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In this study, we constructed an eight-gene metabolic related signature for LUAD. The eight-gene prognostic signature (including PLAUR, F2, UGT2B17, GNG7, IDO2, ST3GAL6, PIK3CG, and GLS2) exhibited a good prognostic value in the TCGA LUAD training dataset and testing dataset. In addition, the risk score based on the signature model was significantly correlated with immune cell infiltration and expression levels of immune markers in LUAD patients. LUAD cohorts from GEO were used to validate the model, indicating the usefulness of the model. In summary, we developed and validated an eight-gene signature model for LUAD, which can reflect the immune microenvironment characteristics and predict the prognostic outcomes for LUAD patients.

Responding to the public health significance of Alzheimer's Disease (AD) and lack of information on AD-related issues in Asian Americans, the present study examined the prevalence and predictors of three stigmatising beliefs about AD (beliefs that associate AD with a normal process of ageing, family embarrassment and social avoidance) using a sample of Asian Americans representing diverse ethnic groups (Chinese, Asian Indian, Korean, Vietnamese, Filipino and other Asians) and a broad age range (18-98). Guided by the Sociocultural Health Belief Model, multivariate regression models of stigmatising beliefs about AD examined the effect of demographic characteristics (age, gender, marital status, education and ethnicity) and immigration and culture-related variables (proportion of life in the U.S., English proficiency and acculturation). Data came from the 2015 Asian American Quality of Life survey (N = 2,609). Results indicate that the prevalence of stigmatising beliefs about AD varied across ethnicities. More than 63% of Vietnamese associated AD with a normal process of ageing, and about 10% of Chinese reported that they would feel embarrassed if their family member had AD. Logistic regression analyses demonstrated that advanced age, male gender, low education and limited English proficiency increased the odds of reporting one or more stigmatising beliefs about AD. The findings suggest varying degrees of AD-related misconceptions and stigmatisation and call attention to the need for culturally sensitive AD education in Asian communities.

Objective: To explore the association between role overload (RO) and sleep maintenance insomnia (SMI), and the moderation effects of social support and social engagement (SE). Methods: We report a cross-sectional study using data drawn from the 2015 National Health and Aging Trends Study and National Study of Caregiving. We used multiple regression and controlled for demographics and potential confounders. Results: Nearly 45% of caregivers reported suffering from SMI during “some” and “more” nights within the past month with one half reporting “almost” or “every” night. RO was found positively associated with the risk of SMI. Instrumental support moderated the effect of RO on SMI overall, although moderation was limited to a subsample of adult children caregivers. Discussion: The sleep quality of dementia caregivers may be affected by RO, particularly for adult children caregivers. Increasing instrumental support may be beneficial to caregiver's sleep quality.

Background: Acute myeloid leukemia (AML) remains the most common type of hematopoietic malignancy in adults and has an unfavorable outcome. Herein, we aimed to construct an N6-methylandenosine (m6A)-related long noncoding RNAs (lncRNAs) signature to accurately predict the prognosis of patients with AML using the data downloaded from The Cancer Genome Atlas (TCGA) database.Methods: The RNA-seq and clinical data were obtained from the TCGA AML cohort. First, Pearson correlation analysis was performed to identify the m6A-related lncRNAs. Next, univariate Cox regression analysis was used to determine the candidate lncRNAs with prognostic value. Then, feature selection was carried out by Least absolute shrinkage and selection operator (LASSO) analysis, and seven eligible m6A-related lncRNAs were included to construct the prognostic risk signature. Kaplan–Meier and receiver operating characteristic (ROC) curve analyses were performed to evaluate the predictive capacity of the risk signature both in the training and testing datasets. A nomogram was used to predict 1-year, 2-year, and 3-year overall survival (OS) of AML patients. Next, the expression levels of lncRNAs in the signature were validated in AML samples by qRT-PCR. Functional enrichment analyses were carried out to identify probable biological processes and cellular pathways. The ceRNA network was developed to explore the downstream targets and mechanisms of m6A-related lncRNAs in AML.Results: Seven m6A-related lncRNAs were identified as a prognostic signature. The low-risk group hold significantly prolonged OS. The nomogram showed excellent accuracy of the signature for predicting 1-year, 2-year and 3-year OS (AUC = 0.769, 0.820, and 0.800, respectively). Moreover, the risk scores were significantly correlated with enrichment in cancer hallmark- and malignancy-related pathways and immunotherapy response in AML patients.Conclusion: We developed and validated a novel risk signature with m6A-related lncRNAs which could predict prognosis accurately and reflect the immunotherapy response in AML patients.

Background: Bladder cancer (BLCA) ranks 10th in incidence among malignant tumors and 6th in incidence among malignant tumors in males. With the application of immune therapy, the overall survival (OS) rate of BLCA patients has greatly improved, but the 5-year survival rate of BLCA patients is still low. Furthermore, not every BLCA patient benefits from immunotherapy, and there are a limited number of biomarkers for predicting the immunotherapy response. Therefore, novel biomarkers for predicting the immunotherapy response and prognosis of BLCA are urgently needed.Methods: The RNA sequencing (RNA-seq) data, clinical data and gene annotation files for The Cancer Genome Atlas (TCGA) BLCA cohort were extracted from the University of California, Santa Cruz (UCSC) Xena Browser. The BLCA datasets GSE31684 and GSE32894 from the Gene Expression Omnibus (GEO) database were extracted for external validation. Immune-related genes were extracted from InnateDB. Significant differentially expressed genes (DEGs) were identified using the R package “limma,” and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for the DEGs were performed using R package “clusterProfiler.” Least absolute shrinkage and selection operator (LASSO) regression analysis were used to construct the signature model. The infiltration level of each immune cell type was estimated using the single-sample gene set enrichment analysis (ssGSEA) algorithm. The performance of the model was evaluated with receiver operating characteristic (ROC) curves and calibration curves.Results: In total, 1,040 immune-related DEGs were identified, and eight signature genes were selected to construct a model using LASSO regression analysis. The risk score of BLCA patients based on the signature model was negatively correlated with OS and the immunotherapy response. The ROC curve for OS revealed that the model had good accuracy. The calibration curve showed good agreement between the predictions and actual observations.Conclusions: Herein, we constructed an immune-related eight-gene signature that could be a potential biomarker to predict the immunotherapy response and prognosis of BLCA patients.

Background: Bladder cancer (BLCA) is a common malignant tumor of the genitourinary system, and there is a lack of specific, reliable, and non-invasive tumor biomarker tests for diagnosis and prognosis evaluation. Homeobox genes play a vital role in BLCA tumorigenesis and development, but few studies have focused on the prognostic value of homeobox genes in BLCA. In this study, we aim to develop a prognostic signature associated with the homeobox gene family for BLCA.Methods: The RNA sequencing data, clinical data, and probe annotation files of BLCA patients were downloaded from the Gene Expression Omnibus database and the University of California, Santa Cruz (UCSC), Xena Browser. First, differentially expressed homeobox gene screening between tumor and normal samples was performed using the “limma” and robust rank aggregation (RRA) methods. The mutation data were obtained with the “TCGAmutation” package and visualized with the “maftools” package. Kaplan–Meier curves were plotted with the “survminer” package. Then, a signature was constructed by logistic regression analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using “clusterProfiler.” Furthermore, the infiltration level of each immune cell type was estimated using the single-sample gene set enrichment analysis (ssGSEA) algorithm. Finally, the performance of the signature was evaluated by receiver-operating characteristic (ROC) curve and calibration curve analyses.Results: Six genes were selected to construct this prognostic model: TSHZ3, ZFHX4, ZEB2, MEIS1, ISL1, and HOXC4. We divided the BLCA cohort into high- and low-risk groups based on the median risk score calculated with the novel signature. The overall survival (OS) rate of the high-risk group was significantly lower than that of the low-risk group. The infiltration levels of almost all immune cells were significantly higher in the high-risk group than in the low-risk group. The average risk score for the group that responded to immunotherapy was significantly lower than that of the group that did not.Conclusion: We constructed a risk prediction signature with six homeobox genes, which showed good accuracy and consistency in predicting the patient’s prognosis and response to immunotherapy. Therefore, this signature can be a potential biomarker and treatment target for BLCA patients.

A conductometric immunoassay protocol was designed for the sensitive detection of a liver cancer biomarker, alpha-fetoprotein (AFP), in biological fluids by using enzyme-conjugated nanometer-sized enzyme-doped silica beads. Initially, urease molecules were doped into nanosilica particles by using the reverse micelle method. Thereafter, arginase-labeled anti-AFP antibodies were covalently conjugated onto the surface of the synthesized nanoparticles. The immunoreaction was carried out in a monoclonal anti-AFP capture antibody-coated microplate with a sandwich-type assay format by using bienzyme-functionalized silica nanobeads as the recognition elements. Upon l-arginine introduction, the substrate was cleaved into urea and l-ornithine on the basis of the arginase enzymatic reaction, and the as-produced urea was then decomposed into ammonia (NH4+) and bicarbonate (HCO3-) ions by the doped urease, thus causing the variation in the local conductivity of the detection solution on an interdigitated conductometric transducer. Under optimal conditions, the developed immunosensing system exhibited good conductometric responses toward target AFP within a dynamic linear range of 0.01-100 ng mL-1 at a relatively low detection limit of 4.8 pg mL-1 based on the 3sB criterion. Importantly, good reproducibility, high specificity and acceptable method accuracy were acquired for the analysis of human serum specimens in liver cancer patients.

Jumping with controllable elevation is significant for insect-scale robots to improve terrain adaptability and to escape from risks. However, jumping robots based on soft materials with low stiffness cannot transmit displacement precisely, exhibiting poor control of jumping. Here, we propose a modified two-bars catapult mechanism combined with an asynchronous sequential releasing strategy to realize elevation controllable jumping. In this work, an 80 mg prototype robot, 56 (long) × 29 mm (wide) × 3 mm (high) mm in size, is designed with the controllable elevation range from 63° to 112°. The soft robot is mainly composed of a shape memory alloy actuator and four electrostatic pads acting as the lock/release structures. Elevation control is realized by asynchronously releasing the electrostatic pads in a small time interval (about 10 ms). A maximum jump height of 62 mm and a maximum half-distance of 41 mm are also achieved.

Background: Butyrophilin (BTN) and butyrophilin-like (BTLN) belong to immunoglobulin superfamily, and also pertain the B7 co-stimulatory molecules family, which has multiple roles in immune modulation. Whether the BTNL9 expression in lung adenocarcinoma (LUAD) correlates with outcome has not been evaluated.Materials and methods: Oncomine and GEPIA were used to analyze the BTNL9, while its mRNA expression in LUAD and corresponding adjacent tissues was investigated using TIMER. The clinical prognosis of BTNL9 was assessed in the GEPIA, Kaplan-Meier plotter, and OncoLnc. Besides, the correlation between BTNL9 and tumor-infiltrating immune cells (TILs) was analyzed using TIMER and GEPIA. The correlation between BTNL9 expression and drug response was analyzed using CARE.Results: BNL9 expression was significantly low in LUAD. Low BTNL9 expression was associated with poor OS, and its expression was found to be regulated by both epigenetic regulation and post-transcriptional modification. BTNL9 expression was significant positively correlated with ImmuneScore and ESTIMATEScore. Moreover, BTNL9 expression was positively correlated with infiltrating levels of B cells, CD4+T, and macrophages, but Kaplan-Meier analysis showed that BTNL9 expression in B cells and DCs was significantly associated with OS. Furthermore, BTNL9 expression has significant positive CARE scores. Conclusions: Low BTNL9 expression can prevent the infiltration of naïve B cells and DCs in the tumor microenvironment and worsen the outcome in LUAD patients. Besides, these findings also suggest the potential role of BTNL9 as a prognostic biomarker and a new immuno-target.

Background Lung adenocarcinoma (LUAD) is the most prevalent non-small cell lung cancer (NSCLC). Patients with LUAD have a poor 5-year survival rate. The use of immune checkpoint inhibitors (ICIs) for the treatment of LUAD has been on the rise in the past decade. This study explored the prognostic role of butyrophilin-like 9 (BTNL9) in LUAD. Methods Gene expression profile of buytrophilins (BTNs) was determined using the GEPIA database. The effect of BTNL9 on the survival of LUAD patients was assessed using Kaplan-Meier plotter and OncoLnc. Correlation between BTNL9 expression and tumor-infiltrating immune cells (TILs) was explored using TIMER and GEPIA databases. Further, the relationship between BTNL9 expression and drug response was evaluated using CARE. Besides, construction and evaluation of nomogram based on BTNL9 expression and TNM stage. Results BTNL9 expression was downregulated in LUAD and was associated with a poor probability of 1, 3, 5-years overall survival (OS). In addition, BTNL9 expression was regulated at epigenetic and post-transcriptional modification levels. Moreover, BTNL9 expression was significantly positively correlated with ImmuneScore and ESTIMATEScore. Furthermore, BTNL9 expression was positively associated with infiltration levels of B cells, CD4+ T cells, and macrophages. Kaplan-Meier analysis showed that BTNL9 expression in B cells and dendritic cells (DCs) was significantly associated with OS. BTNL9 expression was significantly positively correlated with CARE scores. Conclusions These findings show that BTNL9 is a potential prognostic biomarker for LUAD. Low BTNL9 expression levels associated with low infiltration levels of naïve B cells, and DCs in the tumor microenvironment are unfavorable for OS in LUAD patients.

OBJECTIVES: HIV-positive people who use illicit drugs (PWUD) experience elevated rates of HIV-associated morbidity and mortality compared with members of other key affected populations. Although suboptimal levels of access and adherence to antiretroviral therapy (ART) are common among HIV-positive PWUD, there is a need for studies investigating the possible biological impacts of noninjection illicit drug use among people living with HIV in real-world settings. METHODS: We accessed data from the ACCESS study, an ongoing prospective cohort of illicit drug users with systematic HIV viral load monitoring in a setting with universal care and ART dispensation records. We used multivariable generalized linear mixed models to estimate the longitudinal associations between noninjection use of crack cocaine, powder cocaine, opioids, methamphetamine, cannabis and alcohol on plasma HIV-1 RNA viral load, adjusted for ART exposure and relevant confounders. RESULTS: Between 2005 and 2018, 843 individuals from the ACCESS cohort were included and contributed to 8698 interviews. At baseline, the mean age was 43 years, 566 (67%) reported male sex and 659 (78%) used crack cocaine in the previous 6 months. In multivariable models adjusted for ART exposure, only crack cocaine use in the last 6 months was found to be significantly associated with higher HIV viral load. CONCLUSION: We observed significantly higher HIV viral load during periods of crack cocaine use independent of ART exposure. Our findings support further research to investigate the possible biological mechanisms of this effect.

The aim of this study was to analyze the prevalence of mild cognitive impairment (MCI) among the aging population (60 years of age and above) in China. Epidemiological investigations on MCI in online Chinese journals were identified manually using the CQVIP, CNKI, and Wanfang databases. Articles from journals published in English were identified using PubMed and Web of Science. Original studies that included prevalence surveys of MCI were selected. Forty-eight relevant studies were included in the analysis, covering 22 provinces in China. Our results showed that the pooled prevalence of MCI in the older Chinese population was 14.71% (95% confidence interval [CI], 14.50-14.92%). The prevalence was 16.72% (95% CI, 15.68-17.71%) in clinical samples vs. 14.61% (95% CI, 14.40-14.83%) in nonclinical samples (χ2=16.60, P

Corneal neovascularization (CNV) is a common pathological change in ocular surface diseases. It is not only the factor that affects vision but also the main cause for corneal graft failure. Based on the mechanism of CNV, many achievements have been developed to suppress the formation of CNV. There are certain novel drugs such as aflibercept, gold nanoparticles, and netrins which provide us with new clues to treat CNV. However, we still need to develop more effective therapeutic drugs. It is of great significance to develop new effective drugs to suppress the occurrence of CNV. In this paper, recent drug therapies for CNV are reviewed. The electronic database (PubMed) is searched for relative basic research and randomized clinical trials that are published recently.

Respiration is an important physiological function in which air is moved into and out of the lungs. This paper designed a flexible patch for continuous detections of respiration, especially for evaluating nasal symmetry. The monitoring system comprises an integrated hot-film and two time-of-flight flow sensors on a flexible polyimide (PI) substrate. The 5 μm-thick polyimide substrate is flexible to construct wearable devices while the hot-wire flow sensor can monitor the respiration conditions and states, such as different breathing patterns of mouth or nose modes, normal or deep forms, and happy or nervous signs. Moreover, the power consumption of the system is low for possible long-term monitoring applications of sleep and medical diagnosis. The two time-of-flight sensors are aligned symmetrically on different sides of the hot-film heating element for differential signal readings, which used asymmetrical thermal distribution to reveal breathing state differences of two nostrils and they don’t require additional energy since they are located near the heating element. The system serves as a noninvasive wearable device to monitor respiratory airflow while avoiding uncomfortable nasal cannula. Various testing results show the feasibility and effectiveness of the proposed system.

Background Following trauma, the elbow is the most susceptible to restricted motion among all joints. Open arthrolysis is often performed for post-traumatic elbow stiffness if that stiffness does not improve with non-operative management. However, the optimal timing for performing an open arthrolysis remains controversial. The purpose of this study was to compare the outcome (elbow motion and function) and the rate of complications among patients who had undergone early, median and late release procedures to establish an optimal time interval following the injury, after which, an effective open arthrolysis can be performed. Methods In this retrospective cohort study, we included total 133 patients, who had undergone open arthrolysis for post-traumatic elbow stiffness. The subjects were divided into 3 groups, with 31 patients in the early release group (arthrolysis performed at 6–10 months after injury), 78 patients in the median release group (at 11–20 months), and 24 patients in the late release group (at > 20 months). The release procedure in all patients was performed by the same surgeon, using the same technique. The general data, functional performance, and complications, if any, were retrospectively documented for all patients and statistically analysed. Results The demographic data and disease characteristics of all patients were comparable at baseline. Postoperatively, no significant differences were found among the three groups with respect to the range of motion (p = 0.067), Mayo Elbow Performance Score (p = 0.350) and its ratings (p = 0.329), visual analog scale score for pain (p = 0.227), Dellon classification for ulnar nerve symptoms (p = 0.497), and each discrete complication (all p values > 0.05). Conclusions At the final follow-up, our results showed no significant difference in the postoperative elbow motion capacities, functional scores and the rates of complications among patients who had undergone an early, median, and late release. Therefore, we have recommended that an early arthrolysis would be preferable due to its multiple advantages, and the conventionally observed interval of > 1 year after the injury, could be shortened. Level of evidence Level III; Retrospective Cohort Design; Therapeutic Study.

Volume 8, no. 2, e00157-17, 2017, [https://doi.org/10.1128/mBio.00157-17][1]. One source of funding for our study was not properly acknowledged. The acknowledgment section of the manuscript should read: We regret to note that Mark A. Wainberg passed away in April 2017. Therefore, the corresponding

Human immunodeficiency virus (HIV) infection persists despite decades of active antiretroviral therapy (ART), effectively preventing viral eradication. Treatment decreases plasma viral RNA, but viral DNA persists, mostly integrated within the genome of nucleated blood cells. Viral DNA blood levels correlate with comorbidities and the rapidity of viral rebound following treatment interruption. To date, no intervention aiming at decreasing HIV DNA levels below those attained through ART has been successful. This includes use of some integrase inhibitors either as part of ART or in treatment intensification studies. We have argued that using the integrase inhibitor dolutegravir (DTG) in similar studies may yield better results, but this remains to be studied. In treatment-experienced individuals, the most frequent substitution associated with failure with dolutegravir is R263K in integrase. R263K decreases integration both in cell-free and tissue culture assays. We investigated here how integrated DNA levels evolve over time during prolonged infections with R263K viruses. To investigate a potential defect in reverse transcription with R263K, the levels of reverse transcripts were measured by quantitative PCR. We measured HIV type 1 (HIV-1) integration in Jurkat cells over the course of 4-week infections using Alu-mediated quantitative PCR. The results show that R263K did not decrease reverse transcription. Prolonged infections with R263K mutant viruses led to less HIV-1 integrated DNA over time compared to wild-type viruses. These tissue culture results help to explain the absence of the R263K substitution in most individuals experiencing failure with DTG and support studies aiming at longitudinally measuring the levels of integrated DNA in individuals treated with this drug., IMPORTANCE Antiretroviral treatment decreases plasma viral RNA, but HIV DNA persists for decades within infected cells. Studies of nonhuman primates have suggested that reducing retroviral DNA levels might represent a path to eradication. The integrase inhibitor dolutegravir is less susceptible than any other anti-HIV drug to the emergence of resistance in treatment-naive individuals. In treatment-experienced individuals, in contrast, rare cases of treatment failure were commonly associated with emergence of an R263K integrase substitution that confers low-level resistance to dolutegravir. It is unclear why this substitution is not more common in individuals experiencing failure with dolutegravir. We report here that R263K progressively diminishes the levels of integrated HIV-1 DNA in tissue culture over multiple cycles of infection. Our results help to explain aspects of the clinical efficacy of dolutegravir and suggest that this drug may be able to reduce HIV DNA levels within infected individuals compared to other drugs.

Caregivers tend to report role overload and nighttime awakening given the burden of caregiving. Previous research has not explored the association between role overload and nighttime awakening among dementia caregivers. Social engagement has been found to be associated with physical and psychological health outcomes of caregivers. Thus, the present study aims to examine whether role overload is associated with nighttime awakening of dementia caregivers and whether social engagement will mediate the association. We conducted a cross-sectional study by using the 2015 National Health and Aging Trends Study (NHATS) and National Study of Caregiving (NSOC). Six-hundred-and-sixty-nine dementia caregivers were included in the analysis. Relevant factors were controlled as covariates including age, gender, race/ethnicity, etc. Nearly 45% of participants reported suffering from nighttime awakening at “some” or “more” nights within one month, among which half of them reported “almost” or “every” night. Role overload was found associated with caregivers’ nighttime awakening (β=.135, 95%CI: .094 to.176, p

Greater impairment in autoregulation is seen in male versus female piglets following fluid percussion injury (FPI). This is partially mediated by a greater upregulation of extracellular signal-related kinase mitogen-activated protein kinase (ERK MAPK). We hypothesized that these trends would be reflected by the pressure reactivity index (PRx), a clinical measure of autoregulation. We further hypothesized that PRx values would correlate well with pial artery dilatory responses to hypotension. Male and female piglets were subjected to FPI and treated with a vehicle or ERK MAPK antagonist U 0126 (1 mg/kg IV) 30 min post-injury. FPI led to upregulation of CSF ERK MAPK in untreated piglets of both sexes, however significantly higher PRx values were seen in male versus female piglets. Following administration of U 0126, elevation of ERK MAPK levels was blocked in both sexes and PRx values were significantly improved in the male. A strong correlation was seen between the PRx and pial artery vasomotor activity. These data support previous observations that male piglets demonstrate reversible ERK MAPK-mediated impairment in autoregulation following FPI, which is reflected by the PRx. The strong correlation between the PRx and pial artery vasomotor activity supports the practice of continuously monitoring cerebrovascular autoregulation in patients using this index.

The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and has been reported for several treatment-experienced individuals receiving DTG as part of salvage therapy. The R263K substitution seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG-treated individuals. Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand transfer and 3′ processing activities were measured biochemically. The combination of the R263K and T66I substitutions decreased HIV-1 infectivity, replicative capacity, and strand transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL. These findings suggest that the presence of the T66I substitution will not compromise the activity of DTG and may also help to prevent the additional generation of the R263K mutation. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution.IMPORTANCEThe integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus type 1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I substitution, whereas R263K is a signature resistance substitution against dolutegravir. In order to determine how different combinations of integrase resistance mutations can influence the outcome of therapy, we report here the effects of the T66I, E138K, and R263K substitutions, alone and in combination, on viral replicative capacity and resistance to integrase inhibitors. Our results show that the addition of R263K to the T66I substitution diminishes viral replicative capacity and strand transfer activity while not compromising susceptibility to dolutegravir. This supports the use of dolutegravir in second-line therapy for patients failing elvitegravir therapy who harbor the T66I resistance substitution.

Objectives Dolutegravir is an integrase strand-transfer inhibitor that has shown unprecedented robustness against the emergence of HIV drug-resistant strains in treatment-naive individuals. The R263K substitution in integrase was identified through culture selection as a resistance-associated substitution for dolutegravir and was recently detected in two treatment-experienced participants in the SAILING clinical trial, who experienced dolutegravir-based treatment failure, one of whom was infected by a subtype C virus. The objective of this study was to characterize the R263K substitution in HIV-1 subtype C integrase. Design and methods We used cell-free strand transfer assays and tissue culture experiments to characterize the R263K substitution in HIV-1 subtype C integrase in comparison with subtype B. Results Cell-free biochemical assays showed that the R263K substitution diminished subtype C integrase strand-transfer activity by decreasing the affinity of integrase for target DNA. Similarly, both viral infectiousness and replication capacity were reduced by the R263K substitution in tissue culture. Decrease in enzyme activity and viral infectiousness exceeded 35 and 50%, respectively - significantly more than in HIV-1 subtype B. R263K in HIV-1 subtype C also conferred low levels of resistance against dolutegravir and high levels of cross-resistance against elvitegravir, but not raltegravir. Conclusions The R263K substitution is more deleterious to integrase strand-transfer activity and viral infectiousness in HIV-1 subtype C than in subtype B. Our results suggest that cross-resistance may prevent treatment-experienced individuals who are experiencing treatment failure with dolutegravir from being subsequently treated with elvitegravir.

Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α-deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α-dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α-dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.

We employ diffuse correlation and reflectance spectroscopies to monitor perfusion and oxygenation in mice after hindlimb ischemia. Perfusion results were compared with laser Doppler flowmetry and validated as new tools to assess limb perfusion.