SpatialSort: a Bayesian model for clustering and cell population annotation of spatial proteomics data.

MOTIVATION: Recent advances in spatial proteomics technologies have enabled the profiling of dozens of proteins in thousands of single cells in situ. This has created the opportunity to move beyond quantifying the composition of cell types in tissue, and instead probe the spatial relationships between cells. However, most current methods for clustering data from these assays only consider the expression values of cells and ignore the spatial context. Furthermore, existing approaches do not account for prior information about the expected cell populations in a sample. RESULTS: To address these shortcomings, we developed SpatialSort, a spatially aware Bayesian clustering approach that allows for the incorporation of prior biological knowledge. Our method is able to account for the affinities of cells of different types to neighbour in space, and by incorporating prior information about expected cell populations, it is able to simultaneously improve clustering accuracy and perform automated annotation of clusters. Using synthetic and real data, we show that by using spatial and prior information SpatialSort improves clustering accuracy. We also demonstrate how SpatialSort can perform label transfer between spatial and nonspatial modalities through the analysis of a real world diffuse large B-cell lymphoma dataset. AVAILABILITY AND IMPLEMENTATION: Source code is available on Github at: https://github.com/Roth-Lab/SpatialSort.

A BMPR2/YY1 Signaling Axis Is Required for Human Cytomegalovirus Latency in Undifferentiated Myeloid Cells.

Human cytomegalovirus (HCMV) presents a major health burden in the immunocompromised and in stem cell transplant medicine. A lack of understanding about the mechanisms of HCMV latency in undifferentiated CD34+ stem cells, and how latency is broken for the virus to enter the lytic phase of its infective cycle, has hampered the development of essential therapeutics. Using a human induced pluripotent stem cell (iPSC) model of HCMV latency and patient-derived myeloid cell progenitors, we demonstrate that bone morphogenetic protein receptor type 2 (BMPR2) is necessary for HCMV latency. In addition, we define a crucial role for the transcription factor Yin Yang 1 (YY1) in HCMV latency; high levels of YY1 are maintained in latently infected cells as a result of BMPR2 signaling through the SMAD4/SMAD6 axis. Activation of SMAD4/6, through BMPR2, inhibits TGFbeta receptor signaling, which leads to the degradation of YY1 via induction of a cellular microRNA (miRNA), hsa-miR-29a. Pharmacological targeting of BMPR2 in progenitor cells results in the degradation of YY1 and an inability to maintain latency and renders cells susceptible to T cell killing. These data argue that BMPR2 plays a role in HCMV latency and is a new potential therapeutic target for maintaining or disrupting HCMV latency in myeloid progenitors. IMPORTANCE Understanding the mechanisms which regulate HCMV latency could allow therapeutic targeting of the latent virus reservoir from where virus reactivation can cause severe disease. We show that the BMPR2/TGFbeta receptor/YY1 signaling axis is crucial to maintain HCMV latency in undifferentiated cells and that pharmacological reduction of BMPR2 in latently infected cells leads to reactivation of the viral lytic transcription program, which renders the infected cell open to immune detection and clearance in infected individuals. Therefore, this work identifies key host-virus interactions which regulate HCMV latent infection. It also demonstrates a potential new therapeutic approach to reduce HCMV reactivation-mediated disease by the treatment of donor stem cells/organs prior to transplantation, which could have a major impact in the transplant disease setting.

Oxalate contents of commonly used Chinese medicinal herbs.

OBJECTIVE: To assess the total and soluble oxalate contents of commonly used Chinese medicinal herbs. METHODS: Twenty-two Chinese medicinal herbs were extracted in both acid and water prior to determination of total and soluble oxalate, respectively. Oxalate was assayed in herbal extracts using a well-established enzymatic procedure. RESULTS: Among the 22 medicinal herbs, there was significant variation in oxalate content; Houttuynia cordata contained the highest amount of soluble oxalate (2146 mg/100 g) and Selaginella doederleinii contained the lowest amount (71 mg/ 100 g). CONCLUSION: The results indicated that different Chinese medicinal herbs, even from the same family, contain significantly different amounts of oxalate. In susceptible individuals, the use of medicinal herbs with the highest oxalate contents could increase risk of kidney stone formation.

Unstable dorsal proximal interphalangeal joint fracture-dislocations treated with extension-block pinning.

Unstable proximal interphalangeal (PIP) joint fracture-dislocations, which can cause significant disability, can be treated with multiple techniques. Extension-block pinning (EBP) allows for early motion and is less technically demanding than alternative surgical treatments. In the study reported here, 12 patients with unstable dorsal PIP fracture-dislocations were treated with closed reduction of the PIP joint followed by percutaneous insertion of a Kirschner wire (K-wire) into the distal aspect of the proximal phalanx. For these patients, extent of articular surface involvement averaged 43% (range, 25%-75%). Active motion was initiated early after surgery, and the K-wire was removed a mean of 25 days after pinning. Radiographic reduction of joint dislocation was achieved and maintained for 11 of the 12 patients at a mean follow-up of 35.5 months. At follow-up, mean visual analog scale (VAS) score was 0.64 (scale, 0-10). Mean score on the Quick Disabilities of the Arm, Shoulder, and Hand (QuickDASH) questionnaire was 5.7, suggesting minimal functional impairment. Mean PIP active motion was 84° (range, 50°-110°). Grip strength was equal between operative and contralateral hands. Patient satisfaction most closely correlated with low VAS and QuickDASH scores. One patient developed a malunion, which was treated with corrective osteotomy. EBP is a simple, safe, and reproducible technique for unstable PIP fracture-dislocations. This technique yields outcomes similar to those reported for more complex surgical procedures.

Identification of New SRF Binding Sites in Genes Modulated by SRF Over-Expression in Mouse Hearts.

BACKGROUND: To identify in vivo new cardiac binding sites of serum response factor (SRF) in genes and to study the response of these genes to mild over-expression of SRF, we employed a cardiac-specific, transgenic mouse model, with mild over-expression of SRF (Mild-O SRF Tg). METHODOLOGY: Microarray experiments were performed on hearts of Mild-O-SRF Tg at 6 months of age. We identified 207 genes that are important for cardiac function that were differentially expressed in vivo. Among them the promoter region of 192 genes had SRF binding motifs, the classic CArG or CArG-like (CArG-L) elements. Fifty-one of the 56 genes with classic SRF binding sites had not been previously reported. These SRF-modulated genes were grouped into 12 categories based on their function. It was observed that genes associated with cardiac energy metabolism shifted toward that of carbohydrate metabolism and away from that of fatty acid metabolism. The expression of genes that are involved in transcription and ion regulation were decreased, but expression of cytoskeletal genes was significantly increased. Using public databases of mouse models of hemodynamic stress (GEO database), we also found that similar altered expression of the SRF-modulated genes occurred in these hearts with cardiac ischemia or aortic constriction as well. CONCLUSION AND SIGNIFICANCE: SRF-modulated genes are actively regulated under various physiological and pathological conditions. We have discovered that a large number of cardiac genes have classic SRF binding sites and were significantly modulated in the Mild-O-SRF Tg mouse hearts. Hence, the mild elevation of SRF protein in the heart that is observed during typical adult aging may have a major impact on many SRF-modulated genes, thereby affecting cardiac structure and performance. The results from our study could help to enhance our understanding of SRF regulation of cellular processes in the aged heart.

Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFalpha and IL-13.

BACKGROUND: During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFalpha, cytokines known to be important in asthma. METHOD: Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma. RESULTS: PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFalpha) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFalpha had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma. CONCLUSION: Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.

Alternative splicing and nonsense-mediated mRNA decay regulate gene expression of serum response factor.

Serum response factor (SRF) is an important transcription factor that regulates a variety of genes in many tissues during development, maturation and aging. The SRF protein also controls the expression of SRF target genes, including the SRF gene itself. However, it is incompletely established how SRF isoforms contribute to the regulation of SRF gene expression. In the present study, we report the identification of three novel SRF isoforms in human tissue. We found that one novel isoform, SRF-triangle up3, contained a premature termination codon (PTC), which was a target of nonsense-mediated mRNA decay (NMD). By contrast, the SRF-triangle up345 isoform protein was able to specifically bind to the serum response element, and to repress the SRF gene promoter activity. Therefore, we propose that SRF isoforms regulate expression of the SRF gene via two different mechanisms. One mechanism is to reduce the abundance of SRF transcripts via coupled alternative splicing and NMD, the other one is to regulate the SRF gene expression via a feedback mechanism in which the SRF isoform proteins bind to the SRF gene promoter region. Analysis of hundreds of SRF cDNA clones derived from human hearts of fetuses, young adults, old and very old individuals revealed that SRF isoform transcripts were increased in the human heart with advancing age. Our data indicate that the SRF isoforms were differentially expressed in the human versus mouse cardiac muscle. Alternative splicing and NMD likely maintain a delicate balance of SRF transcripts and/or proteins among the full-length SRF form and various SRF isoforms that are critical to the regulation of many SRF target genes, including the SRF gene itself.

The effect of IL-13 and IL-13R130Q, a naturally occurring IL-13 polymorphism, on the gene expression of human airway smooth muscle cells.

BACKGROUND: Growing evidence shows that interleukin 13 (IL-13) may play an essential role in the development of airway inflammation and bronchial hyper-responsiveness (BHR), two defining features of asthma. Although the underlying mechanisms remain unknown, a number of reports have shown that IL-13 may exert its deleterious effects in asthma by directly acting on airway resident cells, including epithelial cells and airway smooth muscle cells. In this report, we hypothesize that IL-13 may participate in the pathogenesis of asthma by activating a set of "pro-asthmatic" genes in airway smooth muscle (ASM) cells. METHODS: Microarray technology was used to study the modulation of gene expression of airway smooth muscle by IL-13 and IL-13R130Q. TaqMan Real Time PCR and flow cytometry was used to validate the gene array data. RESULTS: IL-13 and the IL-13 polymorphism IL-13R130Q (Arg130Gln), recently associated with allergic asthma, seem to modulate the same set of genes, which encode many potentially interesting proteins including vascular cellular adhesion molecule (VCAM)-1, IL-13Ralpha2, Tenascin C and Histamine Receptor H1, that may be relevant for the pathogenesis of asthma. CONCLUSIONS: The data supports the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma.

Assessment of recovery following a novel partial nerve lesion in a rat model.

Partial nerve lesions with a varying degree of retained function and often a painful neuroma pose a dilemma for the clinician. Surgical treatment of partial nerve lesion is perilous because of possible damage to intact axons and subsequent loss of retained function. We present a new rat model of a partial nerve lesion, allowing further study to improve treatment for this condition. A partial (50%) lesion of the tibial portion of the rat sciatic nerve was created and compared to standard crush and neurectomy control lesions. The extent of lost function and the progress of postoperative recovery following the three lesions were compared using serial walking track analyses and end-point muscle weight ratios for atrophy as outcome measures. All groups had tibial functional indices (TFI) significantly different from one another after 1 week. TFIs for the crush group returned to normal by 4 weeks, whereas the neurectomy group showed no recovery. The partial lesion group gradually improved, reaching a plateau of 44% by 7 weeks. Gastrocnemius muscle weight ratios for the partial, crush, and neurectomy lesions at 9 weeks were 0.63, 0.87, and 0.32, respectively. There was a strong correlation between the TFI and muscle weight ratios (r(2) = 0.89; P < 0.001) suggesting that these outcome measures are highly predictive of function. In conclusion, the partial lesion showed a gradual but incomplete functional recovery with a complementary degree of muscle atrophy. The model may prove useful in the evaluation of proposed treatments for partial nerve lesions and the associated painful state.

A measure of curve fitting error for noise filtering diffusion tensor MRI data.

A parameter, chi2p, based on the fitting error was introduced as a measure of reliability of DT-MRI data, and its properties were investigated in simulations and human brain data. Its comparison with the classic chi2 revealed its sensitivity to both the goodness of fit and the pixel signal-to-noise-ratio (SNR), unlike the classic chi2, which is sensitive only to the goodness of fit. The new parameter was thus able to separate effectively pixels with coherent signals (having small fitting error and/or high SNR) from those with random signals (having inconsistent fitting and/or low SNR). A practical advantage of chi2p over the classic chi2 was that chi2p is quantified directly from the data of each pixel, without requiring accurate estimation of data-dependent parameters (such as noise variance), which often makes application of the classic chi2 problematic. Analytical approximations of chi2p enabled an objective (data-independent) and automated calculation of a threshold value, used for internal scaling of the chi2p map. Apart from assessing data reliability on a pixel-by-pixel basis, chi2p was used to develop an objective and generic methodology for the exclusion of pixels with unreliable DT information by discarding pixels with chi2p values exceeding the threshold. Pixels corresponding to very low SNR, and poorly fitted cerebrospinal fluid and surrounding brain tissue, had increased chi2p values and were successfully excluded, providing DT anisotropy maps free from artifactual anisotropic appearance.

Expression of bcl-2 protein is associated with shorter survival in nonsmall cell lung carcinoma.

BACKGROUND: The aim of this study was to assess the relationship between the expression of the 27-kilodalton (kD) bcl-2 protein and survival among nonsmall cell lung carcinoma (NSCLC) patients. METHODS: Paired tissue samples of histologically confirmed tumor and uninvolved lung of 91 randomly selected NSCLC patients were used in this study. The expression of 27-kD bcl-2 proteins in uninvolved lung and tumor tissue specimens was analyzed using Western blot analysis. Overall survival was estimated using the Kaplan-Meier method. The relation between patient survival and expression of the 27-kD bcl-2 protein in uninvolved lung, tumor, and in uninvolved lung and/or tumor tissue specimens was assessed using log-rank tests and Cox proportional hazards multiple regression. RESULTS: The 27-kD bcl-2 protein was expressed in 54% of the uninvolved lung tissue specimens, in 53% of the tumor tissue specimens, and in 70% of the uninvolved lung and/or tumor tissue specimens. When NSCLC patient survival was considered with respect to the expression of the 27-kD bcl-2 protein, a statistically significant shorter survival was observed for patients whose tissue samples expressed the 27-kD bcl-2 protein in the uninvolved lung and in the uninvolved lung and/or tumor (log-rank test, P = 0.008 and P = 0.001, respectively). Cox proportional hazards multiple regression models also showed statistically significant associations between shorter survival and positive 27-kD protein expression in the uninvolved lung (hazard ratio of 2.27; P = 0.05) and in the uninvolved lung and/or tumor (hazard ratio of 5.58, P = 0.008) after controlling for tumor size, stage, the type of surgery received, smoking status, age, and cell type. CONCLUSIONS: In the current study, patients with nonsmall cell lung carcinoma with positive expression of the 27-kD protein bcl-2, either in the uninvolved lung or in uninvolved lung and/or tumor, were found to have significantly poorer postresection survival.

Study of the effect of CSF suppression on white matter diffusion anisotropy mapping of healthy human brain.

Healthy human brain diffusion anisotropy maps derived from standard spin echo diffusion tensor imaging (DTI) were compared with those using fluid-attenuated inversion recovery (FLAIR) preparation prior to DTI to null the signal from cerebrospinal fluid (CSF). Consistent comparisons entailed development of DTI postprocessing methods, image masking based on fitting quality, and an objective region-of-interest-based method for assessment of white matter extent. FLAIR DTI achieved an extended delineation of major white-matter tracts (genu, splenium, and body of the corpus callosum) close to large CSF-filled spaces (lateral ventricles), but did not affect representation of tracts remote from CSF (internal and external capsules and coronal radiation). This result, which was detectable qualitatively (visual inspection), was verified quantitatively by analyses of the relative anisotropy (RA) distribution over white matter structures for 11 subjects. FLAIR DTI thus suppresses the CSF signal that otherwise masks underlying anisotropic parenchymal tissue through partial volume averaging.