptase PCR on an open access analyzer and evaluated detection of macrolide resistance-mediated mutation (MRM) within 23S rRNA in a clinical specimen set. Initial use of 1.2 µM M. genitalium primer and 0.8 µM M. genitalium detection probe concentrations yielded an 80% false-positive detection rate when challenged with 10,000 copies of wild-type RNA. Optimization experiments showed that lowering primer/detection probe and MgCl2 concentrations minimized these false-detections of wild-type 23S rRNA, while higher levels of KCl increased rates of MRM detection with concomitant lower cycle threshold values and higher fluorescence emission. Lower limit of A2058G mutation detection was 5000 copies/mL (180 copies/reaction; 20/20 detections). Utilization of a baseline correction slope limit of 250 units further mitigated false-detection from wild-type 23S rRNA at challenges up to 3.3 billion copies/mL. MRM was detected in 583/866 (67.3%) clinical specimens initially positive for M. genitalium by commercial transcription-mediated amplification. These data included 392/564 detections (69.5%) from M. genitalium-positive swab specimens and 191/302 (63.2%) from M. genitalium-positive-positive first-void urine specimens (P = 0.06). Overall resistance detection rates did not vary by gender (P = 0.76). Specificity of the M. genitalium macrolide resistance ASR was 100% (141 urogenital determinations). MRM detection by the ASR was confirmed at a concordance rate of 90.9% by Sanger sequencing of a clinical specimen subset.

Downregulation of KEAP1 in melanoma promotes resistance to immune checkpoint blockade.

Immune checkpoint blockade (ICB) has demonstrated efficacy in patients with melanoma, but many exhibit poor responses. Using single cell RNA sequencing of melanoma patient-derived circulating tumor cells (CTCs) and functional characterization using mouse melanoma models, we show that the KEAP1/NRF2 pathway modulates sensitivity to ICB, independently of tumorigenesis. The NRF2 negative regulator, KEAP1, shows intrinsic variation in expression, leading to tumor heterogeneity and subclonal resistance.

Correlation of Abnormal Radular Secretion with Tissue Degrowth During Stress Periods in Helisoma trivolvis (Pulmonata, Basommatophora).

Laboratory experiments on starvation stress in Helisoma trivolvis elucidate a relationship between modifications of radular secretion and tissue degrowth resulting from stress. Tissue losses in starved adults ranged from 4.5% at 40 days to 27.7% at 160 days, with negligible mortality (<2%). Modifications in radular secretion that paralleled tissue loss involved not only abnormal secretion of individual teeth and of tooth rows, but especially an increased "packing" of radular rows per unit ribbon length. Radular length remained constant during experimental trials, however the mean number of tooth rows increased by almost 47% after 120 days of food deprivation. Radular patterns reflecting degrowth observed in these experiments were paralleled in radulae taken from overwintered animals sampled from natural populations. Rates of radular turnover averaged between 2.3% new growth per day (43 days to turnover) and 4.0% new growth per day (25 days to turnover). Radular samples could provide for post hoc detection of recent periods of tissue degrowth in snails, just as evidence of longer periods of tissue degrowth can be detected in the shells of long-lived bivalves.

Reproductive effort in molluscs.

A survey of the available molluscan literature shows that reproductive effort is higher in semelparous species (29.90%) than in iteroparous species (18.21%), and that in iteroparous species reproductive effort increases with successive breeding seasons. Oviparous species were found to divert considerably more into reproduction than viviparous species, with 24.24% and 5.25% channeled respectively.

Temperature relations of aerial and aquatic respiration in six littoral snails in respiration in six littoral snails in relation to their vertical zonation.

Aerial and aquatic rates of oxygen consumption were determined over a range of 5 degrees to 45 degrees C at 5 degrees C intervals for six species of marine littoral snails: including the sublittoral species, Acmaea testudinalis, Mitrella lunata, and Lacuna vincta; and the truly intertidal species, Littorina obtusata, L. littorea, and L. saxatilis. Polarographic oxygen electrodes were used with normally active snails collected from populations on Nobska and Manomet Points, Massachusetts. Three subtidal species, A. testudinalis, Lacuna vincta, and M. lunata, do not display any metabolic adjustment to increasing temperature, with thermal limits reached at 30 degrees to 35 degrees C. Aerial respiration in A. testudinalis is similar to aquatic O2 uptake, but rates average only 36.4% of aquatic rates. The intertidal congeners, Littorina obtusata, L. littorea and L. saxatilis, have varying degrees of aerial and aquatic metabolic regulation with increasing temperature. L. obtusata, a low intertidal snail exposed to air for 15% to 45% of the tidal cycle, displays a respiratory pattern of "passive endurance" to high temperatures both in air and in water. L. littorea, the dominant snail of the midlittoral region, remains active when exposed to air (30% to 75% of the tidal cycle) and has a zone of metabolic regulation between 20 degrees C and 30 degrees C. Over this, the normal ambient temperature range, the Q10 closely approximates one, and nearly equivalent O2 uptake rates occur in air and in water. L. saxatilis from the upper littoral region is exposed to air for 70% to 95% of the tidal cycle and is characterized by reduced aerial and aquatic O2 uptake rates above 25 degrees C, representing a reversible torpor up to its thermal maximum at 44 degrees C. For these six snail species, respiratory responses to increasing temperature are thus directly related to the pattern of vertical distribution in the intertidal environment. Discussion of this relationship stresses that the evolution of other nearterrestrial structures and functions in littoral snails has proceeded in a discontinuous fashion. Despite this, the temperature responses in respiration parallel the functional morphology of the pallial structures and the physiological patterns of response to low oxygen stress, as well as adaptive features of reproduction, larval development, water-control, and nitrogenous excretion.

EARLY LIFE-HISTORY OF MELAMPUS AND THE SIGNIFICANCE OF SEMILUNAR SYNCHRONY.

1. The salt-marsh pulmonate snail, Melampus bidentatus, is placed in the Ellobiidae which family encompasses the most primitive of living Pulmonata and is regarded as not far removed from the ancestral stem-group of both modern land snails and freshwater pulmonates. Inhabiting the higher levels of salt marshes. Melampus is "amphibious": although an air-breather with a gill-less vascularized mantle-cavity functioning as a lung, if retains an archetypic pattern of reproduction with small eggs and a free-swimming veliger larva. 2. Field and laboratory studies over several years (based on natural populations at Little Sippewisset, Cape Cod, Massachusetts) have shown that egg-laying, hatching, and larval settlement are each confined to cycles of about four days in phase with the spring high tides. Adaptively such semilunar synchronies ensure that these processes occur only during the 2.3% to 4% of each month when the Melampus habitat in the upper 12% of the intertidal zone is bathed by seawater. 3. The annual reproductive period extends from late May or early June through early July. with either three or four cycles of egg-laying occurring at two-week intervals in phase with the tides of new and of full moon. Synchrony of egg-laying (and of the patterned aggregation and copulation which precede it) is obligate. Stocks of Melampus brought into the laboratory in spring will maintain the same semilunar rhythm of reproductive behavior during the summer period. 4. Eggs are small (about 109 ng organic carbon) and are laid in gelatinous egg-masses averaging 850 eggs. Mean numerical fecundity is 33,150 eggs per snail per year. For most freshwater pulmonates fecundity would lie in the range 8-800 eggs per snail per year. At 18° C, development to a well-differentiated and active veliger within the egg-shell takes 11 days. 5. Hatching shows semilunar synchrony in the field: enormous numbers of newly hatched veligers can be collected on the flood of appropriate spring tides. A series of experiments with laboratory-laid egg-masses showed that eclosion normally occurs in response to a sequence of about 4 tidal floodings in under 50 hours. Hatching can occur from egg-masses from 10 to 24 days after laying. Being facultative, the process allows better survival and overlap of cohorts but also reestablishes the synchronization with spring tides. 6. Veligers feed actively and grow from shell length 127 µ to 280 µ during their time in the plankton, deduced to be 14 ±2 days. The bulk of the settlement is into the exact vertical zone occupied by adult Melampus. 7. A period as a crawling, radula-feeding postlarva (after loss of velar lobes and operculum) is followed by an abrupt metamorphosis of the mantle and shell. Postmetamorphic spat grow rapidly. In terms of organic carbon or ash-free dry weight, growth extends through two orders of magnitude during veliger and early spat life, through more than three during the first eleven weeks, and six in the entire 3-4year life-span. In contrast, similar biomass growth measures in freshwater pulmonates involve only two to three orders in their life-span. 8. In Melampus, the shells of late veligers and of post-larvae show sinistral coiling, and those of metamorphosed spat and of adults dextral coiling. There is a metamorphosis of mantle and shell alone; throughout development, larval and spat stages, the internal organs are in their adult dextral arrangement. Such a metamorphosis from a hyperstrophic shell condition to an orthostrophic one is known to occur in the ectocommensal opisthobranch family Pyramidellidae and in certain other snails with planktonic larvae. The present study provides the first description of the succession of shell stages and metamorphosis for any pulmonate. 9. In conclusion, the small eggs, the mantle-shell metamorphosis, and the semilunar synchrony are discussed in their evolutionary setting. "Primitive" reproduction with small eggs, as retained in Melampus, confers advantages in dispersal and genetic potential. Evolution of larger eggs, as in the freshwater pulmonates, may have involved selection pressures to reduce the temporal extent of immature growth in seasonally variable environments. Mantle-shell changes in Melampus, including the metamorphosis, can be interpreted simply in terms of the changing needs for protective containment at different stages in the life-cycle. Semilunar synchrony of reproductive and of larval stages has evolved in response to the concursion of specialized aerial respiration and the primitive pattern of spawning large numbers of small eggs. The combination of both obligate and facultative processes in producing these synchronies is thought to be significant in relation to the long evolutionary history which can be hypothesized for these semilunar rhythms.