Automated Cell Lineage Reconstruction using Label-Free 4D Microscopy.

Here we describe embGAN, a deep learning pipeline that addresses the challenge of automated cell detection and tracking in label-free 3D time lapse imaging. embGAN requires no manual data annotation for training, learns robust detections that exhibits a high degree of scale invariance and generalizes well to images acquired in multiple labs on multiple instruments.

Influence of environmental temperature on mouth-form plasticity in Pristionchus pacificus acts through daf-11-dependent cGMP signaling.

Mouth-form plasticity in the nematode Pristionchus pacificus has become a powerful system to identify the genetic and molecular mechanisms associated with developmental (phenotypic) plasticity. In particular, the identification of developmental switch genes that can sense environmental stimuli and reprogram developmental processes has confirmed long-standing evolutionary theory. However, how these genes are involved in the direct sensing of the environment, or if the switch genes act downstream of another, primary environmental sensing mechanism, remains currently unknown. Here, we study the influence of environmental temperature on mouth-form plasticity. We find that environmental temperature does influence mouth-form plasticity in most of the 10 wild isolates of P. pacificus tested in this study. We used one of these strains, P. pacificus RSA635, for detailed molecular analysis. Using forward and reverse genetic technology including CRISPR/Cas9, we show that mutations in the guanylyl cyclase Ppa-daf-11, the Ppa-daf-25/AnkMy2, and the cyclic nucleotide-gated channel Ppa-tax-2 eliminate the response to elevated temperatures. Together, our study indicates that DAF-11, DAF-25, and TAX-2 have been co-opted for environmental sensing during mouth-form plasticity regulation in P. pacificus.

Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Dauer Development Modulates Olfactory Behavior.

Reproductive adults and developmentally arrested larvae often occupy different ecological niches and thus are expected to respond differently to environmental stimuli. To understand the genes that coordinate dauer development and olfactory behavior, we examined adult and dauer C. elegans in wild-type and dauer constitutive mutants (Daf-c). We found all dauers showed decreased attraction to all three odorants tested compared to adults, with daf-7 dauer larva (DL) exhibiting a concentration-dependent preference shift towards isoamyl alcohol, suggesting that the TGF-ß pathway is involved in both dauer regulation and dauer-specific odortaxis.

Steroid hormone pathways coordinate developmental diapause and olfactory remodeling in Pristionchus pacificus.

Developmental and behavioral plasticity allow animals to prioritize alternative genetic programs during fluctuating environments. Behavioral remodeling may be acute in animals that interact with host organisms, since reproductive adults and the developmentally arrested larvae often have different ethological needs for chemical stimuli. To understand the genes that coordinate the development and host-seeking behavior, we used the entomophilic nematode Pristionchus pacificus to characterize dauer-constitutive mutants (Daf-c) that inappropriately enter developmental diapause to become dauer larvae. We found two Daf-c loci with dauer-constitutive and cuticle exsheathment phenotypes that can be rescued by the feeding of Δ7-dafachronic acid, and that are dependent on the conserved canonical steroid hormone receptor Ppa-DAF-12. Specifically at one locus, deletions in the sole hydroxysteroid dehydrogenase (HSD) in P. pacificus resulted in Daf-c phenotypes. Ppa-hsd-2 is expressed in the canal-associated neurons (CANs) and excretory cells whose homologous cells in Caenorhabditis elegans are not known to be involved in the dauer decision. While in wildtype only dauer larvae are attracted to host odors, hsd-2 mutant adults show enhanced attraction to the host beetle pheromone, along with ectopic activation of a marker for putative olfactory neurons, Ppa-odr-3. Surprisingly, this enhanced odor attraction acts independently of the Δ7-DA/DAF-12 module, suggesting that Ppa-HSD-2 may be responsible for several steroid hormone products involved in coordinating the dauer decision and host-seeking behavior in P. pacificus.

Evolution of neuronal anatomy and circuitry in two highly divergent nematode species.

The nematodes C. elegans and P. pacificus populate diverse habitats and display distinct patterns of behavior. To understand how their nervous systems have diverged, we undertook a detailed examination of the neuroanatomy of the chemosensory system of P. pacificus. Using independent features such as cell body position, axon projections and lipophilic dye uptake, we have assigned homologies between the amphid neurons, their first-layer interneurons, and several internal receptor neurons of P. pacificus and C. elegans. We found that neuronal number and soma position are highly conserved. However, the morphological elaborations of several amphid cilia are different between them, most notably in the absence of 'winged' cilia morphology in P. pacificus. We established a synaptic wiring diagram of amphid sensory neurons and amphid interneurons in P. pacificus and found striking patterns of conservation and divergence in connectivity relative to C. elegans, but very little changes in relative neighborhood of neuronal processes. These findings demonstrate the existence of several constraints in patterning the nervous system and suggest that major substrates for evolutionary novelty lie in the alterations of dendritic structures and synaptic connectivity.

Comparison of Different Treatment Schedules of Mitomycin C Intravesical Instillation in High-Risk Superficial Bladder Cancer Patients.

ZIEL: Diese Studie erfolgte zum Vergleich der Wirksamkeit einer intravesikalen Instillation von Mitomycin C (MMC) zur Prävention eines nicht muskelinvasiven Ta- oder T1-High-Risk-Harnblasenkarzinoms (NMIBC) unter Verwendung verschiedener Schemata. MATERIAL UND METHODEN: Diese retrospektive Kohortenstudie wurde bei 152 Patienten durchgeführt, die zwischen April 2009 und September 2016 mit einer intravesikalen MMC-Injektion behandelt wurden. Der mittlere Nachbeobachtungszeitraum lag bei 32,67 Monaten. Alle Patienten unterzogen sich einer vollständigen transurethralen Resektion des Blasentumors (TURBT), an die sich innerhalb von 24 Stunden eine postoperative Instillation von MMC anschloss. Die Patienten wurden in 4 Behandlungsgruppen unterteilt: Bei Gruppe 1 erfolgte die Nachbeobachtung ohne MMC-Erhaltungsdosis; Gruppe 2 erhielt in den ersten 8 Wochen einmal pro Woche eine MMC-Instillation; Gruppe 3 erhielt in den ersten 8 Wochen einmal pro Woche und in den darauffolgenden 6 Monaten einmal pro Monat eine MMC-Instillation; Gruppe 4 erhielt in den ersten 8 Wochen einmal pro Woche und in den darauffolgenden 12 Monaten einmal pro Monat eine MMC-Instillation. ERGEBNISSE: Die allgemeine Rezidivrate lag bei 27,6 %. Gruppe 1 zeigte eine signifikant hohe (p < 0,05) Rezidivrate von 50 %, während sich bei den Rezidivraten der übrigen 3 Schemata kein Unterschied fand (Gruppe 2: 15 %; Gruppe 3: 24,1 %; Gruppe 4: 27,2 %). Darüber hinaus zeigte sich zwischen diesen Patientengruppen kein statistischer Unterschied bei den Rezidivraten von Ta- oder T1-Tumoren sowie niedrig- oder hochgradigen Tumoren. SCHLUSSFOLGERUNG: Unser Vergleich der verschiedenen Schemata einer intravesikalen MMC-Instillation ergab bei einer einzigen MMC-Instillation nach TURBT eine signifikant höhere Rezidivrate als bei Patienten, die nach 8 Wochen, 6 Monaten und 12 Monaten eine Erhaltungsdosis erhielten. Zeitlich fanden sich beim MMC-Erhaltungsschema keine signifikanten Unterschiede zwischen der 8. Woche und dem 12. Monat. Daraus folgern wir, dass bei T1- oder Ta-High-Risk-NMIBC nach TURBT einmalig eine MMC-Instillation mit anschließender Erhaltungstherapie mit einmal wöchentlicher Verabreichung über 8 Wochen durchgeführt werden kann.

Robot-assisted radical prostatectomy may induce inguinal hernia within the first 2 years: An 11-year single-surgeon experience of >400 cases.

At present, robot-assisted radical prostatectomy (RARP) is a gold standard in radical prostatectomy. The aim of this study was to evaluate the incidence, risk factors, and timing of occurrence of inguinal hernia (IH) after RARP.We included 427 patients with prostate cancer who underwent RARP by a single surgeon from February 2006 to August 2017. Incidence, clinical, and pathological factors were investigated to assess relationship with the development of IH.Postoperative IH occurred in 29 cases (6.79% of all RARP patients), whereas 22 cases (75.9% of all IH patients) occurred within the first 2 years. The median follow-up period was 5.2 years, and the median age of patients was 65 years. Postoperative IH occurrence was significantly associated with body mass index (BMI), smoking history, and low surgeon experience (P = .036, .023, and .048, respectively). However, low surgeon experience did not reach statistical significance after multivariate analysis.The overall incidence of IH after RARP was significantly associated with BMI and smoking history. With obvious incidence of IH within the first 2 years after operation which was not observed at the open prostatectomy, RARP itself may play a role in the development of IH.

Resonance in modulation instability from non-instantaneous nonlinearities.

To explore resonance phenomena in the nonlinear region, we show by experimental measurements and theoretical analyses that resonance happens in modulation instability from non-instantaneous nonlinearities in photorefractive crystals. With a temporally periodic modulation in the external bias voltage, corresponding to a modulation in the nonlinear strength, an enhancement in the visibility of MI at resonant frequency is reported through spontaneous optical pattern formations. Theoretical curves obtained from a nonlinear non-instantaneous Schrödinger equation give good agreement to experimental data.

A species-specific nematocide that results in terminal embryogenesis.

Nematode-insect interactions are ubiquitous, complex and constantly changing as the host and nematode coevolve. The entomophilic nematode Pristionchus pacificus is found on a myriad beetle species worldwide, although the molecular dynamics of this relationship are largely unknown. To better understand how host cues affect P. pacificus embryogenesis, we characterized the threshold of sensitivity to the pheromone (Z)-7-tetradecen-2-one (ZTDO) by determining the minimum exposure duration and developmental window that results in P. pacificus embryonic lethality. We found early-stage embryos exposed to volatile ZTDO for as few as 4 h all display terminal embryogenesis, characterized by punctuated development up to 48 h later, with abnormal morphology and limited cavity formation. To determine if the pheromone arrests pre-hatching development by suffocating or permeabilizing the eggshells, we raised embryos under anoxic conditions and also examined eggshell permeability using the lipophilic dye FM4-64. We found that asphyxiating the embryos arrested embryogenesis in a reversible manner but did not phenocopy the effects of ZTDO exposure, whereas the ZTDO-induced disruption of embryogenesis did correlate with increased eggshell permeability. The effects of ZTDO are also highly specific, as other lipid insect compounds do not produce any detectable embryocidal effect. The high specificity and unusual teratogenic effect of ZTDO may be important in mediating the host-nematode relationship by regulating P. pacificus development.

The bacterial community of entomophilic nematodes and host beetles.

Insects form the most species-rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode-insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle-nematode-bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five-year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high-throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate-reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect-associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate-reducing bacteria suggests a possible link between beetle-bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.

Control modulation instability in photorefractive crystals by the intensity ratio of background to signal fields.

By experimental measurements and theoretical analyses, we demonstrate the control of modulation instability in photorefractive crystals though the intensity ratio of coherent background to signal fields. Appearance, suppression, and disappearance of modulated stripes are observed in a series of spontaneous optical pattern formations, as the intensity of input coherent beam increases. Theoretical curves based on the band transport model give good agreement to experimental data, both for different bias voltages and different intensity ratios.

Lasing on surface states in vertical-cavity surface-emission lasers.

We report experimental observation of lasing on surface states, in the form of standing waves at the termination of a defect-free photonic crystal on top of vertical-cavity surface-emission lasers. Direct images of lasing modes at the truncated periodic potential, along one side of a square lattice, are demonstrated by collecting near-field radiation patterns, as well as in numerical simulations. Our results provide a step toward realizing surface and edge states in optical cavities.

A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus.

Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.

Conserved behavioral and genetic mechanisms in the pre-hatching molt of the nematode Pristionchus pacificus.

BACKGROUND: During development, juvenile nematodes undergo four molts. Although the number of molts appears to be constant within the Nematoda, the timing of the first molt can occur either before or after hatching. A previous study indicates that, as in some parasitic nematode lineages, a pre-hatching juvenile stage also exists in Diplogastrid nematodes. A detailed description of these sequence of events has yet to be shown for any single species. FINDINGS: To delineate the timing of the pre-hatching molt in the beetle-associated Pristionchus pacificus, we tracked individual mid-J1 stage worms inside the eggshell through the J1-J2 transition and hatching. We found that active movement ended 21 hours after egg-laying, followed by lethargus and hatching. We inferred that lethargus behavior represents the onset of the first molt, which precedes each post-hatching molt in C. elegans and P. pacificus. The onset of the J1-J2 molt was also marked by the upregulation of the P. pacificus molting marker Ppa-pnhr-1. We further corroborated the pre-hatching molt with the isolation of two genetic mutants that exhibited aberrant molting both inside the egg and after hatching, as characterized by protracted and often-aborted shedding of the old cuticle. CONCLUSION: Our results describe in detail the pre-hatching juvenile molt in P. pacificus, provide strong visual evidence of a pre-hatching molt, and show support for common genetic mechanisms regulating molting in the pre-hatching and post-hatching developmental stages. Our findings support the hypothesis that the evolution of pre-hatching development in Diplogastrid nematodes is likely due to a heterochronic shift between the timing of the first molt and hatching.

Bidirectional thermotaxis in Caenorhabditis elegans is mediated by distinct sensorimotor strategies driven by the AFD thermosensory neurons.

The nematode Caenorhabditis elegans navigates toward a preferred temperature setpoint (Ts) determined by long-term temperature exposure. During thermotaxis, the worm migrates down temperature gradients at temperatures above Ts (negative thermotaxis) and performs isothermal tracking near Ts. Under some conditions, the worm migrates up temperature gradients below Ts (positive thermotaxis). Here, we analyze positive and negative thermotaxis toward Ts to study the role of specific neurons that have been proposed to be involved in thermotaxis using genetic ablation, behavioral tracking, and calcium imaging. We find differences in the strategies for positive and negative thermotaxis. Negative thermotaxis is achieved through biasing the frequency of reorientation maneuvers (turns and reversal turns) and biasing the direction of reorientation maneuvers toward colder temperatures. Positive thermotaxis, in contrast, biases only the direction of reorientation maneuvers toward warmer temperatures. We find that the AFD thermosensory neuron drives both positive and negative thermotaxis. The AIY interneuron, which is postsynaptic to AFD, may mediate the switch from negative to positive thermotaxis below Ts. We propose that multiple thermotactic behaviors, each defined by a distinct set of sensorimotor transformations, emanate from the AFD thermosensory neurons. AFD learns and stores the memory of preferred temperatures, detects temperature gradients, and drives the appropriate thermotactic behavior in each temperature regime by the flexible use of downstream circuits.

The cGMP signaling pathway affects feeding behavior in the necromenic nematode Pristionchus pacificus.

BACKGROUND: The genetic tractability and the species-specific association with beetles make the nematode Pristionchus pacificus an exciting emerging model organism for comparative studies in development and behavior. P. pacificus differs from Caenorhabditis elegans (a bacterial feeder) by its buccal teeth and the lack of pharyngeal grinders, but almost nothing is known about which genes coordinate P. pacificus feeding behaviors, such as pharyngeal pumping rate, locomotion, and fat storage. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed P. pacificus pharyngeal pumping rate and locomotion behavior on and off food, as well as on different species of bacteria (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus). We found that the cGMP-dependent protein kinase G (PKG) Ppa-EGL-4 in P. pacificus plays an important role in regulating the pumping rate, mouth form dimorphism, the duration of forward locomotion, and the amount of fat stored in intestine. In addition, Ppa-EGL-4 interacts with Ppa-OBI-1, a recently identified protein involved in chemosensation, to influence feeding and locomotion behavior. We also found that C. crescentus NA1000 increased pharyngeal pumping as well as fat storage in P. pacificus. CONCLUSIONS: The PKG EGL-4 has conserved functions in regulating feeding behavior in both C. elegans and P. pacificus nematodes. The Ppa-EGL-4 also has been co-opted during evolution to regulate P. pacificus mouth form dimorphism that indirectly affect pharyngeal pumping rate. Specifically, the lack of Ppa-EGL-4 function increases pharyngeal pumping, time spent in forward locomotion, and fat storage, in part as a result of higher food intake. Ppa-OBI-1 functions upstream or parallel to Ppa-EGL-4. The beetle-associated omnivorous P. pacificus respond differently to changes in food state and food quality compared to the exclusively bacteriovorous C. elegans.

RNAi mediated gene knockdown and transgenesis by microinjection in the necromenic Nematode Pristionchus pacificus.

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis and Pristionchus pacificus. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F(1) progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.