Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacity

The ability of bone marrow stromal cells (BMSCs) to differentiate into osteoblasts is being exploited in cell-based therapy for repair of bone defects. However, the phenotype of ex vivo cultured BMSCs predicting their bone-forming capacity is not known. Thus we employed DNA microarrays comparing two human bone marrow stromal cell (hBMSC) populations: One is capable of in vivo heterotopic bone formation (hBMSC-TERT(+Bone)), and the other is not (hBMSC-TERT(-Bone)). Compared with hBMSC-TERT(-Bone), the hBMSC-TERT(+Bone) cells had an increased overrepresentation of extracellular matrix genes (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor-binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT(-Bone) cells expressed a larger number of immune-response-related genes (26% versus 8%). In order to test for the predictive value of these markers, we studied the correlation between their expression levels in six different hBMSC-derived clones and the ability to form bone in vivo. We found a significant correlation for decorin, lysyl oxidase-like 4, natriuretic peptide receptor C, and tetranectin. No significant positive correlation was found for canonical osteoblastic markers Runx2, alkaline phosphatase, collagen type I, osteopontin, and bone sialoprotein. Prospective isolation of four additional hBMSC clones based on their expression levels of the molecular markers correlated with their in vivo bone-formation ability. In conclusion, our data suggest an in vitro molecular signature predictive for hBMSCs' in vivo bone-formation ability. Identifying more of these predictive markers would be very useful in the quality control of osteoblastic cells before use in therapy

Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts

Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination

The HUSH complex is a gatekeeper of type I interferon through epigenetic regulation of LINE-1s

The Human Silencing Hub (HUSH) complex is necessary for epigenetic repression of LINE-1 elements. We show that HUSH-depletion in human cell lines and primary fibroblasts leads to induction of interferon-stimulated genes (ISGs) through JAK/STAT signaling. This effect is mainly attributed to MDA5 and RIG-I sensing of double-stranded RNAs (dsRNAs). This coincides with upregulation of primate-conserved LINE-1s, as well as increased expression of full-length hominid-specific LINE-1s that produce bidirectional RNAs, which may form dsRNA. Notably, LTRs nearby ISGs are derepressed likely rendering these genes more responsive to interferon. LINE-1 shRNAs can abrogate the HUSH-dependent response, while overexpression of an engineered LINE-1 construct activates interferon signaling. Finally, we show that the HUSH component, MPP8 is frequently downregulated in diverse cancers and that its depletion leads to DNA damage. These results suggest that LINE-1s may drive physiological or autoinflammatory responses through dsRNA sensing and gene-regulatory roles and are controlled by the HUSH complex

Absence of Inhibin Alpha and Retinoblastoma Protein Leads to Early Sertoli Cell Dysfunction

Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin α knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10–14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice

Assistive Smart Cane (ASCane) for fall detection: first advances

The development of fall detection systems with the capability of real-time monitoring is necessary considering that a large amount of people die and suffer severe consequences from falls. Due to their advantages, daily life accessories can be a solution to embed fall-related systems, and canes are no exception. In this paper, it is presented a cane with fall detection abilities. The ASCane is instrumented with an inertial sensor which data will be tested with three different fixed multi-threshold fall detection algorithms, one dynamic multi-threshold and machine learning methods from the literature. They were tested and modified to account the use of a cane. The best performance resulted in a sensitivity and specificity of 96.90% and 98.98%, respectively.This work is supported by the FCT - Fundação para a Ciência e Tecnologia - with the scholarship reference PD/BD/141515/2018, with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 - Programa Operacional Competitividade e Internacionalização (POCI) - with the reference project POCI-01-0145-FEDER-006941

Alterations of nocturnal activity in rats following subchronic oral administration of the neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline

1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) is neurotoxic when administered to the brain and alters motor behaviour following intraperitoneal administration. We have assessed the long-term effects of oral TaClo administration on nocturnal motor behaviour in rats. Two groups of rats received TaClo orally at a dose of either 0.2 or 0.4 mg/kg twice daily for 7 weeks. The control group was given saline. No change in locomotor activity was observed 4–9 days after the end of the 7-week administration of TaClo. In addition, the spontaneous motor activity was altered dose-dependently 9 months after oral TaClo administration, with an increase in the low-dose TaClo group and a decrease in the high-dose group. Oral administration of TaClo in rats may be useful in investigating the hypothesis that in Parkinson’s disease, an unknown pathogenic factor crossing the intestinal mucosa barrier can induce neurodegenerative processes eventually affecting the entire brain

Assessment of strategies for switching patients from olanzapine to risperidone: A randomized, open-label, rater-blinded study

<p>Abstract</p> <p>Background</p> <p>In clinical practice, physicians often need to change the antipsychotic medications they give to patients because of an inadequate response or the presence of unacceptable or unsafe side effects. However, there is a lack of consensus in the field as to the optimal switching strategy for antipsychotics, especially with regards to the speed at which the dose of the previous antipsychotic should be reduced. This paper assesses the short-term results of strategies for the discontinuation of olanzapine when initiating risperidone.</p> <p>Methods</p> <p>In a 6-week, randomized, open-label, rater-blinded study, patients with schizophrenia or schizoaffective disorder, on a stable drug dose for more than 30 days at entry, who were intolerant of or exhibiting a suboptimal symptom response to more than 30 days of olanzapine treatment, were randomly assigned to the following switch strategies (common risperidone initiation scheme; varying olanzapine discontinuation): (i) abrupt strategy, where olanzapine was discontinued at risperidone initiation; (ii) gradual 1 strategy, where olanzapine was given at 50% entry dose for 1 week after risperidone initiation and then discontinued; or (iii) gradual 2 strategy, where olanzapine was given at 100% entry dose for 1 week, then at 50% in the second week, and then discontinued.</p> <p>Results</p> <p>The study enrolled 123 patients on stable doses of olanzapine. Their mean age was 40.3 years and mean (± standard deviation (SD)) baseline Positive and Negative Syndrome Scale (PANSS) total score of 75.6 ± 11.5. All-cause treatment discontinuation was lowest (12%) in the group with the slowest olanzapine dose reduction (gradual 2) and occurred at half the discontinuation rate in the other two groups (25% in abrupt and 28% in gradual 1). The relative risk of early discontinuation was 0.77 (confidence interval 0.61–0.99) for the slowest dose reduction compared with the other two strategies. After the medication was changed, improvements at endpoint were seen in PANSS total score (-7.3; <it>p </it>< 0.0001) and in PANSS positive (-3.0; <it>p </it>< 0.0001), negative (-0.9; <it>p </it>= 0.171) and anxiety/depression (-1.4; <it>p </it>= 0.0005) subscale scores. Severity of movement disorders and weight changes were minimal.</p> <p>Conclusion</p> <p>When switching patients from olanzapine to risperidone, a gradual reduction in the dose of olanzapine over 2 weeks was associated with higher rates of retention compared with abrupt or less gradual discontinuation. Switching via any strategy was associated with significant improvements in positive and anxiety symptoms and was generally well tolerated.</p> <p>Trial registration</p> <p>ClinicalTrials.gov NCT00378183</p

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation