Cancer-Associated Fibroblast Heterogeneity in Malignancy with Focus on Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) remains an understudied and significant global cancer killer and dismal survival rates have not changed in decades. A better understanding of the molecular basis of OSCC progression and metastasis is needed to develop new approaches for treating this disease. The supportive network surrounding cancer tumor cells known as the tumor microenvironment (TME) has gained increasing interest lately since it performs essential protumorigenic functions. Cancer-associated fibroblasts (CAFs) are one of the main cell types in the TME and are known to play a key role in influencing the biological behavior of tumors. CAFs present a heterogeneity both in phenotype as well as functions, leading to the suggestion of different CAF subtypes in several cancer forms. The task to subtype CAFs in OSCC has, however, just begun, and there is today no united way of subtyping CAFs in this disease. This review aims to define the features of CAFs and to summarize CAF subtype research in malignancy with focus on OSCC including aspects as disease prognosis and therapeutic opportunities.

Real-world outcomes with immuno-oncology therapies in advanced melanoma: final results of the OPTIMIzE registry study.

Aim: The OPTIMIzE registry study evaluated real-world outcomes in patients with advanced melanoma receiving immuno-oncology therapies. Materials and methods: Data were collected for patients treated with anti-programmed death 1 (PD-1) monotherapy (nivolumab or pembrolizumab; n = 147) or nivolumab plus ipilimumab (n = 81) from 2015-2017 and followed for ≥3 years. Results: Nivolumab plus ipilimumab versus anti-PD-1 monotherapy was associated with a nonsignificantly lower risk of death (adjusted HR: 0.83; 95% CI: 0.54-1.28; p = 0.41), higher disease control rate (72 vs 56%; p = 0.04), and stable quality of life, but more grade 3-4 treatment-related adverse events (54 vs 26%; p < 0.0001). Conclusion: These results support the use of immuno-oncology therapy in advanced melanoma.

Melanoma is a serious form of skin cancer that develops from melanocytes, which are pigment cells that give the skin, hair, and other tissues their color. At advanced stages of spread, melanoma can be life-threatening. However, immunotherapy, a type of therapy that helps the body's immune system to destroy cancer cells, allows some patients with advanced melanoma to live longer. The OPTIMIzE study looked at how well patients with advanced melanoma did when treated with different immunotherapies. These patients were treated in a real-world setting, such as a doctor's office, and were not participating in a clinical trial. Compared with clinical trials, real-world studies like the OPTIMIzE study may include a more varied group of patients because of the less selective study enrollment requirements. In the OPTIMIzE study, patients were treated with either a single immunotherapy (nivolumab or pembrolizumab alone) or a combination of two immunotherapies (nivolumab plus ipilimumab). Both single and combination immunotherapies were effective and tolerable. Patients receiving nivolumab plus ipilimumab had greater tumor shrinkage than patients receiving nivolumab or pembrolizumab alone, but with more side effects from their treatment. Despite the occurrence of side effects with both single and combination immunotherapies, patients reported that their quality of life remained stable while being treated. The OPTIMIzE study shows that immunotherapy is effective and tolerable for patients with advanced melanoma in the real-world setting. This information may help doctors with selecting treatments for their patients with advanced melanoma. Clinical Trial Registration: NCT02780089 (ClinicalTrials.gov).

Extracellular vesicles promote activation of pro-inflammatory cancer-associated fibroblasts in oral cancer.

Introduction: Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer and has a survival rate of ∼50% over 5 years. New treatment strategies are sorely needed to improve survival rates-and a better understanding of the mechanisms underlying tumorigenesis is needed to develop these strategies. The role of the tumor microenvironment (TME) has increasingly been identified as crucial in tumor progression and metastasis. One of the main constituents of the TME, cancer-associated fibroblasts (CAFs), plays a key role in influencing the biological behavior of tumors. Multiple mechanisms contribute to CAF activation, such as TGFß signaling, but the role of extracellular vesicles (EVs) in CAF activation in OSCC is poorly understood. Assessing the impact of oral cancer-derived EVs on CAF activation will help to better illuminate OSCC pathophysiology and may drive development of novel treatments options. Methods: EVs were isolated from OSCC cell lines (Cal 27, SCC-9, SCC-25) using differential centrifugation. Nanoparticle tracking analysis was used for EV characterization, and Western blot to confirm the presence of EV protein markers. Oral fibroblasts were co-cultured with enriched EVs, TGFß, or PBS over 72 h to assess activation. Flow cytometry was used to evaluate CAF markers. RNA collected from fibroblasts was extracted and the transcriptome was sequenced. Conditioned media from the co-cultures was evaluated with cytokine array profiling. Results: OSCC-derived EVs can activate oral fibroblasts into CAFs that are different from those activated by TGFß, suggesting different mechanisms of activation and different functional properties. Gene set enrichment analysis showed several upregulated inflammatory pathways in those CAFs exposed to OSCC-derived EVs. Marker genes for inflammatory CAF subtypes were also upregulated, but not in CAFs activated by TGFß. Finally, cytokine array analysis on secreted proteins revealed elevated levels of several pro-inflammatory cytokines from EV-activated CAFs, for instance IL-8 and CXCL5. Discussion: Our results reveal the ability of OSCC-derived EVs to activate fibroblasts into CAFs. These CAFs seem to have unique properties, differing from TGFß-activated CAFs. Gaining an understanding of the interplay between EVs and stromal cells such as CAFs could lead to further insights into OSCC tumorigenesis and potential novel therapeutics.

Induced Vascular Normalization-Can One Force Tumors to Surrender to a Better Microenvironment?

Immunotherapy has changed the way many cancers are being treated. Researchers in the field of immunotherapy and tumor immunology are investigating similar questions: How can the positive benefits achieved with immunotherapies be enhanced? Can this be achieved through combinations with other agents and if so, which ones? In our view, there is an urgent need to improve immunotherapy to make further gains in the overall survival for those patients that should benefit from immunotherapy. While numerous different approaches are being considered, our team believes that drug delivery methods along with appropriately selected small-molecule drugs and drug candidates could help reach the goal of doubling the overall survival rate that is seen in some patients that are given immunotherapeutics. This review article is prepared to address how immunotherapies should be combined with a second treatment using an approach that could realize therapeutic gains 10 years from now. For context, an overview of immunotherapy and cancer angiogenesis is provided. The major targets in angiogenesis that have modulatory effects on the tumor microenvironment and immune cells are highlighted. A combination approach that, for us, has the greatest potential for success involves treatments that will normalize the tumor's blood vessel structure and alter the immune microenvironment to support the action of immunotherapeutics. So, this is reviewed as well. Our focus is to provide an insight into some strategies that will engender vascular normalization that may be better than previously described approaches. The potential for drug delivery systems to promote tumor blood vessel normalization is considered.

Clinical outcomes of a novel porcine small intestinal submucosa patch for full-thickness hand skin defects: a retrospective investigation.

OBJECTIVE: To investigate the clinical outcomes of a novel soft tissue repair patch (porcine small intestinal submucosa patch, SIS patch) in the treatment of full-thickness hand skin defects. METHODS: From January 2017 to July 2019, 80 patients with hand soft tissue defects, who met the inclusion criteria, were retrospectively reviewed and divided into two groups. After debridement, patients in group A were treated with the novel SIS patch to cover the wound, and patients in group B were treated with autologous skin graft. The dimensions of skin defect area and healing outcome were evaluated and recorded. Scar assessment was carried out using Scar Cosmesis Assessment and Rating Scale (SCAR scale) at the last follow-up postoperation, and the recovery of wound sensation was assessed at the same time using British Medical Research Council (BMRC) grading of sensorimotor recovery. All the data were collected and statistically analyzed. RESULTS: A total of 80 patients were enrolled in the study with 40 patients in each group. Four patients in group A and 5 patients in group B were excluded due to wound infection and lost to follow-up. There were 36 patients in group A and 35 patients in group B finally got follow-up postoperation with mean interval of 12.75 ± 5.61 months in group A and 14.11 ± 5.42 months in group B. The dimensions of skin defect area in group A ranged from 7.5 to 87.5 cm2 (mean 25.97 ± 18.66 cm2) and in group B ranged from 7.5 to 86.25 cm2 (mean 33.61 ± 19.27 cm2) which have no significant difference (P > 0.05). SCAR scale results of group A and group B were 10.98 ± 0.33 and 9.49 ± 0.35, respectively, and the difference was statistically significant (P < 0.05). BMRC grading results showed 6 cases of S4, 11 cases of S3+, 5 cases of S3, 6 cases of S2, 6 cases of S1 and 2 cases of S0 in group A, and 8 cases of S4, 10 cases of S3+, 7 cases of S3, 4 cases of S2, 5 cases of S1, and 1 case of S0 in group B, which had no significant difference between them (P > 0.05). CONCLUSIONS: The novel SIS patch is an applicable biological material in the treatment of hand skin defect, which could achieve a better cosmetic appearance of the newborn skin tissue.

High birth weight and greater gestational age at birth in singletons born after frozen compared to fresh embryo transfer.

OBJECTIVE: While many studies agree that the fetal birth weight is higher after frozen embryo transfer (FET), few studies have explored the difference in fetal weight change during such pregnancies. This cohort study was to identify the difference in fetal birth weight and gestational age at birth between singletons born following fresh ET and those born following FET. MATERIALS AND METHODS: This was a hospital-based cohort study using clinical data from the Kaohsiung Chang Gung Memorial Hospital Obstetric and Neonatal Database from January 1, 2007, to December 1, 2018. A sample of 784 eligible women who had singleton pregnancies and live-born deliveries after 428 fresh ET or 356 FET between January 2007 and December 2018. RESULTS: Compared with those in the fresh ET group, singletons in the FET group had higher birth weight (3137 g [2880-3441 g] vs. 3060 g [2710-3340 g], p < 0.05), were born later (39.0 weeks of gestation [38.0-40.0 weeks] vs. 38.0 weeks of gestation [37.0-39.0 weeks], p < 0.05), and had a lower incidence of preterm birth (10.4% vs. 15.2%, p < 0.05). The difference in birth weight was not associated with maternal body weight (BW) or body mass index, increase in maternal BW in the third trimester, but related to the total increase in maternal BW during pregnancy. CONCLUSIONS: The birthweight of singletons born following FET and fresh ET became significant in the late third trimester. The main reason is that singletons conceived from FET were at a lower relative risk of preterm delivery and had a higher gestational age at birth.

Characterizing SARS-CoV-2 Transcription of Subgenomic and Genomic RNAs During Early Human Infection Using Multiplexed Droplet Digital Polymerase Chain Reaction.

BACKGROUND: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission requires understanding SARS-CoV-2 replication dynamics. METHODS: We developed a multiplexed droplet digital polymerase chain reaction (ddPCR) assay to quantify SARS-CoV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from genomic RNAs (gRNAs). We applied the assay to specimens from 144 people with single nasopharyngeal samples and 27 people with >1 sample. Results were compared to quantitative PCR (qPCR) and viral culture. RESULTS: sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio sgRNA:gRNA was stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Daily testing of 6 persons revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. sgRNA:gRNA is constant during infection despite changes in viral culture. CONCLUSIONS: Ct values from qPCR correlate with active viral replication. More work is needed to understand why some cultures are negative despite presence of sgRNA.

Treatment Patterns, Survival, Quality of Life, and Healthcare Resource Use Among Patients With Triple-Class Refractory Multiple Myeloma in US Clinical Practice: Findings From the Connect MM Disease Registry.

BACKGROUND: Adults with triple-class refractory (TCR) multiple myeloma (MM) have limited treatment options and poor prognosis, but the burden of TCR MM has not been well characterized. This study evaluated treatment patterns, overall survival (OS), health-related quality of life (HRQoL), and healthcare resource use (HCRU) among patients with TCR MM in US clinical practice. PATIENTS AND METHODS: Patients with TCR MM in the Connect MM Registry (NCT01081028; a large, US, multicenter, prospective observational cohort study of patients with newly diagnosed MM) were included. Patient characteristics, treatment patterns, HRQoL, and HCRU were analyzed using descriptive statistics. OS was calculated using Kaplan-Meier methodology for the overall cohort and for patients with/without ≥1 post-TCR line of therapy (LOT). RESULTS: A total of 232 patients with TCR MM were included; 155 (67%) had ≥1 post-TCR LOT (post-TCR-Treated subgroup; median 9.9 months of follow-up). Most common post-TCR treatments were carfilzomib (47%), pomalidomide (40%), and daratumumab (26%); median treatment duration was 3.3 months. Median OS was 9.9 months in the overall population, 10.8 months in post-TCR-Treated patients, and 2.6 months for those with no new post-TCR LOT. HRQoL deteriorated and pain increased over 1 year of follow-up, with clinically meaningfully changes in EQ-5D (mean, -0.06 points) and FACT-G (mean, -9.9 points). 124 (53%) patients had ≥1 all-cause hospitalization and 58 (25%) had ≥1 MM-related hospitalization; median annualized length of stay was 35.3 and 42.9 days, respectively. CONCLUSION: The burden of TCR MM is substantial, emphasizing the need for more effective treatment options in the TCR setting.

Higher Risk of Tumor Recurrence in NASH-Related Hepatocellular Carcinoma Following Curative Resection.

The outcomes for patients with NASH-related HCC after curative resection have not been clarified. This study compared the overall survival (OS), time-to-tumor recurrence (TTR), and recurrence-free survival (RFS) associated with NASH-related HCC and virus-related HCC after resection. Methods: Patients with HCC who underwent curative resection were retrospectively enrolled. Baseline characteristics, including disease etiologies and clinical and tumor features, were reviewed. The primary outcomes were OS, TTR, and RFS. Results: Two hundred and six patients were enrolled (HBV: n = 121, HCV: n = 54, NASH: n = 31). Of those with virus-related HCC, 84.0% achieved viral suppression. In both the overall and propensity-score-matched cohorts, those with NASH-related HCC experienced recurrence significantly earlier than those with virus-related HCC (median TTR: 1108 days vs. non-reached; p = 0.03). Through multivariate analysis, NASH-related HCC (hazard ratio (HR), 2.27; 95% confidence interval (CI), 1.25-4.12) was independently associated with early recurrence. The unadjusted RFS rate of the NASH-related HCC group was lower than the virus-related HCC group. There was no difference in the OS between the two groups. Conclusions: NASH-related HCC was associated with earlier tumor recurrence following curative resection compared to virus-related HCC. Post-surgical surveillance is crucial for detecting early recurrence in patients with NASH-related HCC.

Validation of a Deep Learning-based Automatic Detection Algorithm for Measurement of Endotracheal Tube-to-Carina Distance on Chest Radiographs.

BACKGROUND: Improper endotracheal tube (ETT) positioning is frequently observed and potentially hazardous in the intensive care unit. The authors developed a deep learning-based automatic detection algorithm detecting the ETT tip and carina on portable supine chest radiographs to measure the ETT-carina distance. This study investigated the hypothesis that the algorithm might be more accurate than frontline critical care clinicians in ETT tip detection, carina detection, and ETT-carina distance measurement. METHODS: A deep learning-based automatic detection algorithm was developed using 1,842 portable supine chest radiographs of 1,842 adult intubated patients, where two board-certified intensivists worked together to annotate the distal ETT end and tracheal bifurcation. The performance of the deep learning-based algorithm was assessed in 4-fold cross-validation (1,842 radiographs), external validation (216 radiographs), and an observer performance test (462 radiographs) involving 11 critical care clinicians. The performance metrics included the errors from the ground truth in ETT tip detection, carina detection, and ETT-carina distance measurement. RESULTS: During 4-fold cross-validation and external validation, the median errors (interquartile range) of the algorithm in ETT-carina distance measurement were 3.9 (1.8 to 7.1) mm and 4.2 (1.7 to 7.8) mm, respectively. During the observer performance test, the median errors (interquartile range) of the algorithm were 2.6 (1.6 to 4.8) mm, 3.6 (2.1 to 5.9) mm, and 4.0 (1.7 to 7.2) mm in ETT tip detection, carina detection, and ETT-carina distance measurement, significantly superior to that of 6, 10, and 7 clinicians (all P < 0.05), respectively. The algorithm outperformed 7, 3, and 0, 9, 6, and 4, and 5, 5, and 3 clinicians (all P < 0.005) regarding the proportions of chest radiographs within 5 mm, 10 mm, and 15 mm error in ETT tip detection, carina detection, and ETT-carina distance measurement, respectively. No clinician was significantly more accurate than the algorithm in any comparison. CONCLUSIONS: A deep learning-based algorithm can match or even outperform frontline critical care clinicians in ETT tip detection, carina detection, and ETT-carina distance measurement.

Graft Inflow Modulation in Living-Donor Liver Transplantation: Hepatic Hemodynamic Changes in Splenic Artery Ligation and Splenectomy.

BACKGROUND Excessive portal flow to an allograft was a key mechanism for small-for-size syndrome in living-donor liver transplantation (LDLT). Good outcomes in LDLT by graft inflow modulation (GIM) using a small graft were reported, but the effect on hepatic hemodynamics is undefined. This report summarizes our experience with GIM and compares the effects of splenic artery ligation (SAL) and splenectomy on hepatic hemodynamic changes. MATERIAL AND METHODS Ninety-nine patients who underwent adult-to-adult LDLT from June 2014 to December 2020 were included in this study. GIM was performed in 36 patients (17 patients with SAL and 19 with splenectomy). RESULTS The GIM group had lower graft-to-recipient weight compared to the no-modulation group (median, 0.91% versus 1.04%, P=0.022). Initial portal venous flow (PVF) was higher in the GIM group (median, 311 versus 156 ml/min/100 g, P<0.001). After GIM, PVF decreased to 224 ml/min/100 g. One-year graft survival with GIM was 89.9%, and for the no-modulation group it was 86.6% (P=0.945). In the subgroup analysis, the efficacy of decompressing PVF was higher in the splenectomy subgroup (median, 14.3% versus 41.8%, P=0.002). CONCLUSIONS GIM was useful for grafts with high PVF. Splenectomy modulated excessive PVF more effectively than did SAL. Perioperative hepatic hemodynamic changes could assist surgeons in selecting different GIM strategies.

Characterizing SARS-CoV-2 transcription of subgenomic and genomic RNAs during early human infection using multiplexed ddPCR

Control of SARS-CoV-2 (SCV-2) transmission is a major priority that requires understanding SCV-2 replication dynamics. We developed and validated novel droplet digital PCR (ddPCR) assays to quantify SCV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from full-length genomic RNAs (gRNAs) in a multiplexed format. We applied this multiplex ddPCR assay to 144 cross-sectional nasopharyngeal samples. sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio of sgRNA:gRNA was remarkably stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Interestingly, adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Longitudinal daily testing of 6 persons for up to 14 days revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. Further, sgRNA:gRNA is constant during infection despite changes in viral culture. These data indicate stable viral transcription during infection. More work is needed to understand why cultures are negative despite persistence of viral RNAs.

Transiently hypoxic tumour cell turnover and radiation sensitivity in human tumour xenografts.

BACKGROUND: Solid tumour perfusion can be unstable, creating transiently hypoxic cells that can contribute to radiation resistance. We investigated the in vivo lifetime of transiently hypoxic tumour cells and chronically hypoxic tumour cells during tumour growth and following irradiation. METHODS: Hypoxic cells in SiHa and WiDr human tumour xenografts were labelled using pimonidazole and EF5, and turnover was quantified as the loss of labelled cells over time. The perfusion-modifying drug pentoxifylline was used to reoxygenate transiently hypoxic cells prior to hypoxia marker administration or irradiation. RESULTS: Chronically hypoxic cells constantly turnover in SiHa and WiDr tumours, with half-lives ranging from 42-82 h and significant numbers surviving >96 h. Transiently hypoxic cells constitute 26% of the total hypoxic cells in WiDr tumours. These transiently hypoxic cells survive at least 24 h, but then rapidly turnover with a half-life of 34 h and are undetectable 72 h after labelling. Transiently hypoxic cells are radiation-resistant, although vascular dysfunction induced by 10 Gy of ionising radiation preferentially kills transiently hypoxic cells. CONCLUSIONS: Transiently hypoxic tumour cells survive up to 72 h in WiDr tumours and are radiation-resistant, although transiently hypoxic cells are sensitive to vascular dysfunction induced by high doses of ionising radiation.

Characteristics of Optic Disc and Visual Field Changes in Patients with Thyroid-Associated Orbitopathy and Open-Angle Glaucoma.

This study aimed to characterize the changes in the visual field (VF) patterns and disc morphology of patients with thyroid-associated orbitopathy (TAO) and open-angle glaucoma (OAG). A retrospective review of the medical records at the Tri-Service General Hospital in Taiwan identified 396 eyes of 198 patients with thyroid-associated glaucoma. A final follow-up of VF examination in 140 eyes revealed 114 eyes with VF defects, indicating disease progression. The characteristics of and changes in disc morphology, optical coherence tomography findings, and VF defects were statistically analyzed. The most common VF defects at the initial diagnosis and the end of the follow-up period were inferior partial arcuate (17%) and paracentral (15%) defects, respectively. The most common VF defect in patients with unspecific disc signs was an unspecific scotoma (13%). The most common optic disc feature was disc cupping (51%), followed by parapapillary atrophy (48%). The most frequent location of nerve fiber layer thinning was the inferotemporal region (48%). VF defects showed a significantly more pronounced progression in the non-nerve fiber bundle group than in the nerve fiber bundle group (p < 0.001). This study details the characteristics and progression of disc morphology and VF defects in patients with TAO and OAG.

Germinal center hypoxia in tumor-draining lymph nodes negatively regulates tumor-induced humoral immune responses in mouse models of breast cancer.

Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.

Enterovirus A71 Induces Neurological Diseases and Dynamic Variants in Oral Infection of Human SCARB2-Transgenic Weaned Mice.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae, poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling.

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.

Comparison of anatomic and non-anatomic resections for very early-stage hepatocellular carcinoma: The importance of surgical resection margin width in non-anatomic resection.

BACKGROUND: The superiority of anatomic resection (AR) over non-anatomic resection (NAR) for very early-stage hepatocellular carcinoma (HCC) has remained a topic of debate. Thus, this study aimed to compare the prognosis after AR and NAR for single HCC less than 2 cm in diameter. METHODS: Consecutive patients with single HCC of diameter less than 2 cm who underwent curative hepatectomy between 1997 and 2017 were included in this retrospective study. RESULTS: In total, 159 patients were included in this study. Of these, 52 patients underwent AR (AR group) and 107 patients underwent NAR (NAR group). No significant differences were noted in recurrence-free survival (RFS) and overall survival (OS) between the AR and NAR groups (P = 0.236 and P = 0.363, respectively). Multivariate analysis revealed that low preoperative platelet count and presence of satellite nodules were independent prognostic factors of RFS and OS. Wide surgical resection margin did not affect RFS (P = 0.692) in the AR group; however, in the NAR group, RFS was found to be higher with surgical resection margin widths ≥1 cm than with surgical resection margin widths <1 cm (P = 0.038). CONCLUSIONS: Prognosis was comparable between the NAR and AR groups for very early-stage HCC with well-preserved liver function. For better oncologic outcomes, surgeons should endeavor in keeping the surgical resection margin widths during NAR ≥1 cm.