Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Performance comparison of homologous and heterologous Newcastle disease virus in vaccines and antibody tests.

Antigenic differences between commercial Newcastle Disease Virus (NDV) vaccine and circulating field virus reduce vaccine efficacy. Fifty-layer chickens were divided into five groups: three vaccinated chicken groups using killed LaSota (Genotype II/GII), Mega, or VD (Genotype VII/GVII) viral strains, negative, and positive control groups. On day 28, Hemagglutination Inhibition (HI) serology of vaccinated chickens was performed using whole virus antigens of RIVS, LaSota, Mega, and VD strains. Sera were also tested with an alternative antigen, using an ELISA to detect antibody for the cleavage site F protein peptide from GII and GVII NDV strains. Vaccinated and unvaccinated positive control birds underwent infectious challenges using VD and Mega strains. HI testing showed that antibody titers were higher when tested using homologous antigens than heterologous antigens. ELISA performed with alternative antigens did not perform as well as the established HI test using homologous strains. Viral shedding was reduced by vaccination that was homologous to the infectious challenge in comparison with vaccination using the LaSota strain virus. We conclude that superior results are obtained when serological testing, vaccinations, and vaccine challenge experiments all use circulating strains of ND virus. Implementation of this recommendation would likely reduce viral shedding by vaccinated chickens and be more effective in preventing outbreaks of virulent NDV.

Clinical evaluation of a Hepatitis C Virus whole-genome sequencing pipeline for genotyping and resistance testing.

OBJECTIVES: We sought to evaluate clinically a hepatitis C virus (HCV) whole-genome, next-generation sequencing (NGS) pipeline that is agnostic to viral genotype. METHODS: Performance of the NGS pipeline was assessed through comparison of results with Sanger sequencing (SS) of partial HCV genomes. RESULTS: There was 98.7% (376/381) concordance for viral subtype between SS and NGS. The positive and negative per cent agreements for determination of resistance-associated substitutions were 97.8% (95% CI 92.5-99.4%) and 99.9% (95% CI 99.5-100.0%), respectively. The NGS pipeline was also able to detect novel subtypes, mixtures, recombinants, transiently occurring resistance mutations and distinguish re-infection with the same subtype from relapse. DISCUSSION: Particular scenarios where NGS may be used include settings without universal access to pan-genotypic antiviral regimens, those infected with a 'rare' subtype or who have been failed by first-line therapy, and in cases of suspected re-infection.

Liquid repellency enabled antipathogen coatings.

Currently, Coronavirus Disease 2019 (COVID-19)-a respiratory contagion spreading through expiratory droplets-has evolved into a global pandemic, severely impacting the public health. Importantly, the emerging of immune evasion SARS-CoV-2 variants and the limited effect of current antivirals against SARS-CoV-2 in clinical trials suggested that alternative strategies in addition to the conventional vaccines and antivirals are required to successfully control the COVID-19 pandemic. Here, we propose to use liquid-repellent coatings to prevent the spread of the disease in the absence of effective vaccines, antimicrobial agents, or therapeutics, wherein the deposition and penetration of pathogen droplets are prohibited. We use SARS-CoV-2 as a model pathogen and find that SARS-CoV-2 remnants are reduced by seven orders of magnitude on coated surfaces, yielding a repelling efficacy far outperforming the inactivation rate of disinfectants. The SARS-CoV-2 remnant scales exponentially with the liquid/solid adhesion, uncovering the mechanism and effective means for minimizing pathogen attachment. The antipathogen coating that both repels and inactivates pathogens is demonstrated by incorporating the super-liquid-repellent coating with antipathogen additives. Together with its versatility over a wide range of substrates and pathogens, the novel antipathogen coating is of considerable value for infection control in everyday life as well as during pandemics.

Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway.

Choice of direct acting antiviral (DAA) therapy for Hepatitis C Virus (HCV) in the United Kingdom and similar settings usually requires knowledge of the genotype and, in some cases, antiviral resistance (AVR) profile of the infecting virus. To determine these, most laboratories currently use Sanger technology, but next-generation sequencing (NGS) offers potential advantages in throughput and accuracy. However, NGS poses unique technical challenges, which require idiosyncratic development and technical validation approaches. This applies particularly to virology, where sequence diversity is high and the amount of starting genetic material is low, making it difficult to distinguish real data from artifacts. We describe the development and technical validation of a sequence capture-based HCV whole genome sequencing (WGS) assay to determine viral genotype and AVR profile. We use clinical samples of known subtypes and viral loads, and simulated FASTQ datasets to validate the analytical performances of both the wet laboratory and bioinformatic pipeline procedures. We show high concordance of the WGS assay compared to current "gold standard" Sanger assays. Specificity was 92.3 and 96.1% for AVR and genotyping, respectively. Discordances were due to the inability of Sanger assays to assign the correct subtype or accurately call mixed drug-resistant variants. We show high repeatability and reproducibility with >99.8% sequence similarity between sequence runs as well as high precision for variant frequency detection at >98.8% in the 95th percentile. Post-sequencing bioinformatics quality control workflows allow the accurate distinction between mixed infections, cross-contaminants and recombinant viruses at a threshold of >5% for the minority population. The sequence capture-based HCV WGS assay is more accurate than legacy AVR and genotyping assays. The assay has now been implemented in the clinical pathway of England's National Health Service HCV treatment programs, representing the first validated HCV WGS pipeline in clinical service. The data generated will additionally provide granular national-level genomic information for public health policy making and support the WHO HCV elimination strategy.

Granzyme B PET Imaging of Immune Checkpoint Inhibitor Combinations in Colon Cancer Phenotypes.

PURPOSE: Immune checkpoint inhibitor (ICI) monotherapy and combination regimens are being actively pursued as strategies to improve durable response rates in cancer patients. However, the biology surrounding combination therapies is not well understood and may increase the likelihood of immune-mediated adverse events. Accurate stratification of ICI response by non-invasive PET imaging may help ensure safe therapy management across a wide number of cancer phenotypes. PROCEDURES: We have assessed the ability of a fluorine-labelled peptide, [18F]AlF-mNOTA-GZP, targeting granzyme B, to stratify ICI response in two syngeneic models of colon cancer, CT26 and MC38. In vivo tumour uptake of [18F]AlF-mNOTA-GZP following ICI monotherapy, or in combination with PD-1 was characterised and correlated with changes in tumour-associated immune cell populations. RESULTS: [18F]AlF-mNOTA-GZP showed good predictive ability and correlated well with changes in tumour-associated T cells, especially CD8+ T cells; however, overall uptake and response to monotherapy or combination therapies was very different in the CT26 and MC38 tumours, likely due to the immunostimulatory environment imbued by the MSI-high phenotype in MC38 tumours. CONCLUSIONS: [18F]AlF-mNOTA-GZP uptake correlates well with changes in CD8+ T cell populations and is able to stratify tumour response to a range of ICIs administered as monotherapies or in combination. However, tracer uptake can be significantly affected by preexisting phenotypic abnormalities potentially confusing data interpretation.

Hexane exposure and persistent peripheral neuropathy in automotive technicians.

Automotive technicians are commonly exposed to organic and chlorinated solvents, particularly through use of cleaning products. Mainly during the period 1989-2002, n-hexane was a component of some of these products. In other occupational contexts, n-hexane has been shown to be a cause of peripheral neuropathy. The purpose of the present study was to investigate whether previous exposures to low concentrations of n-hexane were a cause of persistent peripheral neuropathy in automotive technicians. Enrolled in the study were 830 San Francisco Bay Area automotive technicians. Each participant underwent a battery of tests to investigate peripheral nervous system impairment. Test results regressed against estimated hexane and total solvent exposures showed only limited evidence of association with solvent exposures. Exposures to both hexane and general solvents were well below their occupational exposure limits. Generally, our results provide reassurance about persistent peripheral neuropathic effects in automotive technicians who previously used hexane-containing automotive cleaning products. This may reflect repair processes, since the exposures occurred some years previous to the study. However, we cannot exclude the possibility that the absence of observed effect in this study may be attributable to low exposures, exposure misclassification and/or the healthy worker survivor effect.

Very early lineage-specific chimerism after reduced intensity stem cell transplantation is highly predictive of clinical outcome for patients with myeloid disease.

BACKGROUND: The importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival. METHODS: 144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. 'Full donor chimerism' was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with 'split' chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells. RESULTS: In patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma. CONCLUSION: The pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.

Analysis of 7,815 cancer exomes reveals associations between mutational processes and somatic driver mutations.

Driver mutations are the genetic variants responsible for oncogenesis, but how specific somatic mutational events arise in cells remains poorly understood. Mutational signatures derive from the frequency of mutated trinucleotides in a given cancer sample, and they provide an avenue for investigating the underlying mutational processes that operate in cancer. Here we analyse somatic mutations from 7,815 cancer exomes from The Cancer Genome Atlas (TCGA) across 26 cancer types. We curate a list of 50 known cancer driver mutations by analysing recurrence in our cohort and annotations of known cancer-associated genes from the Cancer Gene Census, IntOGen database and Cancer Genome Interpreter. We then use these datasets to perform binary univariate logistic regression and establish the statistical relationship between individual driver mutations and known mutational signatures across different cancer types. Our analysis led to the identification of 39 significant associations between driver mutations and mutational signatures (P < 0.004, with a false discovery rate of < 5%). We first validate our methodology by establishing statistical links for known and novel associations between driver mutations and the mutational signature arising from Polymerase Epsilon proofreading deficiency. We then examine associations between driver mutations and mutational signatures for AID/APOBEC enzyme activity and deficient mismatch repair. We also identify negative associations (odds ratio < 1) between mutational signatures and driver mutations, and here we examine the role of aging and cigarette smoke mutagenesis in the generation of driver mutations in IDH1 and KRAS in brain cancers and lung adenocarcinomas respectively. Our study provides statistical foundations for hypothesised links between otherwise independent biological processes and we uncover previously unexplored relationships between driver mutations and mutagenic processes during cancer development. These associations give insights into how cancers acquire advantageous mutations and can provide direction to guide further mechanistic studies into cancer pathogenesis.

Efficacy and tolerability of trastuzumab emtansine in advanced human epidermal growth factor receptor 2-positive breast cancer.

INTRODUCTION: The management of human epidermal growth factor receptor 2 (HER2)-positive breast cancer has changed dramatically with the introduction and widespread use of HER2-targeted therapies. There is, however, relatively limited real-world information about the effectiveness and safety of trastuzumab emtansine (T-DM1) in Hong Kong Chinese patients. We assessed the efficacy and toxicity profiles among local patients with HER2-positive advanced breast cancer who had received T-DM1 therapy in the second-line setting and beyond. METHODS: This retrospective study involved five local centres that provide service for over 80% of the breast cancer population in Hong Kong. The study period was from December 2013 to December 2015. Patients were included if they had recurrent or metastatic histologically confirmed HER2+ breast cancer who had progressed after at least one line of anti-HER2 therapy including trastuzumab. Patients were excluded if they received T-DM1 as first-line treatment for recurrent or metastatic HER2+ breast cancer. Patient charts including biochemical and haematological profiles were reviewed for background information, T-DM1 response, and toxicity data. Adverse events were documented during chemotherapy and 28 days after the last dose of medication. RESULTS: Among 37 patients being included in this study, 28 (75.7%) had two or more lines of anti-HER2 agents and 26 (70.3%) had received two or more lines of palliative chemotherapy. Response assessment revealed that three (8.1%) patients had a complete response, eight (21.6%) a partial response, 11 (29.7%) a stable disease, and 12 (32.4%) a progressive disease; three patients could not be assessed. The median duration of response was 17.3 (95% confidence interval, 8.4-24.8) months. The clinical benefit rate (complete response + partial response + stable disease, ≥12 weeks) was 37.8% (95% confidence interval, 22.2%-53.5%). The median progression-free survival was 6.0 (95% confidence interval, 3.3- 9.8) months and the median overall survival had not been reached by the data cut-off date. Grade 3 or 4 toxicities included thrombocytopaenia (13.5%), raised alanine transaminase (8.1%), anaemia (5.4%), and hypokalaemia (2.7%). No patient died as a result of toxicities. CONCLUSIONS: In patients with HER2-positive advanced breast cancer who have been heavily pretreated with anti-HER2 agents and cytotoxic chemotherapy, T-DM1 is well tolerated and provided a meaningful progression-free survival of 6 months and an overall survival that has not been reached. Further studies to identify appropriate patient subgroups are warranted.

Enhancing Specific Disruption of Intracellular Protein Complexes by Hydrocarbon Stapled Peptides Using Lipid Based Delivery.

Linear peptides can mimic and disrupt protein-protein interactions involved in critical cell signaling pathways. Such peptides however are usually protease sensitive and unable to engage with intracellular targets due to lack of membrane permeability. Peptide stapling has been proposed to circumvent these limitations but recent data has suggested that this method does not universally solve the problem of cell entry and can lead to molecules with off target cell lytic properties. To address these issues a library of stapled peptides was synthesized and screened to identify compounds that bound Mdm2 and activated cellular p53. A lead peptide was identified that activated intracellular p53 with negligible nonspecific cytotoxicity, however it still bound serum avidly and only showed a marginal improvement in cellular potency. These hurdles were overcome by successfully identifying a pyridinium-based cationic lipid formulation, which significantly improved the activity of the stapled peptide in a p53 reporter cell line, principally through increased vesicular escape. These studies underscore that stapled peptides, which are cell permeable and target specific, can be identified with rigorous experimental design and that these properties can be improved through use with lipid based formulations. This work should facilitate the clinical translation of stapled peptides.

Ambient geothermal hydrogen sulfide exposure and peripheral neuropathy.

The mechanism of toxicity of hydrogen sulfide (H2S) gas is thought mainly to operate through effects on the nervous system. The gas has high acute toxicity, but whether chronic exposure causes effects, including peripheral neuropathy, is yet unclear. The city of Rotorua, New Zealand, sits on an active geothermal field and the population has some of the highest measured ambient H2S exposures. A previous study in Rotorua provided evidence that H2S is associated with peripheral neuropathy. Using clinical methods, the present study sought to investigate and possibly confirm this association in the Rotorua population. The study population comprised 1635 adult residents of Rotorua, aged 18-65. Collected data relevant to the peripheral neuropathy investigation included symptoms, ankle stretch reflex, vibration sensitivity, as measured by the timed-tuning fork test and a Bio-Thesiometer (Bio-Medical Instrument Co., Ohio), and light touch sensitivity measured by monofilaments. An exposure metric, estimating time-weighted H2S exposure across the last 30 years was used. Principal components analysis was used to combine data across the various indicators of possible peripheral neuropathy. The main data analysis used linear regression to examine associations between the peripheral nerve function indicators and H2S exposure. None of the peripheral nerve function indicators were associated with H2S exposure, providing no evidence that H2S exposure at levels found in Rotorua is a cause of peripheral neuropathy. The earlier association between H2S exposure and peripheral neuropathy diagnoses may be attributable to the ecological study design used. The possibility that H2S exposure misclassification could account for the lack of association found cannot be entirely excluded.

Beyond Reproduction: Pituitary Hormone Actions on Bone.

The long-held belief that pituitary hormones act solely on master targets was first questioned when we documented G protein-coupled receptors for thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotrophic hormone, oxytocin, and vasopressin on bone cells. These evolutionarily conserved hormones and their receptors are known to have primitive roles, and exist in invertebrate species as far down as coelenterates. It is not surprising therefore that each such hormone has multiple hitherto unrecognized functions in mammalian integrative physiology, and hence, becomes a potential target for therapeutic intervention. Here we discuss the skeletal actions of pituitary hormones.

Practice guideline update summary: Botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity, and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology.

OBJECTIVE: To update the 2008 American Academy of Neurology (AAN) guidelines regarding botulinum neurotoxin for blepharospasm, cervical dystonia (CD), headache, and adult spasticity. METHODS: We searched the literature for relevant articles and classified them using 2004 AAN criteria. RESULTS AND RECOMMENDATIONS: Blepharospasm: OnabotulinumtoxinA (onaBoNT-A) and incobotulinumtoxinA (incoBoNT-A) are probably effective and should be considered (Level B). AbobotulinumtoxinA (aboBoNT-A) is possibly effective and may be considered (Level C). CD: AboBoNT-A and rimabotulinumtoxinB (rimaBoNT-B) are established as effective and should be offered (Level A), and onaBoNT-A and incoBoNT-A are probably effective and should be considered (Level B). Adult spasticity: AboBoNT-A, incoBoNT-A, and onaBoNT-A are established as effective and should be offered (Level A), and rimaBoNT-B is probably effective and should be considered (Level B), for upper limb spasticity. AboBoNT-A and onaBoNT-A are established as effective and should be offered (Level A) for lower-limb spasticity. Headache: OnaBoNT-A is established as effective and should be offered to increase headache-free days (Level A) and is probably effective and should be considered to improve health-related quality of life (Level B) in chronic migraine. OnaBoNT-A is established as ineffective and should not be offered for episodic migraine (Level A) and is probably ineffective for chronic tension-type headaches (Level B).

Cost-effectiveness of 'Program We Care' for patients with chronic obstructive pulmonary disease: A case-control study.

OBJECTIVES: To evaluate the effectiveness of a discharge program for patients with chronic obstructive pulmonary disease (COPD) patients on discharge from an emergency medical ward on discharge home rate, hospital length of stay (LOS), inpatient admission rate and cost. BACKGROUND: Frequent visits to the emergency department (ED) and subsequent hospital admission are common among patients with COPD, which adds a burden to ED and hospital care. A discharge program was implemented in an ED emergency medical ward. The program consisted of multidisciplinary care, discharge planning, discharge health education on disease management, and continued support from the community nursing services. METHODS: A retrospective case-control study was used. Data were retrieved and compared between 478 COPD program cases and 478 COPD non-program cases. RESULTS: No significant difference was found in age, gender, and triage category, LOS in ED, and readmission rate between the program and non-program groups. The program group demonstrated a significantly higher discharge home rate from the ED (33.89% vs. 20.08%) and fewer medical admissions (40.59% vs. 55.02%) compared with the non-program group, resulting in lower total medical costs after the program was implemented. CONCLUSION: The program provides insight on the strategic planning for discharge care in a short stay unit of emergency department.

Detection of Nasopharyngeal Carcinoma by MR Imaging: Diagnostic Accuracy of MRI Compared with Endoscopy and Endoscopic Biopsy Based on Long-Term Follow-Up.

BACKGROUND AND PURPOSE: Our previous nasopharyngeal carcinoma detection study, comparing MR imaging, endoscopy, and endoscopic biopsy, showed that MR imaging is a highly sensitive test that identifies nasopharyngeal carcinomas missed by endoscopy. However, at the close of that study, patients without biopsy-proved nasopharyngeal carcinoma nevertheless had shown suspicious abnormalities on endoscopy and/or MR imaging. The aim of this study was to determine whether there were any patients with undiagnosed nasopharyngeal carcinoma by obtaining long-term follow-up and to use these data to re-evaluate the diagnostic performance of MR imaging. MATERIALS AND METHODS: In the previous study, 246 patients referred to a hospital ear, nose, and throat clinic with suspected nasopharyngeal carcinoma, based on a wide range of clinical indications, had undergone MR imaging, endoscopy, and endoscopic biopsy, and 77 had biopsy-proved nasopharyngeal carcinoma. One hundred twenty-six of 169 patients without biopsy-proved nasopharyngeal carcinoma underwent re-examination of the nasopharynx after a minimum of 3 years, including 17 patients in whom a previous examination (MR imaging = 11; endoscopy = 7) had been positive for nasopharyngeal carcinoma, but the biopsy had been negative for it. Patients with nasopharyngeal carcinoma were identified by biopsy obtained in the previous and this follow-up study; patients without nasopharyngeal carcinoma were identified by the absence of a tumor on re-examination of the nasopharynx. The sensitivity and specificity of the previous investigations were updated and compared by using the Fisher exact test. RESULTS: One patient with a previous positive MR imaging finding was subsequently proved to have nasopharyngeal carcinoma. Nasopharyngeal carcinomas were not found in the remaining 125 patients at follow-up, and the previous positive findings for nasopharyngeal carcinoma on MR imaging and endoscopy were attributed to benign lymphoid hyperplasia. The diagnostic performances for the previous MR imaging, endoscopy, and endoscopic biopsy were 100%, 88%, and 94%, respectively, for sensitivity, and 92%, 94%, and 100%, respectively, for specificity; the differences between MR imaging and endoscopy were significant for sensitivity (P = .003) but not specificity (P = .617). CONCLUSIONS: MR imaging detected the 12% of nasopharyngeal carcinomas that were endoscopically invisible, including 1 cancer that remained endoscopically occult for several years. Lymphoid hyperplasia reduced the specificity of MR imaging.

Migrating intrathecal high-velocity projectile.

Bullet injuries to the spine can cause significant damage to surrounding tissues and cause serious neurological sequelae. These cases are often associated with neurological deficits. We present a case of a gunshot injury to the spine with a migrating intrathecal bullet which subsequently developed neurological deficits. Initially, the patient did not exhibit any neurological symptoms when first assessed soon after the injury. Subsequently, the patient developed signs of neurological injury as a result of spinal intrathecal migration of the projectile.

The oxytocin-bone axis.

We recently demonstrated a direct action of oxytocin (OT) on skeletal homeostasis, mainly mediated through stimulation of osteoblasts (OBs) formation and through the reciprocal modulation of osteoclast (OCs) formation and function. Thus, mice lacking the hormone or its receptor develop a low turnover osteoporosis that worsens with age in both sexes. The skeletons of OT (Ot) and OT receptor (Oxtr) null mice display a pronounced decrease in vertebral and femoral trabecular volume. At the cellular level, OBs from Ot KO and Oxtr KO mice exhibit lower mineralization activity and, at the mRNA level, all master genes for osteoblast differentiation are down-regulated. Moreover, OT has dual effects on OCs: it increases osteoclast formation both directly, by activating nuclear factor kB (NFkB) and mitogen-activated protein kinase (MAPK) signalling and, indirectly, through the up-regulation of receptor activator nuclear factor-kappaB ligand synthesis by OBs. On the other hand, it inhibits bone resorption by triggering cytosolic Ca(2+) release and nitric oxide synthesis in mature OCs. OT is locally produced by osteoblasts acting as paracrine-autocrine regulators of bone formation modulated by oestrogens. The oestrogen signal involved in this feedforward circuit is nongenomic because it requires an intact MAPK kinase signal transduction pathway, instead of the classical nuclear translocation of oestrogen receptor. The ability of oestrogen to increase bone mass in vivo is to some extent OXTR-dependent. Thus, Oxtr KO mice injected 17ß-oestradiol did not show any effects on bone formation parameters, whereas the same treatment increases trabecular and cortical bone in wild-type mice. An intact OT autocrine-paracrine circuit appears to be essential for optimal skeletal remodelling.

Maintenance of mitochondrial morphology by autophagy and its role in high glucose effects on chronological lifespan of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, mitochondrial morphology changes when cells are shifted between nonfermentative and fermentative carbon sources. Here, we show that cells of S. cerevisiae grown in different glucose concentrations display different mitochondrial morphologies. The morphology of mitochondria in the cells growing in 0.5% glucose was similar to that of mitochondria in respiring cells. However, the mitochondria of cells growing in higher glucose concentrations (2% and 4%) became fragmented after growth in these media, due to the production of acetic acid; however, the fragmentation was not due to intracellular acidification. From a screen of mutants involved in sensing and utilizing nutrients, cells lacking TOR1 had reduced mitochondrial fragmentation, and autophagy was found to be essential for this reduction. Mitochondrial fragmentation in cells grown in high glucose was reversible by transferring them into conditioned medium from a culture grown on 0.5% glucose. Similarly, the chronological lifespan of cells grown in high glucose medium was reduced, and this phenotype could be reversed when cells were transferred to low glucose conditioned medium. These data indicate that chronological lifespan seems correlated with mitochondrial morphology of yeast cells and that both phenotypes can be influenced by factors from conditioned medium of cultures grown in low glucose medium.