Development and validation of a clinical prediction model for early ventilator weaning in post-cardiac surgery.

Background: Weaning patients from mechanical ventilation is a critical clinical challenge post cardiac surgery. The effective liberation of patients from the ventilator significantly improves their recovery and survival rates. This study aimed to develop and validate a clinical prediction model to evaluate the likelihood of successful extubation in post-cardiac surgery patients. Method: A predictive nomogram was constructed for extubation success in individual patients, and receiver operating characteristic (ROC) and calibration curves were generated to assess its predictive capability. The superior performance of the model was confirmed using Delong's test in the ROC analysis. A decision curve analysis (DCA) was conducted to evaluate the clinical utility of the nomogram. Results: Among 270 adults included in our study, 107 (28.84%) experienced delayed extubation. A predictive nomogram system was derived based on five identified risk factors, including the proportion of male patients, EuroSCORE II, operation time, pump time, bleeding during operation, and brain natriuretic peptide (BNP) level. Based on the predictive system, five independent predictors were used to construct a full nomogram. The area under the curve values of the nomogram were 0.880 and 0.753 for the training and validation cohorts, respectively. The DCA and clinical impact curves showed good clinical utility of this model. Conclusion: Delayed extubation and weaning failure, common and potentially hazardous complications following cardiac surgery, vary in timing based on factors such as sex, EuroSCORE II, pump duration, bleeding, and postoperative BNP reduction. The nomogram developed and validated in this study can accurately predict when extubation should occur in these patients. This tool is vital for assessing risks on an individual basis and making well-informed clinical decisions.

Application of neutrophil-to-lymphocyte-to-monocyte ratio in predicting mortality risk in adult patients with septic shock: A retrospective cohort study conducted at a single center.

Background: Sepsis is a life-threatening condition characterized by an aberrant host response to infection, resulting in multi-organ dysfunction. The application of currently available prognostic indicators for sepsis in primary hospitals is challenging. In this retrospective study, we established a novel index, the neutrophil-to-lymphocyte-to-monocyte ratio (NLMR), based on routine blood examination upon admission, and assessed its prognostic value for early mortality risk in adult patients with septic shock. Methods: This study included clinical data from adult patients with septic shock who were admitted to the hospital between January 1, 2018, and December 31, 2022. Training and validation sets were constructed, and patients were categorized into "survival" and "death" groups based on their survival status within the 28-day hospitalization period. Baseline data, including demographic characteristics and comorbidities, and laboratory results, such as complete blood count parameters, were collected for analysis. The Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were documented.The NLMR was determined through the utilization of multivariate binary logistic regression analysis, leading to the development of a risk model aimed at predicting early mortality in adult patients suffering from septic shock. Results: Overall, 112 adult patients with septic shock were enrolled in this study, with 84 and 28 patients in the training and validation sets, respectively. Multivariate binary logistic analysis revealed that the neutrophil, lymphocyte, and monocyte counts independently contributed to the mortality risk (odds ratios = 1.22, 0.08, and 0.16, respectively). The NLMR demonstrated an area under the receiver operating characteristic curve (ROC-AUC) of 0.83 for internal validation in the training set and 0.97 for external validation in the validation set. Both overall model quality values were significantly high at 0.74 and 0.91, respectively (P < 0.05). NLMR exhibited a higher ROC-AUC value of 0.88 than quick SOFA (ROC-AUC = 0.71), SOFA (ROC-AUC = 0.83), and APACHE II (ROC-AUC = 0.78). Conclusion: NLMR may be a potential marker for predicting the risk of early death in adult patients with septic shock, warranting further exploration and verification.

Skeletal abnormalities in mice with Dnmt3a missense mutations.

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.

Rice-fish coculture without phosphorus addition improves paddy soil nitrogen availability by shaping ammonia-oxidizing archaea and bacteria in subtropical regions of South China.

Rice-fish coculture (RFC), as a traditional agricultural strategy in China, can optimally utilize the scarce resource, especially in subtropical regions where phosphorus (P) deficiency limits agricultural production. However, ammonia-oxidizing archaea (AOA) and bacteria (AOB) are involved in the ammonia oxidation, but it remains uncertain whether their community compositions are related to the RFC combined with and without P addition that improves soil nitrogen (N) use efficiency. Here, a microcosm experiment was conducted to assess the impacts of RFC combined with and without inorganic P (0 and 50 mg P kg-1 as KH2PO4) addition on AOA and AOB community diversities, enzyme activities and N availability. The results showed that RFC significantly increased available N content without P addition compared with P addition. Moreover, RFC significantly increased urease activity and AOA shannon diversity, and reduced NAG activity and AOB shannon diversity without P addition, respectively. Higher diversity of AOA compared with that of AOB causes greater competition for resources and energy within their habitats, thereby resulting in lower network complexity. Our findings indicated that the abundances of AOA and AOB are influenced through the introduction of fish and/or P availability, of which AOB is linked to N availability. Overall, RFC could improve paddy soil N availability without P addition in subtropical region, which provides a scientific reference for promoting the practices that reduce N fertilizer application in RFC.

Influence of nitrogen substitution at an equivalent total nitrogen level on bacterial and fungal communities, as well as enzyme activities of the ditch bottom soil in a rice-fish coculture system.

BACKGROUND: Rice-fish coculture system (RFS) operates through effectively utilizing water and land resources in a complementary form, but it requires more efficient utilization of fertilizer and feed without compromising rice yield. However, the knowledge of how to regulate the proportion of nitrogen (N) supplied from fertilizer and feed at an equivalent total N level to improve the benefits of RFS remains limited. Therefore, four treatments (S0: RFS with 0% N from fertilizer and 100% N from feed; S25: RFS with 25% N from fertilizer and 75% N from feed; S50: RFS with 50% N from fertilizer and 50% N from feed; S75: RFS with 75% N from fertilizer and 25% N from feed) were conducted to assess the variation of ditch bottom soil properties, microbial communities and enzyme activities, as well as to obtain the optimal ratio of N supplied from fish feed and fertilizer. RESULTS: The experiments showed that the contents of soil organic matter, total carbon and total N, and the activities of urease, N-acetyl-ß-D-glucosaminidase, protease, ß-1,4-glucosidase and catalase in the ditch bottom soil significantly reduced in S25 treatment, compared with the other three treatments. Ammonium N content decreased with increasing percentage of the basal fertilizer, whereas nitrate N content and pH value showed an adverse trend. However, the bacterial and fungal communities were unaffected by the ratio shifts between fertilizer-N and feed-N, but their dominant phyla were influenced by the ditch bottom soil N level. Moreover, the bacterial community composition was positively related to nitrate N, whereas fungal diversity was positively correlated with pH, ammonium N and nitrate N, and urease. We also found that the treatment of N input with 25% N from fertilizer and 75% N from feed can reduce N deposition in the ditch bottom soil in the rice-fish coculture system. CONCLUSION: Our findings indicate that under the equivalent total N input level, the relative higher ratio of N from fish feed increased (S0 treatment) or reduced (S25 treatment) the deposition of N in the ditch bottom soil, and improved fish production, but decreased rice yield; while the higher ratio of N from basal fertilizer increased the transportation of nutrients into the ditch bottom soil and rice yield, but reduced fish production. So when considering multi-balance and multiple benefits, we recommend that a selective substitution ratio within 50% ~ 75% from fish feed to substitute for the basal fertilizer under the equivalent total N input may achieve a good balance of rice and fish production improvement, and reduce nutrients wastage to the ditch bottom, as well as alleviate the potential of non-point source pollution. This study also provides an evidence for regulating and optimizing the ratio of N supplied from fertilizer and fish feed at an equivalent total N level through monitoring the nutrient accumulation in ditch bottom soil in the rice-fish coculture system. © 2024 Society of Chemical Industry.

Inhibition of colon C5a/C5a receptor signalling pathway confers protection against LPS-induced acute kidney injury via gut microbiota-kidney axis.

Acute kidney injury (AKI) is a critical condition often associated with systemic inflammation and dysregulated gut microbiota. This study aimed to investigate the effects of the C5a receptor antagonist W54011 on lipopolysaccharide (LPS)-induced AKI, focusing on the colon's C5a/C5a receptor pathway, intestinal barrier integrity, and gut microbiota. Our findings demonstrate that W54011 effectively ameliorated kidney injury in the LPS-induced AKI model by selectively inhibiting the colon's C5a/C5a receptor signalling pathway. Additionally, C5a receptor blockade resulted in the inhibition of colonic inflammation and the reconstruction of the intestinal mucosal barrier. Furthermore, W54011 administration significantly impacted the composition and stability of the gut microbiota, restoring the abundance of dominant bacteria to levels observed in the normal state of the intestinal flora and reducing the abundance of potentially harmful bacterial groups. In conclusion, W54011 alleviates LPS-induced AKI by modulating the interplay between the colon, gut microbiota, and kidneys. It preserves the integrity of the intestinal barrier and reinstates gut microbiota, thereby mitigating AKI symptoms. These findings suggest that targeting the colon and gut microbiota could be a promising therapeutic strategy for AKI treatment.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.

Nanocellulose-graphene composites: Preparation and applications in flexible electronics.

In recent years, the pursuit of high-performance nano-flexible electronic composites has led researchers to focus on nanocellulose-graphene composites. Nanocellulose has garnered widespread interest due to its exceptional properties and unique structure, such as renewability, biodegradability, and biocompatibility. However, nanocellulose materials are deficient in electrical conductivity, which limits their applications in flexible electronics. On the other hand, graphene boasts remarkable properties, including a high specific surface area, robust mechanical strength, and high electrical conductivity, making it a promising carbon-based nanomaterial. Consequently, research efforts have intensified in exploring the preparation of graphene-nanocellulose flexible electronic composites. Although there have been studies on the application of nanocellulose and graphene, there is still a lack of comprehensive information on the application of nanocellulose/graphene in flexible electronic composites. This review examines the recent developments in nanocellulose/graphene flexible electronic composites and their applications. In this review, the preparation of nanocellulose/graphene flexible electronic composites from three aspects: composite films, aerogels, and hydrogels are first introduced. Next, the recent applications of nanocellulose/graphene flexible electronic composites were summarized including sensors, supercapacitors, and electromagnetic shielding. Finally, the challenges and future directions in this emerging field was discussed.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

Genetic and phenotypic frequency distribution of ACE, ADRB1, AGTR1, CYP2C9*3, CYP2D6*10, CYP3A5*3, NPPA and factors associated with hypertension in Chinese Han hypertensive patients.

We analyzed the polymorphisms of 7 antihypertensive drugs-related genes and the factors associated with hypertension in hypertensive patients of Han ethnicity in Qingyang, China. A total of 354 hypertensive patients of Han ethnicity were enrolled from Qingyang, China. The ACE (I/D), ADRB1 (1165G > C), AGTR1 (1166A > C), CYP2C9*3, CYP2D6*10, CYP3A5*3 and NPPA (T2238C) polymorphisms were assessed. Clinical data of patients was also obtained. The influencing factors of hypertension were evaluated. The genotype frequencies of ACE, ADRB1, AGTR1, CYP2C9, CYP3A5 and NPPA loci were in Hardy-Weinberg equilibrium, with mutation frequencies of 39.27%, 74.29%, 6.21%, 4.80%, 72.46% and 0.71%, respectively. CYP2D6 locus was not in Hardy-Weinberg equilibrium. There was no statistical difference in allele frequencies between different genders (P > .05). There was significant difference in the frequencies of ACE (I/D) and NPPA (T2238C) loci among different regions of China (P < .05). Gender, ACE (I/D) and ADRB1 (1165G > C) gene polymorphism, smoking, homocysteine and HDL levels were associated hypertension. The mutation frequencies of ADRB1 (1165G > C) and CYP3A5*3 were high in hypertensive patients of Han ethnicity in Qingyang, suggesting these patients may be more sensitive to beta-blockers and calcium ion antagonists. Meanwhile, hypertension was associated with gender, ACE (I/D) and ADRB1 (1165G > C) gene polymorphisms, smoking, homocysteine and HDL levels.

Modulation of MicroRNA Expression During In Vitro Chondrogenesis.

Since their discovery in 1993, microRNAs (miRNAs) are now recognized as important epigenetic regulators of many mammalian cellular processes including proliferation, apoptosis, metabolism, and differentiation. These small non-coding RNAs function by interacting with specific regions in the 3'-untranslated region of mRNAs, thereby resulting in mRNA degradation or suppression of translation. Since miRNAs have the ability to target many mRNAs within a given cell type, a number of cellular pathways and networks may be regulated as a result. To study the function of miRNAs, a number of methods can be used to modulate their activity in cells such as synthetic mimics or antagomirs for short-term assays or viral-based approaches for longer-term experiments such as cell differentiation assays. In this chapter, we provide our methodology to constitutively overexpress a desired miRNA during in vitro chondrogenesis of human cartilage progenitor cells (CPCs). Specifically, we describe how we obtain CPCs from human articular cartilage specimens, how we generate and titrate lentivirus engineered to overexpress a precursor miRNA, how we transduce CPCs with lentivirus and differentiate them toward the chondrocyte lineage, and how we extract RNA and measure expression levels of the miRNA of interest during in vitro chondrogenesis. We also provide some data from our laboratory demonstrating that we can achieve and maintain miRNA overexpression for up to 14 days in cartilage pellet cultures. We predict that these lentiviral-based approaches will also be useful to study how miRNA modulation of progenitor cells affects cell differentiation and extracellular matrix production within three-dimensional biomaterial scaffolds.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9-10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

Gasdermin D deficiency attenuates arthritis induced by traumatic injury but not autoantibody-assembled immune complexes.

BACKGROUND: Gasdermin D (GSDMD) is cleaved by several proteases including by caspase-1, a component of intracellular protein complexes called inflammasomes. Caspase-1 also converts pro-interleukin-1ß (pro-IL-1ß) and pro-IL-18 into bioactive IL-1ß and IL-18, respectively. GSDMD amino-terminal fragments form plasma membrane pores, which mediate the secretion of IL-1ß and IL-18 and cause the inflammatory form of cell death pyroptosis. Here, we tested the hypothesis that GSDMD contributes to joint degeneration in the K/BxN serum transfer-induced arthritis (STIA) model in which autoantibodies against glucose-6-phosphate isomerase promote the formation of pathogenic immune complexes on the surface of myeloid cells, which highly express the inflammasomes. The unexpected outcomes with the STIA model prompted us to determine the role of GSDMD in the post-traumatic osteoarthritis (PTOA) model caused by meniscus ligamentous injury (MLI) based on the hypothesis that this pore-forming protein is activated by signals released from damaged joint tissues. METHODS: Gsdmd +/+ and Gsdmd-/- mice were injected with K/BxN mouse serum or subjected to MLI to cause STIA or PTOA, respectively. Paw and ankle swelling and DXA scanning were used to assess the outcomes in the STIA model whereas histopathology and micro-computed tomography (µCT) were utilized to monitor joints in the PTOA model. Murine and human joint tissues were also examined for GSDMD, IL-1ß, and IL-18 expression by qPCR, immunohistochemistry, or immunoblotting. RESULTS: GSDMD levels were higher in serum-inoculated paws compared to PBS-injected paws. Unexpectedly, ablation of GSDMD failed to reduce joint swelling and osteolysis, suggesting that GSDMD was dispensable for the pathogenesis of STIA. GSDMD levels were also higher in MLI compared to sham-operated joints. Importantly, ablation of GSDMD attenuated MLI-associated cartilage degradation (p = 0.0097), synovitis (p = 0.014), subchondral bone sclerosis (p = 0.0006), and subchondral bone plate thickness (p = 0.0174) based on histopathological and µCT analyses. CONCLUSION: GSDMD plays a key role in the pathogenesis of PTOA, but not STIA, suggesting that its actions in experimental arthropathy are tissue context-specific.

Migrating Progenitor Cells Derived From Injured Cartilage Surface Respond to Damage-Associated Molecular Patterns.

OBJECTIVE: To delineate the response of migrating chondrogenic progenitor cells (CPCs) that arose from the surface of mechanically injured articular cartilage to proinflammatory damage-associated-molecular-patterns (DAMPs). DESIGN: Bovine CPCs and non-CPC chondrocytes isolated from either impacted or scratched articular cartilage were studied. Those 2 types of cells were treated with mitochondrial DAMPs (MTDs; 10 nM fMLF and 10 µg/mL CpG DNA), or 10 nM HMGB1, or 10 ng/mL IL-1b for 24 hours. At the end of experiments, conditioned media and cell lysates were collected for analysis of expression levels of matrix metalloproteinases (MMPs), chemokines, and cytokines that are associated with cartilage degeneration with Western blotting and quantitative polymerase chain reaction. The difference of expression levels was compared by Welch's t-test. RESULTS: Our data indicated that HMGB1 and MTDs remarkably upregulated pro-MMP-13 expression in CPCs. Compared with non-CPCs, CPCs expressed significantly more baseline mRNAs of MMP-13, CXCL12, and IL-6. MTDs greatly increased the expression of MMP-13 and IL-6 in CPCs by over 100-fold (P < 0.001). MTDs also significantly increased IL-8 expression in CPCs to a similar extent (P < 0.001). However, when IL-1b was present, CPCs expressed less MMP-3 and active MMP-13 proteins as well as less CCL2 and IL-6 than did non-CPCs. CONCLUSIONS: We concluded that CPCs were more sensitive than non-CPCs in response to DAMPs, especially MTDs. The proinflammatory nature of CPCs implied their critical role in the early phase of posttraumatic osteoarthritis development.

SAGE1: a Potential Target Antigen for Lung Cancer T-Cell Immunotherapy.

A fundamental understanding of cancer-specific antigens is crucial for successful T-cell immunotherapy. Sarcoma antigen 1 (SAGE1) is a cancer/testis antigen that has not yet been verified for T-cell immunotherapy applications. Here, we examined SAGE1 RNA expression and carried out IHC analyses, revealing that SAGE1 is expressed in 50% of non-small cell lung-cancer samples (n = 40). To verify the immunogenicity of SAGE1, we discovered a novel HLA-A*24:02 (HLA-A24)-restricted SAGE1 epitope (SAGE1597-606, VFSTAPPAFI) using mass spectrometry and identified SAGE1597-606-specific T-cell clones and T-cell receptors (TCR) from peripheral bloods of HLA-A24+ donors. The highest affinity TCR VF3 (KD = 4.3 µM) demonstrated the highest antitumor potency. Moreover, VF3-transduced T cells mediated the efficient killing of HLA-A24+/SAGE1+ tumor cells in vitro and effectively inhibited the growth of lung cancer xenografts in mice. Together, our data suggest that SAGE1 could be a target for T-cell immunotherapies against lung cancer, while its specific TCRs could be candidates for developing reagents to treat SAGE1+ tumors.

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model.

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (µCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo µCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion. © 2021 American Society for Bone and Mineral Research (ASBMR).

Expression profiling of mitochondria-associated microRNAs during osteogenic differentiation of human MSCs.

Small non-coding microRNAs (miRNAs) have the ability to target and bind to many mRNAs within the cytosol resulting in reduced protein expression and modulation of a number of cellular pathways and networks. In addition to the cytosol, miRNAs have been identified in other cellular compartments and organelles, including the mitochondria. While a few mitochondria-associated miRNAs (mitomiRs) are predicted to be derived from the mitochondrial genome, the majority appear to be transcribed from nuclear DNA and somehow transported into the mitochondria. These findings raise interesting questions about why miRNAs are located in the mitochondria and if they play a role in regulating processes within these organelles. Previously published work from our laboratory showed that miR-181a/b can regulate osteogenesis, in part, by enhancing mitochondrial metabolism. In other published studies, miR-181 paralogs and many other miRNAs have been identified in mitochondrial extracts derived from common cell lines and specific primary cells and tissues. Taken together, we were motivated to identify mitomiR expression profiles during in vitro osteogenesis. Specifically, we obtained RNA from purified mitochondrial extracts of human bone marrow-derived mesenchymal stem/stromal cells (MSCs) and from whole cell extracts of MSCs at day 0 or following osteogenic induction for 3, 7 and 14 days. Utilizing Affymetrix GeneChip™ miRNA 4.0 arrays, mitomiR expression signatures were determined at each time point. Based on the Affymetrix detection above background algorithm, the total number of miRNAs detected in MSC mitochondria extracts was 527 (non-induced MSCs), 627 (day 3 induced), 372 (day 7 induced) and 498 (day 14 induced). In addition, we identified significantly differentially-expressed mitomiRs at day 7 and day 14 of osteogenic induction when compared to day 0 (fold change ≥1.5; adjusted p value <0.05). In general, the most pronounced and highly significant changes in mitomiR expression during osteogenesis were observed at the day 7 time point. Interestingly, most miRNAs found to be differentially-expressed in mitochondria extracts did not show significantly altered expression in whole cell extracts at the same time points during osteoblast differentiation. This array study provides novel information on miRNAs associated with the mitochondria in MSCs during differentiation toward the osteoblast phenotype. These findings will guide future research to identify new miRNA candidates that may function in regulating mitochondrial function and/or bone formation, homeostasis or repair.

Delineation of the 1q24.3 microdeletion syndrome provides further evidence for the potential role of non-coding RNAs in regulating the skeletal phenotype.

Microdeletions within 1q24 have been associated with growth deficiency, varying intellectual disability, and skeletal abnormalities. The candidate locus responsible for the various phenotypic features of this syndrome has previously been predicted to lie in the area of 1q24.3, but molecular evidence of the causative gene remains elusive. Here, we report two additional patients carrying the smallest reported 1q24 deletion to date. Patient 1 exhibited intrauterine growth retardation, shortening of the long bones, frontal bossing, microstomia, micrognathia, and a language acquisition delay. Her mother, Patient 2, displayed a broad forehead and nasal bridge, thick supraorbital ridges, and toe brachydactyly, along with learning disability and language acquisition delay. The microdeletion encompasses a 94 Kb region containing exon 14 and portions of the surrounding introns of the gene encoding dynamin 3 (DNM3), resulting in an in-frame loss of 38 amino acids. This microdeletion site also contains a long non-coding RNA (DNM3OS) and three microRNAs (miR-214, miR-199A2, and miR-3120). Following culture of patient-derived and control fibroblasts, molecular analyses were performed to determine expression levels of genes affected by the heterozygous deletion. Results show decreased expression of DNM3OS and miR-214-3p in patient fibroblasts cultured in an osteogenic induction medium. Overall, our data provide further evidence to support a functional role for non-coding RNAs in regulating the skeletal phenotype, and the potential of a functionally-impaired DNM3 protein causing the non-skeletal disease pathogenesis.

RNA Isolation from Articular Cartilage Tissue.

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3 µg of total RNA (including small noncoding RNAs) from 100 mg of human OA cartilage with a RIN = 7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

[Analysis of Genomic Landscape in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the gene mutation occurved in AML patients with 29 kinds of fusion genes and 51 kinds of tumor gene. METHODS: Next-generation sequencing (NGS) was used to detected the 49 kinds of targeted gene. FLT3 internal tandem duplication (FLT3-ITD), CALR, NPM1 and CEBPA mutation were detected by DNA-based PCR and Sanger sequencing. Twenty-nine kinds of fusion genes were dected by multiplex nested RT-PCR. RESULTS: The total gene mutation rate was 91% (109/121) in all the 121 patients. On average, 2.1 mutated genes per patient were identified, among these 121 patients, coexistence of ≥ 3 mutations was frequent (34.7%). The most commonly mutated genes were NRAS (23.96%, n=29), followed by NPM1 (14.04%, n=17), CEBPA double mutations (14.04%, n=17), KRAS (11.57%, n=14)，FLT3-ITD (10.74%, n=13), CSF3R (10.74%, n=13), TET2 (9.92%, n=12) and IDH1 (9.1%, n=11). Overall, fusion genes were detected in 47 (37.3%) patients, including AML/ETO (n=12), CBFß/MYH11 (n=11), PML/RARa (n=12), MLL rearranagement realated mutation MLL-X (n=10). TLS/ERG (n=1) and DEK/CAN (n=1) in an order of decreasing frequency. Patients with normal karyotype (NK)- AML exhibited more mutations in CEBPA, NPM1, TET2, RUNX1 and IDH1, comparing with abnormal karyotype patients. KRAS mutation in abnormal kayotype patients was significantly higher than that in normal kayotype patients (P=0.014). TP53 mutations were predominantly associated with complex cytogenetics (P=0.199). KRAS mutations were more frequent in core binding factor (CBF) acute myeloid leukemia (AML) and 11q23/MLL rearrangement leukemia, compared with NK-AML (P=0.006 and 0.003, respectively). KIT mutations predominated in CBF-AML (P=0.006). JAK2V617F mutations were detected in two patients and co-occurred with AML-ETO fusions. CONCLUSION: At least one mutation is observed in more than 90% patients. On average, more than 2 mutated genes per patient are identified. Some gene mutations are associated with gene rearrangement.