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Runt‐related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2‐deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown‐based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme‐like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)‐mediated proteasome degradation. Wild‐type Hakai but not Hakai‐C109A inhibited Smurf2 protein levels through proteasome‐mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2‐mediated ubiquitin‐proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis. [ABSTRACT FROM AUTHOR]

Nitroimidazoles comprise a class of broad-spectrum anti-microbial drugs with efficacy against parasites, mycobacteria, and anaerobic Gram-positive and Gram-negative bacteria. Among these drugs, metronidazole (MTZ) is commonly used with other antibiotics to prevent infection in open fractures. However, the effect of MTZ on bone remains understudied. In this paper, we evaluated six nitroimidazole drugs for their impact on osteoblast differentiation and identified MTZ as having the highest osteogenic effect. MTZ enhanced bone regeneration at the femur osteotomy site in osteopenic ovariectomized (OVX) rats at the human equivalent dose. Moreover, in OVX rats, MTZ significantly improved bone mass and strength and improved microarchitecture compared to the vehicle-treated rats, which was likely achieved by an osteogenic mechanism attributed to the stimulation of the Wnt pathway in osteoblasts. To mitigate the reported neurological and genotoxic effects of MTZ, we designed an injectable sustained-release in situ gel formulation of the drug that improved fracture healing efficacy by 3.5-fold compared to oral administration. This enhanced potency was achieved through a significant increase in the circulating half-life and bioavailability of MTZ. We conclude that MTZ exhibits osteogenic effects, further accentuated by our sustained-release delivery system, which holds promise for enhancing bone regeneration in open fractures. [ABSTRACT FROM AUTHOR]

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-transretinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML. [ABSTRACT FROM AUTHOR]

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin‐proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme‐like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18‐58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome‐dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin‐mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Ormeloxifene (ORM) (3,4‐trans‐2,2‐dimethyl‐3‐phenyl‐4‐p‐(β‐pyrrolidinoethoxy) phenyl‐7‐methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan‐caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c‐Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells. [ABSTRACT FROM AUTHOR]

Imparting epithelial to mesenchymal transition (EMT) during cellular transformation, a major driving force behind tumor progression, is one of the notorious oncogenic activities of transforming growth factor β (TGFβ); however, the secretary factors released during TGFβ‐induced EMT that may have role in potentiating EMT and tumor progression are poorly known. This study was undertaken to identify such secreted protein factors from TGFβ‐induced A549 cells cultured in serum‐free chemically defined medium (FreestyleTM) using Matrix Assisted Laser Desorption Ionization‐Time of flight/Time of flight (MALDI‐TOF/TOF) mass spectrometry. We identified some of the potential factors such as ESR, ANXA2, ALDH1A, TGFβ‐induced protein ig‐h3, and PAI‐1 that were not only secreted but some were also elevated in TGFβ‐induced A549 cells. Interestingly, these factors are widely reported to play crucial role in EMT induction and progression, which not only validates our findings but also opens avenues for further investigation, if upon secretion they act exogenously through certain receptors to potentiate cellular signaling involved in EMT induction and tumor progression. [ABSTRACT FROM AUTHOR]

CCAAT/enhancer‐binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine‐protein kinase NLK, also known as Nemo‐like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin‐mediated degradation. Individual phospho‐point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML. [ABSTRACT FROM AUTHOR]

Introduction: This study was conducted to assess fluid responsiveness in critically ill patients to avoid various complications of fluid overload. Material and methods: This study was done in an ICU of a tertiary care hospital after approval from the institute ethical committee over 18 months. A total of 54 consenting adult patients were included in the study. Patients were hemodynamically unstable requiring mechanical ventilation, had acute circulatory failure, or those with at least one clinical sign of inadequate tissue perfusion. All patients were ventilated using tidal volume of 6--8 mL/kg, RR--12--15/minutes, positive end expiratory pressure (PEEP)--5 cm of water, and plateau pressure was kept below 30 cm water. They were sedated throughout the study. The arterial line and the central venous catheter were placed and connected to Vigileo-FloTrac transducer (Edward Lifesciences). Patients were classified into responder and nonresponder groups on the basis of the cardiac index (CI) after fluid challenge of 10 mL/kg of normal saline over 30 minutes. Pulse pressure variation (PPV), stroke volume variation (SVV), and systolic pressure variation (SPV) were assessed and compared at baseline, 30 minutes, and 60 minutes. Results: In our study we found that PPV and SVV were significantly lower among responders than nonresponders at 30 minutes and insignificant at 60 minutes. Stroke volume variation was 10.28 ± 1.76 in the responder compared to 12.28 ± 4.42 (p = 0.02) at 30 minutes and PPV was 15.28 ± 6.94 in responders while it was 20.03 ± 4.35 in nonresponders (p = 0.01). We found SPV was insignificant at all time periods among both groups. Conclusion: We can conclude that initial assessment for fluid responsiveness in critically ill mechanically ventilated patients should be based on PPV and SVV to prevent complications of fluid overload and their consequences. [ABSTRACT FROM AUTHOR]

Context: Resuscitation of critically ill patients requires an accurate assessment of the patient's intravascular volume status. Passive leg raise cause auto transfusion of fluid to the thoracic cavity. Aims: This study aims to assess and compare the efficacy of superior vena cava (SVC) and inferior vena cava (IVC) diameter changes in response to passive leg raise (PLR) in predicting fluid responsiveness in mechanically ventilated hemodynamically unstable critically ill patients. Methods: We enrolled 30 patients. Predictive indices were obtained by transesophageal and transthoracic echocardiography and were calculated as follows: (Dmax − Dmin)/Dmax for collapsibility index of SVC (cSVC) and (Dmax − Dmin)/Dmin for distensibility index of IVC (dIVC), where Dmax and Dmin are the maximal and minimal diameters of SVC and IVC. Measurements were performed at baseline and 1 min after PLR. Patients were divided into responders (increase in cardiac index (CI) ≥10%) and nonresponders (NR) (increase in CI <10% or no increase in CI). Results: Among those included, 24 (80%) patients were R and six were NR. There was significant rise in mean arterial pressure, decrease in heart rate, and decrease in mean cSVC from baseline to 1 min after PLR among responders. The best threshold values for discriminating R from NR was 35% for cSVC, with sensitivity and specificity of being 100%, and 25% for dIVC, with 54% sensitivity and 86.7% specificity. The areas under the receiver operating characteristic curves for cSVC and dIVC regarding the assessment of fluid responsiveness were 1.00 and 0.66, respectively. Conclusions: cSVC had better sensitivity and specificity than dIVC in predicting fluid responsiveness. [ABSTRACT FROM AUTHOR]

Tibialis anterior tendon ruptures are rare but debilitating injuries. A high index of suspicion is warranted in patients presenting with atraumatic anterior ankle pain, especially in conjunction with diabetes or inflammatory disease. The authors present a case report of bilateral sequential rupture of tibialis anterior tendons, a discussion of management, and a review of the literature. [ABSTRACT FROM AUTHOR]

Tamoxifen-mediated endocrine therapy has been standard treatment for ER+ breast cancers; however, majority of them acquire resistance leading to disease relapse. Although numerous substrates of E3 ligase FBW7 are known, only a handful of factors that regulate FBW7 expression and function are reported. In particular, there remains a lack of in-depth understanding of FBW7 transcriptional regulation. Luciferase reporter assay was performed after cloning full length and truncated FBW7 promoters followed by Chromatin immunoprecipitation assay to validate binding of SOX4 on FBW7 promoter. Transcriptional regulation of FBW7 by SOX4 and their biological consequences with respect to ER+ breast cancer was then evaluated using immunoblotting and other cell based assays. SOX4 positively regulates FBW7 at transcriptional level by binding to three putative SOX4 biding sites within 3.1 kb long FBW7 promoter. Analysis of publicly available RNAseq datasets also showed a positive correlation between SOX4 and FBW7 mRNA in cancer cell lines and patient samples. qPCR and Immunoblotting confirmed that transiently or stably expressed SOX4 induced both endogenous FBW7 mRNA and protein levels. Our findings further demonstrated that increased levels of SOX4 and FBW7 in MCF7 mammospheres promoted cancer stemness and tumor cell dormancy. We further showed that both MCF7 mammospheres and MCFTAMR cells had elevated SOX4 levels which apparently enhanced FBW7 to potentiate GATA3 degradation leading to enhanced stemness, tumor dormancy and Tamoxifen resistance in MCF7TAMR as well as patients with ER+ breast cancers. Targeting SOX4-FBW7-GATA3 axis may overcome tamoxifen resistance in ER+ breast cancers. Graphical model depicts that SOX4 positively regulates FBW7 levels in ER+ breast cancers where it promotes stemness, tumor dormancy and TAM resistance by targeting GATA3 and c-Myc. [Display omitted] [ABSTRACT FROM AUTHOR]