Maintenance of hematopoietic stem cells by tyrosine-unphosphorylated STAT5 and JAK inhibition.

Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells, a process regulated by extracellular cues including cytokines. Many cytokines signal through the conserved JAK/STAT pathway, in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate haematopoiesis but its function in the HSC compartment remains poorly understood. Here, we show that STAT5-deficient HSCs exhibit an unusual phenotype: reduced multi-lineage repopulation and self-renewal, combined with reduced exit from quiescence and increased differentiation. This was driven not only by loss of canonical pSTAT5 signalling, but also by loss of distinct transcriptional functions mediated by STAT5 lacking canonical tyrosine phosphorylation (uSTAT5). Consistent with this concept, expression of an unphosphorylatable STAT5 mutant constrained wild-type HSC differentiation, promoted their maintenance and upregulated transcriptional programs associated with quiescence and stemness. The JAK1/2 inhibitor, ruxolitinib, which increased the uSTAT5:pSTAT5 ratio, had similar effects on murine HSC function: it constrained HSC differentiation and proliferation, promoted HSC maintenance and upregulated transcriptional programs associated with stemness. Ruxolitinib also enhanced serial replating of normal human HSPCs, CALR-mutant murine HSCs and HSPCs obtained from patients with myelofibrosis. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function and highlight JAK inhibition as a potential strategy for enhancing HSC function during ex vivo culture. Increased levels of uSTAT5 may also contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.

Comparing the Efficiency of Single versus Dual Ultrasonic Devices for Metallic Post Removal in Endodontic Retreatment: A Clinical Study.

Introduction: Ultrasonic vibration for metallic post removal seems to be a unanimous choice between endodontists and general practitioners for providing the best results and having the highest safety. This study compared the time required by ultrasonic vibration for removing metallic post (MP) when 1 or 2 ultrasonics devices are used. Materials and Methods: One hundred and fifteen teeth with MPs from 105 patients, indicated for nonsurgical endodontic retreatment were divided into 2 groups according to the number of ultrasonic devices used (G1-1 device) and (G2-2 devices). In G1, the MP was worn with a transmetal bur, alongside the wear of the cement line (around 2 mm deep). Then, an ultrasonic tip attached to an ultrasonic unit, with a power of 100% was activated at the level of the post, with constant water spray at a level of 1 mm above the axial surface of the tooth. The position of the tip was changed between buccal and lingual surfaces every 10 seconds until the MP was removed. In G2 the same procedures were performed as described in G1, but two ultrasonic tips were activated simultaneously at buccal and lingual surfaces until the MP was removed. The vibration time necessary for removing each MP was recorded using a chronometer. Results: The mean time was 131.10±29.68 seconds (mean±standard error of the mean) for MP removal using one ultrasonic device, and 24.86±6.88 seconds for two devices. The time required for MP removal using two ultrasonic devices was significantly less than when using one ultrasonic device (P<0.001). Conclusion: The technique with 2 ultrasonic devices proved to be more efficient than the one using only 1 ultrasonic device.

Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes.

Acute myeloid leukemia (AML) and myeloid neoplasms develop through acquisition of somatic mutations that confer mutation-specific fitness advantages to hematopoietic stem and progenitor cells. However, our understanding of mutational effects remains limited to the resolution attainable within immunophenotypically and clinically accessible bulk cell populations. To decipher heterogeneous cellular fitness to preleukemic mutational perturbations, we performed single-cell RNA sequencing of eight different mouse models with driver mutations of myeloid malignancies, generating 269,048 single-cell profiles. Our analysis infers mutation-driven perturbations in cell abundance, cellular lineage fate, cellular metabolism, and gene expression at the continuous resolution, pinpointing cell populations with transcriptional alterations associated with differentiation bias. We further develop an 11-gene scoring system (Stem11) on the basis of preleukemic transcriptional signatures that predicts AML patient outcomes. Our results demonstrate that a single-cell-resolution deep characterization of preleukemic biology has the potential to enhance our understanding of AML heterogeneity and inform more effective risk stratification strategies.

Bioceramic Cements: Supporting in Remineralization of Osteolytic Lesions in Endodontic-periodontal Diseases: A Report of Two Cases.

Bioceramic cements used for filling root canals in cases of endo-perio lesion of endodontic origin seem to be promising due to having the potential of promoting faster and more predictable healing of the periapical lesion as they stimulate osteogenesis. An effective treatment plan depends on the precise diagnosis of endo-perio lesions. The origin of an infection, being exclusive to the root canal, from the periodontium, or both, is extremely important for devising the treatment plan. In both cases, no clinical evidence of periodontal disease (bleeding, calculus, etc.) was found; however, primary endodontic lesions with the possibility of drainage through the gingival crevice were present. In addition to the disinfection strategies used during the root canal treatments, the bioceramics Bio C Sealer, Bio C Repair and Bio Root RCS were used to fill in the root canals. Both cases presented an impressive bone gain within 8 months for case 1 and 5 months for case 2. Regarding case 1, in the palatal root canal an apical plug with a bioceramic repair cement was used. Based on the literature studied, it can be concluded that after adequate disinfection of the root canals, using bioceramic cements in filling the root canals shows the potential of supporting capabilities in remineralization of osteolytic lesions in endo-perio diseases.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion.

Adult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil-induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.

Arabidopsis sirtuins and poly(ADP-ribose) polymerases regulate gene expression in the day but do not affect circadian rhythms.

Nicotinamide-adenine dinucleotide (NAD) is involved in redox homeostasis and acts as a substrate for NADases, including poly(ADP-ribose) polymerases (PARPs) that add poly(ADP-ribose) polymers to proteins and DNA, and sirtuins that deacetylate proteins. Nicotinamide, a by-product of NADases increases circadian period in both plants and animals. In mammals, the effect of nicotinamide on circadian period might be mediated by the PARPs and sirtuins because they directly bind to core circadian oscillator genes. We have investigated whether PARPs and sirtuins contribute to the regulation of the circadian oscillator in Arabidopsis. We found no evidence that PARPs and sirtuins regulate the circadian oscillator of Arabidopsis or are involved in the response to nicotinamide. RNA-seq analysis indicated that PARPs regulate the expression of only a few genes, including FLOWERING LOCUS C. However, we found profound effects of reduced sirtuin 1 expression on gene expression during the day but not at night, and an embryo lethal phenotype in knockouts. Our results demonstrate that PARPs and sirtuins are not associated with NAD regulation of the circadian oscillator and that sirtuin 1 is associated with daytime regulation of gene expression.

The stem/progenitor landscape is reshaped in a mouse model of essential thrombocythemia and causes excess megakaryocyte production.

Frameshift mutations in CALR (calreticulin) are associated with essential thrombocythemia (ET), but the stages at and mechanisms by which mutant CALR drives transformation remain incompletely defined. Here, we use single-cell approaches to examine the hematopoietic stem/progenitor cell landscape in a mouse model of mutant CALR-driven ET. We identify a trajectory linking hematopoietic stem cells (HSCs) with megakaryocytes and prospectively identify a previously unknown intermediate population that is overrepresented in the disease state. We also show that mutant CALR drives transformation primarily from the earliest stem cell compartment, with some contribution from megakaryocyte progenitors. Last, relative to wild-type HSCs, mutant CALR HSCs show increases in JAK-STAT signaling, the unfolded protein response, cell cycle, and a previously undescribed up-regulation of cholesterol biosynthesis. Overall, we have identified a novel megakaryocyte-biased cell population that is increased in a mouse model of ET and described transcriptomic changes linking CALR mutations to increased HSC proliferation and megakaryopoiesis.

Procedimento endodôntico regenerativo usando fibrina rica em plaquetas e leucócitos associada à cirurgia apical: relato de caso / Regenerative endodontic procedure using leukocyte- and platelet-rich fibrin associated with apical surgery: case report

ntrodução: A cirurgia parendodôntica é uma alternativa ao tratamento endodôntico convencional, principalmente em casos envolvendo lesões perirradiculares associadas a processos osteolíticos, em que a terapia endodôntica convencional ou seu retratamento não obtiveram sucesso. Novos métodos terapêuticos têm ganhado espaço na clínica odontológica para esse tipo de caso, entre os quais se destaca a fibrina rica em plaquetas e leucócitos, que, ao concentrar e permitir uma liberação mais prolongada dos fatores de crescimento, promove uma modulação do processo reparador. Esse processo é baseado na otimização da regeneração tecidual através da inserção da membrana de fibrina. Objetivo: O objetivo do presente trabalho é apresentar, por meio do relato de um caso clínico, a utilização de fibrina rica em plaquetas e leucócitos em cirurgia parendodôntica, com intuito de reparo ósseo. Resultados: O acompanhamento clínico e tomográfico foi realizado após 190 dias, momento no qual foi constatada ausência de sintomatologia, bem como completo reparo da lesão e restabelecimento do osso cortical vestibular, além de ausência de lesão detectável em tomografia. Conclusão: No caso clínico relatado, houve uma completa regeneração tecidual da área envolvida na lesão (AU).

Introduction: Parendodontic surgery is an alternative to conventional endodontic treatment, especially in cases involving periradicular lesions associated with osteolytic processes, in which conventional endodontic therapy or retreatment was not successful. New therapeutic methods for the treatment of these cases have ground in clinical dentistry . For example, leukocyteand platelet-rich fibrin which has a higher concentration and more prolonged release of growth factors, modulates the repair process. This process is based on the optimization of tissue regeneration through the insertion of fibrin membrane. Objective: The objective of this study is to report a case in which leukocyte- and platelet-rich fibrin associated with parendodontic surgery was used for bone repair. Results: Clinical and tomographic follow-up was performed after 72 days, which revealed absence of any symptoms, complete repair of the lesion, buccal cortical bone regeneration, and absence of detectable lesion on tomography. Conclusion: There was complete tissue regeneration at the lesion site(AU).

The impact of a dental operating microscope on the identification of mesiolingual canals in maxillary first molars.

The aim of this study was to clinically assess the impact of a dental operating microscope (DOM) on the clinician's ability to locate a second canal in the mesiobuccal root (MB2), also known as the mesiolingual canal. An endodontic specialist treated 180 maxillary first molars using the same protocol for all teeth except for the use of the DOM (n = 90 per group). In the teeth treated without the use of a DOM, a mesiolingual canal was located in 26.67% of the cases; when a DOM was used, a mesiolingual canal was located in 77.78% of the cases. Analysis with the Fisher exact test revealed that the magnification provided by a DOM significantly (P < 0.05) improved the identification of MB2 canals in maxillary first molars.

Functional coherence metrics in protein families.

BACKGROUND: Biological sequences, such as proteins, have been provided with annotations that assign functional information. These functional annotations are associations of proteins (or other biological sequences) with descriptors characterizing their biological roles. However, not all proteins are fully (or even at all) annotated. This annotation incompleteness limits our ability to make sound assertions about the functional coherence within sets of proteins. Annotation incompleteness is a problematic issue when measuring semantic functional similarity of biological sequences since they can only capture a limited amount of all the semantic aspects the sequences may encompass. METHODS: Instead of relying uniquely on single (reductive) metrics, this work proposes a comprehensive approach for assessing functional coherence within protein sets. The approach entails using visualization and term enrichment techniques anchored in specific domain knowledge, such as a protein family. For that purpose we evaluate two novel functional coherence metrics, mUI and mGIC that combine aspects of semantic similarity measures and term enrichment. RESULTS: These metrics were used to effectively capture and measure the local similarity cores within protein sets. Hence, these metrics coupled with visualization tools allow an improved grasp on three important functional annotation aspects: completeness, agreement and coherence. CONCLUSIONS: Measuring the functional similarity between proteins based on their annotations is a non trivial task. Several metrics exist but due both to characteristics intrinsic to the nature of graphs and extrinsic natures related to the process of annotation each measure can only capture certain functional annotation aspects of proteins. Hence, when trying to measure the functional coherence of a set of proteins a single metric is too reductive. Therefore, it is valuable to be aware of how each employed similarity metric works and what similarity aspects it can best capture. Here we test the behaviour and resilience of some similarity metrics.

GRYFUN: a web application for GO term annotation visualization and analysis in protein sets.

Functional context for biological sequence is provided in the form of annotations. However, within a group of similar sequences there can be annotation heterogeneity in terms of coverage and specificity. This in turn can introduce issues regarding the interpretation of actual functional similarity and overall functional coherence of such a group. One way to mitigate such issues is through the use of visualization and statistical techniques. Therefore, in order to help interpret this annotation heterogeneity we created a web application that generates Gene Ontology annotation graphs for protein sets and their associated statistics from simple frequencies to enrichment values and Information Content based metrics. The publicly accessible website http://xldb.di.fc.ul.pt/gryfun/ currently accepts lists of UniProt accession numbers in order to create user-defined protein sets for subsequent annotation visualization and statistical assessment. GRYFUN is a freely available web application that allows GO annotation visualization of protein sets and which can be used for annotation coherence and cohesiveness analysis and annotation extension assessments within under-annotated protein sets.

Cancer diagnosis disclosure from Portuguese physicians.

OBJECTIVE: Doctor-patient communication in oncology, particularly concerning diagnostic disclosure, is a crucial factor related to the quality of the doctor-patient relationship and the psychological state of the patient. The aims of our study were to investigate physicians' opinions and practice with respect to disclosure of a cancer diagnosis and to explore potential related factors. METHOD: A self-report questionnaire developed for our study was responded to by 120 physicians from Coimbra University Hospital Centre and its primary healthcare units. RESULTS: Some 91.7% of physician respondents generally disclosed a diagnosis, and 94.2% were of the opinion that the patient knowing the truth about a diagnosis had a positive effect on the doctor-patient relationship. A need for training about communicating with oncology patients was reported by 85.8% of participants. The main factors determining what information to provide to patients were: (1) patient intellectual and cultural level, (2) patient desire to know the truth, and (3) the existence of family. SIGNIFICANCE OF RESULTS: Our results point to a paradigm shift in communication with cancer patients where disclosure of the diagnosis should be made part of general clinical practice. Nevertheless, physicians still experience difficulties in revealing cancer diagnoses to patients and often lack the skills to deal with a patient's emotional responses, which suggests that more attention needs to be focused on communication skills training programs.

Annotation extension through protein family annotation coherence metrics.

Protein functional annotation consists in associating proteins with textual descriptors elucidating their biological roles. The bulk of annotation is done via automated procedures that ultimately rely on annotation transfer. Despite a large number of existing protein annotation procedures the ever growing protein space is never completely annotated. One of the facets of annotation incompleteness derives from annotation uncertainty. Often when protein function cannot be predicted with enough specificity it is instead conservatively annotated with more generic terms. In a scenario of protein families or functionally related (or even dissimilar) sets this leads to a more difficult task of using annotations to compare the extent of functional relatedness among all family or set members. However, we postulate that identifying sub-sets of functionally coherent proteins annotated at a very specific level, can help the annotation extension of other incompletely annotated proteins within the same family or functionally related set. As an example we analyse the status of annotation of a set of CAZy families belonging to the Polysaccharide Lyase class. We show that through the use of visualization methods and semantic similarity based metrics it is possible to identify families and respective annotation terms within them that are suitable for possible annotation extension. Based on our analysis we then propose a semi-automatic methodology leading to the extension of single annotation terms within these partially annotated protein sets or families.

Mining GO annotations for improving annotation consistency.

Despite the structure and objectivity provided by the Gene Ontology (GO), the annotation of proteins is a complex task that is subject to errors and inconsistencies. Electronically inferred annotations in particular are widely considered unreliable. However, given that manual curation of all GO annotations is unfeasible, it is imperative to improve the quality of electronically inferred annotations. In this work, we analyze the full GO molecular function annotation of UniProtKB proteins, and discuss some of the issues that affect their quality, focusing particularly on the lack of annotation consistency. Based on our analysis, we estimate that 64% of the UniProtKB proteins are incompletely annotated, and that inconsistent annotations affect 83% of the protein functions and at least 23% of the proteins. Additionally, we present and evaluate a data mining algorithm, based on the association rule learning methodology, for identifying implicit relationships between molecular function terms. The goal of this algorithm is to assist GO curators in updating GO and correcting and preventing inconsistent annotations. Our algorithm predicted 501 relationships with an estimated precision of 94%, whereas the basic association rule learning methodology predicted 12,352 relationships with a precision below 9%.

Chemical Entity Recognition and Resolution to ChEBI.

Chemical entities are ubiquitous through the biomedical literature and the development of text-mining systems that can efficiently identify those entities are required. Due to the lack of available corpora and data resources, the community has focused its efforts in the development of gene and protein named entity recognition systems, but with the release of ChEBI and the availability of an annotated corpus, this task can be addressed. We developed a machine-learning-based method for chemical entity recognition and a lexical-similarity-based method for chemical entity resolution and compared them with Whatizit, a popular-dictionary-based method. Our methods outperformed the dictionary-based method in all tasks, yielding an improvement in F-measure of 20% for the entity recognition task, 2-5% for the entity-resolution task, and 15% for combined entity recognition and resolution tasks.

Application of gene ontology to gene identification.

Candidate gene identification deals with associating genes to underlying biological phenomena, such as diseases and specific disorders. It has been shown that classes of diseases with similar phenotypes are caused by functionally related genes. Currently, a fair amount of knowledge about the functional characterization can be found across several public databases; however, functional descriptors can be ambiguous, domain specific, and context dependent. In order to cope with these issues, the Gene Ontology (GO) project developed a bio-ontology of broad scope and wide applicability. Thus, the structured and controlled vocabulary of terms provided by the GO project describing the biological roles of gene products can be very helpful in candidate gene identification approaches. The method presented here uses GO annotation data in order to identify the most meaningful functional aspects occurring in a given set of related gene products. The method measures this meaningfulness by calculating an e-value based on the frequency of annotation of each GO term in the set of gene products versus the total frequency of annotation. Then after selecting a GO term related to the underlying biological phenomena being studied, the method uses semantic similarity to rank the given gene products that are annotated to the term. This enables the user to further narrow down the list of gene products and identify those that are more likely of interest.

Metrics for GO based protein semantic similarity: a systematic evaluation.

BACKGROUND: Several semantic similarity measures have been applied to gene products annotated with Gene Ontology terms, providing a basis for their functional comparison. However, it is still unclear which is the best approach to semantic similarity in this context, since there is no conclusive evaluation of the various measures. Another issue, is whether electronic annotations should or not be used in semantic similarity calculations. RESULTS: We conducted a systematic evaluation of GO-based semantic similarity measures using the relationship with sequence similarity as a means to quantify their performance, and assessed the influence of electronic annotations by testing the measures in the presence and absence of these annotations. We verified that the relationship between semantic and sequence similarity is not linear, but can be well approximated by a rescaled Normal cumulative distribution function. Given that the majority of the semantic similarity measures capture an identical behaviour, but differ in resolution, we used the latter as the main criterion of evaluation. CONCLUSIONS: This work has provided a basis for the comparison of several semantic similarity measures, and can aid researchers in choosing the most adequate measure for their work. We have found that the hybrid simGIC was the measure with the best overall performance, followed by Resnik's measure using a best-match average combination approach. We have also found that the average and maximum combination approaches are problematic since both are inherently influenced by the number of terms being combined. We suspect that there may be a direct influence of data circularity in the behaviour of the results including electronic annotations, as a result of functional inference from sequence similarity.