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Pubmed Article
Sexual healthcare preferences among gay and bisexual men: a qualitative study in San Francisco, California.
PLoS ONE
PUBLISHED: 01-01-2013
Research on gay and other men who have sex with mens (G/MSM) preferences for sexual healthcare services focuses largely on HIV testing and to some extent on sexually transmitted infections (STI). This research illustrates the frequency and location of where G/MSM interface with the healthcare system, but it does not speak to why men seek care in those locations. As HIV and STI prevention strategies evolve, evidence about G/MSMs motivations and decision-making can inform future plans to optimize models of HIV/STI prevention and primary care.
Authors: Hilary C. Rees, Voichita Ianas, Patricia McCracken, Shannon Smith, Anca Georgescu, Tirdad Zangeneh, Jane Mohler, Stephen A. Klotz.
Published: 07-24-2013
ABSTRACT
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age. HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal. The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
26 Related JoVE Articles!
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Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Authors: David Almond, Timothy Cardozo.
Institutions: School of Medicine, New York University.
The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant β-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.
Infection, Issue 43, HIV-1, structure-activity relationships, ab initio simulations, antibody-mediated neutralization, vaccine design
2118
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Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
Authors: Jia Guo, Clinton Enos, Yuntao Wu.
Institutions: George Mason University.
Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.
Infectious Disease, Issue 43, HIV-1, Rev, GFP, lentiviral vector, RRE
2198
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Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
Authors: Benjamin Dale, Gregory P. McNerney, Deanna L. Thompson, Wolfgang Hübner, Thomas Huser, Benjamin K. Chen.
Institutions: Mount Sinai School of Medicine , University of California, Davis, European Molecular Biology Laboratory.
By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.
Cellular Biology, Issue 44, HIV, GFP, live confocal imaging, virological synapse
2061
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Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Authors: Hugh Alley, Christopher D. Owens, Warren J. Gasper, S. Marlene Grenon.
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone. Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia. The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction. There exists a non-invasive, in vivo method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia. This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
52070
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The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis
Authors: Rishu Gupta, Maya Debbaneh, Daniel Butler, Monica Huynh, Ethan Levin, Argentina Leon, John Koo, Wilson Liao.
Institutions: University of Southern California, University of California, San Francisco , University of California Irvine School of Medicine, University of Arizona College of Medicine, Chicago College of Osteopathic Medicine.
Psoriasis is a chronic, immune-mediated inflammatory skin disease affecting approximately 2-3% of the population. The Goeckerman regimen consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). Goeckerman therapy is extremely effective and relatively safe for the treatment of psoriasis and for improving a patient's quality of life. In the following article, we present our protocol for the Goeckerman therapy that is utilized specifically at the University of California, San Francisco. This protocol details the preparation of supplies, administration of phototherapy and application of topical tar. This protocol also describes how to assess the patient daily, monitor for adverse effects (including pruritus and burning), and adjust the treatment based on the patient's response. Though it is one of the oldest therapies available for psoriasis, there is an absence of any published videos demonstrating the process in detail. The video is beneficial for healthcare providers who want to administer the therapy, for trainees who want to learn more about the process, and for prospective patients who want to undergo treatment for their cutaneous disease.
Medicine, Issue 77, Infection, Biomedical Engineering, Anatomy, Physiology, Immunology, Dermatology, Skin, Dermis, Epidermis, Skin Diseases, Skin Diseases, Eczematous, Goeckerman, Crude Coal Tar, phototherapy, psoriasis, Eczema, Goeckerman regimen, clinical techniques
50509
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Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging
Authors: Steven W. Hetts, Maythem Saeed, Alastair Martin, Prasheel Lillaney, Aaron Losey, Erin Jeannie Yee, Ryan Sincic, Loi Do, Lee Evans, Vincent Malba, Anthony F. Bernhardt, Mark W. Wilson, Anand Patel, Ronald L. Arenson, Curtis Caton, Daniel L. Cooke.
Institutions: University of California, San Francisco , University of California, San Francisco , UCSF Medical Center, University of California, San Francisco , Hansen Medical, Mountain View, CA.
X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance. The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections. A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms. The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.
Biomedical Engineering, Issue 74, Medicine, Bioengineering, Molecular Biology, Anatomy, Physiology, Surgery, Delivery of Health Care, Health Services Research, catheter, microcatheter, deflection, navigation, interventional, Magnetic Resonance Imaging, MRI, lithography, imaging, vascular, endovascular procedures, clinical techniques
50299
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Production and Targeting of Monovalent Quantum Dots
Authors: Daeha Seo, Justin Farlow, Kade Southard, Young-wook Jun, Zev J. Gartner.
Institutions: University of California, San Francisco, University of California, Berkeley, Lawrence Berkeley National Laboratory, University of California, San Francisco, University of California, San Francisco, University of California, San Francisco, University of California, San Francisco.
The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.
Bioengineering, Issue 92, monovalent quantum dots, single particle tracking, SNAP tag, steric exclusion, phosphorothioate, DNA, nanoparticle bioconjugation, single molecule imaging
52198
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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Authors: Kevin Wilhelmsen, Katherine Farrar, Judith Hellman.
Institutions: University of California, San Francisco, University of California, San Francisco.
The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.
Immunology, Issue 78, Cellular Biology, Infection, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Endothelium, Vascular, Neutrophils, Inflammation, Inflammation Mediators, Neutrophil, Leukocyte Adhesion, Endothelial cells, assay
50677
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Myo-mechanical Analysis of Isolated Skeletal Muscle
Authors: Peter E. Oishi, Sompob Cholsiripunlert, Wenhui Gong, Anthony J. Baker, Harold S. Bernstein.
Institutions: University of California San Francisco, University of California San Francisco, San Francisco State University, University of California San Francisco , University of California San Francisco.
To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.
Medicine, Issue 48, muscle, twitch, tetanus, force-frequency, fatigue
2582
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In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy
Authors: Dimitrios Davalos, Katerina Akassoglou.
Institutions: University of California, San Francisco , University of California, San Francisco .
In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task. We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.
Neuroscience, Issue 59, Spinal cord imaging, in vivo two photon microscopy, axons, microglia, blood vessels
2760
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Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor
Authors: Miquella G. Chavez, Jimmy Hu, Kerstin Seidel, Chunying Li, Andrew Jheon, Adrien Naveau, Orapin Horst, Ophir D. Klein.
Institutions: University of California, San Francisco, University of California, San Francisco, Zhongshan Hospital of Dalian University, Université Paris Descartes, Sorbonne Paris Cite, UMR S872, Université Pierre et Marie Curie, UMR S872, INSERM U872, University of California, San Francisco, University of California, San Francisco.
Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.
Stem Cell Biology, Issue 87, Epithelial Stem Cells, Adult Stem Cells, Incisor, Cervical Loop, Cell Culture
51266
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Training Synesthetic Letter-color Associations by Reading in Color
Authors: Olympia Colizoli, Jaap M. J. Murre, Romke Rouw.
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
50893
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Peptide-based Identification of Functional Motifs and their Binding Partners
Authors: Martin N. Shelton, Ming Bo Huang, Syed Ali, Kateena Johnson, William Roth, Michael Powell, Vincent Bond.
Institutions: Morehouse School of Medicine, Institute for Systems Biology, Universiti Sains Malaysia.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
Virology, Issue 76, Biochemistry, Immunology, Infection, Infectious Diseases, Molecular Biology, Medicine, Genetics, Microbiology, Genomics, Proteins, Exosomes, HIV, Peptides, Exocytosis, protein trafficking, secretion, HIV-1, Nef, Secretion Modification Region, SMR, peptide, AIDS, assay
50362
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Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Authors: Jennifer A. Juno, Genevieve Boily-Larouche, Julie Lajoie, Keith R. Fowke.
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
51906
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From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Authors: Wen-Ting Tsai, Ahmed Hassan, Purbasha Sarkar, Joaquin Correa, Zoltan Metlagel, Danielle M. Jorgens, Manfred Auer.
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
51673
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An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Authors: Justen Manasa, Siva Danaviah, Sureshnee Pillay, Prevashinee Padayachee, Hloniphile Mthiyane, Charity Mkhize, Richard John Lessells, Christopher Seebregts, Tobias F. Rinke de Wit, Johannes Viljoen, David Katzenstein, Tulio De Oliveira.
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
51242
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Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology
Authors: William S. Phipps, Zhizhong Yin, Candice Bae, Julia Z. Sharpe, Andrew M. Bishara, Emily S. Nelson, Aaron S. Weaver, Daniel Brown, Terri L. McKay, DeVon Griffin, Eugene Y. Chan.
Institutions: DNA Medicine Institute, Harvard Medical School, NASA Glenn Research Center, ZIN Technologies.
Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.
Cellular Biology, Issue 93, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
51743
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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Authors: James Smadbeck, Meghan B. Peterson, George A. Khoury, Martin S. Taylor, Christodoulos A. Floudas.
Institutions: Princeton University.
The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity. To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
50476
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Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Authors: C. R. Gallistel, Fuat Balci, David Freestone, Aaron Kheifets, Adam King.
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
51047
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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Authors: Daniel T. Claiborne, Jessica L. Prince, Eric Hunter.
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
51506
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Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Authors: Helene Mens, Mary Kearney, Ann Wiegand, Jonathan Spindler, Frank Maldarelli, John W. Mellors, John M. Coffin.
Institutions: NCI-Frederick, University of Pittsburgh, Tuffts University.
Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate. The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.
Immunology, Issue 55, single genome sequencing, SGS, real-time PCR, single-copy assay, SCA, HIV-1, ultra-sensitive, RNA extraction
2960
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Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools
Authors: Utkan Demirci.
Institutions: Brigham and Women's Hospital.
Cellular Biology, Issue 8, microfluidics, diagnostics, capture, blood, HIV, bioengineering
314
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Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Authors: Joachim Hauber.
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells. Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells
793
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Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model
Authors: Denise Lawrie, George Janossy, Maarten Roos, Deborah K. Glencross.
Institutions: National Health Laboratory Services (NHLS-SA), University of Witwatersrand, Lightcurve Films.
We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing > 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.
Immunology, Issue 44, Human Immunodeficiency virus (HIV); CD4 lymphocyte count, white cell count, CD45, panleucogating, lymphocyte activation, CD38, HIV viral load, antiretroviral therapy (ART), internal quality control
2312
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Molecular Evolution of the Tre Recombinase
Authors: Frank Buchholz.
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.
Cell Biology, Issue 15, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
791
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Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care
Authors: Laura S. Lorenz, Jon A. Chilingerian.
Institutions: Brandeis University, Brandeis University.
The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.
Medicine, Issue 48, person-centered care, participatory visual methods, photovoice, photo-elicitation, narrative medicine, acquired brain injury, disability, rehabilitation, palliative care
2342
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.