Ultrahigh foraging rates of Baikal seals make tiny endemic amphipods profitable in Lake Baikal.

Understanding what, how, and how often apex predators hunt is important due to their disproportionately large effects on ecosystems. In Lake Baikal with rich endemic fauna, Baikal seals appear to eat, in addition to fishes, a tiny (<0.1 g) endemic amphipod Macrohectopus branickii (the world's only freshwater planktonic species). Yet, its importance as prey to seals is unclear. Globally, amphipods are rarely targeted by single-prey feeding (i.e., nonfilter-feeding) mammals, presumably due to their small size. If M. branickii is energetically important prey, Baikal seals would exhibit exceptionally high foraging rates, potentially with behavioral and morphological specializations. Here, we used animal-borne accelerometers and video cameras to record Baikal seal foraging behavior. Unlike the prevailing view that they predominantly eat fishes, they also hunted M. branickii at the highest rates (mean, 57 individuals per dive) ever recorded for single-prey feeding aquatic mammals, leading to thousands of catches per day. These rates were achieved by gradual changes in dive depth following the diel vertical migration of M. branickii swarms. Examining museum specimens revealed that Baikal seals have the most specialized comb-like postcanine teeth in the subfamily Phocinae, allowing them to expel water while retaining prey during high-speed foraging. Our findings show unique mammal-amphipod interactions in an ancient lake, demonstrating that organisms even smaller than krill can be important prey for single-prey feeding aquatic mammals if the environment and predators' adaptations allow high foraging rates. Further, our finding that Baikal seals directly eat macroplankton may explain why they are so abundant in this ultraoligotrophic lake.

From cancer to rejuvenation: incomplete regeneration as the missing link (part II: rejuvenation circle).

In the first part of our study, we substantiated that the embryonic reontogenesis and malignant growth (disintegrating growth) pathways are the same, but occur at different stages of ontogenesis, this mechanism is carried out in opposite directions. Cancer has been shown to be epigenetic-blocked redifferentiation and unfinished somatic embryogenesis. We formulated that only this approach of aging elimination has real prospects for a future that is fraught with cancer, as we will be able to convert this risk into a rejuvenation process through the continuous cycling of cell dedifferentiation-differentiation processes (permanent remorphogenesis). Here, we continue to develop the idea of looped ontogenesis and formulate the concept of the rejuvenation circle.

From cancer to rejuvenation: incomplete regeneration as the missing link (Part I: the same origin, different outcomes).

Here, we interpret malignant tissue transformation from the aging point of view, that is, as a result of insufficient cell adaptation to the needs of regeneration/repair and proliferation. A consequence of the aging (senescence) process is gradual loss of self-renewal potential. It limits lifespan and leads to death due to the decline of tissue/organ functions, failure of regulatory mechanisms, disruption of endogenous processes and increased susceptibility to exogenous factors. Recapitulation of the embryonic pathway of self-renewal/rejuvenation in adulthood is epigenetically determined. At the postembryonic stage, in the absence of immune privilege, this recapitulation is transformed into cancer (potency expansion of single structures composing the organism to the detriment of the whole organism or disintegrating growth). We suggest that the process of rebirth occurs in the same way as embryonic tissue growth. Thus, the idea to use the potential of the transformed cells to stop the aging process has been proposed.

Drift dives and prolonged surfacing periods in Baikal seals: resting strategies in open waters?

Many pinnipeds frequently rest on land or ice, but some species remain in open waters for weeks or months, raising the question of how they rest. A unique type of dive, called drift dives, has been reported for several pinnipeds with suggested functions of rest, food processing and predator avoidance. Prolonged surfacing periods have also been observed in captive seals and are thought to aid food processing. However, information from other species in a different environment would be required to better understand the nature and function of this behavior. In this study, we attached multi-sensor tags to Baikal seals Pusa sibirica, a rare, freshwater species that has no aquatic predators and few resting grounds during the ice-free season. The seals exhibited repeated drift dives (mean depth, 116 m; duration, 10.1 min) in the daytime and prolonged periods at the surface (mean duration, 1.3 h) mainly around dawn. Drift dives and prolonged surfacing periods were temporally associated and observed between a series of foraging dives, suggesting a similar function, i.e. a combination of resting and food processing. The maximum durations of both drift and foraging dives were 15.4 min, close to the aerobic dive limit of this species; therefore, metabolic rates might not be significantly depressed during drift dives, further supporting the function of food processing rather than purely resting. Our results also show that drift diving can occur in a predator-free environment, and thus predator avoidance is not a general explanation of drift dives in pinnipeds.

4,6-Di-tert-butyl-2,3-di-hydroxy-benzalde-hyde.

The title compound, C15H22O3, crystallizes with two independent mol-ecules in the asymmetric unit. In each mol-ecule, one hy-droxy group (at position 2) is involved in an intra-molecular O-H⋯O hydrogen bond, and another one (at position 3) exhibits bifurcated hydrogen-bonding being involved in intra- and inter-molecular O-H⋯O inter-actions. In the crystal, O-H⋯O hydrogen bonds link alternating independent mol-ecules into chains running along [010].

Selective induction of apoptosis in the hamster flank sebaceous gland organ by a topical liposome 5-alpha-reductase inhibitor: a treatment strategy for acne.

Acne is a very widespread cosmesis problem. Isotretinoin, a synthetic oral retinoid is used to treat acne, which is androgen dependent. Numerous side-effects occur from this treatment. 5-alpha-Reductase plays a critical role in normal and pathological androgen-dependent processes. We have taken the approach to develop a selective, effective, topically-applied 5-alpha-reductase inhibitor to modify unwanted or pathological processes in the pilosebaceous unit such as acne. Toward this goal, we have previously developed a selective liposome hair follicle targeting system. We demonstrate in this report that the 5-alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5alpha-androstane-17beta-carboxamide (4-MA) incorporated into liposomes induces apoptosis and inhibits growth of the dihydrotestosterone (DHT)-dependent hamster flank organ sebaceous gland. We have compared topical application of liposome 4-MA and solvent-formulated 4-MA and observed selective efficacy of topical application of liposome 4-MA by the reduction of size and induction of apoptosis only in the treated hamster flank organ. Apoptosis induced by liposome 4-MA in the treated flank organ sebaceous gland cells was observed both by assays for DNA fragments (transferase deoxytidyl uridine end labeling) and by observation of condensed and fragmented nuclei. When 4-MA was topically applied formulated in ethanol and glycerol without liposomes, the selective efficacy was lost. Liposome 4-MA did not significantly affect prostate weight, testosterone/DHT ratios or bodyweight gain compared to controls indicating safety as well as efficacy of topical application of liposome 4-MA for pathological processes such as acne.

Body density affects stroke patterns in Baikal seals.

Buoyancy is one of the primary external forces acting on air-breathing divers and it can affect their swimming energetics. Because the body composition of marine mammals (i.e. the relative amounts of lower-density lipid and higher-density lean tissue) varies individually and seasonally, their buoyancy also fluctuates widely, and individuals would be expected to adjust their stroke patterns during dives accordingly. To test this prediction, we attached acceleration data loggers to four free-ranging Baikal seals Phoca sibirica in Lake Baikal and monitored flipper stroking activity as well as swimming speed, depth and inclination of the body axis (pitch). In addition to the logger, one seal (Individual 4) was equipped with a lead weight that was jettisoned after a predetermined time period so that we had a set of observations on the same individual with different body densities. These four data sets revealed the general diving patterns of Baikal seals and also provided direct insights into the influence of buoyancy on these patterns. Seals repeatedly performed dives of a mean duration of 7.0 min (max. 15.4 min), interrupted by a mean surface duration of 1.2 min. Dive depths were 66 m on average, but varied substantially, with a maximum depth of 324 m. The seals showed different stroke patterns among individuals; some seals stroked at lower rates during descent than ascent, while the others had higher stroke rates during descent than ascent. When the lead weight was detached from Individual 4, the seal increased its stroke rate in descent by shifting swimming mode from prolonged glides to more stroke-and-glide swimming, and decreased its stroke rate in ascent by shifting from continuous stroking to stroke-and-glide swimming. We conclude that seals adopt different stroke patterns according to their individual buoyancies. We also demonstrate that the terminal speed reached by Individual 4 during prolonged glide in descent depended on its total buoyancy and pitch, with higher speeds reached in the weighted condition and at steeper pitch. A simple physical model allowed us to estimate the body density of the seal from the speed and pitch (1,027-1,046 kg m(-3), roughly corresponding to 32-41% lipid content, for the weighted condition; 1,014-1,022 kg m(-3), 43-47% lipid content, for the unweighted condition).

Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium.

Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo. The strain grows in tumor xenografts. In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice. S. typhimurium A1 is auxotrophic (Leu/Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally. Whether additional genetic lesions are present is not known. In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction. These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. In vivo, the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging. The bacteria, introduced i.v. or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice. By day 15, S. typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing. When the bacteria were injected intratumorally, the tumor completely regressed by day 20. There were no obvious adverse effects on the host when the bacteria were injected by either route. The S. typhimurium A1 strain grew throughout the tumor, including viable malignant tissue. This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy.

High efficiency genetic modification of hair follicles and growing hair shafts.

A technique for genetic modification of hair follicles was developed which results in efficient alteration of the hair shaft phenotype. High-level in vivo transgene expression was maintained in hair follicles such that growing hair shafts were phenotypically altered. Mouse anagen skin fragments, maintained in histoculture, were genetically modified at high efficiency with adenoviral-GFP. The histocultured skin fragments were treated with collagenase which made hair follicles accessible to the adenoviral GFP gene, allowing high-efficiency transduction. These skin fragments were subsequently grafted on to nude mice where GFP was readily visualized in as many as 75% of hair follicles. Most follicles produced GFP-fluorescent growing hair shafts. This technique has produced efficient genetic modification of the hair shaft.

Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression.

There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein (GFP) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFP-expressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities.

Optically imageable metastatic model of human breast cancer.

We report an optically imageable orthotopic metastatic nude mouse model of the human breast cancer MDA-MB-435 expressing green fluorescent protein (GFP). We demonstrate fluorescent imaging of primary and metastatic growth in live tissue and in intact animals. Fragments of tumor tissue expressing GFP were sutured into the pocket in the right second mammary gland. Tumor tissue was strongly fluorescent, enabling whole-body imaging of tumor growth by week 5. Neovascularization of the primary tumor was also visualized by whole-body imaging by contrast of the vessels to the fluorescent tumor. At autopsy, the MDA-MB-435-GFP was found to have metastasized to various organs, including the lung in 55% of the animals, the lymph nodes in 15% of the animals including axillary nodes, and the liver in 10% of the animals. These metastases could be visualized in fresh tissue by fluorescent imaging. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical metastatic breast cancer and provides a powerful model for drug discovery for this disease.

Dissecting p53 tumor suppressor functions in vivo.

Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.

A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy.

p53 and INK4a/ARF mutations promote tumorigenesis and drug resistance, in part, by disabling apoptosis. We show that primary murine lymphomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16(INK4a). Hence, tumors with p53 or INK4a/ARF mutations-but not those lacking ARF alone-respond poorly to cyclophosphamide therapy in vivo. Moreover, tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16(INK4a), and senescence markers, and typically acquire p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senescence have a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.

Parathyroid hormone-related protein as a novel tumor marker in pancreatic adenocarcinoma.

INTRODUCTION: Parathyroid hormone-related protein (PTHrP) can act as an oncoprotein to regulate the growth and proliferation of many common malignancies, including pancreatic cancer. Previous studies have shown that PTHrP is produced by human pancreatic cancer cell lines, can be shown in the cytoplasm and nucleus of paraffin-embedded pancreatic adenocarcinoma tumor specimens, and is secreted into the media of cultured pancreatic adenocarcinoma cells. We hypothesized that PTHrP could serve as a tumor-marker for growth of pancreatic cancer in vivo. AIM AND METHODOLOGY: To test this hypothesis, we used an orthotopic model developed in our laboratory of the PTHrP-producing human pancreatic cancer line, BxPC-3. This tumor was stably transduced with green fluorescence protein (GFP) to facilitate visualization of tumor growth and metastases. At early (5 weeks) and late (13 weeks) time points after surgical orthotopic implantation, serum PTHrP was measured and primary and metastatic tumor burden was determined for each mouse by assessing GFP expression. RESULTS: By 5 weeks after surgical orthotopic implantation (early group), the mean serum PTHrP level was 33.3 pg/mL. In contrast, by 13 weeks after surgical orthotopic implantation (late group), the mean serum PTHrP level increased to 158.5 pg/mL. These differences were highly significant (p < 0.001, Student t test). Numerous metastatic lesions were readily visualized by GFP in the late group. Serum PTHrP levels measured by immunoassay correlated with primary pancreatic tumor weights and serum calcium levels (p <0.01). PTHrP levels were not detectable (<21 pg/mL) in any of the 10 control mice with no tumor. Western blotting of BxPC-3-GFP tumor lysates confirmed the presence of PTHrP. BxPC-3-GFP tumor tissue stained with antibody to PTHrP. CONCLUSION: These results indicate that PTHrP can serve as a tumor marker in animal models of pancreatic cancer and may be a useful tumor marker for clinical pancreatic adenocarcinoma.

Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.

We report here whole-body optical imaging, in real time, of genetically fluorescent pancreatic tumors growing and metastasizing to multiple sites in live mice. The whole-body optical imaging system is external and noninvasive. Human pancreatic tumor cell lines, BxPC-3 and MiaPaCa-2, were engineered to stably express high-levels of the Aequorea victoria green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell lines were surgically orthotopically implanted as tissue fragments in the body of the pancreas of nude mice. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions that developed in the spleen, bowel, portal lymph nodes, omentum, and liver. Intravital images in the opened animal confirmed the identity of whole-body images. The whole-body images were used for real-time, quantitative measurement of tumor growth in each of these organs. Intravital imaging was used for quantification of growth of micrometastasis on the liver and stomach. Whole-body imaging was carried out with either a trans-illuminated epi-fluorescence microscope or a fluorescence light box, both with a thermoelectrically cooled color CCD camera. The simple, noninvasive, and highly selective imaging made possible by the strong GFP fluorescence allowed detailed simultaneous quantitative imaging of tumor growth and multiple metastasis formation of pancreatic cancer. The GFP imaging affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals without the need for anesthesia, substrate injection, contrast agents, or restraint of animals required by other imaging methods. The GFP imaging technology presented in this report will facilitate studies of modulators of pancreatic cancer growth, including inhibition by potential chemotherapeutic agents.

Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model.

Mouse tumor models have undergone profound improvements in the fidelity of emulating human disease. Replacing ectopic s.c. implantation with organ-specific orthotopic implantation reproduces human tumor growth and metastasis. Strong fluorescent labeling with green fluorescent protein along with inexpensive video detectors, positioned externally to the mouse, allows the monitoring of details of tumor growth, angiogenesis, and metastatic spread. However, the sensitivity of external imaging is limited by light scattering in intervening tissue, most especially in skin. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold. The observable depth of tissue is thereby greatly increased and many tumors that were previously hidden are now clearly observable. This report presents tumor images and related quantitative growth data previously impossible to obtain. Single tumor cells, expressing green fluorescent protein, were seeded on the brain image through a scalp skin-flap. Lung tumor microfoci representing a few cells are viewed through a skin-flap over the chest wall, while contralateral micrometastases were imaged through the corresponding skin-flap. Pancreatic tumors and their angiogenic microvessels were imaged by means of a peritoneal wall skin-flap. A skin-flap over the liver allowed imaging of physiologically relevant micrometastases originating in an orthotopically implanted tumor. Single tumor cells on the liver arising from intraportal injection also were detectable. Possible future technical developments are suggested by the image, through a lower-abdominal skin-flap, of an invasive prostate tumor expressing both red and green fluorescent proteins in separate colonies.