White-nose syndrome, winter duration, and pre-hibernation climate impact abundance of reproductive female bats.

White-nose syndrome (WNS) is an infectious disease that disrupts hibernation in bats, leading to premature exhaustion of fat stores. Though we know WNS does impact reproduction in hibernating female bats, we are unsure how these impacts are exacerbated by local climate factors. We compiled data from four southeastern U.S. states and used generalized linear mixed effects models to compare effects of WNS, pre-hibernation climate variables, and winter duration on the number of reproductive females in species across the range of WNS susceptibility. We predicted we would see a decline in the number of reproductive females in WNS-susceptible species, with the effect exaggerated by longer winter durations and pre-hibernation climate variables that lead to reductions in foraging. We found that the number of reproductive females in WNS-susceptible species was positively correlated with pre-hibernation local climate conditions conducive to foraging; however, WNS-susceptible species experienced an overall decline with the presence of WNS and as winter duration increased. Our long-term dataset provides evidence that pre-hibernation climate, specifically favorable summer weather conditions for foraging, greatly influences the reproduction, regardless of WNS status.

Dynamic Covalent Template-Guided Screen for Nucleic Acid-Targeting Agents.

Trinucleotide repeat diseases such as myotonic dystrophy type 1 (DM1) and Huntington's disease (HD) are caused by expanded DNA repeats that can be used as templates to synthesize their own inhibitors. Because it would be particularly advantageous to reversibly assemble multivalent nucleic acid-targeting agents in situ, we sought to develop a target-guided screen that uses dynamic covalent chemistry to identify multitarget inhibitors. We report the synthesis of a library of amine- or aldehyde-containing fragments. The assembly of these fragments led to a diverse set of hit combinations that was confirmed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in the presence of DM1 and HD repeat sequences. Of interest for both diseases, the resulting hit combinations inhibited transcription selectively and in a cooperative manner in vitro, with inhibitory concentration (IC50) values in the micromolar range. This dynamic covalent library and screening approach could be applied to identify compounds that reversibly assemble on other nucleic acid targets.

Selective and Reversible Ligand Assembly on the DNA and RNA Repeat Sequences in Myotonic Dystrophy.

Small molecule targeting of DNA and RNA sequences has come into focus as a therapeutic strategy for diseases such as myotonic dystrophy type 1 (DM1), a trinucleotide repeat disease characterized by RNA gain-of-function. Herein, we report a novel template-selected, reversible assembly of therapeutic agents in situ via aldehyde-amine condensation. Rationally designed small molecule targeting agents functionalized with either an aldehyde or an amine were synthesized and screened against the target nucleic acid sequence. The assembly of fragments was confirmed by MALDI-MS in the presence of DM1-relevant nucleic acid sequences. The resulting hit combinations of aldehyde and amine inhibited the formation of r(CUG)exp in vitro in a cooperative manner at low micromolar levels and rescued mis-splicing defects in DM1 model cells. This reversible template-selected assembly is a promising approach to achieve cell permeable and multivalent targeting via in situ synthesis and could be applied to other nucleic acid targets.

A Novel Minor Groove Binder as a Potential Therapeutic Agent for Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder that is inherited in an autosomal dominant manner. DM1 originates in a (CTG⋅CAG) repeat expansion in the 3'-UTR of the dystrophia myotonic protein kinase (DMPK) gene on chromosome 19. One of the transcripts, r(CUG)exp , is toxic in various ways. Herein we report a rationally designed small molecule with a thiazole peptidomimetic unit that can serve as a minor groove binder for the nucleic acid targets. This peptide unit linked to two triaminotriazine recognition units selectively binds to d(CTG)exp to inhibit the transcription process, and also targets r(CUG)exp selectively to improve representative DM1 pathological molecular features, including foci formation and pre-mRNA splicing defects in DM1 model cells. As such, it represents a new structure type that might serve as a lead compound for future structure-activity optimization.

Versatile Target-Guided Screen for Discovering Bidirectional Transcription Inhibitors of a Trinucleotide Repeat Disease.

Myotonic dystrophy type 1 originates from d(CTG·CAG) repeats that undergo aberrant expansion during normal processing because the d(CTG) repeat forms stable hairpin structures. Bidirectional transcription of d(CTG·CAG) yields two RNA transcripts that undergo repeat-associated non-ATG (RAN) translation to form homopolymeric proteins. Thus, both the r(CUG) transcript and the r(CAG) transcript are known to be toxic. We report a pairwise fragment-based, target-guided approach to screen for proximity-induced click dimers formed on the nucleic acid template. This screen uses an azide/alkyne clickable fragment library of nucleic acid-binding ligands incubated in parallel, pairwise reactions as an alternative to our previously reported one-pot screening method. MALDI-TOF mass spectroscopy was used to detect template assisted click products. Hit compounds inhibited the in vitro transcription of d(CTG·CAG)90 bidirectionally with IC50 values in the low micromolar range. This approach may be broadly applicable to other trinucleotide repeat diseases and in targeting other disease-associated nucleic acid sequences.

Syndapin I Loss-of-Function in Mice Leads to Schizophrenia-Like Symptoms.

Schizophrenia is associated with cognitive and behavioral dysfunctions thought to reflect imbalances in neurotransmission systems. Recent screenings suggested that lack of (functional) syndapin I (PACSIN1) may be linked to schizophrenia. We therefore studied syndapin I KO mice to address the suggested causal relationship to schizophrenia and to analyze associated molecular, cellular, and neurophysiological defects. Syndapin I knockout (KO) mice developed schizophrenia-related behaviors, such as hyperactivity, reduced anxiety, reduced response to social novelty, and an exaggerated novel object response and exhibited defects in dendritic arborization in the cortex. Neuromorphogenic deficits were also observed for a schizophrenia-associated syndapin I mutant in cultured neurons and coincided with a lack of syndapin I-mediated membrane recruitment of cytoskeletal effectors. Syndapin I KO furthermore caused glutamatergic hypofunctions. Syndapin I regulated both AMPAR and NMDAR availabilities at synapses during basal synaptic activity and during synaptic plasticity-particularly striking were a complete lack of long-term potentiation and defects in long-term depression in syndapin I KO mice. These synaptic plasticity defects coincided with alterations of postsynaptic actin dynamics, synaptic GluA1 clustering, and GluA1 mobility. Both GluA1 and GluA2 were not appropriately internalized. Summarized, syndapin I KO led to schizophrenia-like behavior, and our analyses uncovered associated molecular and cellular mechanisms.

Targeting CD74 in multiple myeloma with the novel, site-specific antibody-drug conjugate STRO-001.

STRO-001 is a site-specific, predominantly single-species, fully human, aglycosylated anti-CD74 antibody-drug conjugate incorporating a non-cleavable linker-maytansinoid warhead with a drug-antibody ratio of 2 which was produced by a novel cell-free antibody synthesis platform. We examined the potential pharmacodynamics and anti-tumor effects of STRO-001 in multiple myeloma (MM). CD74 expression was assessed in MM cell lines and primary bone marrow (BM) MM biopsies. CD74 mRNA was detectable in CD138+ enriched plasma cells from 100% (892/892) of patients with newly diagnosed MM. Immunohistochemistry confirmed CD74 expression in 35/36 BM biopsies from patients with newly diagnosed and relapsed/refractory MM. Cytotoxicity assays demonstrated nanomolar STRO-001 potency in 4/6 MM cell lines. In ARP-1 and MM.1S tumor-bearing mice, repeat STRO-001 dosing provided significant antitumor activity with eradication of malignant hCD138+ BM plasma cells and prolonged survival. In a luciferase-expressing MM.1S xenograft model, dose-dependent STRO-001 efficacy was confirmed using bioluminescent imaging and BM tumor burden quantification. Consistent with the intended pharmacodynamic effect, STRO-001 induced dose-responsive, reversible B-cell and monocyte depletion in cynomolgus monkeys, up to a maximum tolerated 10 mg/kg, with no evidence of off-target toxicity. Collectively, these data suggest that STRO-001 is a promising therapeutic agent for the treatment of MM.

Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination.

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

Pulsed Radiation Therapy With Concurrent Cisplatin Results in Superior Tumor Growth Delay in a Head and Neck Squamous Cell Carcinoma Murine Model.

PURPOSE: To assess the efficacy of 3-week schedules of low-dose pulsed radiation treatment (PRT) and standard radiation therapy (SRT), with concurrent cisplatin (CDDP) in a head and neck squamous cell carcinoma xenograft model. METHODS AND MATERIALS: Subcutaneous UT-SCC-14 tumors were established in athymic NIH III HO female mice. A total of 30 Gy was administered as 2 Gy/d, 5 d/wk for 3 weeks, either by PRT (10 × 0.2 Gy/d, with a 3-minute break between each 0.2-Gy dose) or SRT (2 Gy/d, uninterrupted delivery) in combination with concurrent 2 mg/kg CDDP 3 times per week in the final 2 weeks of radiation therapy. Treatment-induced growth delays were defined from twice-weekly tumor volume measurements. Tumor hypoxia was assessed by (18)F-fluoromisonidazole positron emission tomography imaging, and calculated maximum standardized uptake values compared with tumor histology. Tumor vessel density and hypoxia were measured by quantitative immunohistochemistry. Normal tissues effects were evaluated in gut and skin. RESULTS: Untreated tumors grew to 1000 mm(3) in 25.4 days (±1.2), compared with delays of 62.3 days (±3.5) for SRT + CDDP and 80.2 days (±5.0) for PRT + CDDP. Time to reach 2× pretreatment volume ranged from 8.2 days (±1.8) for untreated tumors to 67.1 days (±4.7) after PRT + CDDP. Significant differences in tumor growth delay were observed for SRT versus SRT + CDDP (P=.04), PRT versus PRT + CDDP (P=.035), and SRT + CDDP versus PRT + CDDP (P=.033), and for survival between PRT versus PRT + CDDP (P=.017) and SRT + CDDP versus PRT + CDDP (P=.008). Differences in tumor hypoxia were evident by (18)F-fluoromisonidazole positron emission tomography imaging between SRT and PRT (P=.025), although not with concurrent CDDP. Tumor vessel density differed between SRT + CDDP and PRT + CDDP (P=.011). No differences in normal tissue parameters were seen. CONCLUSIONS: Concurrent CDDP was more effective in combination PRT than SRT at restricting tumor growth. Significant differences in tumor vascular density were evident between PRT and SRT, suggesting a preservation of vascular network with PRT.

Effect of Irradiation on Tumor Microenvironment and Bone Marrow Cell Migration in a Preclinical Tumor Model.

PURPOSE: To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. METHODS AND MATERIALS: Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2 Gy/d for a total dose of 20 Gy using a 5 days on, 2 days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40 Gy in 4 weeks using the same 2-Gy/d regimens. RESULTS: Over the 2 weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31 ± 0.02 mm(3)/d and 0.55 ± 0.04 mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5% ± 3%), hypoxia (10% ± 3.5%), vascular endothelial growth factor (2.3% ± 1%), and stromal-derived factor-1α (2.5% ± 1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8% ± 2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6% ± 0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. CONCLUSIONS: Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.

Modeling of chronic radiation-induced cystitis in mice.

PURPOSE: Radiation cystitis (RC), a severe inflammatory bladder condition, develops as a side-effect of pelvic radiation therapy in cancer patients. There are currently no effective therapies to treat RC, in part due to the lack of preclinical model systems. In this study, we developed a mouse model for RC and used a small animal radiation research platform (SARRP) to simulate the targeted delivery of radiation as used with human patients. METHODS AND MATERIALS: To induce RC, C3H mice received a single radiation dose of 20Gy delivered through two beams. Mice were subjected to weekly micturition measurements to assess changes in urinary frequency. At the end of the study, bladder tissues were processed for histology. RESULTS: Radiation was well tolerated as no change in weight was observed in the weeks post treatment, and there was no hair loss at the irradiation sites. Starting at 17 weeks post treatment, micturition frequency was significantly higher in irradiated mice versus control animals. Pathological changes include fibrosis, inflammation, urothelial thinning and necrosis. At a site of severe insult, we observed telangiectasia, absence of Uroplakin-3 and E-cadherin relocalization. CONCLUSIONS: We developed a RC model that mimics the human pathology and functional changes. Furthermore, radiation exposure attenuates the urothelial integrity long-term allowing for potential continuous irritability of the bladder wall from exposure to urine. Future studies will focus on the underlying molecular changes associated with this condition and investigate novel treatment strategies.

The Effects of Pulsed Radiation Therapy on Tumor Oxygenation in 2 Murine Models of Head and Neck Squamous Cell Carcinoma.

PURPOSE: To evaluate the efficacy of low-dose pulsed radiation therapy (PRT) in 2 head and neck squamous cell carcinoma (HNSCC) xenografts and to investigate the mechanism of action of PRT compared with standard radiation therapy (SRT). METHODS AND MATERIALS: Subcutaneous radiosensitive UT-SCC-14 and radioresistant UT-SCC-15 xenografts were established in athymic NIH III HO female mice. Tumors were irradiated with 2 Gy/day by continuous standard delivery (SRT: 2 Gy) or discontinuous low-dose pulsed delivery (PRT: 0.2 Gy × 10 with 3-min pulse interval) to total doses of 20 Gy (UT14) or 40 Gy (UT15) using a clinical 5-day on/2-day off schedule. Treatment response was assessed by changes in tumor volume, (18)F-fluorodeoxyglucose (FDG) (tumor metabolism), and (18)F-fluoromisonidazole (FMISO) (hypoxia) positron emission tomography (PET) imaging before, at midpoint, and after treatment. Tumor hypoxia using pimonidazole staining and vascular density (CD34 staining) were assessed by quantitative histopathology. RESULTS: UT15 and UT14 tumors responded similarly in terms of growth delay to either SRT or PRT. When compared with UT14 tumors, UT15 tumors demonstrated significantly lower uptake of FDG at all time points after irradiation. UT14 tumors demonstrated higher levels of tumor hypoxia after SRT when compared with PRT as measured by (18)F-FMISO PET. By contrast, no differences were seen in (18)F-FMISO PET imaging between SRT and PRT for UT15 tumors. Histologic analysis of pimonidazole staining mimicked the (18)F-FMISO PET imaging data, showing an increase in hypoxia in SRT-treated UT14 tumors but not PRT-treated tumors. CONCLUSIONS: Differences in (18)F-FMISO uptake for UT14 tumors after radiation therapy between PRT and SRT were measurable despite the similar tumor growth delay responses. In UT15 tumors, both SRT and PRT were equally effective at reducing tumor hypoxia to a significant level as measured by (18)F-FMISO and pimonidazole.

Intravesical Liposomal Tacrolimus Protects against Radiation Cystitis Induced by 3-Beam Targeted Bladder Radiation.

PURPOSE: We primarily determined whether the small animal radiation research platform could create a rat radiation cystitis model via targeted bladder irradiation (phase I). The response to treating early phase radiation cystitis in rats with transurethral catheter instillation of liposomal tacrolimus was also examined (phase II). MATERIALS AND METHODS: In phase I 16 adult female Sprague Dawley® rats were used. Metabolic urination patterns were analyzed before and after exposure to 20, 30 or 40 Gy radiation. In phase II irradiated rats were randomly assigned to receive a single instillation of saline or liposomal tacrolimus. RESULTS: The 40 Gy radiation dose induced statistically significant reductions in the intermicturition interval compared to the lower radiation doses. By approximately 20 minutes 40 Gy radiation caused a significant decrease in the mean intermicturition interval (p < 0.0001). Histological analysis revealed degenerative epithelial changes and urothelial swelling with evidence of pseudocarcinomatous epithelial hyperplasia. Therefore, 40 Gy were chosen for the phase II efficacy study. There was no measurable change in total voided urine volume after irradiation, or after liposomal tacrolimus or saline instillation. Liposomal tacrolimus significantly increased the post-irradiation intermicturition interval by approximately 30 minutes back to baseline (p < 0.001). CONCLUSIONS: The radiation cystitis rat model showed a dose dependent decrease in the intermicturition interval without inducing short-term skin or gastrointestinal damage. This study demonstrates that liposomal tacrolimus may be a promising new intravesical therapy for the rare, serious condition of radiation cystitis.

Hematopoietic stem and progenitor cell migration after hypofractionated radiation therapy in a murine model.

PURPOSE: To characterize the recruitment of bone marrow (BM)-derived hematopoietic stem and progenitor cells (HSPCs) within tumor microenvironment after radiation therapy (RT) in a murine, heterotopic tumor model. METHODS AND MATERIALS: Lewis lung carcinoma tumors were established in C57BL/6 mice and irradiated with 30 Gy given as 2 fractions over 2 days. Tumors were imaged with positron emission tomography/computed tomography (PET/CT) and measured daily with digital calipers. The HSPC and myelomonocytic cell content was assessed via immunofluorescent staining and flow cytometry. Functionality of tumor-associated HSPCs was verified in vitro using colony-forming cell assays and in vivo by rescuing lethally irradiated C57BL/6 recipients. RESULTS: Irradiation significantly reduced tumor volumes and tumor regrowth rates compared with nonirradiated controls. The number of CD133(+) HSPCs present in irradiated tumors was higher than in nonirradiated tumors during all stages of regrowth. CD11b(+) counts were similar. PET/CT imaging and growth rate analysis based on standardized uptake value indicated that HSPC recruitment directly correlated to the extent of regrowth and intratumor cell activity after irradiation. The BM-derived tumor-associated HSPCs successfully formed hematopoietic colonies and engrafted irradiated mice. Finally, targeted treatment with a small animal radiation research platform demonstrated localized HSPC recruitment to defined tumor subsites exposed to radiation. CONCLUSIONS: Hypofractionated irradiation resulted in a pronounced and targeted recruitment of BM-derived HSPCs, possibly as a mechanism to promote tumor regrowth. These data indicate for the first time that radiation therapy regulates HSPC content within regrowing tumors.

Pulsed versus conventional radiation therapy in combination with temozolomide in a murine orthotopic model of glioblastoma multiforme.

PURPOSE: To evaluate the efficacy of pulsed low-dose radiation therapy (PLRT) combined with temozolomide (TMZ) as a novel treatment approach for radioresistant glioblastoma multiforme (GBM) in a murine model. METHODS AND MATERIALS: Orthotopic U87MG hGBM tumors were established in Nu-Foxn1(nu) mice and imaged weekly using a small-animal micropositron emission tomography (PET)/computed tomography (CT) system. Tumor volume was determined from contrast-enhanced microCT images and tumor metabolic activity (SUVmax) from the F18-FDG microPET scan. Tumors were irradiated 7 to 10 days after implantation with a total dose of 14 Gy in 7 consecutive days. The daily treatment was given as a single continuous 2-Gy dose (RT) or 10 pulses of 0.2 Gy using an interpulse interval of 3 minutes (PLRT). TMZ (10 mg/kg) was given daily by oral gavage 1 hour before RT. Tumor vascularity and normal brain damage were assessed by immunohistochemistry. RESULTS: Radiation therapy with TMZ resulted in a significant 3- to 4-week tumor growth delay compared with controls, with PLRT+TMZ the most effective. PLRT+TMZ resulted in a larger decline in SUVmax than RT+TMZ. Significant differences in survival were evident. Treatment after PLRT+TMZ was associated with increased vascularization compared with RT+TMZ. Significantly fewer degenerating neurons were seen in normal brain after PLRT+TMZ compared with RT+TMZ. CONCLUSIONS: PLRT+TMZ produced superior tumor growth delay and less normal brain damage when compared with RT+TMZ. The differential effect of PLRT on vascularization may confirm new treatment avenues for GBM.

Determining if low dose hyper-radiosensitivity (HRS) can be exploited to provide a therapeutic advantage: a cell line study in four glioblastoma multiforme (GBM) cell lines.

PURPOSE: To determine if ultra-fractionation using repeated pulses of radiation (10 × 0.2 Gray [Gy]) would be more cytotoxic than continuously-delivered radiation to the same total dose (2 Gy) in four glioma cell lines. MATERIALS AND METHODS: Human T98G, U373, U87MG and U138MG cells were conventionally X-irradiated with 0.1-8 Gy and clonogenic survival assessed. Next, cells were treated with either a single dose of 2 Gy or 10 pulses of 0.2 Gy using a 3-min inter-pulse interval and DNA (Deoxyribonucleic acid) repair (pHistone H2A.X), G2-phase cell cycle checkpoint arrest (pHistone H3) and apoptosis (caspase-3) compared between the two regimens. A dose of 0.2 Gy was selected as this reflects the hyper- radiosensitivity (HRS)/increased radioresistance (IRR) transition point of the low-dose cell survival curve. RESULTS: T98G, U87MG and U138MG exhibited distinct HRS responses and survival curves were well-described by the Induced Repair model. Despite the prolonged delivery time, ultra-fractionation (10 × 0.2 Gy) was equally effective as a single continuously-delivered 2 Gy dose. However, ultra-fractionation was more effective when given for five consecutive days to a total dose of 10 Gy. The increased effectiveness of ultra-fractionation could not be attributed directly to differences in DNA damage, repair processes or radiation-induced apoptosis. CONCLUSIONS: Ultra-fractionation (10 × 0.2 Gy) is an effective modality for killing glioma cell lines compared with standard 2 Gy dosing when multiple days of treatment are given.

Pulsed low-dose irradiation of orthotopic glioblastoma multiforme (GBM) in a pre-clinical model: effects on vascularization and tumor control.

BACKGROUND AND PURPOSE: To compare dose-escalated pulsed low-dose radiation therapy (PLRT) and standard radiation therapy (SRT). METHODS AND MATERIALS: Intracranial U87MG GBM tumors were established in nude mice. Animals received whole brain irradiation with daily 2-Gy fractions given continuously (SRT) or in ten 0.2-Gy pulses separated by 3-min intervals (PLRT). Tumor response was evaluated using weekly CT and [(18)F]-FDG-PET scans. Brain tissue was subjected to immunohistochemistry and cytokine bead array to assess tumor and normal tissue effects. RESULTS: Median survival for untreated animals was 18 (SE±0.5) days. A significant difference in median survival was seen between SRT (29±1.8days) and PLRT (34.2±1.9days). Compared to SRT, PLRT resulted in a 31% (p<0.01), 38% (p<0.01), and 53% (p=0.01) reduction in normalized tumor volume and a 48% (p<0.01), 51% (p<0.01), and 70% (p<0.01) reduction in tumor growth rate following the administration of 10Gy, 20Gy, and 30Gy, respectively. Compared to untreated tumors, PLRT resulted in similar tumor vascular density, while SRT produced a 40% reduction in tumor vascular density (p=0.05). Compared to SRT, PLRT was associated with a 28% reduction in degenerating neurons in the surrounding brain parenchyma (p=0.05). CONCLUSIONS: Compared to SRT, PLRT resulted in greater inhibition of tumor growth and improved survival, which may be attributable to preservation of vascular density.

Organ growth without cell division: somatic polyploidy in a moth, Ephestia kuehniella.

Organ growth depends on cell division and (or) cell growth. Here, we present a study on two organs whose growth depends entirely on cell growth, once they are formed in the embryo: Malpighian tubules and silk glands of the flour moth, Ephestia kuehniella . Between first and last larval instar, the volume of Malpighian tubule cells increases by a factor of ∼1800 and that of silk gland cells by a factor of ∼3100. We determined the number of endocyles required to reach these stages by Feulgen cytometry. Cells of Malpighian tubules were in the 2C stage in first instar larvae and reached 1024C after 9 endocycles in last instar larvae (1C = 0.45 pg DNA). Silk gland cells already reached a DNA content of 8C-16C in first instar larvae and attained up to 8192C in last instar larvae after a total of 12 endocycles. The nuclei were small and more or less spherical in first instar larvae, but they were huge, flat, and bizarrely branched in last instar larvae. We consider branching as a compensatory adaptation to improve molecular traffic between nucleus and cytoplasm in these excessively large and highly polyploid cells (i) by reducing the mean distance between nucleus and cytoplasm and (ii) by enlarging the surface-to-volume ratio of these nuclei.