Intestine-to-neuronal signaling alters risk-taking behaviors in food-deprived Caenorhabditis elegans.

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

Hypoxia-regulated carbonic anhydrase IX (CAIX) protein is an independent prognostic indicator in triple negative breast cancer.

BACKGROUND: The effect of extracellular microenvironment (hypoxia and pH) has been regarded as a key hallmark in cancer progression. The study aims to investigate the effects of carbonic anhydrase IX (CAIX), a key hypoxia-inducible marker, in triple-negative breast cancer (TNBC) in correlation with clinicopathological parameters and predicting survival outcomes. METHODS: A total of 323 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital from 2003 to 2013 were used. Immunohistochemical staining (IHC) was performed using CAIX antibody and digital mRNA quantification was performed using NanoString assays. CAIX membranous expression was correlated with clinicopathological parameters using Chi-squared test or Fisher's exact tests. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test. RESULTS: Forty percent of TNBCs were observed to express CAIX protein and demonstrated significant association with larger tumour size (P = 0.002), higher histological grade (P < 0.001), and significantly worse disease-free survival (DFS) and overall survival (OS) (after adjustment: HR = 2.99, 95% CI = 1.78-5.02, P < 0.001 and HR = 2.56, 95% CI = 1.41-4.65, P = 0.002, respectively). Gene ontology enrichment analysis revealed six significantly enriched cellular functions (secretion, cellular component disassembly, regulation of protein complex assembly, glycolytic process, cellular macromolecular complex assembly, positive regulation of cellular component biogenesis) associated with genes differentially expressed (CAIX, SETX, WAS, HK2, DDIT4, TUBA4α, ARL1). Three genes (WAS, SETX and DDIT4) were related to DNA repair, indicating that DNA stability may be influenced by hypoxia in TNBC. CONCLUSIONS: Our results demonstrate that CAIX appears to be a significant hypoxia-inducible molecular marker and increased CAIX protein levels are independently associated with poor survival in TNBC. Identification of CAIX-linked seven gene-signature and its relationship with enriched cellular functions further support the implication and influence of hypoxia-mediated CAIX expression in TNBC tumour microenvironment.

Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.

Staging the Tumor and Staging the Host: Pretreatment Combined Neutrophil Lymphocyte Ratio and Modified Glasgow Prognostic Score Is Associated with Overall Survival in Patients with Esophagogastric Cancers Undergoing Treatment with Curative Intent.

BACKGROUND: This study examined whether an innate systemic inflammatory response (SIR) measured by combination neutrophil to lymphocyte ratio (NLR) and modified Glasgow Prognostic Score (mGPS) was associated with overall survival (OS) in patients with esophagogastric cancer (EC) undergoing neoadjuvant chemotherapy (NAC) followed by surgery. METHODS: Patients diagnosed with EC, managed with NAC prior to surgery at a regional referral center, between January 2010 and December 2015, were included. The mGPS and NLR were calculated within 12 weeks before NAC. Patients were grouped by combined NLR/mGPS score into three groups of increasing SIR: NLR ≤ 3 (n = 152), NLR > 3 + mGPS = 0 (n = 55), and NLR > 3 + mGPS > 0 (n = 32). Univariable and multivariable Cox regression was used to analyse OS. RESULTS: Overall, 337 NAC patients were included, with 301 (89%) proceeding to surgery and 215 (64%) having R0 resection. There were 203 deaths, with a median follow-up of those alive at censor of 69 months (range 44-114). Higher combined NLR/mGPS score (n = 239) was associated with poorer OS independent of clinical stage and performance status (hazard ratio 1.28, 95% confidence interval 1.02-1.61; p = 0.032), higher rate of progression on NAC (7% vs. 7% vs. 19%; p = 0.003), and lower proportion of eventual resection (80% vs. 84% vs. 53%; p = 0.003). CONCLUSIONS: The combined NLR/mGPS score was associated with OS and initial treatment outcomes in patients undergoing NAC prior to surgery for EC, stratifying survival in addition to clinical staging and performance status. The host SIR may be a useful adjunct to multidisciplinary decision making.

Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning.

The skin pigment pattern of zebrafish is a good model system in which to study the mechanism of biological pattern formation. Although it is known that interactions between melanophores and xanthophores play a key role in the formation of adult pigment stripes, molecular mechanisms for these interactions remain largely unknown. Here, we show that Delta/Notch signaling contributes to these interactions. Ablation of xanthophores in yellow stripes induced the death of melanophores in black stripes, suggesting that melanophores require a survival signal from distant xanthophores. We found that deltaC and notch1a were expressed by xanthophores and melanophores, respectively. Moreover, inhibition of Delta/Notch signaling killed melanophores, whereas activation of Delta/Notch signaling ectopically in melanophores rescued the survival of these cells, both in the context of pharmacological inhibition of Delta/Notch signaling and after ablation of xanthophores. Finally, we showed by in vivo imaging of cell membranes that melanophores extend long projections towards xanthophores in the yellow stripes. These data suggest that Delta/Notch signaling is responsible for a survival signal provided by xanthophores to melanophores. As cellular projections can enable long-range interaction between membrane-bound ligands and their receptors, we propose that such projections, combined with direct cell-cell contacts, can substitute for the effect of a diffusible factor that would be expected by the conventional reaction-diffusion (Turing) model.

Construct validity, responsiveness, minimal detectable change, and minimal clinically important difference of the stroke self-efficacy questionnaire in individuals receiving stroke rehabilitation.

PURPOSE: To assess the clinimetric properties of the Stroke Self-Efficacy Questionnaire (SSEQ) and estimate the minimal detectable change (MDC) and minimal clinically important difference (MCID) from the database of our randomized controlled trials (RCT) of individuals receiving stroke rehabilitation. METHODS: We retrieved the pre- and post-intervention scores of the SSEQ and Stroke Impact Scale (SIS) from 80 stroke survivors. The analysis of clinimetric properties was performed using: (1) confirmatory factor analysis and item response theory modeling (IRT) for construct validity; (2) standardized response mean and Glass's delta for responsiveness; (3) MDC based on the standard deviation (SD) or standard error of measurement (SEM) of the SSEQ change scores; (4) MCID determined by the external anchor-SIS; (5) conditional MDC (cMDC) derived from the IRT analysis. RESULTS: There was a bi-factorial construct with excellent model-data fit and marked responsiveness. The MDC determined by the SD and SEM were 1.5 and 3.0, respectively, and the MCIDs were 3.3 and 3.7. CONCLUSIONS: This study confirmed that SSEQ is a valid and reliable assessment tool for patients receiving stroke rehabilitation. We also provided practical threshold values, especially demonstrating the benefit of using individualized cMDC, to help clinicians better interpret the change in the SSEQ scores.

This study indicated that the Stroke Self-Efficacy Questionnaire (SSEQ) is reliable and may involve a bi-factor structure.The SSEQ total scale and the activity domain were highly responsive to change.The self-management domain of the SSEQ was moderately responsive.Using conditional minimal detectable change (cMDC) along with MDC may improve the interpretability of treatment change.

Reliability of the Montreal Cognitive Assessment in people with stroke.

This study examined the relative and absolute reliability of the Taiwanese version of the MoCA (MoCA-T) in people with stroke. The study recruited 114 individuals who were at least 3 months after the onset of a first-ever unilateral stroke. The MoCA-T was administered twice, at a 6-week interval, to all participants. The relative reliability was assessed using the intraclass correlation coefficient (ICC), and the absolute reliability was assessed using standard error of measurement (SEM), the smallest real difference (SRD), the SRD percentage, and the Bland-Altman method. The ICC analysis showed the MoCA-T was highly reliable (ICC = 0.85). The absolute reliability was between an acceptable and excellent level, where the SEM and the SRD at the 95% confidence interval were 1.38 and 3.83, respectively. The Bland-Altman analyses showed no systematic bias between repeated measurements. The range of the 95% limits of agreement was narrow, indicating a high level of stability over time. These findings suggest that the MoCA-T has high agreement between repeated measurements without systematic bias. The threshold to detect real change stands between an acceptable and excellent level. The MoCA-T is a reliable tool for cognitive screening in stroke rehabilitation.

Responsiveness and construct validity of two outcome measures of bilateral upper limb function in patients with chronic stroke.

Background: Stroke is a leading cause of long-term disability among stroke survivors. Despite the availability of numerous stroke rehabilitative therapies, such as mirror therapy, bilateral arm training, and robot-assisted therapy, the recovery of motor function after stroke remains incomplete. Bilateral arm function is a key component in stroke patients to perform activities of daily living and to reflect their functional autonomy. Objective: This clinimetric study investigated and compared the construct validity and responsiveness of 2 bimanual activity outcome measures, the Chedoke Arm and Hand Activity Inventory (CAHAI) and the ABILHAND Questionnaire, in individuals receiving stroke rehabilitation. Methods: The present study is a secondary analysis following the framework of the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN). Individuals with chronic stroke (N = 113) were recruited from outpatient rehabilitation settings. Participants received 18 to 20 sessions of robot-assisted therapy, mirror therapy, combined therapy, or conventional rehabilitation for 4 to 6 weeks. The CAHAI, ABILHAND Questionnaire, and a comparison instrument, the Motor Activity Log (MAL), were administered twice at a 4- to 6-week interval to all participants. ABILHAND scores, in logits, were converted from raw ordinal scores into a linear measure. Results: There was medium to large correlation of the CAHAI and the MAL (ρ = 0.60-0.62, p < 0.01) as well as the ABILHAND Questionnaire and the MAL (ρ = 0.44-0.51, p < 0.01). Change scores from the initial measurement to the post-intervention measurement demonstrated small to medium correlation of the CAHAI and the MAL (ρ = 0.27-0.31, p < 0.01) and medium to large correlation of the ABILHAND Questionnaire and the MAL (ρ = 0.37-0.41, p < 0.01). Overall, 7 of 8 hypotheses were supported. The hypothesis testing regarding the construct validity and responsiveness of the CAHAI and ABILHAND Questionnaire was confirmed. Conclusion: The CAHAI and ABILHAND Questionnaire are both responsive and suitable to detect changes in bilateral arm functional daily activities in individuals with chronic stroke. Patient-reported outcome measures are recommended to use along with therapist-rated outcome measures for upper limb capacity evaluation in stroke rehabilitation. Further study with a prospective study design to capture specific clinical features of participants and the use of body-worn sensors, such as the arm accelerometer, is suggested.

Secular trends of salted fish consumption and nasopharyngeal carcinoma: a multi-jurisdiction ecological study in 8 regions from 3 continents.

BACKGROUND: Despite salted fish being a classical risk factor of Nasopharyngeal Carcinoma (NPC), whether secular trends in salted fish consumption worldwide accounted for changes in NPC rates were unknown. The relationship between vegetable and cigarette consumption to NPC risk worldwide were also largely uncertain. We investigated the longitudinal trends in standardised NPC incidence/mortality rates across 8 regions and their associations with secular trends in salted fish, vegetable and tobacco consumptions. METHODS: Age standardised mortality rate (ASMR) and age standardised incidence rate (ASIR) of NPC were obtained from the WHO cancer mortality database and Hong Kong Cancer Registry. Per capita consumption of salted fish, tobacco and vegetables in Hong Kong and 7 countries (China, Finland, Japan, Portugal, Singapore, United Kingdom and United States) were obtained from the Food and Agriculture Organization of the United Nation (FAO) and Hong Kong Trade and Census Statistics. Pearson correlation and multivariate analysis were performed to examine both crude and adjusted associations. RESULTS: There were markedly decreasing trends of NPC ASIR and ASMR in Hong Kong over the past three decades, which were correlated with corresponding secular changes in salted fish consumption per capita (Pearson r for 10 cumulative years : ASIR = 0.729 (male), 0.674 (female); ASMR = 0.943 (male), 0.622 (female), all p < 0.05 except for female ASMR). However such associations no longer correlated with adjustments for decreasing tobacco and increasing vegetable consumption per capita (Pearson r for 10 cumulative years: ASIR = 2.007 (male), 0.339 (female), ASMR = 0.289 (male), 1.992 (female), all p > 0.05). However, there were no clear or consistent patterns in relations between NPC ASIR and ASMR with salted fish consumption across 7 regions in 3 continents. CONCLUSIONS: Our results do not support the notion that changes in salted fish consumption had played an important role in explaining secular trends of NPC rates in Hong Kong and worldwide. Further studies should explore other lifestyle and genetic factors. However, our findings do support the potentially protective effects of vegetable consumption against NPC.

Objective Measures of Two Musical Interpretations of an Excerpt From Berlioz's "La mort d'Ophélie".

OBJECTIVE/HYPOTHESIS: This study aimed to determine objective production differences relative to two emotional interpretations in performing an excerpt from a classical art song. The null hypothesis was proposed. METHODS: The first author recorded an excerpt from an art song. The excerpt was sung with two contrasting musical interpretations: an "empathetic legato" approach, and a "sarcastic" approach characterized by emphatic attacks. Microphone, airflow, and electroglottography signals were digitized. The vowels were analyzed in terms of intensity, long term average spectra, fundamental frequency (fo), airflow vibrato rate and extent, vowel onset slope, intensity comparison of harmonic frequencies, and glottal measures based on electroglottograph waveforms. Four consonant tokens were analyzed relative to airflow, voice onset time, and production duration. RESULTS & CONCLUSIONS: The emphatic performance had faster vowel onset, increased glottal adduction, increased intensity of harmonics in 2-3 kHz, increased intensity in the fourth and fifth formants, inferred subglottal pressure increase, increased airflow for /f/, and greater aspiration airflow for /p, t/. Vibrato extents for intensity, fo, and airflow were wider in the emphatic approach. Findings revealed larger EGGW25 and peak-to-peak amplitude values of the electroglottography waveform, suggesting greater vocal fold contact area and longer glottal closure for the emphatic approach. Long-term average spectrum analyses of the entire production displayed minor variation across all formant frequencies, suggesting an insignificant change in vocal tract shaping between the two approaches. This single-case objective study emphasizes the reality of physiological, aerodynamic, and acoustic production differences in the interpretive and pedagogical aspects of art song performance.

The experiences of women from culturally and linguistically diverse backgrounds with gestational diabetes mellitus: A mixed methods systematic review.

BACKGROUND: Gestational diabetes mellitus (GDM) is experienced at a higher rate in women from culturally and linguistically diverse (CALD) backgrounds. The aim of this systematic review is to describe the experiences of women with GDM from CALD backgrounds and compare their experiences to women with GDM from non-CALD backgrounds. MATERIALS AND METHODS: MEDLINE, EMBASE, PsycINFO, Scopus, WOS and CINAHL databases were searched for qualitative and quantitative studies which included data on the experiences of CALD background women with GDM during all stages of pregnancy. Quality appraisal utilized checklists for analytical cross-sectional studies and qualitative research. Thematic analysis was performed using nVivo software. RESULTS: Of the 3054 studies identified, 24 studies met the inclusion criteria. Data synthesis produced five key themes: (1) Response to diagnosis, (2) Experiences with self-management, (3) Interactions with the healthcare system, (4) Mental health challenges and (5) Facilitators and barriers to support. Women with GDM from CALD and non-CALD backgrounds similarly expressed mental health challenges, feeling burdened by recommendations, and challenges interacting with healthcare professionals (HCP). The major difference in experience was the cultural relevance of recommendations, especially related to diet recommendations. CONCLUSION: Gestational diabetes mellitus is a burdensome diagnosis for CALD and non-CALD women, with CALD women uniquely experiencing a lack of culturally relevant recommendations for self-management. The similarities and differences in experience call for optimisation of GDM management and support for women with GDM.

Improving women's experiences with gestational diabetes from culturally and linguistically diverse backgrounds in Australia: a qualitative study.

Introduction: Gestational diabetes mellitus (GDM) is the fastest growing type of diabetes in many countries worldwide, including Australia. Although studies have explored the experiences of women with GDM from ethnic minority groups, few have compared their experiences with women from Anglosphere backgrounds. Objective: To investigate the responses to diagnosis, the management of GDM, and the experiences of healthcare services among women with GDM from different culturally and linguistically diverse (CALD) backgrounds. Methods: Participants were recruited via convenience sampling by advertisement posted around antenatal clinics of three hospitals in NSLHD: Royal North Shore, Hornsby, and Manly Hospitals. The interviews were semi-structured, one-on-one, and in-person conducted by a trained female volunteer. The interviews were audio-recorded, transcribed into text. The data was analyzed via an inductive and descriptive coding approach. The codes were then categorized into main themes and sub-themes. Results: 30 women (7 Australian-born, 11 Chinese, 8 Indians, and 4 Koreans) partook the semi-structured interviews and 5 themes were identified: (1) Reaction to diagnosis; (2) Management issues; (3) Roles of friends and family; (4) Information access; and (5) Experience with healthcare services. The lack of culturally tailored dietary information, social support and language barriers were the main factors underpinning the differences in GDM experiences among women from CALD backgrounds versus Australian-born. Conclusion: Healthcare models should provide more emotional support upon diagnosis, culturally tailored guidelines for lifestyle modifications, and involve friends and family in care and management to enhance the experience of GDM for women from CALD backgrounds.

PI3K/mTOR signaling regulates prostatic branching morphogenesis.

Prostatic branching morphogenesis is an intricate event requiring precise temporal and spatial integration of numerous hormonal and growth factor-regulated inputs, yet relatively little is known about the downstream signaling pathways that orchestrate this process. In this study, we use a novel mesenchyme-free embryonic prostate culture system, newly available mTOR inhibitors and a conditional PTEN loss-of-function model to investigate the role of the interconnected PI3K and mTOR signaling pathways in prostatic organogenesis. We demonstrate that PI3K levels and PI3K/mTOR activity are robustly induced by androgen during murine prostatic development and that PI3K/mTOR signaling is necessary for prostatic epithelial bud invasion of surrounding mesenchyme. To elucidate the cellular mechanism by which PI3K/mTOR signaling regulates prostatic branching, we show that PI3K/mTOR inhibition does not significantly alter epithelial proliferation or apoptosis, but rather decreases the efficiency and speed with which the developing prostatic epithelial cells migrate. Using mTOR kinase inhibitors to tease out the independent effects of mTOR signaling downstream of PI3K, we find that simultaneous inhibition of mTORC1 and mTORC2 activity attenuates prostatic branching and is sufficient to phenocopy combined PI3K/mTOR inhibition. Surprisingly, however, mTORC1 inhibition alone has the reverse effect, increasing the number and length of prostatic branches. Finally, simultaneous activation of PI3K and downstream mTORC1/C2 via epithelial PTEN loss-of-function also results in decreased budding reversible by mTORC1 inhibition, suggesting that the effect of mTORC1 on branching is not primarily mediated by negative feedback on PI3K/mTORC2 signaling. Taken together, our data point to an important role for PI3K/mTOR signaling in prostatic epithelial invasion and migration and implicates the balance of PI3K and downstream mTORC1/C2 activity as a critical regulator of prostatic epithelial morphogenesis.

Homologous peptides derived from influenza A, B and C viruses induce variable CD8+ T cell responses with cross-reactive potential.

Objective: Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally, infecting humans and causing widespread morbidity and mortality. Here, we investigate the T cell response towards an immunodominant IAV epitope, NP265-273, and its IBV and ICV homologues, presented by HLA-A*03:01 molecule expressed in ~ 4% of the global population (~ 300 million people). Methods: We assessed the magnitude (tetramer staining) and quality of the CD8+ T cell response (intracellular cytokine staining) towards NP265-IAV and described the T cell receptor (TCR) repertoire used to recognise this immunodominant epitope. We next assessed the immunogenicity of NP265-IAV homologue peptides from IBV and ICV and the ability of CD8+ T cells to cross-react towards these homologous peptides. Furthermore, we determined the structures of NP265-IAV and NP323-IBV peptides in complex with HLA-A*03:01 by X-ray crystallography. Results: Our study provides a detailed characterisation of the CD8+ T cell response towards NP265-IAV and its IBV and ICV homologues. The data revealed a diverse repertoire for NP265-IAV that is associated with superior anti-viral protection. Evidence of cross-reactivity between the three different influenza virus strain-derived epitopes was observed, indicating the discovery of a potential vaccination target that is broad enough to cover all three influenza strains. Conclusion: We show that while there is a potential to cross-protect against distinct influenza virus lineages, the T cell response was stronger against the IAV peptide than IBV or ICV, which is an important consideration when choosing targets for future vaccine design.

Human myostatin negatively regulates human myoblast growth and differentiation.

Myostatin, a member of the transforming growth factor-ß superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.

Defective adult oligodendrocyte and Schwann cell development, pigment pattern, and craniofacial morphology in puma mutant zebrafish having an alpha tubulin mutation.

The processes of myelination remain incompletely understood but are of profound biomedical importance owing to the several dysmyelinating and demyelinating disorders known in humans. Here, we analyze the zebrafish puma mutant, isolated originally for pigment pattern defects limited to the adult stage. We show that puma mutants also have late-arising defects in Schwann cells of the peripheral nervous system, locomotor abnormalities, and sex-biased defects in adult craniofacial morphology. Using methods of positional cloning, we identify a critical genetic interval harboring two alpha tubulin loci, and we identify a chemically induced missense mutation in one of these, tubulin alpha 8-like 3a (tuba8l3a). We demonstrate tuba8l3a expression in the central nervous system (CNS), leading us to search for defects in the development of oligodendrocytes, the myelinating cells of the CNS. We find gross reductions in CNS myelin and oligodendrocyte numbers in adult puma mutants, and these deficits are apparent already during the larval-to-adult transformation. By contrast, analyses of embryos and early larvae reveal a normal complement of oligodendrocytes that nevertheless fail to localize normal amounts of myelin basic protein (mbp) mRNA in cellular processes, and fail to organize these processes as in the wild-type. This study identifies the puma mutant as a valuable model for studying microtubule-dependent events of myelination, as well as strategies for remyelination in the adult.

Optimization of separation of heavy metals by capillary electrophoresis with contactless conductivity detection.

The separation of four toxic metal ions (Cr(3+) , Pb(2+) , Hg(2+) , Ni(2+) ) was achieved by optimizing the composition of the histidine/tartaric acid background electrolyte. An on-column preconcentration technique, viz. field amplified sample injection, was performed to improve the sensitivity. This method afforded an enhancement factor of up to 91,800 fold with the LODs ranging from 0.005 to 2.32 µg/L, which were well below the maximum contaminant levels set by the United States Environmental Protection Agency. The robustness of this method was demonstrated with its application to the analysis of real samples including tap water, drain water, and reservoir water with recoveries between 90 and 120%.

Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol.

The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model.

Local Recurrence After Minimally Invasive Curettage For Primary Giant Cell Tumor of Bone With Perioperative Bisphosphonate Is Comparable to Open Curettage: Retrospective Comparison With 9-Year Follow-Up.

PURPOSE: To compare the long-term oncological outcome of minimally invasive curettage (MIC) with conventional open curettage (OC). METHODS: We studied patients with primary giant cell tumor of bone (GCTB) of extremities who underwent intralesional tumor curettage and cementation and perioperative bisphosphonates from February 2003 to June 2016. All cases were histology-confirmed diagnoses of GCTB. Recurrent GCTB, malignant GCTB, cases in the axial skeleton (pelvis and spine), or cases with bone grafting of the curetted cavity were excluded. The local recurrence-free (LR-free) estimates of the OC and MIC groups were compared. The hazard ratio of a local recurrence was calculated for the various factors of the patients, disease, and treatment. RESULTS: At a mean follow-up of 8.8 years, the overall LR rate was 24.2% (8 out of 33 patients). There was no statistical difference in LR in MIC and OC groups (27.8 % vs 20%; P = .6). The mean time to LR was 33.1 months (8 to 75). The operative time was comparable in both MIC and OC groups. None of the risk factors studied led to a significantly higher hazard of LR. CONCLUSIONS: At a long-term follow-up of 9 years, MIC showed similar LR-free survival to OC. Combining bisphosphonates and MIC with a less invasive approach showed reasonable LR-free survival in long-term follow-up. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Osteonecrosis of the hip: is there a difference in the survivorship of total hip arthroplasty with or without previous vascular iliac bone grafting?

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a debilitating condition. Vascularized iliac bone graft (VIBG) is a joint-preserving surgery to improve blood supply to the avascular portion of the femoral head which may delay secondary osteoarthritis and total hip arthroplasty (THA). However, whether VIBG will affect the subsequent THA survivorship and outcomes are still uncertain. METHODS: Implant survivorship and clinical outcomes were compared between 27 patients who had undergone prior VIBG and 242 patients who had only undergone THA for ONFH. Baseline characteristics and the postoperative Harris Hip Score (HHS) were also recorded and compared between the two groups. Implant survivorship was determined using Kaplan-Meier survival analysis. RESULTS: The overall implant survival for all patients who had a primary diagnosis of ONFH and eventually underwent THA was 92.9%. There was no significant difference in the implant survivorship between the group who directly received THA (survivorship of 93%) and the group which failed VIBG and was subsequently converted to THA (survivorship of 91.9%) (p = 0.71). In addition, higher THA revision rates were associated with smokers and drinkers. CONCLUSIONS: VIBG may be a reasonable option as a "buy-time" procedure for ONFH. Even if conversion to THA is eventually required, patients may be reassured that the overall survivorship and clinical outcomes may not be compromised. Patients are recommended to give up smoking and binge drinking prior to THA to increase implant survival rate.