Gold Nanorods and Nanoprisms Mediate Different Photothermal Cell Death Mechanisms In Vitro and In Vivo.

Photothermal therapy (PTT) is an efficient method of inducing localized hyperthermia and can be achieved using gold nanoparticles as photothermal agents. However, there are many hurdles to get over before this therapy can safely reach the clinics, including nanoparticles' optimal shape and the accurate prediction of cellular responses. Here, we describe the synthesis of gold nanorods and nanoprisms with similar surface plasmon resonances in the near-infrared (NIR) and comparable photothermal conversion efficiencies and characterize the response to NIR irradiation in two biological systems, melanoma cells and the small invertebrate Hydra vulgaris. By integrating animal, cellular, and molecular biology approaches, we show a diverse outcome of nanorods and nanoprisms on the two systems, sustained by the elicitation of different pathways, from necrosis to programmed cell death mechanisms (apoptosis and necroptosis). The comparative multilevel analysis shows great accuracy of in vivo invertebrate models to predict overall responses to photothermal challenging and superior photothermal performance of nanoprisms. Understanding the molecular pathways of these responses may help develop optimized nanoheaters that, safe by design, may improve PTT efficacy for clinical purposes.

Analysis of the conserved NER helicases (XPB and XPD) and UV-induced DNA damage in Hydra.

BACKGROUND: Nucleotide excision repair (NER) pathway is an evolutionarily conserved mechanism of genome maintenance. It detects and repairs distortions in DNA double helix. Xeroderma Pigmentosum group B (XPB) and group D (XPD) are important helicases in NER and are also critical subunits of TFIIH complex. We have studied XPB and XPD for the first time from the basal metazoan Hydra which exhibits lack of organismal senescence. METHODS: In silico analysis of proteins was performed using MEGA 6.0, Clustal Omega, Swiss Model, etc. Gene expression was studied by in situ hybridization and qRT-PCR. Repair of CPDs was studied by DNA blot assay. Interactions between proteins were determined by co- immunoprecipitation. HyXPB and HyXPD were cloned in pET28b, overexpressed and helicase activity of purified proteins was checked. RESULTS: In silico analysis revealed presence of seven classical helicase motifs in HyXPB and HyXPD. Both proteins revealed polarity-dependent helicase activity. Hydra repairs most of the thymine dimers induced by UVC (500 J/m2) by 72 h post-UV exposure. HyXPB and HyXPD transcripts, localized all over the body column, remained unaltered post-UV exposure indicating their constitutive expression. In spite of high levels of sequence conservation, XPB and XPD failed to rescue defects in human XPB- and XPD-deficient cell lines. This was due to their inability to get incorporated into the TFIIH multiprotein complex. CONCLUSIONS: Present results along with our earlier work on DNA repair proteins in Hydra bring out the utility of Hydra as model system to study evolution of DNA repair mechanisms in metazoans.

UV induced foot duplication in regenerating hydra is mediated by metalloproteinases and modulation of the Wnt pathway.

We have shown earlier that irradiation with UV induces duplication of foot in regenerating middle pieces of hydra. The present study was undertaken to elucidate the underlying mechanism(s) leading to this curious phenomenon. UV irradiation induced duplicated foot in about 30% of regenerating middle pieces. Metalloproteinases are important in foot formation, while Wnt pathway genes are important in head formation in hydra. The effect of UV irradiation on expression of these genes was studied by in situ hybridization and q-PCR. In whole polyps and middle pieces, UV irradiation led to up-regulation of HMP2 and HMMP, the two metalloproteinases involved in foot formation in hydra. HMP2 expression was significantly increased starting from 30 min post exposure to UV at 254 nm (500 J/m(2)), while HMMP showed significant up-regulation 6 h post UV exposure onwards. In middle pieces, increased expression of both metalloproteinases was observed only at 48 h. In whole polyps as well as in middle pieces, expression of Wnt3 and ß-catenin was detected within 30 min of UV exposure and was accompanied by up-regulation of GSK3ß, DKK3 and DKK1/2/4, inhibitors of the Wnt pathway. These conditions likely lead to inactivation of Wnt signaling. We therefore conclude that duplication of foot due to UV irradiation in regenerating middle pieces of hydra is a combined effect of up-regulation of metalloproteinases and inactivation of the Wnt pathway. Our results suggest that UV irradiation can be employed as a tool to understand patterning mechanisms during foot formation in hydra.

Extraocular spectral photosensitivity in the tentacles of Hydra vulgaris.

Previous electrophysiological studies on the cnidarian Hydra vulgaris have shown that hydra have a highly developed and specific photoresponse despite their lack of any structure recognizable as a traditional photoreceptor. In an effort to identify the site of hydra's photoreceptors, we recorded extracellularly from single excised tentacles and from ablated hypostomes lacking tentacles in absolute darkness and during exposure to light of various wavelengths. During recording, after an initial period of absolute darkness, tentacles or hypostomes were exposed to light from 450nm to 600nm, red, and white light. Exposure to light caused a change in the pattern and frequency of impulses in the tentacles that varied with color. The number of large tentacle pulses (TPs) increased at 550 and 600nm relative to darkness, whereas the number of small tentacle pulses (STPs) tended to decrease in 500nm light. Impulse frequency was significantly different among the different wavelengths. In addition to bursts of tentacle contraction pulses, long trains of pulses were observed. A change in lighting caused a switch from bursting to trains or vice versa. In contrast to excised tentacles, no change in electrical activity was seen in ablated hypostomes at any of the wavelengths relative to each other or relative to darkness. These results indicate that isolated tentacles can distinguish among and respond to various colors across the visible spectrum and suggest that electromagnetic information is transmitted from the tentacles to the hypostome where it may be integrated by the hypostomal nervous system, ultimately contributing to hydra's photoreceptive behavior.

Ultraviolet irradiation initiates ectopic foot formation in regenerating hydra and promotes budding.

We have studied the effects of ultraviolet-C (UVC) and Ultraviolet-B (UVB) on growth and pattern formation in Pelmatohydra oligactis. UVC brings about a significant increase in budding in intact hydra while UVB does not exhibit such an effect. Excessive budding could be a response for survival at wavelengths that damage biological tissues. If the head or base piece of a bisected hydra is irradiated and recombined with the unirradiated missing part, regeneration proceeds normally indicating that exposure of a body part with either an intact head or foot to UVC does not influence pattern formation. Most significantly, in the middle piece, but not in the head or the base piece of a trisected hydra, UVC leads to initiation of ectopic feet formation in almost one third of the cases. Thus, UV irradiation interferes with pattern formation in regenerating hydra, possibly by changing positional values, and promotes budding in intact hydra. This is the first report on induction of ectopic feet formation by UV in regenerating hydra and opens up the possibility of using UV irradiation as a tool to understand pattern formation in the enigmatic hydra.

Photobehaviour of Hydra (Cnidaria, Hydrozoa) and correlated mechanisms: a case of extraocular photosensitivity.

The morpho-functional organization correlated to photosensitivity in Cnidaria is that of ocelli and extraocular photoreception. Several examples of the second type of organization are reported. The photosensitivity of the cnidarian Hydra is of the extraocular (neural or dermal) type. The effects of photic stimulation (applied according to various experimental protocols: steady condition; step stimulus; single, twin, or repetitive pulses; different polarities and chromaticities of steady, step and pulse stimulation and different phases of pulse application) on the modulation of various bioelectric events linked to the periodic behaviour of the animal are reviewed. The mechanisms correlated with the photobehaviour of Hydra, as well as the problems still open on the molecular mechanisms of phototransduction, are discussed.

Effects of ultraviolet light (2535 A) and dactinomycin upon hydra (Pelmatohydra oligactis).

Hydras exposed to ultraviolet light (2535 A, 12 erg/mm-2/s-2) for 15 minutes suffer a depression immediately, individuals that survived recover quickly and lastingly. Hydras treated with dactinomycin of specific concentrations do not suffer a depression until several days later, do not recover and die eventually. The results are also comparable when hydras are exposed to light and treated with cytostatic of such concentration. In even milder solution of dactinomycin all unexpected hydras recover and survive. But, almost all of the hydras exposed to ultraviolet light and treated with this solution die eventually. The effect of ultraviolet light upon them is then distinctly adverse. Buds are much less damaged and they do not die in this concentration. They grow and reproduce normally. This paper deals with the above mentioned phenomena.

Regeneration of proximal and distal body part in hydra exposed to ultraviolet light (2535 angstroms).

Pelmatohydra oligactis was amputated in the central part of the gastral region and exposed to radiation of ultraviolet rays (2535 angstroms, 12 erg mm(-2)s(-2) for 7, 15 and 20 minutes. The regeneration of the proximal and distal part of the animal which was fixed 8, 24, 48, 72 and 96 hours after the cutting and radiation has been studied cito-histologically. The regeneration of the wounds caused by cutting and those caused by radiation have been compared. It has been found out that the wounds caused by radiation heal much harder and that the radiation-destroyed hypostome needs a longer period to regenerate than the cutting-removed hypostome. It is assumed that radiation-destroyed parts have an inhibitory effect upon environment. But, cito-histological changes concord to a great degree in both cases. The foot regeneration in the animals cut and exposed almost entirely concords the regeneration in the control animals which were cut but not exposed. Namely, both of them, as a rule, remain permanently without a foot. In the paper we have tried to explain these results and brought out the conclusion that hydras do not regenerate the foot because in the bud region there are many zimogen and interstitial cells which are not characteristic of a foot and that is why hydra has a directed growth exclusively toward the distal part, never the opposite. The growth is localized to the hypostome and the bud region. Radiation does not inhibit the process of the budding that has already begun. It is assumed that undamaged cell material travels from the gastroderm toward the bud and serves its formation.