A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

High-efficiency quantitative control of mitochondrial transfer based on droplet microfluidics and its application on muscle regeneration.

Mitochondrial transfer is a spontaneous process to restore damaged cells in various pathological conditions. The transfer of mitochondria to cell therapy products before their administration can enhance therapeutic outcomes. However, the low efficiency of previously reported methods limits their clinical application. Here, we developed a droplet microfluidics-based mitochondrial transfer technique that can achieve high-efficiency and high-throughput quantitative mitochondrial transfer to single cells. Because mitochondria are essential for muscles, myoblast cells and a muscle injury model were used as a proof-of-concept model to evaluate the proposed technique. In vitro and in vivo experiments demonstrated that C2C12 cells with 31 transferred mitochondria had significant improvements in cellular functions compared to those with 0, 8, and 14 transferred mitochondria and also had better therapeutic effects on muscle regeneration. The proposed technique can considerably promote the clinical application of mitochondrial transfer, with optimized cell function improvements, for the cell therapy of mitochondria-related diseases.

Family satisfaction with telemedicine follow-up after pediatric plastic surgery.

OBJECTIVE: One issue faced at institutions that serve a vast area is patients' ability to travel for perioperative care. Telemedicine is an innovative way of providing care while removing the inconvenience of travel or the hindrance of cost associated with travel. We initiated telemedicine as an option for certain postoperative encounters and assessed patient family satisfaction with this novel approach. METHODS: Our practice offers telemedicine visits to patients who have had simple surgical procedures, identified by a fixed list of CPT codes. Visits are scheduled 7 to 14 days after surgery. Families completed a satisfaction survey after their encounter. RESULTS: A pilot program was initiated from January 2019 to March 2020 using this method of postoperative follow-up. The initial response from families (N = 60) was extremely positive. CONCLUSION: We anticipate the option for telemedicine visits will make postoperative follow-ups more amendable to families, increase adherence rates, and increase access to care.

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

Caspases from scleractinian coral show unique regulatory features.

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

School-located immunization programs: do parental preferences predict behavior?

BACKGROUND: Little is known about parental attitudes regarding school-located immunization programs and their effect on program participation behaviors. OBJECTIVE: To determine the relationship between attitudes of middle school parents regarding school-located immunization programs and subsequent consent behaviors when such a program becomes available. METHODS: Primarily Hispanic, middle school parents completed questionnaires about school-located immunization programs. After questionnaire collection, immunization consent/refusal packets (English/Spanish) for a program providing Tdap and MCV4 vaccines were distributed at five Houston middle schools in low-income, urban areas. Responses regarding demographics, enrollment in a medical home, immunization location preferences, and knowledge of immunization recommendations were analyzed from questionnaires returned by those who later returned consent or refusal forms for school-located program participation. Frequency and chi square statistics were calculated using SPSS 18.0. RESULTS: Of 475 parents who completed the questionnaire and later sent a consent or refusal form, 289 (61%) consented to ≥1 vaccines for their child. Among those who consented: 71% were enrolled in a medical home; 42% had previously indicated that they did NOT prefer school as an immunization location; 32% had stated that they wanted to be present for their child's shots. Of those who sent refusal forms indicating they would access the vaccines from their own providers, 70% stated they wanted to be present for their child's vaccination. CONCLUSIONS: A significant proportion of Hispanic, low-income middle school parents participating in a school-located immunization program had previously indicated that schools were not a preferred immunization site. Despite the availability of a medical home, a lack of preference for schools as a site, and the desire to be present during their child's injections when asked prior to program availability, these parents participated in the program when it was made available. Preferences noted in pre-program questionnaires may not predict parental consent behaviors for school-located immunizations.

Urban middle school parent perspectives: the vaccines they are willing to have their children receive using school-based immunization programs.

PURPOSE: With new vaccination recommendations for adolescents, school-based immunization programs become a valuable alternative site for immunization. This study seeks to determine factors associated with parental willingness to utilize school-based programs for immunizations. METHODS: A questionnaire was distributed to the parents of 11-14-year-olds attending 7 middle schools in a large, urban public school district. Participants were asked multiple questions including medical home enrollment, primary language spoken at home, site of last immunization, and comfort with their child receiving specific vaccines during school hours. Frequencies, chi-square analyses, and logistic regression analyses were performed using SPSS 17.0. RESULTS: A total of 615 parent questionnaires were included in the analyses; 81% of parents were Hispanic, 16% black, 39% spoke primarily English at home, and 77% indicated that they had a medical home for their child. Regarding specific vaccines, the largest percentage of parents were willing to have their child receive influenza vaccine (57%) and the smallest percentage were willing to have the human papillomavirus vaccine (27%) at school during school hours. Parents who had used a school-based clinic for their child's last immunization were more willing to receive each vaccine at school. CONCLUSIONS: This study indicates that there is significant interest and willingness among predominantly lower income, Hispanic middle school parents to have their children receive specific vaccines during school hours through school-based immunization programs. More study is needed among a more diverse population of parents to help target the various needs of parents and adolescents and ultimately increase adolescent immunization rates.

At what sites are parents willing to have their 11 through 14-year-old adolescents immunized?

INTRODUCTION: Parental preferences of alternative immunization sites for adolescents must be evaluated to determine optimal delivery strategies. METHODS: Analyses were performed on data from middle-school parent questionnaires in low income areas. RESULTS: 1838 questionnaires were returned; 86.5% of the participants were Hispanic, 65% spoke primarily Spanish at home. Among those with a medical home, 32% had the last immunization at an alternative location. When checking all that apply, 65% of parents selected the medical home as a desirable location for immunization; 41% selected a school-based program. CONCLUSIONS: Despite lack of exposure to school-based immunization programs, school-based programs are a desirable immunization site among middle-school parents.