MSCProfiler: a single cell image processing workflow to investigate mesenchymal stem cell heterogeneity.

Single cell cytometry has demonstrated plausible immuno-heterogeneity of mesenchymal stem cells (MSCs) owing to their multivariate stromal origin. To contribute successfully to next-generation stem cell therapeutics, a deeper understanding of their cellular morphology and immunophenotype is important. In this study, the authors describe MSCProfiler, an image analysis pipeline developed using CellProfiler software. This workflow can extract geometrical and texture features such as shape, size, eccentricity and entropy, along with intensity values of the surface markers from multiple single cell images obtained using imaging flow cytometry. This screening pipeline can be used to analyze geometrical and texture features of all types of MSCs across different passages hallmarked by enhanced feature extraction potential from brightfield and fluorescent images of the cells.

Measuring kindergarteners' motivational beliefs about writing: a mixed-methods exploration of alternate assessment formats.

There have been a handful of studies on kindergarteners' motivational beliefs about writing, yet measuring these beliefs in young children continues to pose a set of challenges. The purpose of this exploratory, mixed-methods study was to examine how kindergarteners understand and respond to different assessment formats designed to capture their motivational beliefs about writing. Across two studies, we administered four assessment formats - a 4-point Likert-type scale survey, a binary choice survey, a challenge preference task, and a semi-structured interview - to a sample of 114 kindergarteners engaged in a larger writing intervention study. Our overall goals were to examine the benefits and challenges of using these assessment formats to capture kindergarteners' motivational beliefs and to gain insight on future directions for studying these beliefs in this young age group. Many participants had a difficult time responding to the 4-point Likert-type scale survey, due to challenges with the response format and the way the items were worded. However, more simplified assessment formats, including the binary choice survey and challenge preference task, may not have fully captured the nuances and complexities of participants' motivational beliefs. The semi-structured interview leveraged participants' voices and highlighted details that were overlooked in the other assessment formats. Participants' interview responses were deeply intertwined with their local, everyday experiences and pushed back on common assumptions of what constitutes negatively oriented motivational beliefs about writing. Overall, our results suggest that kindergarteners' motivational beliefs appear to be multifaceted, contextually grounded, and hard to quantify. Additional research is needed to further understand how motivational beliefs are shaped during kindergarten. We argue that motivational beliefs must be studied in context rather than in a vacuum, in order to work toward a fair and meaningful understanding of motivational beliefs about writing that can be applied to school settings.

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development.

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development.

Macula segmentation and fovea localization employing image processing and heuristic based clustering for automated retinal screening.

BACKGROUND AND OBJECTIVE: Macula segmentation and fovea localization is one of the primary tasks in retinal analysis as they are responsible for detailed vision. Existing approaches required segmentation of retinal structures viz. optic disc and blood vessels for this purpose. METHOD: This work avoids knowledge of other retinal structures and attempts data mining techniques to segment macula. Unsupervised clustering algorithm is exploited for this purpose. Selection of initial cluster centres has a great impact on performance of clustering algorithms. A heuristic based clustering in which initial centres are selected based on measures defining statistical distribution of data is incorporated in the proposed methodology. The initial phase of proposed framework includes image cropping, green channel extraction, contrast enhancement and application of mathematical closing. Then, the pre-processed image is subjected to heuristic based clustering yielding a binary map. The binary image is post-processed to eliminate unwanted components. Finally, the component which possessed the minimum intensity is finalized as macula and its centre constitutes the fovea. RESULTS: The proposed approach outperforms existing works by reporting that 100%,of HRF, 100% of DRIVE, 96.92% of DIARETDB0, 97.75% of DIARETDB1, 98.81% of HEI-MED, 90% of STARE and 99.33% of MESSIDOR images satisfy the 1R criterion, a standard adopted for evaluating performance of macula and fovea identification. CONCLUSION: The proposed system thus helps the ophthalmologists in identifying the macula thereby facilitating to identify if any abnormality is present within the macula region.

Genetic portrait of Tamil non-tribal and Irula tribal population using Y chromosome STR markers.

The 17 Y chromosomal short tandem repeat loci included in the AmpFlSTR® Yfiler™ PCR Amplification Kit were used to analyse the genetic diversity of 517 unrelated males representing the non-tribal and Irula tribal population of Tamil Nadu. A total of 392 unique haplotypes were identified among the 400 non-tribal samples whereas 111 were observed among the 117 Irula tribal samples. Rare alleles for the loci DYS458, DYS635 and YGATAH4.1 were also observed in both population. The haplotype diversity for the non-tribal and Irula tribal population were found to be 0.9999, and the gene diversity ranged from 0.2041 (DYS391) to 0.9612 (DYS385). Comparison of the test population with 26 national and global population using principal coordinate analysis (PCoA) and determination of the genetic distance matrix using phylogenetic molecular analysis indicate a clustering of the Tamil Nadu non-tribal and Irula tribal population away from other unrelated population and proximity towards some Indo-European (IE) and Asian population. Data are available in the Y chromosome haplotype reference database (YHRD) under accession number YA004055 for Tamil non-tribal and YA004056 for the Irula tribal group.

Physicians' career satisfaction, quality of care and patients' trust: the role of community uninsurance.

There is evidence that health care providers located in communities with relatively large uninsured populations face financial difficulties because of low service demand and high levels of uncompensated care. Data on 4,920 physicians from the 2000-2001 Community Tracking Study Physician Survey and from 25,637 adults from the 2003 Community Tracking Study Household Survey were used to analyze whether the relative size of the local uninsured population is associated with the level of career satisfaction and the quality of care provided by physicians and to assess whether patient trust is associated with the level of community uninsurance. The results indicate that the proportion of uninsured adults in a given community is negatively related to physicians' career satisfaction and the perceived quality of health care provided. Community uninsurance is also negatively related to patient trust in their doctor and positively related to whether insured patients believed that their doctor was influenced by rules from health insurance companies. Physicians in communities with relatively large uninsured populations may have lower career satisfaction and lower perceptions of the quality of care provided due to financial difficulties. Patients in these communities are also less likely to trust their physician.

Haematological malignancies developing in previously healthy individuals who received haematopoietic growth factors: report from the Research on Adverse Drug Events and Reports (RADAR) project.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) and granulocyte colony-stimulating factor (G-CSF) promote haematopoietic progenitor cell maturation. We reviewed the findings for healthy volunteers/donors who developed haematological malignancies following PEG-rHuMGDF or G-CSF administration. Information was reviewed for three of 538 volunteers who received PEG-rHuMGDF in clinical trials and two of 200 donors who underwent G-CSF mobilised stem cell harvesting procedures for sibling stem cell transplants. Mantle cell, diffuse large B-cell lymphoma and chronic lymphocytic leukaemia were diagnosed 1-5 years after PEG-rHuMGDF exposure among three volunteers. For one patient, thrombocytopenia due to autoantibodies to PEG-rHuMGDF developed shortly after PEG-rHuMGDF administration and persisted until chemotherapy was administered. All three achieved complete remission, although one patient relapsed. Acute myeloid leukaemia was diagnosed 4 and 5 years after G-CSF mobilisation in two donors who underwent peripheral blood stem cell donation for sibling allogeneic haematopoietic stem cell transplantation. Following intensive chemotherapy, one died from acute leukaemia and the second is in complete remission. Controversy exists over the appropriateness of administering haematopoietic growth factors to healthy individuals. While a causal relationship with haematological malignancies cannot be demonstrated, long-term follow-up among healthy individuals who receive haematopoietic growth factors is needed.

Targeting angiogenesis for the treatment of sarcoma.

PURPOSE OF REVIEW: More therapeutic options are needed for bone and soft tissue sarcomas, especially for patients with metastatic disease. Recent randomized clinical trials conducted in colon, breast and lung cancer have shown the anti-vascular endothelial growth factor agent, bevacizumab, alone or in combination with chemotherapy, improves response and survival. Preclinical studies have demonstrated the anti-tumor effects of varied anti-angiogenic agents in sarcoma cell lines and tumor models. RECENT FINDINGS: Preclinical studies in sarcomas have evaluated the role of targeted agents including platelet-derived growth factor, matrix metalloproteinases, urokinase receptor and varied small-molecule tyrosine kinase inhibitors. Novel angiogenesis inhibitors are being studied in the treatment of sarcoma, including monoclonal antibodies against vascular endothelial growth factor, cis- and trans-retinoic acids, thalidomide, and tyrosine kinase inhibitors. Phase I, II and III clinical trials continue to evaluate these agents alone, in combinations together and combined with standard chemotherapy. We review herein the preclinical rationale and clinical trial results of anti-angiogenesis therapy in the treatment of soft tissue and bone sarcoma. SUMMARY: Preclinical mechanistic study and clinical trials are continuing in order to evaluate the therapeutic role and ultimately validate the efficacy of the varied anti-angiogenesis agents in soft tissue and bone sarcoma.

Role of the p38 mitogen-activated protein kinase pathway in cytokine-mediated hematopoietic suppression in myelodysplastic syndromes.

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by IFNs and other cytokines to mediate signals for important cellular functions, including transcriptional regulation and apoptosis. We examined the role of the p38 pathway in the generation of the effects of myelosuppressive cytokines on human hematopoiesis. Pharmacologic inhibition of p38 using BIX-01208 resulted in reversal of IFN-, tumor necrosis factor-alpha (TNF-alpha)-, and transforming growth factor-beta (TGF-beta)-mediated suppression of human erythroid (blast-forming unit-erythroid) and myeloid (granulocyte-macrophage colony-forming unit) colony formation, consistent with a key role for p38 in the generation of myelosuppressive signals by different cytokines. Similarly, the myelosuppressive effects of TNF-alpha and TGF-beta were reversed by small interfering RNAs targeting p38alpha expression, further establishing the requirement of this kinase in the induction of myelosuppressive responses. As TNF overproduction has been implicated in the pathophysiology of bone marrow failure states, we determined whether pharmacologic inhibition of p38 reverses the hematopoietic defects seen in bone marrows from patients with myelodysplastic syndromes (MDS) and the anemia of chronic disease. Addition of pharmacologic inhibitors of p38 on such bone marrows resulted in increased numbers of erythroid and myeloid progenitors. Similarly, inhibition of the activity of the downstream effectors of p38, MAPK activated protein kinase-2, and mitogen and stress activated kinase 1 partially restored the hematopoietic defect seen in these bone marrows. Taken altogether, our data implicate the p38 MAPK in the pathophysiology of myelodysplasias and suggest that p38 pharmacologic inhibitors may have therapeutic applications in the treatment of MDS.

Motexafin gadolinium induces oxidative stress and apoptosis in hematologic malignancies.

Redox mechanisms have been shown to be important in malignant cell survival and are a system that may be modified for the treatment of hematologic malignancies. Motexafin gadolinium (MGd) is a synthetic expanded porphyrin that selectively accumulates in tumor cells and oxidizes various intracellular metabolites, including ascorbate, nicotinamide adenine dinucleotide phosphate, glutathione, and protein thiols, to generate reactive oxygen species in a process known as futile redox cycling. The rationale for its use in hematologic malignancies is that, like naturally occurring porphyrins, it tends to concentrate selectively in cancer cells, and it has a novel mechanism of action of inducing redox stress and triggering apoptosis in a broad range of malignancies. MGd induces apoptosis in B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and highly resistant myeloma cell lines. Furthermore, MGd is additive or synergistic with ionizing radiation, several chemotherapy agents, and rituximab in vitro and in vivo tumor models. Through gene expression profiling, various stress-related genes are upregulated in response to MGd, including genes encoding metallothioneins, heat shock proteins, and heme oxygenase. Preliminary results from clinical trials with MGd in hematopoietic malignancies have shown that it is well tolerated, with minimal hematologic side effects in both; it has single agent activity in very heavily pretreated chronic lymphocytic leukemia /small lymphocytic lymphoma patients, and it has induced prompt complete remissions in combination with 90Yttrium-ibritumomab (Y-90 Zevalin; Biogen Idec Inc., Cambridge, MA) for relapsed non-Hodgkin's lymphoma in the first two cohorts of patients enrolled. Various clinical trials studying MGd as a single agent and in combination with radiation and/or chemotherapy for the treatment of hematologic malignancies are ongoing.

Expression of connective tissue growth factor after glaucoma filtration surgery in a rabbit model.

PURPOSE: Connective tissue growth factor (CTGF) appears to play a significant role in mediating fibrosis in several tissues. To gain further understanding of the role of CTGF in the scar formation that occurs after glaucoma filtering surgery (GFS), experiments were performed in a rabbit model. METHODS: . Three experiments were performed: (1) CTGF and transforming growth factor (TGF)-beta expression were measured quantitatively after GFS, using ELISA. (2) After GFS conjunctival bleb tissues were immunostained for the presence of CTGF and TGF-beta. (3) Exogenous CTGF was injected into mitomycin-C (MMC)-treated filtering blebs and the scaring response compared to TGF-beta and physiological saline-injected blebs. RESULTS: CTGF and TGF-beta were expressed maximally by day 5 after surgery and were both shown to be present in the bleb tissues after GFS. The addition of exogenous CTGF and TGF-beta increased the rate of failure of GFS blebs. CONCLUSIONS: These data support the hypothesis that CTGF plays an important role in scarring and wound contracture after GFS. Inhibition of CTGF synthesis or its action may help prevent bleb failure and improve long-term GFS outcomes.