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1.  Acute two-photon imaging of the neurovascular unit in the cortex of active mice 
In vivo two-photon scanning fluorescence imaging is a powerful technique to observe physiological processes from the millimeter to the micron scale in the intact animal. In neuroscience research, a common approach is to install an acute cranial window and head bar to explore neocortical function under anesthesia before inflammation peaks from the surgery. However, there are few detailed acute protocols for head-restrained and fully awake animal imaging of the neurovascular unit during activity. This is because acutely performed awake experiments are typically untenable when the animal is naïve to the imaging apparatus. Here we detail a method that achieves acute, deep-tissue two-photon imaging of neocortical astrocytes and microvasculature in behaving mice. A week prior to experimentation, implantation of the head bar alone allows mice to train for head-immobilization on an easy-to-learn air-supported ball treadmill. Following just two brief familiarization sessions to the treadmill on separate days, an acute cranial window can subsequently be installed for immediate imaging. We demonstrate how running and whisking data can be captured simultaneously with two-photon fluorescence signals with acceptable movement artifacts during active motion. We also show possible applications of this technique by (1) monitoring dynamic changes to microvascular diameter and red blood cells in response to vibrissa sensory stimulation, (2) examining responses of the cerebral microcirculation to the systemic delivery of pharmacological agents using a tail artery cannula during awake imaging, and (3) measuring Ca2+ signals from synthetic and genetically encoded Ca2+ indicators in astrocytes. This method will facilitate acute two-photon fluorescence imaging in awake, active mice and help link cellular events within the neurovascular unit to behavior.
doi:10.3389/fncel.2015.00011
PMCID: PMC4318346
two-photon; acute; non-anesthetised; fully awake; cranial window; mice; neurovascular unit; astrocyte
2.  A High Performance, Cost-Effective, Open-Source Microscope for Scanning Two-Photon Microscopy that Is Modular and Readily Adaptable 
PLoS ONE  2014;9(10):e110475.
Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems.
doi:10.1371/journal.pone.0110475
PMCID: PMC4204885  PMID: 25333934
3.  Emerging trend in second messenger communication and myoendothelial feedback 
Over the past decade, second messenger communication has emerged as one of the intriguing topics in the field of vasomotor control. Of particular interest has been the idea of second messenger flux from smooth muscle to endothelium initiating a feedback response that attenuates constriction. Mechanistic details of the precise signaling cascade have until recently remained elusive. In this perspective, we introduce readers to how myoendothelial gap junctions could enable sufficient inositol trisphosphate flux to initiate endothelial Ca2+ events that activate Ca2+ sensitive K+ channels. The resulting hyperpolarizing current would in turn spread back through the same myoendothelial gap junctions to moderate smooth muscle depolarization and constriction. In discussing this defined feedback mechanism, this brief manuscript will stress the importance of microdomains and of discrete cellular signaling.
doi:10.3389/fphys.2014.00243
PMCID: PMC4074893  PMID: 25071588
gap junctions; calcium wavelets; constriction; inositol trisphosphate; potassium channels
5.  Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner 
PLoS ONE  2012;7(9):e46483.
Background
Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.
Methodology/Principal Findings
Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.
Conclusions/Significance
Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.
doi:10.1371/journal.pone.0046483
PMCID: PMC3459928  PMID: 23029528
7.  What Do We Need beyond Hemoglobin A1c to Get the Complete Picture of Glycemia in People with Diabetes? 
Hemoglobin A1c (HbA1c) is currently the most commonly used marker for the determination of the glycemic status in people with diabetes and it is frequently used to guide therapy and especially medical treatment of people with diabetes. The measurement of HbA1c has reached a high level of analytical quality and, therefore, this biomarker is currently also suggested to be used for the diagnosis of diabetes. Nevertheless, it is crucial for people with diabetes and their treating physicians to be aware of possible interferences during its measurement as well as physiological or pathological factors that contribute to the HbA1c concentration without being related to glycemia, which are discussed in this review. We performed a comprehensive review of the literature based on PubMed searches on HbA1c in the treatment and diagnosis of diabetes including its most relevant limitations, glycemic variability and self-monitoring of blood glucose (SMBG). Although the high analytical quality of the HbA1c test is widely acknowledged, the clinical relevance of this marker regarding risk reduction of cardiovascular morbidity and mortality is still under debate. In this respect, we argue that glycemic variability as a further risk factor should deserve more attention in the treatment of diabetes.
doi:10.7150/ijms.4520
PMCID: PMC3465850  PMID: 23055818
Hemoglobin A1C; diabetes mellitus; hemoglobinopathies; glycemic control; glycemic variability; diabetes complications.
8.  Early Exclusive Breastfeeding and Maternal Attitudes Towards Infant Feeding in a Population of New Mothers in San Francisco, California 
Breastfeeding Medicine  2010;5(1):9-15.
Abstract
Background
Positive parental attitudes towards infant feeding are an important component in child nutritional health. Previous studies have found that participants in the Special Supplemental Women, Infants, and Children (WIC) Program have lower breastfeeding rates and attitudes that do not contribute towards healthy infant feeding in spite of breastfeeding and nutrition education programs targeting WIC participants. The objective of this study was to assess the frequency of exclusive breastfeeding in the early postpartum period and maternal attitudes towards breastfeeding in a population of mothers at two San Francisco hospitals and in relation to WIC participation status.
Methods
We interviewed women who had recently delivered a healthy newborn using a structured interview.
Results
A high percentage (79.8%) of our sample was exclusively breastfeeding at 1–4 days postpartum. We did not find any significant differences in rates of formula or mixed feeding by WIC participant status. Independent risk factors for mixed or formula feeding at 1–3 days postpartum included Asian/Pacific Islander ethnicity (odds ratio [OR] 2.90, 95% confidence interval [CI] 1.17–7.19). Being a college graduate was associated with a decreased risk of formula/mixed feeding (OR 0.28, 95% CI 0.10–0.79). We also found that thinking breastfeeding was physically painful and uncomfortable was independently associated with not breastfeeding (OR 1.41, 95% CI 1.06–1.89).
Conclusions
Future studies should be conducted with Asian-Americans and Pacific Islanders to better understand the lower rates of exclusive breastfeeding in this population and should address negative attitudes towards breastfeeding such as the idea that breastfeeding is painful or uncomfortable.
doi:10.1089/bfm.2009.0003
PMCID: PMC2936253  PMID: 19772374
9.  A Clinic-Based Lifestyle Intervention for Pediatric Obesity: Efficacy and Behavioral and Biochemical Predictors of Response 
Aim
To examine efficacy and predictors of response to a lifestyle intervention for obese youth.
Methods
Retrospective chart review of 214 children and adolescents aged 8-19 years. Linear regression identified baseline predictors of response (Δ BMI z-score) at first and ultimate follow-up visits.
Results
Mean Δ BMI z-score from baseline was -0.04 (p <0.001) at first follow-up and -0.09 (p <0.001) at ultimate follow-up (median time 10 mo) among 156 children and adolescents. Higher baseline BMI z-score predicted poor response at first and ultimate follow-up, explaining 10% of variance in response. Fasting insulin explained 6% of response variance at first follow-up. Δ BMI z-score at the first visit along with baseline BMI z-score explained up to 50% of variance in response at ultimate visit.
Conclusion
Clinic-based interventions improve weight status. Baseline variables predict only a small proportion of response; response at the first visit is a more meaningful tool to guide clinical decisions.
PMCID: PMC2921937  PMID: 19960890
obesity; insulin; lifestyle counseling; adolescents; weight loss
10.  The Laminin–Nidogen Complex is a Ligand for a Specific Splice Isoform of the Transmembrane Protein Tyrosine Phosphatase LAR  
The Journal of Cell Biology  1998;141(7):1675-1684.
Leukocyte antigen–related protein (LAR) is a prototype for a family of transmembrane protein tyrosine phosphatases whose extracellular domain is composed of three Ig and several fibronectin type III (FnIII) domains. Complex alternative splicing of the LAR-FnIII domains 4–8 has been observed. The extracellular matrix laminin–nidogen complex was identified as a ligand for the LAR-FnIII domain 5 (Fn5) using a series of GST-LAR-FnIII domain fusion proteins and testing them in in vitro ligand-binding assays. LAR– laminin–nidogen binding was regulated by alternative splicing of a small exon within the LAR-Fn5 so that inclusion of this exon sequence resulted in disruption of the laminin–nidogen-binding activity. Long cellular processes were observed when HeLa cells were plated on laminin–nidogen, but not when plated on a fibronectin surface. Indirect immunofluorescent antibody staining revealed high expression of LAR in a punctate pattern, throughout the length of these cellular processes observed on laminin–nidogen. Antibody-induced cross-linking of LAR inhibited formation of these cellular processes, and inhibition was correlated with changes in cellular actin cytoskeletal structure. Thus, LAR–laminin–nidogen binding may play a role in regulating cell signaling induced by laminin–nidogen, resulting in cell morphological changes.
PMCID: PMC2133008  PMID: 9647658

Results 1-10 (10)