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Background/Objective The deleterious effects of chronic spinal cord injury (SCI) on the skeleton in rats, especially the lower extremities, has been proved previously. However, the long-term skeletal changes after SCI in non-human primates (NHP) have been scarcely studied. This study aimed to evaluate the bone loss in limbs and vertebrae and the bone metabolic changes in NHP after unilateral cervical spinal cord contusion injury. Methods Twelve Macaca fascicularis were randomly divided into the SCI (n=8) and the Sham (n=4) groups. The SCI models were established using hemi-contusion cervical spinal cord injury on fifth cervical vertebra (C5), and were further evaluated by histological staining and neurophysiological monitoring. Changes of bone microstructures, bone biomechanics, and bone metabolism markers were assessed by micro-CT, micro-FEA and serological kit. Results The NHP hemi-contusion cervical SCI model led to consistent unilateral limb dysfunction and potential plasticity in the face of loss of spinal cord. Furthermore, the cancellous bone mass of ipsilateral humerus and radius decreased significantly compared to the contralateral side. The bone volume fraction of humerus and radius were 17.2% and 20.1% on the ipsilateral while 29.0% and 30.1% on the contralateral respectively. Similarly, the thickness of the cortical bone in the ipsilateral forelimbs was significantly decreased, as well as the bone strength of the ipsilateral forelimbs. These changes were accompanied by diminished concentration of osteocalcin and total procollagen type 1 N-terminal propeptide (t-P1NP) as well as increased level of β-C-terminal cross-linking telopeptide of type 1collagen (β-CTX) in serological testing. Conclusions The present study demonstrated that hemi-SCI induced loss of bone mass and compromised biomechanical performance in ipsilateral forelimbs, which could be indicated by both muscle atrophy and serological changes of bone metabolism, and associated with a consistent loss of large-diameter cells of sensory neurons in the dorsal root ganglia. The Translational potential of this Article Our study, for the first time, demonstrated the bone loss in limbs and vertebrae as well as the bone metabolic changes in non-human primates after unilateral spinal cord injury (SCI). This may help to elucidate the role of muscle atrophy, serological changes and loss of sensory neurons in the mechanisms of SCI-induced osteoporosis, which would be definitely better compared with rodent models.

Mounting data have shown that long non‐coding RNAs (lncRNAs) widely participate in tumour initiation, development, progression and glycolysis in a variety of tumours. However, the clinical prognosis and molecular mechanisms of TMEM161B‐AS1 in oesophageal squamous cell carcinoma (ESCC) remain still unknown. Here, TMEM161B‐AS1 and HIF1AN were significantly lower in ESCC tissues than in normal samples, and their low expressions were both related to TNM stage, lymph node metastasis and poor prognosis of ESCC patients. Functionally, TMEM161B‐AS1 overexpression or miR‐23a‐3p depletion suppressed the proliferation, invasion and glycolysis as well as reduced glucose consumption and lactate production in ESCC cells. Mechanistically, TMEM161B‐AS1 manipulated HIF1AN expression by competitively sponging miR‐23a‐3p in ESCC cells. MiR‐23a‐3p mimic and HIF1AN siRNA partly reversed cell phenotypes mediated by TMEM161B‐AS1 in ESCC cells. Collectively, TMEM161B‐AS1, miR‐23a‐3p and HIF1AN may be tightly involved in ESCC development and progression as well as patients’ prognosis, and TMEM161B‐AS1/miR‐23a‐3p/HIF1AN signal axis may be a promising target for the treatment of ESCC patients.

In relation to available polysaccharides, oligosaccharides have a low molecular weight, less viscosity, and complete water solubility. These properties endow oligosaccharides with significant biological properties including the microbiota regulation ability. In this study, a homogeneous oligooctasaccharide, riclinoctaose, was biosynthesized from succinylglycan riclin by enzymatic degradation. Monosaccharide composition, Fourier-transform infrared, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectrometry analysis indicated that riclinoctaose is an oligooctasaccharide consisting of one galactose and seven glucose residues, with a pyruvate group linked to the terminal glucose residue. The effects of dietary riclinoctaose on the gut microbiota of mice were evaluated. We found that the dietary riclinoctaose significantly altered intestinal microbiota with the increased growth of beneficial intestinal bacteria including Bifidobacteria and Lactobacillus and decreased the abundance of pernicious bacteria such as Gammaproteobacteria. The level of short-chain fatty acids (SCFAs) was significantly elevated in the riclinoctaose cecum. Our results suggested that riclinoctaose as a prebiotic may have a great potential application in functional foods.

BACKGROUND: Hyperactivation of PI3K/Akt signaling is implicated in breast cancer (BCa) progression. SETDB1, a methyltransferase, is also implicated in BCa, however, the mechanism remains elusive. Recent studies have shown SETDB1 can methylate non-histone substrates such as Akt1 and contribute to its aberrant activation, thus leading to tumor progression. PELP1 is a proto-oncogene which is overexpressed in BCa and participates in both the nuclear and extra nuclear functions of various nuclear receptors. In a subset of BCa, PELP1 uniquely localizes in the cytoplasm and contributes to endocrine therapy resistance. The objective of this study is to characterize the significance of PELP1 in SETDB1 mediated Akt signaling in BCa progression. METHODS: Using yeast two-hybrid screening, we identified PELP1 as a novel interacting protein of SETDB1. Functional significance of cross-talk was tested using MTT, proliferation, stemness and colony formation assays. Mechanistic studies were conducted using immunoprecipitation, RNA-seq, shRNA, overexpression, Western blotting, and RT-qPCR. Biological significance of PELP1 and SETDB1 in endocrine therapy resistance was also examined. RESULTS: Analyses of TCGA databases showed that SETDB1 is highly expressed in BCa and associated with poor clinical outcome. Further, SETDB1 expression is positively correlated with PELP1 expression in BCa (r=0.30, p CONCLUSIONS: Our study results suggest that the PELP1/SETDB1 interactome plays an important role in aberrant Akt activation. Drugs that target PELP1/SETDB1 axis may be useful in modulating aberrant Akt signaling. Supported by VA grant I01BX004545. Citation Format: Zexuan Liu, Junhao Liu, Weiwei Tang, Uday P. Pratap, Kristin A. Altwegg, Xiaonan Li, Suryavathi Viswanadhapalli, Ratna Vadlamudi. Proto-oncogene PELP1 interactions with SETDB1 contribute to aberrant activation of AKT1 in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-34.

Non-human primates are most suitable for generating cervical experimental models, and it is necessary to study the anatomy of the cervical spine in non-human primates when generating the models. The purpose of this study was to provide the anatomical parameters of the cervical spine and spinal cord in long-tailed macaques (Macaca fascicularis) as a basis for cervical spine-related experimental studies. Cervical spine specimens from 8 male adult subjects were scanned by micro-computed tomography, and an additional 10 live male subjects were scanned by magnetic resonance imaging. The measurements and parameters from them were compared to those of 12 male adult human subjects. Additionally, 10 live male subjects were scanned by magnetic resonance imaging, and the width and depth of the spinal cord and spinal canal and the thickness of the anterior and posterior cerebrospinal fluid were measured and compared to the relevant parameters of 10 male adult human subjects. The tendency of cervical parameters to change with segmental changes was similar between species. The vertebral body, spinal canal, and spinal cord were significantly flatter in the human subjects than in the long-tailed macaques. The cerebrospinal fluid space in the long-tailed macaques was smaller than that in the human subjects. The anatomical features of the cervical vertebrae of long-tailed macaques provide a reference for establishing a preclinical model of cervical spinal cord injury.

Ketogenic diet (KD) has been shown to be beneficial in a range of neurological disorders, with ketone metabolite β-hydroxybutyrate (βOHB) reported to block the nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in bone marrow-derived macrophages. In this study, we show that pretreatment with KD or in situ βOHB suppressed macrophages/microglia activation and the overproduction of inflammatory cytokines, while KD downregulated the expression of NLRP3 inflammasome. Moreover, KD promoted macrophages/microglia transformation from the M1 phenotype to the M2a phenotype following spinal cord injury (SCI) in the in vivo study. Rats in the KD group demonstrated improved behavioral and electrophysiological recovery after SCI when compared to those rats in the standard diet group. The in vitro study performed on BV2 cells indicated that βOHB inhibited an LPS+ATP-induced inflammatory response and decreased NLRP3 protein levels. Our data demonstrated that pretreatment with KD attenuated neuroinflammation following SCI, probably by inhibiting NLRP3 inflammasome and shifting the activation state of macrophages/microglia from the M1 to the M2a phenotype. Therefore, the ketone metabolite βOHB might provide a potential future therapeutic strategy for SCI.

Cancer stem cells (CSCs) are highly tumorigenic, spared by chemotherapy, sustain tumor growth, and are implicated in tumor recurrence after conventional therapies in triple negative breast cancer (TNBC). Lysine-specific histone demethylase 1A (KDM1A) is highly expressed in several human malignancies and CSCs including TNBC. However, the precise mechanistic role of KDM1A in CSC functions and therapeutic utility of KDM1A inhibitor for treating TNBC is poorly understood. The effect of KDM1A inhibition on cell viability, apoptosis, and invasion were examined by Cell Titer Glo, Caspase 3/7 Glo, and matrigel invasion assays, respectively. Stemness and self-renewal of CSCs were examined using mammosphere formation and extreme limiting dilution assays. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, Western blotting and reporter gene assays. Mouse xenograft and patient derived xenograft models were used for preclinical evaluation of KDM1A inhibitor. TCGA data sets indicated that KDM1A is highly expressed in TNBC. CSCs express high levels of KDM1A and inhibition of KDM1A reduced the CSCs enrichment in TNBC cells. KDM1A inhibition reduced cell viability, mammosphere formation, self-renewal and promoted apoptosis of CSCs. Mechanistic studies suggested that IL6-JAK-STAT3 and EMT pathways were downregulated in KDM1A knockdown and KDM1A inhibitor treated cells. Importantly, doxycycline inducible knockout of KDM1A reduced tumor progression in orthotopic xenograft models and KDM1A inhibitor NCD38 treatment significantly reduced tumor growth in patient derived xenograft (PDX) models. Our results establish that KDM1A inhibition mitigates CSCs functions via inhibition of STAT3 and EMT signaling, and KDM1A inhibitor NCD38 may represent a novel class of drug for treating TNBC.

Non-human primate (NHP) spinal cord injury (SCI) models can be informative in the evaluation of treatments that show promise in rodent models prior to translation to humans. In the present study, w...

Investigators who manage multicenter clinical trials need to pay careful attention to patterns of subject accrual, and the prediction of activation time for pending centers is potentially crucial for subject accrual prediction. We propose a Bayesian hierarchical model to predict subject accrual for multicenter clinical trials in which center activation times vary. We define center activation time as the time at which a center can begin enrolling patients in the trial. The difference in activation times between centers is assumed to follow an exponential distribution, and the model of subject accrual integrates prior information for the study with actual enrollment progress. We apply our proposed Bayesian multicenter accrual model to two multicenter clinical studies. The first is the PAIN-CONTRoLS study, a multicenter clinical trial with a goal of activating 40 centers and enrolling 400 patients within 104 weeks. The second is the HOBIT trial, a multicenter clinical trial with a goal of activating 14 centers and enrolling 200 subjects within 36 months. In summary, the Bayesian multicenter accrual model provides a prediction of subject accrual while accounting for both center- and individual patient-level variation.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease. TNBC lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elucidate feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. Here, we examined whether the novel LIFR inhibitor, EC359, has the ability to counteract negative effects of LIFR signaling to enhance HDACIs therapeutic efficacy in the treatment of TNBC. Methods: We tested multiple HDACIs currently in clinical trials including vorinostat, panobinostat, romidepsin, and givinostat using multiple TNBC models. The effect of combination therapy of HDACIs and EC359 on TNBC cell viability and invasion was examined using MTT assays and matrigel invasion assays respectively. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and Caspase 3/7 assays, respectively. Mechanistic studies were performed using Western blotting, qRT-PCR, and reporter gene assays. The efficacy of combination therapy in vivo was examined using Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models. Results: We demonstrated that the treatment of TNBC models with HDACIs increased the expression of LIFR. Immunohistochemistry analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC samples. Knockdown of LIFR or treatment with a small molecule inhibitor of LIFR (EC359) significantly enhanced the efficacy of HDACIs in reducing cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy of HDACIs or EC359 in TNBC cell lines. Mechanistic studies, reporter gene assays and biochemical studies using multiple TNBC models exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359+HDACI combination therapy. Treatment of human breast tumors utilizing PDEX assays showed that EC359 enhanced the ability of HDACIs to decrease the proliferation (Ki-67 positivity) compared to monotherapy. Furthermore, using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides greater therapeutic efficacy than monotherapy. In addition, treatment with EC359 can overcome the feedback activation of LIFR currently observed in the treatment of TNBC with HDACIs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P Pratap, Yiliao Luo, Junhao Liu, Kristin A Altwegg, Xiaonan Li, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R Sareddy, Rajeshwar R Tekmal, Hareesh B Nair, Klaus J Nickisch, Ratna K Vadlamudi. Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-08.

Overweight and obese men face numerous health problems, including type 2 diabetes, subfertility, and even infertility. However, few studies have focused on the effects of nutritional status and obesity-related regulatory signals on fertility deficiency. Our previous observations have shown that the elevation of plasma 5'-adenosine monophosphate (5'-AMP) and the accumulation of adenosine in liver and muscle of obese diabetic db/db mice are related to insulin resistance. Here, we found that adenosine accumulation in testis is a common marker of both genetic obesity and high-fat-diet induced obese mice. An messenger RNA sequencing analysis indicated that 78 upregulated genes and 155 downregulated genes in the testis of 5'-AMP-treated mice overlapped with the same genes in the testis of ob/ob mice, and these genes belonged to the clusters of steroid metabolic process and regulation of hormone levels, respectively. Serum testosterone was reduced in ob/ob and 5'-AMP-treated mice. Metabolomic analysis based on 1 H nuclear magnetic resonance showed that the testicular metabolic profiles of ob/ob mice were similar to those of 5'-AMP treated mice. Exogenous 5'-AMP inhibited the phosphorylation of AKT and mammalian target of rapamycin signal transduction and reduced the proliferating cell nuclear antigen expressions in testes. Our results suggest that the accumulation of adenosine causes metabolic disorders in testes and associates lower testosterone level in obese mice.

Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.

Background The vertebral endplate (VEP) was damaged after spinal instability induced by cervical muscle section (CMS). Whether CMS induces bone formation and mechanical loading change in the vertebra is still obscure. This study was aimed to explore mechanical loading change and endplate damage after CMS. Methods Forty-eight rats were randomly divided into the CMS group and the sham group. The C6/7 segments were harvested at 4, 8, and 12 weeks after surgery. The microarchitectures of the C6 vertebra ​and the vertebral endplate lesions and intervertebral disc height of C6/7 were measured by micro–computed tomography. Micro–finite element analysis was used to evaluate biomechanical properties of the C6 vertebra. Bone remodelling of the C6 vertebra ​and the endplate ​sclerosis and intervertebral disc degeneration of C6/7 were evaluated by histological and immunohistochemical analyses. Results CMS significantly induced bone formation of the C6 ventral vertebra ​and increased the biomechanical properties of mainly the ventral side at 4 weeks, which was gradually rebalanced throughout the rest of the study. CMS also significantly increased protein expression of transforming growth factor-β1 (TGF-β1) and phosphorylated small mothers against decapentaplegic (pSmad)2/3 ​at 4 weeks. Moreover, tartrate-resistant acid phosphatase staining showed that osteoclast-positive cells were slightly in number decreased at 4 weeks, but were obviously increased at 8 weeks. The VEP of the ventral side was abraded earlier followed by calcification in situ later after CMS, consistent with the biomechanical enhancements observed. The degree of endplate degeneration was aggravated with time. Finally, CMS decreased intervertebral disc height and increased disc degeneration scores with time. Conclusions Spinal instability induced by CMS increases bone mass and biomechanical loading of the ventral side of vertebra in the early stage, which might initiate VEP damage and cause intervertebral disc degeneration. The translational potential of this article Our study indicates that vertebral trabecular changes may involve in intervertebral disc degeneration induced by spinal instability. This may help to elucidate the mechanisms by which disc degeneration occur.

Objective: To investigate the neuroprotective effects of trihydroxyethyl rutin in rats with cervical spinal cord hemi-contusion.Methods: Adult male Sprague–Dawley rats were subjected to hemi-contusion at a stroke depth of 1.2 mm, and then intraperitoneally injected with 50 or 100 mg/kg trihydroxyethyl rutin per day for 12 weeks (T50 and T100 groups, respectively). Changes in somatosensory evoked potentials (SEPs), motor evoked potentials (MEPs), and behavior were continuously monitored. At 12 weeks post-injury, immunohistochemical staining was performed to assess changes in cervical spinal cord microvascular morphology. Magnetic resonance imaging (MRI) scans were performed to examine end-stage injury in the cervical spinal cord, and Eriochrome cyanine-stained slices of spinal cord tissue were evaluated for injury.Results: There were no significant differences in biomechanical parameters among the spinal cord injury, T50 and T100 rat groups. At 3 days-post-injury, there was a significant decrease in grip strength. At 12 weeks post-injury, grip strength recovery was significantly better in the T50 and T100 groups than in the injury group. Compared with the injury group, the total limb placement frequency was significantly higher in the T50 group at 2, 4, 6, 10, and 12 weeks post-injury and in the T100 group at 2, 6, 8, and 10 weeks post-injury. Ipsilateral SEPs and MEPs were dynamic, increasing in latency and decreasing in amplitude in the injury compared with sham group. MRI scanning demonstrated that the coronal, sagittal, and transversal lesion areas were smaller in the T50 and T100 groups than in the injury group. Microvascular density showed a greater reduction in the injury group compared with the T50 and T100 groups. Eriochrome cyanine staining showed that the ipsilateral side, residual parenchyma, and gray matter areas were larger in the T50 and T100 groups than in the injury group.Conclusion: Trihydroxyethyl rutin exhibits robust neuroprotective effects, improving limb motor function and nerve electrophysiological parameters after spinal cord injury, maintaining microvascular density, and reducing the area of injury and degree of demyelination.

Histone deacetylase inhibitors (HDACi) are identified as novel therapeutic agents, however, recent clinical studies suggested that they are marginally effective in treating triple negative breast cancer (TNBC). Here, we show that first-in-class Leukemia Inhibitory Factor Receptor (LIFRα) inhibitor EC359 could enhance the therapeutic efficacy of HDACi against TNBC. We observed that both targeted knockdown of LIFR with CRISPR or treatment with EC359 enhanced the potency of four different HDACi in reducing cell viability, cell survival, and enhanced apoptosis compared to monotherapy in TNBC cells. RNA-seq studies demonstrated oncogenic/survival signaling pathways activated by HDACi were attenuated by the EC359 + HDACi therapy. Importantly, combination therapy potently inhibited the growth of TNBC patient derived explants, cell derived xenografts and patient-derived xenografts in vivo. Collectively, our results suggest that targeted inhibition of LIFR can enhance the therapeutic efficacy of HDACi in TNBC., Li, Viswanadhapalli et al utilized multiple in vitro, ex vivo and in vivo models of TNBC to investigate LIFR inhibition. The authors reported that HDAC inhibition in TNBC cells led to an increase of LIFR expression and over activation of the downstream signaling elements (e.g., STAT3, mTOR, AKT), enhancing the aggressive potential of TNBC cells, and an LIFR inhibitor, EC359, synergistically enhanced the efficacy of HADC inhibitors in suppressing TNBC in vitro and in vivo.

Renal ischemia-reperfusion injury is a major trigger of acute kidney injury and leads to permanent renal impairment, and effective therapies remain unresolved. Riclinoctaose is an immunomodulatory octasaccharide composed of glucose and galactose monomers. Here we investigated whether riclinoctaose protects against renal ischemia-reperfusion injury. In mice, pretreatment with riclinoctaose significantly improved renal function, structure, and the inflammatory response after renal ischemia-reperfusion. Flow cytometry analysis revealed that riclinoctaose inhibited ischemia-reperfusion-induced M1 macrophage polarization and facilitated M2 macrophage recruitment into the kidneys. In isolated mouse bone marrow-derived macrophages, pretreatment with riclinoctaose promoted the macrophage polarization toward M2-like phenotype. The inhibitor of Nrf-2/HO-1 brusatol diminished the effects of riclinoctaose on macrophage polarization. In mice, intravenous injection with riclinoctaose-pretreated bone marrow-derived macrophages also protected against renal ischemia-reperfusion injury. Fluorescence-labeled riclinoctaose specifically bound to the membrane of macrophages. Interfering with mDC-SIGN blocked the riclinoctaose function on M2 polarization of macrophages, consequently impairing the renoprotective effect of riclinoctaose. Our results revealed that riclinoctaose is a potential therapeutic agent in preventing renal ischemia-reperfusion injury.

Background: ESR1 mutations are acquired following ERα targeted therapies and are a major determinant of therapy-resistance. These ESR1 mutations maintain ESR1 signaling, albeit in a ligand-independent manner. Effective drugs targeting these mutant (MT) ERα proteins represent a significant unmet clinical need. We had previously shown that ERX-11, an ESR1-coregulator binding inhibitor, could block the function of these MT ERα proteins. In this study, we sought to leverage recently published structures of MT ERα to develop more potent analogues of ERX-11. Methods: Virtual screening of >250,000 derivatives of ERX-11 was performed with simulated docking on the MT ERα to identify and design analogues of ERX-11. Several hundred analogues were synthesized and tested in vitro using multiple BC model cells that express wild type (WT) ESR1 or mutant (MT) ESR1 (Y537S or D538G). Mechanistic studies were performed using RNA-Seq, Western blotting, qRT-PCR and reporter gene assays. The in vivo efficacy of the most potent ERX-11 analogue ERX-245 was examined using xenograft, PDX and metastatic models of MT-ER driven BC. Results: From our virtual and functional screen, we identified an ERX-11 analogue, ERX-245 as the most potent hit to target MT-ERα. Docking studies modeled a better fit of ERX-245 into the ligand binding domain of both the Y537S and D538G MT-ERα. ERX-245 potently reduced (IC50 ~250 nM) the cell viability of both WT-ERα and MT-ERα driven BC cells but not ERα negative BC cells. ERX-245 significantly reduced the growth (colony formation, clonogenic and mammosphere assays) of MT-ERα BC cells. ERX-245 exhibited synergistic activity in combination with CDK4/6 inhibitors. In distinction to classic SERDs like fulvestrant (which degrade ERα with in 4h), ERX-245 treatment decreased MT-ERα protein levels over 24 hours. PK studies indicated that ERX-245 is more polar and has better solubility and pharmacokinetic properties than ERX-11. ERX-245 reduced tumor growth of subcutaneous xenograft and PDX models driven by MT-ERα as well as the proliferation of xenograft derived MT-ERα explant models. ERX-245 significantly reduced the invasive capability of MT-ERα BC cells in vitro and inhibited both the metastatic capability and growth of metastatic tumors derived from MT-ERα BC cells injected by intracardiac or intratibial routes. Conclusions: Taken together, these results indicate that ERX-245 is a potent and pharmacologically translatable analog of ERX-11, with activity against both primary and metastatic tumors driven by MT-ERα. Citation Format: Ganesh V Raj, Suryavathi Viswanadhapalli, Karla Parra, Shihong Ma, Tae-Kyung Lee, Xihui Liu, Kara Kassees, Weiwei Tang, Junhao Liu, Zexuan Liu, Uday P Pratap, Behnam Ebrahimi, Rajeshwar Rao Tekmal, Jung-Mo Ann, Ratna K Vadlamudi. Development of a potent mutant-ESR1 targeted agent, ERX-245, for treating metastatic therapy-resistant breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-09.

Background: Recent studies have shown that rapamycin (Rapa) rescues cancellous bone loss of osteoporosis models, but its effects on cortical bone and the biomechanical properties remain uncertain. This study was aimed to determine whether rapamycin improve the quality of long bones in ovariectomy (OVX)-induced osteoporosis.Methods: Thirty female C57BL/6J mice were randomly divided into Sham, OVX and OVX+Rapa group. Mice in the OVX+Rapa group were injected intraperitoneally with 1.5 mg/kg rapamycin daily after ovariectomy. After 12 weeks, the microstructures of femurs and the vascular canal porosity tibiae were analyzed by micro-CT. The compressive stiffness of the distal femurs was calculated with the micro-finite element method, and the bending strength of the tibiae was evaluated with a three-point bending test. Western blot of LC3, P62 was used to assess autophagy activity of bone. The number of osteoclasts was quantified by tartrate-resistant acid phosphatase (TRAP) staining. ELISA detected the concentration of bone-specific alkaline phosphatase (BALP) in serum.Results: OVX led to a decrease in cross-sectional areas of the mid-diaphyses and distal femoral cortical bones, increasing the cortical bone vascular canal porosity from 1.42% to 4.79%. The rapamycin reduced cancellous bone loss and decreased cortical bone vascular canal porosity by 3.9%, but further reduced the thickness of the distal femoral cortical bone by 16.7%, with no significant effect on the cortical bone in the mid-diaphyses. Compared to the Sham group, OVX mice showed a decrease in distal femoral stiffness to 6497 N/mm and a decrease in tibial maximum load to 6.08 N, while rapamycin intervention did not significantly improve the decreased biomechanical properties. Moreover, rapamycin remarkably reduced the TRAP-positive osteoclasts and the concentration of serum BALP by 80.6% and 30.8%, respectively. Autophagy was activated in the OVX group compared with the sham group.Conclusions: This study has demonstrated that rapamycin ameliorates cancellous bone loss and cortical bone porosity in ovariectomized mice but partly reduces the size of cortical bone, while has no effect on improving the biomechanical performance. Additionally, the present study also proved that OVX led to both cancellous and cortical bone loss, with attenuated biomechanical properties of long bones.

Highlights • Amplification of RAD54B promotes HCC progression by wnt/β-catenin pathway., Liver cancer was reported to be the sixth most frequently diagnosed cancer, and hepatocellular carcinoma (HCC) accounts for 75%-85% of primary liver cancer. Nevertheless, the concrete molecular mechanisms of HCC progression remain obscure, which is essential to elucidate. The expression profile of RAD54B in HCC was measured using qPCR and western blotting. Moreover, the levels of RAD54B in paraffin-embedded samples were evaluated using immunohistochemistry (IHC). The effect of RAD54B on HCC progression was testified by in vitro experiments, and in vivo orthotopic xenograft tumor experiments. The mechanisms of RAD54B promoting HCC progression were investigated through molecular and function experiments. Herein, RAD54B are dramatically upregulated in HCC tissues and cell lines both on mRNA and protein levels, and RAD54B can servers as an independent prognostic parameter of 5-year overall survival and 5-year disease-free survival for patients with HCC. Moreover, up-regulation of RAD54B dramatically increases the capacity for in vitro cell viability and motility, and in vivo intrahepatic metastasis of HCC cells. Mechanistically, RAD54B promotes the HCC progression through modulating the wnt/β-catenin signaling. Notably, blocking the wnt/β-catenin signaling axis can counteract the activating effects of RAD54B on motility of HCC cells. Besides, further analysis illustrates that DNA amplification is one of the mechanisms leading to mRNA overexpression of RAD54B in HCC. Our findings indicate that RAD54B might be a promising potential prognostic marker and a candidate therapeutic target to therapy HCC.

Endometrial cancer (EC) is the fourth most common cancer in women. Advanced-stage EC has limited treatment options with a poor prognosis. There is an unmet need for the identification of actionable drivers for the development of targeted therapies in EC. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a major role in cancer progression, metastasis, stemness, and therapy resistance. However, little is known about the functional significance of the LIF/LIFR axis in EC progression. In this study using endometrial tumor tissue arrays, we identified that expression of LIF, LIFR is upregulated in EC. Knockout of LIFR using CRISPR/Cas9 in two different EC cells resulted in a significant reduction of their cell viability and cell survival. In vivo studies demonstrated that LIFR-KO significantly reduced EC xenograft tumor growth. Treatment of established and primary patient-derived EC cells with a novel LIFR inhibitor, EC359 resulted in the reduction of cell viability with an IC50 in the range of 20–100 nM and induction of apoptosis. Further, treatment with EC359 reduced the spheroid formation of EC cancer stem cells and reduced the levels of cancer stem cell markers SOX2, OCT4, NANOG, and Axin2. Mechanistic studies demonstrated that EC359 treatment attenuated the activation of LIF-LIFR driven pathways, including STAT3 and AKT/mTOR signaling in EC cells. Importantly, EC359 treatment resulted in a significant reduction of the growth of EC patient-derived explants ex vivo, EC cell line-derived xenografts, and patient-derived xenografts in vivo. Collectively, our work revealed the oncogenic potential of the LIF/LIFR axis in EC and support the utility of LIFR inhibitor, EC359, as a novel targeted therapy for EC via the inhibition of LIF/LIFR oncogenic signaling.

Endometrial cancer (EC) often exhibit aberrant activation of PI3K/Akt/mTOR signaling and targeted therapies using mTOR inhibitors showed limited success. The epigenetic modifier, lysine-specific histone demethylase-1A (KDM1A/LSD1) is overexpressed in EC, however, the mechanistic and therapeutic implications of KDM1A in EC are poorly understood. Here, using 119 FDA-approved drugs screen, we identified that KDM1A inhibition is highly synergistic with mTOR inhibitors. Combination therapy of KDM1A and mTOR inhibitors potently reduced the cell viability, survival, and migration of EC cells. Mechanistic studies demonstrated that KDM1A inhibition attenuated the activation of mTOR signaling cascade and abolished rapamycin induced feedback activation of Akt. RNA-seq analysis identified that KDM1A inhibition downregulated the expression of genes involved in rapamycin induced activation of Akt, including the mTORC2 complex. Chromatin immunoprecipitation experiments confirmed KDM1A recruitment to the promoter regions of mTORC2 complex genes and that KDM1A inhibition promoted enrichment of repressive H3K9me2 marks at their promoters. Combination therapy of KDM1A inhibitor and rapamycin reduced the tumor growth in EC xenograft and patient derived xenograft models in vivo and patient derived tumor explants ex vivo. Importantly, in silico analysis of TCGA EC patients data sets revealed that KDM1A expression positively correlated with the levels of PI3K/Akt/mTOR genes. Collectively, our results provide compelling evidence that KDM1A inhibition potentiates the activity of mTOR inhibitors by attenuating the feedback activation of Akt survival signaling. Furthermore, the use of concurrent KDM1A and mTOR inhibitors may be an attractive targeted therapy for EC patients.

Neuroinflammation is recognized as a hallmark of spinal cord injury (SCI). Although neuroinflammation is an important pathogenic factor that leads to secondary injuries after SCI, neuroprotective anti-inflammatory treatments remain ineffective in the management of SCI. Moreover, the molecular signatures involved in the pathophysiological changes that occur during the course of SCI remain ambiguous. The current study investigated the proteins and pathways involved in C5 spinal cord hemi-contusion injury using a rat model by means of 4-D label-free proteomic analysis. Furthermore, two Gene Expression Omnibus (GEO) transcriptomic datasets, Western blot assays, and immunofluorescent staining were used to validate the expression levels and localization of dysregulated proteins. The present study observed that the rat models of SCI were associated with the enrichment of proteins related to the complement and coagulation cascades, cholesterol metabolism, and lysosome pathway throughout the acute and subacute phases of injury. Intriguingly, the current study also observed that 75 genes were significantly altered in both the GEO datasets, including ANXA1, C1QC, CTSZ, GM2A, GPNMB, and PYCARD. Further temporal clustering analysis revealed that the continuously upregulated protein cluster was associated with immune response, lipid regulation, lysosome pathway, and myeloid cells. Additionally, five proteins were further validated by means of Western blot assays and the immunofluorescent staining showed that these proteins coexisted with the F4/80+ reactive microglia and infiltrating macrophages. In conclusion, the proteomic data pertaining to the current study indicate the notable proteins and pathways that may be novel therapeutic targets for the treatment of SCI.

Transcription factors (TFs) are the mainstay of cancer and have a widely reported influence on the initiation, progression, invasion, metastasis, and therapy resistance of cancer. However, the prognostic values of TFs in breast cancer (BC) remained unknown. In this study, comprehensive bioinformatics analysis was conducted with data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. We constructed the co-expression network of all TFs and linked it to clinicopathological data. Differentially expressed TFs were obtained from BC RNA-seq data in TCGA database. The prognostic TFs used to construct the risk model for progression free interval (PFI) were identified by Cox regression analyses, and the PFI was analyzed by the Kaplan-Meier method. A receiver operating characteristic (ROC) curve and clinical variables stratification analysis were used to detect the accuracy of the prognostic model. Additionally, we performed functional enrichment analysis by analyzing the differential expressed gene between high-risk and low-risk group. A total of nine co-expression modules were identified. The prognostic index based on 4 TFs (NR3C2, ZNF652, EGR3, and ARNT2) indicated that the PFI was significantly shorter in the high-risk group than their low-risk counterpart (p < 0.001). The ROC curve for PFI exhibited acceptable predictive accuracy, with an area under the curve value of 0.705 and 0.730. In the stratification analyses, the risk score index is an independent prognostic variable for PFI. Functional enrichment analyses showed that high-risk group was positively correlated with mTORC1 signaling pathway. In conclusion, the TF-related signature for PFI constructed in this study can independently predict the prognosis of BC patients and provide a deeper understanding of the potential biological mechanism of TFs in BC.

Background A graded hemi-contusion spinal cord injury produces complex anatomical deformation of the spinal cord parenchyma. The relationship between lesion severity and behavioral consequences in a novel contusion mouse model remains unknown. Purpose We aimed to establish a graded cervical hemi-contusion spinal cord injury model in mice and investigate the correlation between graded anatomical damage to the spinal cord and resulting behavioral impairments. Methods Thirty-two mice were divided into groups of 1.2 mm, 1.5 mm and sham. to the tip of an impactor with a diameter of 1 mm was utilized to compress the left dorsal cord of C5 by 1.2 mm or 1.5 mm at a speed of 300 mm/s. Forelimb motor function was evaluated using rearing, grooming and grip-strength tests before and after the injuries. Histologically the area of white matter sparing, gray matter sparing and lesion area were quantified at 6-week-post-injury. Results Behavioral assessments showed a more severe forlimb functional deficit in 1.5 mm contusion displacements relative to 1.2 mm contusion displacements after injury. The 1.2 mm hemi-contusion mainly caused damage to the dorsal fasciculus, ventral and dorsal horn, while the 1.5 mm hemi-contusion lead to additional damage extending to ventral fasciculus. Sparing of the gray and white matter at the epicenter was 36.8±2.4% and 12.4±2.6% in the 1.2 mm group, and 27.6±4.0% and 4.1±2.2% in the 1.5 mm group, respectively. Furthermore, the lesion area was 20.8±3.0% and 36.0±2.1% in the 1.2 mm and 1.5 mm groups, respectively. There was a significant correlation between the performance in the grooming test and white matter sparing, and between grip-strength test and gray matter sparing. Conclusion The present study demonstrates that a hemi-contusion cervical spinal cord injury in mice can be graded by contusion displacement and that there is a correlation between anatomical and behavioral outcomes. This study provides a means for determining the severity of lesions in a contusion mouse model.

Background and Aims Iptacopan (LNP023) is a highly selective oral low molecular weight inhibitor of Factor B, a key complement alternative pathway protease. We have previously reported data from an interim analysis (IA) of a Phase 2 study in patients with native C3G (NCT03832114) showing that a 12-week course of iptacopan results in a 49% reduction in proteinuria with no adverse safety findings (ASN 2020 SU-OR39). The aim of this analysis was to determine whether iptacopan treatment altered eGFR slope in this same population. Method Adults with biopsy-proven native C3G received open-label iptacopan for 12w (10-100mg bid during w1-3 then 200mg bid w4-12). All had proteinuria >1g/24h, low plasma C3, were receiving maximally tolerated stable ACEi/ARB and were vaccinated against N. meningitidis, H. influenzae and S. pneumoniae. On study completion, all patients received ongoing iptacopan in a long-term extension study (NCT03955445). Historical data were collected on eGFR (CKD-EPI) for the two-year period prior to patients entering the study or since diagnosis where this was less than two years, and were compared with those obtained in the IA following the commencement of iptacopan. A generalized linear mixed model, with a common intercept, a pre-treatment slope and a change in the slope following iptacopan treatment was used to predict the pre-post iptacopan change in eGFR over time. Results 12 patients (10 male, 2 female; 11 C3GN, 1 DDD) participated in the IA. Study baseline mean (SD) age was 26.1 (12.1) yrs, geometric mean (CV%) urine PCR 397 (56) g/mol and eGFR 57.9 (65.5) ml/min/1.73m2. Complete data sets were collected for all 12 patients; one patient entered the study immediately following biopsy diagnosis. During the two years prior to the iptacopan treatment, the mean eGFR slope was -14.8 ml/min/1.73m2/year (p=0.0016), consistent with the known natural history of native C3G (Nephron 2020; 144: 272–280). Treatment with iptacopan was associated with a mean increase in eGFR of 3.1 ml/min/1.73m2 from baseline to 12w, corresponding to a mean predicted eGFR preservation of 6.4 ml/min/1.73m2 over 12w as a result of iptacopan administration (Figure 1, p=0.0459). Data collected in seven patients who entered the extension study confirmed ongoing eGFR stability until 25 weeks. There were no deaths or SAEs related to iptacopan and no AEs leading to iptacopan discontinuation in this patient cohort. Conclusion 12 weeks treatment with iptacopan 200mg bid in patients with C3G resulted in statistically significant and clinically important improvement in eGFR slope with favourable safety and tolerability in addition to significantly reducing proteinuria. Extended iptacopan treatment up to 25 weeks resulted in ongoing stability of eGFR, suggesting that this may result in clinically relevant prolongation of the time to or even potentially prevention of the development of kidney failure.

Background Monitoring subject recruitment is key to the success of a clinical trial. Accordingly, researchers have developed accrual-monitoring tools to support the design and conduct of trials. At an institutional level, delays in identifying studies with high risk of accrual failure can lead to inefficient and costly trials with little chances of meeting study objectives. Comprehensive accrual monitoring is necessary to the success of the research enterprise. Methods This article describes the design and implementation of the University of Kansas Cancer Center Accrual Prediction Program, a web-based platform was developed to support comprehensive accrual monitoring and prediction for all active clinical trials. The Accrual Prediction Program provides information on accrual, including the predicted completion date, predicted number of accrued subjects during the pre-specified accrual period, and the probability of achieving accrual targets. It relies on a Bayesian accrual prediction model to combine protocol information with real-time trial enrollment data and disseminates results via web application. Results First released in 2016, the Accrual Prediction Program summarizes enrollment information for active studies categorized by various trial attributes. The web application supports real-time evidence-based decision making for strategic resource allocation and study management of over 120 ongoing clinical trials at the University of Kansas Cancer Center. Conclusion The Accrual Prediction Program makes accessing comprehensive accrual information manageable at an institutional level. Cancer centers or even entire institutions can reproduce the Accrual Prediction Program to achieve real-time comprehensive monitoring and prediction of subject accrual to aid investigators and administrators in the design, conduct, and management of clinical trials.

Plasma 5′-AMP (pAMP) is elevated in mouse models of type 2 diabetes. However, the metabolic regulatory role of adenine nucleotides in type 2 diabetes remains unclear. Adenine nucleotides and their metabolites in plasma and liver were examined by HPLC. 1H NMR-based metabolomics analysis was performed to explore the changes of metabolites in mouse models of type 2 diabetes. Na+/K+ ATPase and Na+/H+ exchanger activity were measured in response to adenine nucleotide metabolites. Human recombinant protein tyrosine phosphatase 1B (PTP1B) was used for enzyme kinetic assays. Protein binding assays were performed with microscale thermophoresis. The intracellular pH of hepatocyte AML12 cell lines was measured using the BCECF-AM method. We also analysed pAMP levels in participants with type 2 diabetes. Elevation of pAMP was a universal phenomenon in all mouse models of type 2 diabetes including db/db vs lean mice (13.9 ± 2.3 μmol/l vs 3.7 ± 0.9 μmol/l; p

Aims Risk assessment and treatment stratification for three-vessel disease (3VD) remain challenging. N-terminal pro-B-type natriuretic peptide (NT-proBNP) is an established biomarker for prognostication and treatment in heart failure. The present study aimed to evaluate the prognostic value of NT-proBNP beyond the SYNTAX score II (SSII), and its association with long-term outcome after three strategies [percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and medical therapy (MT)] in a cohort of patients with 3VD. Methods and results A total of 6597 patients with available baseline NT-proBNP data were included in the study. Baseline, procedural, and follow-up data were collected. The primary endpoint was all-cause death. Secondary endpoints included cardiac death and major adverse cardiac and cerebrovascular events (MACCE), a composite of death, myocardial infarction, and stroke. During a median follow-up of 7.0 years, higher NT-proBNP levels were strongly associated with increased risks of all-cause death, cardiac death, and MACCE (all adjusted P Conclusion N-terminal pro-BNP is a potential biomarker for risk stratification and therapeutic decision-making in patients with 3VD. Further randomized studies are needed to confirm these findings.

The angioarchitecture of the spinal cord and microvascular changes after acute and subacute spinal cord injury (SCI) have been reported in rodents. Microvascular changes after chronic SCI have not been explored. We characterized three-dimensional microvascular changes during the chronic phase of cervical hemicontusion SCI in rats.At 12 weeks after 1.2-mm hemicontusion injury, microvascular parameters, including vascular volume, ratio of vascular volume to tissue volume, vascular number, and vascular separation, were measured at the epicenter and each cord segment, and the percentage and volume of spinal vessels with different diameters were measured by micro computed tomography at the injury segment.The 1.2-mm hemicontusion injury applied a compressive force of 1.050 ± 0.103 N to the cord, resulting in a cavity and a significant decrease in microvasculature at the epicenter. The vascular volume, ratio of vascular volume to tissue volume, and vascular number of the C5 cord decreased by 40%, 38%, and 36% at 12 weeks after SCI, whereas vascular separation increased by 54% compared with the control group. In the chronic phase after SCI, the percentage and volume of spinal microvessels at the contusion segment decreased significantly (especially vessels with diameters40 μm).Blood supply to the cervical spinal cord is insufficient during the chronic phase of cervical hemicontusion SCI, especially in microvessels with diameters40 μm. These results may provide a basis to explore microvascular changes of SCI during the chronic phase.

OBJECTIVEThe aim of this study was to evaluate the effect of lumbar sacralization on the level of vertebral slip and disc degeneration in patients with L4 spondylolysis.METHODSThe authors analyzed data from 102 cases in which patients underwent surgical treatment for L4 spondylolysis and spondylolisthesis at their institution between March 2007 and September 2016. Lumbar sacralization was characterized by the presence of pseudarthrosis and/or bony fusion between the L5 transverse process and sacrum, and the type of lumbosacral transitional vertebra (LSTV) was evaluated with the Castellvi classification. The amount of vertebral slippage was measured using the Taillard technique and Meyerding grade. Degeneration of the L4–5 segment was quantified using the Pfirrmann and Modic classifications. Patients were divided into 2 groups based on the presence or absence of sacralization, and the amount of vertebral slip and degeneration of the L4–5 segment was compared between groups.RESULTSLumbar sacralization was present in 37 (36%) of 102 patients with L4 spondylolysis. The LSTV was type IIa in 10 cases, type IIb in 7, type IIIa in 2, and type IIIb in 18. The levels of vertebral slip and disc degeneration in the group of patients with sacralization were significantly greater than in the group without sacralization. No significant difference was found between the 2 groups with respect to Modic changes.CONCLUSIONSThe increased stability between a sacralized L5 and the sacrum may predispose the L4–5 segment to greater instability and disc degeneration in patients with L4 spondylolysis.

Hyperuricemia has been associated with mortality in patients with coronary artery disease (CAD). However, its prognostic value remains unknown in the context of severe CAD with heavy atherosclerotic burden in all 3 vessels. We used data from a large cohort of consecutive patients with severe CAD. The primary end point was all-cause death. Propensity score matching was used to identify 2 cohorts of patients with similar baseline characteristics. A total of 8,529 patients with available serum uric acid data were included in the study. Hyperuricemia was present in 1,207 (14.2%) patients. At baseline, hyperuricemic patients had more co-morbidities, and more often received medical therapy alone. During the median follow-up of 7.5 years, significantly more deaths occurred in hyperuricemic patients compared with normouricemic patients (22.5% vs 13.7%; p0.001). Multivariable analyses showed that hyperuricemia was associated with an increased risk of mortality (hazard ratio 1.33; 95% confidence interval 1.15 to 1.53; p0.001). Propensity score matching yielded similar results (hazard ratio 1.33; 95% confidence interval 1.11 to 1.61; p = 0.003). The association was relatively consistent across subgroups, except for an interaction between age and hyperuricemia. Addition of uric acid to SYNTAX score II provided significant improvements of reclassification and discrimination for mortality prediction. In conclusion, hyperuricemia is relatively common among patients with severe CAD and is independently associated with mortality. Moreover, uric acid can improve the predictability of a well-established risk score.

A ketogenic diet (KD) is composed of low-carbohydrate, high-fat and adequate levels of protein. It has been used for decades as a method to treat pediatric refractory epilepsy. However, recently, its side effects on the bones have received increasing attention. In order to comprehensively evaluate the effect of KD on the microstructures and mechanical properties of the skeleton, 14 male Sprague-Dawley rats were equally divided into two groups and fed with a KD (ratio of fat to carbohydrate and protein, 3:1) or a standard diet for 12 weeks. Body weight, as well as blood ketone and glucose levels, were monitored during the experiment. Bone morphometric analyses via micro-computerized tomography were performed on cortical and trabecular bone at the middle L4 vertebral body, the proximal humerus and tibia. The compressive stiffness and strength of scanned skeletal areas were calculated using micro-finite element analysis. The KD led to higher ketone levels and lower glucose levels, with reduced body weight and total bone mineral density (TBMD). After 12 weeks, the diet reduced the bone volume fraction, the trabecular number of cancellous bone, cortical thickness, total cross-sectional area inside the periosteal envelope and the bone area of cortical bone in the tibia and humerus, while increasing trabecular separation. However, KD may not affect the L4 vertebral body. The serum calcium or phosphate concentrations in the blood remained unchanged. In addition, bone stiffness and strength were clearly decreased by the KD, and significantly correlated with the BMD and bone area at all scanned sites. In conclusion, KD led to significant bone loss and reduced biomechanical function in appendicular bones, with a lesser impact on axial bones.

STUDY DESIGN Retrospective analysis of computed tomographic images of 92 normal pediatric cervical vertebrae. OBJECTIVE The aim of this study was to ascertain the feasibility of the pedicle, intralaminar, and lateral mass screws fixation in children. SUMMARY OF BACKGROUND DATA Fixation techniques of C7 vertebrae are technically challenging in the pediatric population. Although the techniques offer alternatives to screws placement at C7 spine in adult, there is no consensus to apply these screws in children. METHODS A total of 92 patients (59 males and 33 females) from 3- to 16-year old divided into three groups (group 1, 1-6 years; group2, 7-10 years; group 3, 11-16 years) were included. Measurements were taken through computed tomography (CT) imaging without evidence of spinal trauma. Length, width, and angle of lamina and pedicle were measured, and lateral mass length was also assessed. RESULTS There was no statistical difference between the left and right sides, as well as the sexes. In 1 to 6 years' age group, 83.3% of the laminas and 87.5% of the pedicles measured have thicknesses of >3.5 mm. In 7 to 10 years' age group, 84.0% of the laminas and all the pedicles observed have thicknesses of >3.5 mm. In 11 to 16 years' age group, 88.4% of the laminas and 97.7% of the pedicles measured have thicknesses of >3.5 mm. Most of the spinolaminar angles were between 42.5° and 56°. There are three cases (3.3%) having lateral mass length >12 mm. CONCLUSION It is feasible to insert a screw (O 3.5 mm) in C7 lamina in children, and the intralaminar screw fixation is a safe and reliable technique. Around 49°, the spinolaminar angle with axial position, can be used as reference for placing screws in the C7 lamina. Preoperative CT scan will help surgeons to insert the laminar screw safely and accurately. LEVEL OF EVIDENCE N/A.

Background Glioblastomas (GBMs) are the most lethal primary brain tumors. Estrogen receptor β (ESR2/ERβ) function as a tumor suppressor in GBM, however, ERβ expression is commonly suppressed during glioma progression. In this study, we examined whether drugs that reverse epigenetic modifications will enhance ERβ expression and augment ERβ agonist-mediated tumor suppression. Methods We tested the utility of epigenetic drugs which act as an inhibitor of histone deacetylases (HDACs), histone methylases, and BET enzymes. Mechanistic studies utilized RT-qPCR, chromatin immunoprecipitation (ChIP), and western blotting. Cell viability, apoptosis, colony formation, and invasion were measured using in vitro assays. An orthotopic GBM model was used to test the efficacy of in vivo. Results Of all inhibitors tested, HDACi (panobinostat and romidepsin) showed the potential to increase the expression of ERβ in GBM cells. Treatment with HDACi uniquely upregulated ERβ isoform 1 expression that functions as a tumor suppressor but not ERβ isoform 5 that drives oncogenic functions. Further, combination therapy of HDACi with the ERβ agonist, LY500307, potently reduced cell viability, invasion, colony formation, and enhanced apoptosis. Mechanistic studies showed that HDACi induced ERβ is functional, as it enhanced ERβ reporter activities and ERβ target genes expression. ChIP analysis confirmed alterations in the histone acetylation at the ERβ and its target gene promoters. In orthotopic GBM model, combination therapy of panobinostat and LY500307 enhanced survival of tumor-bearing mice. Conclusions Our results suggest that the combination therapy of HDACi and LY500307 provides therapeutic utility in overcoming the suppression of ERβ expression that commonly occurs in GBM progression.

Monitoring and reporting of drug safety during a clinical trial is essential to its success. More recent attention to drug safety has encouraged statistical methods development for monitoring and detecting potential safety signals. This paper investigates the potential impact of the process of the blinded investigator identifying a potential safety signal, which should be further investigated by the Data and Safety Monitoring Board with an unblinded safety data analysis.In this paper, two-stage Bayesian hierarchical models are proposed for safety signal detection following a pre-specified set of interim analyses that are applied to efficacy. At stage 1, a hierarchical blinded model uses blinded safety data to detect a potential safety signal and at stage 2, a hierarchical logistic model is applied to confirm the signal with unblinded safety data.Any interim safety monitoring analysis is usually scheduled via negotiation between the trial sponsor and the Data and Safety Monitoring Board. The proposed safety monitoring process starts once 53 subjects have been enrolled into an eight-arm phase II clinical trial for the first interim analysis. Operating characteristics describing the performance of this proposed workflow are investigated using simulations based on the different scenarios.The two-stage Bayesian safety procedure in this paper provides a statistical view to monitor safety during the clinical trials. The proposed two-stage monitoring model has an excellent accuracy of detecting and flagging a potential safety signal at stage 1, and with the most important feature that further action at stage 2 could confirm the safety issue.

Objective To investigate the effects of ketogenic metabolism on macrophage polarization, inflammation inhibition, and function recovery after acute spinal cord injury (SCI) in rats. Methods Sixty-four adult male Sprague-Dawley rats were randomly and equally divided into sham, standard diet (SD), ketone diet (KD), and 1, 3-butanediol (BD) groups. All animals underwent C5 unilateral laminectomy, whereas the SD, KD, and BD groups underwent C5 spinal cord hemi-contusion. The impact rod with a diameter of 1.5 mm was aligned 22.5° to the left and 1.4 mm to the midline, and then triggered to deliver a set displacement of 1.5 mm at a speed of 100 mm/s. The gene expression of inflammatory factors as well as the protein expression of inducible nitric oxide synthase, arginase-1, and inflammatory factors were measured at 1 week post-injury. Serum ketone and behavior were evaluated every second week for 12 weeks. Then, histological analyses of the gray and white matter at the epicenter were conducted at 12 weeks post-injury. Results The serum ketone levels of the KD and BD groups were significantly increased when compared with the SD group. The gene and protein expression of TNF-α and IL-1β tended to increase after the SCI, but were inhibited in the KD and BD groups. The protein expression of inducible nitric oxide synthase, marker of M1 macrophage, was inhibited in the KD and BD groups; on the other hand, the expression of arginase-1, marker of M2 macrophage, was boosted in the KD and BD groups. The usage of the ipsilateral forelimb was higher in the KD group than in the SD group. The hemi-contusive injury resulted in an obvious ipsilateral lesion area at the epicenter, and there was no significant difference between groups regarding the lesion size. However, the spared gray matter area was significantly greater in the KD group than in the SD and BD groups. Conclusion The present study suggests that ketogenic metabolism promotes macrophage polarization to M2, inhibits an inflammatory response, and alleviates the loss of gray matter after SCI. A higher ketone level, such as that induced by the ketogenic diet, seems to benefit function recovery after SCI.

Protecting white matter is one of the key treatment strategies for spinal cord injury (SCI), including alleviation of myelin loss and promotion of remyelination. Rapamycin has been shown neuroprotective effects against SCI and cardiotoxic effects while enhancing autophagy. However, specific neuroprotection of rapamycin for the white matter after cervical SCI has not been reported. Therefore, we aim to evaluate the role of rapamycin in neuroprotection after hemi-contusion SCI in mice. Forty-six 8-week-old mice were randomly assigned into the rapamycin group (n = 16), vehicle group (n = 16), and sham group (n = 10). All mice of the rapamycin and vehicle groups received a unilateral contusion with 1.2-mm displacement at C5 followed by daily intraperitoneal injection of rapamycin or dimethyl sulfoxide solution (1.5 mg⋅kg–1⋅day–1). The behavioral assessment was conducted before the injury, 3 days and every 2 weeks post-injury (WPI). The autophagy-related proteins, the area of spared white matter, the number of oligodendrocytes (OLs) and axons were evaluated at 12 WPI, as well as the glial scar and the myelin sheaths formed by Schwann cells at the epicenter. The 1.2 mm contusion led to a consistent moderate–severe SCI in terms of motor function and tissue damage. Rapamycin administration promoted autophagy in spinal cord tissue after injury and reduced the glial scar at the epicenter. Additionally, rapamycin increased the number of OLs and improved motor function significantly than in the vehicle group. Furthermore, the rapamycin injection resulted in an increase of Schwann cell-mediated remyelination and weight loss. Our results suggest that rapamycin can enhance autophagy, promote Schwann cell myelination and motor function recovery by preserved neural tissue, and reduce glial scar after hemi-contusive cervical SCI, indicating a potential strategy for SCI treatment.

Natural polysaccharides have attracted considerable interests due to diverse biological activities. Succinoglycan is an extracellular polysaccharide produced by most Agrobacterium strains. Here, we confirmed riclin was a typical succinoglycan by NMR and methylation analysis, and investigated the antitumor effects of riclin in sarcoma 180 tumor-bearing mice. The results showed that riclin inhibited the tumor growth significantly as well as cyclophosphamide (CTX). While CTX caused serious damage to spleen structure, riclin increased the spleen index and promoted lymphocytes proliferation in peripheral blood, spleen and lymph nodes. Riclin decreased splenocytes apoptosis as evidenced by alterations of B-cell lymphoma-2 family proteins and Cleaved Caspase-3 protein. Moreover, 1H nuclear magnetic resonance (NMR)-based metabolomics analysis revealed that riclin partially altered the metabolic profiles of splenocytes. In conclusion, riclin is a succinoglycan that performed strong immunogenicity and suppressed sarcoma growth in mice. Succinoglycan riclin could be a potential antitumor agent for functional food and pharmaceutical purpose.

Background: Risk stratification for three-vessel coronary artery disease (3VD) remains an important clinical challenge. In this study, we utilized machine learning (ML), which can address the limitations of traditional regression-based models, to develop a novel model to assess mortality risk in patients with 3VD. Methods: This study was based on a prospective cohort of 8943 participants with 3VD consecutively enrolled between 2004 and 2011. A ML-derived random forest model was trained and tested to predict 4-year mortality. The predictability of the model was compared with that of an established model, the Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery score II (SSII), among 3VD patients undergoing percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and medical therapy (MT) alone. Findings: The all-cause mortality was 7.5% (667 patients) over the 4-year follow-up period. The correlation-based feature selection algorithm selected 18 of the 94 features to develop the ML model. In the testing dataset, the ML-derived model achieved an area under the curve of 0.81 for 4-year mortality prediction. Its predictability was significantly better than that of the SSII among patients undergoing PCI (0.80 vs 0.70, respectively P < 0.001) or CABG (0.80 vs. 0.67, respectively, P < 0.001). The model also outperformed the SSII in patients receiving MT alone (ML: 0.75 vs. SSII for PCI: 0.70 or SSII for CABG: 0.66, P < 0.001). Interpretation: This ML-based approach exhibited better performance in risk stratification for 3VD compared with the conventional method, indicating its potential utility in clinical practice. Funding Statement: Chinese Academy of Medical Sciences, Beijing Municipal Natural Science Foundation. Declaration of Interests: None declared. Ethics Approval Statement: The study complied with the principles of the Declaration of Helsinki and was approved by the Review Board of Fuwai Hospital. All participants provided written informed consent.

Objective The feasibility of anterior transarticular crossing screws (ATCS) was confirmed in cadaveric specimens and it has been used in clinic. However, no study has documented the morphometric characteristics of ATCS. This study seeks to determine the morphometric characteristics of ATCS in C1-C2 fixation. Methods A total of 100 patients without abnormality of C1-2 were enrolled. The range of screw lateral angles (LAs) and the screw lengths of ATCS on coronal images were measured on multiplanar computed tomography images, as well as the screw incline angles (IAs) in the sagittal plane. The ideal trajectory was designed as toward to the posterosuperior points of the opposite C1 lateral mass, which had the longest screw. Results The LAs were relatively small in men (31.4°–45.3°) compared with women (32.6°–46.0°). In the sagittal plane, the IAs were ranged from 41.0° to 68.4° in men and from 44.4° to 68.1° in women. The overall screw lengths were longer in men (34.7–44.8 mm) than in women (32.2–39.6 mm). In the ideal path, the LA and IA were 38.4° and 41.0° in men and 39.6° and 44.4° in women, and the screw lengths were 44.8 mm in men and 39.6 mm in women. Conclusions This study provides the range of screw angles and lengths of ATCS, which will help surgeons to perform ATCS safely and accurately.

Objective To evaluate the longitudinal somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs) characterization from acute to chronic injury following cervical hemi-contusion spinal cord injury (SCI) in rats, and correlate the MEPs & SEPs to the behavioral outcomes. Methods Fifteen adult male Sprague-Dawley rats were subjected to the hemi-contusion spinal cord injury at C5. Forelimb MEPs & SEPs were applied to 5 animals before injury and 3h, 1d, 3d, 1w, 2w, 4w and 8w after injury respectively. Forelimb functional assessments, including Montoya staircase task and cylinder rearing test, were conducted on another 10 animals before injury and at 2w, 4w and 8w after injury respectively, as well as histological analysis of the cord at 8w after injury. A group correlation was performed between the MEPs & SEPs and behavioral outcomes. Results The hemi-contusion injury resulted in unilateral tissue damage at the epicenter with loss of the ventral horns and lateral funiculus. Both ipsilateral and contralateral forelimb MEPs showed latency prolongation and amplitude reduction at 3 h after injury. The MEPs amplitude increased with time after injury, but the ipsilateral amplitude was persistently lower than the contralateral amplitude. The ipsilateral MEPs latency increased with time after injury and was significantly longer than the contralateral MEPs latency. The ipsilateral SEPs amplitude dropped after injury and stayed at a lower level up to 8 weeks. There was no difference in the SEPs latency among time points and between sides. At 8 weeks after injury, the ipsilateral forelimb grasped 30% pellets while the contralateral forelimb close to 81%. An obvious decreased usage of the ipsilateral paw and increased usage of the contralateral paw were observed in rearing test after injury. The MEPs latency and amplitude correlated significantly with the forelimb motor function. Conclusion Cervical hemi-contusion SCI led to persistent changes in MEPs & SEPs of the ipsilateral forelimb, ipsilateral impairment in motor function and unilateral cord tissue damage. Reliable electrophysiology assessment was obtained in chronic phase due to unstable MEPs & SEPs of bilateral forelimb immediately after injury, which might reflect the underlying pathological processes. The present study further confirmed the link of the MEPs to the behavioral outcomes, supporting the longitudinal electrophysiology assessment for neurological impairment after SCI.

Background: Endometrial cancer (EC) is the fourth most common cancer in women. Approximately 80% of EC belong to the endometroid-EC subtype and are driven by estrogen signaling. Advanced-stage EC has limited treatment options with poor prognosis. There is an urgent need for the identification of actionable drivers as new targets for treating advance stage EC. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF plays a major role in cancer progression, metastasis, stemness, and therapy resistance. Published and our preliminary data suggest a critical role of the LIF-LIFR signaling axis in EC progression. The objective of this study is to test the utility of targeting the LIF/LIFR axis using a novel LIFR inhibitor, EC359. Methods: We used multiple established and primary EC cells to test the utility LIFR inhibitor, EC359 in treating EC. CRISPR/Cas9 system was used to generate LIFR KO EC cells. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. EC cell-derived xenograft (CDX) and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: EC359 treatment of seven EC cells showed anti-proliferative effects in MTT cell viability assays with an IC50 of 25-100 nM. Further, EC359 treatment reduced invasiveness, stemness, and promoted apoptosis of EC cells. The activity of EC359 is dependent on LIF/LIFR expression in EC cells. CRISPR mediated knockout of LIFR significantly abolished EC359 activity. In vivo xenograft studies using Ishikawa-vector or LIFR-KO cells demonstrated that LIFR-KO significantly reduced EC tumor growth, and tumor weights. Further, EC359 treatment attenuated the activation of LIF/LIFR driven pathways, including STAT3, AKT-mTOR signaling. Mechanistic studies using RNA-seq revealed that EC359 significantly upregulated 213 genes and down regulated 126 genes. Pathway analyses of differential genes revealed enrichment in the apoptotic pathways upon EC359 treatment. EC359 (5mg/kg body weight) treatment significantly reduced CDX tumor progression and reduced proliferation in PDEX models. Conclusions: Collectively, these data support EC359 as a novel targeted therapy for EC by inhibiting LIF/LIFR oncogenic signaling pathway. Citation Format: Weiwei Tang, Kumaraguruparan Ramasamy, Sureshkumar M. Pillai, Bindu Santhamma, Swapna Konda, Prabhakar P. Vekata, Logan Blankenship, Junhao Liu, Zexuan Liu, Kristin A. Altwegg, Behnam Ebrahimi, Uday P. Pratap, Xiaonan Li, Edward Kost, Gangadhara R. Sareddy, Ratna K. Vadlamudi, Hareesh B. Nair, Rajeshwar R. Tekmal, Suryavathi Viswanadhapalli. Therapeutic targeting of endometrial cancer with novel LIFR inhibitor EC359 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1253.

Background: Women with triple-negative breast cancer (TNBC) have a more aggressive clinical course, with a higher propensity to metastasize and a worse outcome due to a lack of effective therapies and significant intratumoral and intertumoral heterogeneity. Development of novel therapeutic strategies represents a clear unmet need. We have developed a first-in-class compound, an oligobenzamide, ERX-41, that has anti-proliferative activity against all six molecular subtypes of TNBC. Methods: In vitro activity of ERX-41 on TNBC cell lines was tested using CellTiter glo, MTT, and apoptosis assays. Efficacy of ERX-41 was tested using TNBC patient derived explants (PDEs) ex vivo, cell line-derived xenografts (CDXs), and patient derived xenografts (PDX) in vivo. To examine the mechanism, we conducted mass spec analyses using total lysates of TNBC cells treated with vehicle or ERX-41. Results: ERX-41 demonstrated potent activity in both blocking proliferation and inducing apoptosis in 30 distinct cell line models of TNBC, (representing all six molecular subtypes of TNBC), with an IC50 that ranges from 50-250nM. Incubation of ERX-41 with PDEs from primary TNBC patient tumors ex vivo caused a significant reduction in proliferation indices, as measured by Ki67 staining. ERX-41 also decreased proliferation and increased apoptosis in explants from TNBC CDX and PDX tumors cultured ex vivo. Oral administration of ERX-41 (10 mg/kg/daily) was shown to be non-toxic and dramatically limited the growth of CDX tumors derived from MDA-MB-231, SUM-159 or D2A1 syngeneic tumors. Importantly, ERX-41 treatment also significantly reduced tumor progression in four TNBC PDX (PDX-1, PDX-89, PDX-96 and PDX-98) models compared to the vehicle treated control group. Our ultrastructural and molecular studies indicate that ERX-41 induces significant endoplasmic reticulum (ER) stress within TNBC cells but not in primary epithelial cells. Global mass spectrometry studies indicated that ERX-41 treatment resulted in the alteration [down regulation (265 proteins) or upregulation (218 proteins)] of 483 proteins out of ~4000 proteins quantified with two or more peptides. Reactome pathway analysis indicated that the top pathways modulated by ERX-41 included Intra-Golgi and retrograde Golgi-to-ER traffic, membrane trafficking and TP53 mediated apoptosis. ER stress induced by ERX-41 blocks de novo protein synthesis, and triggers ER-assisted degradation (ERAD) pathways, causing TNBC apoptotic cell death. Conclusions: ERX-41 is orally bioavailable, non-toxic, and demonstrated activity in primary PDEs, CDXs and PDXs. The ability of ERX-41 to induce ER stress and apoptotic cell death in multiple types of TNBC suggests that this drug targets a fundamental weakness in TNBC cells (the high basal level of ER stress) and can effectively overcome the heterogeneity of TNBC. These studies strongly support the further clinical translation of ERX-41. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Shi-Hong Ma, Tae-Kyung Lee, Mengxing Li, Weiwei Tang, Junhao Liu, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ratna K. Vadlamudi, Ganesh V. Raj. Preclinical evaluation of ERX-41 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1237.

Background: Breast cancer (BC) is composed of distinct molecular subtypes, such as ER+ BC and triple negative BC (TNBC). Development of novel effective therapies for patients with TNBC remains the highest unmet need in patient treatment and survivorship. PELP1 plays an essential role in several pathways including hormonal signaling, cell cycle progression, ribosomal biogenesis, and DNA damage response. PELP1 expression is an independent prognostic predictor of shorter BC-specific survival and an independent prognostic factor for predicting poor survivorship in TNBC patients. Recently, we generated a small molecule inhibitor of PELP1 (SMIP34) that binds and inhibits PELP1 oncogenic signaling. The objective of this study is to test the utility of SMIP34 as a novel therapeutic for treating TNBC. Methods: We have selected seven TNBC model cell lines (BT549, MDA-MB-453, MDA-MB-231, MDA-MB-468, SUM-159, HCC1806, and HCC1937) and one human mammary epithelial (HMEC) cell line in this study. In vitro activity was assessed using Cell Titer Glo, MTT, colony formation, and matrigel invasion assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq. Xenograft tumor-derived explant (XDEX) assays and patient-derived tumor explant (PDEX) assays were used for preclinical evaluation. Results: Using a panel of BC model cell lines, we found that SMIP34 treatment reduced cell viability with an IC50 of 3-8µM with no activity in HMEC cells. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, confirming its target specificity. We confirmed the physical interaction of SMIP34 to PELP1 using biotin-SMIP34 and MST assays. SMIP34 treatment significantly reduced the invasiveness and colony formation of TNBC cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. Further, RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. Western analyses confirmed SMIP34 treatment significantly reduced known PELP1 downstream signaling. Mechanistic studies using global RNA-seq identified that SMIP34 treatment alters known PELP1 modulated pathways (ribosomal biogenesis). Using MDA-MB-231 xenograft and PDX tumor tissues in explant assays, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Accordingly, in xenograft models, SMIP34 (20mg/kg/IP) treatment resulted in a significant reduction in tumor volume compared to vehicle control. Conclusion: Our results using in vitro, ex vivo, and in vivo studies demonstrated that the PELP1 inhibitor SMIP34 has therapeutic efficacy against TNBC. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin A. Altwegg, Suryavathi Viswanadhapalli, Junhao Liu, Zexuan Liu, Uday P. Pratap, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Evaluation of a novel PELP1 inhibitor for treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1333.

BACKGROUND: PELP1 is commonly overexpressed in breast cancer (BCa) and is implicated in endocrine therapy resistance. However, the mechanism by which PELP1 contributes to therapy resistance remains elusive. Our ongoing studies using yeast two hybrid screen identified histone methyltransferase SETDB1 as a novel interactor of PELP1. The objective of this study is to determine the significance of PELP1 interaction with SETDB1 in BCa endocrine therapy resistance. METHODS: We used two ER+ BCa cell lines (MCF7 and ZR75) and three endocrine therapy resistant cell lines (Tamoxifen resistant MCF7-TamR, Fulvestrant resistant MCF7-FR, Tamoxifen resistant MCF7-PELP1-cyto). Additional BCa models with stable overexpression and under expression of SETDB1 and PELP1 (MCF7-SETDB1-PELP1shRNA, ZR75-SETDB1-PELP1shRNA, ZR75-SETDB1, MCF7-SETDB1, MCF7-TamR-SETDB1shRNA, MCF7-FR-SETDB1shRNA) were generated using lentiviral transduction. Functional significance of cross-talk was tested using MTT, soft agar, and colony formation assays. Mechanistic studies were conducted using immunoprecipitation, Western blotting, and RT-qPCR. RESULTS: Analyses of TCGA databases showed that SETDB1 expression is positively correlated with PELP1 expression in ER+ BCa (r=0.30, p CONCLUSIONS: The results of study suggest that the PELP1/SETDB1 interactions contribute to endocrine therapy resistance via aberrant activation of Akt signaling. Targeting PELP1/SETDB1 axis could represent a new therapeutic avenue to mitigate endocrine resistance. Supported by VA grant I01BX004545 (R.K. Vadlamudi) Citation Format: Zexuan Liu, Junhao Liu, Weiwei Tang, Uday P. Pratap, Kristin A. Altwegg, Behnam Ebrahimi, Xiaonan Li, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. Significance of PELP1/SETDB1 axis in endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 733.

To clarify osteoporotic effects of ketogenic diet (KD) on cancellous and cortical bone compared with ovariectomy (OVX) in mice. Forty female C57BL/6J 8-week-old mice were randomly divided into SD+Sham, SD+OVX, KD+Sham, and KD+OVX groups, and fed for 12 weeks. The distal femur of trabecular bone and the middle femur of cortical bone were evaluated with Micro-CT scanning. The maximum bending force and stiffness of the tibia were calculated using a three-point bending test. Osteoblast and osteoclast expression of femur were identified using tartrate-resistant acid phosphatase (TRAP), collagen type I (CoLI), and osteocalcin (OCN) staining. A 2-factor analysis of variance was used to evaluate effects of KD and OVX on radiological, biomechanical, and histological parameters. KD resulted in not only remarkable cancellous bone decline comparable to OVX, but also unique cortical bone reduction. The maximum bending force and stiffness decreased in the KD+Sham and KD+OVX groups but did not change in the SD+OVX group. The KD+OVX led to significantly higher expression in TRAP and noticeably lower expression in CoLI when compared with other groups. Both KD+Sham and SD+OVX prominently increased expression in TRAP, but decreased expression in CoLI. There was no significant difference in OCN among the four groups. The present results suggest that KD compromises both the cancellous and cortical bone architecture of long bones while OVX only in cancellous bone architecture. A combination of KD and OVX may lead to more bone loss.

Increased plasma free fatty acids (FFA) level plays a central role in the development of type 2 diabetes. Our previous studies have shown that plasma 5′-adenosine monophosphate (5′-AMP) elevates and acts as a potential upstream regulator of hyperglycemia in diabetic db/db mice. The relationship between FFA and plasma adenosine nucleotides in type 2 diabetes remains unclear. Here we found that plasma 5′-AMP level was also increased in diabetic mice induced by a high-fat diet and streptozotocin (HFD-STZ), as observed in diabetic db/db mice. The metabolites of adenosine nucleotides in plasma were increased in obese mice compared to lean mice. An acute oil gavage to lean mice increased both FFA and plasma purine metabolites, accompanying with glucose intolerance. 5′-AMP administration resulted in an increase in dose-dependent purine metabolites and different levels of glucose intolerance. FFA induced a release of adenine nucleotides from cultural human umbilical vein endothelial cells (HUVECs) prior to induction of their apoptosis. FFA also reduced red blood cells (RBCs) resistance to reactive oxygen species (ROS), leading to hemolysis, thereby increasing plasma nucleotides. Our results suggest that plasma adenine nucleotides play an intermediary role in FFA-induced glucose intolerance and hyperglycemia in obese mice.

Aim: To evaluate the prognostic value of plasma big endothelin-1 level in the context of three-vessel disease (TVD) with heavy atherosclerotic burden. Methods: A total of 6,150 patients with TVD and available big endothelin-1 data were included in the study. Participants were divided into two groups according to the optimal cutoff value of big endothelin-1 for mortality prediction. The primary endpoint was all-cause death. C-index, net reclassification improvement (NRI), and integrated discrimination improvement (IDI) were calculated to evaluate the added prognostic value of plasma big endothelin-1 level beyond the SYNTAX score II. Results: On the basis of the optimal cutoff value of 0.79 pmol/L, 1,984 patients were assigned to the high big endothelin-1 group. During a median follow-up of 6.8 years, 818 patients experienced all-cause death. Plasma big endothelin-1 level was significantly higher in patients who died than in patients who survived. Multivariable analysis found that high big endothelin-1 level was independently associated with an increased risk of mortality (hazard ratio: 1.36, 95% confidence interval: 1.18–1.57, P < 0.001). The association of big endothelin-1 with all-cause death was relatively consistent across subgroups with no significant interactions. The predictive ability of the SYNTAX score II was significantly enhanced by addition of plasma big endothelin-1 level (C-index: 0.723 vs. 0.715, P = 0.029; NRI: 0.304, P < 0.001; IDI: 0.009, P < 0.001). Conclusions: Plasma big endothelin-1 level is an independent predictor of long-term mortality in patients with TVD. It can improve the discrimination and reclassification of the SYNTAX score II for mortality prediction.

Purpose. Barium sulfate and lead oxide are commonly used for angiographic studies, but there is no report on the comparison of two contrast agents in angiography of cervical spinal cord. This study was aimed to compare the microvascular architecture of cervical spinal cord in rats after angiography with the barium sulfate agent to the lead oxide agent. Methods. Twelve adult Sprague-Dawley rats were randomly divided into the barium sulfate group (n=6) and the lead oxide group (n=6). Each rat was perfused under the same protocol using either two contrast agents. The angiography was evaluated with the vascular number at different ranks. The cervical spinal cord samples were scanned using micro-CT with low resolution and high resolution. The microvascular parameters, including ratio of vascular volume to tissue volume (VV/TV), vascular number (V.N), diameter (V.Dm), separation (V.Sp), connectivity density (Conn.D), structure model index (SMI), percentage, and volume of vessels at different diameters were measured. Results. The perfusion was better in the barium sulfate group, with more blood vessel trees of rank II and III visible compared to the lead oxide group. Low-resolution micro-CT analysis showed no difference in microvascular parameters except SMI between the two groups. High-resolution micro-CT analysis results showed that V.N and Conn.D of barium sulfate group were 60% and 290% more than those of the lead oxide group; however, V.Sp was 41% less than the lead oxide group. The percentage of vessels with diameter of 10 μm and 20 μm, and the volume of vessels with diameter of less than 100 μm was higher in the barium sulfate group than in the lead oxide group. The SMI index in the barium sulfate group was higher than that in the lead oxide group at both low resolution and high resolution. Conclusions. Compared with lead oxide, barium sulfate is more suitable for perfusion of cervical spinal cord microvessels, and cheap and nontoxic with high resolution.