H/ACA snoRNA levels are regulated during stem cell differentiation.

H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

The transition from stem cell to progenitor spermatogonia and male fertility requires the SHP2 protein tyrosine phosphatase.

SHP2 is a widely expressed protein tyrosine phosphatase required for signal transduction from multiple cell surface receptors. Gain and loss of function SHP2 mutations in humans are known to cause Noonan and LEOPARD syndromes, respectively, that are characterized by numerous pathological conditions including male infertility. Using conditional gene targeting in the mouse, we found that SHP2 is required for maintaining spermatogonial stem cells (SSCs) and the production of germ cells required for male fertility. After deleting SHP2, spermatogenesis was halted at the initial step during which transit-amplifying undifferentiated spermatogonia are produced from SSCs. In the absence of SHP2, proliferation of SSCs and undifferentiated spermatogonia was inhibited, thus germ cells cannot be replenished and SSCs cannot undergo renewal. However, germ cells beyond the undifferentiated spermatogonia stage of development at the time of SHP2 knockout were able to complete their maturation to become sperm. In cultures of SSCs and their progeny, inhibition of SHP2 activity reduced growth factor-mediated intracellular signaling that regulates SSC proliferation and cell fate. Inhibition of SHP2 also decreased the number of SSCs present in culture and caused SSCs to detach from supporting cells. Injection of mice with an SHP2 inhibitor blocked the production of germ cells from SSCs. Together, our studies show that SHP2 is essential for SSCs to maintain fertility and indicates that the pathogenesis of infertility in humans with SHP2 mutations is due to compromised SSC functions that block spermatogenesis.

The Burden of Burns: An Analysis of Public Health Measures.

Accurate analysis of injuries is paramount when allocating resources for prevention, research, education, and legislation. As burn mortality has improved over recent decades, the societal burden of burn injuries has grown ambiguous to the public while a scarcity of investigational funding for survivors has led to a gap in understanding lifelong sequela. We aim to compare national references reporting the incidence of burn injuries in the United States. The American Burn Association Burn Injury Summary Report (ABA-BISR), American Burn Association Fact Sheet, Centers for Disease Control and Prevention (CDC) Web-based Injury Statistics Query and Reporting (WISQARS) database, the CDC National Center for Health Statistics' National Hospital Ambulatory Medical Care Survey (NHAMCS), National Inpatient Sample (NIS), National Emergency Department Sample (NEDS), and commercially available claims databases were queried for 2020 or the most recent data available. The BISR estimated 30,135 burn admissions in 2022. The 2016 ABA Fact Sheet reported 486,000 burns presented to US emergency departments (ED). In 2020, CDC's WISQARS database reported 3,529 fatal, and 287,926 non-fatal, burn injuries. The 2020 NEDS reported 438,185 ED visits while the 2020 NIS estimated 103,235 inpatients. The NHAMCS reported 359,000 ED visits for burn injuries in the same period, and an analysis of ICD-10 burn codes demonstrated over 698,555 claims. Our study demonstrates a large variability in the reported incidence of burn injury by the ABA, CDC, national samples, and claims databases. Per our analyses, we estimate that 600,000 individuals annually suffer a burn injury which merits emergent care in the United States.

Development of the national injury resource database (NIRD).

INTRODUCTION: Lack of an accurate, publicly available database of burn/trauma resources creates challenges in providing burn care. In response to this gap, our group developed the National Injury Resource Database (NIRD), a comprehensive database of all US burn centers (BC) and trauma centers (TC) and their capabilities. METHODS: Lists of all national BC and TC were obtained from the American Burn Association (ABA), the American College of Surgeons, and every state department of health. Data was cross-checked and included BC/TC were linked with a 7-digit identification number using the American Hospital Association Quick Search guide. Each center's resources and verification status were validated with electronic or telephonic communications. RESULTS: The final database includes 135 BC and 617 TC, of which 18 are BC-only, 500 are TC-only, and 117 are combined BC/TC. ABA-verified BC (n = 76) are only found in Washington DC and 31 states, and 8 states have no BC. In the last 10 years, a net increase of 7 burn centers was found nationally. The ABA's online BC directory is outdated. CONCLUSIONS: NIRD represents the only up-to-date, comprehensive listing of BC and TC in existence. It categorizes all currently operating BC and TC across myriad classifications of designation and capabilities.

Setting the Standard: Using the ABA Burn Registry to Benchmark Risk Adjusted Mortality.

Reports of single center experience and studies of larger databases have identified several predictors of burn center mortality, including age, burn size, and inhalation injury. None of these analyses has been broad enough to allow benchmarking across burn centers. The purpose of this study was to derive a reliable, risk-adjusted, statistical model of mortality based on real-life experience at many burn centers in the U.S. We used the American Burn Association 2020 Full Burn Research Dataset, from the Burn Center Quality Platform (BCQP) to identify 130,729 subjects from July 2015 through June 2020 across 103 unique burn centers. We selected 22 predictor variables, from over 50 recorded in the dataset, based on completeness (at least 75% complete required) and clinical significance. We used gradient-boosted regression, a form of machine learning, to predict mortality and compared this to traditional logistic regression. Model performance was evaluated with AUC and PR curves. The CatBoost model achieved a test AUC of 0.980 with an average precision of 0.800. The logistic regression produced an AUC of 0.951 with an average precision of 0.664. While AUC, the measure most reported in the literature, is high for both models, the CatBoost model is markedly more sensitive, leading to a substantial improvement in precision. Using BCQP data, we can predict burn mortality allowing comparison across burn centers participating in BCQP.

Using a National Burn Registry to Develop a Model for Risk-Adjusted Length of Stay Benchmarking.

Length of stay (LOS) is a frequently reported outcome after a burn injury. LOS benchmarking will benefit individual burn centers as a way to measure their performance and set expectations for patients. We sought to create a nationwide, risk-adjusted model to allow for LOS benchmarking based on the data from a national burn registry. Using data from the American Burn Association's Burn Care Quality Platform, we queried admissions from 7/2015 to 6/2020 and identified 130,729 records reported by 103 centers. Using 22 predictor variables, comparisons of unpenalized linear regression and Gradient boosted (CatBoost) regressor models were performed by measuring the R2 and concordance correlation coefficient on the application of the model to the test dataset. The CatBoost model applied to the bootstrapped versions of the entire dataset was used to calculate O/E ratios for individual burn centers. Analyses were run on 3 cohorts: all patients, 10-20% TBSA, >20% TBSA. The CatBoost model outperformed the linear regression model with a test R2 of 0.67 and CCC of 0.81 compared with the linear model with R2=0.50, CCC=0.68. The CatBoost was also less biased for higher and lower LOS durations. Gradient-boosted regression models provided greater model performance than traditional regression analysis. Using national burn data, we can predict LOS across contributing burn centers while accounting for patient and center characteristics, producing more meaningful O/E ratios. These models provide a risk-adjusted LOS benchmarking using a robust data source, the first of its kind, for burn centers.

Genes involved in post-transcriptional regulation are overrepresented in stem/progenitor spermatogonia of cryptorchid mouse testes.

Gene expression and consequent biological activity of adult tissue stem cells are regulated by signals emanating from the local microenvironment (niche). To gain insights into the molecular regulation of spermatogonial stem cells (SSCs), gene expression was characterized from SSCs isolated from their cognate niches of cryptorchid (stem cell-enriched), wild-type, and busulfan-treated (stem cell-depleted) mouse testes. Quantitative assessment of stem cell activity in each testis model was determined using an in vivo functional assay and correlated with gene expression using Affymetrix MGU74Av2 microarrays and the ChipStat algorithm optimized to detect gene expression from rare cells in complex tissues. We identified 389 stem/progenitor spermatogonia candidate genes, which exhibited significant overlap with genes expressed by embryonic, hematopoietic, and neural stem cells; enriched spermatogonia; and cultured SSCs identified in previous studies. Candidate cell surface markers identified by the microarray may facilitate the isolation and enrichment of stem and/or progenitor spermatogonia. Flow cytometric analyses confirmed the expression of chemokine receptor 2 (Ccr2) and Cd14 on a subpopulation cryptorchid testis cells (alpha6-integrin+, side scatter(lo)) enriched for SSCs. These cell surface molecules may mark progenitor spermatogonia but not SSCs because Ccr2+ and Cd14+ fractions failed to produce spermatogenesis upon transplantation to recipient testes. Functional annotation of candidate genes and subsequent immunohistochemistry revealed that proteins involved in post-transcriptional regulation are overrepresented in cryptorchid testes that are enriched for SSCs. Comparative analyses indicated that this is a recurrent biological theme among stem cells.

Mass spectrometric identification of candidate RNA-binding proteins associated with Transition Nuclear Protein mRNA in the mouse testis.

Spermatogenesis is a differentiation process that requires dramatic changes to DNA architecture, a process governed in part by Transition Nuclear Proteins 1 and 2 (TNP1 and TNP2). Translation of Tnp1 and Tnp2 mRNAs is temporally disengaged from their transcription. We hypothesized that RNA regulatory proteins associate specifically with Tnp mRNAs to control the delayed timing of their translation. To identify potential regulatory proteins, we isolated endogenous mRNA/protein complexes from testis extract and identified by mass spectrometry proteins that associated with one or both Tnp transcripts. Five proteins showed strong association with Tnp transcripts but had low signal when Actin mRNA was isolated. We visualized the expression patterns in testis sections of the five proteins and found that each of the proteins was detected in germ cells at the appropriate stages to regulate Tnp RNA expression.

Comparison of injury patient information from hospitals with records in both the national trauma data bank and the nationwide inpatient sample.

BACKGROUND: Administrative and registry databases are useful for researchers given their availability and size, yet their limitations for specific applications remain undefined. We compared injury records from a large administrative database and the National Trauma Data Bank (NTDB) with the goal of furthering the understanding of their respective limitations. METHODS: The study hospitals had submitted records to both the NTDB and the Nationwide Inpatient Sample (NIS) for patients admitted during 2002. Record inclusion criteria for comparison included nonelective admissions with a primary diagnosis of injury (excluding isolated hip fractures). Numbers of cases and variables common to both databases were compared. RESULTS: Twenty-four hospitals had records both in the NTDB (24,619 records) and in the NIS (25,586 records). We found less missing cost and payer information in the NIS compared with the NTDB (0% and 0.1% vs. 30.5% and 24%, respectively), higher mean number of comorbidities per record in the NIS (0.77 vs. 0.18), and a lower crude case fatality rate in the NIS (3.5% vs. 5.2%). CONCLUSIONS: The main differences between the databases reflected the different motives for data collection and the inclusion or exclusion criteria imposed by trauma registries. These differences require consideration when using either database to investigate injury-related questions.

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Discovery of a Phosphoinositide 3-Kinase (PI3K) ß/δ Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3Kß Potency in a PI3Kδ-Selective Template by Targeting Nonconserved Asp856.

Phosphoinositide 3-kinase (PI3K) ß signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kß/δ inhibitors in which PI3Kß potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kß/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

Germline stem cells: toward the regeneration of spermatogenesis.

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

Spermatogonial stem cell regulation and spermatogenesis.

This article will provide an updated review of spermatogonial stem cells and their role in maintaining the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs) will be described, along with research using these tools to enhance our understanding of stem cell biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity to manipulate these cells for practical application. The chapter concludes with a discussion of future directions for fundamental investigation and practical applications of SSCs.

The utility of bronchoscopy after inhalation injury complicated by pneumonia in burn patients: results from the National Burn Repository.

There are no guidelines to determine when bronchoscopy is appropriate in patients with inhalation injury complicated by pneumonia. We reviewed the National Burn Repository from 1998 to 2007 to determine if there is any difference in outcome in burn patients with inhalation injury and pneumonia who did and did not undergo bronchoscopy. Three hundred fifty-five patients with pneumonia did not undergo bronchoscopy, 173 patients underwent one bronchoscopy, and 96 patients underwent more than one bronchoscopy. Patients with a 30 to 59% surface area burn and pneumonia who underwent bronchoscopy had a decreased duration of mechanical ventilation compared with those who did not (21 days, 95% CI: 19-23 days vs 28 days, 95% CI: 25-31 days, P=.0001). When compared with patients who did not undergo bronchoscopy, patients having a single bronchoscopy had a significantly shorter length of intensive care unit stay and hospital stay (35+/-3 vs 39+/-2, P=.04, and 45+/-3 vs 49+/-2, P=.009). The hospital charges were on average much higher in those patients who did not undergo bronchoscopy, compared with those who did ($473,654+/-44,944 vs $370,572+/-36,602, P=.12). When compared with patients who did not undergo bronchoscopy, patients who did have one or more bronchoscopies showed a reduced risk of death by 18% (OR=0.82, 95% CI: 0.53-1.27, P=.37). Patients with inhalation injury complicated by pneumonia seem to benefit from bronchoscopy. This benefit can be seen in a decreased duration of mechanical ventilation, decreased length of intensive care unit stay, and decreased overall hospital cost. In addition, there was a trend toward an improvement in mortality. The aggressive use of bronchoscopy after inhalation injury may be justified.