Incorporating ideas from computer-supported cooperative work

Many information systems have failed when deployed into complex health-care settings. We believe that one cause of these failures is the difficulty in systematically accounting for the collaborative and exception-filled nature of medical work. In this methodological review paper, we highlight research from the field of computer-supported cooperative work (CSCW) that could help biomedical informaticists recognize and design around the kinds of challenges that lead to unanticipated breakdowns and eventual abandonment of their systems. The field of CSCW studies how people collaborate with each other and the role that technology plays in this collaboration for a wide variety of organizational settings. Thus, biomedical informaticists could benefit from the lessons learned by CSCW researchers. In this paper, we provide a focused review of CSCW methods and ideas-we review aspects of the field that could be applied to improve the design and deployment of medical information systems. To make our discussion concrete, we use electronic medical record systems as an example medical information system, and present three specific principles from CSCW: accounting for incentive structures, understanding workflow, and incorporating awareness.     

Dual-label immunohistochemical study of interleukin-4-and interferon-gamma-expressing cells within the pancreas of the NOD mouse during disease acceleration with cyclophosphamide

Beta cell destruction has been shown to occur when rodent or human islets are exposed in vitro to inflammatory cytokines, such as interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Other cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10), when given to NOD mice, prevent insulin-dependent diabetes mellitus (IDDM). In this study, we have employed immunofluorescence histochemistry to study the expression of IFN-gamma and IL-4 in the pancreas of female NOD mice at various time-points (days 0, 4, 7, 11 and at onset of diabetes) following disease acceleration with cyclophosphamide (Cy). Dual-label confocal and light microscopy were employed to determine the precise cellular sources of the two cytokines. IL-4 immunolabelling was observed in a few immune cells at days 0, 4, and 7 within the pancreatic islets but in larger numbers at day 11 and at onset of diabetes. The cytokine was co-localized predominantly in CD4 cells, while only a small minority of CD8 cells and macrophages also expressed IL-4. At days 0, 4, 7 and 11, weak to moderate immunolabelling for IL-4 was also observed in beta cells. In contrast, immunolabelling for IFN-gamma within the islets was not observed until day 11 and this labelling persisted at onset of diabetes. It was immunolocalized in macrophages and to a lesser extent in CD4 cells. Only a few CD8 cells were immunopositive for IFN-gamma. At day 11, a proportion of beta cells showed weak immunolabelling for IFN-gamma. During the study period, immunolabelling for IFN-gamma was also observed in a proportion of endothelial cells located in the intra-islet and exocrine regions of Cy and diluent-treated mice. From day 11 onwards, both the cytokines were observed in some of the peri-vascular regions. Our results demonstrate that during Cy-induced diabetes, there is increasing expression of both IL-4 and IFN-gamma in specific immune cells within the inflamed islets in the late prediabetic stage and at onset of diabetes. Further studies are required to correlate our protein immunohistochemical findings with in situ cytokine gene expression and to determine whether there is a clear Th1 cytokine protein bias at clinical onset of diabetes and immediately preceding it.     

Skeletal muscle sodium channel gating in mice deficient in myotonic dystrophy protein kinase

Myotonic dystrophy, a progressive autosomal dominant disorder, is associated with an expansion of a CTG repeat tract located in the 3'-untranslated region of a serine/threonine protein kinase, DMPK. DMPK modulates skeletal muscle Na channels in vitro, and thus we hypothesized that mice deficient in DMPK would have altered muscle Na channel gating. We measured macroscopic and single channel Na currents from cell-attached patches of skeletal myocytes from mice heterozygous (DMPK(+/-)) and homozygous (DMPK(-/-)) for DMPK loss. In DMPK(-/-) myocytes, Na current amplitude was reduced because of reduced channel number. Single channel recordings revealed Na channel reopenings, similar to the gating abnormality of human myotonic muscular dystrophy (DM), which resulted in a plateau of Na current. The gating abnormality deteriorated with increasing age. In DMPK(+/-) muscle there was reduced Na current amplitude and increased Na channel reopenings identical to those in DMPK(-/-) muscle. Thus, these mouse models of complete and partial DMPK deficiency reproduce the Na channel abnormality of the human disease, providing direct evidence that DMPK deficiency underlies the Na channel abnormality in DM.     

Correcting signal biases and detecting regulatory elements in STARR-seq data

High-throughput reporter assays such as self-transcribing active regulatory region sequencing (STARR-seq) have made it possible to measure regulatory element activity across the entire human genome at once. The resulting data, however, present substantial analytical challenges. Here, we identify technical biases that explain most of the variance in STARR-seq data. We then develop a statistical model to correct those biases and to improve detection of regulatory elements. This approach substantially improves precision and recall over current methods, improves detection of both activating and repressive regulatory elements, and controls for false discoveries despite strong local correlations in signal.

Gene regulation is of foundational importance to nearly all biological processes, and variation in gene regulatory activity plays a major role in human disease risk (Lee and Young 2013; Parker et al. 2013; Finucane et al. 2015). A major step toward measuring regulatory activity across the human genome has been the development of high-throughput reporter assays such as STARR-seq (Arnold et al. 2013) that allow regulatory element activity to be quantified with high-throughput sequencing rather than with optical detection of a fluorescent or luminescent signal.High-throughput reporter assays create substantial analytical challenges that are distinct from other sequencing-based genomic assays. There is significant local variation in high-throughput reporter assay signal. We show here that, across data from several laboratories, most of that variation can be explained by features of the underlying genomic sequence and experimental procedures rather than by regulatory element activity. For example, nucleotide composition can alter PCR efficiency leading to under- and overrepresentation of some sequences. Meanwhile, highly repetitive sequences often do not align uniquely to the human reference genome, also biasing signal estimates. Additional analytical challenges include that STARR-seq signals can be both positive and negative, reflecting activation and repression, and the boundaries of regulatory elements are typically unknown and must therefore be estimated from the data. Those challenges together impact signal representations, hinder estimation of regulatory element activity, and cause false positives and false negatives when left unaddressed.Taken together, key requirements of statistical methods to analyze STARR-seq data are the ability to identify and estimate the effect of both activating and repressing regulatory elements while also correcting for underlying sequence biases in high-throughput reporter assays. A statistical model was recently introduced that corrects technical biases and detects regulatory elements in STARR-seq, but the model is limited to detecting only activating regulatory elements (Lee et al. 2020). Considering repression is a crucial gene regulation mechanism (Courey and Jia 2001), overlooking repressive elements may limit understanding of gene regulation with STARR-seq. To overcome that challenge, our correcting reads and analysis of differentially active elements (CRADLE) model takes a two-step approach. First, CRADLE uses a generalized linear regression model to estimate and correct major biases that we have identified in STARR-seq data. Next, CRADLE detects regions with statistically significant regulatory activity from the bias-corrected signals while rigorously controlling FDR. In doing so, CRADLE substantially improves the use of STARR-seq by providing a robust estimation of regulatory activity and improved visualization of raw signals.     

Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats

Kidney aging has been recognized as a chronic process of compromised renal function and structural changes in the tubulointerstitium and glomerulus. Cell senescence is associated with alterations in cell structure and function, including expression of cytokines and structural and regulatory components of extracellular matrix proteins. In this investigation, we tested the hypothesis that senescent renal cells may accumulate in vivo with advancing age. We also evaluated the expression of transforming growth factor (TGF)-beta1 and p21WAF1/CIP1 in aging kidneys. Sprague-Dawley rats at the ages of 3, 12, and 24 months were used for this study. Renal tissues were processed for morphometric and senescence analysis. Expression of TGF-beta1 and p21WAF1/CIP1 was evaluated by Northern or Western blot analysis and immunohistochemistry. Substantial tubulointerstitial injury occurred at the age of 12 months, but significant glomerular structure alteration was observed at the age of 24 months. Tubular cells developed senescence, which was detected by beta-galactosidase staining. This staining increased in frequency and intensity with age. Renal cortices showed a significant increase in the mRNA expression for TGF-beta1 and protein level for p21WAF1/CIP1. The enhanced expression of TGF-beta1 and p21WAF1/CIP1 was localized in the tubulointersititial cells. These data suggest that tubular cells undergo senescence and express increased TGF-beta1 and p21WAF1/CIP1 with advancing age. These age-related cellular and molecular alterations may play an important role in the initiation and/or progression of tubulointerstitial fibrosis and glomerulosclerosis in aging.     

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis

The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system (CNS) of tadpoles and adult frogs. This cDNA (xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal (Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine (His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cell-specific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15-16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.     

Intracellular potentials of microperfused human sweat duct cells

Intracellular potentials of cells from isolated segments of microperfused human sweat ducts were measured in order to determine the electrical profiles of these cells under resting, transporting, and inhibited conditions. Even though the cells are relatively small (ca. 6–8 m), continuous recordings of intracellular potentials from the same impalement were stable for up to 2 h. In the resting condition in normal Ringer's solution when the lumen of the duct was collapsed and not perfused, the intracellular potential measured across the basal membrane was 34.6±1.5 mV (n=31; mean±SE). In the same bathing medium, when the duct lumen was also perfused with normal Ringer's solution, the basolateral membrane potential (V _b), the apical membrane potential (V _a) and transepithelial potential (V _t) was –33.8±0.47 mV, –23.7±0.48 mV and –9.6±0.9 mV (n=73), respectively. The average input impedence (R _i) of these cells was 19.6±0.4 M (n=36). The frequency distribution ofV _b was unimodal suggesting that only one functional cell type exists in this tissue. Amiloride (0.1 mM) in the lumen hyperpolarized bothV _a andV _b by –40.5±3.6 mV and –33.2±3.7 mV (n=15), respectively, with a slight but significant increase inR _i (15%), while abolishingV _t. Removing luminal Cl^– depolarizedV _a by +37.0±4.2 mV and hyperpolarizedV _b by –19.0±4.2 mV (n=11). Removing Cl^– from the bath hyperpolarizedV _a by –3.3±2.3 mV and depolarizedV _b by +24.3±2.7 mV (n=15). Ouabain caused an initial fast depolarization (+8 mV) followed by a prolonged slow depolarization ofV _b, and an increase inR _i of about 84%. These results not only provide the first electrical profile of the human sweat duct tissue, but they also show that its cell membrane potentials are unusually low. This unusual property of this epithelium appears to be due to the combination of a significant Na⁺ conductance at the apical membrane and a remarkably high tissue Cl^– conductance.     

Prostate cancer: results of external irradiation.

From 1975 to 1982, 205 patients with local prostate cancer were treated at the radiation oncology department, the University of Kansas Medical Center, Kansas City, Kansas. Patients'' median age was 73 years. All of the patients were staged according to American Urologic staging criteria. Twenty-eight patients had stage A2 cancer, 91 patients had stage B cancer, and 86 patients had stage C cancer. All patients were treated using megavoltage radiation (dosage range: 6000 cGy to 7100 cGy). The follow-up period ranged from a minimum of 8 years to a maximum of 15 years (median: 9.4 years). The clinical local control was 96% for stage A2, 94% for stage B, and 90% for stage C disease. The overall and disease-free survival rates were 71% and 60%, respectively. Fourteen patients developed moderate complications with one patient (0.5%) requiring surgical intervention. The local control and survival rates reported in this study are comparable with surgical results, suggesting that external beam irradiation in prostate cancer is safe and effective.