Stitching and registering highly multiplexed whole-slide images of tissues and tumors using ASHLAR.

MOTIVATION: Stitching microscope images into a mosaic is an essential step in the analysis and visualization of large biological specimens, particularly human and animal tissues. Recent approaches to highly multiplexed imaging generate high-plex data from sequential rounds of lower-plex imaging. These multiplexed imaging methods promise to yield precise molecular single-cell data and information on cellular neighborhoods and tissue architecture. However, attaining mosaic images with single-cell accuracy requires robust image stitching and image registration capabilities that are not met by existing methods. RESULTS: We describe the development and testing of ASHLAR, a Python tool for coordinated stitching and registration of 103 or more individual multiplexed images to generate accurate whole-slide mosaics. ASHLAR reads image formats from most commercial microscopes and slide scanners, and we show that it performs better than existing open-source and commercial software. ASHLAR outputs standard OME-TIFF images that are ready for analysis by other open-source tools and recently developed image analysis pipelines. AVAILABILITY AND IMPLEMENTATION: ASHLAR is written in Python and is available under the MIT license at https://github.com/labsyspharm/ashlar. The newly published data underlying this article are available in Sage Synapse at https://dx.doi.org/10.7303/syn25826362; the availability of other previously published data re-analyzed in this article is described in Supplementary Table S4. An informational website with user guides and test data is available at https://labsyspharm.github.io/ashlar/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Second-Opinion Program for the Care of Youths Prescribed Five or More Psychotropics in Washington State.

This retrospective study describes a second-opinion review program for the care of children in Washington State who received Medicaid coverage and who were prescribed five or more psychotropic medications, primarily by mental health specialists. In total, 136 second-opinion reviews from 2013 and 169 from 2018 were included in this study. Attention-deficit hyperactivity disorder (ADHD), behavioral difficulties, anxiety, and trauma were prevalent among these children, and participants were commonly prescribed ADHD medications, selective serotonin reuptake inhibitors, and second-generation antipsychotics. The incidence of reviews remained stable over the two periods, but psychosocial treatment increased significantly over this time. This study sheds light on the initiation, maintenance, and identification of polypharmacy psychotropic regimens and highlights psychosocial treatment as an intervention that increases best practice care for at-risk patients.

Rare Occurrence of a Poorly Differentiated Neuroendocrine Tumor of the Bladder.

Neuroendocrine tumors rarely occur in the urinary bladder. They can be carcinomatous, subdivided into small cell and large cell pathology. Small cell carcinoma of the bladder is a rarity that may present at an advanced pathologic stage. No treatment regimens have been standardized for local or metastatic disease. Review of the recent literature shows equivalent survival data for localized disease treated with chemoradiotherapy combined with either bladder sparing surgery or radical cystectomy. Patients with significant comorbidities are an additional challenge. We report a case of poorly differentiated neuroendocrine tumor of the bladder, which could not be classified as small or large cell carcinoma, complicated by significant comorbidities. After management with transurethral resection of the tumor, adjuvant chemotherapy, and radiation, the patient is alive and asymptomatic nearly 1 year after initial TURBT with no evidence of disease recurrence.

Robot-assisted Partial Cystectomy for Treatment of Inflammatory Myofibroblastic Tumor of the Bladder.

Inflammatory myofibroblastic tumors rarely occur in the urinary bladder. These masses follow an indolent course, but due to their histologic similarities to more malignant types of bladder masses, they must be differentiated with immunohistochemical staining. Once diagnosed, the mainstay of treatment for these masses is surgical resection. Due to advancements in robotic surgery, new surgical techniques can be employed to treat these masses with fewer perioperative complications. We report a case of inflammatory myofibroblastic tumor of the urinary bladder in a 29-year-old male treated with robot-assisted partial cystectomy.

Implications on clinical management of the small renal mass in patients 80 years of age and older based on a retrospective review of the SEER database.

PURPOSE: Incidental detection of small renal masses has increased in recent years with increased use of various imaging modalities, and a substantial number of diagnoses are made in the elderly population. Minimally invasive surgical procedures have previously been established as options with excellent long-term oncological results, but surveillance strategies have more recently been introduced as alternatives for surgical intervention. This study reviews the outcomes for elderly patients treated with observation or surgery for small renal masses in order to better elucidate optimal management strategies. METHODS: A total of 4647 patients from the SEER database met criteria for inclusion in this study. Cumulative incidences of RCC-specific mortality and non-RCC-related mortality were estimated, and frequency distributions by tumor size and surgical status were calculated. RESULTS: No difference in RCC-related mortality was observed among all treatment groups, including surveillance, for tumors 1-30 mm in size. RCC-related mortality was significantly lower for surgically treated patients for all other tumor size groups. Mortality unrelated to RCC was significantly higher in patients undergoing surveillance compared to those undergoing surgical intervention for tumor sizes 1-30 or 1-40 mm. CONCLUSIONS: A small renal mass in patients of 80+ years of age is best defined as up to 3 cm in size. For these patients, observation appears be a valid, if not preferential strategy. Patients 80+ years of age with renal masses greater than 3 cm still appear to benefit from surgical intervention.

Molecular mechanisms in the pyrolysis of unsaturated chlorinated hydrocarbons: formation of benzene rings. 1. Quantum chemical studies.

Analogues of important aromatic growth mechanisms in hydrocarbon pyrolysis and combustion systems are extended to chlorinated systems. We consider the addition of C2Cl2 to both C4Cl3 and C4Cl5 radicals at the M06-2X/6-311+G(3df,3p)//B3LYP/6-31G(d) level of theory, and we demonstrate that these reaction systems have much in common with those of nonchlorinated species. In particular, we find that these radicals appear to lead preferentially to fulvenes, and not to the observed aromatic products, as is found in nonchlorinated systems. We have therefore also considered nonradical C4/C2 channels by way of Diels-Alder cyclization of C4Cl4/C2Cl2 and C4H2Cl2/C2HCl pairs to describe aromatic formation. While the latter pair readily leads to the formation of partially chlorinated benzenes, the fully chlorinated congeners are sterically prohibited from ring closing directly; this leads to a series of novel rearrangement processes which predict the formation of hexachloro-1,5-diene-3-yne, in addition to hexachlorobenzene, in good agreement with experiment. This suggests, for the first time, that facile nonradical routes to aromatic formation are operative in partially and fully chlorinated pyrolysis and combustion systems.

Molecular mechanisms in the pyrolysis of unsaturated chlorinated hydrocarbons: formation of benzene rings. 2. Experimental and kinetic modeling studies.

The mechanism of formation of benzene rings during the pyrolysis of dichloro- and trichloroethylenes has been investigated by the method of laser powered homogeneous pyrolysis coupled with product analysis by gas chromatography. Additionally, selected (co)pyrolyses between the chlorinated ethylenes, CH2Cl2, C4Cl4, C4Cl6, and C2H2 have been performed to explicitly probe the roles of 2C3 and C4/C2 reaction pairs in aromatic growth. The presence of odd-carbon products in neat C4Cl6 pyrolyses indicates that 2C3 processes are operative in these systems; however, comparison with product yields from C2HCl3 suggests that C4/C2 processes dominate most other systems. This is further evidenced by an absence of C3 and other odd-carbon species in (co)pyrolyses with dichloromethane which should seed C3-based growth. The reactions of perchlorinated C4 species C4Cl5, C4Cl3, and C4Cl4 with C2Cl2 were subsequently explored through extensive kinetic simulations of the possible reaction pathways based on previous kinetic models and the exhaustive quantum chemical investigations of our preceding work. The experimental and theoretical results strongly suggest that, at moderate temperatures, aromatic ring formation from chlorinated ethylenes normally follows a Diels-Alder coupling of C4 and C2 molecular units followed by internal shifts; the one exception is the C4Cl4 + C2Cl2 system, where steric factors lead to the formation of nonaromatic products. There is little evidence for radical-based routes in these systems.

GIMI: the past, the present and the future.

In keeping with the theme of this year's e-Science All Hands Meeting--past, present and future--we consider the motivation for, the current status of, and the future directions for, the technologies developed within the GIMI (Generic Infrastructure for Medical Informatics) project. This analysis provides insights into how some key problems in data federation may be addressed. GIMI was funded by the UK's Technology Strategy Board with the intention of developing a service-oriented framework to facilitate the secure sharing and aggregation of heterogeneous data from disparate sources to support a range of healthcare applications. The project, which was led by the University of Oxford, involved collaboration from the National Cancer Research Institute Informatics Initiative, Loughborough University, University College London, t+ Medical, Siemens Molecular Imaging and IBM UK.

Role of arachidonic acid, lipoxygenase, and mitochondrial depolarization in reperfusion arrhythmias.

We have sought evidence that arachidonic acid (AA) induces mitochondrial depolarization in isolated myocytes by a lipoxygenase (LOX)-dependent mechanism and that such depolarization might contribute to arrhythmogenesis following ischemia-reperfusion injury. A method was developed for measuring mitochondrial depolarization in isolated adult rat myocytes in suspension, using tetramethylrhodamine ethyl ester. The addition of AA to myocytes resulted in mitochondrial depolarization that was inhibited by the LOX inhibitor baicalein, by the reactive oxygen species (ROS) scavenger mercaptoproprionylglycine, and by the anion channel inhibitor diisothiocyanatostilbene-disulfonic acid (DIDS). AA induced mitochondrial uncoupling and mitochondrial ATPase activity in myocytes, but both were insensitive to baicalein. We conclude that the metabolic effect of AA in myocytes puts mitochondria into an energetically compromised state where membrane potential is easily changed by the DIDS-sensitive LOX/ROS-mediated opening of an inner membrane anion channel. In an in vivo anesthetized rat model of coronary artery occlusion, baicalein was found to strongly inhibit arrhythmias induced by ischemia-reperfusion injury. Arrhythmias following ischemia-reperfusion injury have been previously associated with DIDS-sensitive ROS-mediated mitochondrial depolarization, and free fatty acids including AA were previously found to accumulate during such injury. We therefore conclude that arrhythmias following ischemia-reperfusion injury might originate from mitochondrial depolarization mediated by LOX and AA.

Gabapentin responsive audiovestibular paroxysmia.

Trigeminal neuralgia and hemifacial spasm are well-documented vascular compression syndromes involving the 5th and 7th cranial nerves. Drugs that stabilize the irritated nerves and vascular decompression surgery are accepted treatments. By contrast, the diagnosis and treatment of a comparable syndrome involving the 8th cranial nerve is controversial. We describe two patients with brief, spontaneous, recurrent attacks of tinnitus and vertigo that responded to low dose gabapentin and we argue that this clinical presentation represents the prototypical 8th nerve vascular compression syndrome.

Host limits to accurate human growth hormone production in multiple plant systems.

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.

Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity.

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses. Studies show that high Akt activity in breast carcinoma is associated with a poor pathophenotype, as well as hormone and chemotherapy resistance. Additionally, high Akt activity is associated with other features of poor prognosis. Thus, a chemotherapeutic agent directed specifically toward tumors with high Akt activity could prove extremely potent in treating those breast tumors with the most aggressive phenotypes. Several studies have demonstrated that rapamycin, which inhibits mammalian target of rapamycin (mTOR), a downstream target of Akt, sensitizes certain resistant cancer cells to chemotherapeutic agents. This study evaluated the efficacy of mTOR inhibition in the treatment of tamoxifen-resistant breast carcinoma characterized by high Akt activity. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Cotreatment with the mTOR inhibitor rapamycin in vitro, or the ester of rapamycin, CCI-779 (Wyeth) in vivo, inhibited mTOR activity and restored sensitivity to tamoxifen, suggesting that Akt-induced tamoxifen resistance is mediated in part by signaling through the mTOR pathway. Although the mechanism underlying the synergism remains to be understood, the results were associated with rapamycin's ability to block transcriptional activity mediated by estrogen receptor alpha, as assessed by reporter gene assays with estrogen-responsive element luciferase. These data corroborate prior findings indicating that Akt activation induces resistance to tamoxifen in breast cancer cells. Importantly, these data indicate a novel mechanism for tamoxifen resistance and suggest that blockage of the phosphatidylinositol 3'-kinase/Akt signaling pathway by mTOR inhibition effectively restores the susceptibility of these cells to tamoxifen. These data may have implication for future clinical studies of mTOR inhibition in breast carcinoma.

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed.

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.

Conservative vs. invasive treatment of aortocoronary saphenous vein graft aneurysms: Treatment algorithm based upon a large series.

BACKGROUND: The development of a saphenous vein graft aneurysm (SVGA) after coronary artery bypass graft surgery is a rare occurrence. There are approximately 60 cases reported in the literature, the majority being single case reports. There is no consensus on the treatment of SVGA. METHODS: Retrospective analysis of the patients treated with SVGA was performed at our institution. Demographic and co-morbidity data were acquired on the patients. Patients who underwent surgical treatment were compared to those treated conservatively with the primary outcome being survival time from diagnosis of the SVGA. RESULTS: Thirteen patients with 15 SVGA were identified. The average age at the time of the most recent coronary artery bypass grafting (CABG) was similar in the conservative and the surgically treated groups (55 vs. 56.5 years, respectively). The average number of grafts per patient at the most recent CABG was similar (3.83 vs. 4.0, respectively). The average time from CABG to diagnosis was similar in both the groups (12.6 vs. 15 years, respectively). The average survival from diagnosis was similar in both the groups (2.3 vs. 1.5 years, respectively, p>0.05). CONCLUSIONS: Early surgical treatment of SVGA does not provide longer short-term survival compared with conservative management. A treatment algorithm for SVGA based upon patient co-morbidities and aneurysm characteristics is proposed.

A randomized, controlled trial of the use of pulmonary-artery catheters in high-risk surgical patients.

BACKGROUND: Some observational studies suggest that the use of pulmonary-artery catheters to guide therapy is associated with increased mortality. METHODS: We performed a randomized trial comparing goal-directed therapy guided by a pulmonary-artery catheter with standard care without the use of a pulmonary-artery catheter. The subjects were high-risk patients 60 years of age or older, with American Society of Anesthesiologists (ASA) class III or IV risk, who were scheduled for urgent or elective major surgery, followed by a stay in an intensive care unit. Outcomes were adjudicated by observers who were unaware of the treatment-group assignments. The primary outcome was in-hospital mortality from any cause. RESULTS: Of 3803 eligible patients, 1994 (52.4 percent) underwent randomization. The base-line characteristics of the two treatment groups were similar. A total of 77 of 997 patients who underwent surgery without the use of a pulmonary-artery catheter (7.7 percent) died in the hospital, as compared with 78 of 997 patients in whom a pulmonary-artery catheter was used (7.8 percent)--a difference of 0.1 percentage point (95 percent confidence interval, -2.3 to 2.5). There was a higher rate of pulmonary embolism in the catheter group than in the standard-care group (8 events vs. 0 events, P=0.004). The survival rates at 6 months among patients in the standard-care and catheter groups were 88.1 and 87.4 percent, respectively (difference, -0.7 percentage point [95 percent confidence interval, -3.6 to 2.2]; negative survival differences favor standard care); at 12 months, the rates were 83.9 and 83.0 percent, respectively (difference, -0.9 percentage point [95 percent confidence interval, -4.3 to 2.4]). The median hospital stay was 10 days in each group. CONCLUSIONS: We found no benefit to therapy directed by pulmonary-artery catheter over standard care in elderly, high-risk surgical patients requiring intensive care.

Oxygen abstraction by laser pyrolysis of W(CO)6; a mild route to gas-phase carbene chemistry.

SF6-photosensitiZed IR laser pyrolysis of W(CO)6 in the gas phase at moderate temperatures leads to unsaturated W(CO)n (n < 6) species; these prove to be effective abstractors of O atoms from a range of organic substrates, and afford a simple, clean and low-energy route from carbonyl compounds into gas phase carbene chemistry.