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Bone marrow adipose tissue (MAT) accounts for up to 70% of bone marrow volume in healthy adults and increases further in clinical conditions of altered skeletal or metabolic function. Perhaps most strikingly, and in stark contrast to white adipose tissue, MAT has been found to increase during caloric restriction (CR) in humans and many other species. Hypoleptinemia may drive MAT expansion during CR but this has not been demonstrated conclusively. Indeed, MAT formation and function are poorly understood; hence, the physiological and pathological roles of MAT remain elusive. We recently revealed that MAT contributes to hyperadiponectinemia and systemic adaptations to CR. To further these observations, we have now performed CR studies in rabbits to determine whether CR affects adiponectin production by MAT. Moderate or extensive CR decreased bone mass, white adipose tissue mass, and circulating leptin but, surprisingly, did not cause hyperadiponectinemia or MAT expansion. Although this unexpected finding limited our subsequent MAT characterization, it demonstrates that during CR, bone loss can occur independently of MAT expansion; increased MAT may be required for hyperadiponectinemia; and hypoleptinemia is not sufficient for MAT expansion. We further investigated this relationship in mice. In females, CR increased MAT without decreasing circulating leptin, suggesting that hypoleptinemia is also not necessary for MAT expansion. Finally, circulating glucocorticoids increased during CR in mice but not rabbits, suggesting that glucocorticoids might drive MAT expansion during CR. These observations provide insights into the causes and consequences of CR-associated MAT expansion, knowledge with potential relevance to health and disease.

The serotonergic neurons of the dorsal raphe (DR) nucleus in the CNS are involved in fear, anxiety and depression. Depression and anxiety occur quite frequently in postmenopausal women, but estrogen replacement to correct these CNS disorders is at present not favored because estrogen carries with it an increased risk for breast cancer. Serotonin synthesis, release and reuptake in the DR are targets of pharmaceuticals in the treatment of depression. In the present study we have examined by immunohistochemistry, the expression of two nuclear receptors, that is, the estrogen receptors ERα and ERβ. We found that ERβ but not ERα is strongly expressed in the DR and there is no sex difference and no change with ageing in the number of tryptophan hydroxylase (TPH)-positive neurons in the DR of wild-type (WT) mice. However, in ovariectomized (OVX) WT and in ERβ(-/-) mice, there was a marked reduction in the number of TPH-positive normal-looking neurons and a marked increase in TPH-positive spindle-shaped cells. These neuronal changes were prevented in mice 1-3 weeks (but not 10 weeks) after OVX by the selective ERβ agonist, LY3201, given as continuous release pellets for 3 days. The ERβ agonist had no effects on glucose homeostasis. Thus, the onset of action of the ERβ agonist is rapid but there is a limited window in time after estrogen loss when the drug is useful. We conclude that, rather than estradiol, ERβ agonists could be useful pharmaceuticals in maintaining functional DR neurons to treat postmenopausal depression.

An estrogen receptor (ER) β ligand (LY3201) with a preference for ERβ over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17β is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD + terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP + astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERβ was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERβ agonist results in changes opposite to those reported in estradiol-17β–treated mice and suggests that ERα and ERβ play opposing roles in the brain.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.

Nanoimprint lithography (NIL) has developed from an emerging nanoreplication technology into a mature and industrially viable manufacturing technology. It is now by far the fastest and most cost-efficient nanoreplication method available. This technology has been successfully implemented in the manufacturing of High Brightness LED's. However, manufacturing of HBLED's in high volume using nanoimprint lithography faces a few particular challenges. Perhaps the most significant is to produce perfectly imprinted nanostructures on the epitaxially grown substrates with high quality and yield. This is especially important since the substrates are expensive and since the imprint step is close to the end of the production process. This means that the value of the processed substrates is very high. In this work, 8000 imprints were produced. Measured data from the imprinted substrates shows consistent results. It is also shown that the 8000 imprints have been performed using the same stamp without significant degradation. After 8000 imprints, the yield from the nanoimprint lithography step is 99.15%.

Background: Incidental durotomy is among the most common complications of spine surgery with reported incidence ranging from 1.7% to 16%. Various management options including primary repair, fascial or fat graft, epidural blood patch, fibrin glue sealant, etc., have been proposed. The purpose of this study is to evaluate the incidence of incidental durotomy and the efficacy of different management options during a five year period at a tertiary care center.Methods: All patients who underwent various surgical procedures in thoracic and lumbar spine from January 2006 - December 2010 in our centre were retrospectively reviewed. Data on demographics, primary diagnosis, associated co morbidities, details of surgical procedure, training level of the operating surgeon, details of the incidental durotomy, the treatment, complications and the postoperative stay were recorded.Results: Of 2270 patients, 1401 patients were included in the study. The incidence of incidental durotomy was 3.49% (49 patients). We found a very high incidence of 33.33% incidental durotomies among patients who underwent revision procedures as compared to 3.23% for patients who underwent primary surgeries. 5.10% of incidental durotomies were caused by fellows under training, 4.27% by junior consultants and 2.92% by senior consultants. Of 49 durotomies, complication were 5 cases of intracranial hypotension, 5 postoperative neurological deficits, 2 deep wound infection, 2 pseudomeningocele and 1 meningitis.Conclusions: The risk of incidental durotomy in thoracolumbar surgeries is high in revision surgeries and when performed by fellows in training. Intraoperative identification and primary repair with suturing or sealant reduces postoperative complications.

The status of the tumor-immune microenvironment plays a critical role in determining the response to immunotherapy. The omentum, a multifunctional organ that serves as the central regulator of peritoneal homeostasis and plays a central role in immune surveillance, is the most common site of metastasis in early stage ovarian cancer. We analyzed the human omental immune microenvironment that support ovarian cancer metastases. In experimental models, ovarian cancer cells that colonize the omentum are invariably found in association with immune aggregates known as milky spots. We hypothesize that ovarian cancer cells exploit the unique environment of these omental-immune clusters to promote their own survival and growth. Thus, we formulated a systematic approach to evaluate normal and ovarian cancer-populated omental tissues to characterize the cellular components of human omental milky spots. We performed immunohistochemistry and flow cytometry to profile immune cell populations present in omental immune clusters. To facilitate the quantitation of immunohistochemical staining, we developed a computational image processing workflow that enables quantitation of multiple immune-biomarkers from serial sections of formalin-fixed paraffin-embedded tissue. In a complementary approach, we used comprehensive multi-color flow cytometry staining panels detecting 28 markers that collectively encompass all innate and adaptive immune cell populations. We analyzed eight human omental samples (four omenta involved with serous carcinomas and four omenta from benign disease) by immunohistochemistry and found that M2-subtype macrophage (CD163+CD68+) infiltration predominate in high-grade cancer-involved omentum. Conversely, T-regulatory cells (CD25+) predominate cell type in the uninvolved omentum. Hi-D flow cytometry of three cancer-involved omentum samples revealed tumor-associated macrophages (20-40%), PMN myeloid-derived suppressor cells (2-3%), “double positive” CD3+CD4+CD8+ T-cells (3-4%), and “double negative” CD3+CD4-CD8- T-cells (0.9-1%). In addition, we found omental T-cells (Th, Tc, Treg) express immune checkpoint receptors CTLA4 and PD-1, and that omental B-cells express IgD, IgA, and/or IgM, and may be enriched for Ig lambda light chain expression. As this study is ongoing, we expect to present data for omenta from a larger cohort of patients with either benign or neoplastic disease. However, our findings are already sufficient to show that activated T cells, B cells and macrophages infiltrate omental milky spots in patients with high grade serous ovarian cancers. Citation Format: Venkatesh Krishnan, Paul A. Raju, Koah Vierkoetter, Sonia Patel, Justin Youngyunpipatkul, Supreeti Tallapragada, Bruce Schaar, Ann K. Folkins, Leonore A. Herzenberg, Oliver Dorigo. Omentum immune microenvironment: Metastatic niche for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5074.

Hematogenous septic arthritis of the lumbar facet is a well-recognized although rare1-5 primary infectious entity of the spine. Traditionally, the most commonly implicated organism has been Staphylococcus aureus1,2,6 . Although there have been reports of methicillin-resistant Staphylococcus aureus (MRSA) spondylodiscitis7, methicillin-resistant Staphylococcus aureus as a cause of hematogenous facet joint septic arthritis has not been described, to our knowledge. We report our experience with the treatment of two cases of facet joint septic arthritis due to methicillin-resistant Staphylococcus aureus . The patients were informed that data concerning these cases would be submitted for publication, and they consented. Case 1. A fifty-three-year-old man presented with an eighteen-day history of severe back pain radiating to the left lower limb. He was unable to walk because of the intensity of pain. He had retention of urine, for which he required catheterization. Apart from being a chronic smoker, he had no medical comorbidities. Clinical examination revealed severe tenderness over the left side of the lower back. Neurological examination of the left lower extremity revealed grade-2 (of 5) strength of the extensor hallucis longus and tibialis anterior muscles. Lower extremity sensation and deep tendon reflexes were normal. Rectal examination revealed a lax anal sphincter and an absent bulbocavernosus reflex. Hematological examination showed an erythrocyte sedimentation rate of 77 mm/hr (normal, 6 to 12 mm/hr), a total white blood-cell count of 17.4 × 109/L (normal, 4.3 to 10.8 × 109/L), and a C-reactive protein level of 60 mg/L (normal

Recent clinical data on selective estrogen receptor modulators (SERMs) have provided the basis for reassessment of the SERM concept. The molecular basis of SERM activity involves binding of the ligand SERM to the estrogen receptor (ER), causing conformational changes which facilitate interactions with coactivator or corepressor proteins, and subsequently initiate or suppress transcription of target genes. SERM activity is intrinsic to each ER ligand, which accomplishes its unique profile by specific interactions in the target cell, leading to tissue selective actions. We discuss the estrogenic and anti-estrogenic effects of early SERMs, such as clomiphene citrate, used for treatment of ovulation induction, and the triphenylethylene, tamoxifen, which has ER antagonist activity in the breast, and is used for prevention and treatment of ER-positive breast cancer. Since the development of tamoxifen, other triphenylethylene SERMs have been studied for breast cancer prevention, including droloxifene, idoxifene, toremifene, and ospemifene. Other SERMs have entered clinical development more recently, including benzothiophenes (raloxifene and arzoxifene), benzopyrans (ormeloxifene, levormeloxifene, and EM-800), lasofoxifene, pipendoxifene, bazedoxifene, HMR-3339, and fulvestrant, an anti-estrogen which is approved for breast cancer treatment. SERMs have effects on tissues containing ER, such as the breast, bone, uterine and genitourinary tissues, and brain, and on markers of cardiovascular risk. Current evidence indicates that each SERM has a unique array of clinical activities. Differences in the patterns of action of SERMs suggest that each clinical end point must be evaluated individually, and conclusions about any particular SERM can only be established through appropriate clinical trials. Target Audience: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After completion of this article, the reader should be able to summarize the complex function and interactions of selective estrogen receptor modulators (SERMs); recall their suppression or initiation of target genes leading them to their actions; and explain their important roles in breast cancer treatment and possible side effects on bone, the genitourinary system, and on markers of cardiovascular risk.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER receptor subtypes alpha and beta in opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substitution at the 8-position of the benzopyran A-ring to disrupt binding to ERalpha, thus improving ERbeta subtype selectivity. X-ray cocrystal structures with ERalpha and ERbeta are supportive of this approach to improve selectivity in this structural class.

Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. Introduction: Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. Materials and Methods: Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, μCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b−/− mice. Results: Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b−/− mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. Conclusions: High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

The bone is an amazing organ that grows and remodels itself over a lifetime. It is generally accepted that bone sculpting in response to stress and force is carried out by groups of cells contained within bone multicellular units that are coordinated to degrade existing bone and form new bone. Because of the nature of bone and the extensiveness of the skeleton, it is difficult to study bone remodeling in vivo. On the other hand, because the bone contains a complex environment of many cell types, is it possible to study bone remodeling in vitro? We propose that one can at minimum study the interaction between osteoblasts (bone formation) and osteoclasts (bone degradation) in a three dimensional (3D) "bioreactor". Furthermore, one can add bone degrading metastatic cancer cells, and study how they contribute to and take part in the bone degradation process. We have primarily cultured and differentiated MC3T3-E1 osteoblasts for long periods (2-10 months) before addition of bone marrow osteoclasts and/or metastatic (MDA-MB-231), metastasis suppressed (MDA-MB-231BRMS1) or non-metastatic (MCF-7) breast cancer cells. In the co-culture of osteoblasts and osteoclasts there was clear evidence of matrix degradation. Loss of matrix was also evident after co-culture with metastatic breast cancer cells. Tri-culture permitted an evaluation of the interaction of the three cell types. The 3D system holds promise for further studies of cancer dormancy, hormone, and cytokine effects and matrix manipulation.

A highly error resilient mode of the newly standardized 3GPP EVS speech codec is described. Compared to the AMR-WB codec and other conversational codecs, the EVS channel aware mode offers significantly improved error resilience in voice communication over packet-switched networks such as Voice-over-IP (VoIP) and Voice-over-LTE (VoLTE). The error resilience is achieved using a form of in-band forward error correction. Source-controlled coding techniques are used to identify candidate speech frames for bitrate reduction, leaving spare bits for transmission of partial copies of prior frames such that a constant bit rate is maintained. The self-contained partial copies are used to improve the error robustness in case the original primary frame is lost or discarded due to late arrival. Subjective evaluation results from ITU-T P.800 Mean Opinion Score (MOS) tests are provided, showing improved quality under channel impairments as well as negligible impact to clean channel performance.

This paper describes the time-domain bandwidth extension (TBE) framework employed to code wideband and super-wideband speech in the newly standardized 3GPP EVS codec. The TBE algorithm uses a nonlinear harmonic modeling technique that incorporates principles of time-domain envelope-modulated noise mixing. At 13.2 kbps, the super-wideband coding of speech uses as low as 1.55 kbps for encoding the spectral content from 6.4–14.4 kHz. Subjective evaluation results from ITU-T P.800 Mean Opinion Score (MOS) tests are provided, showing significantly improved quality compared to the other standardized SWB codecs under both clean speech and speech with background noise.

This paper presents a low bit-rate MDCT coder, which is adopted as a part of the recently standardized codec for Enhanced Voice Services. To maximize codec performance for NB to SWB input signals for low bit-rates (7.2 to 16.4 kbps), new adaptive bit-allocation and spectrum quantization schemes, which emphasize perceptually important spectrum while efficiently coding full spectrum, was introduced into the low bit-rate MDCT coder. Further, small symbol switched Huffman coding is exploited for reducing the bits consumption for quantizing band energies of the spectrum. Finally, the performance of the coder is illustrated with some listening test results.

Wnt proteins are a family of secreted proteins that regulate many aspects of cell growth, differentiation, function, and death. Considerable progress has been made in our understanding of the molecular links between Wnt signaling and bone development and remodeling since initial reports that mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) are causally linked to alterations in human bone mass. Of the pathways activated by Wnts, it is signaling through the canonical (i.e., Wnt/beta-catenin) pathway that increases bone mass through a number of mechanisms including renewal of stem cells, stimulation of preosteoblast replication, induction of osteoblastogenesis, and inhibition of osteoblast and osteocyte apoptosis. This pathway is an enticing target for developing drugs to battle skeletal diseases as Wnt/beta-catenin signaling is composed of a series of molecular interactions that offer potential places for pharmacological intervention. In considering opportunities for anabolic drug discovery in this area, one must consider multiple factors, including (a) the roles of Wnt signaling for development, remodeling, and pathology of bone; (b) how pharmacological interventions that target this pathway may specifically treat osteoporosis and other aspects of skeletal health; and (c) whether the targets within this pathway are amenable to drug intervention. In this Review we discuss the current understanding of this pathway in terms of bone biology and assess whether targeting this pathway might yield novel therapeutics to treat typical bone disorders.

We present a new hardware adaptive filter architecture for very high throughput LMS adaptive filters using distributed arithmetic (DA). DA uses bit-serial operations and look-up tables (LUTs) to implement high throughput filters that use only about one cycle per bit of resolution regardless of filter length. However, building adaptive DA filters requires recalculating the LUTs for each adaptation which can negate any performance advantages of DA filtering. By using an auxiliary LUT with special addressing, the efficiency and throughput of DA adaptive filters can be of the same order as fixed DA filters. In this paper, we discuss a new hardware adaptive filter structure for very high throughput LMS adaptive filters. We describe the development of DA adaptive filters and show that practical implementations of DA adaptive filters have very high throughput relative to multiply and accumulate architectures. We also show that DA adaptive filters have a potential area and power consumption advantage over digital signal processing microprocessor architectures.

In this issue of Cell Metabolism, Mounier et al. (2013) show that AMPKα1 is a crucial contributor to the regeneration of damaged muscle tissues, acting in macrophages at the nexus between proinflammatory debris removal and resolution of muscle tissue inflammation.

The direct use of vector quantization (VQ) to encode LPC parameters in a communication system suffers from the following two limitations: 1) complexity of implementation for large vector dimensions and codebook sizes and 2) sensitivity to errors in the received indices due to noise in the communication channel. In the past, these issues have been simultaneously addressed by designing channel matched multistage vector quantizers (CM-MSVQ). A sub-optimal sequential design procedure has been used to train the codebooks of the CM-MSVQ. In this paper, a novel channel-optimized multistage vector quantization (CO-MSVQ) codec is presented, in which the stage codebooks are jointly designed. The proposed codec uses a source and channel-dependent distortion measure to encode line spectral frequencies derived from segments of a speech signal. Extensive simulation results are provided to demonstrate the consistent reduction in both the mean and the variance of the spectral distortion obtained using the proposed codec relative to the conventional sequentially designed CM-MSVQ. Furthermore, the perceptual quality of the reconstructed speech using the proposed codec was found to be better than that obtained using the sequentially designed CM-MSVQ.

Recently, novel vector quantization techniques in multiple nonorthogonal domains for both waveform and model-based signal characterization that give an improved qualitative and quantitative signal coding performance as compared to vector quantization in single domain have been reported. In these techniques, vectors are formed either directly from the signal waveform or from the model parameters extracted from the signal. Then, the vectors are represented in multiple nonorthogonal domains. The encoder chooses the domain that best represents the vector according to a predetermined criterion. An iterative codebook enhancement algorithm, applicable to both waveform and model-based vector quantization in nonorthogonal domains is developed and presented in this paper. In this algorithm, each set of codebooks in a given domain is retrained by the vectors that were best represented by that particular set of codebooks in the most recent iteration. The algorithm is applied successfully and extensive simulation results yield considerable performance enhancement of the vector quantization in nonorthogonal domains, for a given bit rate. Sample results are provided which demonstrate the improved performance.

Linear prediction (LP) is a powerful technique for efficient source-system model based representation of signals, such as speech, and video, with useful applications including compression, and recognition. This has been found to be particularly true when vector quantization is used to code the linear predictor coefficients. Recently, signal processing in multiple nonorthogonal domains has been reported that further enhances the efficiency of signal representation. In this contribution, a novel LP model based coding technique is presented where the advantages of multiple nonorthogonal domain representations of the LP coefficients and the prediction residuals are exploited in conjunction with vector quantization to yield considerable LP coding enhancement. The proposed signal coding technique is applied to one of the most commonly used signals, namely, speech. The resulting performance improvement is clearly demonstrated in terms of reconstruction quality for the same bit rate compared to the existing single domain vector quantization techniques.

The Cbfa1/Runx2 (referred to herein as Cbfa1) transcription factor has been shown to be essential for osteoblast differentiation and bone formation during embryogenesis. PTH given intermittently is a proven bone anabolic agent. Here, we investigated whether PTH regulates the expression and/or activity of Cbfa1 in osteoblastic cells and in a rat metatarsal organ culture assay. PTH was found to regulate Cbfa1 mRNA in the rat osteosarcoma cell line UMR106 in a concentration-dependent manner. The effect of PTH was mimicked by forskolin, an activator of adenylate cyclase leading to the protein kinase A pathway. PTH administered intermittently for 5 d in vivo was found to stimulate Cbfa1 protein in the rat proximal tibiae metaphysis. To demonstrate PTH regulation of Cbfa1 activity, a construct containing six tandem Cbfa1 binding elements fused to luciferase was shown to be rapidly stimulated in response to PTH. This stimulation preceded the effects on mRNA regulation and resulted from a protein kinase A-mediated increase in Cbfa1 activity. Finally, using a neonate rat metatarsal organ culture system, we demonstrated dose-dependent anabolic responsiveness to PTH and to Cbfa1 overexpression from an adenoviral construct. We further showed that Cbfa1 antisense oligonucleotides that blocked adenoviral Cbfa1-induced anabolic effects in this organ culture model also abolished the PTH-mediated anabolic increase. These findings suggest a requirement for Cbfa1 in mediating the anabolic effects of PTH. Thus, regulation of Cbfa1 expression or activity is an important mechanism by which PTH controls osteoblast function.

The convergence of Web technology, wireless networks, and portable client devices provides new design opportunities for computer/communications systems. In the HP Labs' Cooltown project we have been exploring these opportunities through an infrastructure to support Web presence for people, places and things. We put Web servers into things like printers and put information into Web servers about things like artwork; we group physically related things into places embodied in Web servers. Using URLs for addressing, physical URL beaconing and sensing of URLs for discovery, and localized Web servers for directories, we can create a location-aware but ubiquitous system to support nomadic users. On top of this infrastructure we can leverage Internet connectivity to support communications services. Web presence bridges the World Wide Web and the physical world we inhabit, providing a model for supporting nomadic users without a central control point.

BACKGROUND: Metastatic ovarian cancer remains an urgent clinical problem. The homing and invasion of cancer cells into the omental adipose tissue, which is the preferred site of ovarian cancer metastasis, is a critical step in disease progression. Improving patient outcomes requires a mechanistic understanding of how cancer cells colonize the omentum and ultimately give rise to widespread peritoneal metastases. Unlike other peritoneal adipose tissues, omental adipose contains immune aggregates known as milky spots, which contain macrophages, B, T, natural killer (NK), and stromal cells within capillary nests. Our data shows that milky spots play an active role in ovarian cancer metastatic colonization to the omentum. We hypothesize that macrophages within the milky spots play a crucial role in promoting ovarian cancer progression. METHODS AND RESULTS: Immunocompetent mice were injected with syngeneic murine ID8 and immunocompromised mice were injected with human (SKOV3ip.1, CaOV3, HeyA8), ovarian cancer cells. After seven days post-injection, mice were sacrificed and analyzed for the distribution of cancer cells. We found metastases in omental milky spots and not in adipose lacking milky spots. Use of genetic models (C57BL/6, Athymic Nude, Beige Nude, Rag1-/-, Igh6-/- mice) ruled out a requirement for B, T, and NK cells in ovarian cancer cell homing to milky spots, suggesting a critical role for macrophages in this process. Our recent data shows that depletion of omental adipose tissue macrophages abrogates cancer cell colonization of milky spots. Furthermore, factors secreted by omental macrophages have an enhanced ability to promote ovarian cancer migration in vitro, compared to factors secreted by other peritoneal adipose tissue macrophages. Screening omental adipose conditioned medium with a cytokine array revealed higher concentration of macrophage-secreted cytokines (e.g., MCSF-1, IL10) than control medium conditioned by adipose lacking milky spots. Examination of naïve human omentum confirmed the presence of macrophages in immune clusters. Clinical samples from patients with high-grade serous cancer showed the incidence of microscopic metastases within omental immune clusters. Importantly, RNA-Seq of CD45+CD11b+F4/80+ flow-sorted macrophages from omentum and mesentery of naïve and cancer-bearing mice revealed that omental macrophages have a very distinct gene expression signature. CONCLUSION: We demonstrate that omental milky spots are the preferential sites for cancer colonization of peritoneal adipose. Further, we have identified omental macrophages as the key mediators of colonization. Future studies are focused on understanding the cancer cell-macrophage interactions that can be targeted therapeutically to disrupt metastatic growth and extend disease-free survival. Citation Format: Venkatesh Krishnan, Bruce Schaar, Carrie Rinker-Schaeffer, Oliver Dorigo. OMENTAL MACROPHAGES REGULATE OVARIAN CANCER METASTATIC COLONIZATION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-028.

The high regenerative capacity of adult skeletal muscle relies on a self-renewing depot of adult stem cells, termed muscle satellite cells (MSCs). Androgens, known mediators of overall body composition and specifically skeletal muscle mass, have been shown to regulate MSCs. The possible overlapping function of androgen regulation of muscle growth and MSC activation has not been carefully investigated with regards to muscle regeneration. Therefore, the aim of this study was to examine coinciding androgen-mediated genetic changes in an in vitro MSC model and clinically relevant in vivo models. A gene signature was established via microarray analysis for androgen-mediated MSC engagement and highlighted several markers including follistatin (FST), IGF-1, C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF) and glucocorticoid receptor (GR/Nr3c1). In an in vivo muscle atrophy model, androgen re-supplementation significantly increased muscle size and expression of IGF-1, FST, and HGF, while significantly decreasing expression of GR. Biphasic gene expression profiles over the 7-day re-supplementation period identifed temporal androgen regulation of molecular targets involved in satellite cell engagement into myogenesis. In a muscle injury model, removal of androgens resulted in delayed muscle recovery and regeneration. Modifications in the androgen signaling gene signature, along with reduced Pax7 and MyoD expression, suggested that limited MSC activation and increased inflammation contributed to the delayed regeneration. However, enhanced MSC activation in the androgen-deplete mouse injury model was driven by an androgen receptor (AR) agonist. These results provide novel in vitro and in vivo evidence describing molecular targets of androgen signaling, while also increasing support for translational use of AR agonists in skeletal muscle recovery and regeneration.

This chapter presents an overview of recent developments in conversational speech coding technologies, important new algorithmic advances, and recent standardization activities in ITU-T, 3GPP, 3GPP2, MPEG and IETF that offer a significantly improved user experience during voice calls on existing and future communication systems. User experience is determined by speech quality, hence network operators are very concerned about quality of speech coders. Operators are also concerned about capacity, hence coding efficiency is another important measure. Advanced speech coding technologies provide the capability to both improve coding efficiency and user experience. One option to improve quality is to extend the audio bandwidth from traditional narrowband to wideband (16 kHz sampling) and super-wideband (32 kHz sampling). Another method is in increasing the robustness of the coder against transmission errors. Error concealment algorithms are used which substitute the missing parts of the audio signal as far as possible. In packet-switched applications (VoIP systems), special mechanisms are included in jitter buffer management (JBM) algorithms to maximize sound quality. It is of high importance to ensure standardization and deployment of speech coders that meet quality expectations. As an example of this, we refer to the Enhanced Voice Services (EVS) project in 3GPP that is developing the next generation speech coder in 3GPP. The basic motivation for 3GPP to start the EVS project was to extend the path of codec evolution by providing super-wideband experience at around 13 kb/s and better quality for music and mixed content in conversational applications. Optimized behavior in VoIP applications is achieved through the introduction of high error robustness, jitter buffer management, inclusion of source-controlled variable bit rate operation, support of various audio bandwidths, and stereo.

Follistatin (FST) is a member of the tissue growth factor β family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.

Nutritional or pharmacological perturbations during perinatal growth can cause persistent effects on the function of white adipose tissue, altering susceptibility to obesity later in life. Previous studies have established that saccharin, a nonnutritive sweetener, inhibits lipolysis in mature adipocytes and stimulates adipogenesis. Thus, the current study tested whether neonatal exposure to saccharin via maternal lactation increased susceptibility of mice to diet-induced obesity. Saccharin decreased body weight of female mice beginning postnatal week 3. Decreased liver weights on week 14 corroborated this diminished body weight. Initially, saccharin also reduced male mouse body weight. By week 5, weights transiently rebounded above controls, and by week 14, male body weights did not differ. Body composition analysis revealed that saccharin increased lean and decreased fat mass of male mice, the latter due to decreased adipocyte size and epididymal, perirenal, and sc adipose weights. A mild improvement in glucose tolerance without a change in insulin sensitivity or secretion aligned with this leaner phenotype. Interestingly, microcomputed tomography analysis indicated that saccharin also increased cortical and trabecular bone mass of male mice and modified cortical bone alone in female mice. A modest increase in circulating testosterone may contribute to the leaner phenotype in male mice. Accordingly, the current study established a developmental period in which saccharin at high concentrations reduces adiposity and increases lean and bone mass in male mice while decreasing generalized growth in female mice.

Functional expression of sweet taste receptors (T1R2 and T1R3) has been reported in numerous metabolic tissues, including the gut, pancreas, and, more recently, in adipose tissue. It has been suggested that sweet taste receptors in these nongustatory tissues may play a role in systemic energy balance and metabolism. Smaller adipose depots have been reported in T1R3 knockout mice on a high carbohydrate diet, and sweet taste receptors have been reported to regulate adipogenesis in vitro. To assess the potential contribution of sweet taste receptors to adipose tissue biology, we investigated the adipose tissue phenotypes of T1R2 and T1R3 knockout mice. Here we provide data to demonstrate that when fed an obesogenic diet, both T1R2 and T1R3 knockout mice have reduced adiposity and smaller adipocytes. Although a mild glucose intolerance was observed with T1R3 deficiency, other metabolic variables analyzed were similar between genotypes. In addition, food intake, respiratory quotient, oxygen consumption, and physical activity were unchanged in T1R2 knockout mice. Although T1R2 deficiency did not affect adipocyte number in peripheral adipose depots, the number of bone marrow adipocytes is significantly reduced in these knockout animals. Finally, we present data demonstrating that T1R2 and T1R3 knockout mice have increased cortical bone mass and trabecular remodeling. This report identifies novel functions for sweet taste receptors in the regulation of adipose and bone biology, and suggests that in these contexts, T1R2 and T1R3 are either dependent on each other for activity or have common independent effects in vivo.

With the discoveries of different death mechanisms, an emerging definition of apoptosis is the process of cell death associated with caspase activation or caspase-mediated cell death. This definition accepts that caspases represent the final common mechanistic pathway in apoptosis. Apoptosis may be triggered either by activation events that target mitochondria or endoplasmic reticulum or by activation of cell surface "death receptors," for example, those in the tumor necrosis factor (TNF) superfamily. In the postnatal and adult skeleton, apoptosis is integral to physiological bone turnover, repair, and regeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time. Although apoptosis has been recorded in many studies of bone, the selective mechanisms invoked in the different models studied rarely have been identified. This review offers a broad overview of the current general concepts and controversies in apoptosis research and then considers specific examples of osteoblast apoptosis pertinent to skeletal development and to the regulation of bone turnover. In reviewing selected work on interdigital apoptosis in the developing skeleton, we discuss the putative roles of the bone morphogenetic proteins (BMPs), Msx2, RAR-gamma, and death inducer obliterator 1 (DIO-1). In reviewing factors regulating apoptosis in the postnatal skeleton, we discuss roles of cytokines, growth factors, members of the TNF pathway, and the extracellular matrix (ECM). Finally, the paradoxical effects of parathyroid hormone (PTH) on osteoblast apoptosis in vivo are considered in the perspective of a recent hypothesis speculating that this may be a key mechanism to explain the anabolic effects of the hormone. An improved understanding of the apoptotic pathways and their functional outcomes in bone turnover and fracture healing may facilitate development of more targeted therapeutics to control bone balance in patients with osteoporosis and other skeletal diseases.

A neonatal rat metatarsal organ culture model has been employed to study the anabolic effects of Sonic/Indian hedgehog and BMP-4. In this culture system, a significant increase in endochondral ossification is measured by an increase in length of mineralized bone, after daily treatment for 7 days with Sonic hedgehog protein (Shh-N). Previous evidence indicated that PTH related protein (PTHrP) is a critical target of hedgehog function in endochondral ossification. Using a PTHrP blocking antibody, it is shown that hedgehog mediates this activity via pathways other than through PTHrP. A dose-related increase in endochondral ossification is observed when metatarsals are treated with 25 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4). However, this activity is not evident at higher doses of BMP-4 (200 ng/ml). High doses of BMP-4 resulted in increased expression of noggin messenger RNA and cotreatment of noggin and Shh-N resulted in reversal of the anabolic action of Shh-N. This observation suggests that BMP-4 signaling can influence the Shh-N mediated increase in endochondral ossification. Finally, we show that the Shh-N and BMP-4 mediated increase in endochondral ossification is reversed by treatment with antisense oligonucleotides targeted against Cbfa1. Thus, this report identifies Shh-N as an inducer of endochondral ossification that mediates its effect via BMP-4 and Cbfa1-dependent pathways.

Just-In-Time (JIT) Compilation is a technology used to improve speed of virtual machines that support dynamic loading of applications. It is better known as a technique that accelerates Java programs. Current JIT compilers are either huge in size or compile complete methods of the application requiring large amounts of memory for their functioning. This has made Java Virtual Machines for embedded systems devoid of JIT compilers. This paper explains a simple technique of combining interpretation with compilation to get a hybrid interpretation strategy. It also describes a new code generation technique that works using its self-code. The combination gives a JIT compiler that is very small (10K) and suitable for Java Virtual Machines for embedded systems.

Background: Assessment of bone formation activity is an important component of pharmacologic efficacy and toxicity evaluations for compounds in development for osteoporosis therapies. Antemortem biomarkers of bone formation and remodeling in rodents are uncommon. While the periosteal alkaline phosphatase (ALP) assay is a postmortem and laborious means of testing bone-building activity, the semiautomated ALP isoenzyme assay is an antemortem assay that is performed on an automated chemistry analyzer after 2 simple dilutions of the initial serum sample and a short incubation. Objectives: The goal of our investigation was to determine if the serum bone ALP (BALP) data obtained from the semiautomated ALP isoenzyme assay had a similar pattern of response when compared with the periosteal ALP (PALP) assay for use in pharmacologic screening in rats. Methods: Serum and bone tissue samples were obtained from orchidectomized Wistar rats, a model of clinically induced osteoporosis. Subsequent bone formation was initiated via treatment with one of several compounds. In study 1, orchidectomized male rats were given either vehicle, dihydrotestosterone or a testosterone derivative subcutaneously every 4 days for 28 days. In study 2, orchidectomized male rats were given either vehicle or compounds A, B, or C by oral gavage daily for 15 days. Blood and tibias were collected at necropsy. Serum was analyzed for BALP activity using a semiautomated ALP assay. Tibias from the same rats were analyzed for PALP activity. Results: Serum BALP activity paralleled PALP activity within each group when compared with the controls. Conclusion: Our data indicate that the semiautomated serum BALP isoenzyme assay may be used as a biomarker of bone-building potential in rat models of osteoporosis. This assay affords many advantages to investigators of musculoskeletal diseases, including the potential to measure multiple data points in a single study.

Goods movement is an important aspect of the transportation system. Freight flow, complemented with the much-researched passenger movement, provides a way for understanding the complete vehicle flow scenario on the highways. Commodity movement prediction has not received much attention because of the lack of sufficient and easily accessible data sources. Most data sources give aggregated commodity movements and, because of the heterogeneity of freight, accurate predictions of truck flows have not been possible. A methodology for calculating the truck flows on the various highways in Massachusetts from interstate commodity flow data is presented. Freight tons originating and ending in Massachusetts have been converted to truck numbers by using a quantitative procedure and distributed to different areas in the state by using employment as an economic indicator variable. The truck flow is assigned to the important highways and validated against existing survey counts. On comparison, a large percentage of the roads show the estimated truck counts are within a tolerable error margin. The research also shows that statewide analyses need to be refined near urban areas because of a variety of complexities involved.

Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor beta (ERbeta) in vitro assays. X-ray crystal structures of SERBA-1 in ERalpha and beta help explain observed beta-selectivity of this ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate in CD-1 mice (ERbeta effect), while having no effect on gonadal hormone levels (ERalpha effect) at 10x the efficacious dose, consistent with in vitro properties of this molecule.

Pseudomeningocele is an extradural cerebrospinal fluid collection arising from a dural defect, that may be congenital, traumatic, or more commonly as a result of postoperative complication. Majority of the postoperative pseudomeningoceles occurring after lumbar spine surgeries are small and resolve spontaneously. However, large pseudomeningoceles are rare and spontaneous resolution of such pseudomeningoceles has not been described. We report four cases of postoperative large lumbar pseudomeningoceles that presented as asymptomatic soft fluctuant swelling over the back which resolved spontaneously. We also reviewed the related literatures and operative records of these patients to find the possible mechanism of occurrence, their management, prevention, and reasons for spontaneous resolution. We conclude that nonoperative management under close observation can be employed for asymptomatic postoperative large lumbar pseudomeningoceles. Surgical exploration and repair should be reserved for symptomatic cases presenting with clinical features of intracranial hypotension, worsening neurology, external fistula or infection, thereby avoiding morbidity and potential complications associated with surgical treatment.

A therapeutic goal in the treatment of certain CNS diseases, including multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson disease, is to down-regulate inflammatory pathways. Inflammatory molecules produced by microglia are responsible for removal of damaged neurons, but can cause collateral damage to normal neurons located close to defective neurons. Although estrogen can inactivate microglia and inhibit the recruitment of T cells and macrophages into the CNS, there is controversy regarding which of the two estrogen receptors (ERs), ERα or ERβ, mediates the beneficial effects in microglia. In this study, we found that ERβ, but not ERα, is expressed in microglia. Using the experimental autoimmune encephalomyelitis (EAE) model in SJL/J mice, we evaluated the benefit of an ERβ agonist as a modulator of neuroinflammation. Treatment of EAE mice with LY3201, a selective ERβ agonist provided by Eli Lilly, resulted in marked reduction of activated microglia in the spinal cord. LY3201 down-regulated the nuclear transcription factor NF - κB, as well as the NF-κB–induced gene inducible nitric oxide synthase in microglia and CD3 + T cells. In addition, LY3201 inhibited T-cell reactivity through regulation of indoleamine-2,3-dioxygenase. In the EAE model, treatment with LY3201 decreased mortality in the first 2 wk after disease onset, and also reduced the severity of symptoms in mice surviving for 4 wk. Our data show that ERβ-selective agonists, by modulating the immune system in both microglia and T cells, offer promise as a useful class of drugs for treating degenerative diseases of the CNS.

COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in several species, from Drosophila to man. The vertebrate COUP-TFs can be classified into four subgroups according to sequence homology in their ligand-binding domain. COUP-TFs bind to AGGTCA direct repeats or palindromes with various spacings. These include the response elements of several other members of the superfamily, the vitamin D receptor, the thyroid hormone receptor, the retinoic acid receptor, the retinoid X receptor, the peroxisome proliferation activated regulator, and the hepatocyte nuclear factor-4. COUP-TF response elements have been identified in the promoters of many genes and COUP-TFs have been shown to act as negative regulators both in vitro and in vivo. They can compete with the above mentioned receptors for binding to the common response elements. The ratio of COUP-TF and the other positive regulator determines the transcriptional state of the particular gene in any given moment. COUP-TFs are expressed in the developing central nervous system of mouse and zebra-fish. In addition, they are also expressed in many organs during mouse organogenesis. The expression pattern and profile of COUP-TFs favor the hypothesis that they are involved in development and differentiation. The expression of COUP-TFs are also highly regulated. P19 embryonal carcinoma cells have been used as a model system to study COUP-TF regulation. COUP-TFs are up-regulated in retinoic acid (RA) treated P19 cells. Transient transfection assay showed that mouse COUP-TFII promoter directly responded to RA treatment, suggesting that COUP-TF expression is directly regulated by RA signaling pathway.

Human follistatin is a regulatory glycoprotein with widespread biological functions including anti-inflammatory activities, wound healing properties and muscle stimulating effects. The role of follistatin in a wide range of biological activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential disease modifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic and pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315. Our data indicates that the intrinsic PK/PD properties of native follistatin-315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Herein, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing follistatin-315 to a murine IgG1 Fc and removing the intrinsic heparan sulfate binding activity of follistatin. The engineered variant molecule had ~100- and ~1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native follistatin-315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy towards the development of a follistatin therapeutic with potential utility in patients affected with skeletal muscle diseases.

Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the "vicious cycle" of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue, it is difficult to study this system in vivo. In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases.

Introduction: The omentum is the primary site of metastasis in both ovarian cancer models and clinical disease. It is composed predominantly of adipose studded with lymphoreticular organs (milky spots), distinguishing it from other peritoneal adipose. Milky spots are specialized for immune cell trafficking and peritoneal surveillance. We and others have shown that ovarian cancer cells exploit the physiologic function(s) of these structures for omental metastatic colonization. The purpose of this study was to identify cellular and molecular mechanisms responsible for ovarian cancer homing to and growth within milky spot structures. Experimental Procedures: Quantitative in vivo and ex vivo assays were used to evaluate human (SKOV3ip.1, HEYA8, and CaOV3) and murine (ID8) ovarian cancer cell localization to milky spots on the murine omental fat band. Assays were conducted using C57/Bl6 mice or those lacking B cells (Igh6-/-); T cells (Nude); B and T cells (Rag1-/-); or B, T, and NK cells (BN XID). In vitro assays were used to assess the migration-promoting activity of omentum- and macrophage-conditioned media. Standard approaches were used to assess protein expression, cell growth and viability, etc. Rationale: Milky spots provide resident tissue macrophages and lymphocytes needed for peritoneal homeostasis. Macrophages and stromal cells secrete chemokines promoting peritoneal lymphocyte homing to the omentum. In response to peritoneal irritants, activated CD11b+ milky spot macrophages organize coordinated expansion of vascular and stromal compartments. The increase in both the number and size of milky spots is needed to process particulates, resolve infections, and encapsulate foreign bodies. Hypothesis: CD11b+ cells secrete homeostatic chemokines promoting G-protein-dependent migration of ovarian cancer cells to milky spots. Ovarian cancer cell binding to adhesion molecules on the milky spot surface activates CD11b+ cell-dependent tissue remodeling, creating a microenvironment promoting ovarian cancer growth. New Findings: Consistent with our hypothesis, in vivo assays showed that macrophage depletion prior to injection of ID8 and SKOV3ip.1 cells prevented microscopic metastasis formation. In vitro assays found that macrophages are required for ovarian cancer cell localization to milky spots. These data prompt the hypothesis that CD11b+ cells produce a factor(s) responsible for the migration-promoting ability of omentum-conditioned media. To test this, media was conditioned by omental adipose isolated from mice after macrophage depletion. In support of our hypothesis, the migration-promoting activity of macrophage-depleted omentum-conditioned media was on a par with that of media conditioned by milky spot-deficient adipose. Further, media conditioned by CD11b+ cells recapitulates the migration-promoting activity exhibited by omentum-conditioned media. In vitro and ex vivo assays were used to test whether ovarian cancer cells utilize mechanisms analogous to lymphocyte trafficking for milky spot homing. Specifically, cells were pretreated with pertussis toxin or vehicle alone, and then evaluated for migration in response to omentum-conditioned media and milky spot localization ex vivo. Consistent with data reported for lymphocyte homing, pertussis toxin pre-treatment caused a 40% to 50% reduction in ovarian cancer cell homing. Conclusions and Current Efforts: Our data support a model in which CD11b+ macrophages secrete one or more chemokines promoting G-protein receptor-dependent ovarian cancer cell migration, and potentially integrin activation, which mediate milky spot homing. Current experiments focus on identification of integrin-ligand interactions, CD11b+ cell activation, and defining the link between ovarian cancer cell growth and increase in milky spot size and number. Citation Format: Venkatesh Krishnan, Kelly Mitchell, Jason Miska, Sophia George, Patricia Shaw, Victoria Seewaldt, Maciej Lesniak, Marina Chekmareva, Lev Becker, Vinita Parkash, Cindy Miranti, Carrie Rinker-Schaeffer. Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A82.