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Assessment of intratumoral heterogeneity and tumor-host interaction within the tumor microenvironment is becoming increasingly important for innovative cancer therapy decisions because of the unique information it can generate about the state of the disease. However, its assessment and quantification are limited by ambiguous definitions of the tumor-host interface and by human cognitive capacity in current pathology practice. Advances in machine learning and artificial intelligence have opened the field of digital pathology to novel tissue image analytics and feature extraction for generation of high-capacity computational disease management models. A particular benefit is expected from machine-learning applications that can perform extraction and quantification of subvisual features of both intratumoral heterogeneity and tumor microenvironment aspects. These methods generate information about cancer cell subpopulation heterogeneity, potential tumor-host interactions, and tissue microarchitecture, derived from morphologically resolved content using both explicit and implicit features. Several studies have achieved promising diagnostic, prognostic, and predictive artificial intelligence models that often outperform current clinical and pathology criteria. However, further effort is needed for clinical adoption of such methods through development of standardizable high-capacity workflows and proper validation studies., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Despite the continued high prevalence of faltering growth, height monitoring remains limited in many low- and middle-income countries. Objective: The objective of this study was to test whether providing parents with information on their child's height can improve children's height and developmental outcomes. Design: Villages in Chipata District, Zambia ( n = 127), were randomly assigned with equal probability to 1 of 3 groups: home-based growth monitoring (HBGM), community-based growth monitoring including nutritional supplementation for children with stunted growth (CBGM+NS), and control. Primary study outcomes were individual height-for-age z score (HAZ) and overall child development assessed with the International Fetal and Newborn Growth Consortium for the 21st Century Neurodevelopment Assessment tool. Secondary outcomes were weight-for-age z score (WAZ), protein consumption, breastfeeding, and general dietary diversity. Results: We enrolled a total of 547 children with a median age of 13 mo at baseline. Estimated mean difference (β) in HAZ was 0.127 (95% CI: -0.107, 0.361) for HBGM and -0.152 (95% CI: -0.341, 0.036) for CBGM+NS. HBGM had no impact on child development [β: -0.017 (95% CI: -0.133, 0.098)]; CBGM+NS reduced overall child development scores by -0.118 SD (95% CI: -0.230, -0.006 SD). Both interventions had larger positive effects among children with stunted growth at baseline, with estimated interaction effects of 0.503 (95% CI: 0.160, 0.846) and 0.582 (95% CI: 0.134, 1.030) for CBGM+NS and HBGM, respectively. HBGM increased mean WAZ [β = 0.183 (95% CI: 0.037, 0.328)]. Both interventions improved parental reports of children's protein intake. Conclusions: The results from this trial suggest that growth monitoring has a limited effect on children's height and development, despite improvements in self-reported feeding practices. HBGM had modest positive effects on children with stunted growth. Given its relatively low cost, this intervention may be a cost-effective tool for increasing parental efforts toward reducing children's physical growth deficits. This trial was registered at clinicaltrials.gov as NCT02242539.

We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins, including most GPCRs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The A2A adenosine receptor (AdR) subtype has emerged as an attractive target in the pursuit of improved therapy for Parkinson's disease (PD). This report focuses on characterization of zebrafish a2 AdRs. By mining the zebrafish EST and genomic sequence databases, we identified two zebrafish a2a (adora2a.1 and adora2a.2) genes and one a2b (adora2b) AdR gene. Sequence comparisons indicate that the predicted zebrafish A2 AdR polypeptides share 62-74% amino acid identity to mammalian A2 AdRs. We mapped the adora2a.1 gene to chromosome 8, the adora2a.2 gene to chromosome 21, and the adora2b gene to chromosome 5. Whole mount in situ hybridization analysis indicates zebrafish a2 AdR genes are expressed primarily within the central nervous system (CNS). Zebrafish are known to be sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes selective loss of dopaminergic neurons and PD-like symptoms in humans as well as in animal models. Here we show that caffeine, an A2A AdR antagonist, is neuroprotective against the adverse effects of MPTP in zebrafish embryos. These results suggest that zebrafish AdRs may serve as useful targets for testing novel therapeutic strategies for the treatment of PD.

Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.

Orientation with respect to gravity is essential for the survival of complex organisms. The gravity receptor is one of the phylogenetically oldest sensory systems, and special adaptations that enhance sensitivity to gravity are highly conserved. The fish inner ear contains three large extracellular biomineral particles, otoliths, which have evolved to transduce the force of gravity into neuronal signals. Mammalian ears contain thousands of small particles called otoconia that serve a similar function. Loss or displacement of these structures can be lethal for fish and is responsible for benign paroxysmal positional vertigo (BPPV) in humans. The distinct morphologies of otoconial particles and otoliths suggest divergent developmental mechanisms. Mutations in a novel gene Otopetrin 1 (Otop1), encoding multi-transmembrane domain protein, result in nonsyndromic otoconial agenesis and a severe balance disorder in mice. Here we show that the zebrafish, Danio rerio, contains a highly conserved gene, otop1, that is essential for otolith formation. Morpholino-mediated knockdown of zebrafish Otop1 leads to otolith agenesis without affecting the sensory epithelium or other structures within the inner ear. Despite lack of otoliths in early development, otolith formation partially recovers in some fish after 2 days. However, the otoliths are malformed, misplaced, lack an organic matrix, and often consist of inorganic calcite crystals. These studies demonstrate that Otop1 has an essential and conserved role in the timing of formation and the size and shape of the developing otolith.

Background: Pennsylvania state law requires licensed child care centers (CCCs) to document that each enrolled child is up to date (UTD) for routine immunizations within 60 days of enrollment. This study evaluates the law's impact on immunization coverage among children aged

We have identified and characterized cDNAs encoding a novel zebrafish Na,K-ATPase alpha subunit. The full-length cDNA encodes a 1023-amino-acid-long peptide which shows greatest homology to zebrafish alpha1 polypeptides. Radiation hybrid mapping localized the new gene (atp1a1a.5) to linkage group 1 in close proximity to the previously identified cluster of Na,K-ATPase alpha1 genes. The expression of atp1a1a.5 in zebrafish embryos was analyzed using whole-mount in situ hybridization. From mid-somitogenesis through 48 h post fertilization (hpf), atp1a1a.5 transcripts were detected in the pronephric duct, ear, and mucous cells. This expression pattern continues through 108 hpf, when high levels of expression were also detected in the intestinal bulb.

We have identified and characterized cDNAs encoding a novel zebrafish Na,K-ATPase alpha subunit. The full-length cDNA encodes a 1,023-amino-acid-long peptide which shows greatest homology to zebrafish alpha1 polypeptides. Radiation hybrid mapping localized the new gene (atp1a1a.5) to linkage group 1 in close proximity to the previously identified cluster of Na,K-ATPase alpha1 genes. The expression of atp1a1a.5 in zebrafish embryos was analyzed using whole-mount in situ hybridization. From mid-somitogenesis through 48 h post fertilization (hpf), atp1a1a.5 transcripts were detected in the pronephric duct, ear, and mucous cells. This expression pattern continues through 108 hpf, when high levels of expression were also detected in the intestinal bulb.

We have used in situ hybridization to analyze Na,K-ATPase alpha and beta subunit gene expression during zebrafish embryogenesis. The most striking finding is that each of the 14 Na,K-ATPase genes exhibits a distinct expression profile. All alpha and beta subunit genes are expressed in the nervous system, although the pattern of expression in different regions varies dramatically. In peripheral tissues, three of the five alpha1-like genes are expressed in pronephros and mucous cells, one is expressed in heart, and one is predominant in skeletal muscle. The alpha2 gene is expressed in brain and heart but is most prominent in skeletal muscle, while the two alpha3 genes are restricted in their expression to the nervous system. Of the six beta subunit genes, beta1a is expressed at highest abundance in lens, pronephros, and heart, while beta1b transcripts are abundant in mucous cells. The two beta2-like genes are differentially expressed in the nervous system. One beta3 gene is expressed exclusively in brain while the other is abundantly expressed in skeletal muscle. Based on these expression patterns, we predict that at least 14 alpha/beta subunit pairs are likely to be formed in different tissues.

Psychosocial interventions have been shown to improve quality of life (QOL) for many cancer patients. A pilot study was conducted to assess the feasibility of a psychoeducational intervention for cancer patients receiving chemotherapy. Eight patients receiving chemotherapy for colorectal carcinoma participated. The intervention is based on a modification of Interpersonal Therapy. It consisted of four sessions, administered biweekly, using a manual format, by a psychiatric nurse clinician over the telephone. The participants also completed a set of QOL measures by telephone to assess tolerance of the planned assessment. The patients received assistance with treatment-related side effects, reported improved ability to communicate with their physician, and gained an understanding of the stresses they discussed. The patients felt satisfied with the emotional support and medical information provided. A randomized trial is planned to test the intervention for patients participating in a cooperative trial sponsored by the National Cancer Institute.

A major obstacle to full utilization of the powerful technique of two-dimensional (2-D) gel electrophoresis is the expense and complexity of quantifying the results. Using an analog-to-digital converter already present in the widely available Commodore 64 or Commodore 128 microcomputer, we have developed a 2-D gel densitometer (GELSCAN) which adds only $20.00 to the cost of the Commodore system (currently around $700.00). The system is designed to work with autoradiograms of 2-D gels. Spots of interest are identified visually and then positioned manually over a light source. A pinhole photoelectric sensor mounted in a hand-held, Plexiglas holder, or "mouse," is briefly rubbed over each spot. Maximum density of the spot is determined and its value is converted to counts per minute via an internal calibration curve which corrects for the nonlinear response of film to radiation. Local spot backgrounds can be subtracted and values can be normalized between gels to adjust for variation in amount of radioactivity applied or in exposure time. Reproducibility is excellent and the technique has some practical as well as theoretical advantages over other more complicated approaches to 2-D gel densitometry. In addition, the GELSCAN system can also be used for scanning individual bands in 1-D gels, quantitation of "dot-blot" autoradiograms and other tasks involving transmission densitometry.