Expression Level of Kita, Kitb, Kitla, and Kitlb in Zebrafish Gastrointestional Tract

Gastrointestinal (GI) motility is the muscular contractions that move intestinal contents in an anterograde (mouth to anus) direction and is necessary for nutrient absorption and elimination of waste. GI motility is highly coordinated and rhythmic contraction patterns. Interstitial cells of Cajal (ICC), enteric neurons, and smooth muscle cells all regulate GI motility. ICC function as pacemaker cells and determine contraction frequency. ICC growth and development is influenced by Kit, a tyrosine kinase receptor located on the plasma membrane of ICC. TMEM16A is a calcium activated chloride channel which contributes to the slow wave in the GI tract. Constipation, delayed gastric emptying, and bloating have been correlated with deficits of ICC in GI tissues. A functional Kit receptor and stimulation of Kit with Kit ligand is necessary for ICC growth and development. However, little is known about ICC development in adults or in developing GI tissue. The objective for this project is to determine the relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb in the zebrafish model system at several developmental time points. Understanding the temporal and relative expression pattern of these genes is the first step towards a more complete understanding of ICC development and turnover. The zebrafish model system is anatomically similar to the human GI tract and at early time points the zebrafish is transparent. One advantage to this model system is that GI motility may be examined in the intact larvae. RNA was isolated from dissected zebrafish GI tissues and used as template for reverse transcriptase reactions to make eDNA. Relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb was determined at 5 days post fertilization (dpf), 7 dpf, 11 dpf, 28dpf, and in adult gut tissues using eDNA as template for real time PCR. Kita and Kitla were confirmed as a functional receptor/ligand pair which was first identified in melanocyte migration19. The relative expression data suggests that Kitb and Kitlb are also a functional receptor/ligand pair. Temporal expression data shows high expression of Kitb early in development (5dpf). Besides the early high expression of Kitb, gene expression for all genes of interest peak at 11 dpf. TMEM16A (also called ANOI) was identified as a more accurate marker for gastrointestinal stromal tumors (GIST) than Kit24. RNA isolated from dissected zebrafish GI tract was used to make eDNA which became the template for reverse transcriptase (RT)-PCR and real-tin1e PCR (q-PCR). Anti-ANOI antibodies were used to identify TMEM16A in dissected, fixed zebrafish GI tract. RT-PCR showed that TMEM16A, B, and Care expressed in the zebrafish GI tract. Immunohistochemistry identifies a network of cells in the zebrafish GI tract that is similar in morphology and location to ICC stained by Kit antibodies. Relative and temporal expression was determined using samples isolated at 5, 7, 11, 28dpf, and adult time points. Expression of TMEM16B dominates TMEM16A and B at 28dpf and adult time points

Sharing culture in a tech world: Grandparent–grandchild cultural exchanges over video chat.

Grandparents who were separated from their infant grandchildren during COVID-19 sought other ways to connect, including video chat. Video chat supports learning, and its features (e.g., contingent responsiveness) may allow for cultural exchange. However, technological problems may disrupt these exchanges. In a semi-naturalistic, longitudinal study, 47 families submitted up to three video chats and surveys. Families were predominantly White/Caucasian, highly-educated, and lived between 1 and 2700 miles apart. Multilevel models were used to predict the proportion of the sessions devoted to exchanging culture (e.g., holidays, parenting advice) and managing tech problems. Culture exchange did not change as a function of infant age, video chat experience, or when encountering tech problems. Although only marginally statistically significant, culture exchange increased as distance increased. Tech problems changed as a function of tech talk. A qualitative analysis revealed that cultural transmission occurred via a culture of care and sharing of information across video chat, that families adapted their behaviors to the new technology, and that technology disruptions rarely interfered with the flow of information. These findings demonstrate the ability to share culture when physically separated and in the presence of tech disruptions. Further, this study supports previous work on the emerging culture of video chat. Families adapted to being separated, and grandparents and infants successfully communicated through a new modality. Because video chat supports family relationships, equitable access to high-speed internet should be a priority to enable more families to use it

Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.This work was supported by National Science Foundation (NSF) Grant MCB0956027 and National Institutes of Health Grant R03 MH081231-01 from the Molecular Libraries Program (to A. W. N.); University of New Mexico Center for Molecular Discovery Molecular Libraries Probe Production Centers (UNMCMD MLPCN) National Institutes of Health Grants U54MH084690 and R01HL081062 (to L. A. S.); UNM National Center for Research Resources (NCRR) Grant 5P20RR016480 (to L. G. H.); National Institutes of Health Grant R21 CA170375-01 through the NCI (to A. W. N., L. G. H., and J. E. G.); National Institutes of Health Grants NS066429 and AI092130 (to T. B.); and University of Kansas Specialized Chemistry Center (KUSCC) MLPCN National Institutes of Health Grant U54HG005031 (to J. A.)

Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFPtagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors

Quality of care for hypertension in the United States

BACKGROUND: Despite heavy recent emphasis on blood pressure (BP) control, many patients fail to meet widely accepted goals. While access and adherence to therapy certainly play a role, another potential explanation is poor quality of essential care processes (QC). Yet little is known about the relationship between QC and BP control. METHODS: We assessed QC in 12 U.S. communities by reviewing the medical records of a randomly selected group of patients for the two years preceding our study. We included patients with either a diagnosis of hypertension or two visits with BPs of ≥140/90 in their medical records. We used 28 process indicators based on explicit evidence to assess QC. The indicators covered a broad spectrum of care and were developed through a modified Delphi method. We considered patients who received all indicated care to have optimal QC. We defined control of hypertension as BP < 140/90 in the most recent reading. RESULTS: Of 1,953 hypertensive patients, only 57% received optimal care and 42% had controlled hypertension. Patients who had received optimal care were more likely to have their BP under control at the end of the study (45% vs. 35%, p = .0006). Patients were more likely to receive optimal care if they were over age 50 (76% vs. 63%, p < .0001), had diabetes (77% vs. 71%, p = .0038), coronary artery disease (87% vs. 69%, p < .0001), or hyperlipidemia (80% vs. 68%, p < .0001), and did not smoke (73% vs. 66%, p = .0005). CONCLUSIONS: Higher QC for hypertensive patients is associated with better BP control. Younger patients without cardiac risk factors are at greatest risk for poor care. Quality measurement systems like the one presented in this study can guide future quality improvement efforts

Sick and tired: mood, fatigue, and inflammation in cancer.

Cancer patients commonly experience depression and fatigue before, during, and after treatment. Symptoms can be debilitating, and the risks associated with unrecognized or inadequately treated depression are substantial. Inflammation may be important in the genesis of depression and fatigue in cancer patients; potential neurobiological mechanisms of inflammation-related behavioral symptoms are reviewed. Randomized studies of pharmacologic treatments for depression in cancer populations are limited, but available data are generally encouraging. Studies of pharmacologic treatments for cancer-related fatigue have been more numerous but with mixed results. A practical approach to pharmacologic treatment of depression and fatigue in cancer patients involves weighing the potential risks and benefits of specific agents, including potential for adverse or advantageous side effects. Progress in understanding the neurobiological mechanisms underlying inflammation-related behavioral symptoms will provide opportunities for the development of novel and targeted treatments

Sick and tired: mood, fatigue, and inflammation in cancer.

Cancer patients commonly experience depression and fatigue before, during, and after treatment. Symptoms can be debilitating, and the risks associated with unrecognized or inadequately treated depression are substantial. Inflammation may be important in the genesis of depression and fatigue in cancer patients; potential neurobiological mechanisms of inflammation-related behavioral symptoms are reviewed. Randomized studies of pharmacologic treatments for depression in cancer populations are limited, but available data are generally encouraging. Studies of pharmacologic treatments for cancer-related fatigue have been more numerous but with mixed results. A practical approach to pharmacologic treatment of depression and fatigue in cancer patients involves weighing the potential risks and benefits of specific agents, including potential for adverse or advantageous side effects. Progress in understanding the neurobiological mechanisms underlying inflammation-related behavioral symptoms will provide opportunities for the development of novel and targeted treatments