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Right atrial thrombosis, which occurs alone, is rare in clinical practice. Its incidence and mechanism are unclear, but susceptibility factors are usually present at its occurrence: ischemic heart disease, heart failure, atrial fibrillation, and chronic kidney disease. Complete isolated right atrial thrombosis rarely occurs. We report here a 47-year-old male patient with a right atrial mass on cardiac ultrasound and chest computed tomography (CT) and a history of previous right heart surgery with type 2 diabetes mellitus and atrial fibrillation, complaining of “chest tightness and shortness of breath after activity for half a month.” The patient was admitted to the hospital and underwent right atrial mass resection, and the postoperative pathology showed “right atrial thrombus.” As right atrial thrombus is very rare and can be a serious threat to life when it occurs in the heart, the prevention and treatment of right atrial thrombus are very important. Based on the analysis of this case, we believe that for patients with special medical history such as “post right heart surgery and atrial fibrillation,” we need to be vigilant for atrial thrombosis.

Background Brain metastasis is an important cause of breast cancer-related death. Aim We evaluated the relationships between breast cancer subtype and prognosis among patients with brain metastasis at the initial diagnosis. Methods The Surveillance, Epidemiology, and End Results database was searched to identify patients with brain metastasis from breast cancer between 2010 and 2015. Multivariable Cox proportional hazard models were used to identify factors that were associated with survival among patients with initial brain metastases. The Kaplan–Meier method was used to compare survival outcomes according to breast cancer subtype. Results Among 752 breast cancer patients with brain metastasis at diagnosis, 140 patients (18.6%) underwent primary surgery and 612 patients (81.4%) did not undergo surgery, while 460 patients (61.2%) received chemotherapy and 292 patients (38.8%) did not receive chemotherapy. Multivariable analysis revealed that, relative to HR+/HER2– breast cancer, HR–/HER2– breast cancer was associated with significantly poorer overall survival (hazard ratio: 2.52, 95% confidence interval: 1.99–3.21), independent of age, sex, race, marital status, insurance status, grade, liver involvement, lung involvement, primary surgery, radiotherapy, and chemotherapy. The median overall survival intervals were 12 months for HR+/HER2−, 19 months for HR+/HER2+, 11 months for HR−/HER2+, and 6 months for HR–/HER2– ( P < .0001). Relative to HR+/HER2– breast cancer, HR–/HER2– breast cancer was associated with a significantly higher risk of mortality among patients, and the association was stronger among patients who received chemotherapy ( p for interaction = .005). Conclusions Breast cancer subtype significantly predicted overall survival among patients with brain metastasis at diagnosis.

Objective: The objective of this work is to discuss and analyze the related factors of lower extremity fracture complicated by preoperative deep vein thrombosis (DVT). Methods: A total of 11,891 patients with closed fractures of lower extremities were selected. By analyzing each patient’s gender, age, presence or absence of diabetes and hypertension, preoperative plasma D-dimer level, and color Doppler ultrasound of the lower extremity vein, the pertinent factors of the patients with lower extremity fractures complicated by preoperative DVT were analyzed. Results: A total of 578 with preoperative DVT were detected, displaying a total incidence of 4.86%. All patients were categorized into either the DVT group or non-DVT group. The results demonstrate that there were statistically significant differences between the 2 groups in age, the presence of diabetes and hypertension, the fracture site, and the preoperative plasma D-dimer level ( P < 0.05). Logistic multivariate analysis revealed that age, the presence of diabetes, and the preoperative plasma D-dimer level of patients were independent risk factors for lower extremity fracture complicated by DVT. Conclusion: Age, the presence of diabetes, the fracture site, and increased D-dimer levels were found to be potential risk factors and indicators for preoperative DVT in patients with lower extremity fractures. In addition, the preoperative plasma D-dimer level has certain guiding significance for the prediction of venous thrombosis after lower extremity fracture, which is conducive to the early prediction and diagnosis of DVT, but it often must be followed with good clinic acumen and examinations.

Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT) occurs more frequently in cancer patients than in the general population. A retrospective cross-sectional study was carried out in patients with solid tumor complicated with VTE admitted to the Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between January 1st, 2008 and December 31th, 2017. The incidence of VTE in hospitalized cancer patients was 1.8%, twice the incidence of VTE in hospitalized non-cancer patients. The annual incidence of cancer-associated VTE in our center varied between 1.6% in 2015 and 0.4% in 2009 with an overall average incidence of 1.3% over the research decade. BMI values of 549(67.7%) cancer patients were within the normal range, but none of patients had BMI greater than 35 kg/m 2 . 747(92.1%) cancer patients had ECOG PS score ≤ 2 and 481(59.3%) had distant metastasis. Patients with pancreatic, bladder, ovarian and endometrial cancer had the highest incidence of VTE. Upper extremity DVT (47.2%) was more common in cancer patients and might be closely associated with CVC (74.9%), while lower extremities DVT (36.1%) intended to PE development (15.0%). The annual incidence rates showed a fluctuating and upward trend over the research decade. VTE occurrence was closely related to tumor stage, tumor site, catheterization and anti-neoplasm therapy in cancer patients.

Background: Whether statins can reduce the incidence of cancers has been an interesting topic in recent years. This meta-analysis aimed to determine the relationship between statin treatment with the risk of hepatocellular carcinoma. Methods: Studies published up to July 2019 were screened from databases. The data from approved studies were pooled. Random-effects or fixed-effects model was used to calculate the relative risk with 95% CIs in the overall group and subgroups. Sensitivity and meta-regression analyses were performed, and publication bias was evaluated. Results: A total of 18 studies involving 1 611 596 patients were included in this meta-analysis. The overall result showed a significantly reduced risk of hepatocellular carcinoma (relative risk = 0.54, 95% CI: 0.42-0.66) in statin users. In comparison to the risk in nonstatin users, the risk of hepatocellular carcinoma was reduced in all subgroups. The dose of statins and their pharmacokinetics can partly explain the heterogeneity in the overall meta-analysis ( I 2 = 94.6%, P = .000). A dose-dependent effect of statin use for the reduced risk of hepatocellular carcinoma was found. Conclusions: Findings from this meta-analysis support that statin use can significantly reduce the incidence of hepatocellular carcinoma.

Maintaining the efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse transcriptase ACTivity crispR (REACTR) for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to the synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

Despite global calls to eliminate cervical cancer, rates of cervical cancer incidence and mortality remain high in resource-limited settings, where it is challenging to implement and sustain screening, diagnosis, and treatment programs. The presence of high-risk HPV mRNA in cervical cells is a sensitive and specific biomarker of cervical precancer. Yet, current testing methods are too costly and complex for use in resource-limited settings. Here, we present a novel method for semi-quantitative detection of HPV16 and HPV18 mRNA with minimal infrastructure requirements. The assay relies on isothermal reverse transcription recombinase polymerase amplification (RT-RPA) with real-time fluorescence readout, demonstrated on rugged, portable, and affordable instruments. We demonstrate adapting the assay from DNA detection to RNA detection, characterizing the test with samples of increasing biological complexity, and ultimately establishing a limit of detection of 1000 HPV16 or HPV18 transcripts per reaction with RNA extracted from cell lines. HPV16 and HPV18 mRNA assays were used to test total RNA from 11 patient samples; results for 10 samples (91%) agreed with the gold standard of RT-qPCR. To reduce cost, the assay was demonstrated with multiplexed detection of HPV16 and HPV18 DNA, validated with a reaction volume that was reduced from 50 to 5 µL with DNA and RNA, and performed using a low-cost, portable reader with DNA and RNA. With incorporation of point-of-care-friendly sample preparation and detection of additional genotypes, this test has the potential to expand global access to HPV testing., Competing Interests: Declarations. Source of biological material: Pre-existing and de-identified cervicovaginal swab samples were provided by the AIDS Malignancy Consortium (AMC). Competing interests: PEC has received HPV tests and assays for research at a reduced or no cost from Roche, Becton Dickinson, Cepheid, and Arbor Vita Corporation. MEN is currently an employee of Orasure Technologies, Inc. (formerly Sherlock Biosciences). JRM has received HPV tests for research at no cost from Becton Dickinson. All other authors have no competing interests to declare. Copyright: Sections of this manuscript were previously published in KAK’s doctoral dissertation [32]., (© 2025. The Author(s).)

Maintaining adequate levels of antiretroviral (ARV) medications is crucial for the efficacy of HIV treatment and prevention regimens. Monitoring ARV levels can predict or prevent adverse health outcomes like treatment failure or drug resistance. However, conventional ARV measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is slow, expensive, and centralized delaying clinical and behavioral interventions. We previously developed a rapid enzymatic assay for measuring nucleotide reverse transcriptase inhibitors (NRTIs) - the backbone of HIV treatment and prevention regimens - based on the drugs' termination of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here, we expand our work to include non-nucleoside reverse transcriptase inhibitors (NNRTIs) - an ARV class used in established and emerging HIV treatment and prevention regimens. We demonstrate that the REverse Transcriptase ACTivity (REACT) assay can detect NNRTIs including medications used in oral and long-acting/extended-release HIV treatment and prevention. We demonstrate that REACT can measure NNRTIs spiked in either buffer or diluted plasma and that fluorescence can be measured on both a traditional plate reader and an inexpensive portable reader that can be deployed in point-of-care (POC) settings. REACT measured clinically relevant concentrations of five NNRTIs spiked in aqueous buffer. REACT measurements showed excellent agreement between the plate reader and the portable reader, with a high correlation in both aqueous buffer (Pearson's r = 0.9807, P < 0.0001) and diluted plasma (Pearson's r = 0.9681, P < 0.0001). REACT has the potential to provide rapid measurement of NNRTIs in POC settings and may help to improve HIV treatment and prevention outcomes., Competing Interests: Declarations. Conflict of interest: A.O. is an inventor on a patient filed (PCT/US2020/037609) based on related work on enzymatic assays for measuring reverse transcriptase inhibitors. Provisional patent applications have been filed on several components of the Harmony reader. N.P. and B.L. are inventors on two patent applications: PCT/US2021/041035 (pending international patent application) and 63/165,029 (pending U.S. provisional patent). N.P. and B.L. hold equity in a startup company that has licensed related technology and supports ongoing work in the B.L. laboratory at the University of Washington. B.L. serves as a scientific advisor for the company. The company played no role in the funding, study design, data analyses, or reporting of results for this study. C.B., M.A.S., J.T., C.C., S.G., and M.M.C. have no conflicts of interest to declare., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

Microfluidics offer user-friendly liquid handling for a range of biochemical applications. 3D printing microfluidics is rapid and cost-effective compared to conventional cleanroom fabrication. Typically, microfluidics are 3D printed using digital light projection (DLP) stereolithography (SLA), but many models in use are expensive (≥$10,000 USD), limiting widespread use. Recent liquid crystal display (LCD) technology advancements have provided inexpensive (<$500 USD) SLA 3D printers with sufficient pixel resolution for microfluidic applications. However, there are only a few demonstrations of microfluidic fabrication, limited validation of print fidelity, and no direct comparisons between LCD and DLP printers. We compared a 40 μm pixel DLP printer (∼$18,000 USD) with a 34.4 μm pixel LCD printer (<$380 USD). Consistent with prior work, we observed linear trends between designed and measured channel widths ≥4 pixels on both printers, so we calculated accuracy above this size threshold. Using a standard IPA-wash resin and optimized parameters for each printer, the average error between designed and measured widths was 2.11 ± 1.26% with the DLP printer and 15.4 ± 2.57% with the 34.4 μm LCD printer. Printing with optimized conditions for a low-cost water-wash resin designed for LCD-SLA printers resulted in an average error of 2.53 ± 0.94% with the 34.4 μm LCD printer and 5.35 ± 4.49% with a 22 μm LCD printer. We characterized additional parameters including surface roughness, channel perpendicularity, and light intensity uniformity, and as an application of LCD-printed devices, we demonstrated consistent flow rates in capillaric circuits for self-regulated and self-powered delivery of multiple liquids. LCD printers are an inexpensive alternative for fabricating microfluidics, with minimal differences in fidelity and accuracy compared with a 40X more expensive DLP printer., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The CandyCollect device is a lollipop-inspired open fluidic oral sampling device designed to provide a comfortable user sampling experience. We demonstrate that the CandyCollect device can be coupled with a rapid antigen detection test (RADT) kit designed for Group A Streptococcus (GAS). Through in vitro experiments with pooled saliva spiked with Streptococcus pyogenes we tested various reagents and elution volumes to optimize the RADT readout from CandyCollect device samples. The resulting optimized protocol uses the kit-provided reagents and lateral flow assay (LFA) while replacing the kit's pharyngeal swab with the CandyCollect device, reducing the elution solution volume, and substituting the tube used for elution to accommodate the CandyCollect device. Positive test results were detected by eye with bacterial concentrations as low as the manufacturer's "minimal detection limit" - 1.5×10 5 CFU/mL. LFA strips were also scanned and quantified with image analysis software to determine the signal-to-baseline ratio (SBR) and categorize positive test results without human bias. We tested our optimized protocol for integrating CandyCollect and RADT using CandyCollect clinical samples from pediatric patients (n=6) who were previously diagnosed with GAS pharyngitis via pharyngeal swabs tested with RADT as part of their clinical care. The LFA results of these CandyCollect devices and interspersed negative controls were determined by independent observers, with positive results obtained in four of the six participants on at least one LFA replicate. Taken together, our results show that CandyCollect devices from children with GAS pharyngitis can be tested using LFA rapid tests., Competing Interests: Conflict of Interest Disclosures Ashleigh B. Theberge, Xiaojing Su, Erwin Berthier, and Sanitta Thongpang filed patent 63/152,103 (International Publication Number: WO 2022/178291 Al) through the University of Washington on the CandyCollect oral sampling device. J. Carlos Sanchez, Timothy R. Robinson, Ayokunle O. Olanrewaju, Erwin Berthier, and Ashleigh B. Theberge filed patent 63/683,571 through the University of Washington on a related platform. Ashleigh B. Theberge reports filing multiple patents through the University of Washington and receiving a gift to support research outside the submitted work from Ionis Pharmaceuticals. Erwin Berthier is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin. Sanitta Thongpang has ownership in Salus Discovery, LLC, and Tasso, Inc. Erwin Berthier has ownership in Salus Discovery, LLC, and Tasso, Inc. and is employed by Tasso, Inc. However, this research is not related to these companies. Sanitta Thongpang, Erwin Berthier, and Ashleigh B. Theberge have ownership in Seabright, LLC, which will advance new tools for diagnostics and clinical research, potentially including the CandyCollect device. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. The other authors have no conflicts of interest to disclose.

Objective: To evaluate the current body of literature pertaining to the use of ocular point-of-care ultrasound (POCUS) in the emergency department., Methods: A comprehensive literature search was conducted on SCOPUS, Web of Science, MEDLINE, and Cochrane CENTRAL. Inclusion criteria included studies written in English only and primary clinical studies involving ocular POCUS scans in an emergency department setting. Exclusion criteria included non-primary studies (e.g. reviews or case reports), studies written in a non-English language, non-human studies, studies performed in a non-emergency setting, studies involving non-POCUS ocular ultrasound modalities, or studies published outside of the last decade. Data extraction was guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations., Results: The initial search yielded 391 results with 153 duplicates. Of the remaining 238 studies selected for retrieval and screening, 24 met inclusion criteria. These 24 included studies encompassed 2448 patients across prospective, retrospective, cross sectional, and case series study designs. We found that a majority of included studies focus on the use of POCUS in the emergency department to measure ONSD as a proxy for papilledema and metabolic aberrations, while a minority use ocular POCUS to assist in the diagnosis of orbital fractures or posterior segment pathology., Conclusion: The vast majority of articles investigating the use of ocular POCUS in recent years emphasize its utility in measuring ONSD and fluctuations in intracranial pressure, though additional outcomes of interest include posterior segment, orbit, and globe pathology.

Isothermal nucleic acid amplification tests have the potential to improve disease diagnosis at the point of care, but it remains challenging to develop multiplexed tests that can detect ≥3 targets or to detect point mutations that may cause disease. These capabilities are critical to enabling informed clinical decision-making for many applications, such as sickle cell disease (SCD). To address this, we describe the development of a multiplexed allele-specific recombinase polymerase amplification (RPA) assay with lateral flow readout. We first characterize the specificity of RPA using primer design strategies employed in PCR to achieve point mutation detection, and demonstrate the utility of these strategies in achieving selective isothermal amplification and detection of genomic DNA encoding for the healthy β A globin allele, or genomic DNA containing point mutations encoding for pathologic β S and β C globin alleles, which are responsible for most sickle cell disorders. We then optimize reaction conditions to achieve multiplexed amplification and identification of the three alleles in a single reaction. Finally, we perform a small pilot study with 20 extracted genomic DNA samples from SCD patients and healthy volunteers - of the 13 samples with valid results, the assay demonstrated 100% sensitivity and 100% specificity for detecting pathologic alleles, and an overall accuracy of 92.3% for genotype prediction. This multiplexed assay is rapid, minimally instrumented, and when combined with point-of-care sample preparation, could enable DNA-based diagnosis of SCD in low-resource settings. The strategies reported here could be applied to other challenges, such as detection of mutations that confer drug resistance.

Immune checkpoint therapy (ICB) has conferred significant and durable clinical benefit to some patients with cancer. However, most patients do not respond to ICB, and reliable biomarkers of ICB response are needed to improve patient stratification. Here, we performed a transcriptome-wide meta-analysis across 1,486 tumors from ICB-treated patients and tumors with expected ICB outcomes based on microsatellite status. Using a robust transcriptome deconvolution approach, we inferred cancer- and stroma-specific gene expression differences and identified cell-type specific features of ICB response across cancer types. Consistent with current knowledge, stromal expression of CXCL9, CXCL13, and IFNG were the top determinants of favorable ICB response. In addition, we identified a group of potential immune-suppressive genes, including FCER1A, associated with poor response to ICB. Strikingly, PD-L1 expression in stromal cells, but not cancer cells, is correlated with ICB response across cancer types. Furthermore, the unbiased transcriptome-wide analysis failed to identify cancer-cell intrinsic expression signatures of ICB response conserved across tumor types, suggesting that cancer cells lack tissue-agnostic transcriptomic features of ICB response., Significance: Our results challenge the prevailing dogma that cancer cells present tissue-agnostic molecular markers that modulate immune activity and ICB response, which has implications on the development of improved ICB diagnostics and treatments., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Introduction: Early diagnosis of patients with dilated cardiomyopathy (DCM) remains challenging. Cardiac MR can correlate myocardial changes with their pathological basis. There have been some previous studies on the effect of T1 mapping in DCM, but there is limited data on the incremental value of T2 mapping for DCM in routine clinical practice. This study will examine whether the combination of MRI T1 and T2 mapping offers greater advantages in the diagnosis of DCM., Methods: The study included 28 patients with DCM and 21 healthy controls. CMR evaluation included late gadolinium enhancement (LGE), T1 mapping, extracellular volume (ECV) fraction and T2 mapping. The DCM group was divided into LGE (+) and LGE (-) subgroups. The main modes of LGE are subendocardial, midwall, subepicardial, or transmural. T1 values, T2 values, and ECV in the 16 segments myocardial levels were measured by post-processing software. Student's t-tests or Mann-Whitney U test was used to compare between two groups, and one-way ANOVA or Kruskal-Wallis H test was used to compare between multiple groups, with p values corrected by Bonferroni. The difference was considered statistically significant at P < 0.05. ROC curve analysis was used to compare the area under the curve (AUC) of each index and its combined value, and the cut-off value, sensitivity and specificity were determined by Jordan's index., Results: Mean native myocardial T1, ECV and T2 were significantly higher in the DCM group compared to controls (p ≤ 0.001, respectively). The best cut-off values for T1, T2 and ECV to discriminate DCM from controls were 1184 ms, 40.9 ms and 29.2%, respectively. The AUC of T1, ECV and T2 were 0.87, 0.89, and 0.83, respectively. The combined AUC of the three values was 0.96., Conclusion: Native T1 value and ECV overcome some of the limitations of LGE, and the T2 helps to understand the extent of myocardial damage. The combination of T1 and T2 mapping techniques can reveal fibrotic and oedematous changes in the early stages of DCM, providing a more comprehensive assessment of DCM and better guidance for individualised clinical management of patients., Implications for Practice: We suggest that the addition of T2 mapping to the routine CMR examination of patients with suspected DCM, and the combined assessment of T1mapping and T2 mapping can provide complementary information about the disease and improve the early diagnosis of DCM., Competing Interests: Conflict of interest statement None., (Copyright © 2024 The College of Radiographers. Published by Elsevier Ltd. All rights reserved.)

Introduction: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) [hereafter, SJS/TEN] are uncommon but severe mucocutaneous reactions. Although they have been described in many populations worldwide, data from Hong Kong are limited. Here, we explored the epidemiology, disease characteristics, aetiology, morbidity, and mortality of SJS/TEN in Hong Kong., Methods: This retrospective cohort study included all hospitalised patients who had been diagnosed with SJS/TEN in Prince of Wales Hospital from 1 January 2004 to 31 December 2020., Results: There were 125 cases of SJS/TEN during the 17-year study period. The annual incidence was 5.07 cases per million. The mean age at onset was 51.4 years. The mean maximal body surface area of epidermal detachment was 23%. Overall, patients in 32% of cases required burns unit or intensive care unit admission. Half of the cases involved concomitant sepsis, and 23.2% of cases resulted in multiorgan failure or disseminated intravascular coagulation. The mean length of stay was 23.9 days. The cause of SJS/TEN was attributed to a drug in 91.9% of cases, including 84.2% that involved anticonvulsants, allopurinol, antibiotics, or analgesics. In most cases, patients received treatment comprising either best supportive care alone (35.2%) or combined with intravenous immunoglobulin (43.2%). The in-hospital mortality rate was 21.6%. Major causes of death were multiorgan failure and/or fulminant sepsis (81.5%)., Conclusion: This study showed that SJS/TEN are uncommon in Hong Kong but can cause substantial morbidity and mortality. Early recognition, prompt withdrawal of offending agents, and multidisciplinary supportive management are essential for improving clinical outcomes., Competing Interests: All authors have disclosed no conflicts of interest.

Human adipose-derived stem cells (ASCs) have shown immense potential for regenerative medicine. Our previous work demonstrated that chitosan nano-deposited surfaces induce spheroid formation and differentiation of ASCs for treating sciatic nerve injuries. However, the underlying cell fate and differentiation mechanisms of ASC-derived spheroids remain unknown. Here, we investigate the epigenetic regulation and signaling coordination of these therapeutic spheroids. During spheroid formation, we observed significant increases in histone 3 trimethylation at lysine 4 (H3K4me3), lysine 9 (H3K9me3), and lysine 27 (H3K27me3), accompanied by increased histone deacetylase (HDAC) activities and decreased histone acetyltransferase activities. Additionally, HDAC5 translocated from the cytoplasm to the nucleus, along with increased nuclear HDAC5 activities. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the chitosan-induced ASC spheroids and discovered distinct cluster subpopulations, cell fate trajectories, differentiation traits, and signaling networks using the 10x Genomics platform, R studio/language, and the Ingenuity Pathway Analysis (IPA) tool. Specific subpopulations were identified within the spheroids that corresponded to a transient reprogramming state (Cluster 6) and the endpoint cell state (Cluster 3). H3K4me3 and H3K9me3 were discovered as key epigenetic regulators by IPA to initiate stem cell differentiation in Cluster 6 cells, and confirmed by qPCR and their respective histone methyltransferase inhibitors: SNDX-5613 (a KMT2A inhibitor for H3K4me3) and SUVi (an SUV39H1 inhibitor for H3K9me3). Moreover, H3K9me3 and HDAC5 were involved in regulating downstream signaling and neuronal markers during differentiation in Cluster 3 cells. These findings emphasize the critical role of epigenetic regulation, particularly H3K4me3, H3K9me3, and HDAC5, in shaping stem cell fate and directing lineage-specific differentiation., (© The Author(s) 2023. Published by Oxford University Press.)

Cervical cancer is a leading cause of death for women in low-resource settings despite being preventable through human papillomavirus (HPV) vaccination, early detection, and treatment of precancerous lesions. The World Health Organization recommends high-risk HPV (hrHPV) as the preferred cervical cancer screening strategy, which is difficult to implement in low-resource settings due to high costs, reliance on centralized laboratory infrastructure, and long sample-to-answer times. To help meet the need for rapid, low-cost, and decentralized cervical cancer screening, we developed tailed primer isothermal amplification and lateral flow detection assays for HPV16, HPV18, and HPV45 DNA. We translated these assays into a self-contained cartridge to achieve multiplexed detection of three hrHPV genotypes in a disposable cartridge. The developed test achieves clinically relevant limits of detection of 50-500 copies per reaction with extracted genomic DNA from HPV-positive cells. Finally, we performed sample-to-answer testing with direct lysates of HPV-negative and HPV-positive cell lines and demonstrated consistent detection of HPV16, HPV18, and HPV45 with 5000-50,000 cells/mL in < 35 min. With additional optimization to improve cartridge reliability, incorporation of additional hrHPV types, and validation with clinical samples, the assay could serve as a point-of-care HPV DNA test that improves access to cervical cancer screening in low-resource settings., (© 2023. The Author(s).)

Competing Interests: Disclosure: The authors declare no conflict of interest.

High-risk human papillomavirus (HPV) DNA testing is widely acknowledged as the most sensitive cervical cancer screening method but has limited availability in resource-limited settings, where the burden of cervical cancer is highest. Recently, HPV DNA tests have been developed for use in resource-limited settings, but they remain too costly for widespread use and require instruments that are often limited to centralized laboratories. To help meet the global need for low-cost cervical cancer screening, we developed a prototype, sample-to-answer, point-of-care test for HPV16 and HPV18 DNA. Our test relies on isothermal DNA amplification and lateral flow detection, two technologies that reduce the need for complex instrumentation. We integrated all test components into a low-cost, manufacturable platform, and performance of the integrated test was evaluated with synthetic samples, provider-collected clinical samples in a high-resource setting in the United States, and self-collected clinical samples in a low-resource setting in Mozambique. We demonstrated a clinically relevant limit of detection of 1000 HPV16 or HPV18 DNA copies per test. The test requires six user steps, yields results in 45 min, and can be performed using a benchtop instrument and minicentrifuge by minimally trained personnel. The projected per-test cost is <$5, and the projected instrumentation cost is <$1000. These results show the feasibility of a sample-to-answer, point-of-care HPV DNA test. With the inclusion of other HPV types, this test has the potential to fill a critical gap for decentralized and globally accessible cervical cancer screening.

The novel coronavirus disease (COVID-19) pandemic caused by the SARS-CoV-2 virus started in December 2019 in the city of Wuhan, in China's Hubei province. This virus quickly spread worldwide, and on March 11, 2020, it was declared a pandemic. Thrombosis, as a hallmark of severe disease, was recognized early as a cause of death; however, the exact pathophysiological mechanism is still not fully understood. We are reporting the case of a 46-year-old patient who presented with multiple arterial thromboses in the setting of an acute COVID-19 infection requiring systemic thrombolytic therapy and thrombectomy., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Guevara et al.)

Prcis: Intraoperative anterior chamber (AC) washout reduces the odds of hypertensive phase (HP) by 95% compared with those not receiving washout during Ahmed glaucoma implant (AGI), suggesting AC washout is an effective prophylaxis for postoperative HP., Purpose: To characterize the incidence of postoperative HP in patients receiving AC washout at the time of AGI., Patients and Methods: A total of 24 patients with medically refractory glaucoma who underwent AGI surgery at a tertiary academic medical center in Southern California from December 2018 through March 2021 were included in this retrospective comparative case series. Patients who received a pediatric implant and underwent concurrent intraocular surgery or did not complete a minimum of 6 months of follow-up were excluded. Nine patient's eyes received AC washout and 15 controls that were analyzed through 6 months postprocedure. HP was defined as intraocular pressure (IOP) above 21 mm Hg within 6 months postprocedure with maximum tolerated medical therapy. AC washout was performed by irrigating the AC with a 5 mL balanced salt solution before placing the tube of the AGI into the AC. The rate of postoperative HP, defined as peak IOP >21 mm Hg, at 6 months follow-up, was observed as the primary outcome measure., Results: Patients included in the study had a high mean preoperative IOP ( X̅ = 44.11, SD = 13.85). There were no baseline differences between the washout and control groups. The odds of HP were significantly reduced (odds ratio: 0.050; 95% CI: 0.004, 0.706; P = 0.027) for patients who underwent intraoperative AC washout compared with those who did not. Bivariate analysis of subject baseline characteristics revealed that only washout status was significantly different in subjects with HP compared with subjects without HP ( P = 0.015). A multivariate logistic regression model using washout status and autoimmune conditions as covariates was significant in predicting HP ( X2 (2) = 12.337, P = 0.002), with washout as a significant predictor when controlling for autoimmune comorbidities ( P = 0.027)., Conclusions: AC washout significantly decreases the odds of HP after AGI surgery. AC washout with balanced salt solution adds minimal time and risks to surgery and therefore may be an effective adjunct during AGI placement., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Pancytopenia is a hematologic condition characterized by a decrease in all three peripheral blood cell lines. There are many causes of pancytopenia, and the proper approach is required for accurate diagnosis. Brucellosis and systemic lupus erythematosus (SLE) are both diseases that can initially present as pancytopenia, both of which require a targeted workup to diagnose. Due to the immune system's complexity, many distinct diseases may have similar symptomatology. Furthermore, infections and rheumatological diseases can stimulate the same molecular pathways and trigger T and B cells. This creates a cross-reactivity between microbial peptides and self-peptides, allowing the spread of microbial-specific T cells that can also respond to self-peptides. Brucellosis has broad clinical manifestations, often mimicking many other diseases, such as rheumatoid arthritis, sarcoidosis, and SLE. In addition, brucellosis-induced autoantibody production has been described as a triggering factor for immunologic reactions, elevating rheumatological markers by a poorly understood mechanism. Finally, SLE is a well-known medical condition that can mimic several medical conditions, including brucellosis.  We present a case of a young patient who was admitted with febrile pancytopenia. The patient also had IgM antibodies positive for brucellosis and high immune markers for SLE. She was treated for both diseases, and afterward, in retrospect, it was confirmed that the patient did not have acute brucellosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Guevara et al.)

Cervical cancer is a leading cause of cancer death for women in low-resource settings. The World Health Organization recommends that cervical cancer screening programs incorporate HPV DNA testing, but available tests are expensive, require laboratory infrastructure, and cannot be performed at the point-of-care. We developed a two-dimensional paper network (2DPN), hybrid-capture, signal amplification assay and a point-of-care sample preparation protocol to detect high-risk HPV DNA from exfoliated cervical cells within an hour. The test does not require expensive equipment and has an estimated cost of <$3 per test without the need for batching. We evaluated performance of the paper HPV DNA assay with short synthetic and genomic HPV DNA targets, HPV positive and negative cellular samples, and two sets of clinical samples. The first set of clinical samples consisted of 16 biobanked, provider-collected cervical samples from a study in El Salvador previously tested with careHPV and subsequently tested in a controlled laboratory environment. The paper HPV DNA test correctly identified eight of eight HPV-negative clinical samples and seven of eight HPV-positive clinical samples. We then performed a field evaluation of the paper HPV DNA test in a hospital laboratory in Mozambique. Cellular controls generated expected results throughout field testing with fully lyophilized sample preparation and 2DPN reagents. When evaluated with 16 residual self-collected cervicovaginal samples previously tested by the GeneXpert HPV assay ("Xpert"), the accuracy of the HPV DNA paper test in the field was reduced compared to testing in the controlled laboratory environment, with positive results obtained for all eight HPV-positive samples as well as seven of eight HPV-negative samples. Further evaluation showed reduction in performance was likely due in part to increased concentration of exfoliated cells in the self-collected clinical samples from Mozambique compared with provider-collected samples from El Salvador. Finally, a formal usability assessment was conducted with users in El Salvador and Mozambique; the assay was rated as acceptable to perform after minimal training. With additional optimization for higher cell concentrations and inclusion of an internal cellular control, the paper HPV DNA assay offers promise as a low-cost, point-of-care cervical cancer screening test in low-resource settings.

Fibroblast growth factor 9 (FGF9) modulates cell proliferation, differentiation and motility for development and tissue repair in normal cells. Growing evidence shows that abnormal activation of FGF9 signaling is associated with tumor malignancy. We have previously reported that FGF9 increases MA-10 mouse Leydig tumor cell proliferation, in vitro , and tumor growth, in vivo . Also, FGF9 promotes the tumor growth and liver metastasis of mouse Lewis lung cancer cells, in vivo . However, the effects of FGF9 in the early stage of tumorigenesis remains elusive. In this study, TM3 mouse Leydig progenitor cells, that are not tumorigenic in immunocompromised mice, were used as a model cell line to investigate the role of FGF9 in tumorigenesis. The results demonstrated that FGF9 significantly induced cell proliferation and activated the MAPK, PI3K and PLCγ signaling pathways in TM3 cells. The percentage of the cell number in G1 phase was reduced and that in S and G2/M phases was increased after FGF9 stimulation in TM3 cells. Cyclin D1, cyclin A1, CDK2, CDK1, and p21 expressions and the phosphorylation level of Rb were all induced in FGF9-treated TM3 cells. In addition, FGF9 increased the expression of FGF receptor 1-4 in TM3 cells, suggesting the positive feedback loop between FGF9 and FGFRs. Furthermore, in the allograft mouse model, FGF9 promoted the tumorigenesis of TM3 cells characterized by higher expression of tumor markers, such as tumor necrosis factor alpha (TNFα) and α-fetoprotein (AFP), in the subcutaneously inoculated TM3 cell tissue. Conclusively, FGF9 induced cell cycle to increase cell proliferation of TM3 cells through FAK, MAPK, PI3K/Akt and PLCγ signaling pathways, in vitro , and promoted the tumorigenesis of TM3 cell allograft tissue, in vivo , which is a potential marker for tumor as well as a target for cancer therapeutic strategies., Competing Interests: None., (AJCR Copyright © 2022.)

Paper-fluidic devices are a popular platform for point-of-care diagnostics due to their low cost, ease of use, and equipment-free detection of target molecules. They are limited, however, by their lack of sensitivity and inability to incorporate more complex processes, such as nucleic acid amplification or enzymatic signal enhancement. To address these limitations, various valves have previously been implemented in paper-fluidic devices to control fluid obstruction and release. However, incorporation of valves into new devices is a highly iterative, time-intensive process due to limited experimental data describing the microscale flow that drives the biophysical reactions in the assay. In this paper, we tested and modeled different geometries of thermally actuated valves to investigate how they can be more easily implemented in an LFIA with precise control of actuation time, flow rate, and flow pattern. We demonstrate that bulk flow measurements alone cannot estimate the highly variable microscale properties and effects on LFIA signal development. To further quantify the microfluidic properties of paper-fluidic devices, micro-particle image velocimetry was used to quantify fluorescent nanoparticle flow through the membranes and demonstrated divergent properties from bulk flow that may explain additional variability in LFIA signal generation. Altogether, we demonstrate that a more robust characterization of paper-fluidic devices can permit fine-tuning of parameters for precise automation of multi-step assays and inform analytical models for more efficient design.

Southern blot analysis is an important molecular biology technique for identifying a specific sequence in DNA samples. Although it is no longer used extensively in recent years, the steps and underlying principles of Southern blot are applicable to modern biology. High sensitivity and limited background are keys to successful Southern blots, whereas obtaining good quality and quantity of genomic DNA as starting materials and detecting a single/low copy target sequence in the genome can be challenging. To ensure student success in performing the technique for the first time, a modified "plasmid-to-plasmid" Southern blot was implemented to confirm the presence of grape nucleotide-binding site (nbs) sequences in cloned plasmids like those described previously. The plasmid DNA and a control plasmid, pSCA7 (T1-T3-W6) containing a known grape nbs sequence, were digested with restriction enzymes, followed by agarose gel electrophoresis. The DNA band corresponding to the nbs sequence of the pSCA7 (T1-T3-W6) was extracted from the gel for PCR digoxigenin (DIG) probe synthesis. At the same time, the cloned plasmid DNA and its digested DNA fragments were blotted from the gel onto nylon membranes to be hybridized with the DIG probe followed by the detection for nbs sequences. Students successfully performed Southern blots to confirm the presence of nbs sequences in their cloned plasmids and wrote up the results following the format of scientific research papers. They learned the principles and applications of Southern blot and gained hands-on experience with associated techniques., (© 2022 International Union of Biochemistry and Molecular Biology.)

Objective: To identify work-environment characteristics associated with Veterans Health Administration (VHA) behavioral health provider (BHP) burnout among psychiatrists, psychologists, and social workers., Data Sources: The 2015-2018 data from Annual All Employee Survey (AES); Mental Health Provider Survey (MHPS); N = 57,397 respondents; facility-level Mental Health Onboard Clinical (MHOC) staffing and productivity data, N = 140 facilities., Study Design: For AES and MHPS separately, we used mixed-effects logistic regression to predict BHP burnout using surveys from year pairs (2015-2016, 2016-2017, 2017-2018; six models). Within each year-pair, we used the earlier year of data to train models and tested the model in the later year, with burnout (emotional exhaustion and/or depersonalization) as the outcome for each survey. We used potentially modifiable work-environment characteristics as predictors, controlling for employee demographic characteristics as covariates, and employment facility as random intercepts., Data Collection/extraction Methods: We included work-environment predictors that appeared in all 4 years (11 in AES; 17 in MHPS)., Principal Findings: In 2015-2018, 31.0%-38.0% of BHPs reported burnout in AES or MHPS. Work characteristics consistently associated with significantly lower burnout were included for AES: reasonable workload; having appropriate resources to perform a job well; supervisors address concerns; given an opportunity to improve skills. For MHPS, characteristics included: reasonable workload; work improves veterans' lives; mental health care provided is well-coordinated; and three reverse-coded items: staffing vacancies; daily work that clerical/support staff could complete; and collateral duties reduce availability for patient care. Facility-level staffing ratios and productivity did not significantly predict individual-level burnout. Workload represented the strongest predictor of burnout in both surveys., Conclusions: This study demonstrated substantial, ongoing impacts that having appropriate resources including staff, workload, and supervisor support had on VHA BHP burnout. VHA may consider investing in approaches to mitigate the impact of BHP burnout on employees and their patients through providing staff supports, managing workload, and goal setting., (© 2022 The Authors. Health Services Research published by Wiley Periodicals LLC on behalf of Health Research and Educational Trust. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats., Competing Interests: The authors have declared that no competing interests exist.

Objective: To evaluate national trends in incident varenicline and nicotine replacement therapy (NRT) prescribing among Department of Veterans Affairs (VA) beneficiaries before and after US Food and Drug Administration (FDA) warnings regarding neuropsychiatric side effects with varenicline use., Methods: All adult VA patients identified as smokers from 2007 to 2019 (N = 3,600,947) were determined and monthly counts of new varenicline and NRT users were calculated. An interrupted time-series analysis estimated the effect of the FDA warnings on varenicline and NRT prescribing overall and among Veterans with and without mental health disorders., Results: The incident use rate of varenicline decreased from a peak of 6.2 per 1,000 veteran smokers in October 2007 to 1.0 by July 2009 following the first FDA warning (pre-warning monthly slope = -0.27; P  = .03). New NRT use increased from 10.7 per 1,000 veteran smokers in October 2007 to a peak of 12.6 per 1,000 in July 2009 (slope change = 0.71; P  = .01), suggesting potential substitution. Following removal of the FDA boxed warning in December 2016, varenicline prescribing increased but did not return to pre-warning levels by December 2019. Among veterans with and without mental health disorders, varenicline use decreased 90% and 88%, respectively, following the first FDA warning, and both groups had comparable rates of new NRT use., Conclusions: Following the first FDA warning, incident use of varenicline declined significantly among veterans both with and without mental health disorders. Despite removal of the FDA boxed warning in December 2016, new use of varenicline had not returned to pre-warning levels 3 years following the removal., (© Copyright 2021 Physicians Postgraduate Press, Inc.)

Traumatic spinal cord injury (SCI) is a devastating condition marked by permanent motor, sensory, and autonomic dysfunction, in which the inflammatory response serves an important and preventable role. High mobility group box-1 (HMGB1) is a potent regulator of inflammation in numerous acute and chronic inflammatory conditions.; however, the role of HMGB1 in SCI remains unclear. The present study aimed to characterize the temporal dynamics of HMGB1 release after SCI, to investigate the role of spinal microglia activation in mediating the effects of HMGB1 on SCI, and to explore the therapeutic potential of intrathecal anti-HMGB1 polyclonal antibody on alleviating SCI. The present study demonstrated that HMGB1 expression was increased immediately after traumatic injury of a primary spinal neuron culture. It was found that neutralizing HMGB1 significantly ameliorated SCI pathogenesis and hind limb paralysis. Moreover, the levels of a number of pro-inflammatory cytokines in the SCI lesion were reduced when local HMGB1 was blocked by anti-HMGB1 antibody. In addition, the injured neuron-derived conditioned medium increased TNF-α secretion and the NF-κB pathway in the BV2 microglia cell line via HMGB1. Collectively, these results indicated that HMGB1 served an important role in SCI inflammation and suggested the therapeutic potential of an anti-HMGB1 antibody for SCI., (©2021 Society for Leukocyte Biology.)

Objective: To reduce delays in referral to specialty mental health care, we evaluated clinical prediction models estimating the likelihood of response to primary care treatment of depression in the VA healthcare system., Methods: We included patients with a primary care depression diagnosis between October 1, 2015 and December 31, 2017, an initial PHQ-9 score ≥ 10 within 30 days, a follow-up PHQ-9 score within 2-8 months, and no specialty mental health care within three months prior to depression diagnosis. We evaluated eight ordinary least squares regression models, each with a different procedure for selecting predictors of percentage change in PHQ-9 score from baseline to follow-up. Predictors included patient characteristics from electronic health records and neighborhood characteristics from US census data. We repeated each modeling procedure 1,000 times, using different training and validation sets of patients. We used R 2 , RMSE, and MAE to evaluate model performance., Results: The final cohort included 3,464 patients. The two best performing models included multiple iterations of backwards stepwise variable selection with R 2 of 0.07, RMSE of 41.45, MAE of 33.30; and R 2 of 0.07, RMSE of 41.39, MAE of 33.28., Limitations: Wide follow-up interval, possibility of misclassification error due to use of EHR data., Conclusions: Model performance did not suggest its use as a guide in clinical decision-making. Future research should explore whether obtaining additional risk factor data from patients (e.g., duration of symptoms) or modeling PHQ-9 scores over a narrower time interval improves performance of clinical risk prediction tools for depression., (Copyright © 2021. Published by Elsevier B.V.)

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis., (Copyright © 2021. Published by Elsevier Inc.)

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types., (© The Author(s) 2021. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.)

Background: Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity-however, most predicted enhancer regions remain to be functionally tested., Methods: We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions., Results: We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity., Conclusions: Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity., (© 2021. The Author(s).)

Frequent and accessible testing is a critical tool to contain the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To develop low-cost rapid tests, many researchers have used reverse transcription loop-mediated isothermal amplification (RT-LAMP) with fluorescent readout. Fluorescent LAMP-based assays can be performed using cost-effective, portable, isothermal instruments that are simpler to use and more rugged than polymerase chain reaction (PCR) instruments. However, false-positive results due to nonspecific priming and amplification have been reported for a number of LAMP-based assays. In this report, we implemented a RT-LAMP assay for SARS-CoV-2 on a portable isothermal fluorimeter and a traditional thermocycler; nonspecific amplification was not observed using the thermocycler but did occur frequently with the isothermal fluorimeter. We explored 4 strategies to optimize the SARS-CoV-2 RT-LAMP assay for use with an isothermal fluorimeter and found that overlaying the reaction with mineral oil and including the enzyme Tte UvrD helicase in the reaction eliminated the problem. We anticipate these results and strategies will be relevant for use with a wide range of portable isothermal instruments., (© 2021 ABRF.)

Introduction: Although the benefits of employment for veterans with mental health conditions are well-known, the effect of veterans' employment on a health system has not been evaluated. The purpose of this study was to evaluate the effect of veterans' employment (versus unemployment) on subsequent health care utilization in the Veterans Health Administration (VHA)., Materials and Methods: This study used a sample of 29,022 veterans with mental health and substance use disorders who were discharged from VHA's employment services programs between fiscal years 2006 and 2010. Veterans' employment status (employed/unemployed) upon discharge from VHA employment programs was ascertained from program discharge forms and linked with VHA administrative health care utilization data for the subsequent 1- and 5-year periods., Results: Multivariable ordinary least-squares and logistic regression models adjusted for site clustering and covariates indicated that employment (versus unemployment) predicted less health care utilization 1 year and 5 years post-discharge from employment services, including fewer outpatient mental health visits, homelessness services visits, employment services visits, primary care visits, and lower odds of mental health hospitalizations, mental health or vocational rehabilitation residential stays, and medical hospitalizations. Employment did not predict emergency department visits., Conclusions: VHA's investment in employment services for veterans with mental health and substance use disorders could reduce health care utilization system wide., (Published by Oxford University Press on behalf of the Association of Military Surgeons of the United States 2021. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Zika virus (ZIKV; Flaviviridae ) is a devastating virus transmitted to humans by the mosquito Aedes aegypti. The interaction of the virus with the mosquito vector is poorly known. The double-stranded RNA (dsRNA)-mediated interruption or activation of immunity-related genes in the Toll, IMD, JAK-STAT, and short interfering RNA (siRNA) pathways did not affect ZIKV infection in A. aegypti. Transcriptome-based analysis indicated that most immunity-related genes were upregulated in response to ZIKV infection, including leucine-rich immune protein (LRIM) genes. Further, there was a significant increment in the ZIKV load in LRIM9- , LRIM10A- , and LIRM10B -silenced A. aegypti, suggesting their function in modulating viral infection. Further, gene function enrichment analysis revealed that viral infection increased global ribosomal activity. Silencing of RpL23 and RpL27 , two ribosomal large subunit genes, increased mosquito resistance to ZIKV infection. In vitro fat body culture assay revealed that the expression of RpL23 and RpL27 was responsive to the Juvenile hormone (JH) signaling pathway. These two genes were transcriptionally regulated by JH and its receptor methoprene-tolerant (Met) complex. Silencing of Met also inhibited ZIKV infection in A. aegypti. This suggests that ZIKV enhances ribosomal activity through JH regulation to promote infection in mosquitoes. Together, these data reveal A. aegypti immune responses to ZIKV and suggest a control strategy that reduces ZIKV transmission by modulating host factors. IMPORTANCE Most flaviviruses are transmitted between hosts by arthropod vectors such as mosquitoes. Since therapeutics or vaccines are lacking for most mosquito-borne diseases, reducing the mosquito vector competence is an effective way to decrease disease burden. We used high-throughput sequencing technology to study the interaction between mosquito Aedes aegypti and ZIKV. Leucine-rich immune protein (LRIM) genes were involved in the defense in response to viral infection. In addition, RNA interference (RNAi) silencing of RpL23 and RpL27 , two JH-regulated ribosomal large subunit genes, suppressed ZIKV infection in A. aegypti. These results suggest a novel control strategy that could block the transmission of ZIKV.

A new ellimmichthyiform, Guiclupea superstes , gen. et sp. nov., from the Oligocene Ningming Formation of Ningming Basin, Guangxi Zhuang Autonomous Region, South China is described herein. With relatively large body size, parietals meeting at the midline, anterior ceratohyal with a beryciform foramen in the center, a complete predorsal scutes series of very high number and about equally-size scutes with radiating ridges on dorsal surface, first preural centrum unfused with first uroneural but fused with the parhypural, and first ural centrum of roughly the same size as the preural centrum, Guiclupea superstes cannot be assigned to the order Clupeiformes. The phylogenetic analyses using parsimony and Bayesian inference methods with Chanos / Elops as outgroup respectively suggests that the new form is closer to ellimmichthyiform genus Diplomystus than to any other fishes, although there are some discrepancies between the two criteria and different outgroups used. It shares with Diplomystus the high supraoccipital crest, pelvic-fin insertion in advance of dorsal fin origin, and the number of predorsal scutes more than 20. The new form represents the youngest ellimmichthyiform fish record in the world. Its discovery indicates that the members of the Ellimmichthyiformes had a wider distribution range and a longer evolutional history than previously known., Competing Interests: The authors declare there are no competing interests., (©2021 Chen et al.)

Profiling of circulating tumor DNA (ctDNA) may offer a non-invasive approach to monitor disease progression. Here, we develop a quantitative method, exploiting local tissue-specific cell-free DNA (cfDNA) degradation patterns, that accurately estimates ctDNA burden independent of genomic aberrations. Nucleosome-dependent cfDNA degradation at promoters and first exon-intron junctions is strongly associated with differential transcriptional activity in tumors and blood. A quantitative model, based on just 6 regulatory regions, could accurately predict ctDNA levels in colorectal cancer patients. Strikingly, a model restricted to blood-specific regulatory regions could predict ctDNA levels across both colorectal and breast cancer patients. Using compact targeted sequencing (<25 kb) of predictive regions, we demonstrate how the approach could enable quantitative low-cost tracking of ctDNA dynamics and disease progression.

The transition of flow microenvironments from veins to arteries in vein graft surgery induces "peel-off" of venous endothelial cells (vECs) and results in restenosis. Recently, arterial laminar shear stress (ALS) and oscillatory shear stress (OS) have been shown to affect the cell cycle and inflammation through epigenetic controls such as histone deacetylation by histone deacetylases (HDACs) and trimethylation on lysine 9 of histone 3 (H3K9me3) in arterial ECs. However, the roles of H3K9me3 and HDAC in vEC damage under ALS are not known. We hypothesized that the different responses of HDACs and H3K9me3 might cause vEC damage under the transition of venous flow to arterial flow. We found that arterial ECs showed high expression of H3K9me3 protein and were retained in the G0 phase of the cell cycle after being subjected to ALS. vECs became round under ALS with a decrease in the expression of H3K9me3, HDAC3, and HDAC5, and an increase in the expression of vascular cell adhesion molecule 1 (VCAM-1). Inhibition of HDACs activity by a specific inhibitor, phenylbutyrate, in arterial ECs caused similar ALS-induced inflammation and cell loss as observed in vECs. Activation of HDACs and H3K9me3 by ITSA-1, an HDAC activator, could prevent ALS-induced peel-off and reduced VCAM-1 expression in vECs. Moreover, shear stress modulates EC morphology by the regulation of focal adhesion kinase (FAK) expression. ITSA-1 or EGF could increase phosphorylated (p)-FAK expression in vECs under ALS. We found that perturbation of the activity of p-FAK and increase in p-FAK expression restored ALS-induced H3K9me3 expression in vECs. Hence, the abnormal mechanoresponses of H3K9me3 and HDAC in vECs after being subjected to ALS could be reversed by ITSA-1 or EGF treatment: this offers a strategy to prevent vein graft failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Chang, Li, Huang, Chien and Wu.)