Empowering Engineered Muscle in Biohybrid Pump by Extending Connexin 43 Duration with Reduced Graphene Oxides

Engineered skeletal muscle act as therapeutics invaluable to treat injured or diseased muscle and a living material essential to assemble biological machinery. For normal development, skeletal myoblasts should express connexin 43, one of the gap junction proteins that promote myoblast fusion and myogenesis, during the early differentiation stage. However, myoblasts cultured in vitro often down-regulate connexin 43 before differentiation, limiting myogenesis and muscle contraction. This study demonstrates that tethering myoblasts with reduced graphene oxide (rGO) slows connexin 43 regression during early differentiation and increases myogenic mRNA synthesis. The whole RNA sequencing also confirms that the rGO on cells increases regulator genes for myogenesis, including troponin, while decreasing negative regulator genes. The resulting myotubes generated a three-fold larger contraction force than the rGO-free myotubes. Accordingly, a valveless biohybrid pump assembled with the rGO-tethered muscle increased the fluid velocity and flow rate considerably. The results of this study would provide an important foundation for developing physiologically relevant muscle and powering up biomachines that will be used for various bioscience studies and unexplored applications.

Dilated Odontoma Arising in the Mandibular Third Molar Germ: Report of a Case of an Unusual Lesion in an Uncommon Site

Dilated odontoma is the most severe variant of dens invaginatus. It is extremely uncommon in the posterior mandible. It is thought to originate during the morpho-differentiation stage of dental development. However, its etiology and pathogenesis remain obscure. We report here the clinical and pathologic findings of an incidentally discovered dilated odontoma arising in the left third mandibular molar germ of an 11-year-old male and a review of the pertinent literature. As dilated odontoma is not established as an independent entity in the current WHO classification of odontogenic tumors and is the result of a well-established developmental anomaly of the tooth (that is, the invagination of the enamel organ into the dental papilla), it should be better identified as dilated dens invaginatus.

Single-Cell Decoding of Minimal Residual Disease in Pediatric B Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, of which B cell ALL (B-ALL) accounts for up to 85% of all cases. Although risk-stratified chemotherapy significantly improves the clinical outcome of affected pediatric patients with B-ALL, relapse still occurs in approximately 10% of children, representing the main cause of pediatric cancer deaths. Minimal residual disease (MRD) that persists after chemotherapy was the most valuable prognostic marker for hematological malignancies and solid cancers. Unfortunately, our understanding of the resistance mechanisms elicited in MRD is limited due to the rarity and heterogeneity of these residual cells. In our study, we employed paired scRNA-seq and single-cell BCR sequencing (scBCR-seq) to study the distinct features of leukemic cells from longitudinal samples obtained at the diagnosis, residual and relapsed stages at the single-cell level. By performing unsupervised clustering of the scRNA-seq data from 16,543 bone marrow CD19 -CD34 +cells and 20,392 CD19 +cells of healthy donors, we successfully defined the cell clusters of different B cell development stages sequentially, from hematopoietic stem cell / lymphoid-primed multipotential progenitors (HSC/LMPP) to common lymphoid progenitor (CLP), proB, preBI, preBII, immature/mature B cells and finally, activated B cells. By referring to the landmarks of normal cells, we then employed scRNA-seq and scBCR-seq to further dissect the phenotypic complexities within and across 4 pediatric B-ALL diagnostic-relapsed pairs at the single cell level. Our study found that BCR states can be used to distinguish leukemic cells and normal cells. From the four relapsed pediatric B-ALL patients, we obtained a total of 104,055 CD19 +cells with paired scRNA-seq and scBCR-seq data encompassing the three stages of B cell differentiation that passed the quality control. By examining the scBCR-seq data at the diagnosis stage to distinguish leukemic and normal (or non-leukemic) B cells based on genome-wide expression patterns rather than a few marker genes, we employed a machine learning approach. To train the classifier, CD19 +cells with non-clonal BCRs in D19 samples were selected as "non-leukemic", and CD19 +cells with clonal BCRs (B265) and without detected BCRs (B590, B069 and B887) in D19 samples were selected as "leukemic". Using this strategy, we compared the gene expression profiles of the leukemic cells at the diagnosis and relapse for each patient, with the aim to identify specific features of relapse stage leukemia cells at the single-cell level. We found that the cell composition profile tended to shift to early differentiation stages in the relapsed samples in all four patients. By analyzing the differentiation stage, cell cycle and gene expression characteristics of relapsed cell samples at the single-cell level, we obtained some unique findings, such as significantly increased expression of CDKN1A in relapsed cells. To understand the basis of such specific differentiation stage and cell cycle transition during chemotherapy, we performed differential expression analysis to identify genes specifically altered at D19 compared to diagnosis and relapse. Then, pathway enrichment analysis applied to the differentially expressed genes revealed that the hypoxia pathway was one of the top hits, being significantly upregulated during intensified chemotherapy. We obtained experimental support by validating the efficacy of the HIF-1a inhibitor PX478 combined with chemotherapy drugs in two B-ALL cell lines and two primary B-ALL cells. In summary, our study leveraged single-cell transcriptomic analysis with paired BCR repertoire profiling to decode the molecular aberrations across the phenotypically heterogeneous disease, B-ALL. We also applied a powerful B cell development classifier and an innovative machine learning model for B-ALL cell identification at single-cell resolution to determine the clonal signatures, and to track residual cell evolution in a longitudinal manner from diagnosis, to post-treatment, and finally relapse. We propose that hypoxia signaling pathway activation might serve as valuable MRD therapeutic target. While this study provided great insights into the transcriptomic characteristics of MRD cells, practically, our analytical approach could readily be applied to other hematological malignancies and solid cancers. Disclosures No relevant conflicts of interest to declare.

A Chinese Child Presented with Early T Cell Precursor Lymphoblastic Lymphoma

T cell lymphoblastic lymphoma (T-LBL) is regarded as the leukemic phase of T cell acute lymphoblastic leukemia (T-ALL). The early T cell precursors ALL/LBL (ETP-LBL/ALL) are derived from thymic cells at the ETP differentiation stage and recognized as a high-risk subgroup of T-ALL/LBL. Most of these cases presented with ALL at the disease onset, but the ETP-LBL phase is uncommon. Here, we report a patient who presented with ETP-LBL at the disease onset. In this case, ALL developed even despite receiving chemotherapy, but the patient achieved a complete remission with intensive chemotherapy.

An Improved Protocol for Asymbiotic Seed Germination and Seedling Development of Paphiopedilum tigrinum

Paphiopedilum tigrinum is an endangered orchid with high ornamental value. However, seed germination and seedling regeneration in P. tigrinum is very difficult in vitro. Little is known about why P. tigrinum seedlings are difficult to propagate or how to improve the seed germination and seedling rates of this species. In this study, we investigated the developmental process of P. tigrinum from asymbiotic seed germination to seedling rooting by comparing it with P. appletoniantum, a much easier species for germination and seedling formation. We found that asymbiotic seed germination in P. tigrinum is limited by severe browning of the protocorm at the seed germination stage, and protocorm rooting at the differentiation stage was also proved to be difficult. The optimal medium for seed germination of P. tigrinum was a modified Harvais (mHa) medium supplemented with 0.5 mg·L−1 kinetin (Kin), 0.1 g·L−1 activated charcoal (AC) and 100 mL·L−1 coconut water (CW). At the protocorm differentiation stage, seedlings with 1–2 leaves were obtained on a 1/4 MS medium supplemented with 1.0 mg·L−1 6-benzylaminopurin (BA), 0.3 g·L−1 AC and 50–100 mL·L−1 CW after culturing for 120 day. At the seedling subculture stage, a 1/2 MS medium supplemented with 0.5–1.5 g·L−1 AC and 100 mL·L−1 CW was better for leaf and root growth of P. tigrinum. At the rooting stage, a 1/2 MS medium supplemented with 1.0 g·L−1 AC, 0.5 g·L−1 dolomite flour, 15 g·L−1 potato homogenate and 30 g·L−1 banana homogenate was most suitable for the growth and rooting of seedlings. This study has established an effective protocol for seed germination and seedling regeneration of P. tigrinum.

Transcriptome Analysis in a Primary Human Muscle Cell Differentiation Model for Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.

Chronic Hepatitis C Pathogenesis: Immune Response in the Liver Microenvironment and Peripheral Compartment

Chronic hepatitis C (CHC) pathogenic mechanisms as well as the participation of the immune response in the generation of liver damage are still a topic of interest. Here, we evaluated immune cell populations and cytokines in the liver and peripheral blood (PB) to elucidate their role in CHC pathogenesis. B, CTL, Th, Treg, Th1, Th17, and NK cell localization and frequency were evaluated on liver biopsies by immunohistochemistry, while frequency, differentiation, and functional status on PB were evaluated by flow cytometry. TNF-α, IL-23, IFN-γ, IL-1β, IL-6, IL-8, IL-17A, IL-21, IL-10, and TGF-β expression levels were quantified in fresh liver biopsy by RT-qPCR and in plasma by CBA/ELISA. Liver CTL and Th1 at the lobular area inversely correlated with viral load (r = −0.469, p =0.003 and r = −0.384, p = 0.040). Treg correlated with CTL and Th1 at the lobular area (r = 0.784, p < 0.0001; r = 0.436, p = 0.013). Th17 correlated with hepatic IL-8 (r = 0.52, p < 0.05), and both were higher in advanced fibrosis cases (Th17 p = 0.0312, IL-8 p = 0.009). Hepatic cytokines were higher in severe hepatitis cases (IL-1β p = 0.026, IL-23 p = 0.031, IL-8 p = 0.002, TGF-β, p= 0.037). Peripheral NK (p = 0.008) and NK dim (p = 0.018) were diminished, while NK bright (p = 0.025) was elevated in patients vs. donors. Naïve Th (p = 0.011) and CTL (p = 0.0007) were decreased, while activated Th (p = 0.0007) and CTL (p = 0.0003) were increased. IFN-γ production and degranulation activity in NK and CTL were normal. Peripheral cytokines showed an altered profile vs. donors, particularly elevated IL-6 (p = 0.008) and TGF-β (p = 0.041). Total hepatic CTLs favored damage. Treg could not prevent fibrogenesis triggered by Th17 and IL-8. Peripheral T-lymphocyte differentiation stage shift, elevated cytokine levels and NK-cell count decrease would contribute to global disease.

Sulfation of O-glycans on mucin-type proteins from serous ovarian epithelial tumors

Despite that sulfated O-linked glycans are abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression to defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers, and with 1-4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or-2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared to tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 were differentially expressed in benign, borderline or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.

BILATERAL FUSION OF PRIMARY MANDIBULAR CANINE AND FIRST MOLAR: A RARE OCCURRENCE

Objective: Fusion is a developmental anomaly of the teeth. It is dened as the union of two independently developing primary or permanent teeth. Aberrations in morpho differentiation stage of tooth development leads to abnormal forms and sizes of teeth. This paper reports a rare case of bilateral fusion of mandibular primary rst molar and canine. A 10 year old boy reported with the chief complaint of difculty in chewing due to mobility of teeth in lower back teeth region. Intraoral, radiographic and histopathological examinations indicated fusion of mandibular deciduous canine and deciduous rst molar on both sides. According to the treatment plan, the fused teeth were extracted. Fusion of teeth is caused by various etiological factors, can be diagnosed by amalgamation of clinical, radiological and histopathological examinations and can be treated by multidisciplinary approach. Accurate diagnosis of dental anomalies helps in prompt treatment, which in turn avoids future orthodontic complications and better prognosis.

Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage

Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for treatment customization. However, the absence of a uniform CTC isolation platform and consensus on an analytical assay has prevented the incorporation of these measurements in routine clinical practice. Here, we present a single-CTC sensitive digital droplet PCR (ddPCR) assay for the quantitation of the two most common AR-Vs, AR-V7, and AR-v567es, using antigen agnostic CTC enrichment. In a cohort of 29 mCRPC patients, we identify AR-V7 in 66% and AR-v567es in 52% of patients. These results are corroborated using another gene expression platform (NanoStringTM) and by analysis of RNA-Seq data from patients with mCRPC (SU2C- PCF Dream Team). We next quantify AR-V expression in matching EpCAM-positive vs EpCAM-negative CTCs, as EpCAM-based CTC enrichment is commonly used. We identify lower AR-V prevalence in the EpCAM-positive fraction, suggesting that EpCAM-based CTC enrichment likely underestimates AR-V prevalence. Lastly, using single CTC analysis we identify enrichment for AR-v567es in patients with neuroendocrine prostate cancer (NEPC) indicating that AR-v567es may be involved in lineage plasticity, which warrants further mechanistic interrogation.