Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice

Enabling structural resilience of street-involved children and youth in Kenya: reintegration outcomes and the Flourishing Community model

IntroductionMillions of children and youth live on city streets across the globe, vulnerable to substance use, abuse, material and structural neglect. Structural resilience, the re-establishment of access to structural goods within a society such as housing, education, and healthcare following some interruption, provides an orientation for research and interventional efforts with street-involved children and youth (SICY). Further, a structural resilience framework supports organizing interactions between levels and sectors of a socio-ecology.MethodsFollowing the expressed interests of Kenyan SICY, and consistent with emerging policy interests at national and global levels, we assess reintegration trajectories of Kenyan SICY (n = 227) participating in a new program intervention and model. The intervention combines two coordinated, parallel programs – one focused on the rescue, rehabilitation, reintegration and resocialization of SICY, and the other focused on empowering families and communities to provide better care for children and youth who are reintegrating from life on the streets to the broader community. Data were collected and analyzed from multiple stages across SICY involvement with the intervention.ResultsWe found 79% of SICY participants reintegrated with the broader community, and 50% reintegrated with families of origin and returned to school. Twenty-five percent of participants reintegrated to a boarding school, polytechnical school, or began a business. Probability of reintegrating successfully was significantly improved among participants whose families participated in the family- and community-oriented program, who were younger, with less street-exposure, expressed more personal interests, and desired to reintegrate with family.DiscussionTo our knowledge, these are the first quantitative data published of successful reintegration of SICY to the broader, non-institutionalized community in any low- or middle-income country. Future research should (1) identify factors across socio-ecological levels and sectors contributing to health and developmental outcomes of reintegrated children and youth, (2) mechanisms to support SICY for whom the interventional strategy did not work, (3) methods to prevent street-migration by children and youth, and (4) system development to coordinate follow-up and relevant investment by institutions, organizations and community leaders to continue reintegration work

Overview of Spirit Microscopic Imager Results

This paper provides an overview of Mars Exploration Rover Spirit Microscopic Imager (MI) operations and the calibration, processing, and analysis of MI data. The focus of this overview is on the last five Earth years (2005-2010) of Spirit's mission in Gusev crater, supplementing the previous overview of the first 450 sols of the Spirit MI investigation. Updates to radiometric calibration using in-flight data and improvements in high-level processing are summarized. Released data products are described, and a table of MI observations, including target/feature names and associated data sets, is appended. The MI observed natural and disturbed exposures of rocks and soils as well as magnets and other rover hardware. These hand-lens-scale observations have provided key constraints on interpretations of the formation and geologic history of features, rocks, and soils examined by Spirit. MI images complement observations by other Spirit instruments, and together show that impact and volcanic processes have dominated the origin and evolution of the rocks in Gusev crater, with aqueous activity indicated by the presence of silica-rich rocks and sulfate-rich soils. The textures of some of the silica-rich rocks are similar to terrestrial hot spring deposits, and observations of subsurface cemented layers indicate recent aqueous mobilization of sulfates in places. Wind action has recently modified soils and abraded many of the rocks imaged by the MI, as observed at other Mars landing sites. Plain Language Summary The Microscopic Imager (MI) on NASA's Spirit rover returned the highest-resolution images of the Martian surface available at the time of the 2004-2010 mission. Designed to survive 90 Mars days (sols) and search for evidence of water in the past, Spirit returned data for 2210 sols, far exceeding all expectations. This paper summarizes the scientific insights gleaned from the thousands of MI images acquired during the last 5years of the mission, supplementing the summary of the first 450 sols of the Spirit MI investigation published previously (Herkenhoff et al., ). Along with data from the other instruments on Spirit, MI images guided the scientific interpretation of the geologic history of the rocks and soils observed in Gusev crater on Mars. We conclude that the geologic history of the area explored by Spirit has been dominated by impacts and volcanism, and that water, perhaps very hot water, was involved in the evolution of some of the rocks and soils. More recently, winds have moved soil particles and abraded rocks, as observed elsewhere on Mars. These results have improved our understanding of Mars' history and informed planning of future missions to Mars.National Aeronautics and Space AdministrationPublic domain articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Cohort profile: seek, test, treat and retain United States criminal justice cohort

Abstract Background The STTR treatment cascade provides a framework for research aimed at improving the delivery of services, care and outcomes of PLWH. The development of effective approaches to increase HIV diagnoses and engage PLWH in subsequent steps of the treatment cascade could lead to earlier and sustained ART treatment resulting in viral suppression. There is an unmet need for research applying the treatment cascade to improve outcomes for those with criminal justice involvement. Methods The Seek, Test, Treat, and Retain (STTR) criminal justice (CJ) cohort combines data from 11 studies across the HIV treatment cascade that focused on persons involved in the criminal justice system, often but not exclusively for reasons related to substance use. The studies were conducted in a variety of CJ settings and collected information across 11 pre-selected domains: demographic characteristics, CJ involvement, HIV risk behaviors, HIV and/or Hepatitis C infections, laboratory measures of CD4 T-cell count (CD4) and HIV RNA viral load (VL), mental illness, health related quality of life (QoL), socioeconomic status, health care access, substance use, and social support. Results The STTR CJ cohort includes data on 11,070 individuals with and without HIV infection who range in age from 18 to 77 years, with a median age at baseline of 37 years. The cohort reflects racial, ethnic and gender distributions in the U.S. CJ system, and 64% of participants are African-American, 12% are Hispanic and 83% are men. Cohort members reported a wide range of HIV risk behaviors including history of injection drug use and, among those who reported on pre-incarceration sexual behaviors, the prevalence of unprotected sexual intercourse ranged across studies from 4% to 79%. Across all studies, 53% percent of the STTR CJ cohort reported recent polysubstance use. Conclusions The STTR CJ cohort is comprised of participants from a wide range of CJ settings including jail, prison, and community supervision who report considerable diversity in their characteristics and behavioral practices. We have developed harmonized measures, where feasible, to improve the integration of these studies together to answer questions that cannot otherwise be addressed

Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells

Strongyloides stercoralis and HTLV-1 coinfection in CD34+ cord blood stem cell humanized mice: Alteration of cytokine responses and enhancement of larval growth.

Viral and parasitic coinfections are known to lead to both enhanced disease progression and altered disease states. HTLV-1 and Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. Independently, HTLV-1 induces a Th1 response and S. stercoralis infection induces a Th2 response. However, coinfection with the two pathogens has been associated with the development of S. stercoralis hyperinfection and an alteration of the Th1/Th2 balance. In this study, a model of HTLV-1 and S. stercoralis coinfection in CD34+ umbilical cord blood hematopoietic stem cell engrafted humanized mice was established. An increased level of mortality was observed in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected group. The mortality was not correlated with proviral loads or total viral RNA. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. HTLV-1 infected and coinfection groups showed a significant, yet non-clonal expansion of the CD4+CD25+ T-cell population. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group and no autoinfective larvae were found. However, infective larvae recovered from the coinfection group showed an enhancement in growth, as was seen in mice with S. stercoralis hyperinfection caused by treatment with steroids. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections in vivo in the context of human immune cells

Cigarette smoke exposure worsens acute lung injury in antibiotic-treated bacterial pneumonia in mice.

Evidence is accumulating that exposure to cigarette smoke (CS) increases the risk of developing acute respiratory distress syndrome (ARDS). Streptococcus pneumoniae is the most common cause of bacterial pneumonia, which in turn is the leading cause of ARDS. Chronic smokers have increased rates of pneumococcal colonization and develop more severe pneumococcal pneumonia than nonsmokers; yet mechanistic connections between CS exposure, bacterial pneumonia, and ARDS pathogenesis remain relatively unexplored. We exposed mice to 3 wk of moderate whole body CS or air, followed by intranasal inoculation with an invasive serotype of S. pneumoniae. CS exposure alone caused no detectable lung injury or bronchoalveolar lavage (BAL) inflammation. During pneumococcal infection, CS-exposed mice had greater survival than air-exposed mice, in association with reduced systemic spread of bacteria from the lungs. However, when mice were treated with antibiotics after infection to improve clinical relevance, the survival benefit was lost, and CS-exposed mice had more pulmonary edema, increased numbers of BAL monocytes, and elevated monocyte and lymphocyte chemokines. CS-exposed antibiotic-treated mice also had higher serum surfactant protein D and angiopoietin-2, consistent with more severe lung epithelial and endothelial injury. The results indicate that acute CS exposure enhances the recruitment of immune cells to the lung during bacterial pneumonia, an effect that may provide microbiological benefit but simultaneously exposes the mice to more severe inflammatory lung injury. The inclusion of antibiotic treatment in preclinical studies of acute lung injury in bacterial pneumonia may enhance clinical relevance, particularly for future studies of current or emerging tobacco products

Cigarette smoke exposure worsens acute lung injury in antibiotic-treated bacterial pneumonia in mice.

Evidence is accumulating that exposure to cigarette smoke (CS) increases the risk of developing acute respiratory distress syndrome (ARDS). Streptococcus pneumoniae is the most common cause of bacterial pneumonia, which in turn is the leading cause of ARDS. Chronic smokers have increased rates of pneumococcal colonization and develop more severe pneumococcal pneumonia than nonsmokers; yet mechanistic connections between CS exposure, bacterial pneumonia, and ARDS pathogenesis remain relatively unexplored. We exposed mice to 3 wk of moderate whole body CS or air, followed by intranasal inoculation with an invasive serotype of S. pneumoniae. CS exposure alone caused no detectable lung injury or bronchoalveolar lavage (BAL) inflammation. During pneumococcal infection, CS-exposed mice had greater survival than air-exposed mice, in association with reduced systemic spread of bacteria from the lungs. However, when mice were treated with antibiotics after infection to improve clinical relevance, the survival benefit was lost, and CS-exposed mice had more pulmonary edema, increased numbers of BAL monocytes, and elevated monocyte and lymphocyte chemokines. CS-exposed antibiotic-treated mice also had higher serum surfactant protein D and angiopoietin-2, consistent with more severe lung epithelial and endothelial injury. The results indicate that acute CS exposure enhances the recruitment of immune cells to the lung during bacterial pneumonia, an effect that may provide microbiological benefit but simultaneously exposes the mice to more severe inflammatory lung injury. The inclusion of antibiotic treatment in preclinical studies of acute lung injury in bacterial pneumonia may enhance clinical relevance, particularly for future studies of current or emerging tobacco products

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice