Bulk RNA sequencing of human pediatric lung cell populations reveals unique transcriptomic signature associated with postnatal pulmonary development.

Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

Factors Affecting Pregnancy Success in the Bitch Following Transcervical Insemination.

Endoscopic-assisted transcervical inseminations (TCIs) have become increasingly popular. The aim of this retrospective clinical study was to evaluate data from the TCIs performed at our facility. We evaluated data from January 2018 through December 2021. This included 137 cases with fresh, 67 cases chilled, and 63 cases using frozen-thawed semen. All bitches underwent breeding management to determine the ideal breeding period. All semen samples were evaluated for total number of sperm, total motility, and progressive motility. Pregnancy was determined by B-mode ultrasonography about 4 weeks after the breeding. Litter size was determined by radiographs performed around the last week of gestation. The pregnancy rate was 83.21% for fresh, 67.16% for chilled, and 66.67% for frozen-thawed semen. There was a significant difference in litter size between fresh semen (6.82 puppies per litter) and both chilled (5.21 puppies per litter) and frozen-thawed (4.59 puppies per litter) semen (P < .05). There was no significant difference in litter size between chilled and frozen-thawed semen. There was no difference in pregnancy rates between clinicians performing the inseminations. Pregnancy rate was not different when sedation was used for the insemination (66.67%) compared to when sedation was not used (74.84%; P > .05). Performing 2 TCIs during the fertile period, regardless of the semen type, resulted in an increase of 6.6% in pregnancy rate (P > .05) and an increase of 0.7 puppies per litter, on average (P > .05). These results can be used to help guide recommendations for breeding clients on the best options to increase both pregnancy rate and litter size for their breeding.

Hybrid donor-acceptor polymer nanoparticles and combination antibiotic for mitigation of pathogenic bacteria and biofilms.

Biofilms pose a significant clinical problem in skin and soft tissue infections. Their resistance to antibiotics has spurred investigations into alternative treatments, such as nanoparticle-mediated photothermal ablation. Non-toxic Hybrid Donor- Acceptor (DA) Polymer nanoParticles (H-DAPPs) were developed for fluorescence imaging (using poly(3-hexylthiophene-2,5 diyl) (P3HT)) and rapid, near-infrared photothermal ablation (NIR- PTA) (using poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe)). H-DAPPs were evaluated alone, and in combination with antibiotics, against planktonic S. aureus and S. pyogenes, and S. aureus biofilms. H-DAPPs NIR-PTA (15-700 µg/ mL) can generate rapid temperature changes of 27.6-73.1 °C, which can eradicate planktonic bacterial populations and reduce biofilm bacterial viability by more than 4- log (> 99.99%) with exposure to 60 s of 800 nm light. Reductions were confirmed via confocal analysis, which suggested that H-DAPPs PTA caused bacterial inactivation within the biofilms, but did not significantly reduce biofilm polysaccharides. SEM imaging revealed structural changes in biofilms after H-DAPPs PTA. S. aureus biofilms challenged with 100 µg/mL of H-DAPPs (H-DAPPs-100) to induce an average temperature of 55.1 °C, and the minimum biofilm eradication concentration (MBEC) of clindamycin, resulted in up to ~3- log decrease in bacterial viability compared to untreated biofilms and those administered H-DAPPs-100 PTA only, and up to ~2- log compared to biofilms administered only clindamycin. This study demonstrates that polymer nanoparticle PTA can mitigate biofilm infection and may improve antimicrobial efficacy.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

Evaluating the Effects of Cell Sorting on Gene Expression.

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

H2S-releasing amphiphilic dipeptide hydrogels are potent S. aureus biofilm disruptors.

As a gasotransmitter, hydrogen sulfide (H2S) has been studied to treat wounds and inflammation, but its potential antimicrobial effects in this context have not been evaluated. An H2S-releasing dipeptide hydrogel (S-FE), and several non-H2S-releasing control dipeptides, (C-FE, C-GE, FBA-FE, and FE where S = S-aroylthiooxime, an H2S donor; C = control, an oxime incapable of H2S release; FBA = 4-formylbenzamide, also incapable of H2S release; and E, F, G = glutamic acid, phenylalanine, and glycine, respectively), were studied to correlate differences in their chemical structures and H2S-releasing abilities with their antimicrobial effects on Staphylococcus aureus bacteria. Dipeptides with Phe (S-FE, C-FE, and FE) self-assembled into nanoribbons in water and displayed ß-sheet formation and enhanced fluorescence, while the other two dipeptides (FBA-FE and C-GE) did not form assemblies in water. In vitro experiments with Staphylococcus aureus, which is a commonly found bacterium associated with wounds, showed significant antimicrobial effects from some of the dipeptides. Dipeptide S-FE inhibited bacterial growth more effectively than any of the controls, thereby limiting biofilm formation or disrupting established biofilms. These antimicrobial H2S-releasing dipeptide hydrogels provide a promising new approach to treat wound infections.

"Provoked" feto-maternal hemorrhage may represent insensible cell exchange in pregnancies from 6 to 22 weeks gestational age.

OBJECTIVE: To quantify spontaneous and provoked fetal to maternal cell exchange in the first half of pregnancy. Transfer of fetal red blood cells (FRBCs) into the maternal circulation during the first half of pregnancy is poorly characterized but of clinical relevance for miscarriage management and invasive procedures. STUDY DESIGN: Prospective, descriptive cohort study of women presenting for surgical termination of pregnancy with sonographically confirmed gestational age (GA). Pre-procedural and post-procedural blood samples were collected to characterize both spontaneous (pre) and provoked (post) cell exchange with analysis via flow cytometry to quantify FRBC count. RESULTS: A total of 100 patients at 6-22 weeks GA contributed 200 matched pre- and post-procedural samples. FRBCs were identified in 69 patients including 4 who exhibited FRBCs pre-procedure only and 9 post-procedure only, for a total of 65 patients having post-procedural FRBCs. Of patients with FRBCs following their procedure, the majority (n=56, 86%) also exhibited evidence of cells before the procedure with just 9 patients (14%) exhibiting FRBCs only after. No dose-response relationship was appreciable between GA and FRBC count. CONCLUSION: After experiencing disruption of the placenta with instrumentation, roughly two thirds of patients had detectable FRBCs in maternal circulation following their procedure but-among those that did-the majority also exhibited cell presence prior to the procedure. This leads to further questions regarding the relationship between risk events and alloimmunization potential in previable pregnancies as the rate of spontaneous transplacental cell exchange may be underappreciated and the magnitude of provoked transfer may be overestimated. IMPLICATIONS: The relationship between feto-maternal hemorrhage risk events and alloimmunization potential in previable pregnancies has previously been poorly characterized but these data reveal spontaneous transplacental cell exchange may be underappreciated and the magnitude of provoked transfer may be overestimated.

Cell type-resolved human lung lipidome reveals cellular cooperation in lung function.

Cell type-resolved proteome analyses of the brain, heart and liver have been reported, however a similar effort on the lipidome is currently lacking. Here we applied liquid chromatography-tandem mass spectrometry to characterize the lipidome of major lung cell types isolated from human donors, representing the first lipidome map of any organ. We coupled this with cell type-resolved proteomics of the same samples (available at Lungmap.net). Complementary proteomics analyses substantiated the functional identity of the isolated cells. Lipidomics analyses showed significant variations in the lipidome across major human lung cell types, with differences most evident at the subclass and intra-subclass (i.e. total carbon length of the fatty acid chains) level. Further, lipidomic signatures revealed an overarching posture of high cellular cooperation within the human lung to support critical functions. Our complementary cell type-resolved lipid and protein datasets serve as a rich resource for analyses of human lung function.

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues.

Human lung morphogenesis begins by embryonic life and continues after birth into early childhood to form a complex organ with numerous morphologically and functionally distinct cell types. Pulmonary organogenesis involves dynamic changes in cell proliferation, differentiation, and migration of specialized cells derived from diverse embryonic lineages. Studying the molecular and cellular processes underlying formation of the fully functional lung requires isolating distinct pulmonary cell populations during development. We now report novel methods to isolate four major pulmonary cell populations from pediatric human lung simultaneously. Cells were dissociated by protease digestion of neonatal and pediatric lung and isolated on the basis of unique cell membrane protein expression patterns. Epithelial, endothelial, nonendothelial mesenchymal, and immune cells were enriched by fluorescence-activated cell sorting. Dead cells and erythrocytes were excluded by 7-aminoactinomycin D uptake and glycophorin-A (CD235a) expression, respectively. Leukocytes were identified by membrane CD45 (protein tyrosine phosphatase, receptor type C), endothelial cells by platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (CD144), and both were isolated. Thereafter, epithelial cell adhesion molecule (CD326)-expressing cells were isolated from the endothelial- and immune cell-depleted population to enrich epithelial cells. Cells lacking these membrane markers were collected as "nonendothelial mesenchymal" cells. Quantitative RT-PCR and RNA sequencing analyses of population specific transcriptomes demonstrate the purity of the subpopulations of isolated cells. The method efficiently isolates major human lung cell populations that we announce are now available through the National Heart, Lung, and Blood Institute Lung Molecular Atlas Program (LungMAP) for their further study.

International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

Molecular typing of epizootic hemorrhagic disease virus serotypes by one-step multiplex RT-PCR.

Epizootic hemorrhagic disease virus (EHDV) causes a highly infectious noncontagious hemorrhagic disease in wild and captive deer (Cervidae) populations in the US. Although rapid and accurate identification of the disease is important, identification of the serotype is equally important for understanding the epidemiology of the disease in white-tailed deer (Odocoileus virginianus) populations. We developed a one-step multiplex reverse transcriptase PCR assay for rapid differentiation and identification of EHDV serotypes 1, 2, and 6 in cell culture and clinical samples by targeting the viral gene segment 2 (L2) that encodes for the structural protein VP2. From 2009 to 2012, 427 clinical samples including tissue and blood (in ethylenediaminetetraacetic acid) from white-tailed deer, found EHDV positive by real-time PCR, were used to evaluate this subtyping assay. Eighteen percent of the positive samples tested were EHDV-1, 59% were EHDV-2, and 21% were EHDV-6; 2% of the samples were positive for more than one subtype, indicating mixed infection. This assay provides a rapid, sensitive, specific diagnostic tool for differentiation and identification of EHDV serotypes in field samples and virus isolates.

Follicular lymphoma tumor-infiltrating T-helper (T(H)) cells have the same polyfunctional potential as normal nodal T(H) cells despite skewed differentiation.

The follicular lymphoma (FL) T-cell microenvironment plays a critical role in the biology of this disease. We therefore determined the lineage, differentiation state, and functional potential of FL-infiltrating CD4(+) T-helper cells (T(H)) compared with reactive and normal lymph node (NLN) T(H) cells. Relative to NLNs, FL cells have decreased proportions of naive and central memory but increased proportions of effector memory T(H) cells. We further show differences in the distribution and anatomical localization of CXCR5(+) T(H) populations that, on the basis of transcription factor analysis, include both regulatory and follicular helper T cells. On Staphylococcus enterotoxin-B stimulation, which stimulates T cells through the T-cell receptor, requires no processing by APCs, and can overcome regulator T cell-mediated suppression, the proportion of uncommitted primed precursor cells, as well as T(H)2 and T(H)17 cells is higher in FL cells than in reactive lymph nodes or NLNs. However, the proportion of T(H)1 and polyfunctional T(H) cells (producing multiple cytokines simultaneously) is similar in FL cells and NLNs. These data suggest that, although T(H)-cell differentiation in FL is skewed compared with NLNs, FL T(H) cells should have the same intrinsic ability to elicit antitumor effector responses as NLN T(H) cells when tumor suppressive mechanisms are attenuated.

An 11-color flow cytometric assay for identifying, phenotyping, and assessing endocytic ability of peripheral blood dendritic cell subsets in a single platform.

Human peripheral blood dendritic cells (PBDC) are a rare population comprised of several distinctive subsets. Analysis of these cells has been hindered by their low frequency. In this study, we report a novel direct ex vivo 11-color flow cytometric assay that combines subset identification with analysis of activation status and endocytic ability of three major PBDC subsets (CD1c(+)CD11c(+) "MDC1," CD141(+)CD11c(+) "MDC2," and CD303(+)CD11c(-) "PDC") within a single platform. This method eliminates the need for DC enrichment, isolation, or prolonged culture. Human peripheral blood mononuclear cells (PBMC) from healthy donors are incubated with FITC-dextran directly ex vivo, prior to cell surface staining with various markers. As expected, PBDC identified by this assay express low levels of CD40 and CD86 directly ex vivo, and significantly upregulate expression of these molecules upon stimulation with toll-like receptor ligands LPS and CpG oligonucleotides. In addition, PDC internalize FITC-labeled dextran poorly in comparison to MDC1 and MDC2 subsets. Specificity of FITC-dextran endocytosis is further verified by imaging flow cytometry. Furthermore, the combination of surface markers used in this assay reveals a previously unreported CD4(+)CD11c(+)CD303(-)CD1c(-)CD141(-) cell population. Taken together, this assay is a rapid and cost-effective method that avoids manipulation of PBDC while providing direct ex vivo high-dimensional flow cytometry data for PBDC studies.